Compositions and Methods for Simultaneously Modulating Expression of Genes

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 18, 2020, is named 57623_701_601_SL.txt and is 326,570 bytes in size.

BACKGROUND

Numerous human diseases and disorders are caused by combinations of higher and/or lower expression levels of certain proteins compared to the expression levels of these proteins in humans without the disease or disorder. Combinatorial therapies to increase the expression and/or secretion of a target protein and to decrease the expression of another, different target protein, may have a therapeutic effect. For example, therapies for coronavirus infection, e.g., COVID-19, the disease caused by infection with the coronavirus SARS-CoV-2, that effectively and specifically decrease production of one or more target gene products and concomitantly increase production of others are needed.

SUMMARY

The present invention relates to modulating expression of two or more proteins or nucleic acid sequences simultaneously using one recombinant polynucleic acid or RNA construct. In some embodiments, the recombinant polynucleic acid or RNA construct of the present invention simultaneously upregulate and downregulate the expression of two or more proteins or nucleic acid sequences by providing a nucleic acid sequence encoding a single or multiple small interfering RNA (siRNA) capable of binding to specific targets and a nucleic acid sequence encoding single or multiple proteins for overexpression. In some embodiments, the present invention is useful to treat diseases and disorders wherein a specific physiological mechanism (e.g., catabolism) can be controlled by siRNA while another physiological mechanism can be activated (e.g., anabolism) by overexpression of a therapeutic protein in parallel.

The invention also provides a recombinant polynucleic acid or RNA construct that comprises a polynucleic acid or RNA that encodes or comprises: one or more small interfering RNAs (siRNAs) that are capable of binding to one or more coronavirus target RNAs and/or one or more RNAs encoding a host protein, e.g., a viral entry element or a proinflammatory cytokine; and a nucleic acid sequence that encodes one or more proteins for overexpression, e.g., a host anti-inflammatory cytokine or a decoy protein, e.g., a soluble Angiotensin Converting Enzyme-2 (ACE2). In some embodiments, the coronavirus target RNA is an mRNA encoding one or more coronavirus proteins, or a noncoding RNA. The present invention thus provides embodiments wherein a single polynucleotide molecule both inhibit a virus and modulate the host inflammatory response.

In some embodiments, (i) and (ii) are comprised in 5′ to 3′ direction. In some embodiments, (i) and (ii) are not comprised in 5′ to 3′ direction. In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a linker. In some embodiments, the nucleic acid sequence encoding or comprising the linker connects (i) and (ii). In some embodiments, the linker comprises a tRNA linker. In some aspects, the nucleic acid sequence encoding or comprising the linker is at least 6 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is up to 80 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is about 6 to about 80 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is about 6 to about 15 nucleic acid residues in length.

In some embodiments, the recombinant polynucleic acid construct is circular. In some embodiments, the recombinant polynucleic acid construct is linear. In some embodiments, the recombinant polynucleic acid construct is DNA. In some embodiments, the recombinant polynucleic acid construct is RNA.

In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a poly(A) tail. In some embodiments, the poly(A) tail comprises 1-220 base pairs of poly(A) (SEQ ID NO: 191). In some embodiments, the recombinant polynucleic acid construct further comprises a 5′ cap. In some embodiments, the 5′ cap comprises an anti-reverse CAP analog, Clean Cap, Cap 0, Cap 1, Cap 2, or Locked Nucleic Acid cap (LNA-cap). In some embodiments, the 5′ cap comprises m₂^7,3′-OG(5′)ppp(5′)G, m7G, m7G(5′)G, m7GpppG, or m7GpppGm. In some embodiments, the recombinant polynucleic acid construct further comprises a promoter. In some embodiments, the promoter is selected from the group consisting of T3, T7, SP6, P60, Syn5, and KP34. In some embodiments, the promoter is a T7 promoter. In some embodiments, the T7 promoter is upstream of the at least one nucleic acid sequence encoding or comprising the siRNA. In some embodiments, the T7 promoter comprises a sequence comprising TAATACGACTCACTATA (SEQ ID NO: 25). In some embodiments, the recombinant polynucleic acid construct further comprises a Kozak sequence.

In some embodiments, the siRNA comprises 1-10 copies of siRNA. In some embodiments, the siRNA comprises a sense siRNA strand. In some embodiments, the siRNA comprises an anti-sense siRNA strand. In some embodiments, the siRNA comprises a sense and an anti-sense siRNA strand. In some embodiments, the siRNA does not affect the expression of the gene of interest. In some embodiments, the siRNA does not inhibit the expression of the gene of interest. In some embodiments, the recombinant polynucleic acid construct comprises two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA. In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a linker. In some embodiments, the nucleic acid sequence encoding or comprising the linker connects each of the two or more nucleic acid sequences encoding or comprising the siRNA capable of binding to the target mRNA. In some embodiments, the linker comprises a tRNA linker. In some embodiments, each of the two or more nucleic acid sequences encodes or comprises an siRNA capable of binding to a same target mRNA. In some embodiments, each of the two or more nucleic acid sequences encodes or comprises an siRNA capable of binding to a different target mRNA. In some embodiments, each of at least two of the two or more nucleic acid sequences encodes or comprises an siRNA capable of binding to the same target mRNA or different target mRNAs.

In some embodiments, the target RNA is an mRNA. In some embodiments, the target mRNA encodes a protein selected from the group consisting of Tumor Necrosis Factor alpha (TNF-alpha), interleukin, Angiotensin Converting Enzyme-2 (ACE2), SARS CoV-2 ORF1ab, SARS CoV-2 S, and SARS CoV-2 N. In some embodiments, the target RNA is an mRNA encoding a protein selected from the group consisting of Tumor Necrosis Factor alpha (TNF-alpha), interleukin, Angiotensin Converting Enzyme-2 (ACE2), SARS CoV-2 ORF1ab, SARS CoV-2 S, SARS CoV-2 N, Superoxide dismutase-1 (SOD1), and Activin receptor-like kinase-2 (ALK2).

In some embodiments, the target RNA is an mRNA encoding a protein selected from the group consisting of Interleukin 8 (IL-8), Interleukin 1 beta (IL-1 beta), Interleukin 17 (IL-17), Tumor Necrosis Factor alpha (TNF-alpha), Interleukin 6 (IL-6), Interleukin 6R (IL-6R), Interleukin 6R-alpha (IL-6R-alpha), Interleukin 6R-beta (IL-6R-beta), Angiotensin Converting Enzyme-2 (ACE2), SARS CoV-2 ORF1ab, SARS CoV-2 S, and SARS CoV-2 N. In some embodiments, the target RNA is an mRNA encoding a protein selected from the group consisting of Interleukin 8 (IL-8), Interleukin 1 beta (IL-1 beta), Interleukin 17 (IL-17), Tumor Necrosis Factor alpha (TNF-alpha), Interleukin 6 (IL-6), Interleukin 6R (IL-6R), Interleukin 6R-alpha (IL-6R-alpha), Interleukin 6R-beta (IL-6R-beta), Angiotensin Converting Enzyme-2 (ACE2), SARS CoV-2 ORF1ab, SARS CoV-2 S, SARS CoV-2 N, Superoxide dismutase-1 (SOD1), and Activin receptor-like kinase-2 (ALK2).

In some embodiments, the target mRNA encodes a protein selected from the group consisting of Interleukin 8 (IL-8), Interleukin 1 beta (IL-1 beta), Interleukin 17 (IL-17), and Tumor Necrosis Factor alpha (TNF-alpha).

In some embodiments, the target RNA is a coronavirus target RNA or a coronavirus host cell target RNA. In some embodiments, the coronavirus target RNA is an mRNA that encodes a coronavirus protein. In some embodiments, the coronavirus target RNA is a coronavirus noncoding RNA. In some embodiments, the coronavirus protein is a Spike protein (S), a Nucleocapsid protein (N), a non-structural protein (NSP), or an ORF1ab (polyprotein PP1ab) protein, e.g., a SARS CoV-2 NSP1 protein. In some embodiments, the coronavirus target RNA is a SARS CoV-2 NSP12 and 13 coding RNA. In some embodiments, the coronavirus host cell target is a host cell protein. In some embodiments, the host cell is a human cell. In some embodiments, the host cell protein is ACE2, IL-6, IL-6R-alpha, or IL-6R-beta.

In some embodiments, the expression of the target RNA is modulated by the siRNA capable of binding to the target RNA. In some embodiments, the expression of the target RNA is downregulated by the siRNA capable of binding to the target RNA. In some embodiments, the expression of the target RNA is modulated by the siRNA capable of binding to the target mRNA. In some embodiments, the expression of the target RNA is downregulated by the siRNA capable of binding to the target RNA. In some embodiments, the expression of the target RNA is modulated by the siRNA capable of specifically binding to the target RNA. In some embodiments, the expression of the target RNA is downregulated by the siRNA capable of specifically binding to the target RNA.

In some embodiments, the recombinant nucleic acid construct comprises two or more nucleic acid sequences encoding a gene of interest. In some embodiments, each of the two or more nucleic acid sequences encodes a same gene of interest. In some embodiments, each of the two or more nucleic acid sequences encodes a different gene of interest. In some embodiments, each of the two or more nucleic acid sequences encoding the gene of interest comprises a nucleic acid sequence encoding a secretory protein. In some embodiments, each of the two or more nucleic acid sequences encoding the gene of interest comprises a nucleic acid sequence encoding an intracellular protein. In some embodiments, each of the two or more nucleic acid sequences encoding the gene of interest comprises a nucleic acid sequence encoding an intraorganelle protein. In some embodiments, each of the two or more nucleic acid sequences encoding the gene of interest comprises a nucleic acid sequence encoding a membrane protein. In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a linker. In some embodiments, the nucleic acid sequence encoding or comprising the linker connects each of the two or more nucleic acid sequences encoding the gene of interest. In some embodiments, the linker comprises a 2A peptide linker or a tRNA linker. In some embodiments, the gene of interest is selected from the group consisting of Insulin-like Growth Factor 1 (IGF-1), Interleukin 4 (IL-4), Interferon beta (IFN beta), Interferon alpha (IFN alpha), ACE2 soluble receptor, Interleukin 37 (IL-37), and Interleukin 38 (IL-38). In some embodiments, the gene of interest is selected from the group consisting of Insulin-like Growth Factor 1 (IGF-1), Interleukin 4 (IL-4), Interferon beta (IFN beta), and ACE2 soluble receptor. In some embodiments, the gene of interest is selected from the group consisting of Insulin-like Growth Factor 1 (IGF-1), Interleukin 4 (IL-4), Interferon beta (IFN beta), ACE2 soluble receptor, and Erythropoietin (EPO). In some embodiments, the gene of interest is selected from the group consisting of Insulin-like Growth Factor 1 (IGF-1), and Interleukin 4.

In some embodiments, the gene of interest encodes a coronavirus host protein. In some embodiments, the host protein encoded by the gene of interest is selected from: an IFN-α, e.g., interferon alpha-n3, interferon alpha-2a, or interferon alpha-2b, an IFN-β, an IFN-δ, an IFN-ε, an IFN-κ, an IFN-ν, an IFN-τ, an IFN-ω, an IFN-γ, an IFN-λ, IL-37, IL-38, and a soluble ACE2 receptor.

In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding a target motif. In some embodiments, the nucleic acid sequence encoding the target motif is operably linked to the at least one nucleic acid sequence encoding the gene of interest. In some embodiments, the target motif comprises a signal peptide, a nuclear localization signal (NLS), a nucleolar localization signal (NoLS), a lysosomal targeting signal, a mitochondrial targeting signal, a peroxisomal targeting signal, a microtubule tip localization signal (MtLS), an endosomal targeting signal, a chloroplast targeting signal, a Golgi targeting signal, an endoplasmic reticulum (ER) targeting signal, a proteasomal targeting signal, a membrane targeting signal, a transmembrane targeting signal, or a centrosomal localization signal (CLS). In some embodiments, the target motif is selected from the group consisting of (a) a target motif heterologous to a protein encoded by the gene of interest; (b) a target motif heterologous to a protein encoded by the gene of interest, wherein the target motif heterologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; (c) a target motif homologous to a protein encoded by the gene of interest, wherein the target motif homologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; and (d) a naturally occurring amino acid sequence which does not have the function of a target motif in nature, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion, and/or substitution of at least one amino acid. In some embodiments, the signal peptide is selected from the group consisting of (a) a signal peptide heterologous to a protein encoded by the gene of interest; (b) a signal peptide heterologous to a protein encoded by the gene of interest, wherein the signal peptide heterologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid, with proviso that the protein is not an oxidoreductase; (c) a signal peptide is homologous to a protein encoded by the gene of interest, wherein the signal peptide is homologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; and (d) a naturally occurring amino acid sequence which does not have the function of a signal peptide in nature, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion, and/or substitution of at least one amino acid. In some embodiments, the amino acids 1-9 of the N-terminal end of the signal peptide have an average hydrophobic score of above 2. In some embodiments, the recombinant polynucleic acid construct is a vector suitable for gene therapy. In some aspects, the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are comprised in a sequential manner. In some aspects, the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are present in a sequential manner. In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is downstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream or downstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the siRNA capable of binding to a target RNA binds to an exon of a target mRNA. In some aspects, the siRNA capable of binding to a target RNA specifically binds to one target RNA. In some aspects, the siRNA capable of binding to a target RNA is not encoded by or comprised of an intron sequence of the gene of interest. In some aspects, the gene of interest is expressed without RNA splicing.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to a target RNA (e.g., mRNA); and (ii) an mRNA encoding a gene of interest; wherein the target RNA is different from the mRNA encoding the gene of interest. In some embodiments, the target RNA is an mRNA.

In some embodiments, (i) and (ii) are comprised in 5′ to 3′ direction. In some embodiments, (i) and (ii) are not comprised in 5′ to 3′ direction. In some embodiments, the recombinant RNA construct further encodes or comprises a linker. In some embodiments, the nucleic acid sequence encoding or comprising the linker connects (i) and (ii). In some embodiments, the linker comprises a tRNA linker.

In some embodiments, the recombinant RNA construct further comprises a poly(A) tail. In some embodiments, the poly(A) tail comprises 1-220 base pairs of poly(A) (SEQ ID NO: 191). In some embodiments, the recombinant RNA construct further comprises a 5′ cap. In some embodiments, the 5′ cap comprises an anti-reverse CAP analog, Clean Cap, Cap 0, Cap 1, Cap 2, or Locked Nucleic Acid cap (LNA-cap). In some embodiments, the 5′ cap comprises m₂^7,3′-OG(5′)ppp(5′)G, m7G, m7G(5′)G, m7GpppG, or m7GpppGm. In some embodiments, the recombinant RNA construct further comprises a Kozak sequence.

In some embodiments, the siRNA comprises 1-10 copies of siRNA. In some embodiments, the siRNA comprises a sense siRNA strand. In some embodiments, the siRNA comprises an anti-sense siRNA strand. In some embodiments, the siRNA comprises a sense and an anti-sense siRNA strand. In some embodiments, the siRNA does not affect the expression of the gene of interest. In some embodiments, the siRNA does not inhibit the expression of the gene of interest. In some embodiments, the recombinant RNA construct comprises two or more nucleic acid sequences comprising an siRNA capable of binding to a target mRNA. In some embodiments, the recombinant RNA construct further comprises a linker. In some embodiments, the linker connects each of the two or more nucleic acid sequences comprising the siRNA capable of binding to the target mRNA. In some embodiments, the linker comprises a tRNA linker. In some embodiments, each of the two or more nucleic acid sequences comprises an siRNA capable of binding to a same target mRNA. In some embodiments, each of the two or more nucleic acid sequences comprises an siRNA capable of binding to a different target mRNA. In some embodiments, at least two of the two or more nucleic acid sequences encodes or comprises an siRNA capable of binding to the same or a different target mRNA.

In some embodiments, the target RNA is an mRNA encoding a protein selected from the group consisting of Tumor Necrosis Factor alpha (TNF-alpha), interleukin, Angiotensin Converting Enzyme-2 (ACE2), SARS CoV-2 ORF1ab, SARS CoV-2 S, and SARS CoV-2 N. In some embodiments, the target RNA is an mRNA encoding a protein selected from the group consisting of Tumor Necrosis Factor alpha (TNF-alpha), interleukin, Angiotensin Converting Enzyme-2 (ACE2), SARS CoV-2 ORF1ab, SARS CoV-2 S, SARS CoV-2 N, Superoxide dismutase-1 (SOD1), and Activin receptor-like kinase-2 (ALK2).

In some embodiments, the target mRNA is selected from the group consisting of Interleukin 8 (IL-8), Interleukin 1 beta (IL-1 beta), Interleukin 17 (IL-17), and Tumor Necrosis Factor alpha (TNF-alpha).

In some embodiments, the expression of the target mRNA is modulated by the siRNA capable of binding to the target mRNA. In some embodiments, the expression of the target mRNA is downregulated by the siRNA capable of binding to the target mRNA.

In some embodiments, the recombinant RNA construct comprises two or more nucleic acid sequences encoding a gene of interest. In some embodiments, each of the two or more nucleic acid sequences encodes a same gene of interest. In some embodiments, each of the two or more nucleic acid sequences encodes a different gene of interest. In some embodiments, each of the two or more nucleic acid sequences encoding the gene of interest comprises a nucleic acid sequence encoding a secretory protein. In some embodiments, each of the two or more nucleic acid sequences encoding the gene of interest comprises a nucleic acid sequence encoding an intracellular protein. In some embodiments, each of the two or more nucleic acid sequences encoding the gene of interest comprises a nucleic acid sequence encoding an intraorganelle protein. In some embodiments, each of the two or more nucleic acid sequences encoding the gene of interest comprises a nucleic acid sequence encoding a membrane protein. In some embodiments, the recombinant RNA construct further comprises a linker or a nucleic acid sequence encoding a linker. In some embodiments, the nucleic acid sequence encoding or comprising the linker connects each of the two or more nucleic acid sequences encoding the gene of interest. In some embodiments, the linker comprises a 2A peptide linker, a tRNA linker or a flexible linker. In some embodiments, the gene of interest is selected from the group consisting of Insulin-like Growth Factor 1 (IGF-1), Interleukin 4 (IL-4), Interferon beta (IFN beta), Interferon alpha (IFN alpha), ACE2 soluble receptor, Interleukin 37 (IL-37), and Interleukin 38 (IL-38). In some embodiments, the gene of interest is selected from the group consisting of Insulin-like Growth Factor 1 (IGF-1), Interleukin 4 (IL-4), Interferon beta (IFN beta), and ACE2 soluble receptor. In some embodiments, the gene of interest is selected from the group consisting of Insulin-like Growth Factor 1 (IGF-1), Interleukin 4 (IL-4), Interferon beta (IFN beta), ACE2 soluble receptor, and Erythropoietin (EPO). In some embodiments, the gene of interest is selected from the group consisting of Insulin-like Growth Factor 1 (IGF-1), and IL-4.

In some embodiments, the gene of interest encodes a coronavirus host protein. In some embodiments, the host protein is selected from: an IFN-α, e.g., interferon alpha-n3, interferon alpha-2a, or interferon alpha-2b, an IFN-β, an IFN-δ, an IFN-ε, an IFN-κ, an IFN-ν, an IFN-τ, an IFN-ω, an IFN-γ, an IFN-λ, IL-37, IL-38, and a soluble ACE2 receptor.

In some embodiments, the recombinant RNA construct further comprises a nucleic acid sequence encoding a target motif. In some embodiments, the nucleic acid sequence encoding the target motif is operably linked to the at least one nucleic acid sequence encoding the gene of interest. In some embodiments, the target motif comprises a signal peptide, a nuclear localization signal (NLS), a nucleolar localization signal (NoLS), a lysosomal targeting signal, a mitochondrial targeting signal, a peroxisomal targeting signal, a microtubule tip localization signal (MtLS), an endosomal targeting signal, a chloroplast targeting signal, a Golgi targeting signal, an endoplasmic reticulum (ER) targeting signal, a proteasomal targeting signal, a membrane targeting signal, a transmembrane targeting signal, or a centrosomal localization signal (CLS). In some embodiments, the target motif is selected from the group consisting of (a) a target motif heterologous to a protein encoded by the gene of interest; (b) a target motif heterologous to a protein encoded by the gene of interest, wherein the target motif heterologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; (c) a target motif homologous to a protein encoded by the gene of interest, wherein the target motif homologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; and (d) a naturally occurring amino acid sequence which does not have the function of a target motif in nature, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion, and/or substitution of at least one amino acid.

In some embodiments, the signal peptide is selected from the group consisting of (a) a signal peptide heterologous to a protein encoded by the gene of interest; (b) a signal peptide heterologous to a protein encoded by the gene of interest, wherein the signal peptide heterologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid, with proviso that the protein is not an oxidoreductase; (c) a signal peptide is homologous to a protein encoded by the gene of interest, wherein the signal peptide is homologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; and (d) a naturally occurring amino acid sequence which does not have the function of a signal peptide in nature, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion, and/or substitution of at least one amino acid. In some embodiments, the amino acids 1-9 of the N-terminal end of the signal peptide have an average hydrophobic score of above 2.

In some aspects, provided herein, is a cell comprising the composition of any recombinant polynucleic acid or RNA construct described herein. In some aspects, provided herein, is a pharmaceutical composition comprising the composition of any recombinant polynucleic acid or RNA construct described herein and a pharmaceutically acceptable excipient. In some aspects, provided herein, is a method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject the pharmaceutical composition described herein. In some embodiments, the disease or the condition is selected from the group consisting of intervertebral disc disease (IVDD), osteoarthritis, and psoriasis. In some embodiments, the disease or the condition is selected from the group consisting of intervertebral disc disease (IVDD), osteoarthritis, psoriasis, fibrodysplasia ossificans progressiva (FOP) and Amyotrophic lateral sclerosis (ALS). In some embodiments, the disease or the condition is selected from the group consisting of intervertebral disc disease (IVDD), osteoarthritis, psoriasis, fibrodysplasia ossificans progressiva (FOP), amyotrophic lateral sclerosis (ALS), and a coronavirus infection, or a disease or condition resulting from or associated with a coronavirus infection. In some embodiments, the subject is a human.

In some aspects, provided herein, is a method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject a pharmaceutical composition described herein. In some embodiments, the disease or condition in the subject is a coronavirus infection, or a disease or condition resulting from or associated with a coronavirus infection. In some embodiments, the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV-2. In some embodiments, the disease or disorder is SARS, MERS, or COVID-19.

In some aspects, provided herein, is a method of simultaneously expressing an siRNA and an mRNA from a single RNA transcript in a cell, comprising introducing into the cell the composition of any recombinant polynucleic acid or RNA construct described herein. In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target messenger RNA (mRNA); and (ii) at least one nucleic acid sequence encoding a gene of interest; wherein the target mRNA is different from an mRNA encoded by the gene of interest, and wherein the expression of the target mRNA and the gene of interest is modulated simultaneously.

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target messenger RNA (mRNA); and (ii) at least one nucleic acid sequence encoding a gene of interest; wherein the target mRNA is different from an mRNA encoded by the gene of interest, and wherein the expression of the target mRNA is downregulated and the expression of the gene of interest is upregulated simultaneously. In some embodiments, the expression of the target mRNA is downregulated by the siRNA capable of binding to the target mRNA. In some embodiments, the expression of the gene of interest is upregulated by expressing an mRNA or a protein encoded by the gene of interest.

In some aspects, provided herein, is a method of producing an RNA construct comprising a small interfering RNA (siRNA) capable of binding to a target messenger RNA (mRNA), and an mRNA encoding a gene of interest, wherein the target mRNA is different from the mRNA encoding the gene of interest, the method comprising: (a) providing, for in vitro transcription reaction: (i) a polynucleic acid construct comprising a promoter, at least one nucleic acid sequence encoding an siRNA capable of binding to a target mRNA, at least one nucleic acid sequence encoding a gene of interest, and a nucleic acid sequence encoding poly(A) tail; (ii) an RNA polymerase; and (iii) a mixture of nucleotide triphosphates (NTPs); and (b) isolating and purifying transcribed RNAs from the in vitro transcription reaction mixture, thus producing the RNA construct. In some embodiments, the RNA polymerase is selected from the group consisting of T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, P60 RNA polymerase, Syn5 RNA polymerase, and KP34 RNA polymerase. In some embodiments, the RNA polymerase is T7 RNA polymerase. In some embodiments, the mixture of NTPs comprises unmodified NTPs. In some embodiments, the mixture of NTPs comprises modified NTPs. In some embodiments, the modified NTPs comprise N¹-methylpseudouridine, Pseudouridine, N¹-Ethylpseudouridine, N¹-Methoxymethylpseudouridine, N¹-Propylpseudouridine, 2-thiouridine, 4-thiouridine, 5-methoxyuridine, 5-methylurdine, 5-carboxymethylesteruridine, 5-formyluridine, 5-carboxyuridine, 5-hydroxyuridine, 5-Bromouridine, 5-lodouridine, 5,6-dihydrouridine, 6-Azauridine, Thienouridine, 3-methyluridine, 1-carboxymethyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, dihydrouridine, dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-methylcytidine, 5-methoxycytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, 5-hydroxycytidine, 5-lodocytidine, 5-Bromocytidine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, 3-methyl-cytidine, N⁴-acetylcytidine, 5-formylcytidine, N⁴-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine, N¹-methyladenosine, N⁶-methyladenosine, N⁶-methyl-2-Aminoadenosine, N⁶-isopentenyladenosine, N⁶,N⁶-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In some embodiments, step (a) further comprises providing a capping enzyme. In some embodiments, isolating and purifying transcribed RNAs comprise column purification.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to Interleukin 8 (IL-8) messenger RNA (mRNA); and (ii) an mRNA encoding Insulin-like Growth Factor 1 (IGF-1). In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to Interleukin 1 beta (IL-1 beta) messenger RNA (mRNA); and (ii) an mRNA encoding Insulin-like Growth Factor 1 (IGF-1). In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to Interleukin 17 (IL-17) messenger RNA (mRNA); and (ii) an mRNA encoding Interleukin 4 (IL-4). In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to Tumor Necrosis Factor alpha (TNF-alpha) messenger RNA (mRNA); and (ii) an mRNA encoding Interleukin 4 (IL-4). In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to Tumor Necrosis Factor alpha (TNF-alpha) messenger RNA (mRNA) and a small interfering RNA (siRNA) capable of binding to Interleukin 17 (IL-17) messenger RNA (mRNA); and (ii) an mRNA encoding Interleukin 4 (IL-4). In related aspects, the composition comprises or encodes at least 2, 3, 4, 5, or 6 siRNAs.

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8.

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to IL-6 mRNA; and (ii) an mRNA encoding Interferon beta (IFN-beta). In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 1 siRNA directed to IL-6 mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to IL-6 mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 29 or 30 (Compound B1 or B2).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to Interleukin 6R (IL-6R) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 1 siRNA directed to IL-6R mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to IL-6R mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 31 (Compound B3).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct encoding or comprising (i) at least one siRNA capable of binding to Interleukin 6R alpha (IL-6R-alpha) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 1 siRNA directed to IL-6R-alpha mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to IL-6R-alpha mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 32 (Compound B4).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct encoding or comprising (i) at least one siRNA capable of binding to Interleukin 6R beta (IL-6R-beta) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 1 siRNA directed to IL-6R-beta mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to IL-6R-beta mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 33 (Compound B5).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct encoding or comprising (i) at least one siRNA capable of binding to ACE2 mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 1 siRNA directed to ACE2 mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to ACE2 mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 34 or 35 (Compound B6 or B7).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct: encoding or comprising (i) at least one siRNA capable of binding to SARS CoV-2 ORF1ab mRNA, at least one siRNA capable of binding to SARS CoV-2 S mRNA, at least one siRNA capable of binding to SARS CoV-2 N mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 3 siRNAs, one directed to SARS CoV-2 ORF1ab mRNA, one directed to SARS CoV-2 S mRNA, and one directed to SARS CoV-2 N mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In certain aspects, such a composition, e.g., a composition comprising Compound B8 (SEQ ID NO: 36) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, SARS CoV-2, or both. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 36.

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct encoding or comprising (i) at least one siRNA capable of binding to SARS CoV-2 N mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 1 siRNA directed to SARS CoV-2 N mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to SARS CoV-2 N mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 38 (Compound B10).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct: encoding or comprising (i) at least one siRNA capable of binding to SARS CoV-2 ORF1ab mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes 1 siRNA directed to SARS CoV-2 ORF1ab mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to SARS CoV-2 ORF1ab mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In certain aspects, such a composition, including a composition comprising Compound B12 (SEQ ID NO: 40) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, MERS-CoV, or both. In certain aspects, such a composition, including a composition comprising Compound B13 (SEQ ID NO: 41) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, SARS CoV-2, and/or MERS-CoV. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in any one of SEQ ID NOs: 40, 41 and 42 (Compounds B12, B13, and B14).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct: encoding or comprising (i) at least one siRNA capable of binding to IL-6 mRNA, at least one siRNA capable of binding to ACE2 mRNA, and at least one siRNA capable of binding to SARS CoV-2 S mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 3 siRNAs, one directed IL-6 mRNA, one directed to ACE2 mRNA, and one directed to SARS CoV-2 S mRNA. In related aspects, the mRNA encoding IFN-beta encodes the native IFN-beta signal peptide, or a modified signal peptide. In related aspects, the modified IFN-beta signal peptide is SP1 or SP2 as described herein (SEQ ID NOs: 52 and 54, respectively). In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in any one of SEQ ID NOs: 43, 44, and 45 (Compounds B15, B16, and B17).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct encoding or comprising (i) at least one small interfering RNA capable of binding to SARS CoV-2 ORF1ab mRNA, at least one siRNA capable of binding to SARS CoV-2 S mRNA, and at least one siRNA capable of binding to SARS CoV-2 N mRNA; and (ii) an mRNA encoding the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 3 siRNAs, one directed to ORF1ab mRNA, one directed to SARS CoV-2 S mRNA, and one directed to SARS CoV-2 N mRNA. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 46 (Compound B18). In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 190 (Compound B18).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct: encoding or comprising (i) at least one siRNA capable of binding to SARS CoV-2 S mRNA; and (ii) an mRNA encoding the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 1 siRNA directed to SARS CoV-2 S mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to SARS CoV-2 S mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 47 (Compound B19).

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of binding to an IL-6 mRNA; and (ii) an mRNA encoding interferon-beta (IFN-beta). In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the recombinant RNA construct comprises at least 1, 2, or 3 siRNAs. In related aspects, the recombinant RNA construct comprises 1 siRNA directed to an IL-6 mRNA. In related aspects, the recombinant RNA construct comprises 3 siRNAs, each directed to an IL-6 mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by a sequence as set forth in SEQ ID NO: 29 or 30.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of binding to an Interleukin 6R (IL-6R) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the recombinant RNA construct comprises at least 1, 2, or 3 siRNAs. In related aspects, the recombinant RNA construct comprises 1 siRNA directed to an IL-6R mRNA. In related aspects, the recombinant RNA construct comprises 3 siRNAs, each directed to an IL-6R mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by a sequence as set forth in SEQ ID NO: 31.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of binding to an Interleukin 6R alpha (IL-6R-alpha) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the recombinant RNA construct comprises at least 1, 2, or 3 siRNAs. In related aspects, the recombinant RNA construct comprises 1 siRNA directed to an IL-6R-alpha mRNA. In related aspects, the recombinant RNA construct comprises 3 siRNAs, each directed to an IL-6R-alpha mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by the sequence as set forth in SEQ ID NO: 32.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of binding to an Interleukin 6R beta (IL-6R-beta) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes an ACE2 soluble receptor. In related aspects, the recombinant RNA construct comprises at least 1, 2, or 3 siRNAs. In related aspects, the recombinant RNA construct comprises 1 siRNA directed to an IL-6R-beta mRNA. In related aspects, the recombinant RNA construct comprises 3 siRNAs, each directed to an IL-6R-beta mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by the sequence as set forth in SEQ ID NO: 33.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of binding to an ACE2 mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the recombinant RNA construct comprises at least 1, 2, or 3 siRNAs. In related aspects, the recombinant RNA construct comprises 1 siRNA directed to an ACE2 mRNA. In related aspects, the recombinant RNA construct comprises 3 siRNAs, each directed to an ACE2 mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by a sequence as set forth in SEQ ID NO: 34 or 35.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of binding to a SARS CoV-2 ORF1ab mRNA, at least one siRNA capable of binding to a SARS CoV-2 S mRNA, at least one siRNA capable of binding to a SARS CoV-2 N mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the recombinant RNA construct comprises at least 1, 2, or 3 siRNAs. In related aspects, the recombinant RNA construct comprises 3 siRNAs, one directed to a SARS CoV-2 ORF1ab mRNA, one directed to a SARS CoV-2 S mRNA, and one directed to a SARS CoV-2 N mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In certain aspects, such a composition, e.g., a composition comprising Compound B8 (SEQ ID NO: 36), is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, SARS CoV-2, or both. In related aspects, the recombinant RNA construct comprises a sequence encoded by the sequence as set forth in SEQ ID NO: 36.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of binding to a SARS CoV-2 S mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the recombinant RNA construct comprises at least 1, 2, or 3 siRNAs. In related aspects, the recombinant RNA construct comprises 1 siRNA directed to a SARS CoV-2 S mRNA. In related aspects, the recombinant RNA construct comprises 3 siRNAs, each directed to a SARS CoV-2 S mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by a sequence as set forth in SEQ ID NO: 37 or 39.

In some aspects, provided herein, is a recombinant RNA construct comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of binding to a SARS CoV-2 N mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the recombinant RNA construct comprises at least 1, 2, or 3 siRNAs. In related aspects, the recombinant RNA construct comprises 1 siRNA directed to a SARS CoV-2 N mRNA. In related aspects, the recombinant RNA construct comprises 3 siRNAs, each directed to a SARS CoV-2 N mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by the sequence as set forth in SEQ ID NO: 38.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of binding to a SARS CoV-2 ORF1ab mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the recombinant RNA construct comprises 1 siRNA directed to a SARS CoV-2 ORF1ab mRNA. In related aspects, the recombinant RNA construct comprises 3 siRNAs, each directed to a SARS CoV-2 ORF1ab mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In certain aspects, such a composition, including a composition comprising Compound B12 (SEQ ID NO: 40) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, MERS, or both. In certain aspects, such a composition, including a composition comprising Compound B13 (SEQ ID NO: 41) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, SARS CoV-2, and/or MERS. In related aspects, the recombinant RNA construct comprises a sequence encoded by a sequence as set forth in any one of SEQ ID NOs: 40, 41 and 42.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of binding to an IL-6 mRNA, at least one siRNA capable of binding to an ACE2 mRNA, and at least one siRNA capable of binding to a SARS CoV-2 S mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the recombinant RNA construct comprises at least 1, 2, or 3 siRNAs. In related aspects, the recombinant RNA construct comprises 3 siRNAs, one directed to an IL-6 mRNA, one directed to an ACE2 mRNA, and one directed to a SARS CoV-2 S mRNA. In related aspects, the mRNA encoding IFN-beta encodes the native IFN-beta signal peptide, or a modified signal peptide. In related aspects, the modified IFN-beta signal peptide is SP1 or SP2 as described herein (SEQ ID NOs: 52 and 54, respectively). In related aspects, the recombinant RNA construct comprises a sequence encoded by a sequence as set forth in any one of SEQ ID NOs: 43, 44, and 45.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one small interfering RNA capable of binding to a SARS CoV-2 ORF1ab mRNA, at least one siRNA capable of binding to a SARS CoV-2 S mRNA, and at least one siRNA capable of binding to a SARS CoV-2 N mRNA; and (ii) an mRNA encoding an ACE2 soluble receptor. In related aspects, the recombinant RNA construct comprises at least 1, 2, or 3 siRNAs. In related aspects, the recombinant RNA construct comprises 3 siRNAs, one directed to an ORF1ab mRNA, one directed to a SARS CoV-2 S mRNA, and one directed to a SARS CoV-2 N mRNA. In related aspects, the recombinant RNA construct comprises a sequence encoded by the sequence as set forth in SEQ ID NO: 46.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of binding to a SARS CoV-2 S mRNA; and (ii) an mRNA encoding an ACE2 soluble receptor. In related aspects, the recombinant RNA construct comprises at least 1, 2, or 3 siRNAs. In related aspects, the recombinant RNA construct comprises 1 siRNA directed to a SARS CoV-2 S mRNA. In related aspects, the recombinant RNA construct comprises 3 siRNAs, each directed to a SARS CoV-2 S mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof.

In related aspects, the recombinant RNA construct comprises a sequence encoded by the sequence as set forth in SEQ ID NO: 47.

In some embodiments, a polynucleic acid construct of the present invention comprises: (i) an siRNA that targets an RNA selected from: an IL-8 mRNA, an IL-1 beta mRNA, an IL-17 mRNA, a TNF-alpha mRNA, a SARS CoV-2 ORF1ab RNA (polyprotein PP1ab, e.g., in a noncoding region or where it encodes a protein that is selected from: a SARS CoV-2 nonstructure protein (NSP), Nsp1, Nsp3 (Nsp3b, Nsp3c, PLpro, and Nsp3e), Nsp7_Nsp8 complex, Nsp9-Nsp10, and Nsp14-Nsp16, 3CLpro, E-channel (E protein), ORF7a, C-terminal RNA binding domain (CRBD), N-terminal RNA binding domain (NRBD), helicase, and RdRp), a SARS CoV-2 Spike protein (S) mRNA, a SARS CoV-2 Nucleocapsid protein (N) mRNA, a tumor necrosis factor alpha (TNF-alpha) mRNA, an interleukin mRNA (including but not limited to interleukin 1 (e.g., IL-1alpha, IL-1beta), interleukin 6 (IL-6), interleukin 6R (IL-6R), interleukin 6R alpha (IL-6R-alpha), interleukin 6R beta (IL-6R-beta), interleukin 18 (IL-18), interleukin 36-alpha (IL-36-alpha), interleukin 36-beta (IL-36-beta), interleukin 36-gamma (IL-36-gamma), interleukin 33 (IL-33)), an Angiotensin Converting Enzyme-2 (ACE2) mRNA, a transmembrane protease, serine 2 (TMPRSS2) mRNA, and a coding NSP12 and 13 RNA; and (ii) at least one gene of interest that encodes, or at least one mRNA that encodes, a protein to be overexpressed, wherein the protein is selected from: IGF-1, IL-4, IGF-1 (including derivatives thereof as described elsewhere herein), carboxypeptidases (e.g., ACE, ACE2, CNDP1, CPA1, CPA2, CPA4, CPA5, CPA6, CPB1, CPB2, CPE, CPN1, CPQ, CPXM1, CPZ, SCPEP1); cytokines (e.g., BMP1, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, C1QTNF4, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL3L3, CCL4, CCL4L, CCL4L2, CCL5, CCL7, CCL8, CD40LG, CER1, CKLF, CLCF1, CNTF, CSF1, CSF2, CSF3, CTF1, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, CXCL17, CXCL2, CXCL3, CXCL5, CXCL8, CXCL9, DKK1, DKK2, DKK3, DKK4, EDA, EBI3, FAM3B, FAM3C, FASLG, FLT3LG, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF5, GDF6, GDF7, GDF9, GPI, GREM1, GREM2, GRN, IFNA1, IFNA13, IFNA10, IFNA14, IFNA16, IFNA17, IFNA2, IFNA21, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNB1, IFNE, IFNG, IFNK, IFNL1, IFNL2, IFNL3, IFNL4, IFNW1, IL10, IL11, IL12A, IL12B, IL13, IL15, IL16, IL17A, IL17B, IL17C, IL17D, IL17F, IL18, IL19, IL1A, IL1B, IL1F10, IL2, IL20, IL21, IL22, IL23A, IL24, IL25, IL26, IL27, IL3, IL31, IL32, IL33, IL34, IL36A, IL36B, IL36G, IL36RN, IL37, IL4, IL5, IL6, IL7, IL9, LEFTY1, LEFTY2, LIF, LTA, MIF, MSTN, NAMPT, NODAL, OSM, PF4, PF4V1, SCGB3A1, SECTM1, SLURP1, SPP1, THNSL2, THPO, TNF, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TSLP, VSTM1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, and XCL2); extracellular ligands and transporters (e.g., APCS, CHI3L1, CHI3L2, CLEC3B, DMBT1, DMKN, EDDM3A, EDDM3B, EFNA4, EMC10, ENAM, EPYC, ERVH48-1, F13B, FCN1, FCN2, GLDN, GPLD1, HEG1, ITFG1, KAZALD1, KCP, LACRT, LEG1, METRN, NOTCH2NL, NPNT, OLFM1, OLFML3, PRB2, PSAP, PSAPL1, PSG1, PSG6, PSG9, PTX3, PTX4, RBP4, RNASE10, RNASE12, RNASE13, RNASE9, RSPRY1, RTBDN, S100A12, S100A13, S100A7, S100A8, SAA2, SAA4, SCG1, SCG2, SCG3, SCGB1C1, SCGB1C2, SCGB1D1, SCGB1D2, SCGB1D4, SCGB2B2, SCGB3A2, SCGN, SCRG1, SCUBE1, SCUBE2, SCUBE3, SDCBP, SELENOP, SFTA2, SFTA3, SFTPA1, SFTPA2, SFTPC, SFTPD, SHBG, SLURP2, SMOC1, SMOC2, SMR3A, SMR3B, SNCA, SPATA20, SPATA6, SOGA1, SPARC, SPARCL1, SPATA20, SPATA6, SRPX2, SSC4D, STX1A, SUSD4, SVBP, TCN1, TCN2, TCTN1, TF, TULP3, TFF2, TFF3, THSD7A, TINAG, TINAGL1, TMEFF2, TMEM25, VWC2L); extracellular matrix proteins (e.g., ABI3BP, AGRN, CCBE1, CHL1, COL15A1, COL19A1, COLEC11, DMBT1, DRAXIN, EDIL3, ELN, EMID1, EMILIN1, EMILIN2, EMILIN3, EPDR1, FBLN1, FBLN2, FBLN5, FLRT1, FLRT2, FLRT3, FREM1, GLDN, IBSP, KERA, KIAA0100, KIRREL3, KRT10, LAMB2, MGP, RPTN, SBSPON, SDC1, SDC4, SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, SIGLEC1, SIGLEC10, SIGLEC6, SLIT1, SLIT2, SLIT3, SLITRK1, SNED1, SNORC, SPACA3, SPACA7, SPON1, SPON2, STATH, SVEP1, TECTA, TECTB, TNC, TNN, TNR, TNXB); glucosidases (AMY1A, AMY1B, AMY1C, AMY2A, AMY2B, CEMIP, CHIA, CHIT1, FUCA2, GLB1L, GLB1L2, HPSE, HYAL1, HYAL3, KL, LYG1, LYG2, LYZL1, LYZL2, MAN2B2, SMPD1, SMPDL3B, SPACA5, SPACA5B); glycosyltransferases (e.g., ARTS, B4GALT1, EXTL2, GALNT1, GALNT2, GLT1D1, MGAT4A, ST3GAL1, ST3GAL2, ST3GAL3, ST3GAL4, ST6GAL1, XYLT1); growth factors (e.g., AMH, ARTN, BTC, CDNF, CFC1, CFC1B, CHRDL1, CHRDL2, CLEC11A, CNMD, EFEMP1, EGF, EGFL6, EGFL7, EGFL8, EPGN, EREG, EYS, FGF1, FGF10, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FRZB, GDNF, GFER, GKN1, HBEGF, HGF, IGF-1, IGF2, INHA, INHBA, INHBB, INHBC, INHBE, INS, KITLG, MANF, MDK, MIA, NGF, NOV, NRG1, NRG2, NRG3, NRG4, NRTN, NTF3, NTF4, OGN, PDGFA, PDGFB, PDGFC, PDGFD, PGF, PROK1, PSPN, PTN, SDF1, SDF2, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5, TDGF1, TFF1, TGFA, TGFB1, TGFB2, TGFB3, THBS4, TIMP1, VEGFA, VEGFB, VEGFC, VEGFD, WISP3); growth factor binding proteins (e.g., CHRD, CYR61, ESM1, FGFBP1, FGFBP2, FGFBP3, HTRA1, GHBP, IGFALS, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7, LTBP1, LTBP2, LTBP3, LTBP4, SOSTDC1, NOG, TWSG1, and WIF1); heparin binding proteins (e.g., ADA2, ADAMTSL5, ANGPTL3, APOB, APOE, APOH, COL5A1, COMP, CTGF, FBLN7, FN1, FSTL1, HRG, LAMC2, LIPC, LIPG, LIPH, LIPI, LPL, PCOLCE2, POSTN, RSPO1, RSPO2, RSPO3, RSPO4, SAA1, SLIT2, SOST, THBS1, VTN); hormones (e.g., ADCYAP1, ADIPOQ, ADM, ADM2, ANGPTL8, APELA, APLN, AVP, C1QTNF12, C1QTNF9, CALCA, CALCB, CCK, CGA, CGB1, CGB2, CGB3, CGB5, CGB8, COPA, CORT, CRH, CSH1, CSH2, CSHL1, ENHO, EPO, ERFE, FBN1, FNDC5, FSHB, GAL, GAST, GCG, GH, GH1, GH2, GHRH, GHRL, GIP, GNRH1, GNRH2, GPHA2, GPHB5, IAPP, INS, INSL3, INSL4, INSL5, INSL6, LHB, METRNL, MLN, NPPA, NPPB, NPPC, OSTN, OXT, PMCH, PPY, PRL, PRLH, PTH, PTHLH, PYY, RETN, RETNLB, RLN1, RLN2, RLN3, SCT, SPX, SST, STC1, STC2, TG, TOR2A, TRH, TSHB, TTR, UCN, UCN2, UCN3, UTS2, UTS2B, and VIP); hydrolases (e.g., AADACL2, ABHD15, ACP7, ACPP, ADA2, ADAMTSL1, AOAH, ARSF, ARSI, ARSJ, ARSK, BTD, CHI3L2, ENPP1, ENPP2, ENPP3, ENPP5, ENTPD5, ENTPD6, GBP1, GGH, GPLD1, HPSE, LIPC, LIPF, LIPG, LIPH, LIPI, LIPK, LIPM, LIPN, LPL, PGLYRP2, PLA1A, PLA2G10, PLA2G12A, PLA2G1B, PLA2G2A, PLA2G2D, PLA2G2E, PLA2G2F, PLA2G3, PLA2G5, PLA2G7, PNLIP, PNLIPRP2, PNLIPRP3, PON1, PON3, PPT1, SMPDL3A, THEM6, THSD1, and THSD4); immunoglobulins (e.g., IGSF10, IGKV1-12, IGKV1-16, IGKV1-33, IGKV1-6, IGKV1D-12, IGKV1D-39, IGKV1D-8, IGKV2-30, IGKV2D-30, IGKV3-11, IGKV3D-20, IGKV5-2, IGLC1, IGLC2, IGLC3); isomerases (e.g., NAXE, PPIA, PTGDS); kinases (e.g., ADCK1, ADPGK, FAM20C, ICOS, PKDCC); lyases (e.g., PM20D1, PAM, CA6); metalloenzyme inhibitors (e.g., FETUB, SPOCK3, TIMP2, TIMP3, TIMP4, WFIKKN1, WFIKKN2); metalloproteases (e.g., ADAM12, ADAM28, ADAM9, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, CLCA1, CLCA2, CLCA4, IDE, MEP1B, MMEL1, MMP1, MMP10, MMP11, MMP12, MMP13, MMP16, MMP17, MMP19, MMP2, MMP20, MMP21, MMP24, MMP25, MMP26, MMP28, MMP3, MMP1, MMP8, MMP9, PAPPA, PAPPA2, TLL1, TLL2); milk proteins (e.g., CSN1S1, CSN2, CSN3, LALBA); neuroactive proteins (e.g., CARTPT, NMS, NMU, NPB, NPFF, NPS, NPVF, NPW, NPY, PCSK1N, PDYN, PENK, PNOC, POMC, PROK2, PTH2, PYY2, PYY3, QRFP, TAC1, and TAC3); proteases (e.g., ADAMTS6, C1R, C1RL, C2, CASP4, CELA1, CELA2A, CELA2B, CFB, CFD, CFI, CMA1, CORIN, CTRB1, CTRB2, CTSB, CTSD, DHH, F10, F11, F12, F2, F3, F7, F8, F9, FAP, FURIN, GZMA, GZMK, GZMM, HABP2, HGFAC, HTRA3, HTRA4, IHH, KLK10, KLK11, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLKB1, MASP1, MASP2, MST1L, NAPSA, OVCH1, OVCH2, PCSK2, PCSK5, PCSK6, PCSK9, PGA3, PGA4, PGA5, PGC, PLAT, PLAU, PLG, PROC, PRSS1, PRSS12, PRSS2, PRSS22, PRSS23, PRSS27, PRSS29P, PRSS3, PRSS33, PRSS36, PRSS38, PRSS3P2, PRSS42, PRSS44, PRSS47, PRSS48, PRSS53, PRSS57, PRSS58, PRSS8, PRTN3, RELN, REN, TMPRSS11D, TMPRSS11E, TMPRSS2, TPSAB1, TPSB2, TPSD1); protease inhibitors (e.g., A2M, A2ML1, AMBP, ANOS1, COL28A1, COL6A3, COL7A1, CPAMD8, CST1, CST2, CST3, CST4, CST5, CST6, CST7, CST8, CST9, CST9L, CST9LP1, CSTL1, EPPIN, GPC3, HMSD, ITIH1, ITIH2, ITIH3, ITIH4, ITIH5, ITIH6, KNG1, OPRPN, OVOS1, OVOS2, PAPLN, PI15, PI16, PI3, PZP, R3HDML, SERPINA1, SERPINA10, SERPINA11, SERPINA12, SERPINA13P, SERPINA3, SERPINA4, SERPINA5, SERPINA7, SERPINA9, SERPINB2, SERPINB5, SERPINC1, SERPINE1, SERPINE2, SERPINE3, SERPINF2, SERPING1, SERPINI1, SERPINI2, SPINK1, SPINK13, SPINK14, SPINK2, SPINK4, SPINK5, SPINK6, SPINK7, SPINK8, SPINK9, SPINT1, SPINT3, SPINT4, SPOCK1, SPOCK2, SPP2, SSPO, TFPI, TFPI2, WFDC1, WFDC10A, WFDC13, WFDC2, WFDC3, WFDC5, WFDC6, WFDC8); protein phosphatases (e.g., ACP7, ACPP, PTEN, PTPRZ1); esterases (e.g., BCHE, CEL, CES4A, CES5A, NOTUM, SIAE); transferases (e.g., METTL24, FKRP, CHSY1, CHST9, B3GAT1); vasoactive proteins (e.g., AGGF1, AGT, ANGPT1, ANGPT2, ANGPTL4, ANGPTL6, EDN1, EDN2, EDN3, NTS), a Type I interferon (e.g., an IFN-α, including, but not limited to an interferon alpha-n3, an interferon alpha-2a, and an interferon alpha-2b, an IFN-β, an IFN-δ, an IFN-ε, an IFN-κ, an IFN-ν, an IFN-τ, and an IFN-ω), a Type II interferon (e.g., IFN-γ), a Type III interferon (e.g., IFN-λ) an interleukin, e.g., IL-37, IL-38, and a soluble ACE2 receptor.

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA; and (ii) at least one nucleic acid sequence encoding a gene of interest; wherein the target RNA is different from an mRNA encoded by the gene of interest. In some aspects, the recombinant polynucleic acid construct comprises two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target RNA, wherein each of the two or more nucleic acid sequences encode or comprise an siRNA capable of binding to a same target RNA or a different target RNA. In some embodiments, the recombinant polynucleic acid construct comprises two or more nucleic acid sequences that each encode or comprise an siRNA capable of binding to a target RNA, wherein the respective target RNAs are the same, different, or a combination thereof. In some embodiments, the target RNA is an mRNA. In some embodiments, the target RNA is a noncoding RNA. In some aspects, the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are comprised in a sequential manner. In some aspects, the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are present in a sequential manner. In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is downstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream or downstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the siRNA capable of binding to a target RNA binds to an exon of a target mRNA. In some aspects, the siRNA capable of binding to a target RNA specifically binds to one target RNA. In some aspects, the siRNA capable of binding to a target RNA is not encoded by or comprised of an intron sequence of the gene of interest. In some aspects, the gene of interest is expressed without RNA splicing. In some aspects, the target RNA is an mRNA encoding a protein selected from the group consisting of Tumor Necrosis Factor alpha (TNF-alpha), interleukin, Angiotensin Converting Enzyme-2 (ACE2), SARS CoV-2 ORF1ab, SARS CoV-2 S, and SARS CoV-2 N. In some aspects, the target RNA is an mRNA encoding a protein selected from the group consisting of Interleukin 8 (IL-8), Interleukin 1 beta (IL-1 beta), Interleukin 17 (IL-17), Tumor Necrosis Factor alpha (TNF-alpha), Interleukin 6 (IL-6), Interleukin 6R (IL-6R), Interleukin 6R-alpha (IL-6R-alpha), Interleukin 6R-beta (IL-6R-beta), Angiotensin Converting Enzyme-2 (ACE2), SARS CoV-2 ORF1ab, SARS CoV-2 S, and SARS CoV-2 N. In some aspects, the target RNA is an mRNA encoding a protein selected from the group consisting of: Interleukin 8 (IL-8), Interleukin 1 beta (IL-1 beta), Interleukin 17 (IL-17), and Tumor Necrosis Factor alpha (TNF-alpha). In some aspects, the recombinant polynucleic acid construct comprises two or more nucleic acid sequences encoding a gene of interest, wherein each of the two or more nucleic acid sequences encodes a same gene of interest or a different gene of interest. In some embodiments, the recombinant polynucleic acid construct comprises two or more nucleic acid sequences that each encode or a gene of interest, wherein the respective genes of interest are the same, different, or a combination thereof. In some aspects, the gene of interest comprises a nucleic acid sequence encoding a protein selected from the group consisting of a secretory protein, an intracellular protein, an intraorganelle protein, and a membrane protein. In some aspects, the gene of interest is selected from the group consisting of Insulin-like Growth Factor 1 (IGF-1), and Interleukin 4 (IL-4). In some aspects, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding a target motif operably linked to the at least one nucleic acid sequence encoding the gene of interest, wherein the target motif comprises a signal peptide, a nuclear localization signal (NLS), a nucleolar localization signal (NoLS), a lysosomal targeting signal, a mitochondrial targeting signal, a peroxisomal targeting signal, a microtubule tip localization signal (MtLS), an endosomal targeting signal, a chloroplast targeting signal, a Golgi targeting signal, an endoplasmic reticulum (ER) targeting signal, a proteasomal targeting signal, a membrane targeting signal, a transmembrane targeting signal, or a centrosomal localization signal (CLS). In some aspects, the target motif is selected from the group consisting of: (a) a target motif heterologous to a protein encoded by the gene of interest; (b) a target motif heterologous to a protein encoded by the gene of interest, wherein the target motif heterologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; (c) a target motif homologous to a protein encoded by the gene of interest, wherein the target motif homologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; and (d) a naturally occurring amino acid sequence which does not have the function of a target motif in nature, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion, and/or substitution of at least one amino acid. In some aspects, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a poly(A) tail, a nucleic acid sequence encoding or comprising a 5′ cap, a nucleic acid sequence encoding or comprising a promoter, or a nucleic acid sequence encoding or comprising a Kozak sequence. In some aspects, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a linker. In some aspects, the nucleic acid sequence encoding or comprising the linker connects (a) the at least one nucleic acid sequence encoding or comprising an siRNA capable of binding to a target mRNA and the at least one nucleic acid sequence encoding a gene of interest, (b) each of the two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA, and/or (c) each of the two or more nucleic acid sequences encoding a gene of interest. In some aspects, the linker comprises a tRNA linker, a 2A peptide linker or a flexible linker. In some aspects, the linker is at least 6 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is up to 50 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is up to 80 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is about 6 to about 50 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is about 6 to about 80 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is about 6 to about 15 nucleic acid residues in length. In some aspects, the recombinant polynucleic acid construct comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8. In some aspects, the composition comprises a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to a target RNA; and (ii) an mRNA encoding a gene of interest; wherein the target RNA is different from the mRNA encoding the gene of interest. In some aspects, the composition is for use in simultaneously modulating the expression of two or more genes in a cell. In some aspects, the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are comprised in a sequential manner. In some aspects, the composition is for use in simultaneously modulating the expression of two or more genes in a cell. In some aspects, the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are present in a sequential manner. In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is downstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream or downstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the siRNA capable of binding to a target RNA binds to an exon of a target mRNA. In some aspects, the siRNA capable of binding to a target RNA specifically binds to one target RNA. In some aspects, the siRNA capable of binding to a target RNA is not encoded by or comprised of an intron sequence of the gene of interest. In some aspects, the gene of interest is expressed without RNA splicing.

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct for treatment or prevention of a viral disease or condition in a subject, the construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA; and (ii) at least one nucleic acid sequence encoding or comprising an mRNA of a gene of interest; wherein the target RNA is different from an mRNA encoded by the gene of interest. In some aspects, the siRNA does not affect the expression of and/or is not capable of binding to the mRNA of the gene of interest. In some aspects, the recombinant polynucleic acid construct comprises two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target RNA, wherein each of the two or more nucleic acid sequences encode or comprise an siRNA capable of binding to a same target RNA or a different target RNA. In some aspects, the recombinant polynucleic acid construct comprises three or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target RNA, wherein at least two nucleic acid sequences encode or comprise an siRNA capable of binding to the same target RNA and at least one nucleic acid sequence encodes or comprises an siRNA capable of binding to a different target RNA. In some embodiments, the target RNA is an mRNA. In some embodiments, the target RNA is a noncoding RNA. In some embodiments, each target RNA is the same, or different. In some embodiments, the target RNA is an mRNA encoding a protein selected from the group consisting of: interleukin, Angiotensin Converting Enzyme-2 (ACE2); SARS CoV-2 ORF1ab; SARS CoV-2 S, and SARS CoV-2 N. In some embodiments, the interleukin is selected from the group consisting of: IL-1alpha, IL-1beta, IL-6, IL-6R, IL-6R-alpha, interleukin IL-6R-beta, IL-18, IL-36-alpha, IL-36-beta; IL-36-gamma, and IL-33. In some embodiments, the target mRNA is an mRNA encoding a protein selected from the group consisting of: IL-6, IL-6R, IL-6R-alpha, IL-6R-beta, Angiotensin Converting Enzyme-2 (ACE2); SARS CoV-2 ORF1ab; SARS CoV-2 S, and SARS CoV-2 N. In some embodiments, the composition comprises in (ii) two or more nucleic acid sequences, each encoding a gene of interest. In some embodiments, each mRNA is the same or different. In some embodiments, at least two mRNAs are the same and at least one mRNA is different from the at least two same mRNAs. In some embodiments, the gene of interest of (ii) is selected from the group of genes encoding: IFN alpha-n3, IFN alpha-2a, IFN alpha-2b, IFN beta-1a, IFN beta-1b, ACE2 soluble receptor, IL-37, and IL-38. In some embodiments, the gene of interest of (ii) is selected from the group of genes encoding: IFN beta and ACE2 soluble receptor. In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding a target motif operably linked to the at least one nucleic acid sequence encoding the mRNA of the gene of interest, wherein the target motif comprises a signal peptide, a nuclear localization signal (NLS), a nucleolar localization signal (NoLS), a lysosomal targeting signal, a mitochondrial targeting signal, a peroxisomal targeting signal, a microtubule tip localization signal (MtLS), an endosomal targeting signal, a chloroplast targeting signal, a Golgi targeting signal, an endoplasmic reticulum (ER) targeting signal, a proteasomal targeting signal, a membrane targeting signal, a transmembrane targeting signal, or a centrosomal localization signal (CLS). In some embodiments, the target motif is selected from the group consisting of: (a) a target motif heterologous to a protein encoded by the gene of interest; (b) a target motif heterologous to a protein encoded by the gene of interest, wherein the target motif heterologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; (c) a target motif homologous to a protein encoded by the gene of interest, wherein the target motif homologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; and (d) a naturally occurring amino acid sequence which does not have the function of a target motif in nature, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion, and/or substitution of at least one amino acid. In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a poly(A) tail, a nucleic acid sequence encoding or comprising a 5′ cap, a nucleic acid sequence encoding or comprising a promoter, or a nucleic acid sequence encoding or comprising a Kozak sequence. In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a linker. In some embodiments, the nucleic acid sequence encoding or comprising the linker connects (a) the at least one nucleic acid sequence encoding or comprising the siRNA capable of binding to the target mRNA and the at least one nucleic acid sequence encoding the gene of interest, (b) each of the two or more nucleic acid sequences encoding or comprising the siRNA capable of binding to the target mRNA, and/or (c) each of the two or more nucleic acid sequences encoding the gene of interest. In some embodiments, the linker comprises a tRNA linker, a 2A peptide linker, or a flexible linker. In some aspects, the nucleic acid sequence encoding or comprising the linker is at least 6 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is up to 50 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is up to 80 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is about 6 to about 50 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is about 6 to about 80 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is about 6 to about 15 nucleic acid residues in length. In some embodiments, the recombinant polynucleic acid construct is a vector suitable for gene therapy. In some embodiments, the recombinant polynucleic acid construct comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 29-47. In some aspects, the composition comprises a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to a target RNA; and (ii) an mRNA encoding a gene of interest; wherein the target RNA is different from the mRNA encoding the gene of interest. In some embodiments, the composition is for use in simultaneously modulating the expression of two or more genes in a cell. In some embodiments, the composition is present in an amount sufficient to treat or prevent a viral disease or condition in the subject. In some aspects, the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are comprised in a sequential manner. In some aspects, the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are present in a sequential manner. In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is downstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream or downstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the siRNA capable of binding to a target RNA binds to an exon of a target mRNA. In some aspects, the siRNA capable of binding to a target RNA specifically binds to one target RNA. In some aspects, the siRNA capable of binding to a target RNA is not encoded by or comprised of an intron sequence of the gene of interest. In some aspects, the gene of interest is expressed without RNA splicing.

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct, the construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA; and (ii) at least one nucleic acid sequence encoding or comprising an mRNA of a gene of interest; wherein the target RNA of (i) is different from the mRNA of (ii). In some embodiments, the siRNA does not affect the expression of and/or is not capable of binding to the mRNA of the gene of interest. In some aspects, the recombinant polynucleic acid construct comprises two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target RNA, wherein each of the two or more nucleic acid sequences encode or comprise an siRNA capable of binding to a same target RNA or a different target RNA. In some aspects, the recombinant polynucleic acid construct comprises three or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target RNA, wherein at least two nucleic acid sequences encode or comprise an siRNA capable of binding to the same target RNA and at least one nucleic acid sequence encodes or comprises an siRNA capable of binding to a different target RNA. In some embodiments, the target RNA is an mRNA. In some embodiments, the target RNA is a noncoding RNA. In some embodiments, each target RNA is the same, or different. In some embodiments, the target is an mRNA encoding a protein selected from the group consisting of: IL-8 mRNA, an IL-1 beta mRNA, an IL-17 mRNA, a TNF-alpha mRNA, a SARS CoV-2 ORF1ab RNA (polyprotein PP1ab, e.g., in a noncoding region or where it encodes a protein that is selected from: a SARS CoV-2 nonstructure protein (NSP), Nsp1, Nsp3 (Nsp3b, Nsp3c, PLpro, and Nsp3e), Nsp7 Nsp8 complex, Nsp9-Nsp10, and Nsp14-Nsp16, 3CLpro, E-channel (E protein), ORF7a, C-terminal RNA binding domain (CRBD), N-terminal RNA binding domain (NRBD), helicase, and RdRp), a SARS CoV-2 Spike protein (S) mRNA, a SARS CoV-2 Nucleocapsid protein (N) mRNA, a tumor necrosis factor alpha (TNF-alpha) mRNA, an interleukin mRNA (including but not limited to interleukin 1 (e.g., IL-1alpha, IL-1beta), interleukin 6 (IL-6), interleukin 6R (IL-6R), interleukin 6R alpha (IL-6R-alpha), interleukin 6R beta (IL-6R-beta), interleukin 18 (IL-18), interleukin 36-alpha (IL-36-alpha), interleukin 36-beta (IL-36-beta), interleukin 36-gamma (IL-36-gamma), interleukin 33 (IL-33)), an Angiotensin Converting Enzyme-2 (ACE2) mRNA, a transmembrane protease, serine 2 (TMPRSS2) mRNA, and a coding NSP12 and 13 RNA. In some embodiments, the composition comprises in (ii) two or more nucleic acid sequences, each encoding an mRNA of a gene of interest. In some embodiments, each mRNA is the same or different. In some embodiments, at least two mRNAs are the same and at least one mRNA is different from the at least two same mRNAs. In some embodiments, the gene of interest of (ii) is selected from the group of genes encoding a protein selected from: IGF-1, IL-4, IGF-1 (including derivatives thereof as described elsewhere herein), carboxypeptidases (e.g., ACE, ACE2, CNDP1, CPA1, CPA2, CPA4, CPA5, CPA6, CPB1, CPB2, CPE, CPN1, CPQ, CPXM1, CPZ, SCPEP1); cytokines (e.g., BMP1, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, C1QTNF4, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL3L3, CCL4, CCL4L, CCL4L2, CCL5, CCL7, CCL8, CD40LG, CER1, CKLF, CLCF1, CNTF, CSF1, CSF2, CSF3, CTF1, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, CXCL17, CXCL2, CXCL3, CXCL5, CXCL8, CXCL9, DKK1, DKK2, DKK3, DKK4, EDA, EBI3, FAM3B, FAM3C, FASLG, FLT3LG, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF5, GDF6, GDF7, GDF9, GPI, GREM1, GREM2, GRN, IFNA1, IFNA13, IFNA10, IFNA14, IFNA16, IFNA17, IFNA2, IFNA21, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNB1, IFNE, IFNG, IFNK, IFNL1, IFNL2, IFNL3, IFNL4, IFNW1, IL10, IL11, IL12A, IL12B, IL13, IL15, IL16, IL17A, IL17B, IL17C, IL17D, IL17F, IL18, IL19, IL1A, IL1B, IL1F10, IL2, IL20, IL21, IL22, IL23A, IL24, IL25, IL26, IL27, IL3, IL31, IL32, IL33, IL34, IL36A, IL36B, IL36G, IL36RN, IL37, IL4, IL5, IL6, IL7, IL9, LEFTY1, LEFTY2, LIF, LTA, MIF, MSTN, NAMPT, NODAL, OSM, PF4, PF4V1, SCGB3A1, SECTM1, SLURP1, SPP1, THNSL2, THPO, TNF, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TSLP, VSTM1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, and XCL2); extracellular ligands and transporters (e.g., APCS, CHI3L1, CHI3L2, CLEC3B, DMBT1, DMKN, EDDM3A, EDDM3B, EFNA4, EMC10, ENAM, EPYC, ERVH48-1, F13B, FCN1, FCN2, GLDN, GPLD1, HEG1, ITFG1, KAZALD1, KCP, LACRT, LEG1, METRN, NOTCH2NL, NPNT, OLFM1, OLFML3, PRB2, PSAP, PSAPL1, PSG1, PSG6, PSG9, PTX3, PTX4, RBP4, RNASE10, RNASE12, RNASE13, RNASE9, RSPRY1, RTBDN, S100A12, S100A13, S100A7, S100A8, SAA2, SAA4, SCG1, SCG2, SCG3, SCGB1C1, SCGB1C2, SCGB1D1, SCGB1D2, SCGB1D4, SCGB2B2, SCGB3A2, SCGN, SCRG1, SCUBE1, SCUBE2, SCUBE3, SDCBP, SELENOP, SFTA2, SFTA3, SFTPA1, SFTPA2, SFTPC, SFTPD, SHBG, SLURP2, SMOC1, SMOC2, SMR3A, SMR3B, SNCA, SPATA20, SPATA6, SOGA1, SPARC, SPARCL1, SPATA20, SPATA6, SRPX2, SSC4D, STX1A, SUSD4, SVBP, TCN1, TCN2, TCTN1, TF, TULP3, TFF2, TFF3, THSD7A, TINAG, TINAGL1, TMEFF2, TMEM25, VWC2L); extracellular matrix proteins (e.g., ABI3BP, AGRN, CCBE1, CHL1, COL15A1, COL19A1, COLEC11, DMBT1, DRAXIN, EDIL3, ELN, EMID1, EMILIN1, EMILIN2, EMILIN3, EPDR1, FBLN1, FBLN2, FBLN5, FLRT1, FLRT2, FLRT3, FREM1, GLDN, IBSP, KERA, KIAA0100, KIRREL3, KRT10, LAMB2, MGP, RPTN, SBSPON, SDC1, SDC4, SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, SIGLEC1, SIGLEC10, SIGLEC6, SLIT1, SLIT2, SLIT3, SLITRK1, SNED1, SNORC, SPACA3, SPACA7, SPON1, SPON2, STATH, SVEP1, TECTA, TECTB, TNC, TNN, TNR, TNXB); glucosidases (AMY1A, AMY1B, AMY1C, AMY2A, AMY2B, CEMIP, CHIA, CHIT1, FUCA2, GLB1L, GLB1L2, HPSE, HYAL1, HYAL3, KL, LYG1, LYG2, LYZL1, LYZL2, MAN2B2, SMPD1, SMPDL3B, SPACA5, SPACA5B); glycosyltransferases (e.g., ARTS, B4GALT1, EXTL2, GALNT1, GALNT2, GLT1D1, MGAT4A, ST3GAL1, ST3GAL2, ST3GAL3, ST3GAL4, ST6GAL1, XYLT1); growth factors (e.g., AMH, ARTN, BTC, CDNF, CFC1, CFC1B, CHRDL1, CHRDL2, CLEC11A, CNMD, EFEMP1, EGF, EGFL6, EGFL7, EGFL8, EPGN, EREG, EYS, FGF1, FGF10, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FRZB, GDNF, GFER, GKN1, HBEGF, HGF, IGF-1, IGF2, INHA, INHBA, INHBB, INHBC, INHBE, INS, KITLG, MANF, MDK, MIA, NGF, NOV, NRG1, NRG2, NRG3, NRG4, NRTN, NTF3, NTF4, OGN, PDGFA, PDGFB, PDGFC, PDGFD, PGF, PROK1, PSPN, PTN, SDF1, SDF2, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5, TDGF1, TFF1, TGFA, TGFB1, TGFB2, TGFB3, THBS4, TIMP1, VEGFA, VEGFB, VEGFC, VEGFD, WISP3); growth factor binding proteins (e.g., CHRD, CYR61, ESM1, FGFBP1, FGFBP2, FGFBP3, HTRA1, GHBP, IGFALS, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7, LTBP1, LTBP2, LTBP3, LTBP4, SOSTDC1, NOG, TWSG1, and WIF1); heparin binding proteins (e.g., ADA2, ADAMTSL5, ANGPTL3, APOB, APOE, APOH, COL5A1, COMP, CTGF, FBLN7, FN1, FSTL1, HRG, LAMC2, LIPC, LIPG, LIPH, LIPI, LPL, PCOLCE2, POSTN, RSPO1, RSPO2, RSPO3, RSPO4, SAA1, SLIT2, SOST, THBS1, VTN); hormones (e.g., ADCYAP1, ADIPOQ, ADM, ADM2, ANGPTL8, APELA, APLN, AVP, C1QTNF12, C1QTNF9, CALCA, CALCB, CCK, CGA, CGB1, CGB2, CGB3, CGB5, CGB8, COPA, CORT, CRH, CSH1, CSH2, CSHL1, ENHO, EPO, ERFE, FBN1, FNDC5, FSHB, GAL, GAST, GCG, GH, GH1, GH2, GHRH, GHRL, GIP, GNRH1, GNRH2, GPHA2, GPHB5, IAPP, INS, INSL3, INSL4, INSL5, INSL6, LHB, METRNL, MLN, NPPA, NPPB, NPPC, OSTN, OXT, PMCH, PPY, PRL, PRLH, PTH, PTHLH, PYY, RETN, RETNLB, RLN1, RLN2, RLN3, SCT, SPX, SST, STC1, STC2, TG, TOR2A, TRH, TSHB, TTR, UCN, UCN2, UCN3, UTS2, UTS2B, and VIP); hydrolases (e.g., AADACL2, ABHD15, ACP7, ACPP, ADA2, ADAMTSL1, AOAH, ARSF, ARSI, ARSJ, ARSK, BTD, CHI3L2, ENPP1, ENPP2, ENPP3, ENPP5, ENTPD5, ENTPD6, GBP1, GGH, GPLD1, HPSE, LIPC, LIPF, LIPG, LIPH, LIPI, LIPK, LIPM, LIPN, LPL, PGLYRP2, PLA1A, PLA2G10, PLA2G12A, PLA2G1B, PLA2G2A, PLA2G2D, PLA2G2E, PLA2G2F, PLA2G3, PLA2G5, PLA2G7, PNLIP, PNLIPRP2, PNLIPRP3, PON1, PON3, PPT1, SMPDL3A, THEM6, THSD1, and THSD4); immunoglobulins (e.g., IGSF10, IGKV1-12, IGKV1-16, IGKV1-33, IGKV1-6, IGKV1D-12, IGKV1D-39, IGKV1D-8, IGKV2-30, IGKV2D-30, IGKV3-11, IGKV3D-20, IGKV5-2, IGLC1, IGLC2, IGLC3); isomerases (e.g., NAXE, PPIA, PTGDS); kinases (e.g., ADCK1, ADPGK, FAM20C, ICOS, PKDCC); lyases (e.g., PM20D1, PAM, CA6); metalloenzyme inhibitors (e.g., FETUB, SPOCK3, TIMP2, TIMP3, TIMP4, WFIKKN1, WFIKKN2); metalloproteases (e.g., ADAM12, ADAM28, ADAM9, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, CLCA1, CLCA2, CLCA4, IDE, MEP1B, MMEL1, MMP1, MMP10, MMP11, MMP12, MMP13, MMP16, MMP17, MMP19, MMP2, MMP20, MMP21, MMP24, MMP25, MMP26, MMP28, MMP3, MMP1, MMP8, MMP9, PAPPA, PAPPA2, TLL1, TLL2); milk proteins (e.g., CSN1S1, CSN2, CSN3, LALBA); neuroactive proteins (e.g., CARTPT, NMS, NMU, NPB, NPFF, NPS, NPVF, NPW, NPY, PCSK1N, PDYN, PENK, PNOC, POMC, PROK2, PTH2, PYY2, PYY3, QRFP, TAC1, and TAC3); proteases (e.g., ADAMTS6, C1R, C1RL, C2, CASP4, CELA1, CELA2A, CELA2B, CFB, CFD, CFI, CMA1, CORIN, CTRB1, CTRB2, CTSB, CTSD, DHH, F10, F11, F12, F2, F3, F7, F8, F9, FAP, FURIN, GZMA, GZMK, GZMM, HABP2, HGFAC, HTRA3, HTRA4, IHH, KLK10, KLK11, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLKB1, MASP1, MASP2, MST1L, NAPSA, OVCH1, OVCH2, PCSK2, PCSK5, PCSK6, PCSK9, PGA3, PGA4, PGA5, PGC, PLAT, PLAU, PLG, PROC, PRSS1, PRSS12, PRSS2, PRSS22, PRSS23, PRSS27, PRSS29P, PRSS3, PRSS33, PRSS36, PRSS38, PRSS3P2, PRSS42, PRSS44, PRSS47, PRSS48, PRSS53, PRSS57, PRSS58, PRSS8, PRTN3, RELN, REN, TMPRSS11D, TMPRSS11E, TMPRSS2, TPSAB1, TPSB2, TPSD1); protease inhibitors (e.g., A2M, A2ML1, AMBP, ANOS1, COL28A1, COL6A3, COL7A1, CPAMD8, CST1, CST2, CST3, CST4, CST5, CST6, CST7, CST8, CST9, CST9L, CST9LP1, CSTL1, EPPIN, GPC3, HMSD, ITIH1, ITIH2, ITIH3, ITIH4, ITIH5, ITIH6, KNG1, OPRPN, OVOS1, OVOS2, PAPLN, PI15, PI16, PI3, PZP, R3HDML, SERPINA1, SERPINA10, SERPINA11, SERPINA12, SERPINA13P, SERPINA3, SERPINA4, SERPINA5, SERPINA7, SERPINA9, SERPINB2, SERPINB5, SERPINC1, SERPINE1, SERPINE2, SERPINE3, SERPINF2, SERPING1, SERPINI1, SERPINI2, SPINK1, SPINK13, SPINK14, SPINK2, SPINK4, SPINK5, SPINK6, SPINK7, SPINK8, SPINK9, SPINT1, SPINT3, SPINT4, SPOCK1, SPOCK2, SPP2, SSPO, TFPI, TFPI2, WFDC1, WFDC10A, WFDC13, WFDC2, WFDC3, WFDC5, WFDC6, WFDC8); protein phosphatases (e.g., ACP7, ACPP, PTEN, PTPRZ1); esterases (e.g., BCHE, CEL, CES4A, CES5A, NOTUM, SIAE); transferases (e.g., METTL24, FKRP, CHSY1, CHST9, B3GAT1); vasoactive proteins (e.g., AGGF1, AGT, ANGPT1, ANGPT2, ANGPTL4, ANGPTL6, EDN1, EDN2, EDN3, NTS), a Type I interferon (e.g., an IFN-α, including, but not limited to an interferon alpha-n3, an interferon alpha-2a, and an interferon alpha-2b, an IFN-β, an IFN-δ, an IFN-ε, an IFN-κ, an IFN-ν, an IFN-τ, and an IFN-ω), a Type II interferon (e.g., IFN-γ), a Type III interferon (e.g., IFN-λ), an interleukin, e.g., IL-37, IL-38, and a soluble ACE2 receptor. In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding a target motif operably linked to the at least one nucleic acid sequence encoding the mRNA of the gene of interest, wherein the target motif comprises a signal peptide, a nuclear localization signal (NLS), a nucleolar localization signal (NoLS), a lysosomal targeting signal, a mitochondrial targeting signal, a peroxisomal targeting signal, a microtubule tip localization signal (MtLS), an endosomal targeting signal, a chloroplast targeting signal, a Golgi targeting signal, an endoplasmic reticulum (ER) targeting signal, a proteasomal targeting signal, a membrane targeting signal, a transmembrane targeting signal, or a centrosomal localization signal (CLS). In some embodiments, the target motif is selected from the group consisting of: (a) a target motif heterologous to a protein encoded by the gene of interest; (b) a target motif heterologous to a protein encoded by the gene of interest, wherein the target motif heterologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; (c) a target motif homologous to a protein encoded by the gene of interest, wherein the target motif homologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; and (d) a naturally occurring amino acid sequence which does not have the function of a target motif in nature, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion, and/or substitution of at least one amino acid. In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a poly(A) tail, a nucleic acid sequence encoding or comprising a 5′ cap, a nucleic acid sequence encoding or comprising a promoter, or a nucleic acid sequence encoding or comprising a Kozak sequence. In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a linker. In some embodiments, the nucleic acid sequence encoding or comprising the linker connects (a) the at least one nucleic acid sequence encoding or comprising the siRNA capable of binding to the target mRNA and the at least one nucleic acid sequence encoding the gene of interest, (b) each of the two or more nucleic acid sequences encoding or comprising the siRNA capable of binding to the target mRNA, and/or (c) each of the two or more nucleic acid sequences encoding the gene of interest. In some embodiments, the linker comprises a tRNA linker, a 2A peptide linker, or a flexible linker. In some embodiments, the nucleic acid sequence encoding or comprising the linker is at least 6 nucleic acid residues in length. In some embodiments, the nucleic acid sequence encoding or comprising the linker is up to 50 nucleic acid residues in length. In some embodiments, the nucleic acid sequence encoding or comprising the linker is up to 80 nucleic acid residues in length. In some embodiments, the nucleic acid sequence encoding or comprising the linker is about 6 to about 50 nucleic acid residues in length. In some embodiments, the nucleic acid sequence encoding or comprising the linker is about 6 to about 80 nucleic acid residues in length. In some embodiments, the nucleic acid sequence encoding or comprising the linker is about 6 to about 15 nucleic acid residues in length. In some embodiments, the recombinant polynucleic acid construct is a vector suitable for gene therapy. In some embodiments, the composition is useful for simultaneously modulating the expression of two or more genes in a cell.

In some embodiments, the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are comprised in a sequential manner. In some embodiments, the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are present in a sequential manner. In some embodiments, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some embodiments, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is downstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some embodiments, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream or downstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some embodiments, the siRNA capable of binding to a target RNA binds to an exon of a target mRNA. In some embodiments, the siRNA capable of binding to a target RNA specifically binds to one target RNA. In some embodiments, the siRNA capable of binding to a target RNA is not encoded by or comprised of an intron sequence of the gene of interest. In some embodiments, the gene of interest is expressed without RNA splicing. In some embodiments, the composition is present or administered in an amount sufficient to treat or prevent a viral infection, disease or condition, or a disease or condition selected from the group consisting of intervertebral disc disease (IVDD), osteoarthritis, and psoriasis. In some embodiments, the composition is present or administered in an amount sufficient to treat or prevent a viral infection, disease or condition, or a disease or condition selected from the group consisting of intervertebral disc disease (IVDD), osteoarthritis, and psoriasis, fibrodysplasia ossificans progressiva (FOP), and amyotrophic lateral sclerosis (ALS), In some embodiments, the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are comprised in a sequential manner. In some embodiments, the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are present in a sequential manner. In some embodiments, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some embodiments, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is downstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some embodiments, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream or downstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some embodiments, the siRNA capable of binding to a target RNA binds to an exon of a target mRNA. In some embodiments, the siRNA capable of binding to a target RNA specifically binds to one target RNA. In some embodiments, the siRNA capable of binding to a target RNA is not encoded by or comprised of an intron sequence of the gene of interest. In some embodiments, the gene of interest is expressed without RNA splicing.

In some aspects, a composition of the present invention comprises a polynucleic acid construct comprising an siRNA comprising a sense strand sequence encoded by a sequence selected from SEQ ID NOs: 80-109 and SEQ ID NOs: 140-145. In some embodiments, the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 80-109 and SEQ ID NOs: 140-145, and the corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 110-139 and SEQ ID NOs: 146-151. In some aspects, a composition of the present invention comprises a polynucleic acid construct comprising an siRNA comprising a sense strand sequence encoded by a sequence selected from SEQ ID NOs: 80-92. In some embodiments, the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 80-92, and the corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 110-122. In some aspects, a composition of the present invention comprises a polynucleic acid construct comprising an siRNA comprising a sense strand sequence encoded by a sequence selected from SEQ ID NOs: 93-109. In some embodiments, the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 93-109, and the corresponding antisense strand encoded by a sequence selected from SEQ ID NOS: 123-139. In some aspects, a composition of the present invention comprises a polynucleic acid construct comprising an siRNA comprising a sense strand sequence encoded by a sequence selected from SEQ ID NOs: 140-145. In some embodiments, the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 140-145, and the corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 146-151.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

FIG. 1 depicts a schematic representation of construct design. T7:T7 promoter, siRNA: small interfering RNA.

FIG. 2A shows the comparison of IGF-1 mRNA construct and Compound A1 (Cpd. 1) in IGF-1 expression in HEK-293 cells while FIG. 2B shows simultaneous RNA interference of Compound A1 which comprises IL-8-targeting siRNA in an IL-8 overexpression model in HEK-293 cells. Control: IL-8 overexpression construct alone.

FIG. 3 shows dose-dependent RNA interference of Compound A1 (Cpd. 1) which comprises IL-8-targeting siRNA in an IL-8 overexpression model in HEK-293 cells.

FIG. 4A shows the modulation of IL-8 expression by Compound A2 (Cpd. 2) in THP-1 cells. Control: IL-8 overexpression construct alone.

FIG. 4B shows the IGF-1 expression of Compound A2 (Cpd. 2) in HEK-293 cells.

FIG. 5A shows the modulation of IL-8 expression by Compound A3 (Cpd. 3) in THP-1 cells. Control: IL-8 overexpression construct alone.

FIG. 5B shows the IGF-1 expression of Compound A3 (Cpd. 3) in HEK-293 cells.

FIG. 6A shows the comparison of Compound A4 (Cpd. 4) and Compound A5 (Cpd. 5) in IL-8 expression in THP-1 cells. Control: IL-8 overexpression construct alone.

FIG. 6B shows the comparison of Compound A3 (Cpd. 3) and Compound A5 (Cpd. 5) in IL-8 expression in THP1 cells. Control: IL-8 overexpression construct alone.

FIG. 7 shows the comparison of Compound A4 (Cpd. 4) and Compound A5 (Cpd. 5) in IL-8 expression in HEK-293 cells. Control: IL-8 overexpression construct alone.

FIG. 8A shows the effect of Compound A6 (Cpd. 6) in endogenous IL-1 beta (IL1b) expression in THP-1 cells. Control: LPS+dsDNA only.

FIG. 8B shows the effect of Compound A6 (Cpd. 6) in endogenous IL-1 beta (IL1b) expression in THP-1 cells. Control: LPS+dsDNA only.

FIG. 8C shows the IGF-1 expression of Compound A6 (Cpd. 6) in HEK-293 cells.

FIG. 9A shows the effect of Compound A7 (Cpd. 7) in endogenous IL-1 beta (IL1b) expression in THP-1 cells. Control: LPS+dsDNA only.

FIG. 9B shows the effect of Compound A7 (Cpd. 7) in endogenous IL-1 beta (IL1b) expression in THP-1 cells. Control: LPS+dsDNA only.

FIG. 9C shows the IGF-1 expression of Compound A7 (Cpd. 7) in HEK-293 cells.

FIG. 10A shows RNA interference of Compound A8 (Cpd. 8) which comprises TNF-α-targeting siRNA in an TNF-α overexpression model in HEK-293 cells. Control: TNF-α overexpression construct alone.

FIG. 10B shows RNA interference of Compound A8 (Cpd. 8) which comprises TNF-α-targeting siRNA in an endogenous TNF-α expression model in THP-1 cells. Control: LPS+R848 only.

FIG. 10C shows IL-4 expression of Compound A8 (Cpd. 8) in the same cell (HEK-293) culture as in FIG. 10A.

FIG. 10D shows IL-4 expression of Compound A8 (Cpd. 8) in the same cell (THP-1) culture supernatant as in FIG. 10B.

FIG. 11 depicts a phylogenetic analysis of three coronaviruses that lead to human outbreaks in the last two decades, MERS-CoV (at top), SARS-CoV-2 (middle), and SARS-CoV (bottom). The genomic sequences are publicly available (obtained from NCBI Nucleotide) and analyzed in Geneious Prime v.2019.2.3 with Tamura-Nei Genetic distance model; the tree was made with UPGMA algorithm.

FIG. 12A shows RNA interference of Compound A9 (Cpd. 9) and Compound A10 (Cpd. 10) which comprise TNF-α-targeting siRNAs in an endogenous TNF-α expression model in THP-1 cells. Control: LPS+R848 only, sc-siRNA: scrambled siRNA. Data represent means±standard error of the mean of 4 replicates. Significance (*, <0.05) was assessed by Student's t-test for siRNA activity. Significance (***, p<0.001) was assessed by one way ANOVA followed by Dunnet's multiple comparing test related to control.

FIG. 12B shows the IL-4 expression of Compound A9 (Cpd. 9) and Compound A10 (Cpd. 10) in THP-1 cells. Data represent means±standard error of the mean of 4 replicates. Significance (**, <0.01) was assessed by Student's t-test for IL-4 expression.

FIG. 13A shows RNA interference of Compound A9 (Cpd. 9) and Compound A10 (Cpd. 10) which comprise TNF-α-targeting siRNAs in an TNF-α overexpression model in HEK-293 cells. Control: TNF-α overexpression construct alone. Data represent means±standard error of the mean of 4 replicates. Significance (**, p<0.01) was assessed by one way ANOVA followed by Dunnet's multiple comparing test related to control.

FIG. 13B shows the IL-4 expression of Compound A9 (Cpd. 9) and Compound A10 (Cpd. 10) in HEK-293 cells. Data represent means±standard error of the mean of 4 replicates. Significance (***, <0.001) was assessed by Student's t-test.

FIG. 14 shows dose-dependent RNA interference of Compound A11 (Cpd. 11) which comprises ALK2-targeting siRNA in an endogenous ALK2 expression model in A549 cells and the IGF-1 expression of Compound A11 (Cpd. 11) in A549 cells. Data represent means±standard error of the mean of 4 replicates.

FIG. 15A shows dose-dependent RNA interference of Compound A12 (Cpd. 12) and Compound 13 (Cpd. 13) which comprise SOD1-targeting siRNA in an endogenous SOD1 expression model in IMR32 cells. Data represent means±standard error of the mean of 3 replicates.

FIG. 15B shows dose-dependent EPO expression of Compound A13 (Cpd. 13) in IMR32 cells. Data represent means±standard error of the mean of 4 replicates.

FIG. 15C shows dose-dependent IGF-1 expression of Compound A12 (Cpd. 12) in IMR32 cells. Data represent means±standard error of the mean of 4 replicates.

FIG. 16A shows RNA interference of Compound A14 (Cpd. 14) and Compound A15 (Cpd. 15) which comprise siRNAs targeting IL-1 beta in an IL-1 beta overexpression model in HEK-293 cells. Control: IL-1 beta overexpression construct alone. Data represent means±standard error of the mean of 4 replicates. Significance (*, <0.05) was assessed by Student's t-test. Significance (***, p<0.001) was assessed by one-way ANOVA followed by Dunnet's multiple comparing test related to control.

FIG. 16B shows the IGF-1 expression of Compound A14 (Cpd. 14) and Compound A15 (Cpd. 15) in HEK-293 cells. Data represent means±standard error of the mean of 4 replicates. Significance (***, <0.001) was assessed by Student's t-test.

FIG. 17A shows the expression of eGFP positive A549 cells transfected with pcDNA3⁺ vector containing a sequence encoding SARS CoV-2 Nucleocapsid protein tagged with eGFP.

FIG. 17B shows the expression of eGFP positive A549 cells co-transfected with pcDNA3⁺ vector containing a sequence encoding SARS CoV-2 Nucleocapsid protein tagged with eGFP and Compound B18 (Cpd. B18) comprising 3 siRNAs, one of which targets SARS CoV-2 Nucleocapsid protein.

FIG. 17C shows RNA interference of Compound B18 (Cpd. B18) which comprise siRNAs targeting SARS CoV-2 Nucleocapsid protein in A549 cells expressing SARS CoV-2 Nucleocapsid protein tagged with eGFP. Control: SARS CoV-2 Nucleocapsid protein-eGFP construct alone. Significance (***, <0.001) was assessed by Student's t-test of Compound B18 (Cpd. B18) compared to a control.

DETAILED DESCRIPTION

Certain specific details of this description are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the present disclosure may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods, and materials are described below.

Definitions

As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. The terms “and/or” and “any combination thereof” and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

The term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

Reference in the specification to “embodiments,” “certain embodiments,” “preferred embodiments,” “specific embodiments,” “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” mean that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

The term “RNA” as used herein includes RNA which encodes an amino acid sequence (e.g., mRNA, etc.) as well as RNA which does not encode an amino acid sequence (e.g., siRNA, shRNA etc.). The RNA as used herein may be a coding RNA, i.e., an RNA which encodes an amino acid sequence. Such RNA molecules are also referred to as mRNA (messenger RNA) and are single-stranded RNA molecules. The RNA as used herein may be a non-coding RNA, i.e., an RNA which does not encode an amino acid sequence or is not translated into a protein. A non-coding RNA can include, but are not limited to, small interfering RNA (siRNA), short or small harpin RNA (shRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), and long non-coding RNA (lncRNA). siRNAs as used herein may comprise a double-stranded RNA (dsRNA) region, a hairpin structure, a loop structure, or a combination thereof. In some embodiments, siRNAs as used herein may comprise at least one shRNA, at least one dsRNA region, or at least one loop structure. In some embodiments, siRNAs as used herein may be processed from a dsRNA or an shRNA. The RNA may be made by synthetic chemical and enzymatic methodology known to one of ordinary skill in the art, or by the use of recombinant technology, or may be isolated from natural sources, or by a combination thereof. The RNA may optionally comprise unnatural and naturally occurring nucleoside modifications known in the art such as e.g., N¹-Methylpseudouridine also referred herein as methylpseudouridine.

The terms “nucleic acid sequence,” “polynucleic acid sequence,” “nucleotide sequence,” and “nucleotide acid sequence” are used herein interchangeably and have the identical meaning herein and refer to preferably DNA or RNA. The terms “nucleic acid sequence,” “nucleotide sequence,” and “nucleotide acid sequence” can be used synonymously with the term “polynucleotide sequence.” In some embodiments, a nucleic acid sequence is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone. The term “nucleic acid sequence” also encompasses modified nucleic acid sequences, such as base-modified, sugar-modified or backbone-modified etc., DNA or RNA.

The recombinant polynucleic acid or RNA construct described herein may include one or more nucleotide variants, including nonstandard nucleotide(s), non-natural nucleotide(s), nucleotide analog(s), and/or modified nucleotides. Examples of modified nucleotides include, but are not limited to diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine and the like. In some cases, nucleotides may include modifications in their phosphate moieties, including modifications to a triphosphate moiety. Non-limiting examples of such modifications include phosphate chains of greater length (e.g., a phosphate chain having, 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., alpha-thiotriphosphate and beta-thiotriphosphates).

The recombinant polynucleic acid or RNA construct described herein may be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and/or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety, or phosphate backbone. In some embodiments, backbone modifications include, but are not limited to, a phosphorothioate, a phosphorodithioate, a phosphoroselenoate, a phosphorodiselenoate, a phosphoroanilothioate, a phosphoraniladate, a phosphoramidate, and a phosphorodiamidate linkage. A phosphorothioate linkage substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone and delay nuclease degradation of oligonucleotides. A phosphorodiamidate linkage (N3′→P5′) allows prevents nuclease recognition and degradation. In some embodiments, backbone modifications include having peptide bonds instead of phosphorous in the backbone structure (e.g., N-(2-aminoethyl)-glycine units linked by peptide bonds in a peptide nucleic acid), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups. Oligonucleotides with modified backbones are reviewed in Micklefield, Backbone modification of nucleic acids: synthesis, structure and therapeutic applications, Curr. Med. Chem., 8 (10): 1157-79, 2001 and Lyer et al., Modified oligonucleotides-synthesis, properties and applications, Curr. Opin. Mol. Ther., 1 (3): 344-358, 1999.

The terms “peptide” refers to a series of amino acid residues connected one to the other, typically by peptide bonds between the α-amino and carboxyl groups of adjacent amino acid residues.

The term “target motif” or “targeting motif” as used herein can refer to any short peptide present in the newly synthesized polypeptides or proteins that are destined to any parts of cell membranes, extracellular compartments, or intracellular compartments except cytoplasm or cytosol. Intracellular compartments include, but are not limited to, intracellular organelles such as nucleus, nucleolus, endosome, proteasome, ribosome, chromatin, nuclear envelope, nuclear pore, exosome, melanosome, Golgi apparatus, peroxisome, endoplasmic reticulum (ER), lysosome, centrosome, microtubule, mitochondria, chloroplast, microfilament, intermediate filament, or plasma membrane. Other terms include, but are not limited to, signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide. The target motif may comprise a signal peptide, a nuclear localization signal (NLS), a nucleolar localization signal (NoLS), a lysosomal targeting signal, a mitochondrial targeting signal, a peroxisomal targeting signal, a microtubule tip localization signal (MtLS), an endosomal targeting signal, a chloroplast targeting signal, a Golgi targeting signal, an endoplasmic reticulum (ER) targeting signal, a proteasomal targeting signal, a membrane targeting signal, a transmembrane targeting signal, or a centrosomal localization signal (CLS).

The term “signal peptide” also referred herein to as signaling peptide or pre-domain is a short peptide (usually 16-40 amino acids long) present at the N-terminus of newly synthesized proteins that are destined towards the secretory pathway. The signal peptide of the present invention is preferably 10-50, more preferably 11-45, even more preferably 12-45, most preferably 13-45, in particular 14-45, more particular 15-45, even more particular 16-40 amino acids long. A signal peptide according to the invention is situated at the N-terminal end of the protein of interest or at the N-terminal end of the pro-protein form of the protein of interest. A signal peptide according to the invention is usually of eukaryotic origin e.g., the signal peptide of a eukaryotic protein, preferably of mammalian origin e.g., the signal peptide of a mammalian protein, more preferably of human origin e.g., the signal peptide of a mammalian protein. In some embodiments the heterologous signal peptide and/or the homologous signal peptide to be modified is the naturally occurring signal peptide of a eukaryotic protein, preferably the naturally occurring signal peptide of a mammalian protein, more preferably the naturally occurring signal peptide of a human protein.

The term “protein” as used herein refers to molecules typically comprising one or more peptides or polypeptides. A peptide or polypeptide is typically a chain of amino acid residues, linked by peptide bonds. A peptide usually comprises between 2 and 50 amino acid residues. A polypeptide usually comprises more than 50 amino acid residues. A protein is typically folded into 3-dimensional form, which may be required for the protein to exert its biological function. The term “protein” as used herein includes a fragment of a protein and fusion proteins. In some embodiments, the protein is mammalian, e.g., of human origin, i.e., is a human protein. In some embodiments, the protein is a protein which is normally secreted from a cell, i.e., a protein which is secreted from a cell in nature, or a protein produced by a virus. In some embodiments, proteins as referred to herein are selected from the group consisting of: carboxypeptidases; cytokines; extracellular ligands and transporters, including receptors; extracellular matrix proteins; glucosidases; glycosyltransferases; growth factors; growth factor binding proteins; heparin binding proteins; hormones; hydrolases; immunoglobulins; isomerases; kinases; lyases; metalloenzyme inhibitors; metalloproteases; milk proteins; neuroactive proteins; proteases; protease inhibitors; protein phosphatases; esterases; transferases; and vasoactive proteins. In some embodiments, the protein is a viral protein, e.g., a coronavirus protein, as described herein.

Carboxypeptidases are proteins which are protease enzymes that hydrolyze (cleave) a peptide bond at the carboxy-terminal (C-terminal) end of a protein; cytokines are proteins which are secreted and act either locally or systemically as modulators of target cell signaling via receptors on their surfaces, often involved in immunologic reactions; extracellular ligands and transporters are proteins that are secreted and act via binding to other proteins or carrying other proteins or other molecules to exert a certain biological function; extracellular matrix proteins are a collection of proteins secreted by support cells that provide structural and biochemical support to the surrounding cells; glucosidases are enzymes involved in breaking down complex carbohydrates such as starch and glycogen into their monomers; glycosyltransferases are enzymes that establish natural glycosidic linkages; growth factors are secreted proteins capable of stimulating cellular growth, proliferation, healing, and cellular differentiation either acting locally or systemically as modulators of target cell signaling via receptors on their surfaces, often involved in trophic reactions and survival or cell homeostasis signaling; growth factor binding proteins are secreted proteins binding to growth factors and thereby modulating their biological activity; heparin binding proteins are secreted proteins that interact with heparin to modulate their biological function, often in conjunction with another binding to a growth factor or hormone; hormones are members of a class of signaling molecules produced by glands in multicellular organisms that are secreted and transported by the circulatory system to target distant organs to regulate physiology and behavior via binding to specific receptors on their target cells; hydrolases are a class of enzymes that biochemically catalyze molecule cleavage by utilizing water to break chemical bonds, resulting in a division of a larger molecule to smaller molecules; immunoglobulins are large, Y-shaped secreted proteins produced mainly by plasma cells that are used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses; isomerases are a general class of enzymes that convert a molecule from one isomer to another, thereby facilitating intramolecular rearrangements in which bonds are broken and formed; kinases are enzymes catalyzing the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates; lyases are enzymes catalyzing the breaking of various chemical bonds by means other than hydrolysis and oxidation, often forming a new double bond or a new ring structure; metalloenzyme inhibitors cellular inhibitors of the Matrix metalloproteases (MMPs); metalloproteases are protease enzymes whose catalytic mechanism involves a metal ion; milk proteins are proteins secreted into milk; neuroactive proteins are secreted proteins that act either locally or via distances to support neuronal function, survival and physiology; proteases (also called peptidases or proteinases) are enzymes that perform proteolysis by hydrolysis of peptide bonds; protease inhibitors are proteins that inhibit the function of proteases; protein phosphatases are enzymes that remove phosphate groups from phosphorylated amino acid residues of their substrate protein; esterases are enzymes that split esters into an acid and an alcohol in a chemical reaction with water at an amino acid residue; transferases are a class of enzymes that catalyze the transfer of specific functional groups (e.g., a methyl or glycosyl group) from one molecule (called the donor) to another (called the acceptor); vasoactive proteins are secreted proteins that biologically affect function of blood vessels. Carboxypeptidases; cytokines; extracellular ligands and transporters; extracellular matrix proteins; glucosidases; glycosyltransferases; growth factors; growth factor binding proteins; heparin binding proteins; hormones; hydrolases; immunoglobulins; isomerases; kinases; lyases; metalloenzyme inhibitors; metalloproteases; milk proteins; neuroactive proteins; proteases; protease inhibitors; protein phosphatases; esterases; transferases; and vasoactive proteins as referred to herein can be found in the UniProt database.

In some embodiments, proteins as referred to herein are, e.g., cytokines, proteins that are secreted and act either locally or systemically as modulators of target cell signaling via receptors on their surfaces, often involved in immunologic reactions, other host proteins involved in viral infection, and virus proteins. Nucleotide and amino acid sequences of proteins useful in the context of the present invention, including proteins that are encoded by a gene of interest, are known in the art and available in the literature, e.g., in the UniProt database.

The terms “fragment,” or “fragment of a sequence” which have the identical meaning herein is a shorter portion of a full-length sequence of e.g., a nucleic acid molecule like DNA or RNA or a protein. Accordingly, a fragment, typically, consists of a sequence that is identical to the corresponding stretch within the full-length sequence. A preferred fragment of a sequence in the context of the present invention, consists of a continuous stretch of entities, such as nucleotides or amino acids corresponding to a continuous stretch of entities in the molecule the fragment is derived from, which represents at least 5%, usually at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, and most preferably at least 80% of the total (i.e., full-length) molecule, from which the fragment is derived.

The term “vector” or “expression vector” as used herein refers to naturally occurring or synthetically generated constructs for uptake, proliferation, expression or transmission of nucleic acids in a cell, e.g., plasmids, minicircles, phagemids, cosmids, artificial chromosomes/mini-chromosomes, bacteriophages, viruses such as baculovirus, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, bacteriophages. Vectors can either integrate into the genome of the host cell or remain as autonomously replicating construct within the host cell. Methods used to construct vectors are well known to a person skilled in the art and described in various publications. In particular techniques for constructing suitable vectors, including a description of the functional and regulatory components such as promoters, enhancers, termination and polyadenylation signals, selection markers, origins of replication, and splicing signals, are known to the person skilled in the art. The eukaryotic expression vectors will typically contain also prokaryotic sequences that facilitate the propagation of the vector in bacteria such as an origin of replication and antibiotic resistance genes for selection in bacteria which might be removed before transfection of eukaryotic cells. A variety of eukaryotic expression vectors, containing a cloning site into which a polynucleotide can be operably linked, are well known in the art and some are commercially available from companies such as Agilent Technologies, Santa Clara, Calif.; Invitrogen, Carlsbad, Calif.; Promega, Madison, Wis. or Invivogen, San Diego, Calif.

The term “transcription unit,” “expression unit,” or “expression cassette” as used herein refers a region within a vector, construct or polynucleotide sequence that contains one or more genes to be transcribed, wherein the genes contained within the segment are operably linked to each other. They are transcribed from a single promoter and transcription is terminated by at least one polyadenylation signal. As a result, the different genes are at least transcriptionally linked. More than one protein or product can be transcribed and expressed from each transcription unit (multicistronic transcription unit). Each transcription unit will comprise the regulatory elements necessary for the transcription and translation of any of the selected sequence that are contained within the unit. And each transcription unit may contain the same or different regulatory elements. For example, each transcription unit may contain the same terminator. IRES element or introns may be used for the functional linking of the genes within a transcription unit. A vector or polynucleotide sequence may contain more than one transcription unit.

The term “skeletal muscle injury” as used herein refers to any injuries and ruptures of skeletal muscle, preferably ruptures of skeletal muscle, induced by eccentric muscle contractions, elongations and muscle overload. In principle any skeletal muscle can be affected by such injury or rupture. Preferably skeletal muscle injury are injuries and ruptures of skeletal muscle wherein the skeletal muscles are selected from the muscle groups of the head, the neck, the thorax, the back, the abdomen, the pelvis, the arms, the legs and the hip.

More preferably skeletal muscle injury are injuries and ruptures wherein the skeletal muscles are selected from the group consisting of plantaris, temporal, papillary, pectoralis major, tibialis posterior, tibialis anterior, gastrocnemius, coracobrachialis, diaphragma, palmaris longus, rectus abdominis, external anal sphincter, internal anal sphincter, subscapularis, biceps, triceps, quadriceps, calf, groin, hamstring, deltoid, teres major, rotator cuff supraspinatus, rotator cuff infraspinatus, rotator cuff teres minor, rotator cuff subscapularis, rectus femoralis, rectus abdominis, abdominal external oblique, masseter, trapezius, latissimus, pectoralis, erector spinae, iliocostalis, longissimus, spinalis, latissimus dorsi, transversospinales, semispinalis dorsi, semispinalis cervices, semispinalis capitis, multifidus, rotatores, interspinales, intertransversarii, splenius capitis, splenius cervices, intercostals, subcostales, transversus thoracis, levatores costarum, serratus posterior inferior, serratus posterior superior, Transversus abdominis, rectus abdominis, pyramidalis, cremaster, quadratus lumborum, external oblique, internal oblique. Even more preferably skeletal muscle injury are injuries and ruptures wherein the skeletal muscles are selected from the group consisting of plantaris, temporal, papillary, pectoralis major, tibialis posterior, tibialis anterior, gastrocnemius, coracobrachialis, diaphragma, palmaris longus, rectus abdominis, external anal sphincter, internal anal sphincter, subscapularis, biceps, triceps, quadriceps, calf, groin, hamstring, deltoid, teres major, rotator cuff supraspinatus, rotator cuff infraspinatus, rotator cuff teres minor, rotator cuff subscapularis, rectus femoralis, rectus abdominis, abdominal external oblique, masseter, trapezius, latissimus, pectoralis.

Preferably any injuries and ruptures of skeletal muscle, preferably ruptures of skeletal muscle, induced by eccentric muscle contraction, elongation or muscle overload are treated by the method of the present invention.

The term “subject” or “patient” encompasses mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. In one aspect, the mammal is a human. The term “animal” as used herein comprises human beings and non-human animals. In one embodiment, a “non-human animal” is a mammal, for example a rodent such as rat or a mouse. In one embodiment, a non-human animal is a mouse.

The terms “pharmaceutical composition” and “pharmaceutical formulation” (or “formulation”) are used interchangeably and denote a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients to be administered to a subject, e.g., a human in need thereof.

The term “pharmaceutically acceptable” denotes an attribute of a material which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and is acceptable for veterinary as well as human pharmaceutical use. “Pharmaceutically acceptable” can refer to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

The terms “pharmaceutically acceptable excipient”, “pharmaceutically acceptable carrier” and “therapeutically inert excipient” can be used interchangeably and denote any pharmaceutically acceptable ingredient in a pharmaceutical composition having no therapeutic activity and being non-toxic to the subject administered, such as disintegrators, binders, fillers, solvents, buffers, tonicity agents, stabilizers, antioxidants, surfactants, carriers, diluents, excipients, preservatives or lubricants used in formulating pharmaceutical products.

The term “recombinant polynucleic acid” or “recombinant RNA” can refer to a polynucleic acid or RNA that are not naturally occurring and are synthesized or manipulated in vitro. A recombinant polynucleic acid or RNA can be synthesized in a laboratory and can be prepared by using recombinant DNA or RNA technology by using enzymatic modification of DNA or RNA, such as enzymatic restriction digestion, ligation, and cloning. A recombinant polynucleic acid can be transcribed in vitro to produce a messenger RNA (mRNA) and the recombinant mRNA can be isolated, purified, and used for transfection. A recombinant polynucleic acid or RNA used herein can encode a protein, polypeptide, a target motif, a signal peptide, and/or a non-coding RNA such as small interfering RNA (siRNA). Under suitable conditions, a recombinant polynucleic acid or RNA can be incorporated into a cell and expressed within the cell.

The term “expression” of a polynucleic acid, gene, DNA, or RNA, as used herein, can refer to transcription and/or translation of the polynucleic acid, gene, DNA, or RNA. The term “modulating,” “increasing,” “upregulating,” “decreasing,” or “downregulating” the expression of a polynucleic acid, gene such as a gene of interest, DNA, or RNA such as a target mRNA, as used herein, can refer to modulating, increasing, upregulating, decreasing, downregulating the level of protein encoded by a polynucleic acid, gene such as a gene of interest, DNA, or RNA such as a target mRNA by affecting transcription and/or translation of the polynucleic acid, gene such as a gene of interest, DNA, or RNA such as a target mRNA. The term “inhibiting” the expression of a polynucleic acid, gene such as a gene of interest, DNA, or RNA such as a target mRNA can refer to affect transcription and/or translation of the polynucleic acid, gene such as a gene of interest, DNA, or RNA such as a target mRNA such that the level of protein encoded by the polynucleic acid, gene such as a gene of interest, DNA, or RNA such as a target mRNA is reduced or abolished.

The term “operably linked” can refer to a functional relationship between two or more nucleic acid sequences, e.g., a functional relationship of a transcriptional regulatory or signal sequence to a transcribed sequence. For example, a target motif or a nucleic acid encoding a target motif is operably linked to a coding sequence if it is expressed as a preprotein that participates in targeting the polypeptide encoded by the coding sequence to a cell membrane, intracellular, or an extracellular compartment. For example, a signal peptide or a nucleic acid encoding a signal peptide is operably linked to a coding sequence if it is expressed as a preprotein that participates in the secretion of the polypeptide encoded by the coding sequence. For example, a promoter is operably linked if it stimulates or modulates the transcription of the coding sequence.

The term “Kozak sequence,” “Kozak consensus sequence,” or “Kozak consensus” can refer to a nucleic acid sequence motif that functions as the protein translation initiation site. Kozak sequences are described at length in the literature, e.g., by Kozak, M., Gene 299(1-2):1-34, incorporated herein by reference herein in its entirety.

Construct Design

The present invention disclosed herein refers to a composition comprising a polynucleic acid or RNA construct to express (i) siRNAs capable of binding to one or more target RNA (e.g., mRNA) and (ii) one or more genes of interest from a single RNA transcript. The present invention provides a means to express (i) siRNAs capable of binding to one or more target mRNA and (ii) one or more protein of interest simultaneously from a single RNA transcript. The present invention provides a means to modulate expression of two or more genes simultaneously. In some embodiments, siRNA capable of binding to a target mRNA in the composition downregulates the expression of the target mRNA while simultaneously the gene of interest is expressed or overexpressed to increase the level of protein encoded by the gene of interest. In some embodiments, the recombinant polynucleic acid or RNA construct of the present invention comprises (i) siRNAs that can target multiple mRNAs and multiple genes of interest, (ii) multiple copies of siRNAs that can target one mRNA and multiple copies of the same gene of interest, or (iii) combination of the (i) and (ii). In some embodiments, the recombinant polynucleic acid or RNA construct of the present invention comprise siRNAs that target multiple mRNAs and multiple copies of the same gene of interest. In some embodiments, the recombinant polynucleic acid or RNA construct of the present invention comprise multiple copies of siRNAs that can target one mRNA and multiple genes of interest.

(SEQ ID NO: 24)

AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTA

CAGACCCGGGTTCGATTCCCGGCTGGTGCA.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to a target messenger RNA (mRNA); and (ii) an mRNA encoding a gene of interest; wherein the target mRNA is different from the mRNA encoding the gene of interest.

In some embodiments, (i) and (ii) may be comprised in 5′ to 3′ direction. In some embodiments, (i) and (ii) may not be comprised in 5′ to 3′ direction. In some embodiments (i) and (ii) may be comprised in 3′ to 5′ direction. In some embodiments, (i) and (ii) may not be comprised or present in a sequential manner. In some embodiments, (i) and (ii) may be comprised or present in a sequential manner. In some aspects, the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are comprised or present in a sequential manner. In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is downstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream or downstream of the at least one nucleic acid sequence encoding a gene of interest (ii). In some aspects, the siRNA capable of binding to a target RNA binds to an exon of a target mRNA. In some aspects, the siRNA capable of binding to a target RNA specifically binds to one target RNA. In some aspects, the siRNA capable of binding to a target RNA is not encoded by or comprised of an intron sequence of the gene of interest. In some aspects, the gene of interest is expressed without RNA splicing. In some embodiments, (i) and (ii) may be separated. In some embodiments, (i) and (ii) may be arranged in tandem. In some embodiments, the siRNA capable of binding to the target RNA and the mRNA encoding the gene of interest are separated. In some embodiments, the siRNA capable of binding to the target RNA and the mRNA encoding the gene of interest are arranged in tandem. For example, the siRNA capable of binding to the target RNA is located either upstream or downstream of the mRNA encoding the gene of interest in the composition.

In some embodiments, the expression of the gene of interest is increased when the composition comprises a nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA downstream of (or 3′ to) a nucleic acid sequence encoding a gene of interest, compared to the expression of the gene of interest from a composition comprising a nucleic acid sequence encoding or comprising an siRNA capable of binding to a target RNA upstream of (or 5′ to) a nucleic acid sequence encoding a gene of interest. In some embodiments, the expression of the gene of interest is increased when the composition comprises a nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA and a nucleic acid sequence encoding a gene of interest in 3′ to 5′ direction, compared to the expression of the gene of interest from a composition comprising a nucleic acid sequence encoding or comprising an siRNA capable of binding to a target RNA and a nucleic acid sequence encoding a gene of interest in 5′ to 3′ direction.

As described herein, in some embodiments, in a composition comprising a recombinant polynucleic acid construct, the at least one nucleic acid sequence encoding or comprising the at least one small interfering RNA (siRNA) capable of binding to a target RNA and the at least one nucleic acid sequence encoding a gene of interest are comprised in a sequential manner. In some embodiments, in a composition comprising a recombinant polynucleic acid construct, the at least one nucleic acid sequence encoding or comprising the at least one small interfering RNA (siRNA) capable of binding to a target RNA and the at least one nucleic acid sequence encoding a gene of interest are present in a sequential manner. In some embodiments, the composition comprises the at least one nucleic acid sequence encoding or comprising two or more, preferably 2 to 10, more preferably 2 to 6, small interfering RNAs (siRNAs) capable of binding to a target RNA and the at least one nucleic acid sequence encoding a gene of interest in a sequential manner. In some embodiments, the expression of the gene of interest is decreased when the composition comprises a nucleic acid sequence encoding or comprising two or more, preferably 2 to 10, more preferably 2 to 6, siRNAs capable of binding to a target RNA and a nucleic acid sequence encoding a gene of interest in 5′ to 3′ direction, compared to the expression of the gene of interest from a composition comprising a nucleic acid sequence encoding or comprising two or more siRNAs capable of binding to a target RNA and a nucleic acid sequence encoding a gene of interest in 3′ to 5′ direction. In some embodiments, the expression of the gene of interest is decreased when the composition comprises a nucleic acid sequence encoding or comprising two or more, preferably 2 to 10, more preferably 2 to 6, siRNAs capable of binding to a target RNA upstream of (or 5′ to) the nucleic acid sequence encoding a gene of interest, compared to the expression of the gene of interest from a composition comprising a nucleic acid sequence encoding or comprising two or more siRNAs capable of binding to a target RNA downstream of (or 3′ to) a nucleic acid sequence encoding a gene of interest. In some embodiments, the expression of the gene of interest is decreased when the sequential manner comprises the at least one nucleic acid sequence encoding a gene of interest positioned downstream of (3′ to), the at least one nucleic acid sequence encoding or comprising the two or more, preferably 2 to 10, more preferably 2 to 6, siRNAs, relative to the expression of the gene of interest when the sequential manner comprises the at least one nucleic acid sequence encoding a gene of interest positioned upstream of (5′ to), the at least one nucleic acid sequence encoding or comprising the two or more siRNAs.

In some embodiments, the expression of the gene of interest is increased when the composition comprises a nucleic acid sequence encoding or comprising two or more, preferably 2 to 10, more preferably 2 to 6, siRNAs capable of binding to a target RNA and a nucleic acid sequence encoding a gene of interest in 3′ to 5′ direction, compared to the expression of the gene of interest from a composition comprising a nucleic acid sequence encoding or comprising two or more siRNAs capable of binding to a target RNA and a nucleic acid sequence encoding a gene of interest in 5′ to 3′ direction. In some embodiments, the expression of the gene of interest is increased when the composition comprises a nucleic acid sequence encoding or comprising two or more, preferably 2 to 10, more preferably 2 to 6, siRNAs capable of binding to a target RNA downstream of (or 5′ to) the nucleic acid sequence encoding a gene of interest, compared to the expression of the gene of interest from a composition comprising a nucleic acid sequence encoding or comprising two or more siRNAs capable of binding to a target RNA upstream of (or 3′ to) a nucleic acid sequence encoding a gene of interest. In some embodiments, the expression of the gene of interest is increased when the sequential manner comprises the at least one nucleic acid sequence encoding a gene of interest positioned upstream of (5′ to), the at least one nucleic acid sequence encoding or comprising the two or more, preferably 2 to 10, more preferably 2 to 6, siRNA, relative to the expression of the gene of interest when the sequential manner comprises the at least one nucleic acid sequence encoding a gene of interest positioned downstream of (3′ to), the at least one nucleic acid sequence encoding or comprising the two or more siRNA.

In some embodiments, the downregulation of the target RNA is enhanced when the composition comprises a nucleic acid sequence encoding or comprising two or more, preferably 2 to 10, more preferably 2 to 6, small interfering RNAs (siRNAs) capable of binding to a target RNA downstream of (or 3′ to) a nucleic acid sequence encoding a gene of interest, compared to the downregulation of the target RNA from a composition comprising a nucleic acid sequence encoding or comprising two or more siRNAs capable of binding to a target RNA upstream of (or 5′ to) a nucleic acid sequence encoding a gene of interest. In some embodiments, the downregulation of the target RNA is enhanced when the composition comprises a nucleic acid sequence encoding or comprising two or more, preferably 2 to 10, more preferably 2 to 6, siRNAs capable of binding to a target RNA and a nucleic acid sequence encoding a gene of interest in 3′ to 5′ direction, compared to the downregulation of the target RNA from a composition comprising a nucleic acid sequences encoding or comprising two or more siRNA capable of binding to a target RNA and a nucleic acid sequence encoding a gene of interest in 5′ to 3′ direction. In some embodiments, the downregulation of the target RNA is enhanced when the sequential manner comprises the at least one nucleic acid sequence encoding or comprising two or more, preferably 2 to 10, more preferably 2 to 6, siRNAs positioned downstream of (3′ to), the at least one nucleic acid sequence encoding the gene of interest, relative to the downregulation of the target RNA when the sequential manner comprises the at least one nucleic acid sequence encoding or comprising two or more siRNAs positioned upstream of (5′ to), the at least one nucleic acid sequence encoding the gene of interest.

In some embodiments, the downregulation of the target RNA is reduced when the composition comprises a nucleic acid sequence encoding or comprising two or more, preferably 2 to 10, more preferably 2 to 6, small interfering RNAs (siRNAs) capable of binding to a target RNA upstream of (or 5′ to) a nucleic acid sequence encoding a gene of interest, compared to the downregulation of the target RNA from a composition comprising a nucleic acid sequence encoding or comprising two or more siRNAs capable of binding to a target RNA downstream of (or 3′ to) a nucleic acid sequence encoding a gene of interest. In some embodiments, the downregulation of the target RNA is reduced when the composition comprises a nucleic acid sequence encoding or comprising two or more, preferably 2 to 10, more preferably 2 to 6, siRNAs capable of binding to a target RNA and a nucleic acid sequence encoding a gene of interest in 5′ to 3′ direction, compared to the downregulation of the target RNA from a composition comprising a nucleic acid sequence encoding or comprising two or more siRNAs capable of binding to a target RNA and a nucleic acid sequence encoding a gene of interest in 3′ to 5′ direction. In some embodiments, the downregulation of the target RNA is reduced when the sequential manner comprises the at least one nucleic acid sequence encoding or comprising two or more, preferably 2 to 10, more preferably 2 to 6, siRNAs positioned upstream of (5′ to), the at least one nucleic acid sequence encoding the gene of interest, relative to the downregulation of the target RNA when the sequential manner comprises the at least one nucleic acid sequence encoding or comprising two or more siRNAs positioned downstream of (3′ to), the at least one nucleic acid sequence encoding the gene of interest.

In some embodiments, the expression of the gene of interest is increased, and the downregulation of the target RNA is enhanced, when the sequential manner comprises the at least one nucleic acid sequence encoding a gene of interest positioned upstream of (5′ to), the at least one nucleic acid sequence encoding or comprising two or more, preferably 2 to 10, more preferably 2 to 6, siRNAs, relative to the expression of the gene of interest when the sequential manner comprises the at least one nucleic acid sequence encoding a gene of interest positioned downstream of (3′ to), the at least one nucleic acid sequence encoding or comprising two or more siRNAs.

In some embodiments, the relative increase in the expression of the gene of interest is about 2-fold to about 30-fold. In some embodiments, the relative increase in the expression of the gene of interest is about 2 fold to about 30 fold. In some embodiments, the relative increase in the expression of the gene of interest is about 2 fold to about 5 fold, about 2 fold to about 10 fold, about 2 fold to about 15 fold, about 2 fold to about 17 fold, about 2 fold to about 18 fold, about 2 fold to about 19 fold, about 2 fold to about 20 fold, about 2 fold to about 21 fold, about 2 fold to about 22 fold, about 2 fold to about 25 fold, about 2 fold to about 30 fold, about 5 fold to about 10 fold, about 5 fold to about 15 fold, about 5 fold to about 17 fold, about 5 fold to about 18 fold, about 5 fold to about 19 fold, about 5 fold to about 20 fold, about 5 fold to about 21 fold, about 5 fold to about 22 fold, about 5 fold to about 25 fold, about 5 fold to about 30 fold, about 10 fold to about 15 fold, about 10 fold to about 17 fold, about 10 fold to about 18 fold, about 10 fold to about 19 fold, about 10 fold to about 20 fold, about 10 fold to about 21 fold, about 10 fold to about 22 fold, about 10 fold to about 25 fold, about 10 fold to about 30 fold, about 15 fold to about 17 fold, about 15 fold to about 18 fold, about 15 fold to about 19 fold, about 15 fold to about 20 fold, about 15 fold to about 21 fold, about 15 fold to about 22 fold, about 15 fold to about 25 fold, about 15 fold to about 30 fold, about 17 fold to about 18 fold, about 17 fold to about 19 fold, about 17 fold to about 20 fold, about 17 fold to about 21 fold, about 17 fold to about 22 fold, about 17 fold to about 25 fold, about 17 fold to about 30 fold, about 18 fold to about 19 fold, about 18 fold to about 20 fold, about 18 fold to about 21 fold, about 18 fold to about 22 fold, about 18 fold to about 25 fold, about 18 fold to about 30 fold, about 19 fold to about 20 fold, about 19 fold to about 21 fold, about 19 fold to about 22 fold, about 19 fold to about 25 fold, about 19 fold to about 30 fold, about 20 fold to about 21 fold, about 20 fold to about 22 fold, about 20 fold to about 25 fold, about 20 fold to about 30 fold, about 21 fold to about 22 fold, about 21 fold to about 25 fold, about 21 fold to about 30 fold, about 22 fold to about 25 fold, about 22 fold to about 30 fold, or about 25 fold to about 30 fold. In some embodiments, the relative increase in the expression of the gene of interest is about 2 fold, about 5 fold, about 10 fold, about 15 fold, about 17 fold, about 18 fold, about 19 fold, about 20 fold, about 21 fold, about 22 fold, about 25 fold, or about 30 fold. In some embodiments, the relative increase in the expression of the gene of interest is at least about 2 fold, about 5 fold, about 10 fold, about 15 fold, about 17 fold, about 18 fold, about 19 fold, about 20 fold, about 21 fold, about 22 fold, or about 25 fold. In some embodiments, the relative increase in the expression of the gene of interest is at most about 5 fold, about 10 fold, about 15 fold, about 17 fold, about 18 fold, about 19 fold, about 20 fold, about 21 fold, about 22 fold, about 25 fold, or about 30 fold.

In embodiments, the relative enhancement of target RNA downregulation is about 1.1 fold to about 5 fold. In embodiments, the relative enhancement of target RNA downregulation is about 1.1 fold to about 1.75 fold, about 1.1 fold to about 2 fold, about 1.1 fold to about 2.25 fold, about 1.1 fold to about 2.5 fold, about 1.1 fold to about 3 fold, about 1.1 fold to about 3.5 fold, about 1.1 fold to about 4 fold, about 1.1 fold to about 4.5 fold, about 1.1 fold to about 5 fold, about 1.5 fold to about 1.75 fold, about 1.5 fold to about 2 fold, about 1.5 fold to about 2.25 fold, about 1.5 fold to about 2.5 fold, about 1.5 fold to about 3 fold, about 1.5 fold to about 3.5 fold, about 1.5 fold to about 4 fold, about 1.5 fold to about 4.5 fold, about 1.5 fold to about 5 fold, about 1.75 fold to about 2 fold, about 1.75 fold to about 2.25 fold, about 1.75 fold to about 2.5 fold, about 1.75 fold to about 3 fold, about 1.75 fold to about 3.5 fold, about 1.75 fold to about 4 fold, about 1.75 fold to about 4.5 fold, about 1.75 fold to about 5 fold, about 2 fold to about 2.25 fold, about 2 fold to about 2.5 fold, about 2 fold to about 3 fold, about 2 fold to about 3.5 fold, about 2 fold to about 4 fold, about 2 fold to about 4.5 fold, about 2 fold to about 5 fold, about 2.25 fold to about 2.5 fold, about 2.25 fold to about 3 fold, about 2.25 fold to about 3.5 fold, about 2.25 fold to about 4 fold, about 2.25 fold to about 4.5 fold, about 2.25 fold to about 5 fold, about 2.5 fold to about 3 fold, about 2.5 fold to about 3.5 fold, about 2.5 fold to about 4 fold, about 2.5 fold to about 4.5 fold, about 2.5 fold to about 5 fold, about 3 fold to about 3.5 fold, about 3 fold to about 4 fold, about 3 fold to about 4.5 fold, about 3 fold to about 5 fold, about 3.5 fold to about 4 fold, about 3.5 fold to about 4.5 fold, about 3.5 fold to about 5 fold, about 4 fold to about 4.5 fold, about 4 fold to about 5 fold, or about 4.5 fold to about 5 fold. In embodiments, the relative enhancement of target RNA downregulation is about 1.5 fold, about 1.75 fold, about 2 fold, about 2.25 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 4.5 fold, or about 5 fold. In embodiments, the relative enhancement of target RNA downregulation is at least about 1.5 fold, about 1.75 fold, about 2 fold, about 2.25 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, or about 4.5 fold. In embodiments, the relative enhancement of target RNA downregulation is at most about 1.75 fold, about 2 fold, about 2.25 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 4.5 fold, or about 5 fold.

In some embodiments, the expression of the gene of interest is increased by about 2-fold to about 30-fold, and the downregulation of the target RNA is enhanced by about 1.1 fold to about 5 fold, when the sequential manner comprises the at least one nucleic acid sequence encoding a gene of interest positioned upstream of (5′ to), the at least one nucleic acid sequence encoding or comprising the two or more siRNAs, relative to the expression of the gene of interest when the sequential manner comprises the at least one nucleic acid sequence encoding a gene of interest positioned downstream of (3′ to), the at least one nucleic acid sequence encoding or comprising the two or more siRNAs.

In some embodiments, the composition comprising a recombinant RNA construct further encodes or comprises a linker. In some embodiments, the nucleic acid sequence encoding or comprising the linker connects (i) and (ii). In some embodiments, the nucleic acid sequence encoding or comprising the linker connects the small interfering RNA (siRNA) capable of binding to a target messenger RNA (mRNA) and the mRNA encoding a gene of interest. In some embodiments, the linker comprises a tRNA linker. In some embodiments, the tRNA linker may comprise a nucleic acid sequence comprising

(SEQ ID NO: 24)

AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTA

CAGACCCGGGTTCGATTCCCGGCTGGTGCA.

In some embodiments, the recombinant polynucleic acid construct encodes a linker. In some embodiments, the encoded linker is a 2A peptide linker. In some aspects, the linker encoded or comprised by the recombinant nucleic acid construct is at least 6 nucleic acid residues in length. In some aspects, the linker encoded or comprised by the recombinant polynucleic acid construct is at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40, nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is up to 50 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is up to 80 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is up to 10, up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, up to 50, up to 55, up to 60, up to 65, up to 70, or up to 75 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is about 6 nucleic acid residues in length to about 50 nucleic acid residues in length. In some aspects, the nucleic acid sequence encoding or comprising the linker is about 6 nucleic acid residues in length to about 80 nucleic acid residues in length. In some aspects, the linker is about 6 nucleic acid residues in length to about 8 nucleic acid residues in length, about 6 nucleic acid residues in length to about 10 nucleic acid residues in length, about 6 nucleic acid residues in length to about 12 nucleic acid residues in length, about 6 nucleic acid residues in length to about 15 nucleic acid residues in length, about 6 nucleic acid residues in length to about 20 nucleic acid residues in length, about 6 nucleic acid residues in length to about 25 nucleic acid residues in length, about 6 nucleic acid residues in length to about 30 nucleic acid residues in length, about 6 nucleic acid residues in length to about 35 nucleic acid residues in length, about 6 nucleic acid residues in length to about 40 nucleic acid residues in length, about 6 nucleic acid residues in length to about 45 nucleic acid residues in length, about 6 nucleic acid residues in length to about 50 nucleic acid residues in length, about 6 nucleic acid residues in length to about 60 nucleic acid residues in length, about 6 nucleic acid residues in length to about 70 nucleic acid residues in length, about 6 nucleic acid residues in length to about 80 nucleic acid residues in length, about 8 nucleic acid residues in length to about 10 nucleic acid residues in length, about 8 nucleic acid residues in length to about 12 nucleic acid residues in length, about 8 nucleic acid residues in length to about 15 nucleic acid residues in length, about 8 nucleic acid residues in length to about 20 nucleic acid residues in length, about 8 nucleic acid residues in length to about 25 nucleic acid residues in length, about 8 nucleic acid residues in length to about 30 nucleic acid residues in length, about 8 nucleic acid residues in length to about 35 nucleic acid residues in length, about 8 nucleic acid residues in length to about 40 nucleic acid residues in length, about 8 nucleic acid residues in length to about 45 nucleic acid residues in length, about 8 nucleic acid residues in length to about 50 nucleic acid residues in length, about 10 nucleic acid residues in length to about 12 nucleic acid residues in length, about 10 nucleic acid residues in length to about 15 nucleic acid residues in length, about 10 nucleic acid residues in length to about 20 nucleic acid residues in length, about 10 nucleic acid residues in length to about 25 nucleic acid residues in length, about 10 nucleic acid residues in length to about 30 nucleic acid residues in length, about 10 nucleic acid residues in length to about 35 nucleic acid residues in length, about 10 nucleic acid residues in length to about 40 nucleic acid residues in length, about 10 nucleic acid residues in length to about 45 nucleic acid residues in length, about 10 nucleic acid residues in length to about 50 nucleic acid residues in length, about 12 nucleic acid residues in length to about 15 nucleic acid residues in length, about 12 nucleic acid residues in length to about 20 nucleic acid residues in length, about 12 nucleic acid residues in length to about 25 nucleic acid residues in length, about 12 nucleic acid residues in length to about 30 nucleic acid residues in length, about 12 nucleic acid residues in length to about 35 nucleic acid residues in length, about 12 nucleic acid residues in length to about 40 nucleic acid residues in length, about 12 nucleic acid residues in length to about 45 nucleic acid residues in length, about 12 nucleic acid residues in length to about 50 nucleic acid residues in length, about 15 nucleic acid residues in length to about 20 nucleic acid residues in length, about 15 nucleic acid residues in length to about 25 nucleic acid residues in length, about 15 nucleic acid residues in length to about 30 nucleic acid residues in length, about 15 nucleic acid residues in length to about 35 nucleic acid residues in length, about 15 nucleic acid residues in length to about 40 nucleic acid residues in length, about 15 nucleic acid residues in length to about 45 nucleic acid residues in length, about 15 nucleic acid residues in length to about 50 nucleic acid residues in length, about 20 nucleic acid residues in length to about 25 nucleic acid residues in length, about 20 nucleic acid residues in length to about 30 nucleic acid residues in length, about 20 nucleic acid residues in length to about 35 nucleic acid residues in length, about 20 nucleic acid residues in length to about 40 nucleic acid residues in length, about 20 nucleic acid residues in length to about 45 nucleic acid residues in length, about 20 nucleic acid residues in length to about 50 nucleic acid residues in length, about 25 nucleic acid residues in length to about 30 nucleic acid residues in length, about 25 nucleic acid residues in length to about 35 nucleic acid residues in length, about 25 nucleic acid residues in length to about 40 nucleic acid residues in length, about 25 nucleic acid residues in length to about 45 nucleic acid residues in length, about 25 nucleic acid residues in length to about 50 nucleic acid residues in length, about 30 nucleic acid residues in length to about 35 nucleic acid residues in length, about 30 nucleic acid residues in length to about 40 nucleic acid residues in length, about 30 nucleic acid residues in length to about 45 nucleic acid residues in length, about 30 nucleic acid residues in length to about 50 nucleic acid residues in length, about 35 nucleic acid residues in length to about 40 nucleic acid residues in length, about 35 nucleic acid residues in length to about 45 nucleic acid residues in length, about 35 nucleic acid residues in length to about 50 nucleic acid residues in length, about 40 nucleic acid residues in length to about 45 nucleic acid residues in length, about 40 nucleic acid residues in length to about 50 nucleic acid residues in length, or about 45 nucleic acid residues in length to about 50 nucleic acid residues in length. In some aspects, the linker is about 6 nucleic acid residues in length, about 8 nucleic acid residues in length, about 10 nucleic acid residues in length, about 12 nucleic acid residues in length, about 15 nucleic acid residues in length, about 20 nucleic acid residues in length, about 25 nucleic acid residues in length, about 30 nucleic acid residues in length, about 35 nucleic acid residues in length, about 40 nucleic acid residues in length, about 45 nucleic acid residues in length, or about 50 nucleic acid residues in length. In some aspects, the linker is at least about 6 nucleic acid residues in length, about 8 nucleic acid residues in length, about 10 nucleic acid residues in length, about 12 nucleic acid residues in length, about 15 nucleic acid residues in length, about 20 nucleic acid residues in length, about 25 nucleic acid residues in length, about 30 nucleic acid residues in length, about 35 nucleic acid residues in length, about 40 nucleic acid residues in length, or about 45 nucleic acid residues in length. In some aspects, the linker is at most about 8 nucleic acid residues in length, about 10 nucleic acid residues in length, about 12 nucleic acid residues in length, about 15 nucleic acid residues in length, about 20 nucleic acid residues in length, about 25 nucleic acid residues in length, about 30 nucleic acid residues in length, about 35 nucleic acid residues in length, about 40 nucleic acid residues in length, about 45 nucleic acid residues in length, or about 50 nucleic acid residues in length.

In some aspects, the nucleic acid sequence encoding or comprising the linker is about 6 to about 15 nucleic acid residues in length. In some aspects, the linker is about 6 nucleic acid residues in length to about 7 nucleic acid residues in length, about 6 nucleic acid residues in length to about 8 nucleic acid residues in length, about 6 nucleic acid residues in length to about 9 nucleic acid residues in length, about 6 nucleic acid residues in length to about 10 nucleic acid residues in length, about 6 nucleic acid residues in length to about 11 nucleic acid residues in length, about 6 nucleic acid residues in length to about 12 nucleic acid residues in length, about 6 nucleic acid residues in length to about 13 nucleic acid residues in length, about 6 nucleic acid residues in length to about 14 nucleic acid residues in length, about 6 nucleic acid residues in length to about 15 nucleic acid residues in length, about 7 nucleic acid residues in length to about 8 nucleic acid residues in length, about 7 nucleic acid residues in length to about 9 nucleic acid residues in length, about 7 nucleic acid residues in length to about 10 nucleic acid residues in length, about 7 nucleic acid residues in length to about 11 nucleic acid residues in length, about 7 nucleic acid residues in length to about 12 nucleic acid residues in length, about 7 nucleic acid residues in length to about 13 nucleic acid residues in length, about 7 nucleic acid residues in length to about 14 nucleic acid residues in length, about 7 nucleic acid residues in length to about 15 nucleic acid residues in length, about 8 nucleic acid residues in length to about 9 nucleic acid residues in length, about 8 nucleic acid residues in length to about 10 nucleic acid residues in length, about 8 nucleic acid residues in length to about 11 nucleic acid residues in length, about 8 nucleic acid residues in length to about 12 nucleic acid residues in length, about 8 nucleic acid residues in length to about 13 nucleic acid residues in length, about 8 nucleic acid residues in length to about 14 nucleic acid residues in length, about 8 nucleic acid residues in length to about 15 nucleic acid residues in length, about 9 nucleic acid residues in length to about 10 nucleic acid residues in length, about 9 nucleic acid residues in length to about 11 nucleic acid residues in length, about 9 nucleic acid residues in length to about 12 nucleic acid residues in length, about 9 nucleic acid residues in length to about 13 nucleic acid residues in length, about 9 nucleic acid residues in length to about 14 nucleic acid residues in length, about 9 nucleic acid residues in length to about 15 nucleic acid residues in length, about 10 nucleic acid residues in length to about 11 nucleic acid residues in length, about 10 nucleic acid residues in length to about 12 nucleic acid residues in length, about 10 nucleic acid residues in length to about 13 nucleic acid residues in length, about 10 nucleic acid residues in length to about 14 nucleic acid residues in length, about 10 nucleic acid residues in length to about 15 nucleic acid residues in length, about 11 nucleic acid residues in length to about 12 nucleic acid residues in length, about 11 nucleic acid residues in length to about 13 nucleic acid residues in length, about 11 nucleic acid residues in length to about 14 nucleic acid residues in length, about 11 nucleic acid residues in length to about 15 nucleic acid residues in length, about 12 nucleic acid residues in length to about 13 nucleic acid residues in length, about 12 nucleic acid residues in length to about 14 nucleic acid residues in length, about 12 nucleic acid residues in length to about 15 nucleic acid residues in length, about 13 nucleic acid residues in length to about 14 nucleic acid residues in length, about 13 nucleic acid residues in length to about 15 nucleic acid residues in length, or about 14 nucleic acid residues in length to about 15 nucleic acid residues in length. In some aspects, the linker is about 6 nucleic acid residues in length, about 7 nucleic acid residues in length, about 8 nucleic acid residues in length, about 9 nucleic acid residues in length, about 10 nucleic acid residues in length, about 11 nucleic acid residues in length, about 12 nucleic acid residues in length, about 13 nucleic acid residues in length, about 14 nucleic acid residues in length, or about 15 nucleic acid residues in length. In some aspects, the linker is at least about 6 nucleic acid residues in length, about 7 nucleic acid residues in length, about 8 nucleic acid residues in length, about 9 nucleic acid residues in length, about 10 nucleic acid residues in length, about 11 nucleic acid residues in length, about 12 nucleic acid residues in length, about 13 nucleic acid residues in length, or about 14 nucleic acid residues in length. In some aspects, the linker is at most about 7 nucleic acid residues in length, about 8 nucleic acid residues in length, about 9 nucleic acid residues in length, about 10 nucleic acid residues in length, about 11 nucleic acid residues in length, about 12 nucleic acid residues in length, about 13 nucleic acid residues in length, about 14 nucleic acid residues in length, or about 15 nucleic acid residues in length.

In some embodiments, the recombinant polynucleic acid construct is circular. In some embodiments, the recombinant polynucleic acid construct is linear. In some embodiments, the recombinant polynucleic acid is DNA. In some embodiments, the recombinant polynucleic acid is RNA.

In some embodiments, the recombinant polynucleic acid construct further comprises a promoter. In some embodiments, the promoter is upstream of the at least one nucleic acid sequence encoding or comprising the siRNA. Non-limiting examples of promoters include T3, T7, SP6, P60, Syn5, and KP34, etc. In some embodiments, the recombinant polynucleic acid construct comprises a T3 promoter. In some embodiments, the recombinant polynucleic acid construct comprises a SP6 promoter. In some embodiments, the recombinant polynucleic acid construct comprises a P60 promoter. In some embodiments, the recombinant polynucleic acid construct comprises a Syn5 promoter. In some embodiments, the recombinant polynucleic acid construct comprises a KP34 promoter. In a preferred embodiment, the recombinant polynucleic acid construct comprises a T7 promoter. In some embodiments, the T7 promoter comprises a sequence comprising TAATACGACTCACTATA (SEQ ID NO: 25). In some embodiments, the recombinant polynucleic acid or RNA construct further comprises a Kozak sequence.

In some embodiments, the recombinant polynucleic acid or RNA construct may be codon-optimized. In some embodiments, the recombinant polynucleic acid used in the present invention to transcribe the recombinant RNA construct of the present invention and the recombinant RNA construct of the present invention are codon-optimized. In general, codon optimization refers to a process of modifying a nucleic acid sequence for expression in a host cell of interest by replacing at least one codon (e.g., more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of a native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Codon usage tables are readily available, for example, at the “Codon Usage Database,” and these tables can be adapted in a number of ways. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge® (Aptagen, Pa.) and GeneOptimizer® (ThermoFischer, Mass.). In some embodiments, the recombinant polynucleic acid or RNA construct may not be codon-optimized.

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid or RNA construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target messenger RNA (mRNA); and (ii) at least one nucleic acid sequence encoding a gene of interest; wherein the target mRNA is different from an mRNA encoded by the gene of interest. In some embodiments, the recombinant polynucleic acid or RNA construct may comprise two or more nucleic acid sequences encoding or comprising a small interfering RNA (siRNA) capable of binding to a target messenger RNA (mRNA) and two or more nucleic acid sequences encoding a gene of interest. In some embodiments, the recombinant nucleic acid or RNA construct may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding an siRNA capable of binding to a target mRNA. In this embodiment, each of the two or more nucleic acid sequences may encode or comprise an siRNA capable of binding to a same target mRNA or a different target mRNA. In one embodiment, each of the two or more nucleic acid sequences may encode or comprise an siRNA capable of binding to a same target mRNA. In another embodiment, each of the two or more nucleic acid sequences may encode or comprise an siRNA capable of binding to a different target mRNA. In some embodiments, the recombinant nucleic acid or RNA construct may comprise two or more nucleic acid sequences encoding a gene of interest. In some embodiments, the recombinant nucleic acid or RNA construct may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding a gene of interest. In this embodiment, each of the two or more nucleic acid sequences may encode a same gene of interest or a different gene of interest, wherein the mRNA encoded by the same or the different gene of interest is different from the siRNA target mRNA. In one embodiment, each of the two or more nucleic acid sequences may encode a same gene of interest, wherein the mRNA encoded by the same gene of interest is different from the siRNA target mRNA. In another embodiment, each of the two or more nucleic acid sequences may encode a different gene of interest, wherein the mRNA encoded by the different gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and at least one nucleic acid sequence encoding a gene of interest, wherein each of the two or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a same target mRNA, wherein the at least one nucleic acid sequence encoding a gene of interest encodes a same gene of interest, and wherein the mRNA encoded by the same gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and at least one nucleic acid sequence encoding a gene of interest, wherein each of the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a same target mRNA, wherein the at least one nucleic acid sequence encoding a gene of interest encodes a same gene of interest, and wherein the mRNA encoded by the same gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and at least one nucleic acid sequence encoding a gene of interest, wherein each of the two or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a different target mRNA, wherein the at least one nucleic acid sequence encoding a gene of interest encodes a same gene of interest, and wherein the mRNA encoded by the same gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and at least one nucleic acid sequence encoding a gene of interest, wherein each of the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a different target mRNA, wherein the at least one nucleic acid sequence encoding a gene of interest encodes a same gene of interest, and wherein the mRNA encoded by the same gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and two or more nucleic acid sequences encoding a gene of interest, wherein each of the two or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a same target mRNA, wherein each of the two or more nucleic acid sequences encoding a gene of interest encodes a same gene of interest, and wherein the mRNA encoded by the same gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and two or more nucleic acid sequences encoding a gene of interest, wherein each of the two or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a different target mRNA, wherein each of the two or more nucleic acid sequences encoding a gene of interest encodes a same gene of interest, and wherein the mRNA encoded by the same gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and two or more nucleic acid sequences encoding a gene of interest, wherein each of the two or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a same target mRNA, wherein each of the two or more nucleic acid sequences encoding a gene of interest encodes a different gene of interest, and wherein the mRNA encoded by the different gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and two or more nucleic acid sequences encoding a gene of interest, wherein each of the two or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a different target mRNA, wherein each of the two or more nucleic acid sequences encoding a gene of interest encodes a different gene of interest, and wherein the mRNA encoded by the different gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and two or more nucleic acid sequences encoding a gene of interest, wherein each of the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a same target mRNA, wherein each of the two or more nucleic acid sequences encoding a gene of interest encodes a same gene of interest, and wherein the mRNA encoded by the same gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and two or more nucleic acid sequences encoding a gene of interest, wherein each of the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a different target mRNA, wherein each of the two or more nucleic acid sequences encoding a gene of interest encodes a same gene of interest, and wherein the mRNA encoded by the same gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and two or more nucleic acid sequences encoding a gene of interest, wherein each of the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a same target mRNA, wherein each of the two or more nucleic acid sequences encoding a gene of interest encodes a different gene of interest, and wherein the mRNA encoded by the different gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and two or more nucleic acid sequences encoding a gene of interest, wherein each of the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a different target mRNA, wherein each of the two or more nucleic acid sequences encoding a gene of interest encodes a different gene of interest, and wherein the mRNA encoded by the different gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding a gene of interest, wherein each of the two or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a same target mRNA, wherein each of the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding a gene of interest encodes a same gene of interest, and wherein the mRNA encoded by the same gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding a gene of interest, wherein each of the two or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a different target mRNA, wherein each of the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding a gene of interest encodes a same gene of interest, and wherein the mRNA encoded by the same gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding a gene of interest, wherein each of the two or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a same target mRNA, wherein each of the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding a gene of interest encodes a different gene of interest, and wherein the mRNA encoded by the different gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding a gene of interest, wherein each of the two or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a different target mRNA, wherein each of the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding a gene of interest encodes a different gene of interest, and wherein the mRNA encoded by the different gene of interest is different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise three or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and at least one nucleic acid sequence encoding a gene of interest, wherein one or more of the three or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a first target mRNA, another one or more of the three or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a second target mRNA, and the other one or more of the three or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a third target mRNA, wherein the first, the second, and the third target mRNAs are different, wherein the at least one nucleic acid sequence encoding a gene of interest encodes a same gene of interest, and wherein the mRNA encoded by the same gene of interest is different from the first, the second, and the third target mRNA that the siRNA is capable of binding to. For example, the recombinant polynucleic acid or RNA construct of the present invention may comprise five nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and at least one nucleic acid sequence encoding a gene of interest, wherein two of the five nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a first target mRNA, one of the five nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a second target mRNA, and one of the five nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a third target mRNA, wherein the first target mRNA, the second target mRNA, and the third target mRNA are different, wherein the at least one nucleic acid sequence encoding a gene of interest encodes a same gene of interest, and wherein the mRNA encoded by the same gene of interest is different from the first, the second, and the third target mRNA that the siRNA is capable of binding to.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise at least one nucleic acid sequence encoding or comprising an siRNA capable of binding to a target mRNA and three or more nucleic acid sequences encoding a gene of interest, wherein at least one nucleic acid sequence encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a same target mRNA, wherein one or more of the three or more nucleic acid sequences encoding a gene of interest encodes a first gene of interest and one or more of the three or more nucleic acid sequences encoding a gene of interest encodes a second gene of interest, wherein the first gene of interest and the second gene of interest are different, and wherein the mRNAs encoded by the first gene of interest and the second gene of interest are different from the siRNA target mRNA. For example, the recombinant polynucleic acid or RNA construct of the present invention may comprise at least one nucleic acid sequence encoding or comprising an siRNA capable of binding to a target mRNA and five nucleic acid sequences encoding a gene of interest, wherein at least one nucleic acid sequence encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a same target mRNA, wherein three of the five nucleic acid sequences encoding a gene of interest encodes a first gene of interest and two of the five nucleic acid sequences encoding a gene of interest encodes a second gene of interest, wherein the first gene of interest and the second gene of interest are different, and wherein the mRNAs encoded by the first gene of interest and the second gene of interest are different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise at least one nucleic acid sequence encoding or comprising an siRNA capable of binding to a target mRNA and three or more nucleic acid sequences encoding a gene of interest, wherein at least one nucleic acid sequence encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a same target mRNA, wherein one or more of the three or more nucleic acid sequences encoding a gene of interest encodes a first gene of interest, one or more of the three or more nucleic acid sequences encoding a gene of interest encodes a second gene of interest, and one or more of the three or more nucleic acid sequences encoding a gene of interest encodes a third gene of interest, wherein the first gene of interest, the second gene of interest, and the third gene of interest are different, and wherein the mRNAs encoded by the first gene of interest, the second gene of interest, and the third gene of interest are different from the siRNA target mRNA. For example, the recombinant polynucleic acid or RNA construct of the present invention may comprise at least one nucleic acid sequence encoding or comprising an siRNA capable of binding to a target mRNA and five nucleic acid sequences encoding a gene of interest, wherein at least one nucleic acid sequence encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a same target mRNA, wherein three of the five nucleic acid sequences encoding a gene of interest encodes a first gene of interest, one of the five nucleic acid sequences encoding a gene of interest encodes a second gene of interest, and one of the five nucleic acid sequences encoding a gene of interest encodes a third gene of interest, wherein the first gene of interest, the second gene of interest, and the third gene of interest are different, and wherein the mRNAs encoded by the first gene of interest, the second gene of interest, and the third gene of interest are different from the siRNA target mRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise three or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and three or more nucleic acid sequences encoding a gene of interest, wherein one or more of the three or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a first target mRNA and one or more of the three or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a second target mRNA, wherein the first and the second target mRNA are different, wherein the one or more of the three or more nucleic acid sequences encoding a gene of interest encodes a first gene of interest and one or more of the three or more nucleic acid sequences encoding a gene of interest encodes a second gene of interest, wherein the first gene of interest and the second gene of interest are different, and wherein the mRNAs encoded by the first gene of interest and the second gene of interest are different from the first and the second target mRNA that the siRNA is capable of binding to. For example, the recombinant polynucleic acid or RNA construct of the present invention may comprise five nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and five nucleic acid sequences encoding a gene of interest, wherein three of the five nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a first target mRNA and the other two of the five nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a second target mRNA, wherein the first and the second target mRNA are different, wherein three of the five nucleic acid sequences encoding a gene of interest encodes a first gene of interest and two of the five nucleic acid sequences encoding a gene of interest encodes a second gene of interest, wherein the first gene of interest and the second gene of interest are different, and wherein the mRNAs encoded by the first gene of interest and the second gene of interest are different from the first and the second target mRNA that the siRNA is capable of binding to.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise three or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and three or more nucleic acid sequences encoding a gene of interest, wherein one or more of the three or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a first target mRNA, another one or more of the three or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a second target mRNA, and the other one or more of the three or more nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a third target mRNA, wherein the first, the second, and the third target mRNAs are different, wherein one or more of the three or more nucleic acid sequences encoding a gene of interest encodes a first gene of interest, one or more of the three or more nucleic acid sequences encoding a gene of interest encodes a second gene of interest, and one or more of the three or more nucleic acid sequences encoding a gene of interest encodes a third gene of interest, wherein the first gene of interest, the second gene of interest, and the third gene of interest are different, and wherein the mRNAs encoded by the first gene of interest, the second gene of interest, and the third gene of interest are different from the first, the second, and the third target mRNA that the siRNA is capable of binding to. For example, the recombinant polynucleic acid or RNA construct of the present invention may comprise five nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA and five nucleic acid sequences encoding a gene of interest, wherein two of the five nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a first target mRNA, one of the five nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a second target mRNA, and one of the five nucleic acid sequences encoding or comprising an siRNA encodes or comprises an siRNA capable of binding to a third target mRNA, wherein the first target mRNA, the second target mRNA, and the third target mRNA are different, and wherein three of the five nucleic acid sequences encoding a gene of interest encodes a first gene of interest, one of the five nucleic acid sequences encoding a gene of interest encodes a second gene of interest, and one of the five nucleic acid sequences encoding a gene of interest encodes a third gene of interest, wherein the first gene of interest, the second gene of interest, and the third gene of interest are different, and wherein the mRNAs encoded by the first gene of interest, the second gene of interest, and the third gene of interest are different from the first, the second, and the third target mRNA that the siRNA is capable of binding to.

In some embodiments wherein multiple genes of interest are encoded by a polynucleotide construct, all genes of interest encode the same protein. In some embodiments, all genes of interest encode different proteins. In some embodiments, more than one gene of interest encodes the same protein and at least one gene of interest encodes a different protein. In some embodiments, wherein multiple siRNAs are encoded or comprised by a polynucleotide construct, all siRNAs encoded or comprised by a polynucleotide construct are capable of binding to the same RNA. In some embodiments, all siRNAs are capable of binding to different target RNAs. In some embodiments, more than one siRNA is capable of binding to the same target RNA and at least one siRNA is capable of binding to a different target RNA. In some embodiments, the target RNA is an mRNA. In some embodiments, the target RNA is a noncoding RNA. In some embodiments, wherein multiple siRNAs encoded or comprised by the polynucleotide construct are capable of binding to the same target RNA, all or some of the siRNAs are capable of binding to the same or different target RNA binding sites.

Recombinant RNA Construct

In one embodiment of the present invention, the recombinant polynucleic acid construct is a recombinant RNA construct. In some embodiments, the recombinant RNA construct is naked RNA. In a preferred embodiment, the recombinant RNA construct comprises a 5′ cap (e.g., an anti-reverse CAP analog, Clean Cap, Cap 0, Cap 1, Cap 2, or Locked Nucleic Acid cap (LNA-cap), etc.), an internal ribosome entry site (IRES), and/or a poly(A) tail at the 3′ end in a particular in order to improve translation. In some embodiments, the recombinant RNA construct has further regions promoting translation known to any skilled artisan. In some embodiments, the 5′ cap comprises an anti-reverse CAP analog, Clean Cap, Cap 0, Cap 1, Cap 2, or Locked Nucleic Acid cap (LNA-cap). In some embodiments, 5′ cap comprises m2^7,3′-OG(5′)ppp(5′)G, m7G, m7G(5′)G, m7GpppG, or m7GpppGm.

In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding a poly(A) tail. In some embodiments, the recombinant RNA construct comprises a poly(A) tail.

In some embodiments, the poly(A) tail comprises 1, 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, or 220 base pairs of poly(A) (SEQ ID NO: 192). In some embodiments, the poly(A) tail comprises 1 to 220 base pairs of poly(A) (SEQ ID NO: 191). In some embodiments, the poly(A) tail comprises 1 to 20, 1 to 40, 1 to 60, 1 to 80, 1 to 100, 1 to 120, 1 to 140, 1 to 160, 1 to 180, 1 to 200, 1 to 220, 20 to 40, 20 to 60, 20 to 80, 20 to 100, 20 to 120, 20 to 140, 20 to 160, 20 to 180, 20 to 200, 20 to 220, 40 to 60, 40 to 80, 40 to 100, 40 to 120, 40 to 140, 40 to 160, 40 to 180, 40 to 200, 40 to 220, 60 to 80, 60 to 100, 60 to 120, 60 to 140, 60 to 160, 60 to 180, 60 to 200, 60 to 220, 80 to 100, 80 to 120, 80 to 140, 80 to 160, 80 to 180, 80 to 200, 80 to 220, 100 to 120, 100 to 140, 100 to 160, 100 to 180, 100 to 200, 100 to 220, 120 to 140, 120 to 160, 120 to 180, 120 to 200, 120 to 220, 140 to 160, 140 to 180, 140 to 200, 140 to 220, 160 to 180, 160 to 200, 160 to 220, 180 to 200, 180 to 220, or 200 to 220 base pairs of poly(A) (SEQ ID NO: 194). In some embodiments, the poly(A) tail comprises 1, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, or 220 base pairs of poly(A) (SEQ ID NO: 195). In some embodiments, the poly(A) tail comprises at least 1, 20, 40, 60, 80, 100, 120, 140, 160, 180, or 200 base pairs of poly(A) (SEQ ID NO: 199). In some embodiments, the poly(A) tail comprises at most 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, or 220 base pairs of poly(A) (SEQ ID NO: 196). In a preferred embodiment, the poly(A) tail comprises 120 base pairs of poly(A) (SEQ ID NO: 193).

In one embodiment of the present invention, the recombinant RNA construct may contain a combination of modified and unmodified nucleotides. In a preferred embodiment, in such a modified recombinant RNA construct, 1 to 100%, preferably 10 to 100%, more preferably 50 to 100%, even more preferably 90 to 100%, most preferably 100% of the uridine nucleotides may be modified. In some embodiments, recombinant RNA constructs transcribed from any DNA constructs described herein may comprise modified uridines. In a preferred embodiment, 100% of uridine nucleotides in recombinant RNA constructs transcribed from any DNA constructs described herein are modified. In some embodiments, the adenosine-, guanosine-, and cytidine-containing nucleotides are unmodified or partially modified, and they are preferably present in unmodified form. Preferably the content of the modified uridine nucleotides in the recombinant RNA construct may lie in a range from 5 to 25%. Non-limiting examples of the modified uridine nucleotides may comprise pseudouridines, N¹-Methylpseudouridines, or N1-methylpseudo-UTP and any modified uridine nucleotides known in the art may be utilized. In some embodiments, the recombinant RNA construct may contain a combination of modified and unmodified nucleotides, wherein in such a modified recombinant RNA construct, 1 to 100%, preferably 10 to 100%, more preferably 50 to 100%, even more preferably 90 to 100%, most preferably 100% of the uridine nucleotides may comprise pseudouridines, N¹-Methylpseudouridines, N1-methylpseudo-UTP, or any other modified uridine nucleotide known in the art. In some embodiments, the recombinant RNA construct may contain a combination of modified and unmodified nucleotides, wherein in such a modified recombinant RNA construct, 1 to 100%, preferably 10 to 100%, more preferably 50 to 100%, even more preferably 90 to 100%, most preferably 100% of the uridine nucleotides may comprise N¹-Methylpseudouridines. In some embodiments, recombinant RNA constructs transcribed from any DNA constructs described herein may comprise N¹-Methylpseudouridines. In a preferred embodiment, 100% of uridine nucleotides in recombinant RNA constructs transcribed from any DNA constructs described herein are modified to N¹-Methylpseudouridines.

In some embodiments, the recombinant RNA construct may be codon-optimized. In general, codon optimization refers to a process of modifying a nucleic acid sequence for expression in a host cell of interest by replacing at least one codon (e.g., more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of a native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Codon usage tables are readily available, for example, at the “Codon Usage Database,” and these tables can be adapted in a number of ways. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge® (Aptagen, Pa.) and GeneOptimizer® (ThermoFischer, Mass.) which is preferred. In some embodiments, the recombinant RNA construct may not be codon-optimized.

In a preferred embodiment the present invention comprises a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to Interleukin 8 (IL-8) messenger RNA (mRNA); and (ii) an mRNA encoding Insulin-like Growth Factor 1 (IGF-1).

In another preferred embodiment the present invention comprises a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to Interleukin 1 beta (IL-1 beta) messenger RNA (mRNA); and (ii) an mRNA encoding Insulin-like Growth Factor 1 (IGF-1).

In another preferred embodiment the present invention comprises a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to Interleukin 17 (IL-17) messenger RNA (mRNA); and (ii) an mRNA encoding Interleukin 4 (IL-4).

In another preferred embodiment the present invention comprises a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to Tumor Necrosis Factor alpha (TNF-alpha or TNF-α) messenger RNA (mRNA); and (ii) an mRNA encoding Interleukin 4 (IL-4).

In another preferred embodiment the present invention comprises a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to Tumor Necrosis Factor alpha (TNF-alpha) messenger RNA (mRNA) and a small interfering RNA (siRNA) capable of binding to Interleukin 17 (IL-17) messenger RNA (mRNA); and (ii) an mRNA encoding Interleukin 4 (IL-4).

In another preferred embodiment the present invention is a composition comprising a polynucleic acid construct comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8.

In another preferred embodiment the present invention is a composition comprising a polynucleic acid construct, e.g., a recombinant RNA construct, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 29-47.

In another preferred embodiment the present invention comprises a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to Activin receptor-like kinase-2 (ALK2) messenger RNA (mRNA); and (ii) an mRNA encoding Insulin-like Growth Factor 1 (IGF-1).

In another preferred embodiment the present invention comprises a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to Superoxide dismutase-1 (SOD1) messenger RNA (mRNA); and (ii) an mRNA encoding Insulin-like Growth Factor 1 (IGF-1).

In another preferred embodiment the present invention comprises a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to Superoxide dismutase-1 (SOD1) messenger RNA (mRNA); and (ii) an mRNA encoding Erythropoietin (EPO).

In some embodiments, the recombinant polynucleic acid construct described herein comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 99% sequence identity to any one of SEQ ID NOs: 177-189. In some embodiments, the recombinant polynucleic acid construct described herein comprises a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 99% sequence identity to SEQ ID NO: 190.

In another preferred embodiment the present invention is a composition comprising a polynucleic acid construct comprising a nucleic acid sequence of SEQ ID NO: 190.

In some aspects, provided herein, is a method of producing an RNA construct comprising an siRNA capable of binding to a target mRNA and mRNA encoding a gene of interest. In some embodiments, the RNA construct is produced by in vitro transcription. In this embodiment, (i) a polynucleic acid construct comprising a promoter, at least one nucleic acid sequence encoding an siRNA capable of binding to a target mRNA, at least one nucleic acid sequence encoding a gene of interest, and a nucleic acid sequence encoding poly(A) tail; (ii) an RNA polymerase; and (iii) a mixture of nucleotide triphosphates (NTPs) is provided for the in vitro (“cell free”) transcription. Details of producing RNA using in vitro transcription as well as isolating and purifying transcribed RNAs is well known in the art and can be found, for example, in Beckert & Masquida ((2011) Synthesis of RNA by In vitro Transcription. RNA. Methods in Molecular Biology (Methods and Protocols), vol 703. Humana Press). A non-limiting list of in vitro transcript kits includes MEGAscript™ T3 Transcription Kit, MEGAscript T7 kit, MEGAscript™ SP6 Transcription Kit, MAXIscript™ T3 Transcription Kit, MAXIscript™ T7 Transcription Kit, MAXIscript™ SP6 Transcription Kit, MAXIscript™ T7/T3 Transcription Kit, MAXIscript™ SP6/T7 Transcription Kit, mMESSAGE mMACHINE™ T3 Transcription Kit, mMESSAGE mMACHINE™ T7 Transcription Kit, mMESSAGE mMACHINE™ SP6 Transcription Kit, MEGAshortscript™ T7 Transcription Kit, HiScribe™ T7 High Yield RNA Synthesis Kit, HiScribe™ T7 In Vitro Transcription Kit, AmpliScribe™ T7-Flash™ Transcription Kit, AmpliScribe™ T7 High Yield Transcription Kit, AmpliScribe™ T7-Flash™ Biotin-RNA Transcription Kit, T7 Transcription Kit, HighYield T7 RNA Synthesis Kit, DuraScribe® T7 Transcription Kit, etc.

In some embodiments, the polynucleic acid construct may be linear. The in vitro transcription reaction can further comprise a transcription buffer system, nucleotide triphosphates (NTPs), and an RNase inhibitor. In some embodiments, the transcription buffer system may comprise dithiothreitol (DTT) and magnesium ions. The NTPs can be naturally occurring or non-naturally occurring (modified) NTPs. Non-limiting examples of non-naturally occurring (modified) NTPs include N¹-methylpseudouridine, Pseudouridine, N¹-Ethylpseudouridine, N¹-Methoxymethylpseudouridine, N¹—Propylpseudouridine, 2-thiouridine, 4-thiouridine, 5-methoxyuridine, 5-methylurdine, 5-carboxymethylesteruridine, 5-formyluridine, 5-carboxyuridine, 5-hydroxyuridine, 5-Bromouridine, 5-Iodouridine, 5,6-dihydrouridine, 6-Azauridine, Thienouridine, 3-methyluridine, 1-carboxymethyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, dihydrouridine, dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-methylcytidine, 5-methoxycytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, 5-hydroxycytidine, 5-Iodocytidine, 5-Bromocytidine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, 3-methyl-cytidine, N⁴-acetylcytidine, 5-formylcytidine, N⁴-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine, N¹-methyladenosine, N⁶-methyladenosine, N⁶-methyl-2-Aminoadenosine, N⁶-isopentenyladenosine, N⁶,N⁶-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. Non-limiting examples of DNA-dependent RNA polymerase include T3, T7, SP6, P60, Syn5, and KP34 RNA polymerases. In some embodiments, the RNA polymerase is selected from the group consisting of T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, P60 RNA polymerase, Syn5 RNA polymerase, and KP34 RNA polymerase. In some embodiments, the RNA polymerase is T3 RNA polymerase. In some embodiments, the RNA polymerase is SP6 RNA polymerase. In some embodiments, the RNA polymerase is P60 RNA polymerase. In some embodiments, the RNA polymerase is Syn5 RNA polymerase. In some embodiments, the RNA polymerase is KP34 RNA polymerase. In a preferred embodiment, the RNA polymerase is T7 RNA polymerase.

In further embodiments, transcribed RNAs may be isolated and purified from the in vitro transcription reaction mixture. In this embodiments, transcribed RNAs may be isolated and purified using column purification. Details of isolating and purifying transcribed RNAs from in vitro transcription reaction mixture is well known in the art and any commercially available kits may be used. A non-limiting list of RNA purification kits includes MEGAclear kit, Monarch® RNA Cleanup Kit, EasyPure® RNA Purification Kit, NucleoSpin® RNA Clean-up, etc.

Recombinant Polynucleic Acid Construct for Treating a Viral Disease or Condition

The recombinant polynucleic acid construct of the present invention can be directed toward treatment of diseases and conditions related to virus infection. In these embodiments, the recombinant polynucleic acid construct can simultaneously downregulate the expression of one or more proteins and upregulate the expression of one or more proteins by providing a nucleic acid sequence encoding or comprising a single or multiple small interfering RNA (siRNA) species capable of binding to a specific target(s), and a nucleic acid sequence encoding single or multiple proteins for overexpression. In some embodiments, the recombinant polynucleic acid is DNA. In some embodiments, the recombinant polynucleic acid is RNA.

In some embodiments, (i) and (ii) are oriented in a 5′ to 3′ direction (the elements of (i) are upstream of the elements of (ii)). In some embodiments, (i) and (ii) are not oriented in a 5′ to 3′ direction (e.g., the element(s) of (ii) are upstream of the elements of (i)). In some embodiments, the at least one nucleic acid sequence encoding or comprising the small interfering RNA (siRNA) capable of specifically binding to the target RNA (e.g., an mRNA or a noncoding RNA) is upstream of the at least one nucleic acid sequence encoding the gene of interest. In some embodiments, the at least one nucleic acid sequence encoding or comprising the small interfering RNA (siRNA) capable of specifically binding to the target RNA (e.g., an mRNA or a noncoding RNA) is downstream of the at least one nucleic acid sequence encoding the gene of interest. In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a linker. In some embodiments, the nucleic acid sequence encoding or comprising the linker connects (i) and (ii). In some embodiments, the nucleic acid sequence encoding or comprising the linker connects the at least one nucleic acid sequence encoding or comprising the small interfering RNA (siRNA) capable of specifically binding to the target RNA (e.g., an mRNA or a noncoding RNA) and the at least one nucleic acid sequence encoding the gene of interest. In some embodiments, the linker comprises a tRNA linker. In some embodiments, the recombinant polynucleic acid construct is circular. In some embodiments, the recombinant polynucleic acid construct is linear. In some embodiments, the recombinant polynucleic acid construct is DNA. In some embodiments, the recombinant polynucleic acid construct is RNA. In some embodiments, the recombinant polynucleic acid construct comprises a nucleic acid sequence as set forth in one of SEQ ID NOs: 1-8 or 29-47. In some embodiments, the recombinant polynucleic acid construct comprises a nucleic acid sequence as set forth in one of SEQ ID NOs: 152-158. In some embodiments, the recombinant polynucleic acid construct comprises a nucleic acid sequence as set forth in one of SEQ ID NOs: 177-190.

In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a poly(A) tail. In some embodiments, the poly(A) tail comprises 1-220 A residues (SEQ ID NO: 191). In some embodiments, the recombinant polynucleic acid construct further comprises a 5′ cap. In some embodiments, the 5′ cap comprises an anti-reverse CAP analog, Clean Cap, Cap 0, Cap 1, Cap 2, or Locked Nucleic Acid cap (LNA-cap). In some embodiments, the 5′ cap comprises m2^7,3′-OG(5′)ppp(5′)G, m7G, m7G(5′)G, m7GpppG, or m7GpppGm. In some embodiments, the recombinant polynucleic acid construct further comprises a promoter. In some embodiments, the promoter is selected from the group consisting of T3, T7, SP6, P60, Syn5, and KP34. In some embodiments, the promoter is a T7 promoter. In some embodiments, the T7 promoter is upstream of the at least one nucleic acid sequence encoding or comprising the siRNA. In some embodiments, the T7 promoter is upstream of the at least one nucleic acid sequence encoding or comprising the gene of interest. In some embodiments, the T7 promoter comprises a sequence TAATACGACTCACTATA (SEQ ID NO: 25). In some embodiments, the recombinant polynucleic acid construct further comprises a Kozak sequence. In some embodiments, the Kozak sequence is GCCACC (SEQ ID NO: 26).

In some embodiments, the recombinant polynucleic acid construct encodes or comprises 1-10 siRNA species. In some embodiments, the siRNA species are the same. In some embodiments, the siRNA species are different. In some embodiments, some siRNA species are the same and some are different. In some embodiments, the siRNA comprises a sense siRNA strand. In some embodiments, the siRNA comprises an anti-sense siRNA strand. In some embodiments, the siRNA comprises a sense and an anti-sense siRNA strand. In some embodiments, the siRNA does not affect the expression of the gene of interest. In some embodiments, the siRNA does not inhibit the expression of the gene of interest. In some embodiments, the recombinant polynucleic acid construct comprises two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA. In some embodiments, the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a linker. In some embodiments, the nucleic acid sequence encoding or comprising the linker connects each of the two or more nucleic acid sequences encoding or comprising the siRNA capable of binding to the target mRNA. In some embodiments, the linker comprises a tRNA linker. In some embodiments, each of the two or more nucleic acid sequences encodes or comprises an siRNA capable of binding to a same target mRNA. In some embodiments, each of the two or more nucleic acid sequences encodes or comprises an siRNA capable of binding to a different target mRNA.

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of specifically binding to IL-6 mRNA; and (ii) an mRNA encoding Interferon beta (IFN-beta). In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 1 siRNA directed to IL-6 mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to IL-6 mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 29 or 30 (Compound B1 or B2).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of specifically binding to Interleukin 6R (IL-6R) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 1 siRNA directed to IL-6R mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to IL-6R mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 31 (Compound B3).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct encoding or comprising (i) at least one siRNA capable of specifically binding to Interleukin 6R alpha (IL-6R-alpha) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 1 siRNA directed to IL-6R-alpha mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to IL-6R-alpha mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 32 (Compound B4).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct encoding or comprising (i) at least one siRNA capable of specifically binding to Interleukin 6R beta (IL-6R-beta) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 1 siRNA directed to IL-6R-beta mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to IL-6R-beta mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 33 (Compound B5).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct encoding or comprising (i) at least one siRNA capable of specifically binding to ACE2 mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 1 siRNA directed to ACE2 mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to ACE2 mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 34 or 35 (Compound B6 or B7).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct: encoding or comprising (i) at least one small interfering RNA (siRNA) capable of specifically binding to SARS CoV-2 ORF1ab mRNA, at least one siRNA capable of specifically binding to SARS CoV-2 S mRNA, at least one siRNA capable of specifically binding to SARS CoV-2 N mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 3 siRNAs, one directed to SARS CoV-2 ORF1ab mRNA, one directed to SARS CoV-2 S mRNA, and one directed to SARS CoV-2 N mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In certain aspects, such a composition, e.g., a composition comprising Compound B8 (SEQ ID NO: 36) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, SARS CoV-2, or both. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 36.

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct encoding or comprising (i) at least one siRNA capable of specifically binding to SARS CoV-2 N mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 1 siRNA directed to SARS CoV-2 N mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to SARS CoV-2 N mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 38 (Compound B10).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct: encoding or comprising (i) at least one siRNA capable of specifically binding to SARS CoV-2 ORF1ab mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes 1 siRNA directed to SARS CoV-2 ORF1ab mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to SARS CoV-2 ORF1ab mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In certain aspects, such a composition, including a composition comprising Compound B12 (SEQ ID NO: 40) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, MERS-CoV, or both. In certain aspects, such a composition, including a composition comprising Compound B13 (SEQ ID NO: 41) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, SARS CoV-2, and/or MERS-CoV. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in any one of SEQ ID NOs: 40, 41 and 42 (Compounds B12, B13, and B14).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct: encoding or comprising (i) at least one siRNA capable of specifically binding to IL-6 mRNA, at least one siRNA capable of specifically binding to ACE2 mRNA, and at least one siRNA capable of specifically binding to SARS CoV-2 S mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the recombinant polynucleic acid construct in (ii) encodes or further encodes the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 3 siRNAs, one directed IL-6 mRNA, one directed to ACE2 mRNA, and one directed to SARS CoV-2 S mRNA. In related aspects, the mRNA encoding IFN-beta encodes the native IFN-beta signal peptide, or a modified signal peptide. In related aspects, the modified IFN-beta signal peptide is SP1 or SP2 as described herein (SEQ ID NOs: 52 and 54, respectively). In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in any one of SEQ ID NOs: 43, 44, and 45 (Compounds B15, B16, and B17).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct encoding or comprising (i) at least one small interfering RNA capable of specifically binding to SARS CoV-2 ORF1ab mRNA, at least one siRNA capable of specifically binding to SARS CoV-2 S mRNA, and at least one siRNA capable of specifically binding to SARS CoV-2 N mRNA; and (ii) an mRNA encoding the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 3 siRNAs, one directed to ORF1ab mRNA, one directed to SARS CoV-2 S mRNA, and one directed to SARS CoV-2 N mRNA. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 46 (Compound B18). In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 190 (Compound B18).

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid construct: encoding or comprising (i) at least one siRNA capable of specifically binding to SARS CoV-2 S mRNA; and (ii) an mRNA encoding the ACE2 soluble receptor. In related aspects, the composition comprises or encodes at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises or encodes 1 siRNA directed to SARS CoV-2 S mRNA. In related aspects, the composition comprises or encodes 3 siRNAs, each directed to SARS CoV-2 S mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 47 (Compound B19).

In some aspects, the IFN-beta construct comprises a modified signal peptide as described herein. In some aspects, the present invention provides a composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 29-47. In some aspects, the present invention provides a composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence set forth in SEQ ID NO: 190. In some aspects, the composition comprising the recombinant polynucleic acid construct is useful in the treatment of a viral infection, disease or condition. In some aspects, the composition is present or administered in an amount sufficient to treat or prevent a viral infection, disease or condition.

In some embodiments, the present invention provides a composition and related methods, wherein the composition comprises a recombinant polynucleic acid construct encoding or comprising: at least one siRNA capable of binding to a target RNA; and an mRNA encoding a gene of interest; wherein: the siRNA targets an RNA selected from: an IL-8 mRNA, an IL-1 beta mRNA, an IL-17 mRNA, a TNF-alpha mRNA, a SARS CoV-2 ORF1ab RNA (polyprotein PP1ab, e.g., in a noncoding region or where it encodes a protein that is selected from: a SARS CoV-2 nonstructure protein (NSP), Nsp1, Nsp3 (Nsp3b, Nsp3c, PLpro, and Nsp3e), Nsp7 Nsp8 complex, Nsp9-Nsp10, and Nsp14-Nsp16, 3CLpro, E-channel (E protein), ORF7a, C-terminal RNA binding domain (CRBD), N-terminal RNA binding domain (NRBD), helicase, and RdRp), a SARS CoV-2 Spike protein (S) mRNA, a SARS CoV-2 Nucleocapsid protein (N) mRNA, a tumor necrosis factor alpha (TNF-alpha) mRNA, an interleukin mRNA (including but not limited to interleukin 1 (e.g., IL-1alpha, IL-1beta), interleukin 6 (IL-6), interleukin 6R (IL-6R), interleukin 6R alpha (IL-6R-alpha), interleukin 6R beta (IL-6R-beta), interleukin 18 (IL-18), interleukin 36-alpha (IL-36-alpha), interleukin 36-beta (IL-36-beta), interleukin 36-gamma (IL-36-gamma), interleukin 33 (IL-33)), an Angiotensin Converting Enzyme-2 (ACE2) mRNA, a transmembrane protease, serine 2 (TMPRSS2) mRNA, and a coding NSP12 and 13 RNA; and the gene of interest encodes a protein selected from: IGF-1, IL-4, IGF-1 (including derivatives thereof as described elsewhere herein), carboxypeptidases (e.g., ACE, ACE2, CNDP1, CPA1, CPA2, CPA4, CPA5, CPA6, CPB1, CPB2, CPE, CPN1, CPQ, CPXM1, CPZ, SCPEP1); cytokines (e.g., BMP1, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, C1QTNF4, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL3L3, CCL4, CCL4L, CCL4L2, CCL5, CCL7, CCL8, CD40LG, CER1, CKLF, CLCF1, CNTF, CSF1, CSF2, CSF3, CTF1, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, CXCL17, CXCL2, CXCL3, CXCL5, CXCL8, CXCL9, DKK1, DKK2, DKK3, DKK4, EDA, EBI3, FAM3B, FAM3C, FASLG, FLT3LG, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF5, GDF6, GDF7, GDF9, GPI, GREM1, GREM2, GRN, IFNA1, IFNA13, IFNA10, IFNA14, IFNA16, IFNA17, IFNA2, IFNA21, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNB1, IFNE, IFNG, IFNK, IFNL1, IFNL2, IFNL3, IFNL4, IFNW1, IL10, IL11, IL12A, IL12B, IL13, IL15, IL16, IL17A, IL17B, IL17C, IL17D, IL17F, IL18, IL19, IL1A, IL1B, IL1F10, IL2, IL20, IL21, IL22, IL23A, IL24, IL25, IL26, IL27, IL3, IL31, IL32, IL33, IL34, IL36A, IL36B, IL36G, IL36RN, IL37, IL4, IL5, IL6, IL7, IL9, LEFTY1, LEFTY2, LIF, LTA, MIF, MSTN, NAMPT, NODAL, OSM, PF4, PF4V1, SCGB3A1, SECTM1, SLURP1, SPP1, THNSL2, THPO, TNF, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TSLP, VSTM1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, and XCL2); extracellular ligands and transporters (e.g., APCS, CHI3L1, CHI3L2, CLEC3B, DMBT1, DMKN, EDDM3A, EDDM3B, EFNA4, EMC10, ENAM, EPYC, ERVH48-1, F13B, FCN1, FCN2, GLDN, GPLD1, HEG1, ITFG1, KAZALD1, KCP, LACRT, LEG1, METRN, NOTCH2NL, NPNT, OLFM1, OLFML3, PRB2, PSAP, PSAPL1, PSG1, PSG6, PSG9, PTX3, PTX4, RBP4, RNASE10, RNASE12, RNASE13, RNASE9, RSPRY1, RTBDN, S100A12, S100A13, S100A7, S100A8, SAA2, SAA4, SCG1, SCG2, SCG3, SCGB1C1, SCGB1C2, SCGB1D1, SCGB1D2, SCGB1D4, SCGB2B2, SCGB3A2, SCGN, SCRG1, SCUBE1, SCUBE2, SCUBE3, SDCBP, SELENOP, SFTA2, SFTA3, SFTPA1, SFTPA2, SFTPC, SFTPD, SHBG, SLURP2, SMOC1, SMOC2, SMR3A, SMR3B, SNCA, SPATA20, SPATA6, SOGA1, SPARC, SPARCL1, SPATA20, SPATA6, SRPX2, SSC4D, STX1A, SUSD4, SVBP, TCN1, TCN2, TCTN1, TF, TULP3, TFF2, TFF3, THSD7A, TINAG, TINAGL1, TMEFF2, TMEM25, VWC2L); extracellular matrix proteins (e.g., ABI3BP, AGRN, CCBE1, CHL1, COL15A1, COL19A1, COLEC11, DMBT1, DRAXIN, EDIL3, ELN, EMID1, EMILIN1, EMILIN2, EMILIN3, EPDR1, FBLN1, FBLN2, FBLN5, FLRT1, FLRT2, FLRT3, FREM1, GLDN, IBSP, KERA, KIAA0100, KIRREL3, KRT10, LAMB2, MGP, RPTN, SBSPON, SDC1, SDC4, SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, SIGLEC1, SIGLEC10, SIGLEC6, SLIT1, SLIT2, SLIT3, SLITRK1, SNED1, SNORC, SPACA3, SPACA7, SPON1, SPON2, STATH, SVEP1, TECTA, TECTB, TNC, TNN, TNR, TNXB); glucosidases (AMY1A, AMY1B, AMY1C, AMY2A, AMY2B, CEMIP, CHIA, CHIT1, FUCA2, GLB1L, GLB1L2, HPSE, HYAL1, HYAL3, KL, LYG1, LYG2, LYZL1, LYZL2, MAN2B2, SMPD1, SMPDL3B, SPACA5, SPACA5B); glycosyltransferases (e.g., ARTS, B4GALT1, EXTL2, GALNT1, GALNT2, GLT1D1, MGAT4A, ST3GAL1, ST3GAL2, ST3GAL3, ST3GAL4, ST6GAL1, XYLT1); growth factors (e.g., AMH, ARTN, BTC, CDNF, CFC1, CFC1B, CHRDL1, CHRDL2, CLEC11A, CNMD, EFEMP1, EGF, EGFL6, EGFL7, EGFL8, EPGN, EREG, EYS, FGF1, FGF10, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FRZB, GDNF, GFER, GKN1, HBEGF, HGF, IGF-1, IGF2, INHA, INHBA, INHBB, INHBC, INHBE, INS, KITLG, MANF, MDK, MIA, NGF, NOV, NRG1, NRG2, NRG3, NRG4, NRTN, NTF3, NTF4, OGN, PDGFA, PDGFB, PDGFC, PDGFD, PGF, PROK1, PSPN, PTN, SDF1, SDF2, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5, TDGF1, TFF1, TGFA, TGFB1, TGFB2, TGFB3, THBS4, TIMP1, VEGFA, VEGFB, VEGFC, VEGFD, WISP3); growth factor binding proteins (e.g., CHRD, CYR61, ESM1, FGFBP1, FGFBP2, FGFBP3, HTRA1, GHBP, IGFALS, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7, LTBP1, LTBP2, LTBP3, LTBP4, SOSTDC1, NOG, TWSG1, and WIF1); heparin binding proteins (e.g., ADA2, ADAMTSL5, ANGPTL3, APOB, APOE, APOH, COL5A1, COMP, CTGF, FBLN7, FN1, FSTL1, HRG, LAMC2, LIPC, LIPG, LIPH, LIPI, LPL, PCOLCE2, POSTN, RSPO1, RSPO2, RSPO3, RSPO4, SAA1, SLIT2, SOST, THBS1, VTN); hormones (e.g., ADCYAP1, ADIPOQ, ADM, ADM2, ANGPTL8, APELA, APLN, AVP, C1QTNF12, C1QTNF9, CALCA, CALCB, CCK, CGA, CGB1, CGB2, CGB3, CGB5, CGB8, COPA, CORT, CRH, CSH1, CSH2, CSHL1, ENHO, EPO, ERFE, FBN1, FNDC5, FSHB, GAL, GAST, GCG, GH, GH1, GH2, GHRH, GHRL, GIP, GNRH1, GNRH2, GPHA2, GPHB5, IAPP, INS, INSL3, INSL4, INSL5, INSL6, LHB, METRNL, MLN, NPPA, NPPB, NPPC, OSTN, OXT, PMCH, PPY, PRL, PRLH, PTH, PTHLH, PYY, RETN, RETNLB, RLN1, RLN2, RLN3, SCT, SPX, SST, STC1, STC2, TG, TOR2A, TRH, TSHB, TTR, UCN, UCN2, UCN3, UTS2, UTS2B, and VIP); hydrolases (e.g., AADACL2, ABHD15, ACP7, ACPP, ADA2, ADAMTSL1, AOAH, ARSF, ARSI, ARSJ, ARSK, BTD, CHI3L2, ENPP1, ENPP2, ENPP3, ENPP5, ENTPD5, ENTPD6, GBP1, GGH, GPLD1, HPSE, LIPC, LIPF, LIPG, LIPH, LIPI, LIPK, LIPM, LIPN, LPL, PGLYRP2, PLA1A, PLA2G10, PLA2G12A, PLA2G1B, PLA2G2A, PLA2G2D, PLA2G2E, PLA2G2F, PLA2G3, PLA2G5, PLA2G7, PNLIP, PNLIPRP2, PNLIPRP3, PON1, PON3, PPT1, SMPDL3A, THEM6, THSD1, and THSD4); immunoglobulins (e.g., IGSF10, IGKV1-12, IGKV1-16, IGKV1-33, IGKV1-6, IGKV1D-12, IGKV1D-39, IGKV1D-8, IGKV2-30, IGKV2D-30, IGKV3-11, IGKV3D-20, IGKV5-2, IGLC1, IGLC2, IGLC3); isomerases (e.g., NAXE, PPIA, PTGDS); kinases (e.g., ADCK1, ADPGK, FAM20C, ICOS, PKDCC); lyases (e.g., PM20D1, PAM, CA6); metalloenzyme inhibitors (e.g., FETUB, SPOCK3, TIMP2, TIMP3, TIMP4, WFIKKN1, WFIKKN2); metalloproteases (e.g., ADAM12, ADAM28, ADAM9, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, CLCA1, CLCA2, CLCA4, IDE, MEP1B, MMEL1, MMP1, MMP10, MMP11, MMP12, MMP13, MMP16, MMP17, MMP19, MMP2, MMP20, MMP21, MMP24, MMP25, MMP26, MMP28, MMP3, MMP1, MMP8, MMP9, PAPPA, PAPPA2, TLL1, TLL2); milk proteins (e.g., CSN1S1, CSN2, CSN3, LALBA); neuroactive proteins (e.g., CARTPT, NMS, NMU, NPB, NPFF, NPS, NPVF, NPW, NPY, PCSK1N, PDYN, PENK, PNOC, POMC, PROK2, PTH2, PYY2, PYY3, QRFP, TAC1, and TAC3); proteases (e.g., ADAMTS6, C1R, C1RL, C2, CASP4, CELA1, CELA2A, CELA2B, CFB, CFD, CFI, CMA1, CORIN, CTRB1, CTRB2, CTSB, CTSD, DHH, F10, F11, F12, F2, F3, F7, F8, F9, FAP, FURIN, GZMA, GZMK, GZMM, HABP2, HGFAC, HTRA3, HTRA4, IHH, KLK10, KLK11, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLKB1, MASP1, MASP2, MST1L, NAPSA, OVCH1, OVCH2, PCSK2, PCSK5, PCSK6, PCSK9, PGA3, PGA4, PGA5, PGC, PLAT, PLAU, PLG, PROC, PRSS1, PRSS12, PRSS2, PRSS22, PRSS23, PRSS27, PRSS29P, PRSS3, PRSS33, PRSS36, PRSS38, PRSS3P2, PRSS42, PRSS44, PRSS47, PRSS48, PRSS53, PRSS57, PRSS58, PRSS8, PRTN3, RELN, REN, TMPRSS11D, TMPRSS11E, TMPRSS2, TPSAB1, TPSB2, TPSD1); protease inhibitors (e.g., A2M, A2ML1, AMBP, ANOS1, COL28A1, COL6A3, COL7A1, CPAMD8, CST1, CST2, CST3, CST4, CST5, CST6, CST7, CST8, CST9, CST9L, CST9LP1, CSTL1, EPPIN, GPC3, HMSD, ITIH1, ITIH2, ITIH3, ITIH4, ITIH5, ITIH6, KNG1, OPRPN, OVOS1, OVOS2, PAPLN, PI15, PI16, PI3, PZP, R3HDML, SERPINA1, SERPINA10, SERPINA11, SERPINA12, SERPINA13P, SERPINA3, SERPINA4, SERPINA5, SERPINA7, SERPINA9, SERPINB2, SERPINB5, SERPINC1, SERPINE1, SERPINE2, SERPINE3, SERPINF2, SERPING1, SERPINI1, SERPINI2, SPINK1, SPINK13, SPINK14, SPINK2, SPINK4, SPINK5, SPINK6, SPINK7, SPINK8, SPINK9, SPINT1, SPINT3, SPINT4, SPOCK1, SPOCK2, SPP2, SSPO, TFPI, TFPI2, WFDC1, WFDC10A, WFDC13, WFDC2, WFDC3, WFDC5, WFDC6, WFDC8); protein phosphatases (e.g., ACP7, ACPP, PTEN, PTPRZ1); esterases (e.g., BCHE, CEL, CES4A, CES5A, NOTUM, SIAE); transferases (e.g., METTL24, FKRP, CHSY1, CHST9, B3GAT1); vasoactive proteins (e.g., AGGF1, AGT, ANGPT1, ANGPT2, ANGPTL4, ANGPTL6, EDN1, EDN2, EDN3, NTS), a Type I interferon (e.g., an IFN-α, including, but not limited to an interferon alpha-n3, an interferon alpha-2a, and an interferon alpha-2b, an IFN-β, an IFN-δ, an IFN-ε, an IFN-κ, an IFN-ν, an IFN-τ, and an IFN-ω), a Type II interferon (e.g., IFN-γ), a Type III interferon (e.g., IFN-λ) an interleukin, e.g., IL-37, IL-38, and a soluble ACE2 receptor. In some aspects, the composition comprising the recombinant polynucleic acid construct is useful in the treatment of a viral infection, disease or condition. In some aspects, the composition is present or administered in an amount sufficient to treat or prevent a viral infection, disease or condition. In some embodiments, the disease or the condition is selected from the group consisting of intervertebral disc disease (IVDD), osteoarthritis, and psoriasis.

Recombinant RNA Construct for Treating a Viral Disease or Condition

As described above, in some aspects, the recombinant polynucleic acid construct is a recombinant RNA construct. In some aspects, the recombinant polynucleic acid construct or recombinant RNA construct is useful in a composition for treating or preventing a viral infection, disease, or condition. In some aspects, the invention provides a composition comprising a recombinant RNA construct comprising: (i) a small interfering RNA (siRNA) capable of binding to a target RNA (e.g., mRNA); and (ii) an mRNA of a gene of interest; wherein the target mRNA is different from the mRNA encoding the gene of interest.

In some embodiments, the recombinant RNA construct comprises 1-10 siRNA species. In some embodiments, the siRNA species are the same, e.g., capable of binding to the same target mRNA. In some embodiments, the siRNA species are different, e.g., capable of binding to different target mRNAs. In some embodiments, some siRNA species are the same and some are different. In some embodiments, the siRNA comprises a sense siRNA strand. In some embodiments, the siRNA comprises an anti-sense siRNA strand. In some embodiments, the siRNA comprises a sense and an anti-sense siRNA strand. In some embodiments, the siRNA does not affect the expression of the gene of interest. In some embodiments, the siRNA does not inhibit the expression of the gene of interest. In some embodiments, the recombinant RNA construct comprises two or more nucleic acid sequences comprising an siRNA capable of binding to a target mRNA. In some embodiments, the recombinant RNA construct further comprises or encodes a linker. In some embodiments, the nucleic acid sequence encoding or comprising the linker connects each of the two or more nucleic acid sequences comprising the siRNA capable of binding to the target mRNA. In some embodiments, the linker comprises a tRNA linker. In some embodiments, the linker comprises a 2A peptide linker. In some embodiments, each of the two or more nucleic acid sequences comprises an siRNA capable of binding to a same target mRNA. In some embodiments, each of the two or more nucleic acid sequences comprises an siRNA capable of binding to a different target mRNA.

In some embodiments, the recombinant RNA construct comprises a nucleic acid sequence comprising a gene of interest (and thereby encoding an mRNA of interest and/or a protein of interest corresponding to the gene of interest). In some embodiments, the recombinant RNA construct comprises two or more nucleic acid sequences, each comprising a gene of interest and thereby each encoding an mRNA of interest and/or a protein of interest corresponding to the gene.

In some embodiments, each of the two or more nucleic acid sequences comprises the same gene of interest. In some embodiments, each of the two or more nucleic acid sequences encodes the same mRNA and/or protein of interest. In some embodiments, the recombinant RNA construct comprises three or more nucleic acid sequences, each comprising a gene of interest and thereby each encoding an mRNA of interest and/or a protein of interest corresponding to the gene. In some embodiments, each of the three or more nucleic acid sequences can comprise the same gene of interest, encode the same mRNA of interest, and/or encode the same protein of interest. In some embodiments, each of the three or more nucleic acid sequences can comprise different genes of interest, encode different mRNAs of interest, and/or encode different proteins of interest. In some embodiments, two or more of the three or more nucleic acid sequences can comprise the same gene of interest, encode the same mRNA of interest, and/or encode the same protein of interest, while one or more of the three or more nucleic acid sequences comprises a different gene of interest, encodes a different mRNA of interest, and/or encodes a different protein of interest from the two or more of the three or more nucleic acid sequences.

In some embodiments, the expression level of the gene or protein of interest is modulated by expressing an mRNA or a protein encoded by the gene of interest. In some embodiments, the expression level of the gene of interest is upregulated by expressing an mRNA or a protein encoded by the gene of interest. In some embodiments, the recombinant RNA construct is codon-optimized. In some embodiments, the recombinant RNA construct is not codon-optimized.

In some embodiments, the recombinant RNA construct further comprises a nucleic acid sequence encoding a target motif, also referred to as a targeting motif. In some embodiments, the nucleic acid sequence encoding the target motif is operably linked to the at least one nucleic acid sequence encoding the gene of interest. In some embodiments, the target motif comprises a signal peptide, a nuclear localization signal (NLS), a nucleolar localization signal (NoLS), a lysosomal targeting signal, a mitochondrial targeting signal, a peroxisomal targeting signal, a microtubule tip localization signal (MtLS), an endosomal targeting signal, a chloroplast targeting signal, a Golgi targeting signal, an endoplasmic reticulum (ER) targeting signal, a proteasomal targeting signal, a membrane targeting signal, a transmembrane targeting signal, or a centrosomal localization signal (CLS). In some embodiments, the target motif is selected from the group consisting of (a) a target motif heterologous to a protein encoded by the gene of interest; (b) a target motif heterologous to a protein encoded by the gene of interest, wherein the target motif heterologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; (c) a target motif homologous to a protein encoded by the gene of interest, wherein the target motif homologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; and (d) a naturally occurring amino acid sequence which does not have the function of a target motif in nature, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion, and/or substitution of at least one amino acid.

In some embodiments, the signal peptide is selected from the group consisting of (a) a signal peptide heterologous to a protein encoded by the gene of interest; (b) a signal peptide heterologous to a protein encoded by the gene of interest, wherein the signal peptide heterologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid, with proviso that the protein is not an oxidoreductase; (c) a signal peptide homologous to a protein encoded by the gene of interest, wherein the signal peptide homologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; and (d) a naturally occurring amino acid sequence which does not have the function of a signal peptide in nature, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion, and/or substitution of at least one amino acid. In some embodiments, the amino acids 1-9 of the N-terminal end of the signal peptide have an average hydrophobic score of above 2.

In some aspects, provided herein, is a cell comprising the composition of any recombinant polynucleic acid or RNA construct described herein. In some aspects, provided herein, is a pharmaceutical composition comprising the composition of any recombinant polynucleic acid or RNA construct described herein and a pharmaceutically acceptable excipient. In some aspects, provided herein, is a method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject the pharmaceutical composition described herein. In some embodiments, the disease or condition is COVID-19. In some embodiments, the disease or condition is SARS (severe acute respiratory syndrome) caused by infection with SARS-CoV-1 or SARS-CoV-2. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is an adult, a child, or an infant. In some embodiments, the subject is a companion animal. In some embodiments, the subject is feline, canine, or a rodent. In some embodiments, the subject is a dog or a cat.

In some aspects, provided herein, is a method of simultaneously expressing an siRNA and an mRNA from a single RNA transcript in a cell, comprising introducing into the cell the composition of any recombinant polynucleic acid or RNA construct described herein. In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target messenger RNA (mRNA); and (ii) at least one nucleic acid sequence of a gene of interest; wherein the target mRNA is different from an mRNA encoded by the gene of interest, and wherein the expression of the target mRNA and the gene of interest is modulated simultaneously.

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target messenger RNA (mRNA); and (ii) at least one nucleic acid sequence of a gene of interest; wherein the target mRNA is different from an mRNA encoded by the gene of interest, and wherein the expression of the target mRNA is downregulated and the expression of the gene of interest is upregulated simultaneously. In some embodiments, the expression of the target mRNA is downregulated by the siRNA capable of binding to the target mRNA. In some embodiments, the expression of the gene of interest is upregulated by expressing an mRNA or a protein encoded by the gene of interest.

In some aspects, provided herein, is a method of producing an RNA construct comprising a small interfering RNA (siRNA) capable of binding to a target messenger RNA (mRNA), and an mRNA of a gene of interest, wherein the target mRNA is different from the mRNA encoding the gene of interest, the method comprising: (a) providing, for in vitro transcription reaction: (i) a polynucleic acid construct comprising a promoter, at least one nucleic acid sequence encoding an siRNA capable of binding to a target mRNA, at least one nucleic acid sequence comprising a gene of interest, and a nucleic acid sequence encoding a poly(A) tail; (ii) an RNA polymerase; and (iii) a mixture of nucleotide triphosphates (NTPs); and (b) isolating and purifying transcribed RNAs from the in vitro transcription reaction mixture, thus producing the RNA construct. In some embodiments, the RNA polymerase is selected from the group consisting of T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, P60 RNA polymerase, Syn5 RNA polymerase, and KP34 RNA polymerase. In some embodiments, the RNA polymerase is T7 RNA polymerase. In some embodiments, the mixture of NTPs comprises unmodified NTPs. In some embodiments, the mixture of NTPs comprises modified NTPs. In some embodiments, the modified NTPs comprise N¹-methylpseudouridine, Pseudouridine, N¹-Ethylpseudouridine, N¹-Methoxymethylpseudouridine, N¹-Propylpseudouridine, 2-thiouridine, 4-thiouridine, 5-methoxyuridine, 5-methylurdine, 5-carboxymethylesteruridine, 5-formyluridine, 5-carboxyuridine, 5-hydroxyuridine, 5-Bromouridine, 5-lodouridine, 5,6-dihydrouridine, 6-Azauridine, Thienouridine, 3-methyluridine, 1-carboxymethyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, dihydrouridine, dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-methylcytidine, 5-methoxycytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, 5-hydroxycytidine, 5-lodocytidine, 5-Bromocytidine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, 3-methyl-cytidine, N⁴-acetylcytidine, 5-formylcytidine, N⁴-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine, N¹-methyladenosine, N⁶-methyladenosine, N⁶-methyl-2-Aminoadenosine, N⁶-isopentenyladenosine, N⁶,N⁶-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In some embodiments, step (a) further comprises providing a capping enzyme. In some embodiments, isolating and purifying transcribed RNAs comprise column purification.

In some embodiments, specific binding of an siRNA to its mRNA target results in interference with the normal function of the target mRNA to cause a modulation, e.g., downregulation, of function and/or activity, and wherein there is a sufficient degree of complementarity to avoid non-specific binding of the siRNA to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of specifically binding to IL-6 mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the composition comprises at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises 1 siRNA directed to IL-6 mRNA. In related aspects, the composition comprises 3 siRNAs, each directed to IL-6 mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by a sequence as set forth in SEQ ID NO: 29 or 30 (Compound B1 or B2).

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of specifically binding to Interleukin 6R (IL-6R) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the composition comprises at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises 1 siRNA directed to IL-6R mRNA. In related aspects, the composition comprises 3 siRNAs, each directed to IL-6R mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by the sequence as set forth in SEQ ID NO: 31 (Compound B3).

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of specifically binding to Interleukin 6R alpha (IL-6R-alpha) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the composition comprises at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises 1 siRNA directed to IL-6R-alpha mRNA. In related aspects, the composition comprises 3 siRNAs, each directed to IL-6R-alpha mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by the sequence as set forth in SEQ ID NO: 32 (Compound B4).

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of specifically binding to Interleukin 6R beta (IL-6R-beta) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the composition comprises at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises 1 siRNA directed to IL-6R-beta mRNA. In related aspects, the composition comprises 3 siRNAs, each directed to IL-6R-beta mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by the sequence as set forth in SEQ ID NO: 33 (Compound B5).

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of specifically binding to ACE2 mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the composition comprises at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises 1 siRNA directed to ACE2 mRNA. In related aspects, the composition comprises 3 siRNAs, each directed to ACE2 mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by a sequence as set forth in SEQ ID NO: 34 or 35 (Compound B6 or B7).

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one small interfering RNA (siRNA) capable of specifically binding to SARS CoV-2 ORF1ab mRNA, at least one siRNA capable of specifically binding to SARS CoV-2 S mRNA, at least one siRNA capable of specifically binding to SARS CoV-2 N mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the composition comprises at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises 3 siRNAs, one directed to SARS CoV-2 ORF1ab mRNA, one directed to SARS CoV-2 S mRNA, and one directed to SARS CoV-2 N mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In certain aspects, such a composition, e.g., a composition comprising Compound B8 (SEQ ID NO: 36) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, SARS CoV-2, or both. In related aspects, the recombinant RNA construct comprises a sequence encoded by the sequence as set forth in SEQ ID NO: 36.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of specifically binding to SARS CoV-2 S mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the composition comprises at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises 1 siRNA directed to SARS CoV-2 S mRNA. In related aspects, the composition comprises 3 siRNAs, each directed to SARS CoV-2 S mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by a sequence as set forth in SEQ ID NO: 37 or 39 (Compound B9 or B11).

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of specifically binding to SARS CoV-2 N mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the composition comprises at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises 1 siRNA directed to SARS CoV-2 N mRNA. In related aspects, the composition comprises 3 siRNAs, each directed to SARS CoV-2 N mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by the sequence as set forth in SEQ ID NO: 38 (Compound B10).

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of specifically binding to SARS CoV-2 ORF1ab mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the composition comprises 1 siRNA directed to SARS CoV-2 ORF1ab mRNA. In related aspects, the composition comprises 3 siRNAs, each directed to SARS CoV-2 ORF1ab mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In certain aspects, such a composition, including a composition comprising Compound B12 (SEQ ID NO: 40) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, MERS, or both. In certain aspects, such a composition, including a composition comprising Compound B13 (SEQ ID NO: 41) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, SARS CoV-2, and/or MERS. In related aspects, the recombinant RNA construct comprises a sequence encoded by a sequence as set forth in any one of SEQ ID NOs: 40, 41 and 42 (Compounds B12, B13 and B14).

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of specifically binding to IL-6 mRNA, at least one siRNA capable of specifically binding to ACE2 mRNA, and at least one siRNA capable of specifically binding to SARS CoV-2 S mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the composition comprises at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises 3 siRNAs, one directed IL-6 mRNA, one directed to ACE2 mRNA, and one directed to SARS CoV-2 S mRNA. In related aspects, the mRNA encoding IFN-beta encodes the native IFN-beta signal peptide, or a modified signal peptide. In related aspects, the modified IFN-beta signal peptide is SP1 or SP2 as described herein (SEQ ID NOS: 52 and 54, respectively). In related aspects, the recombinant RNA construct comprises a sequence encoded by a sequence as set forth in any one of SEQ ID NOS: 43, 44, and 45 (Compounds B15, B16, and B17).

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one small interfering RNA capable of specifically binding to SARS CoV-2 ORF1ab mRNA, at least one siRNA capable of specifically binding to SARS CoV-2 S mRNA, and at least one siRNA capable of specifically binding to SARS CoV-2 N mRNA; and (ii) an mRNA encoding an ACE2 soluble receptor. In related aspects, the composition comprises at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises 3 siRNAs, one directed to ORF1ab mRNA, one directed to SARS CoV-2 S mRNA, and one directed to SARS CoV-2 N mRNA. In related aspects, the recombinant RNA construct comprises a sequence encoded by the sequence as set forth in SEQ ID NO: 46 (Compound B18). In related aspects, the recombinant RNA construct comprises a sequence as set forth in SEQ ID NO: 190.

In some aspects, provided herein, is a composition comprising a recombinant RNA construct comprising: (i) at least one siRNA capable of specifically binding to SARS CoV-2 S mRNA; and (ii) an mRNA encoding ACE2 soluble receptor. In related aspects, the composition comprises at least 1, 2, or 3 siRNAs. In related aspects, the composition comprises 1 siRNA directed to SARS CoV-2 S mRNA. In related aspects, the composition comprises 3 siRNAs, each directed to SARS CoV-2 S mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant RNA construct comprises a sequence encoded by the sequence as set forth in SEQ ID NO: 47 (Compound B19).

In some aspects, the IFN-beta construct comprises a modified signal peptide as described herein.

In some aspects, the present invention provides a composition comprising a recombinant RNA construct comprising a nucleic acid sequence encoded by a sequence selected from the group consisting of SEQ ID NOs: 29-47. In some aspects, the present invention provides a composition comprising a recombinant RNA construct comprising a nucleic acid sequence as set forth in SEQ ID NO: 190.

In some embodiments, the present invention provides a composition and related methods, wherein the composition comprises a recombinant RNA construct comprising: at least one siRNA capable of binding to a target RNA; and an mRNA encoding a gene of interest; wherein: the siRNA targets an RNA selected from: an IL-8 mRNA, an IL-1 beta mRNA, an IL-17 mRNA, a TNF-alpha mRNA, a SARS CoV-2 ORF1ab RNA (polyprotein PP1ab, e.g., in a noncoding region or where it encodes a protein that is selected from: a SARS CoV-2 nonstructure protein (NSP), Nsp1, Nsp3 (Nsp3b, Nsp3c, PLpro, and Nsp3e), Nsp7 Nsp8 complex, Nsp9-Nsp10, and Nsp14-Nsp16, 3CLpro, E-channel (E protein), ORF7a, C-terminal RNA binding domain (CRBD), N-terminal RNA binding domain (NRBD), helicase, and RdRp), a SARS CoV-2 Spike protein (S) mRNA, a SARS CoV-2 Nucleocapsid protein (N) mRNA, a tumor necrosis factor alpha (TNF-alpha) mRNA, an interleukin mRNA (including but not limited to interleukin 1 (e.g., IL-1alpha, IL-1beta), interleukin 6 (IL-6), interleukin 6R (IL-6R), interleukin 6R alpha (IL-6R-alpha), interleukin 6R beta (IL-6R-beta), interleukin 18 (IL-18), interleukin 36-alpha (IL-36-alpha), interleukin 36-beta (IL-36-beta), interleukin 36-gamma (IL-36-gamma), interleukin 33 (IL-33)), an Angiotensin Converting Enzyme-2 (ACE2) mRNA, a transmembrane protease, serine 2 (TMPRSS2) mRNA, and a coding NSP12 and 13 RNA; and the gene of interest encodes a protein selected from: IGF-1, IL-4, IGF-1 (including derivatives thereof as described elsewhere herein), carboxypeptidases (e.g., ACE, ACE2, CNDP1, CPA1, CPA2, CPA4, CPA5, CPA6, CPB1, CPB2, CPE, CPN1, CPQ, CPXM1, CPZ, SCPEP1); cytokines (e.g., BMP1, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, C1QTNF4, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL3L3, CCL4, CCL4L, CCL4L2, CCL5, CCL7, CCL8, CD40LG, CER1, CKLF, CLCF1, CNTF, CSF1, CSF2, CSF3, CTF1, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, CXCL17, CXCL2, CXCL3, CXCL5, CXCL8, CXCL9, DKK1, DKK2, DKK3, DKK4, EDA, EBI3, FAM3B, FAM3C, FASLG, FLT3LG, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF5, GDF6, GDF7, GDF9, GPI, GREM1, GREM2, GRN, IFNA1, IFNA13, IFNA10, IFNA14, IFNA16, IFNA17, IFNA2, IFNA21, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNB1, IFNE, IFNG, IFNK, IFNL1, IFNL2, IFNL3, IFNL4, IFNW1, IL10, IL11, IL12A, IL12B, IL13, IL15, IL16, IL17A, IL17B, IL17C, IL17D, IL17F, IL18, IL19, IL1A, IL1B, IL1F10, IL2, IL20, IL21, IL22, IL23A, IL24, IL25, IL26, IL27, IL3, IL31, IL32, IL33, IL34, IL36A, IL36B, IL36G, IL36RN, IL37, IL4, IL5, IL6, IL7, IL9, LEFTY1, LEFTY2, LIF, LTA, MIF, MSTN, NAMPT, NODAL, OSM, PF4, PF4V1, SCGB3A1, SECTM1, SLURP1, SPP1, THNSL2, THPO, TNF, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TSLP, VSTM1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, and XCL2); extracellular ligands and transporters (e.g., APCS, CHI3L1, CHI3L2, CLEC3B, DMBT1, DMKN, EDDM3A, EDDM3B, EFNA4, EMC10, ENAM, EPYC, ERVH48-1, F13B, FCN1, FCN2, GLDN, GPLD1, HEG1, ITFG1, KAZALD1, KCP, LACRT, LEG1, METRN, NOTCH2NL, NPNT, OLFM1, OLFML3, PRB2, PSAP, PSAPL1, PSG1, PSG6, PSG9, PTX3, PTX4, RBP4, RNASE10, RNASE12, RNASE13, RNASE9, RSPRY1, RTBDN, S100A12, S100A13, S100A7, S100A8, SAA2, SAA4, SCG1, SCG2, SCG3, SCGB1C1, SCGB1C2, SCGB1D1, SCGB1D2, SCGB1D4, SCGB2B2, SCGB3A2, SCGN, SCRG1, SCUBE1, SCUBE2, SCUBE3, SDCBP, SELENOP, SFTA2, SFTA3, SFTPA1, SFTPA2, SFTPC, SFTPD, SHBG, SLURP2, SMOC1, SMOC2, SMR3A, SMR3B, SNCA, SPATA20, SPATA6, SOGA1, SPARC, SPARCL1, SPATA20, SPATA6, SRPX2, SSC4D, STX1A, SUSD4, SVBP, TCN1, TCN2, TCTN1, TF, TULP3, TFF2, TFF3, THSD7A, TINAG, TINAGL1, TMEFF2, TMEM25, VWC2L); extracellular matrix proteins (e.g., ABI3BP, AGRN, CCBE1, CHL1, COL15A1, COL19A1, COLEC11, DMBT1, DRAXIN, EDIL3, ELN, EMID1, EMILIN1, EMILIN2, EMILIN3, EPDR1, FBLN1, FBLN2, FBLN5, FLRT1, FLRT2, FLRT3, FREM1, GLDN, IBSP, KERA, KIAA0100, KIRREL3, KRT10, LAMB2, MGP, RPTN, SBSPON, SDC1, SDC4, SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, SIGLEC1, SIGLEC10, SIGLEC6, SLIT1, SLIT2, SLIT3, SLITRK1, SNED1, SNORC, SPACA3, SPACA7, SPON1, SPON2, STATH, SVEP1, TECTA, TECTB, TNC, TNN, TNR, TNXB); glucosidases (AMY1A, AMY1B, AMY1C, AMY2A, AMY2B, CEMIP, CHIA, CHIT1, FUCA2, GLB1L, GLB1L2, HPSE, HYAL1, HYAL3, KL, LYG1, LYG2, LYZL1, LYZL2, MAN2B2, SMPD1, SMPDL3B, SPACA5, SPACA5B); glycosyltransferases (e.g., ARTS, B4GALT1, EXTL2, GALNT1, GALNT2, GLT1D1, MGAT4A, ST3GAL1, ST3GAL2, ST3GAL3, ST3GAL4, ST6GAL1, XYLT1); growth factors (e.g., AMH, ARTN, BTC, CDNF, CFC1, CFC1B, CHRDL1, CHRDL2, CLEC11A, CNMD, EFEMP1, EGF, EGFL6, EGFL7, EGFL8, EPGN, EREG, EYS, FGF1, FGF10, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FRZB, GDNF, GFER, GKN1, HBEGF, HGF, IGF-1, IGF2, INHA, INHBA, INHBB, INHBC, INHBE, INS, KITLG, MANF, MDK, MIA, NGF, NOV, NRG1, NRG2, NRG3, NRG4, NRTN, NTF3, NTF4, OGN, PDGFA, PDGFB, PDGFC, PDGFD, PGF, PROK1, PSPN, PTN, SDF1, SDF2, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5, TDGF1, TFF1, TGFA, TGFB1, TGFB2, TGFB3, THBS4, TIMP1, VEGFA, VEGFB, VEGFC, VEGFD, WISP3); growth factor binding proteins (e.g., CHRD, CYR61, ESM1, FGFBP1, FGFBP2, FGFBP3, HTRA1, GHBP, IGFALS, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7, LTBP1, LTBP2, LTBP3, LTBP4, SOSTDC1, NOG, TWSG1, and WIF1); heparin binding proteins (e.g., ADA2, ADAMTSL5, ANGPTL3, APOB, APOE, APOH, COL5A1, COMP, CTGF, FBLN7, FN1, FSTL1, HRG, LAMC2, LIPC, LIPG, LIPH, LIPI, LPL, PCOLCE2, POSTN, RSPO1, RSPO2, RSPO3, RSPO4, SAA1, SLIT2, SOST, THBS1, VTN); hormones (e.g., ADCYAP1, ADIPOQ, ADM, ADM2, ANGPTL8, APELA, APLN, AVP, C1QTNF12, C1QTNF9, CALCA, CALCB, CCK, CGA, CGB1, CGB2, CGB3, CGB5, CGB8, COPA, CORT, CRH, CSH1, CSH2, CSHL1, ENHO, EPO, ERFE, FBN1, FNDC5, FSHB, GAL, GAST, GCG, GH, GH1, GH2, GHRH, GHRL, GIP, GNRH1, GNRH2, GPHA2, GPHB5, IAPP, INS, INSL3, INSL4, INSL5, INSL6, LHB, METRNL, MLN, NPPA, NPPB, NPPC, OSTN, OXT, PMCH, PPY, PRL, PRLH, PTH, PTHLH, PYY, RETN, RETNLB, RLN1, RLN2, RLN3, SCT, SPX, SST, STC1, STC2, TG, TOR2A, TRH, TSHB, TTR, UCN, UCN2, UCN3, UTS2, UTS2B, and VIP); hydrolases (e.g., AADACL2, ABHD15, ACP7, ACPP, ADA2, ADAMTSL1, AOAH, ARSF, ARSI, ARSJ, ARSK, BTD, CHI3L2, ENPP1, ENPP2, ENPP3, ENPP5, ENTPD5, ENTPD6, GBP1, GGH, GPLD1, HPSE, LIPC, LIPF, LIPG, LIPH, LIPI, LIPK, LIPM, LIPN, LPL, PGLYRP2, PLA1A, PLA2G10, PLA2G12A, PLA2G1B, PLA2G2A, PLA2G2D, PLA2G2E, PLA2G2F, PLA2G3, PLA2G5, PLA2G7, PNLIP, PNLIPRP2, PNLIPRP3, PON1, PON3, PPT1, SMPDL3A, THEM6, THSD1, and THSD4); immunoglobulins (e.g., IGSF10, IGKV1-12, IGKV1-16, IGKV1-33, IGKV1-6, IGKV1D-12, IGKV1D-39, IGKV1D-8, IGKV2-30, IGKV2D-30, IGKV3-11, IGKV3D-20, IGKV5-2, IGLC1, IGLC2, IGLC3); isomerases (e.g., NAXE, PPIA, PTGDS); kinases (e.g., ADCK1, ADPGK, FAM20C, ICOS, PKDCC); lyases (e.g., PM20D1, PAM, CA6); metalloenzyme inhibitors (e.g., FETUB, SPOCK3, TIMP2, TIMP3, TIMP4, WFIKKN1, WFIKKN2); metalloproteases (e.g., ADAM12, ADAM28, ADAM9, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, CLCA1, CLCA2, CLCA4, IDE, MEP1B, MMEL1, MMP1, MMP10, MMP11, MMP12, MMP13, MMP16, MMP17, MMP19, MMP2, MMP20, MMP21, MMP24, MMP25, MMP26, MMP28, MMP3, MMP1, MMP8, MMP9, PAPPA, PAPPA2, TLL1, TLL2); milk proteins (e.g., CSN1S1, CSN2, CSN3, LALBA); neuroactive proteins (e.g., CARTPT, NMS, NMU, NPB, NPFF, NPS, NPVF, NPW, NPY, PCSK1N, PDYN, PENK, PNOC, POMC, PROK2, PTH2, PYY2, PYY3, QRFP, TAC1, and TAC3); proteases (e.g., ADAMTS6, C1R, C1RL, C2, CASP4, CELA1, CELA2A, CELA2B, CFB, CFD, CFI, CMA1, CORIN, CTRB1, CTRB2, CTSB, CTSD, DHH, F10, F11, F12, F2, F3, F7, F8, F9, FAP, FURIN, GZMA, GZMK, GZMM, HABP2, HGFAC, HTRA3, HTRA4, IHH, KLK10, KLK11, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLKB1, MASP1, MASP2, MST1L, NAPSA, OVCH1, OVCH2, PCSK2, PCSK5, PCSK6, PCSK9, PGA3, PGA4, PGA5, PGC, PLAT, PLAU, PLG, PROC, PRSS1, PRSS12, PRSS2, PRSS22, PRSS23, PRSS27, PRSS29P, PRSS3, PRSS33, PRSS36, PRSS38, PRSS3P2, PRSS42, PRSS44, PRSS47, PRSS48, PRSS53, PRSS57, PRSS58, PRSS8, PRTN3, RELN, REN, TMPRSS11D, TMPRSS11E, TMPRSS2, TPSAB1, TPSB2, TPSD1); protease inhibitors (e.g., A2M, A2ML1, AMBP, ANOS1, COL28A1, COL6A3, COL7A1, CPAMD8, CST1, CST2, CST3, CST4, CST5, CST6, CST7, CST8, CST9, CST9L, CST9LP1, CSTL1, EPPIN, GPC3, HMSD, ITIH1, ITIH2, ITIH3, ITIH4, ITIH5, ITIH6, KNG1, OPRPN, OVOS1, OVOS2, PAPLN, PI15, PI16, PI3, PZP, R3HDML, SERPINA1, SERPINA10, SERPINA11, SERPINA12, SERPINA13P, SERPINA3, SERPINA4, SERPINA5, SERPINA7, SERPINA9, SERPINB2, SERPINB5, SERPINC1, SERPINE1, SERPINE2, SERPINE3, SERPINF2, SERPING1, SERPINI1, SERPINI2, SPINK1, SPINK13, SPINK14, SPINK2, SPINK4, SPINK5, SPINK6, SPINK7, SPINK8, SPINK9, SPINT1, SPINT3, SPINT4, SPOCK1, SPOCK2, SPP2, SSPO, TFPI, TFPI2, WFDC1, WFDC10A, WFDC13, WFDC2, WFDC3, WFDC5, WFDC6, WFDC8); protein phosphatases (e.g., ACP7, ACPP, PTEN, PTPRZ1); esterases (e.g., BCHE, CEL, CES4A, CES5A, NOTUM, SIAE); transferases (e.g., METTL24, FKRP, CHSY1, CHST9, B3GAT1); vasoactive proteins (e.g., AGGF1, AGT, ANGPT1, ANGPT2, ANGPTL4, ANGPTL6, EDN1, EDN2, EDN3, NTS), a Type I interferon (e.g., an IFN-α, including, but not limited to an interferon alpha-n3, an interferon alpha-2a, and an interferon alpha-2b, an IFN-β, an IFN-δ, an IFN-ε, an IFN-κ, an IFN-ν, an IFN-τ, and an IFN-ω), a Type II interferon (e.g., IFN-γ), a Type III interferon (e.g., IFN-λ) an interleukin, e.g., IL-37, IL-38, and a soluble ACE2 receptor. In some aspects, the composition comprising the recombinant RNA construct is useful in the treatment of a viral infection, disease or condition. In some aspects, the composition is present or administered in an amount sufficient to treat or prevent a viral infection, disease or condition. In some embodiments, the disease or the condition is selected from the group consisting of intervertebral disc disease (IVDD), osteoarthritis, and psoriasis.

In some aspects, the composition comprising the recombinant RNA construct is useful in the treatment of a skin disease or condition. In some aspects, the composition is present or administered in an amount sufficient to treat or prevent a skin disease or condition. In some embodiments, the skin disease or condition comprises an inflammatory skin disorder. In some embodiments, the inflammatory skin disorder comprises psoriasis. In some aspects, the composition comprising the recombinant RNA construct is useful in the treatment of a muscular disease or condition. In some aspects, the composition is present or administered in an amount sufficient to treat or prevent a muscular disease or condition. In some embodiments, the muscular disease or condition comprises a skeletal muscle disorder. In some embodiments, the skeletal muscle disorder comprises fibrodysplasia ossificans progressiva (FOP). In some aspects, the composition comprising the recombinant RNA construct is useful in the treatment of a neurodegenerative disease or condition. In some aspects, the composition is present or administered in an amount sufficient to treat or prevent a neurodegenerative disease or condition. In some embodiments, the neurodegenerative disease or condition comprises a motor neuron disorder. In some embodiments, the motor neuron disorder comprises amyotrophic lateral sclerosis (ALS). In some aspects, the composition comprising the recombinant RNA construct is useful in the treatment of a joint disease or condition. In some aspects, the composition is present or administered in an amount sufficient to treat or prevent a joint disease or condition. In some embodiments, the joint disease or condition comprises a joint degeneration. In some embodiments, the joint degeneration comprises intervertebral disc disease (IVDD) or osteoarthritis (OA).

RNA Interference and Small Interfering RNA (siRNA)

RNA interference (RNAi) or RNA silencing is a process in which RNA molecules inhibit gene expression or translation, by neutralizing target mRNA molecules. RNAi process is described in Mello & Conte (2004) Nature 431, 338-342, Meister & Tuschl (2004) Nature 431, 343-349, Hannon & Rossi (2004) Nature 431, 371-378, and Fire (2007) Angew. Chem. Int. Ed. 46, 6966-6984. Briefly, in a natural process, the reaction initiates with a cleavage of long double-stranded RNA (dsRNA) into small dsRNA fragments or siRNAs with a hairpin or loop structure by a dsRNA-specific endonuclease Dicer. These small dsRNA fragments or siRNAs are then integrated into RNA-induced silencing complex (RISC) and guide the RISC to the target mRNA sequence. During interference, the siRNA duplex unwinds, and the antisense strand remains in complex with RISC to lead RISC to the target mRNA sequence to induce degradation and subsequent suppression of protein translation. Unlike commercially available synthetic siRNA (e.g., Patisiran, etc.), the siRNA in the present invention utilizes endogenous Dicer and RISC pathway in the cytoplasm of a cell to get cleaved from mRNA transcript construct of the present invention and follow the natural process detailed above. In addition, as the rest of the mRNA transcript of the present invention is left intact after cleavage of the siRNA by Dicer, and the desired protein expression from the gene of interest in the mRNA transcript of the present invention is attained.

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid or RNA construct comprising at least one nucleic acid sequence encoding or comprising a siRNA capable of binding to a target mRNA. In some embodiments, the recombinant polynucleic acid or RNA construct comprises a nucleic acid sequence encoding or comprising a sense siRNA strand. In some embodiment, the recombinant polynucleic acid or RNA construct comprises a nucleic acid sequence encoding or comprising an anti-sense siRNA strand. In a preferred embodiment, the recombinant polynucleic acid or RNA construct comprises a nucleic acid sequence encoding or comprising a sense siRNA strand and a nucleic acid sequence encoding or comprising an anti-sense siRNA strand. The details of siRNA comprised in the present invention is described in Cheng, et al. (2018) J. Mater. Chem. B., 6, 4638-4644, which is incorporated by reference herein.

In some embodiments, the recombinant polynucleic acid or RNA construct has at least 1 copy of siRNA, i.e., a nucleic acid sequence encoding or comprising sense strand of siRNA and a nucleic acid sequence encoding or comprising anti-strand of siRNA. 1 copy of siRNA, as described herein, can refer to 1 copy of sense strand siRNA and 1 copy of anti-sense strand siRNA. In some embodiments, the recombinant polynucleic acid or RNA construct has more than 1 copy of siRNA, i.e., more than 1 copy of nucleic acid sequence encoding or comprising sense strand of siRNA and more than 1 copy of nucleic acid sequence encoding or comprising anti-strand of siRNA. In some embodiments, the recombinant polynucleic acid or RNA construct has 1 to 10 copies of siRNA, i.e., 1 to 10 copies of nucleic acid sequence encoding or comprising sense strand of siRNA and 1 to 10 copies of nucleic acid sequence encoding or comprising anti-strand of siRNA. In some embodiments, the recombinant polynucleic acid or RNA construct has 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10 copies of siRNA. In some embodiments, the recombinant polynucleic acid or RNA construct has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of siRNA. In some embodiments, the recombinant polynucleic acid or RNA construct has at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 copies of siRNA. In some embodiments, the recombinant polynucleic acid or RNA construct has at most 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of siRNA.

In some embodiments, the recombinant polynucleic acid or RNA construct further comprises a nucleic acid sequence encoding or comprising a linker. In some embodiments, the nucleic acid sequence encoding or comprising the linker may connect each of the two or more nucleic acid sequences encoding the siRNA. In some embodiments, the linker may be a non-cleavable linker. In a preferred embodiment, the linker may be a cleavable linker. In some embodiments, the linker may be a self-cleavable linker. In some embodiments, the linker may be a tRNA linker. The tRNA system is evolutionarily conserved across living organism and utilizes endogenous RNases P and Z to process multicistronic constructs (Dong et al., 2016). In some embodiments, the tRNA linker may comprise a nucleic acid sequence comprising

(SEQ ID NO: 24)

AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTA

CAGACCCGGGTTCGATTCCCGGCTGGTGCA.

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid or RNA construct comprising at least one nucleic acid sequence encoding or comprising a siRNA capable of binding to a target mRNA. A list of non-limiting examples of the target mRNAs that the siRNA is capable of binding to include an mRNA encoding Interleukin 8 (IL-8), Interleukin 1 beta (IL-1 beta), Interleukin 17 (IL-17), and Tumor Necrosis Factor alpha (TNF-alpha, or TNF-α). A list of additional examples of the target RNAs that the siRNA is capable of binding to includes an mRNA encoding Activin receptor-like kinase-2 (ALK2) and Superoxide dismutase-1 (SOD1).

In some aspects, the siRNA is capable of binding to a target RNA that is a coronavirus RNA. In some embodiments, the coronavirus RNA is a target mRNA that encodes a coronavirus protein. In some embodiments, the coronavirus RNA is a target noncoding RNA. In some embodiments, the coronavirus is an Alphacoronavirus, Betacoronavirus, Gammacoronavirus or a Deltacoronavirus. In some embodiments, the coronavirus target mRNA encodes a protein selected from: SARS CoV-2 ORF1ab (polyprotein PP1ab); SARS CoV-2 Spike protein (S), and SARS CoV-2 Nucleocapsid protein (N). In some embodiments, the siRNA is capable of binding to an ORF1ab mRNA in a region or where it encodes a protein that is selected from: a SARS CoV-2 nonstructure protein (NSP), Nsp1, Nsp3 (Nsp3b, Nsp3c, PLpro, and Nsp3e), Nsp7 Nsp8 complex, Nsp9-Nsp10, and Nsp14-Nsp16, 3CLpro, E-channel (E protein), ORF7a, C-terminal RNA binding domain (CRBD), N-terminal RNA binding domain (NRBD), helicase, and RdRp. In some embodiments, the target coding RNA is SARS CoV-2 NSP12 and 13. In some embodiments, the target mRNA encodes a coronavirus protein that is conserved among coronaviruses, e.g., among SARS-CoV, SARS-CoV-2, and/or MERS-CoV, and the corresponding siRNA is useful in compositions and methods that can be used to treat two or more different diseases or conditions, e.g., two or more diseases or conditions caused by or associated with more than one coronavirus. In some embodiments, the target mRNA encodes SARS-CoV-2 Nsp15, which is 89% identical to the analogous protein of SARS-CoV, and the polynucleic acid construct can be used to treat SARS-CoV and SARS-CoV-2 infection. In some embodiments, the siRNA is capable of binding to an mRNA target or noncoding RNA target common to more than one coronavirus. In some embodiments, the coding RNA target is Nsp12-Nsp13, relating to SARS CoV-2, SARS-CoV and MERS-CoV. In some embodiments, the coronavirus target RNA and any corresponding encoded protein is any one that is known to those of skill in the art or described in the literature, e.g., by Wu, et al., 27 Feb. 2020, Acta Pharmaceutica Sinica, preproof at doi.org/10.1016/j.apsb.2020.02.008, incorporated by reference herein. In some embodiments, the target mRNA encodes a host protein. In some embodiments, the target mRNA encodes a cytokine. In some embodiments, the target mRNA encodes a cytokine selected from the group consisting of: tumor necrosis factor alpha (TNF-alpha), an interleukin (including but not limited to interleukin 1 (e.g., IL-1alpha, IL-1beta), interleukin 6 (IL-6), interleukin 6R (IL-6R), interleukin 6R alpha (IL-6R-alpha), interleukin 6R beta (IL-6R-beta), interleukin 18 (IL-18), interleukin 36-alpha (IL-36-alpha), interleukin 36-beta (IL-36-beta)), interleukin 36-gamma (IL-36-gamma), and interleukin 33 (IL-33)). The role of TNF-alpha in Covid-19 is discussed in the literature, e.g., by Feldmann, et al., 9 Apr. 2020, The Lancet S0140-6736(20)30858-8, incorporated by reference herein. In some embodiments, the target mRNA encodes an inflammatory cytokine. In some embodiments, the target mRNA encodes a host viral entry protein. In some embodiments, the host viral entry protein is an Angiotensin Converting Enzyme-2 (ACE2). In some embodiments, the target mRNA encodes a host enzyme. In some embodiments, the enzyme is transmembrane protease, serine 2 (TMPRSS2).

In some embodiments, the recombinant nucleic acid construct comprises two or more nucleic acid sequences encoding an siRNA capable of binding to a target RNA. In some embodiments, the target RNA is an mRNA. In some embodiments, the target RNA is a noncoding RNA. In some embodiments, the recombinant nucleic acid construct comprises three nucleic acid sequences encoding an siRNA capable of binding to a target mRNA. In some embodiments, the recombinant nucleic acid construct comprises four nucleic acid sequences encoding an siRNA capable of binding to a target mRNA. In some embodiments, the recombinant nucleic acid construct comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding an siRNA capable of binding to a target mRNA. In some embodiments, the recombinant nucleic acid construct comprises 2 to 10 nucleic acid sequences encoding an siRNA capable of binding to a target mRNA. In some embodiments, the recombinant nucleic acid construct comprises 2 to 6 nucleic acid sequences encoding an siRNA capable of binding to a target mRNA. In some embodiments, each of the two or more nucleic acid sequences encodes an siRNA capable of binding to a same target mRNA. In some embodiments, each of the two or more nucleic acid sequences encodes an siRNA capable of binding to a different target mRNA.

In some embodiments, the expression of the target mRNA is modulated by the siRNA capable of binding to the target mRNA. In some embodiments, the siRNA is capable of binding to a target mRNA in its 5′ untranslated region. In some embodiments, the siRNA is capable of binding to a target mRNA in its 3′ untranslated region. In some embodiments, the siRNA is capable of binding to a target mRNA in a translated region. In some embodiments, the expression of the target mRNA is downregulated by the siRNA capable of binding to the target mRNA. In some embodiments, the expression of the target mRNA is inhibited by the siRNA capable of binding to the target mRNA. Inhibition or downregulation of the expression of the target mRNA, as described herein, can refer to, but is not limited to, interference with the target mRNA to interfere with translation of the protein from the target mRNA encoded by or comprised in the recombinant polynucleic acid or RNA construct, respectively; thus, inhibition or downregulation of the expression of the target mRNA can refer to, but is not limited to, a decreased level of the protein expressed from the target mRNA compared to a level of the protein expressed from the target mRNA in the absence of the recombinant polynucleic acid or RNA construct comprising siRNA capable of binding to the target mRNA. The level of protein expression can be measured by using any methods well known in the art and these include, but are not limited to Western-blotting, flow cytometry, ELISAs, RIAs, and various proteomics techniques. An exemplary method to measure or detect a polypeptide is an immunoassay, such as an ELISA. This type of protein quantitation can be based on an antibody capable of capturing a specific antigen, and a second antibody capable of detecting the captured antigen. Exemplary assays for detection and/or measurement of polypeptides are described in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor Laboratory Press.

In some aspects, provided herein, is a composition comprising a recombinant polynucleic acid or RNA construct comprising at least one nucleic acid sequence encoding or comprising a siRNA capable of binding to a target mRNA and at least one nucleic acid sequence encoding a gene of interest wherein the target mRNA is different from an mRNA encoded by the gene of interest. In some embodiments, the siRNA does not affect the expression of the gene of interest. In some embodiments, the siRNA is not capable of binding to the nucleic acid encoding the gene of interest. In a preferred embodiment, the siRNA does not inhibit the expression of the gene of interest. In another preferred embodiment, the siRNA does not downregulate the expression of the gene of interest. Inhibiting or downregulating the expression of the gene of interest, as described herein, can refer to, but is not limited to, interfering with transcription of DNA and/or translation of protein from the recombinant polynucleic acid or RNA construct; thus, inhibiting or downregulating the expression of the gene of interest can refer to, but is not limited to, a decreased level of protein compared to a level of protein expressed in the absence of the recombinant polynucleic acid or RNA construct comprising siRNA capable of binding to the target mRNA. The level of protein expression can be measured by using any methods well known in the art and these include, but are not limited to Western-blotting, flow cytometry, ELISAs, RIAs, and various proteomics techniques. An exemplary method to measure or detect a polypeptide is an immunoassay, such as an ELISA. This type of protein quantitation can be based on an antibody capable of capturing a specific antigen, and a second antibody capable of detecting the captured antigen. Exemplary assays for detection and/or measurement of polypeptides are described in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor Laboratory Press.

In some aspects, the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 80-109. In some aspects, the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 80-109, and the corresponding antisense strand encoded by a sequence selected from SEQ ID NOS: 110-139. In some embodiments, the target RNA is an IL-8 mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 80-83. In some embodiments, the target RNA is an IL-8 mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 80-83, and a corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 110-113, respectively. In some embodiments, the target RNA is an IL-1 beta mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 84-86. In some embodiments, the target RNA is an IL-1 beta mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 84-86, and a corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 114-116, respectively. In some embodiments, the target RNA is a TNF-alpha mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 87-89. In some embodiments, the target RNA is a TNF-alpha mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 87-89, and a corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 117-119, respectively. In some embodiments, the target RNA is an IL-17 mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 90-92. In some embodiments, the target RNA is an IL-17 mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 90-92, and a corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 120-122, respectively. In some embodiments, the target RNA is an IL-6 mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 93-95. In some embodiments, the target RNA is an IL-6 mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 93-95, and a corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 123-125, respectively. In some embodiments, the target RNA is an IL-6R alpha mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 96 and 97. In some embodiments, the target RNA is an IL-6R alpha mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 96 and 97, and a corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 125 and 127, respectively. In some embodiments, the target RNA is an IL-6R beta mRNA, and the siRNA comprises a sense strand encoded by the sequence set forth in SEQ ID NO: 98. In some embodiments, the target RNA is an IL-6R beta mRNA, and the siRNA comprises a sense strand encoded by the sequence set forth in SEQ ID NO: 98, and a corresponding antisense strand encoded by the sequence set forth in SEQ ID NO: 128. In some embodiments, the target RNA is an ACE2 mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 99-101. In some embodiments, the target RNA is an ACE2 mRNA, and the siRNA comprises a sense strand encoded by the sequence set forth in selected from SEQ ID NOs: 99-101, and a corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 129-131, respectively. In some embodiments, the target RNA is a SARS CoV-2 ORF1ab mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 102-105. In some embodiments, the target RNA is a SARS CoV-2 ORF1ab mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 102-105, and a corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 132-135, respectively. In some embodiments, the target RNA is a SARS CoV-2 Spike Protein mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 106-108. In some embodiments, the target RNA is a SARS CoV-2 Spike Protein mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 106-108, and a corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 136-138, respectively. In some embodiments, the target RNA is a SARS CoV-2 Nucleocapsid Protein mRNA, and the siRNA comprises a sense strand encoded by the sequence set forth in SEQ ID NO: 109. In some embodiments, the target RNA is a SARS CoV-2 Nucleocapsid Protein mRNA, and the siRNA comprises a sense strand encoded by the sequence set forth in SEQ ID NO: 109, and a corresponding antisense strand encoded by the sequence set forth in SEQ ID NO: 139. In some aspects, the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 140-145. In some aspects, the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 140-145, and the corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 146-151. In some embodiments, the target RNA is an ALK2 mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 140-142. In some embodiments, the target RNA is an ALK2 mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 140-142, and a corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 146-148, respectively. In some embodiments, the target RNA is a SOD1 mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 143-145. In some embodiments, the target RNA is a SOD1 mRNA, and the siRNA comprises a sense strand encoded by a sequence selected from SEQ ID NOs: 143-145, and a corresponding antisense strand encoded by a sequence selected from SEQ ID NOs: 149-151, respectively.

Gene of Interest

In some embodiments, the recombinant nucleic acid or RNA construct of the present invention may comprise two or more nucleic acid sequences encoding a gene of interest. In some embodiments, the recombinant nucleic acid or RNA construct of the present invention may comprise three nucleic acid sequences encoding a gene of interest. In some embodiments, the recombinant nucleic acid or RNA construct of the present invention may comprise four nucleic acid sequences encoding a gene of interest. In some embodiments, the recombinant nucleic acid or RNA construct of the present invention may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleic acid sequences encoding a gene of interest. In one embodiment, each of the two or more nucleic acid sequences may encode a same gene of interest. In another embodiment, each of the two or more nucleic acid sequences encodes a different gene of interest. In some embodiments, each of the two or more nucleic acid sequences encoding the gene of interest comprises a nucleic acid sequence encoding a secretory protein. In some embodiments, each of the two or more nucleic acid sequences encoding the gene of interest may comprise a nucleic acid sequence encoding an intracellular protein. In some embodiments, each of the two or more nucleic acid sequences encoding the gene of interest comprises a nucleic acid sequence encoding an intraorganelle protein. In some embodiments, each of the two or more nucleic acid sequences encoding the gene of interest comprises a nucleic acid sequence encoding a membrane protein.

In some embodiments, the recombinant polynucleic acid or RNA construct may further comprise a nucleic acid sequence encoding or comprising a linker. In some embodiments, the nucleic acid sequence encoding or comprising the linker may connect each of the two or more nucleic acid sequences encoding the gene of interest. In some embodiments, the linker may be a non-cleavable linker. In a preferred embodiment, the linker may be a cleavable linker. In some embodiments, the linker may be a self-cleavable linker. Non-limiting examples of the linker comprise 2A peptide linker (or 2A self-cleaving peptides) such as T2A, P2A, E2A, or F2A, or tRNA linker, etc. In some embodiments, the linker is a T2A peptide linker. In some embodiments, the linker may be a P2A peptide linker. In some embodiments, the linker may be a E2A peptide linker. In some embodiments, the linker may be a F2A linker. In some embodiments, the linker may be a tRNA linker. The tRNA system is evolutionarily conserved across living organism and utilizes endogenous RNases P and Z to process multicistronic constructs (Dong et al., 2016). In some embodiments, the tRNA linker may comprise a nucleic acid sequence comprising

(SEQ ID NO: 24)

AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTA

CAGACCCGGGTTCGATTCCCGGCTGGTGCA.

In some embodiments, the expression of the gene of interest is modulated by expressing an mRNA or a protein encoded by the gene of interest. In some embodiments, the expression of the gene of interest is upregulated by expressing an mRNA or a protein encoded by the gene of interest. Upregulation of the expression of an mRNA or a protein encoded by the gene of interest, as used herein, can refer to, but is not limited to, increasing the level of protein encoded by the gene of interest. The level of protein expression can be measured by using any methods well known in the art and these include, but are not limited to Western-blotting, flow cytometry, ELISAs, RIAs, and various proteomics techniques. An exemplary method to measure or detect a polypeptide is an immunoassay, such as an ELISA. This type of protein quantitation can be based on an antibody capable of capturing a specific antigen, and a second antibody capable of detecting the captured antigen. Exemplary assays for detection and/or measurement of polypeptides are described in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor Laboratory Press.

In some embodiments, the gene of the interest encodes a protein. In some embodiments, the protein is a therapeutic protein. In a preferred embodiment of the present invention the protein is of human origin i.e., is a human protein. Non-limiting examples of proteins encoded by the gene of interest comprises: carboxypeptidases; cytokines; extracellular ligands and transporters; extracellular matrix proteins; glucosidases; glycosyltransferases; growth factors; growth factor binding proteins; heparin binding proteins; hormones; hydrolases; immunoglobulins; isomerases; kinases; lyases; metalloenzyme inhibitors; metalloproteases; milk proteins; neuroactive proteins; proteases; protease inhibitors; protein phosphatases; esterases; transferases; and vasoactive proteins all of human origin. In a more preferred embodiment of the present invention the protein of the present invention is a human protein selected from the group consisting of human carboxypeptidases; human cytokines; human extracellular ligands and transporters; human extracellular matrix proteins; human glucosidases; human glycosyltransferases; human growth factors; human growth factor binding proteins; human heparin binding proteins; human hormones; human hydrolases; human immunoglobulins; human isomerases; human kinases; human lyases; human metalloenzyme inhibitors; human metalloproteases; human milk proteins; human neuroactive proteins; human proteases; human protease inhibitors; human protein phosphatases; human esterases; human transferases; or human vasoactive proteins.

In one embodiment, the protein is selected from the group consisting of carboxypeptidases, wherein the carboxypeptidases are selected from the group consisting of ACE, ACE2, CNDP1, CPA1, CPA2, CPA4, CPA5, CPA6, CPB1, CPB2, CPE, CPN1, CPQ, CPXM1, CPZ, and SCPEP1; cytokines wherein the cytokines are selected from the group consisting of BMP1, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, C1QTNF4, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL3L3, CCL4, CCL4L, CCL4L2, CCL5, CCL7, CCL8, CD40LG, CER1, CKLF, CLCF1, CNTF, CSF1, CSF2, CSF3, CTF1, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, CXCL17, CXCL2, CXCL3, CXCL5, CXCL8, CXCL9, DKK1, DKK2, DKK3, DKK4, EDA, EBI3, FAM3B, FAM3C, FASLG, FLT3LG, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF5, GDF6, GDF7, GDF9, GPI, GREM1, GREM2, GRN, IFNA1, IFNA13, IFNA10, IFNA14, IFNA16, IFNA17, IFNA2, IFNA21, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNB1, IFNE, IFNG, IFNK, IFNL1, IFNL2, IFNL3, IFNL4, IFNW1, IL10, IL11, IL12A, IL12B, IL13, IL15, IL16, IL17A, IL17B, IL17C, IL17D, IL17F, IL18, IL19, IL1A, IL1B, IL1F10, IL2, IL20, IL21, IL22, IL23A, IL24, IL25, IL26, IL27, IL3, IL31, IL32, IL33, IL34, IL36A, IL36B, IL36G, IL36RN, IL37, IL4, IL5, IL6, IL7, IL9, LEFTY1, LEFTY2, LIF, LTA, MIF, MSTN, NAMPT, NODAL, OSM, PF4, PF4V1, SCGB3A1, SECTM1, SLURP1, SPP1, THNSL2, THPO, TNF, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TSLP, VSTM1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, and XCL2; extracellular ligands and transporters, wherein the extracellular ligands and transporters are selected from the group consisting of APCS, CHI3L1, CHI3L2, CLEC3B, DMBT1, DMKN, EDDM3A, EDDM3B, EFNA4, EMC10, ENAM, EPYC, ERVH48-1, F13B, FCN1, FCN2, GLDN, GPLD1, HEG1, ITFG1, KAZALD1, KCP, LACRT, LEG1, METRN, NOTCH2NL, NPNT, OLFM1, OLFML3, PRB2, PSAP, PSAPL1, PSG1, PSG6, PSG9, PTX3, PTX4, RBP4, RNASE10, RNASE12, RNASE13, RNASE9, RSPRY1, RTBDN, S100A12, S100A13, S100A7, S100A8, SAA2, SAA4, SCG1, SCG2, SCG3, SCGB1C1, SCGB1C2, SCGB1D1, SCGB1D2, SCGB1D4, SCGB2B2, SCGB3A2, SCGN, SCRG1, SCUBE1, SCUBE2, SCUBE3, SDCBP, SELENOP, SFTA2, SFTA3, SFTPA1, SFTPA2, SFTPC, SFTPD, SHBG, SLURP2, SMOC1, SMOC2, SMR3A, SMR3B, SNCA, SPATA20, SPATA6, SOGA1, SPARC, SPARCL1, SPATA20, SPATA6, SRPX2, SSC4D, STX1A, SUSD4, SVBP, TCN1, TCN2, TCTN1, TF, TULP3, TFF2, TFF3, THSD7A, TINAG, TINAGL1, TMEFF2, TMEM25, and VWC2L; extracellular matrix proteins, wherein the extracellular matrix proteins are selected from the group consisting of ABI3BP, AGRN, CCBE1, CHL1, COL15A1, COL19A1, COLEC11, DMBT1, DRAXIN, EDIL3, ELN, EMID1, EMILIN1, EMILIN2, EMILIN3, EPDR1, FBLN1, FBLN2, FBLN5, FLRT1, FLRT2, FLRT3, FREM1, GLDN, IBSP, KERA, KIAA0100, KIRREL3, KRT10, LAMB2, MGP, RPTN, SBSPON, SDC1, SDC4, SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, SIGLEC1, SIGLEC10, SIGLEC6, SLIT1, SLIT2, SLIT3, SLITRK1, SNED1, SNORC, SPACA3, SPACA7, SPON1, SPON2, STATH, SVEP1, TECTA, TECTB, TNC, TNN, TNR and TNXB; glucosidases, wherein the glucosidases are selected from the group consisting of AMY1A, AMY1B, AMY1C, AMY2A, AMY2B, CEMIP, CHIA, CHIT1, FUCA2, GLB1L, GLB1L2, HPSE, HYAL1, HYAL3, KL, LYG1, LYG2, LYZL1, LYZL2, MAN2B2, SMPD1, SMPDL3B, SPACA5, and SPACA5B; glycosyltransferases, wherein the glycosyltransferases are selected from the group consisting of ARTS, B4GALT1, EXTL2, GALNT1, GALNT2, GLT1D1, MGAT4A, ST3GAL1, ST3GAL2, ST3GAL3, ST3GAL4, ST6GAL1, and XYLT1; growth factors, wherein the growth factors are selected from the group consisting of AMH, ARTN, BTC, CDNF, CFC1, CFC1B, CHRDL1, CHRDL2, CLEC11A, CNMD, EFEMP1, EGF, EGFL6, EGFL7, EGFL8, EPGN, EREG, EYS, FGF1, FGF10, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FRZB, GDNF, GFER, GKN1, HBEGF, HGF, IGF-1, IGF2, INHA, INHBA, INHBB, INHBC, INHBE, INS, KITLG, MANF, MDK, MIA, NGF, NOV, NRG1, NRG2, NRG3, NRG4, NRTN, NTF3, NTF4, OGN, PDGFA, PDGFB, PDGFC, PDGFD, PGF, PROK1, PSPN, PTN, SDF1, SDF2, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5, TDGF1, TFF1, TGFA, TGFB1, TGFB2, TGFB3, THBS4, TIMP1, VEGFA, VEGFB, VEGFC, VEGFD, and WISP3; growth factor binding proteins, wherein the growth factor binding proteins are selected from the group consisting of CHRD, CYR61, ESM1, FGFBP1, FGFBP2, FGFBP3, HTRA1, GHBP, IGFALS, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7, LTBP1, LTBP2, LTBP3, LTBP4, SOSTDC1, NOG, TWSG1, and WIF1; heparin binding proteins, wherein the heparin binding proteins are selected from the group consisting of ADA2, ADAMTSL5, ANGPTL3, APOB, APOE, APOH, COL5A1, COMP, CTGF, FBLN7, FN1, FSTL1, HRG, LAMC2, LIPC, LIPG, LIPH, LIPI, LPL, PCOLCE2, POSTN, RSPO1, RSPO2, RSPO3, RSPO4, SAA1, SLIT2, SOST, THBS1, and VTN; hormones, wherein the hormones are selected from the group consisting of ADCYAP1, ADIPOQ, ADM, ADM2, ANGPTL8, APELA, APLN, AVP, C1QTNF12, C1QTNF9, CALCA, CALCB, CCK, CGA, CGB1, CGB2, CGB3, CGB5, CGB8, COPA, CORT, CRH, CSH1, CSH2, CSHL1, ENHO, EPO, ERFE, FBN1, FNDC5, FSHB, GAL, GAST, GCG, GH, GH1, GH2, GHRH, GHRL, GIP, GNRH1, GNRH2, GPHA2, GPHB5, IAPP, INS, INSL3, INSL4, INSL5, INSL6, LHB, METRNL, MLN, NPPA, NPPB, NPPC, OSTN, OXT, PMCH, PPY, PRL, PRLH, PTH, PTHLH, PYY, RETN, RETNLB, RLN1, RLN2, RLN3, SCT, SPX, SST, STC1, STC2, TG, TOR2A, TRH, TSHB, TTR, UCN, UCN2, UCN3, UTS2, UTS2B, and VIP; hydrolases, wherein the hydrolases are selected from the group consisting of AADACL2, ABHD15, ACP7, ACPP, ADA2, ADAMTSL1, AOAH, ARSF, ARSI, ARSJ, ARSK, BTD, CHI3L2, ENPP1, ENPP2, ENPP3, ENPP5, ENTPD5, ENTPD6, GBP1, GGH, GPLD1, HPSE, LIPC, LIPF, LIPG, LIPH, LIPI, LIPK, LIPM, LIPN, LPL, PGLYRP2, PLA1A, PLA2G10, PLA2G12A, PLA2G1B, PLA2G2A, PLA2G2D, PLA2G2E, PLA2G2F, PLA2G3, PLA2G5, PLA2G7, PNLIP, PNLIPRP2, PNLIPRP3, PON1, PON3, PPT1, SMPDL3A, THEM6, THSD1, and THSD4; immunoglobulins, wherein the immunoglobulins are selected from the group consisting of IGSF10, IGKV1-12, IGKV1-16, IGKV1-33, IGKV1-6, IGKV1D-12, IGKV1D-39, IGKV1D-8, IGKV2-30, IGKV2D-30, IGKV3-11, IGKV3D-20, IGKV5-2, IGLC1, IGLC2, and IGLC3; isomerases, wherein the isomerases are selected from the group consisting of NAXE, PPIA, and PTGDS; kinases, wherein the kinases are selected from the group consisting of ADCK1, ADPGK, FAM20C, ICOS, and PKDCC; lyases, wherein the lyases are selected from the group consisting of PM20D1, PAM, and CA6; metalloenzyme inhibitors, wherein the metalloenzyme inhibitors are selected from the group consisting of FETUB, SPOCK3, TIMP2, TIMP3, TIMP4, WFIKKN1, and WFIKKN2; metalloproteases, wherein the metalloproteases are selected from the group consisting of ADAM12, ADAM28, ADAM9, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, CLCA1, CLCA2, CLCA4, IDE, MEP1B, MMEL1, MMP1, MMP10, MMP11, MMP12, MMP13, MMP16, MMP17, MMP19, MMP2, MMP20, MMP21, MMP24, MMP25, MMP26, MMP28, MMP3, MMP1, MMP8, MMP9, PAPPA, PAPPA2, TLL1, and TLL2; milk proteins, wherein the milk proteins are selected from the group consisting of CSN1S1, CSN2, CSN3, and LALBA; neuroactive proteins, wherein the neuroactive proteins are selected from the group consisting of CARTPT, NMS, NMU, NPB, NPFF, NPS, NPVF, NPW, NPY, PCSK1N, PDYN, PENK, PNOC, POMC, PROK2, PTH2, PYY2, PYY3, QRFP, TAC1, and TAC3; proteases, wherein the proteases are selected from the group consisting of ADAMTS6, C1R, C1RL, C2, CASP4, CELA1, CELA2A, CELA2B, CFB, CFD, CFI, CMA1, CORIN, CTRB1, CTRB2, CTSB, CTSD, DHH, F10, F11, F12, F2, F3, F7, F8, F9, FAP, FURIN, GZMA, GZMK, GZMM, HABP2, HGFAC, HTRA3, HTRA4, IHH, KLK10, KLK11, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK9, KLKB1, MASP1, MASP2, MST1L, NAPSA, OVCH1, OVCH2, PCSK2, PCSK5, PCSK6, PCSK9, PGA3, PGA4, PGA5, PGC, PLAT, PLAU, PLG, PROC, PRSS1, PRSS12, PRSS2, PRSS22, PRSS23, PRSS27, PRSS29P, PRSS3, PRSS33, PRSS36, PRSS38, PRSS3P2, PRSS42, PRSS44, PRSS47, PRSS48, PRSS53, PRSS57, PRSS58, PRSS8, PRTN3, RELN, REN, TMPRSS11D, TMPRSS11E, TMPRSS2, TPSAB1, TPSB2, and TPSD1; protease inhibitors, wherein the protease inhibitors are selected from the group consisting of A2M, A2ML1, AMBP, ANOS1, COL28A1, COL6A3, COL7A1, CPAMD8, CST1, CST2, CST3, CST4, CST5, CST6, CST7, CST8, CST9, CST9L, CST9LP1, CSTL1, EPPIN, GPC3, HMSD, ITIH1, ITIH2, ITIH3, ITIH4, ITIH5, ITIH6, KNG1, OPRPN, OVOS1, OVOS2, PAPLN, PI15, PI16, PI3, PZP, R3HDML, SERPINA1, SERPINA10, SERPINA11, SERPINA12, SERPINA13P, SERPINA3, SERPINA4, SERPINA5, SERPINA7, SERPINA9, SERPINB2, SERPINB5, SERPINC1, SERPINE1, SERPINE2, SERPINE3, SERPINF2, SERPING1, SERPINI1, SERPINI2, SPINK1, SPINK13, SPINK14, SPINK2, SPINK4, SPINK5, SPINK6, SPINK7, SPINK8, SPINK9, SPINT1, SPINT3, SPINT4, SPOCK1, SPOCK2, SPP2, SSPO, TFPI, TFPI2, WFDC1, WFDC10A, WFDC13, WFDC2, WFDC3, WFDC5, WFDC6, and WFDC8; protein phosphatases, wherein the protein phosphatases are selected from the group consisting of ACP7, ACPP, PTEN, and PTPRZ1; esterases, wherein the esterases, are selected from the group consisting of BCHE, CEL, CES4A, CES5A, NOTUM, and SIAE; transferases, wherein the transferases, are selected from the group consisting of METTL24, FKRP, CHSY1, CHST9, and B3GAT1; and vasoactive proteins, wherein the vasoactive proteins are selected from the group consisting of AGGF1, AGT, ANGPT1, ANGPT2, ANGPTL4, ANGPTL6, EDN1, EDN2, EDN3, and NTS. In some embodiments, the protein is selected from the group consisting of Insulin-like Growth Factor 1 (IGF-1), Interleukin 4 (IL-4), Interferon beta (IFN beta), Interferon alpha (IFN alpha), ACE2 soluble receptor, Interleukin 37 (IL-37), and Interleukin 38 (IL-38). In some embodiments, the protein is selected from the group consisting of Insulin-like Growth Factor 1 (IGF-1), Interleukin 4 (IL-4), Interferon beta (IFN beta), and ACE2 soluble receptor. In some embodiments, the protein is selected from the group consisting of Insulin-like Growth Factor 1 (IGF-1), Interleukin 4 (IL-4), Interferon beta (IFN beta), ACE2 soluble receptor, and Erythropoietin (EPO). In some embodiments, the protein is selected from the group consisting of Insulin-like Growth Factor 1 (IGF-1), and Interleukin 4 (IL-4). In some embodiments, the protein is IGF-1. In some embodiments, the protein is IL-4. In some embodiments, the protein is Interferon beta (IFN beta). In some embodiments, the protein is ACE2 soluble receptor. In some embodiments, the protein is Erythropoietin (EPO).

In one embodiment of the present invention, the recombinant polynucleic acid or RNA construct comprising a nucleic acid sequence or an mRNA encoding a gene of interest may comprise a nucleic acid sequence encoding human insulin-like growth factor 1 (IGF-1). In another embodiment, the recombinant polynucleic acid or RNA construct can be naked DNA or RNA comprising a nucleic acid sequence encoding IGF-1. In this embodiment of the present invention, the recombinant polynucleic acid or RNA construct may comprise a nucleic acid sequence encoding the mature human IGF-1. In a preferred embodiment of the present invention, the recombinant polynucleic acid or RNA construct may comprise a nucleic acid sequence encoding a propeptide of IGF-1, preferably a propeptide of human IGF-1, and a nucleic acid sequence encoding a mature protein of IGF-1, or preferably a mature protein of human IGF-1, and does not comprise a nucleic acid sequence encoding an E-peptide of IGF-1, preferably does not comprise a nucleic acid sequence encoding a human E-peptide of IGF-1, i.e., IGF-1 with a carboxyl-terminal extension. In a more preferred embodiment of the present invention, the recombinant polynucleic acid or RNA construct may comprise a nucleic acid sequence encoding a propeptide of IGF-1, preferably a propeptide of human IGF-1, a nucleic acid sequence encoding a mature protein of IGF-1, or preferably a mature protein of human IGF-1. Preferably the recombinant polynucleic acid or RNA construct does not comprise a nucleic acid sequence encoding an E-peptide of IGF-1, or more preferably does not comprise a nucleic acid sequence encoding a human E-peptide of IGF-1. In a further preferred embodiment of the present invention, the recombinant polynucleic acid or RNA construct may comprise a nucleic acid sequence encoding a propeptide of IGF-1, preferably a propeptide of human IGF-1, a nucleic acid sequence encoding a mature protein of IGF-1, or preferably a mature protein of human IGF-1 and a nucleic acid sequence encoding the signal peptide of the brain-derived neurotrophic factor (BDNF). Preferably the recombinant polynucleic acid or RNA construct does not comprise a nucleic acid sequence encoding an E-peptide of IGF-1, and more preferably does not comprise a nucleic acid sequence encoding a human E-peptide of IGF-1.

In some embodiments, the recombinant polynucleic acid or RNA construct may comprise a nucleic acid sequence encoding a propeptide (also called pro-domain) of IGF-1, preferably of human IGF-1 having 27 amino acids, and a nucleic sequence encoding a mature IGF-1, preferably a mature human IGF-1 having 70 amino acids, and preferably does not comprise a nucleotide sequence encoding an E-peptide of IGF-1, and preferably does not comprise a nucleic acid sequence encoding a human E-peptide of IGF-1. In some embodiments, the recombinant polynucleic acid or RNA construct may comprise a nucleic acid sequence encoding a propeptide (also called pro-domain) of IGF-1, preferably of human IGF-1 having 27 amino acids, a nucleic sequence encoding a mature IGF-1, preferably a mature human IGF-1 having 70 amino acids and a nucleic acid sequence encoding the signal peptide of the brain-derived neurotrophic factor (BDNF). Preferably the recombinant polynucleic acid or RNA construct does not comprise a nucleic sequence encoding an E-peptide of IGF-1, more preferably does not comprise a nucleic acid sequence encoding a human E-peptide of IGF-1.

In some embodiments, the recombinant polynucleic acid or RNA construct of the present invention may comprise a nucleic acid sequence encoding a propeptide (also called pro-domain) of human IGF-1 having 27 amino acids, and a nucleic acid sequence encoding a mature human IGF-1 having 70 amino acids and preferably does not comprise a nucleic acid sequence encoding an E-peptide (also called E-domain) of human IGF-1, wherein the nucleic acid sequence encoding the propeptide (also called pro-domain) of human IGF-1 having 27 amino acids, and the nucleic acid sequence encoding the mature human IGF-1 having 70 amino acids and the nucleic acid sequence encoding the E-peptides are as referred to in the Uniprot database as UniProtKB—P05019 and in the Genbank database as NM_000618.4, NM_001111285.2 and NM_001111283.2, respectively.

In some embodiments, the gene of interest (which can encode, e.g., an mRNA of interest and/or a protein of interest corresponding to the gene of interest), encodes a protein of interest, wherein the protein of interest is an anti-inflammatory cytokine. In some embodiments, the anti-inflammatory cytokine is an interferon or an interleukin. In some embodiments, the interferon is a Type I interferon (e.g., IFN-α, IFN-δ, IFN-ε, IFN-κ, IFN-ν, IFN-τ, and IFN-ω), a Type II interferon (IFN-γ), or a Type III interferon (IFN-λ). In some embodiments, an alpha interferon is selected from interferon alpha-n3, interferon alpha-2a, and interferon alpha-2b. The activities of interferons against viral infections have been described, e.g., in WO 2004/096852 (Chen, et al.) describing an anti-SARS effect of IFN-ω, and WO 2005/097165 (Klucher, et al.), describing an anti-viral effect of IFN-λ, variants, both incorporated herein by reference. In some embodiments, the cytokine is an interleukin. In some embodiments, the interleukin is an interleukin 1F family member. In some embodiments, the interleukin is interleukin 37 (IL-37, formerly known as the interleukin-1 family member 7 or IL-1F7, and described by, e.g., Yan, et al., 2018, Mediators of Inflammation Volume 2019, Article ID 2650590, and Conti, et al., March-April 2020, Journal of biological regulators and homeostatic agents 34(2), doi: 10.23812/CONTI-E [Epub ahead of print], both incorporated herein by reference). In some embodiments, the interleukin is interleukin 38 (formerly known as IL-1HY2, and described by, e.g., Xu, et al., June 2018, Frontiers in Immunology vol. 9, article. 1462, incorporated herein by reference). In some embodiments, the gene of interest encodes a decoy protein. In some embodiments the decoy protein is a soluble form of the virus host cell receptor. In some embodiments, the decoy protein is soluble ACE2 receptor. In some embodiments, the gene of interest encodes a protein selected from: a Type I interferon, a Type II interferon, a Type III interferon, an interleukin, and a decoy protein. In some embodiments, the gene of interest encodes a protein selected from: an IFN-α, e.g., interferon alpha-n3, interferon alpha-2a, or interferon alpha-2b, an IFN-β, an IFN-δ, an IFN-ε, an IFN-κ, an IFN-ν, an IFN-τ, an IFN-ω, an IFN-γ, an IFN-λ, IL-37, IL-38, and soluble ACE2 receptor.

Target Motif

In some embodiments, the compositions described herein comprise a recombinant polynucleic acid or an RNA construct comprising a target motif. The term “target motif” or “targeting motif” as used herein can refer to any short peptide present in the newly synthesized polypeptides or proteins that are destined to any parts of cell membranes, extracellular compartments, or intracellular compartments. Intracellular compartments include, but are not limited to, intracellular organelles such as nucleus, nucleolus, endosome, proteasome, ribosome, chromatin, nuclear envelope, nuclear pore, exosome, melanosome, Golgi apparatus, peroxisome, endoplasmic reticulum (ER), lysosome, centrosome, microtubule, mitochondria, chloroplast, microfilament, intermediate filament, or plasma membrane. Other terms include, but are not limited to, signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide. In some embodiments, the target motif is operably linked to the at least one nucleic acid sequence encoding the gene of interest. Non-limiting examples of the target motif comprise a signal peptide, a nuclear localization signal (NLS), a nucleolar localization signal (NoLS), a lysosomal targeting signal, a mitochondrial targeting signal, a peroxisomal targeting signal, a microtubule tip localization signal (MtLS), an endosomal targeting signal, a chloroplast targeting signal, a Golgi targeting signal, an endoplasmic reticulum (ER) targeting signal, a proteasomal targeting signal, a membrane targeting signal, a transmembrane targeting signal, a centrosomal localization signal (CLS) or any other signal that targets a protein to a certain part of cell membrane, extracellular compartments, or intracellular compartments.

In some embodiments, the target motif is selected from the group consisting of (a) a target motif heterologous to a protein encoded by the gene of interest; (b) a target motif heterologous to a protein encoded by the gene of interest, wherein the target motif heterologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; (c) a target motif homologous to a protein encoded by the gene of interest, wherein the target motif homologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; and (d) a naturally occurring amino acid sequence which does not have the function of a target motif in nature, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion, and/or substitution of at least one amino acid.

In some embodiments, the target motif is a signal peptide. In some embodiments, the signal peptide is selected from the group consisting of: (a) a signal peptide heterologous to a protein encoded by the gene of interest; (b) a signal peptide heterologous to a protein encoded by the gene of interest, wherein the signal peptide heterologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid, with proviso that the protein is not an oxidoreductase; (c) a signal peptide homologous to a protein encoded by the gene of interest, wherein the signal peptide homologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; and (d) a naturally occurring amino acid sequence which does not have the function of a signal peptide in nature, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion, and/or substitution of at least one amino acid. In some embodiments, the amino acids 1-9 of the N-terminal end of the signal peptide have an average hydrophobic score of above 2.

The term “target motif heterologous to a protein encoded by the gene of interest” or “signal peptide heterologous to a protein encoded by the gene of interest” as used herein refers to a naturally occurring target motif or signal peptide which is different to the naturally occurring target motif or signal peptide of the protein, i.e., the target motif or the signal peptide is not derived from the same gene of the protein. Usually a target motif or a signal peptide heterologous to a given protein is a target motif or a signal peptide from another protein, which is not related to the given protein i.e., which has an amino acid sequence which differs from the target motif or the signal peptide of the given protein, e.g., which has an amino acid sequence which differs from the target motif or the signal peptide of the given protein by more than 50%, preferably by more than 60%, more preferably by more than 70%, even more preferably by more than 80%, most preferably by more than 90%, or in particular by more than 95%. Preferably a target motif or a signal peptide heterologous to a given protein has a sequence identity with the amino acid sequence of the naturally occurring (homologous) target motif or signal peptide of the given protein of less than 95%, preferably less than 90%, more preferably less than 80%, even more preferably less than 70%, most preferably less than 60%, or in particular, less than 50%. Although heterologous sequences may be derived from the same organism, they naturally (in nature) do not occur in the same nucleic acid molecule, such as in the same mRNA. The target motif or the signal peptide heterologous to a protein and the protein to which the target motif or the signal peptide is heterologous can be of the same or different origin and are usually of the same origin, preferably of eukaryotic origin, more preferably of eukaryotic origin of the same eukaryotic organism, even more preferably of mammalian origin, in particular of mammalian origin of the same mammalian organism, or more particular of human origin. For example, a recombinant polynucleic acid or RNA construct comprising a nucleic acid sequence encoding the human BDNF signal peptide and the human IGF-1 gene, i.e., a signal peptide heterologous to a protein wherein the signal peptide and the protein are of the same origin, namely of human origin is disclosed.

The term “target motif homologous to a protein encoded by the gene of interest” or “signal peptide homologous to a protein encoded by the gene of interest” as used herein refers to the naturally occurring target motif or signal peptide of a protein. A target motif or a signal peptide homologous to a protein is the target motif or the signal peptide encoded by the gene of the protein as it occurs in nature. A target motif or a signal peptide homologous to a protein is usually of eukaryotic origin e.g., the naturally occurring target motif or signal peptide of a eukaryotic protein, preferably of mammalian origin e.g., the naturally occurring target motif or signal peptide of a mammalian protein, or more preferably of human origin e.g., the naturally occurring target motif or signal peptide of a human protein.

The term “naturally occurring amino acid sequence which does not have the function of a target motif in nature” or “naturally occurring amino acid sequence which does not have the function of a signal peptide in nature” as used herein refers to an amino acid sequence which occurs in nature and which is not identical to the amino acid sequence of any target motif or signal peptide occurring in nature. The naturally occurring amino acid sequence which does not have the function of a target motif or a signal peptide in nature as referred to in the present invention is preferably between 10-50, more preferably 11-45, even more preferably 12-45, most preferably 13-45, in particular 14-45, more particular 15-45, or even more particular 16-40 amino acids long. Preferably the naturally occurring amino acid sequence which does not have the function of a target motif or a signal peptide in nature of the present invention is of eukaryotic origin and not identical to any target motif or signal peptide of eukaryotic origin, more preferably is of mammalian origin and not identical to any target motif or signal peptide of mammalian origin, or more preferably is of human origin and not identical to any target motif or signal peptide of human origin occurring in nature. A naturally occurring amino acid sequence which does not have the function of a target motif or a signal peptide in nature is usually an amino acid sequence of the coding sequence of a protein. A naturally occurring amino acid sequence which does not have the function of a target motif or a signal peptide in nature according to the present invention is usually of eukaryotic origin, preferably of mammalian origin, or more preferably of human origin. The term “naturally occurring,” “natural,” and “in nature” as used herein have the equivalent meaning.

The term “amino acids 1-9 of the N-terminal end of the signal peptide” as used herein refers to the first nine amino acids of the N-terminal end of the amino acid sequence of a signal peptide. Analogously the term “amino acids 1-7 of the N-terminal end of the signal peptide” as used herein refers to the first seven amino acids of the N-terminal end of the amino acid sequence of a signal peptide and the term “amino acids 1-5 of the N-terminal end of the signal peptide” as used herein refers to the first five amino acids of the N-terminal end of the amino acid sequence of a signal peptide.

The term “amino acid sequence modified by insertion, deletion, and/or substitution of at least one amino acid” as used herein refers to an amino acid sequence which includes an amino acid substitution, insertion, and/or deletion of at least one amino acid within the amino acid sequence. The term “target motif heterologous to a protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid” or “signal peptide heterologous to a protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid” as used herein refers to an amino acid sequence of a naturally occurring target motif or signal peptide heterologous to a protein which includes an amino acid substitution, insertion, and/or deletion of at least one amino acid within its naturally occurring amino acid sequence. The term “target motif homologous to a protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid” or “signal peptide homologous to a protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid” as used herein refers to a naturally occurring target motif or signal peptide homologous to a protein which includes an amino acid substitution, insertion, and/or deletion of at least one amino acid within its naturally occurring amino acid sequence. The term “the naturally occurring amino acid sequence is modified by insertion, deletion, and/or substitution of at least one amino acid” refers to a naturally occurring amino acid sequence which includes an amino acid substitution, insertion, and/or deletion of at least one amino acid within its naturally occurring amino acid sequence. By “amino acid substitution” or “substitution” herein may refer to the replacement of an amino acid at a particular position in a parent protein sequence with another amino acid. For example, the substitution R34K refers to a polypeptide, in which the arginine at position 34 is replaced with a lysine. For the preceding example, 34K indicates the substitution of an amino acid at position 34 with a lysine. For the purposes herein, multiple substitutions are typically separated by a slash. For example, R34K/L78V refers to a double variant comprising the substitutions R34K and L38V. By “amino acid insertion” or “insertion” as used herein may refer to the addition of an amino acid at a particular position in a parent protein sequence. For example, insert −34 designates an insertion at position 34. By “amino acid deletion” or “deletion” as used herein may refer to the removal of an amino acid at a particular position in a parent protein sequence. For example, R34-designates the deletion of arginine at position 34.

Preferably the deleted amino acid is an amino acid with a hydrophobic score of below −0.8, preferably below 1.9. Preferably the substitute amino acid is an amino acid with a hydrophobic score which is higher than the hydrophobic score of the substituted amino acid, more preferably the substitute amino acid is an amino acid with a hydrophobic score of 2.8 and higher, or more preferably with a hydrophobic score of 3.8 and higher. Preferably the inserted amino acid is an amino acid with a hydrophobic score of 2.8 and higher, or more preferably with a hydrophobic score of 3.8 and higher.

Usually between 1 and 15, preferably between 1 and 11 amino acids, more preferably between 1 and 10 amino acids, even more preferably between 1 and 9 amino acids, in particular between 1 and 8 amino acids, more particular between 1 and 7 amino acids, even more particular between 1 and 6 amino acids, particular preferably between 1 and 5 amino acids, more particular preferably between 1 and 4 amino acids, or even more particular preferably between 1 and 2 amino acids in a given amino acid sequence are inserted, deleted, and/or substituted. Usually between 1 and 15, preferably between 1 and 11 amino acids, more preferably between 1 and 10 amino acids, even more preferably between 1 and 9 amino acids, in particular between 1 and 8 amino acids, more particular between 1 and 7 amino acids, even more particular between 1 and 6 amino acids, particular preferably between 1 and 5 amino acids, more particular preferably between 1 and 4 amino acids, or even more particular preferably between 1 and 2 amino acids in a given amino acid sequence are inserted, deleted, and/or substituted usually within the amino acids 1-11, preferably within the amino acids 1-10, more preferably within the amino acids 1-9, even more preferably within the amino acids 1-8, in particular within the amino acids 1-7, more particular within the amino acids 1-6, even more particular within the amino acids 1-5, particular preferably within the amino acids 1-4, more particular preferably within the amino acids 1-3, or even more particular preferably within the amino acids 1-2 of the N-terminal end of the amino acid sequence of the target motif or the signal peptide. Preferably the amino acid sequence is optionally modified by deletion, and/or substitution of at least one amino acid.

Preferably, the average hydrophobic score of the first nine amino acids of the N-terminal end of the amino acid sequence of the modified signal peptide is increased 1.0 unit or above compared to the signal peptide without modification.

The term “insulin-like growth factor 1,” “insulin-like growth factor 1 (IGF1 or IGF-1),” “IGF1,” or “IGF-1” as used herein usually refers to the natural sequence of the IGF-1 protein without the signal peptide and may comprise the propeptide and/or the E-peptide and preferably refers to the natural sequence of the IGF-1 protein without the signal peptide and without the E-peptide. The term “human insulin-like growth factor 1 (IGF-1)” as used herein refers to the natural sequence of human IGF-1 (pro-IGF-1 which is referred to in the Uniprot database as UniProtKB—P05019 and in the Genbank database as NM_000618.4, NM_001111285.2 and NM_001111283.2, or a fragment thereof. The natural DNA sequence encoding human insulin-like growth factor 1 may be codon-optimized. The natural sequence of human IGF-1 consists of the human signal peptide having 21 amino acids (nucleotides 1-63), the human propeptide (also called pro-domain) having 27 amino acids (nucleotides 64-144), the mature human IGF-1 having 70 amino acids (nucleotides 145-354) and the C-terminal domain of human IGF-1 which is the so-called E-peptide (or E-domain). The C-terminal domain of human IGF-1 (so called E-peptide or E-domain) comprises the Ea-, Eb-, or Ec-domain which are generated by alternative splicing events. The Ea-domain consists or 35 amino acids (105 nucleotides), the Eb-domain consists of 77 amino acids (231 nucleotides), and the Ec-domain consists of 40 amino acids (120 nucleotides) (see e.g., Wallis M (2009) New insulin-like growth factor (IGF)-precursor sequences from mammalian genomes: the molecular evolution of IGFs and associated peptides in primates. Growth Horm IGF Res 19(1):12-23. doi: 10.1016/j.ghir.2008.05.001). The term “human insulin-like growth factor 1 (IGF-1)” as used herein usually refers to the natural sequence of the human IGF-1 protein without the signal peptide and may comprise the propeptide and/or the E-peptide and preferably refers to the natural sequence of the human IGF-1 protein without the signal peptide and without the E-peptide. The term “human insulin-like growth factor 1 (IGF-1)” as used herein usually comprises the mature human IGF-1. The term “mature protein” refers to the protein synthesized in the endoplasmic reticulum and secreted via the Golgi apparatus in a cell expressing and

secreting the protein. The term “mature IGF-1” refers to the protein synthesized in the endoplasmic reticulum and secreted via the Golgi apparatus in a cell expressing and secreting IGF-1. The term “mature human IGF-1” refers to the protein synthesized in the endoplasmic reticulum and secreted via the Golgi apparatus in a human cell expressing and secreting human IGF-1 and normally contains the amino acids encoded by nucleotide as shown in SEQ ID NO: 19.

SEQ ID NO: 19

GGACCTGAGACACTTTGTGGCGCTGAACTGGTGGACGCCCTGCAGTT

TGTGTGTGGCGACAGAGGCTTCTACTTCAACAAGCCCACAGGCTACG

GCAGCAGCTCTAGAAGGGCTCCTCAGACCGGAATCGTGGACGAGTGC

TGTTTCAGAAGCTGCGACCTGCGGCGGCTGGAAATGTATTGTGCCCC

TCTGAAGCCTGCCAAGAGCGCC

The term “signal peptide of the Insulin growth factor 1 (IGF-1) Modified,” “modified signal peptide of IGF-1,” or “signal peptide of IGF-1-Modified” as used herein refers to the modified signal peptide of IGF-1 wherein natural signal peptide of IGF-1 which is referred to in the Uniprot database as P05019 and in the Genbank database as NM_000618.4, NM_001111284.1 and NM_001111285.2 is modified by the substitutions G2L/S5L/T9L/Q10L and deletions K3- and C15- and has preferably the amino acid sequence as shown in SEQ ID NO: 20 and/or is preferably encoded by the DNA sequence as shown in SEQ ID NO: 21.

SEQ ID NO: 20

Met-Leu-Ile-Leu-Leu-Leu-Pro-Leu-Leu-Leu-Phe-

Lys-Cys-Phe-Cys-Asp-Phe-Leu-Lys

SEQ ID NO: 21

ATGCTGATTCTGCTGCTGCCCCTGCTGCTGTTCAAGTGCTTCTGCGA

CTTCCTGAAA

The term “Insulin growth factor 1 (IGF-1) pro domain modified,” “modified IGF-1 pro domain,” or “IGF-1-Pro-Modified” as used herein refers to the pro-peptide of IGF-1 which is a naturally occurring amino acid sequence which does not have the function of a signal peptide in nature which is referred to in the Uniprot database as P05019 and in the Genbank database as NM_000618.4, NM_001111284.1 and NM_001111285.2 is modified by deletion of ten amino acid residues (VKMHTMSSSH (SEQ ID NO: 198)) flanking 22-31 in the N-terminal end of pro peptide and has preferably the amino acid sequence as shown in SEQ ID NO: 22 and/or is preferably encoded by the DNA sequence as shown in SEQ ID NO: 23.

SEQ ID NO: 22

Met-Leu-Phe-Tyr-Leu-Ala-Leu-Cys-Leu-Leu-Thr-

Phe-Thr-Ser-Ser-Ala-Thr-Ala

SEQ ID NO: 23

ATGCTGTTCTATCTGGCCCTGTGCCTGCTGACCTTTACCAGCTCTGC

TACCGCC

The term “the mRNA comprises a nucleic acid sequence encoding the propeptide of IGF-1, and a nucleic acid sequence encoding the mature IGF-1 and does not comprise a nucleic acid sequence encoding an E-peptide of IGF-1” as used herein refers usually to a mRNA which comprises a nucleotide sequence encoding the propeptide (also called pro-domain) of human IGF-1 having 27 amino acids, and a nucleotide sequence encoding the mature human IGF-1 having 70 amino acids and which does not comprise a nucleotide sequence encoding an E-peptide (also called E-domain) of human IGF-1 i.e., does not comprise a nucleotide sequence encoding a Ea-, Eb-, or Ec-domain. The nucleotide sequence encoding the propeptide (also called pro-domain) of human IGF-1 having 27 amino acids, and the nucleotide sequence encoding the mature human IGF-1 having 70 amino acids may be codon-optimized.

The term “hydrophobic score” or “hydrophobicity score” is used synonymously to the term “hydropathy score” herein and refers to the degree of hydrophobicity of an amino acid as calculated according to the Kyte-Doolittle scale (Kyte J., Doolittle R. F.; J. Mol. Biol. 157:105-132(1982)). The amino acid hydrophobic scores according to the Kyte-Doolittle scale are as follows:

Amino Acid
One Letter Code
Hydrophobic Score

Isoleucine
I
4.5

Valine
V
4.2

Leucine
L
3.8

Phenylalanine
F
2.8

Cysteine
C
2.5

Methionine
M
1.9

Alanine
A
1.8

Glycine
G
−0.4

Threonine
T
−0.7

Serine
S
−0.8

Tryptophan
W
−0.9

Tyrosine
Y
−1.3

Proline
P
−1.6

Histidine
H
−3.2

Glutamic acid
E
−3.5

Glutamine
Q
−3.5

Aspartic acid
D
−3.5

Asparagine
N
−3.5

Lysine
K
−3.9

Arginine
R
−4.5

The “average hydrophobic score” of an amino acid sequence e.g., the average hydrophobic score of the amino acids 1-9 of the N-terminal end of the amino acid sequence of a signal peptide is calculated by adding the hydrophobic score according to the Kyte-Doolittle scale of each of the amino acid of the amino acid sequence e.g., the hydrophobic score of each of the nine amino acids of the amino acids 1-9 of the N-terminal end, divided by the number of the amino acids, e.g., divided by nine.

The polarity is calculated according to Zimmerman Polarity index (Zimmerman J. M., Eliezer N., Simha R.; J. Theor. Biol. 21:170-201(1968)). The “average polarity” of an amino acid sequence e.g., the average polarity of the amino acids 1-9 of the N-terminal end of the amino acid sequence of a signal peptide is calculated by adding the polarity value calculated according to Zimmerman Polarity index of each of the amino acid of the amino acid sequence e.g., the average polarity of each of the nine amino acids of the amino acids 1-9 of the N-terminal end, divided by the number of the amino acids, e.g., divided by nine. The polarity of amino acids according to Zimmerman Polarity index is as follows:

Amino Acid
One Letter Code
Polarity

Isoleucine
I
0.13

Valine
V
0.13

Leucine
L
0.13

Phenylalanine
F
0.35

Cysteine
C
1.48

Methionine
M
1.43

Alanine
A
0

Glycine
G
0

Threonine
T
1.66

Serine
S
1.67

Tryptophan
W
2.1

Tyrosine
Y
1.61

Proline
P
1.58

Histidine
H
51.6

Glutamic acid
E
49.9

Glutamine
Q
3.53

Aspartic acid
D
49.7

Asparagine
N
3.38

Lysine
K
49.5

Arginine
R
52

Disease and Treatment

In some aspects, provided herein, is a cell comprising the composition of any recombinant polynucleic acid or RNA constructs described herein. In some aspects, provided herein, is a pharmaceutical composition comprising the composition of any recombinant polynucleic acid or RNA constructs described herein and a pharmaceutically acceptable excipient. Pharmaceutical compositions can be formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compounds into preparations that can be used pharmaceutically. A proper formulation is dependent upon the route of administration chosen and a summary of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), herein incorporated by reference. In some embodiments, the pharmaceutical composition facilitates administration of the compound to an organism.

In some aspects, provided herein, is the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct described herein for use a medicament. In some aspects, provided herein, is a method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, described herein. In some aspects, provided herein, is the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct described herein for use in a method of treating a disease or a condition in a subject in need thereof. In some aspects, provided herein, is the use of the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct described herein for the manufacture of a medicament for treating a disease or a condition in a subject in need thereof. In some embodiments, the disease or the condition is selected from the group consisting of SARS (severe acute respiratory syndrome), acute respiratory distress syndrome (ARDS), venous thromboembolism, cardiovascular complications, acute kidney injury, acute liver injury, neurologic complications, cytokine release syndrome, pediatric multisystem inflammatory syndrome, septic shock, disseminated intravascular coagulation, acute respiratory failure, and any combination thereof, intervertebral disc disease (IVDD), osteoarthritis, psoriasis, fibrodysplasia ossificans progressiva (FOP), and amyotrophic lateral sclerosis (ALS). In some embodiments, the disease or the condition is selected from the group consisting of SARS (severe acute respiratory syndrome), acute respiratory distress syndrome (ARDS), venous thromboembolism, cardiovascular complications, acute kidney injury, acute liver injury, neurologic complications, cytokine release syndrome, pediatric multisystem inflammatory syndrome, septic shock, disseminated intravascular coagulation, acute respiratory failure, and any combination thereof, intervertebral disc disease (IVDD), osteoarthritis, and psoriasis. In some embodiments, the disease or the condition is selected from the group consisting of SARS (severe acute respiratory syndrome), intervertebral disc disease (IVDD), osteoarthritis, psoriasis, fibrodysplasia ossificans progressiva (FOP), and amyotrophic lateral sclerosis (ALS). In some embodiments, the disease or the condition is selected from the group consisting of SARS (severe acute respiratory syndrome), intervertebral disc disease (IVDD), osteoarthritis, and psoriasis.

In some embodiments, the disease or the condition is selected from the group consisting of intervertebral disc disease (IVDD), osteoarthritis, psoriasis, fibrodysplasia ossificans progressiva (FOP), and amyotrophic lateral sclerosis (ALS). In some embodiments, the disease or the condition is selected from the group consisting of intervertebral disc disease (IVDD), osteoarthritis, and psoriasis. In some embodiments, the disease or condition comprises a skin disease or condition. In some embodiments, the skin disease or condition comprises an inflammatory skin disorder. In some embodiments, an inflammatory skin disorder comprises psoriasis. In some embodiments, the disease or condition comprises a muscular disease or condition. In some embodiments, the muscular disease or condition comprises a skeletal muscle disorder. In some embodiments, the skeletal muscle disorder comprises fibrodysplasia ossificans progressiva (FOP). In some embodiments, the disease or condition comprises a neurodegenerative disease or condition. In some embodiments, the neurodegenerative disease or condition comprises a motor neuron disorder. In some embodiments, the motor neuron disorder comprises amyotrophic lateral sclerosis (ALS). In some embodiments, the disease or condition comprises a joint disease or condition. In some embodiments, the joint disease or condition comprises a joint degeneration. In some embodiments, the joint degeneration comprises intervertebral disc disease (IVDD) or osteoarthritis (OA).

Intervertebral disc disease (IVDD) is a condition that is estimated to affect about 5% of the population in developed countries each year and characterized by the degeneration of one or more of the discs that separate each vertebra of the spine. The intervertebral discs provide cushioning between vertebrae and absorb pressure put on the spine. Although discs in the lower region of the spine are most often affected in IVDD, any part of the spine can have disc degeneration and thus, this condition causes pain in the back, neck, legs, and arms. Also, depending on the location of the affected disc or discs, IVDD can cause periodic or chronic pain, which can be worse when sitting, bending, twisting, or lifting object. IVDD results from a combination of genetic and environmental factors, most of which remain unknown. Several genes have been identified to have variations that may influence the risk of developing IVDD and these include genes associated with collagen, immune function, and proteins that play roles in the development and maintenance of the intervertebral discs and vertebrae. Nongenetic factors include aging, smoking, obesity, chronic inflammation, and driving for a long period of time. Two of these genes are Insulin-like growth factor 1 (IGF-1) and its receptor (insulin-like growth factor 1 receptor, IGF-1R), which can regulate the extracellular matrix synthesis and play a crucial role in maintaining the normal functions of the intervertebral disc.

Osteoarthritis is a common disease of the joints, characterized by progressive degeneration of articular cartilage, causing pain, stiffness, and restricted movement as the condition gets worse. Areas of bone no longer cushioned by cartilage rub against each other and start to break down, causing further damage such as inflammation as the immune system attempts to repair and rebuild these tissues. In addition, osteophytes (or abnormal growths of bone and other tissue) can also occur and these may be visible as enlarged joints. It is thought that the balance of catabolism and anabolism is lost in osteoarthritis patients, leading to cartilage damage and complete breakdown. The genes of which expression affects osteoarthritis risk are typically involved in the formation and maintenance of bone and cartilage.

In both IVDD and osteoarthritis, decreasing inflammation (e.g., decreasing IL-1 beta, IL-8, etc.) while increasing anabolic signal (e.g., IGF-1, etc.) could have a therapeutic effect. In some aspects, provided herein, is a method of treating intervertebral disc disease (IVDD) in a subject, the method comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, comprising siRNA capable of binding to a target mRNA and an mRNA encoding a gene of interest. In a preferred embodiment, the siRNA is capable of binding to IL-1 beta mRNA. In another preferred embodiment, the siRNA is capable of binding to IL-8 mRNA. In a preferred embodiment, the mRNA encoding the gene of interest encodes IGF-1.

In some aspects, provided herein, is a method of treating a joint disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, comprising siRNA capable of binding to IL-1 beta mRNA and an mRNA encoding IGF-1. In some aspects, provided herein, is a method of treating a joint disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence encoding the siRNA capable of binding to IL-1 beta mRNA and a nucleic acid encoding IGF-1. In some aspects, provided herein, is a method of treating a joint disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant RNA construct comprising siRNA capable of binding to IL-1 beta mRNA and an mRNA encoding IGF-1. In some embodiments, the joint disease or condition is a joint degeneration. In some embodiments, the joint degeneration is intervertebral disc disease (IVDD) or osteoarthritis (OA).

In some aspects, provided herein, is a method of treating intervertebral disc disease (IVDD) in a subject, the method comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, comprising siRNA capable of binding to IL-1 beta mRNA and an mRNA encoding IGF-1. In some aspects, provided herein, is a method of treating intervertebral disc disease (IVDD) in a subject, the method comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, comprising siRNA capable of binding to IL-8 mRNA and an mRNA encoding IGF-1.

In some aspects, provided herein, is a method of treating intervertebral disc disease (IVDD) in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence encoding the siRNA capable of binding to IL-1 beta mRNA and a nucleic acid encoding IGF-1. In some aspects, provided herein, is a method of treating intervertebral disc disease (IVDD) in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence encoding the siRNA capable of binding to IL-8 mRNA and a nucleic acid sequence encoding IGF-1.

In some aspects, provided herein, is a method of treating intervertebral disc disease (IVDD) in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant RNA construct comprising siRNA capable of binding to IL-1 beta mRNA and an mRNA encoding IGF-1. In some aspects, provided herein, is a method of treating intervertebral disc disease (IVDD) in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant RNA construct comprising siRNA capable of binding to IL-8 mRNA and an mRNA encoding IGF-1.

In some aspects, provided herein, is a method of treating osteoarthritis in a subject, the method comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, comprising siRNA capable of binding to a target mRNA and an mRNA encoding a gene of interest. In a preferred embodiment, the siRNA is capable of binding to IL-1 beta mRNA. In another preferred embodiment, the siRNA is capable of binding to IL-8 mRNA. In a preferred embodiment, the mRNA encoding the gene of interest encodes IGF-1.

In some aspects, provided herein, is a method of treating osteoarthritis in a subject, the method comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, comprising siRNA capable of binding to IL-1 beta mRNA and an mRNA encoding IGF-1. In some aspects, provided herein, is a method of treating osteoarthritis in a subject, the method comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, comprising siRNA capable of binding to IL-8 mRNA and an mRNA encoding IGF-1.

In some aspects, provided herein, is a method of treating osteoarthritis in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence encoding the siRNA capable of binding to IL-1 beta mRNA and a nucleic acid encoding IGF-1. In some aspects, provided herein, is a method of treating osteoarthritis in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence encoding the siRNA capable of binding to IL-8 mRNA and a nucleic acid sequence encoding IGF-1.

In some aspects, provided herein, is a method of treating osteoarthritis in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant RNA construct comprising siRNA capable of binding to IL-1 beta mRNA and an mRNA encoding IGF-1. In some aspects, provided herein, is a method of treating osteoarthritis in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant RNA construct comprising siRNA capable of binding to IL-8 mRNA and an mRNA encoding IGF-1.

Psoriasis is a chronic inflammatory skin disorder, characterized by patches of red, irritated skin that are often covered by flaky white scales. Psoriasis patients may also develop psoriatic arthritis, a condition involving joint inflammation. Although the exact cause of this disease is not currently understood, the disease is thought to be an autoimmune disease caused by an immune system problem with T cells (e.g., T cells attacking healthy skin cells) and other white blood cells, such as neutrophils.

In some aspects, provided herein, is a method of treating a skin disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, comprising siRNA capable of binding to IL-1 beta mRNA and an mRNA encoding IGF-1. In some aspects, provided herein, is a method of treating a joint disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence encoding the siRNA capable of binding to IL-1 beta mRNA and a nucleic acid encoding IGF-1. In some aspects, provided herein, is a method of treating a skin disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant RNA construct comprising siRNA capable of binding to IL-1 beta mRNA and an mRNA encoding IGF-1. In some embodiments, the skin disease or condition is an inflammatory skin disorder. In some embodiments, the inflammatory skin disorder is psoriasis.

In some aspects, provided herein, is a method of treating psoriasis in a subject, the method comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, comprising siRNA capable of binding to IL-17 mRNA and an mRNA encoding IL-4. In some aspects, provided herein, is a method of treating psoriasis in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence encoding the siRNA capable of binding to IL-17 mRNA and a nucleic acid encoding IL-4. In some aspects, provided herein, is a method of treating psoriasis in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant RNA construct comprising siRNA capable of binding to IL-17 mRNA and an mRNA encoding IL-4.

In some aspects, provided herein, is a method of treating psoriasis in a subject, the method comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, comprising siRNA capable of binding to TNF-alpha mRNA and an mRNA encoding IL-4. In some aspects, provided herein, is a method of treating psoriasis in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence encoding the siRNA capable of binding to TNF-alpha mRNA and a nucleic acid encoding IL-4. In some aspects, provided herein, is a method of treating psoriasis in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant RNA construct comprising siRNA capable of binding to TNF-alpha mRNA and an mRNA encoding IL-4.

Fibrodysplasia ossificans progressiva (FOP) is a skeletal muscle disorder in which muscle tissues and connective tissues such as tendons and ligaments are gradually ossified, forming extra-skeletal or heterotopic bones that constrains movement. The formation of extra-skeletal bone causes progressive loss of mobility as the joints become affected. Any trauma to the muscles of an individual with FOP such as a fall or an invasive medical procedure can trigger episodes of muscle swelling and inflammation followed by more rapid ossification of muscle and connective tissues in the injured area.

In some aspects, provided herein, is a method of treating a muscular disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, comprising siRNA capable of binding to ALK2 mRNA and an mRNA encoding IGF-1. In some aspects, provided herein, is a method of treating a muscular disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence encoding the siRNA capable of binding to ALK2 mRNA and a nucleic acid encoding IGF-1. In some aspects, provided herein, is a method of treating a muscular disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant RNA construct comprising siRNA capable of binding to ALK2 mRNA and an mRNA encoding IGF-1. In some embodiments, the muscular disease or condition is a skeletal muscle disorder. In some embodiments, the skeletal muscle disorder is fibrodysplasia ossificans progressiva (FOP).

Amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease, is a progressive neurodegenerative disease that affects nerve cells in the brain and spinal cord, causing loss of muscle. It is a motor neuron disease characterized by the degeneration of both upper and lower motor neurons, which leads to muscle weakness and eventual paralysis. The cause of ALS is not yet known, however, some biomarkers and genes associated with ALS, including Superoxide Dismutase 1 (SOD1), have been discovered. There are 2 types of ALS differentiated by genetics: familial and sporadic (idiopathic).

In some aspects, provided herein, is a method of treating a neurodegenerative disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, comprising siRNA capable of binding to SOD1 mRNA and an mRNA encoding IGF-1. In some aspects, provided herein, is a method of treating a neurodegenerative disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence encoding the siRNA capable of binding to SOD1 mRNA and a nucleic acid encoding IGF-1. In some aspects, provided herein, is a method of treating a neurodegenerative disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant RNA construct comprising siRNA capable of binding to SOD1 mRNA and an mRNA encoding IGF-1. In some embodiments, the neurodegenerative disease or condition is a motor neuron disorder. In some embodiments, the motor neuron disorder is amyotrophic lateral sclerosis (ALS).

In some aspects, provided herein, is a method of treating a neurodegenerative disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, comprising siRNA capable of binding to SOD1 mRNA and an mRNA encoding EPO. In some aspects, provided herein, is a method of treating a neurodegenerative disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence encoding the siRNA capable of binding to SOD1 mRNA and a nucleic acid encoding EPO. In some aspects, provided herein, is a method of treating a neurodegenerative disease or condition in a subject, the method comprising administering to the subject the pharmaceutical composition comprising a recombinant RNA construct comprising siRNA capable of binding to SOD1 mRNA and an mRNA encoding EPO. In some embodiments, the neurodegenerative disease or condition is a motor neuron disorder. In some embodiments, the motor neuron disorder is amyotrophic lateral sclerosis (ALS).

In some aspects, provided herein, is a method of treating a disease or a condition relating to infection with a coronavirus in a subject in need thereof, comprising administering to the subject the pharmaceutical composition, the cell, the recombinant polynucleic acid construct, or the recombinant RNA construct, described herein. In some embodiments, the disease or the condition is SARS (severe acute respiratory syndrome) caused by infection with a SARS-associated coronavirus. In some embodiments, the present invention is useful for treating a disease or condition caused by or associated with infection with a coronavirus, including but not limited to a complication of coronavirus infection. In some embodiments, the disease or condition is a respiratory syndrome, e.g., SARS (severe acute respiratory syndrome) caused by infection with a SARS-associated coronavirus. In some embodiments, the disease or condition is selected from, e.g., acute respiratory distress syndrome (ARDS), venous thromboembolism, cardiovascular complications, acute kidney injury, acute liver injury, neurologic complications, cytokine release syndrome, pediatric multisystem inflammatory syndrome, septic shock, disseminated intravascular coagulation, acute respiratory failure, and any combination thereof. In some embodiments, the disease or condition associated with coronavirus infection treated using the compositions or methods of the invention is any known to those of skill in the art and described in the literature. In some embodiments, the present invention is useful for treating such a disease or condition by parallel control and/or downregulation of a specific physiological mechanism by siRNA, and activation and/or increase of another physiological mechanism, e.g., inflammation, by overexpression of a therapeutic protein. In some embodiments, the coronavirus is SARS-CoV (also known as SARS-CoV-1; the virus responsible for 2002-2003 SARS epidemic), SARS-CoV-2 (the virus that causes novel coronavirus disease-2019, or COVID-19), or MERS-CoV (Middle East Respiratory Syndrome virus). In some embodiments, one or more of SARS-CoV, SARS-CoV-2, and MERS is treated using the present invention. These and related viruses are described by, e.g., Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, March 2020, Nature Microbiology 5:536-44), incorporated herein by reference.

In some aspects, the composition administered to the subject comprises a polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to an IL-6 mRNA; and (ii) an mRNA IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct comprises 1 siRNA directed to an IL-6 mRNA. In related aspects, the polynucleic acid construct comprises 3 siRNAs, each directed to an IL-6 mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 29 or 30 (Compound B1 or B2).

In some aspects, the composition administered to the subject comprises a polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to an Interleukin 6R (IL-6R) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the polynucleic acid construct comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct comprises 1 siRNA directed to an IL-6R mRNA. In related aspects, the polynucleic acid construct comprises 3 siRNAs, each directed to an IL-6R mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 31 (Compound B3).

In some aspects, the composition administered to the subject comprises a polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to an Interleukin 6R alpha (IL-6R-alpha) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the polynucleic acid construct comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct comprises 1 siRNA directed to an IL-6R-alpha mRNA. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, each directed to an IL-6R-alpha mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 32 (Compound B4).

In some aspects, the composition administered to the subject comprises a polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to an Interleukin 6R beta (IL-6R-beta) mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct encodes or comprises 1 siRNA directed to an IL-6R-beta mRNA. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, each directed to an IL-6R-beta mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 33 (Compound B5).

In some aspects, the composition administered to the subject comprises a polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to an ACE2 mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct encodes or comprises 1 siRNA directed to an ACE2 mRNA. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, each directed to an ACE2 mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 34 or 35 (Compound B6 or B7).

In some aspects, the composition administered to the subject comprises a recombinant polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to a SARS CoV-2 ORF1ab mRNA, at least one siRNA capable of binding to a SARS CoV-2 S mRNA, at least one siRNA capable of binding to a SARS CoV-2 N mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, one directed to a SARS CoV-2 ORF1ab mRNA, one directed to a SARS CoV-2 S mRNA, and one directed to a SARS CoV-2 N mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In certain aspects, such a composition, including a composition comprising Compound B8 (SEQ ID NO: 36) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, SARS CoV-2, or both. In related aspects, the polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 36.

In some aspects, the composition administered to the subject comprises a polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to a SARS CoV-2 ORF1ab mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises 1 siRNA directed to a SARS CoV-2 ORF1ab mRNA. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, each directed to a SARS CoV-2 ORF1ab mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In certain aspects, such a composition, including a composition comprising Compound B12 (SEQ ID NO: 40) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, MERS, or both. In certain aspects, such a composition, including a composition comprising Compound B13 (SEQ ID NO: 41) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, SARS CoV-2, and/or MERS. In related aspects, the polynucleic acid construct comprises a sequence as set forth in any one of SEQ ID NOs: 40, 41 and 42 (Compounds B12, B13 and B14).

In some aspects, the composition administered to the subject comprises a polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to an IL-6 mRNA, at least one siRNA capable of binding to an ACE2 mRNA, and at least one siRNA capable of binding to a SARS CoV-2 S mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, one directed to an IL-6 mRNA, one directed to an ACE2 mRNA, and one directed to a SARS CoV-2 S mRNA. In related aspects, the mRNA encoding IFN-beta encodes the native IFN-beta signal peptide, or a modified signal peptide. In related aspects, the modified IFN-beta signal peptide is SP1 or SP2 as described herein (SEQ ID NOs: 52 and 54, respectively). In related aspects, the polynucleic acid construct comprises a sequence as set forth in any one of SEQ ID NOs: 43, 44, and 45 (Compounds B15, B16, and B17).

In some aspects, the composition administered to the subject comprises a polynucleic acid construct encoding or comprising: (i) at least one small interfering RNA capable of binding to a SARS CoV-2 ORF1ab mRNA, at least one siRNA capable of binding to a SARS CoV-2 S mRNA, and at least one siRNA capable of binding to a SARS CoV-2 N mRNA; and (ii) an mRNA encoding an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, one directed to an ORF1ab mRNA, one directed to a SARS CoV-2 S mRNA, and one directed to a SARS CoV-2 N mRNA. In related aspects, the polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 46 (Compound B18). In related aspects, the polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 190 (Compound B18).

In some aspects, the composition administered to the subject comprises a polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to a SARS CoV-2 S mRNA; and (ii) an mRNA encoding an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct encodes or comprises 1 siRNA directed to a SARS CoV-2 S mRNA. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, each directed to a SARS CoV-2 S mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In related aspects, the recombinant polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 47 (Compound B19).

In some aspects, the present invention provides a composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 29-47. In some aspects, the present invention provides a composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence of SEQ ID NO: 190.

The compositions of the present invention can be administered to a subject using any suitable methods known in the art. Suitable formulations for use in the present invention and methods of delivery are generally well known in the art. For example, the compositions described herein can be administered to the subject in a variety of ways, including parenterally, intravenously, intradermally, intramuscularly, colonically, rectally, or intraperitoneally. In some embodiments, the compositions described herein is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject. In some embodiments, the compositions described herein can be administered parenterally, intravenously, intramuscularly or orally.

Any of the compositions of the present invention may be provided together with an instruction manual. The instruction manual may comprise guidance for the skilled person or attending physician how to treat (or prevent) a disease or a disorder as described herein (e.g., IVDD, osteoarthritis, psoriasis, or skeletal muscle injury) in accordance with the present invention. In some embodiments, the instruction manual may comprise guidance as to the herein described mode of delivery/administration and delivery/administration regimen, respectively (e.g., route of delivery/administration, dosage regimen, time of delivery/administration, frequency of delivery/administration, etc.). In some embodiments, the instruction manual may comprise the instruction that how the composition of the present invention is to be administrated or injected and/or is prepared for administration or injection. In principle, what has been described herein elsewhere with respect to the mode of delivery/administration and delivery/administration regimen, respectively, may be comprised as respective instructions in the instruction manual.

The composition of the present invention can be used in a gene therapy. In certain some embodiments, the composition comprising the recombinant polynucleic acid or RNA construct described herein can be delivered to a cell in gene therapy vectors. Gene therapy vectors and methods of gene delivery are well known in the art. Non-limiting examples of these methods include viral vector delivery systems including DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell, non-viral vector delivery systems including DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, transposon system (for delivery and integration into the host genomes; Moriarity, et al. (2013) Nucleic Acids Res 41(8), e92, Aronovich, et al., (2011) Hum. Mol. Genet. 20(R1), R14-R20), retrovirus-mediated DNA transfer (e.g., Moloney Mouse Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus; see e.g., Kay et al. (1993) Science 262, 117-119, Anderson (1992) Science 256, 808-813), and DNA virus-mediated DNA transfer including adenovirus, herpes virus, parvovirus and adeno-associated virus (e.g., Ali et al. (1994) Gene Therapy 1, 367-384). Viral vectors also include but are not limited to adeno-associated virus, adenoviral virus, lentivirus, retroviral, and herpes simplex virus vectors. Vectors capable of integration in the host genome include but are not limited to retrovirus or lentivirus.

In some embodiments, the composition comprising the recombinant polynucleic acid or RNA construct described herein can be delivered to a cell via direct DNA transfer (Wolff et al. (1990) Science 247, 1465-1468). The recombinant polynucleic acid or RNA construct can be delivered to cells following mild mechanical disruption of the cell membrane, temporarily permeabilizing the cells. Such a mild mechanical disruption of the membrane can be accomplished by gently forcing cells through a small aperture (Sharei et al. PLOS ONE (2015) 10(4), e0118803). In another embodiment, the composition comprising the recombinant polynucleic acid or RNA construct described herein can be delivered to a cell via liposome-mediated DNA transfer (e.g., Gao & Huang (1991) Biochem. Biophys. Res. Comm. 179, 280-285, Crystal (1995) Nature Med. 1, 15-17, Caplen et al. (1995) Nature Med. 3, 39-46). The term “liposome” can encompass a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. The recombinant polynucleic acid or RNA construct can be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, or complexed with a liposome.

Modulation of Gene Expression

In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target messenger RNA (mRNA); and (ii) at least one nucleic acid sequence encoding a gene of interest; wherein the target mRNA is different from an mRNA encoded by the gene of interest, and wherein the expression of the target mRNA and the gene of interest is modulated simultaneously.

In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to an IL-1 beta mRNA; and (ii) at least one nucleic acid sequence encoding IGF-1; wherein the expression of the IL-1 beta mRNA and the IGF-1 is modulated simultaneously.

In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to an IL-8 mRNA; and (ii) at least one nucleic acid sequence encoding IGF-1; wherein the expression of the IL-8 mRNA and the IGF-1 is modulated simultaneously.

In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to an IL-17 mRNA; and (ii) at least one nucleic acid sequence encoding IL-4; wherein the expression of the IL-17 mRNA and the IL-4 is modulated simultaneously.

In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a TNF-alpha mRNA; and (ii) at least one nucleic acid sequence encoding IL-4; wherein the expression of the TNF-alpha mRNA and the IL-4 is modulated simultaneously.

In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a TNF-alpha mRNA and at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a IL-17 mRNA; and (ii) at least one nucleic acid sequence encoding IL-4; wherein the expression of the TNF-alpha mRNA, the IL-17 mRNA and the IL-4 is modulated simultaneously.

In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to an ALK2 mRNA; and (ii) at least one nucleic acid sequence encoding IGF-1; wherein the expression of the ALK2 mRNA and the IGF-1 is modulated simultaneously.

In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a SOD1 mRNA; and (ii) at least one nucleic acid sequence encoding IGF-1; wherein the expression of the SOD1 mRNA and the IGF-1 is modulated simultaneously.

In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a SOD1 mRNA; and (ii) at least one nucleic acid sequence encoding EPO; wherein the expression of the SOD1 mRNA and the EPO is modulated simultaneously.

In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct encoding or comprising: (i) at least one small interfering RNA (siRNA) capable of binding to a SARS CoV-2 ORF1ab mRNA, at least one siRNA capable of binding to a SARS CoV-2 S mRNA, at least one siRNA capable of binding to a SARS CoV-2 N mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, one directed to a SARS CoV-2 ORF1ab mRNA, one directed to a SARS CoV-2 S mRNA, and one directed to a SARS CoV-2 N mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In certain aspects, such a composition, including a composition comprising Compound B8 (SEQ ID NO: 36) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, SARS CoV-2, or both. In related aspects, the polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 36.

In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to a SARS CoV-2 ORF1ab mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises 1 siRNA directed to a SARS CoV-2 ORF1ab mRNA. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, each directed to a SARS CoV-2 ORF1ab mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In certain aspects, such a composition, including a composition comprising Compound B12 (SEQ ID NO: 40) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, MERS, or both. In certain aspects, such a composition, including a composition comprising Compound B13 (SEQ ID NO: 41) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, SARS CoV-2, and/or MERS. In related aspects, the polynucleic acid construct comprises a sequence as set forth in any one of SEQ ID NOs: 40, 41 and 42 (Compounds B12, B13 and B14).

In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to an IL-6 mRNA, at least one siRNA capable of binding to an ACE2 mRNA, and at least one siRNA capable of binding to a SARS CoV-2 S mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, one directed to an IL-6 mRNA, one directed to an ACE2 mRNA, and one directed to a SARS CoV-2 S mRNA. In related aspects, the mRNA encoding IFN-beta encodes the native IFN-beta signal peptide, or a modified signal peptide. In related aspects, the modified IFN-beta signal peptide is SP1 or SP2 as described herein (SEQ ID NOs: 52 and 54, respectively). In related aspects, the polynucleic acid construct comprises a sequence as set forth in any one of SEQ ID NOs: 43, 44, and 45 (Compounds B15, B16, and B17).

In some aspects, provided herein, is a method of simultaneously modulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct encoding or comprising: (i) at least one small interfering RNA capable of binding to a SARS CoV-2 ORF1ab mRNA, at least one siRNA capable of binding to a SARS CoV-2 S mRNA, and at least one siRNA capable of binding to a SARS CoV-2 N mRNA; and (ii) an mRNA encoding an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, one directed to an ORF1ab mRNA, one directed to a SARS CoV-2 S mRNA, and one directed to a SARS CoV-2 N mRNA. In related aspects, the polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 46 (Compound B18). In related aspects, the polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 190 (Compound B18).

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target messenger RNA (mRNA); and (ii) at least one nucleic acid sequence encoding a gene of interest; wherein the target mRNA is different from an mRNA encoded by the gene of interest, and wherein the expression of the target mRNA is downregulated and the expression of the gene of interest is upregulated simultaneously. In some embodiments, the expression of the target mRNA is downregulated by the siRNA capable of binding to the target mRNA. In some embodiments, the expression of the gene of interest is upregulated by expressing or overexpressing an mRNA or a protein encoded by the gene of interest.

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to an IL-1 beta mRNA; and (ii) at least one nucleic acid sequence encoding IGF-1; wherein the expression of the IL-1 beta mRNA is downregulated and the expression of IGF-1 is upregulated simultaneously. In some embodiments, the expression of the IL-1 beta mRNA is downregulated by the siRNA capable of binding to the IL-1 beta mRNA. In some embodiments, the expression of IGF-1 is upregulated by expressing or overexpressing an IGF-1 mRNA or an IGF-1 protein.

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to an IL-8 mRNA; and (ii) at least one nucleic acid sequence encoding IGF-1; wherein the expression of the IL-8 mRNA is downregulated and the expression of IGF-1 is upregulated simultaneously. In some embodiments, the expression of the IL-8 mRNA is downregulated by the siRNA capable of binding to the IL-8 mRNA. In some embodiments, the expression of IGF-1 is upregulated by expressing or overexpressing an IGF-1 mRNA or an IGF-1 protein.

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to an IL-17 mRNA; and (ii) at least one nucleic acid sequence encoding IL-4; wherein the expression of the IL-17 mRNA is downregulated and the expression of IL-4 is upregulated simultaneously. In some embodiments, the expression of the IL-17 mRNA is downregulated by the siRNA capable of binding to the IL-17 mRNA. In some embodiments, the expression of IL-4 is upregulated by expressing or overexpressing an IL-4 mRNA or an IL-4 protein.

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a TNF-alpha mRNA; and (ii) at least one nucleic acid sequence encoding IL-4; wherein the expression of the TNF-alpha mRNA is downregulated and the expression of IL-4 is upregulated simultaneously. In some embodiments, the expression of the TNF-alpha mRNA is downregulated by the siRNA capable of binding to the TNF-alpha mRNA. In some embodiments, the expression of IL-4 is upregulated by expressing or overexpressing an IL-4 mRNA or an IL-4 protein.

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a TNF-alpha mRNA and at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a IL-17 mRNA; and (ii) at least one nucleic acid sequence encoding IL-4; wherein the expression of the TNF-alpha mRNA and/or the expression of the IL-17 mRNA is downregulated and the expression of IL-4 is upregulated simultaneously. In some embodiments, the expression of the TNF-alpha mRNA and the expression of the IL-17 mRNA is downregulated by the siRNA capable of binding to the TNF-alpha mRNA and the siRNA capable of binding to the IL-17 mRNA. In some embodiments, the expression of IL-4 is upregulated by expressing or overexpressing an IL-4 mRNA or an IL-4 protein.

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to an ALK2 mRNA; and (ii) at least one nucleic acid sequence encoding IGF-1; wherein the expression of the ALK2 mRNA is downregulated and the expression of IGF-1 is upregulated simultaneously. In some embodiments, the expression of the ALK2 mRNA is downregulated by the siRNA capable of binding to the ALK2 mRNA. In some embodiments, the expression of IGF-1 is upregulated by expressing or overexpressing an IGF-1 mRNA or an IGF-1 protein.

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a SOD1 mRNA; and (ii) at least one nucleic acid sequence encoding IGF-1; wherein the expression of the SOD1 mRNA is downregulated and the expression of IGF-1 is upregulated simultaneously. In some embodiments, the expression of the SOD1 mRNA is downregulated by the siRNA capable of binding to the SOD1 mRNA. In some embodiments, the expression of IGF-1 is upregulated by expressing or overexpressing an IGF-1 mRNA or an IGF-1 protein.

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct comprising: (i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a SOD1 mRNA; and (ii) at least one nucleic acid sequence encoding EPO; wherein the expression of the SOD1 mRNA is downregulated and the expression of EPO is upregulated simultaneously. In some embodiments, the expression of the SOD1 mRNA is downregulated by the siRNA capable of binding to the SOD1 mRNA. In some embodiments, the expression of EPO is upregulated by expressing or overexpressing an EPO mRNA or an EPO protein.

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct encoding or comprising: (i) at least one small interfering RNA (siRNA) capable of binding to a SARS CoV-2 ORF1ab mRNA, at least one siRNA capable of binding to a SARS CoV-2 S mRNA, at least one siRNA capable of binding to a SARS CoV-2 N mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, one directed to a SARS CoV-2 ORF1ab mRNA, one directed to a SARS CoV-2 S mRNA, and one directed to a SARS CoV-2 N mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In certain aspects, such a composition, including a composition comprising Compound B8 (SEQ ID NO: 36) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, SARS CoV-2, or both. In related aspects, the polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 36.

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to a SARS CoV-2 ORF1ab mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises 1 siRNA directed to a SARS CoV-2 ORF1ab mRNA. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, each directed to a SARS CoV-2 ORF1ab mRNA. In related aspects, each of the at least 3 siRNAs is the same, different, or a combination thereof. In certain aspects, such a composition, including a composition comprising Compound B12 (SEQ ID NO: 40) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, MERS, or both. In certain aspects, such a composition, including a composition comprising Compound B13 (SEQ ID NO: 41) is contemplated for use in methods described herein, e.g., for modulating or regulating gene expression in relation to infection with SARS CoV, SARS CoV-2, and/or MERS. In related aspects, the polynucleic acid construct comprises a sequence as set forth in any one of SEQ ID NOs: 40, 41 and 42 (Compounds B12, B13 and B14).

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct encoding or comprising: (i) at least one siRNA capable of binding to an IL-6 mRNA, at least one siRNA capable of binding to an ACE2 mRNA, and at least one siRNA capable of binding to a SARS CoV-2 S mRNA; and (ii) an mRNA encoding IFN-beta. In related aspects, the mRNA of ii) encodes or further encodes an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, one directed to an IL-6 mRNA, one directed to an ACE2 mRNA, and one directed to a SARS CoV-2 S mRNA. In related aspects, the mRNA encoding IFN-beta encodes the native IFN-beta signal peptide, or a modified signal peptide. In related aspects, the modified IFN-beta signal peptide is SP1 or SP2 as described herein (SEQ ID NOs: 52 and 54, respectively). In related aspects, the polynucleic acid construct comprises a sequence as set forth in any one of SEQ ID NOs: 43, 44, and 45 (Compounds B15, B16, and B17).

In some aspects, provided herein, is a method of simultaneously upregulating and downregulating the expression of two or more genes in a cell, comprising introducing into the cell a recombinant polynucleic acid construct encoding or comprising: (i) at least one small interfering RNA capable of binding to a SARS CoV-2 ORF1ab mRNA, at least one siRNA capable of binding to a SARS CoV-2 S mRNA, and at least one siRNA capable of binding to a SARS CoV-2 N mRNA; and (ii) an mRNA encoding an ACE2 soluble receptor. In related aspects, the polynucleic acid construct encodes or comprises at least 1, 2, or 3 siRNAs. In related aspects, the polynucleic acid construct encodes or comprises 3 siRNAs, one directed to an ORF1ab mRNA, one directed to a SARS CoV-2 S mRNA, and one directed to a SARS CoV-2 N mRNA. In related aspects, the polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 46 (Compound B18). In related aspects, the polynucleic acid construct comprises a sequence as set forth in SEQ ID NO: 190 (Compound B18).

EXEMPLARY EMBODIMENTS

Embodiment 1. A composition comprising a recombinant polynucleic acid construct comprising:

(i) at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA; and

(ii) at least one nucleic acid sequence encoding a gene of interest;

wherein the target RNA is different from an mRNA encoded by the gene of interest.

Embodiment 2. The composition of embodiment 1, wherein the recombinant polynucleic acid construct comprises two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target RNA, wherein each of the two or more nucleic acid sequences encode or comprise an siRNA capable of binding to a same target RNA or a different target RNA.

Embodiment 3. The composition of embodiment 1 or 2, wherein the target RNA is an mRNA.

Embodiment 4. The composition of embodiment 1 or 2, wherein the target RNA is an mRNA encoding a protein selected from the group consisting of: Interleukin 8 (IL-8), Interleukin 1 beta (IL-1 beta), Interleukin 17 (IL-17), and Tumor Necrosis Factor alpha (TNF-alpha).

Embodiment 5. The composition of any one of embodiments 1-4, wherein the recombinant polynucleic acid construct comprises two or more nucleic acid sequences encoding a gene of interest, wherein each of the two or more nucleic acid sequences encodes a same gene of interest or a different gene of interest.

Embodiment 6. The composition of any one of embodiments 1-5, wherein the gene of interest comprises a nucleic acid sequence encoding a protein selected from the group consisting of a secretory protein, an intracellular protein, an intraorganelle protein, and a membrane protein.

Embodiment 7. The composition of any one of embodiments 1-3, wherein the gene of interest is selected from the group consisting of Insulin-like Growth Factor 1 (IGF-1), and Interleukin 4 (IL-4).

Embodiment 8. The composition of any one of embodiments 1-7, wherein the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding a target motif operably linked to the at least one nucleic acid sequence encoding the gene of interest, wherein the target motif comprises a signal peptide, a nuclear localization signal (NLS), a nucleolar localization signal (NoLS), a lysosomal targeting signal, a mitochondrial targeting signal, a peroxisomal targeting signal, a microtubule tip localization signal (MtLS), an endosomal targeting signal, a chloroplast targeting signal, a Golgi targeting signal, an endoplasmic reticulum (ER) targeting signal, a proteasomal targeting signal, a membrane targeting signal, a transmembrane targeting signal, or a centrosomal localization signal (CLS).

Embodiment 9. The composition of embodiment 8, wherein the target motif is selected from the group consisting of:

(a) a target motif heterologous to a protein encoded by the gene of interest;

(b) a target motif heterologous to a protein encoded by the gene of interest, wherein the target motif heterologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid;

(c) a target motif homologous to a protein encoded by the gene of interest, wherein the target motif homologous to the protein encoded by the gene of interest is modified by insertion, deletion, and/or substitution of at least one amino acid; and

(d) a naturally occurring amino acid sequence which does not have the function of a target motif in nature, wherein the naturally occurring amino acid sequence is optionally modified by insertion, deletion, and/or substitution of at least one amino acid.

Embodiment 10. The composition of any one of embodiments 1-9, wherein the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a poly(A) tail, a nucleic acid sequence encoding or comprising a 5′ cap, a nucleic acid sequence encoding or comprising a promoter, or a nucleic acid sequence encoding or comprising a Kozak sequence.

Embodiment 11. The composition of any one of embodiments 1-9, wherein the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a linker.

Embodiment 12. The composition of any one of embodiments 1-11, wherein the nucleic acid sequence encoding or comprising the linker connects (a) the at least one nucleic acid sequence encoding or comprising an siRNA capable of binding to a target mRNA and the at least one nucleic acid sequence encoding a gene of interest, (b) each of the two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target mRNA, and/or (c) each of the two or more nucleic acid sequences encoding a gene of interest.

Embodiment 13. The composition of embodiment 11 or 12, wherein the linker comprises a tRNA linker, a 2A peptide linker or a flexible linker.

Embodiment 14. The composition of any one of embodiments 11-13, wherein nucleic acid sequence encoding or comprising the linker is at least 6 nucleic acid residues in length.

Embodiment 15. The composition of any one of embodiments 11-13, wherein the nucleic acid sequence encoding or comprising the linker is up to 50 nucleic acid residues in length.

Embodiment 16. The composition of any one of embodiments 11-13, wherein the nucleic acid sequence encoding or comprising the linker is about 6 to about 50 nucleic acid residues in length.

Embodiment 17. The composition any one of embodiments 11-13, wherein the nucleic acid sequence encoding or comprising the linker is about 6 to about 15 nucleic acid residues in length.

Embodiment 18. A composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8.

Embodiment 19. A composition comprising a recombinant RNA construct comprising:

(i) a small interfering RNA (siRNA) capable of binding to a target RNA; and

(ii) an mRNA encoding a gene of interest;

wherein the target RNA is different from the mRNA encoding the gene of interest.

Embodiment 20. The composition of embodiment 19, wherein the target RNA is mRNA.

Embodiment 21. The composition of any one of embodiments 1-20 for use in simultaneously modulating the expression of two or more genes in a cell.

Embodiment 22. The composition of any one of embodiments 1-21, wherein the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are comprised in a sequential manner.

Embodiment 23. The composition of embodiment 22, wherein the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream of the at least one nucleic acid sequence encoding a gene of interest (ii).

Embodiment 24. The composition of embodiment 22, wherein the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is downstream of the at least one nucleic acid sequence encoding a gene of interest (ii).

Embodiment 25. The composition of embodiment 22, wherein the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream or downstream of the at least one nucleic acid sequence encoding a gene of interest (ii).

Embodiment 26. The composition of any one of embodiments 1-25, wherein the siRNA capable of binding to a target RNA binds to an exon of a target mRNA.

Embodiment 27. The composition of any one of embodiments 1-26, wherein the siRNA capable of binding to a target RNA specifically binds to one target RNA.

Embodiment 28. The composition of any one of embodiments 1-27, wherein the siRNA capable of binding to a target RNA is not encoded by or comprised of an intron sequence of the gene of interest.

Embodiment 29. The composition of any one of embodiments 1-28, wherein the gene of interest is expressed without RNA splicing.

Embodiment 30. A composition comprising a recombinant polynucleic acid construct for treatment or prevention of a viral disease or condition in a subject, the construct comprising:

Embodiment 31. The composition of embodiment 30, wherein the recombinant polynucleic acid construct comprises two or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target RNA, wherein each of the two or more nucleic acid sequences encode or comprise an siRNA capable of binding to a same target RNA or a different target RNA.

Embodiment 32. The composition of embodiment 30, wherein the recombinant polynucleic acid construct comprises three or more nucleic acid sequences encoding or comprising an siRNA capable of binding to a target RNA, wherein at least two nucleic acid sequences encode or comprise an siRNA capable of binding to the same target RNA and at least one nucleic acid sequence encodes or comprises an siRNA capable of binding to a different target RNA.

Embodiment 33. The composition of any one of embodiments 30-32, wherein the target RNA is an mRNA.

Embodiment 34. The composition of any one of embodiments 30-32, wherein the target RNA is a noncoding RNA.

Embodiment 35. The composition of any one of embodiments 30-32, wherein the target RNA is an mRNA encoding a protein selected from the group consisting of: interleukin, Angiotensin Converting Enzyme-2 (ACE2); SARS CoV-2 ORF1ab; SARS CoV-2 S, and SARS CoV-2 N.

Embodiment 36. The composition of embodiment 35, wherein the interleukin is selected from the group consisting of: IL-1alpha, IL-1beta, IL-6, IL-6R, IL-6R-alpha, interleukin IL-6R-beta, IL-18, IL-36-alpha, IL-36-beta; IL-36-gamma, and IL-33.

Embodiment 37. The composition of any one of embodiments 30-32, wherein the target RNA is an mRNA encoding a protein selected from the group consisting of: IL-6, IL-6R, IL-6R-alpha, IL-6R-beta, Angiotensin Converting Enzyme-2 (ACE2); SARS CoV-2 ORF1ab; SARS CoV-2 S, and SARS CoV-2 N.

Embodiment 38. The composition of any one of embodiments 30-37, wherein the recombinant polynucleic acid construct comprises two or more nucleic acid sequences encoding a gene of interest, wherein each of the two or more nucleic acid sequences encodes a same gene of interest or a different gene of interest.

Embodiment 39. The composition of any one of embodiments 30-38, wherein the gene of interest of (ii) is selected from the group of genes encoding: IFN alpha-n3, IFN alpha-2a, IFN alpha-2b, IFN beta-1a, IFN beta-1b, ACE2 soluble receptor, IL-37, and IL-38.

Embodiment 40. The composition of any one of embodiments 30-38, wherein the gene of interest of (ii) is selected from the group of genes encoding: IFN beta and ACE2 soluble receptor.

Embodiment 41. The composition of any one of embodiments 30-40, wherein the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding a target motif operably linked to the at least one nucleic acid sequence encoding the gene of interest, wherein the target motif comprises a signal peptide, a nuclear localization signal (NLS), a nucleolar localization signal (NoLS), a lysosomal targeting signal, a mitochondrial targeting signal, a peroxisomal targeting signal, a microtubule tip localization signal (MtLS), an endosomal targeting signal, a chloroplast targeting signal, a Golgi targeting signal, an endoplasmic reticulum (ER) targeting signal, a proteasomal targeting signal, a membrane targeting signal, a transmembrane targeting signal, or a centrosomal localization signal (CLS).

Embodiment 42. The composition of embodiment 41, wherein the target motif is selected from the group consisting of:

Embodiment 43. The composition of any one of embodiments 30-42, wherein the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a poly(A) tail, a nucleic acid sequence encoding or comprising a 5′ cap, a nucleic acid sequence encoding or comprising a promoter, or a nucleic acid sequence encoding or comprising a Kozak sequence.

Embodiment 44. The composition of any one of embodiments 30-43, wherein the recombinant polynucleic acid construct further comprises a nucleic acid sequence encoding or comprising a linker.

Embodiment 45. The composition of embodiment 44, wherein the nucleic acid sequence encoding or comprising the linker connects (a) the at least one nucleic acid sequence encoding or comprising the siRNA capable of binding to the target mRNA and the at least one nucleic acid sequence encoding the gene of interest, (b) each of the two or more nucleic acid sequences encoding or comprising the siRNA capable of binding to the target mRNA, and/or (c) each of the two or more nucleic acid sequences encoding the gene of interest.

Embodiment 46. The composition of embodiment 44 or 45, wherein the linker comprises a tRNA linker, a 2A peptide linker, or a flexible linker.

Embodiment 47. The composition of any one of embodiments 44-46, wherein the nucleic acid sequence encoding or comprising the linker is at least 6 nucleic acid residues in length.

Embodiment 48. The composition of any one of embodiments 44-46, wherein the nucleic acid sequence encoding or comprising the linker is up to 50 nucleic acid residues in length.

Embodiment 49. The composition of any one of embodiments 44-46, wherein the nucleic acid sequence encoding or comprising the linker is about 6 to about 50 nucleic acid residues in length.

Embodiment 50. The composition of any one of embodiments 44-46, wherein the nucleic acid sequence encoding or comprising the linker is about 6 to about 15 nucleic acid residues in length.

Embodiment 51. A composition comprising a recombinant polynucleic acid construct comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 29-47.

Embodiment 55. A composition comprising a recombinant RNA construct for treatment or prevention of a viral disease or condition in a subject, the construct comprising:

Embodiment 53. The composition of any one of embodiments 30-52 for use in simultaneously modulating the expression of two or more genes in a cell.

Embodiment 54. The composition of any one of embodiments 30-53, wherein the composition is present in an amount sufficient to treat or prevent a viral disease or condition in the subject.

Embodiment 55. The composition of any one of embodiments 30-54, wherein the at least one nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) and the at least one nucleic acid sequence encoding a gene of interest (ii) are comprised in a sequential manner.

Embodiment 56. The composition of embodiment 55, wherein the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream of the at least one nucleic acid sequence encoding a gene of interest (ii).

Embodiment 57. The composition of embodiment 55, wherein the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is downstream of the at least one nucleic acid sequence encoding a gene of interest (ii).

Embodiment 58. The composition of embodiment 55, wherein the nucleic acid sequence encoding or comprising a small interfering RNA (siRNA) capable of binding to a target RNA (i) is upstream or downstream of the at least one nucleic acid sequence encoding a gene of interest (ii).

Embodiment 59. The composition of any one of embodiments 30-58, wherein the siRNA capable of binding to a target RNA binds to an exon of a target mRNA.

Embodiment 60. The composition of any one of embodiments 30-59, wherein the siRNA capable of binding to a target RNA specifically binds to one target RNA.

Embodiment 61. The composition of any one of embodiments 30-60, wherein the siRNA capable of binding to a target RNA is not encoded by or comprised of an intron sequence of the gene of interest.

Embodiment 62. The composition of any one of embodiments 30-61, wherein the gene of interest is expressed without RNA splicing.

Embodiment 63. The composition of any one of embodiments 30-62, wherein the siRNA comprises a sense strand sequence selected from SEQ ID NOs: 93-109.

Embodiment 64. The composition of any one of embodiments 1-29, wherein the siRNA comprises a sense strand sequence selected from SEQ ID NOs: 80-92.

Embodiment 65. The composition of any one of embodiments 1-29, wherein the recombinant polynucleic acid construct comprises a sequence with at least 85% sequence identity to any one of SEQ ID NOs: 177-189.

Embodiment 66. The composition of any one of embodiments 1-29, wherein the recombinant polynucleic acid construct comprises a sequence selected from the group consisting of SEQ ID NOs: 177-189.

Embodiment 67. The composition of any one of embodiments 30-63, wherein the recombinant polynucleic acid construct comprises a sequence with at least 85% sequence identity to SEQ ID NO: 190.

Embodiment 68. The composition of any one of embodiments 30-63, wherein the recombinant polynucleic acid construct comprises a sequence of SEQ ID NO: 190.

EXAMPLES

These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

Example 1: Construct Design, Sequence, and Synthesis

Construct Design

The present invention discloses that both siRNAs and any proteins of interest can be simultaneously expressed from a single transcript generated by in vitro transcription. The RNA constructs disclosed herein were designed to include siRNA designs as described in Cheng, et al. (2018) J. Mater. Chem. B., 6, 4638-4644 with one or more genes of interest downstream or upstream of the siRNA sequence (FIG. 1). The construct of the present invention may comprise more than one siRNA sequence sequentially targeting the same or different genes. Likewise, the construct of the present invention may comprise nucleic acid sequences of two or more genes of interest with a linker sequence or linker coding sequence in between (e.g., 2A peptide linker or tRNA linker).

The constructs further include T7 promoter (5′ TAATACGACTCACTATA 3′; SEQ ID NO: 25) sequence upstream of the siRNA sequence for RNA polymerase binding and successful in vitro transcription of both siRNA and the gene of interest. Alternative promoters can be utilized, and alternative promoters include SP6, T3, P60, Syn5, and KP34 promoters, which are equally functional for in vitro transcription.

Construct Synthesis

The designed constructs (Table 1, Compound ID numbers A1-A8) were gene-synthesized from GeneArt, Germany (Thermo Fisher Scientific). The constructs were synthesized as pMA-RQ vector, which contains a T7 RNA polymerase promoter, with codon optimization using GeneOptimizer algorithm. Table 1 summarizes the compounds used in the examples in the present disclosure with their respective siRNA target to downregulate protein expression, and protein target for upregulated protein expression. All uridines in Compounds A1-A8 used in the examples described herein were modified to N¹-methylpseudouridine. For each compound, the position of siRNA sequence is indicated in regard to the gene of interest. For example, “5′ siRNA position” indicates that siRNA sequences are upstream of or 5′ to the gene of interest in the compound. The sequences of the constructs of A1-A8 are shown in Table 2 and annotated as indicated in the table below.

TABLE 1

Summary of Compounds A1-A8

siRNA
# of
Protein Target

Compound ID
siRNA Target
Position
siRNAs
(gene of interest)
Indication

A1
IL-8
5’
1
IGF-1
OA, IVDD

A2
IL-8
5’
1
IGF-1
OA, IVDD

A3
IL-8
5’
3
IGF-1
OA, IVDD

A4
IL-8
5’
1
—
OA, IVDD

A5
IL-8
5’
3
—
OA, IVDD

A6
IL-1 beta
5’
1
IGF-1
OA, IVDD

A7
IL-1 beta
5’
3
IGF-1
OA, IVDD

A8
TNF-alpha/IL-17*
5’
6
IL-4
Psoriasis

OA: Osteoarthritis;

IVDD: Intervertebral disc disease;

*: only the siRNA effect of TNF-α studied

TABLE 2

Sequences of Compounds A1-A8

SEQ ID NO:
Compound #
Sequence (5′ → 3′ direction)

1
Compound A1
ATAGTGAGTCGTATTAACGTACCAACAACAAGGAAGTGCTAAAGAAACT

A1 sense

TG custom-character

TTTATCTTAGAGGCATATCCCTGCCACCA

strand siRNA

TGACCATCCTGTTTCTGACAATGGTCATCAGCTACTTCGGCTGCATGAA

80, antisense

GGCCGTGAAGATGCACACCATGAGCAGCAGCCACCTGTTCTATCTGGCC

110

CTGTGCCTGCTGACCTTTACCAGCTCTGCTACCGCCGGACCTGAGACAC

TTTGTGGCGCTGAACTGGTGGACGCCCTGCAGTTTGTGTGTGGCGACAG

AGGCTTCTACTTCAACAAGCCCACAGGCTACGGCAGCAGCTCTAGAAGG

GCTCCTCAGACCGGAATCGTGGACGAGTGCTGCTTCAGAAGCTGCGACC

TGCGGCGGCTGGAAATGTATTGTGCCCCTCTGAAGCCTGCCAAGAGCGC

CTAATTTATCTTAGAGGCATATCCCT

2
Compound A2
ATAGTGAGTCGTATTAACGTACCAACAACAAGGAGTGCTAAAGAAACTT

A2 sense

G custom-character

TTTATCTTAGAGGCATATCCCTGCCACCATG

strand siRNA

ACCATCCTGTTTCTGACAATGGTCATCAGCTACTTCGGCTGCATGAAGG

81, antisense

CCGTGAAGATGCACACCATGAGCAGCAGCCACCTGTTCTATCTGGCCCT

111

GTGCCTGCTGACCTTTACCAGCTCTGCTACCGCCGGACCTGAGACACTT

TGTGGCGCTGAACTGGTGGACGCCCTGCAGTTTGTGTGTGGCGACAGAG

GCTTCTACTTCAACAAGCCCACAGGCTACGGCAGCAGCTCTAGAAGGGC

TCCTCAGACCGGAATCGTGGACGAGTGCTGCTTCAGAAGCTGCGACCTG

CGGCGGCTGGAAATGTATTGTGCCCCTCTGAAGCCTGCCAAGAGCGCCT

AATTTATCTTAGAGGCATATCCCT

3
Compound A3
ATAGTGAGTCGTATTAACGTACCAACAACAAGGAGTGCTAAAGAAACTT

5′ to 3′:

G custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAA

A3-1 sense

CAAGAGAGTGATTGAGAGTGGACTTG custom-character

TTTAT

strand siRNA

CTTAGAGGCATATCCCTACGTACCAACAAGAGAGCTCTGTCTGGACCAC

81, antisense

TTG custom-character

TTTATCTTAGAGGCATATCCCTGCCACC

111;

ATGACCATCCTGTTTCTGACAATGGTCATCAGCTACTTCGGCTGCATGA

A3-2 sense

AGGCCGTGAAGATGCACACCATGAGCAGCAGCCACCTGTTCTATCTGGC

strand siRNA

CCTGTGCCTGCTGACCTTTACCAGCTCTGCTACCGCCGGACCTGAGACA

82, antisense

CTTTGTGGCGCTGAACTGGTGGACGCCCTGCAGTTTGTGTGTGGCGACA

112;

GAGGCTTCTACTTCAACAAGCCCACAGGCTACGGCAGCAGCTCTAGAAG

A3-3 sense

GGCTCCTCAGACCGGAATCGTGGACGAGTGCTGCTTCAGAAGCTGCGAC

strand siRNA

CTGCGGCGGCTGGAAATGTATTGTGCCCCTCTGAAGCCTGCCAAGAGCG

83, antisense

CCTAATTTATCTTAGAGGCATATCCCT

113

4
Compound A4
ATAGTGAGTCGTATTAACGTACCAACAACAAGGAAGTGCTAAAGAAACT

A4 sense

TG custom-character

TTTATCTTAGAGGCATATCCCT

strand siRNA

80, antisense

110

5
Compound A5
ATAGTGAGTCGTATTAACGTACCAACAACAAGGAGTGCTAAAGAAACTT

A5-1 sense

G custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAA

strand siRNA

CAAGAGAGTGATTGAGAGTGGACTTG custom-character

TTTAT

81, antisense

CTTAGAGGCATATCCCTACGTACCAACAAGAGAGCTCTGTCTGGACCAC

111;

TTG custom-character

TTTATCTTAGAGGCATATCCCT

A5-2 sense

strand siRNA

82, antisense

112;

A5-3 sense

strand siRNA

83, antisense

113

6
Compound A6
ATAGTGAGTCGTATTAACGTACCAACAAGAAAGATGATAAGCCCACTCT

A6 sense

ACTTG custom-character

TTTATCTTAGAGGCATATCCCTG

strand siRNA

CCACC
ATGACCATCCTGTTTCTGACAATGGTCATCAGCTACTTCGGCTG

84, antisense

CATGAAGGCCGTGAAGATGCACACCATGAGCAGCAGCCACCTGTTCTAT

114

CTGGCCCTGTGCCTGCTGACCTTTACCAGCTCTGCTACCGCCGGACCTG

AGACACTTTGTGGCGCTGAACTGGTGGACGCCCTGCAGTTTGTGTGTGG

CGACAGAGGCTTCTACTTCAACAAGCCCACAGGCTACGGCAGCAGCTCT

AGAAGGGCTCCTCAGACCGGAATCGTGGACGAGTGCTGCTTCAGAAGCT

GCGACCTGCGGCGGCTGGAAATGTATTGTGCCCCTCTGAAGCCTGCCAA

GAGCGCCTAATTTATCTTAGAGGCATATCCCT

7
Compound A7
ATAGTGAGTCGTATTAACGTACCAACAAGAAAGATGATAAGCCCACTCT

A7-1 sense

ACTTG custom-character

TTTATCTTAGAGGCATATCCCTA

strand siRNA

CGTACCAACAAGGTGATGTCTGGTCCATATGAACTTG custom-character

84, antisense

custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGATGAT

114;

AAGCCCACTCTAACTTG custom-character

TTATCTTAGAGGC

A7-2 sense

ATATCCCTGCCACCATGACCATCCTGTTTCTGACAATGGTCATCAGCTA

strand siRNA

CTTCGGCTGCATGAAGGCCGTGAAGATGCACACCATGAGCAGCAGCCAC

85, antisense

CTGTTCTATCTGGCCCTGTGCCTGCTGACCTTTACCAGCTCTGCTACCG

115;

CCGGACCTGAGACACTTTGTGGCGCTGAACTGGTGGACGCCCTGCAGTT

A7-3 sense

TGTGTGTGGCGACAGAGGCTTCTACTTCAACAAGCCCACAGGCTACGGC

strand siRNA

AGCAGCTCTAGAAGGGCTCCTCAGACCGGAATCGTGGACGAGTGCTGCT

86, antisense

TCAGAAGCTGCGACCTGCGGCGGCTGGAAATGTATTGTGCCCCTCTGAA

116

GCCTGCCAAGAGCGCCTAATTTATCTTAGAGGCATATCCCT

8
Compound A8
ATAGTGAGTCGTATTAACGTACCAACAAGGCGTGGAGCTGAGAGATAAA

A8-1 sense

CTTG custom-character

TTTATCTTAGAGGCATATCCCTACG

strand siRNA

TACCAACAAGGGCCTGTACCTCATCTACTACTTG custom-character

87, antisense

custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGGTATGAGCC

117;

CATCTATCTACTTG custom-character

TTTATCTTAGAGGCAT

A8-2 sense

ATCCCTACGTACCAACAAGCAATGAGGACCCTGAGAGATACTTG custom-character

strand siRNA

custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACA

88, antisense

AGCTGATGGGAACGTGGACTAACTTG custom-character

TTT

118;

ATCTTAGAGGCATATCCCTACGTACCAACAAGGTCCTCAGATTACTACA

A8-3 sense

AACTTG custom-character

TTTATCTTAGAGGCATATCCCTGC

strand siRNA

CACC
ATGGGACTGACATCTCAACTGCTGCCTCCACTGTTCTTTCTGCTG

89, antisense

GCCTGCGCCGGCAATTTTGTGCACGGCCACAAGTGCGACATCACCCTGC

119;

AAGAGATCATCAAGACCCTGAACAGCCTGACCGAGCAGAAAACCCTGTG

A8-4 sense

CACCGAGCTGACCGTGACCGATATCTTTGCCGCCAGCAAGAACACAACC

strand siRNA

GAGAAAGAGACATTCTGCAGAGCCGCCACCGTGCTGAGACAGTTCTACA

90, antisense

GCCACCACGAGAAGGACACCAGATGCCTGGGAGCTACAGCCCAGCAGTT

120;

CCACAGACACAAGCAGCTGATCCGGTTCCTGAAGCGGCTGGACAGAAAT

A8-5 sense

CTGTGGGGACTCGCCGGCCTGAATAGCTGCCCTGTGAAAGAGGCCAACC

strand siRNA

AGTCTACCCTGGAAAACTTCCTGGAACGGCTGAAAACCATCATGCGCGA

91, antisense

GAAGTACAGCAAGTGCAGCAGCTGATTTATCTTAGAGGCATATCCCT

121;

A8-6 sense

strand siRNA

92, antisense

122

Bold = Sense siRNA strand

Bold and Italics = anti-Sense siRNA strand

Underline = Signal peptide

Italics = Kozak sequence

TABLE 3

Plasmid Sequences for Compounds A1-A8

SEQ

ID NO
Compound #
Sequence (5′ → 3′ direction)

9
Compound A1 in
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATATAGTGAGTCGTATTAACGTACC

AACAACAAGGAAGTGCTAAAGAAACTTGTTCTTTAGCACTTCCTTGTTT

ATCTTAGAGGCATATCCCTGCCACCATGACCATCCTGTTTCTGACAATG

GTCATCAGCTACTTCGGCTGCATGAAGGCCGTGAAGATGCACACCATGA

GCAGCAGCCACCTGTTCTATCTGGCCCTGTGCCTGCTGACCTTTACCAG

CTCTGCTACCGCCGGACCTGAGACACTTTGTGGCGCTGAACTGGTGGAC

GCCCTGCAGTTTGTGTGTGGCGACAGAGGCTTCTACTTCAACAAGCCCA

CAGGCTACGGCAGCAGCTCTAGAAGGGCTCCTCAGACCGGAATCGTGGA

CGAGTGCTGCTTCAGAAGCTGCGACCTGCGGCGGCTGGAAATGTATTGT

GCCCCTCTGAAGCCTGCCAAGAGCGCCTAATTTATCTTAGAGGCATATC

CCT
CTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAGTCGGGA

AACCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGT

ATTGGGCGCTCTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGT

TCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAAAGGCCA

GGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCC

CCCTGAGGAGCATCACAAAAATCGAGGCTCAAGTCAGAGGTGGCGAAAC

CCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCG

TGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTT

TCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTAT

CTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAAC

CCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGA

GTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGT

AACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGA

AGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTG

CGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGA

TCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGC

AGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT

TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATT

TTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATT

AAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTC

TGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGT

CTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTA

CGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCG

AGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCC

GGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCC

AGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAA

TAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGC

TCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGC

GAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGG

TCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATG

GTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGAT

GCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTG

TATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACC

GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTT

CGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGAT

GTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACC

AGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGG

GAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCA

ATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATA

TTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTC

CCCGAAAAGTGCCAC

10
Compound A2 in
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATATAGTGAGTCGTATTAACGTACC

AACAACAAGGAGTGCTAAAGAAACTTGTTCTTTAGCACTCCTTGTTTAT

CTTAGAGGCATATCCCTGCCACCATGACCATCCTGTTTCTGACAATGGT

CATCAGCTACTTCGGCTGCATGAAGGCCGTGAAGATGCACACCATGAGC

AGCAGCCACCTGTTCTATCTGGCCCTGTGCCTGCTGACCTTTACCAGCT

CTGCTACCGCCGGACCTGAGACACTTTGTGGCGCTGAACTGGTGGACGC

CCTGCAGTTTGTGTGTGGCGACAGAGGCTTCTACTTCAACAAGCCCACA

GGCTACGGCAGCAGCTCTAGAAGGGCTCCTCAGACCGGAATCGTGGACG

AGTGCTGCTTCAGAAGCTGCGACCTGCGGCGGCTGGAAATGTATTGTGC

CCCTCTGAAGCCTGCCAAGAGCGCCTAATTTATCTTAGAGGCATATCCC

T
CTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAGTCGGGAAA

CCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTAT

TGGGCGCTCTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTC

GGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAAAGGCCAGG

AACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCC

CTGAGGAGCATCACAAAAATCGAGGCTCAAGTCAGAGGTGGCGAAACCC

GACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTG

CGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTC

TCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCT

CAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCC

CCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGT

CCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAA

CAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAG

TGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCG

CTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC

CGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAG

CAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT

CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTT

GGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAA

AAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTG

ACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCT

ATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACG

ATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAG

AACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGG

AAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAG

TCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATA

GTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTC

GTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGA

GTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTC

CTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGT

TATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGC

TTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTA

TGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGC

GCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCG

GGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGT

AACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAG

CGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGA

ATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAAT

ATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATT

TGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCC

CGAAAAGTGCCAC

11
Compound A3 in
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATATAGTGAGTCGTATTAACGTACC

AACAACAAGGAGTGCTAAAGAAACTTGTTCTTTAGCACTCCTTGTTTAT

CTTAGAGGCATATCCCTACGTACCAACAAGAGAGTGATTGAGAGTGGAC

TTGCCACTCTCAATCACTCTCTTTATCTTAGAGGCATATCCCTACGTAC

CAACAAGAGAGCTCTGTCTGGACCACTTGGGTCCAGACAGAGCTCTCTT

TATCTTAGAGGCATATCCCTGCCACCATGACCATCCTGTTTCTGACAAT

GGTCATCAGCTACTTCGGCTGCATGAAGGCCGTGAAGATGCACACCATG

AGCAGCAGCCACCTGTTCTATCTGGCCCTGTGCCTGCTGACCTTTACCA

GCTCTGCTACCGCCGGACCTGAGACACTTTGTGGCGCTGAACTGGTGGA

CGCCCTGCAGTTTGTGTGTGGCGACAGAGGCTTCTACTTCAACAAGCCC

ACAGGCTACGGCAGCAGCTCTAGAAGGGCTCCTCAGACCGGAATCGTGG

ACGAGTGCTGCTTCAGAAGCTGCGACCTGCGGCGGCTGGAAATGTATTG

TGCCCCTCTGAAGCCTGCCAAGAGCGCCTAATTTATCTTAGAGGCATAT

CCCT
CTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAGTCGGG

AAACCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCG

TATTGGGCGCTCTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCG

TTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAAAGGCC

AGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCC

CCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAA

CCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTC

GTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT

TTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTA

TCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAA

CCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTG

AGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGG

TAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTG

AAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCT

GCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG

ATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAG

CAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCT

TTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGAT

TTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAAT

TAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGT

CTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTG

TCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACT

ACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGC

GAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGC

CGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATC

CAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTA

ATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACG

CTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGG

CGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCG

GTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCAT

GGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGA

TGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGT

GTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATAC

CGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCT

TCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGA

TGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCAC

CAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAG

GGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTC

AATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACAT

ATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTT

CCCCGAAAAGTGCCAC

12
Compound A4 in
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATCAACGAGCTCATAGTGAGTCGTA

TTAACGTACCAACAACAAGGAAGTGCTAAAGAAACTTGTTCTTTAGCAC

TTCCTTGTTTATCTTAGAGGCATATCCCT
GGTACCCTCTGGGCCTCATG

GGCCTTCCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAG

CTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCG

CTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGG

GGTGCCTAATGAGCAAAAGGCGAGCAAAAGGCCAGGAACCGTAAAAAGG

CCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCA

CAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA

AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTC

CGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAG

CGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAG

GTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCG

ACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAG

ACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGA

GCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT

ACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCC

AGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACC

ACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA

GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA

CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTA

TCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTA

AATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATG

CTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCC

ATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCT

TACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACC

GGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGC

AGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTT

GCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGT

TGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATG

GCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCC

CCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGT

CAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTG

CATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTG

GTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAG

TTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGA

ACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCT

CAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGC

ACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA

GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGAGAC

GGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCAT

TTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAG

AAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC

13
Compound A5 in
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATGAAGGGCGCGCCAATAGTGAGTC

GTATTAACGTACCAACAACAAGGAGTGCTAAAGAAACTTGTTCTTTAGC

ACTCCTTGTTTATCTTAGAGGCATATCCCTACGTACCAACAAGAGAGTG

ATTGAGAGTGGACTTGCCACTCTCAATCACTCTCTTTATCTTAGAGGCA

TATCCCTACGTACCAACAAGAGAGCTCTGTCTGGACCACTTGGGTCCAG

ACAGAGCTCTCTTTATCTTAGAGGCATATCCCT
TTTTAATTAACAACCT

GGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAGTCGGGAAACCT

GTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGG

GCGCTCTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGG

TAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAAAGGCCAGGAAC

CGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTG

ACGAGCATCACAAAAATCGAGGCTCAAGTCAGAGGTGGCGAAACCCGAC

AGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGC

TCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCC

CTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAG

TTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCC

GTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA

ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG

GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGG

TGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTC

TGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGG

CAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAG

ATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTA

CGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGT

CATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAA

TGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACA

GTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATT

TCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATA

CGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAAC

CACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAG

GGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT

ATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTT

TGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTC

GTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTT

ACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTC

CGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTAT

GGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT

TCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGC

GGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCC

ACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGG

CGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAAC

CCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGT

TTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATA

AGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATT

ATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGA

ATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGA

AAAGTGCCAC

14
Compound A6 in
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATATAGTGAGTCGTATTAACGTACC

AACAAGAAAGATGATAAGCCCACTCTACTTGAGAGTGGGCTTATCATCT

TTCTTTATCTTAGAGGCATATCCCTGCCACCATGACCATCCTGTTTCTG

ACAATGGTCATCAGCTACTTCGGCTGCATGAAGGCCGTGAAGATGCACA

CCATGAGCAGCAGCCACCTGTTCTATCTGGCCCTGTGCCTGCTGACCTT

TACCAGCTCTGCTACCGCCGGACCTGAGACACTTTGTGGCGCTGAACTG

GTGGACGCCCTGCAGTTTGTGTGTGGCGACAGAGGCTTCTACTTCAACA

AGCCCACAGGCTACGGCAGCAGCTCTAGAAGGGCTCCTCAGACCGGAAT

CGTGGACGAGTGCTGCTTCAGAAGCTGCGACCTGCGGCGGCTGGAAATG

TATTGTGCCCCTCTGAAGCCTGCCAAGAGCGCCTAGTTTATCTTAGAGG

CATATCCCT
CTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAG

TCGGGAAACCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCC

TTGCGTATTGGGCGCTCTCCGCTTCCTCGCTCACTGACTCGCTGCGCTC

GGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAA

AGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCT

CCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGG

CGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCT

CCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTC

CGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGT

AGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGC

ACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG

TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC

ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGT

TCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGG

TATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC

TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTT

GCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTT

GATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAA

GGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTT

TAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAAC

TTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCG

ATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGA

TAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGAT

ACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAG

CCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT

CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCC

AGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTG

TCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGAT

CAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTC

CTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCA

CTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCG

TAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGA

ATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGAT

AATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAAC

GTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG

TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACT

TTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAA

AAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCT

TTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGA

TACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCA

CATTTCCCCGAAAAGTGCCAC

15
Compound A7 in
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATATAGTGAGTCGTATTAACGTACC

AACAAGAAAGATGATAAGCCCACTCTACTTGAGAGTGGGCTTATCATCT

TTCTTTATCTTAGAGGCATATCCCTACGTACCAACAAGGTGATGTCTGG

TCCATATGAACTTGTCATATGGACCAGACATCACCTTTATCTTAGAGGC

ATATCCCTACGTACCAACAAGATGATAAGCCCACTCTAACTTGTAGAGT

GGGCTTATCATCTTTATCTTAGAGGCATATCCCTGCCACCATGACCATC

CTGTTTCTGACAATGGTCATCAGCTACTTCGGCTGCATGAAGGCCGTGA

AGATGCACACCATGAGCAGCAGCCACCTGTTCTATCTGGCCCTGTGCCT

GCTGACCTTTACCAGCTCTGCTACCGCCGGACCTGAGACACTTTGTGGC

GCTGAACTGGTGGACGCCCTGCAGTTTGTGTGTGGCGACAGAGGCTTCT

ACTTCAACAAGCCCACAGGCTACGGCAGCAGCTCTAGAAGGGCTCCTCA

GACCGGAATCGTGGACGAGTGCTGCTTCAGAAGCTGCGACCTGCGGCGG

CTGGAAATGTATTGTGCCCCTCTGAAGCCTGCCAAGAGCGCCTAGTTTA

TCTTAGAGGCATATCCCT
CTGGGCCTCATGGGCCTTCCGCTCACTGCCC

GCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAACATGGTCATA

GCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCGCTCACTGACTC

GCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAG

GCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT

CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGT

CAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCC

CTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGG

ATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGC

TCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGG

GCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG

TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGAGTTATCGCCACTG

GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTG

CTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAAC

AGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGA

GTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTT

TTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGA

AGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAAC

TCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCT

AGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATA

TGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCT

ATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCG

TCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGC

TGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCA

ATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTT

TATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAG

TAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGC

ATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTT

CCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGC

GGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCA

GTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCA

TGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC

ATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCA

ATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCA

TTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT

GAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCA

TCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAA

ATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT

ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTC

ATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGG

TTCCGCGCACATTTCCCCGAAAAGTGCCAC

16
Compound A8 in
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATATAGTGAGTCGTATTAACGTACC

AACAAGGCGTGGAGCTGAGAGATAAACTTGTTATCTCTCAGCTCCACGC

CTTTATCTTAGAGGCATATCCCTACGTACCAACAAGGGCCTGTACCTCA

TCTACTACTTGAGTAGATGAGGTACAGGCCCTTTATCTTAGAGGCATAT

CCCTACGTACCAACAAGGTATGAGCCCATCTATCTACTTGAGATAGATG

GGCTCATACCTTTATCTTAGAGGCATATCCCTACGTACCAACAAGCAAT

GAGGACCCTGAGAGATACTTGATCTCTCAGGGTCCTCATTGCTTTATCT

TAGAGGCATATCCCTACGTACCAACAAGCTGATGGGAACGTGGACTAAC

TTGTAGTCCACGTTCCCATCAGCTTTATCTTAGAGGCATATCCCTACGT

ACCAACAAGGTCCTCAGATTACTACAAACTTGTTGTAGTAATCTGAGGA

CCTTTATCTTAGAGGCATATCCCTGCCACCATGGGACTGACATCTCAAC

TGCTGCCTCCACTGTTCTTTCTGCTGGCCTGCGCCGGCAATTTTGTGCA

CGGCCACAAGTGCGACATCACCCTGCAAGAGATCATCAAGACCCTGAAC

AGCCTGACCGAGCAGAAAACCCTGTGCACCGAGCTGACCGTGACCGATA

TCTTTGCCGCCAGCAAGAACACAACCGAGAAAGAGACATTCTGCAGAGC

CGCCACCGTGCTGAGACAGTTCTACAGCCACCACGAGAAGGACACCAGA

TGCCTGGGAGCTACAGCCCAGCAGTTCCACAGACACAAGCAGCTGATCC

GGTTCCTGAAGCGGCTGGACAGAAATCTGTGGGGACTCGCCGGCCTGAA

TAGCTGCCCTGTGAAAGAGGCCAACCAGTCTACCCTGGAAAACTTCCTG

GAACGGCTGAAAACCATCATGCGCGAGAAGTACAGCAAGTGCAGCAGCT

GATTTATCTTAGAGGCATATCCCT
CTGGGCCTCATGGGCCTTCCGCTCA

CTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAACATG

GTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCGCTCAC

TGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAG

CAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGC

GTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGC

TCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGT

TTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCT

TACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCT

CATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA

AGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTT

ATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCG

CCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAG

GCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAG

AAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGA

AAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCG

GTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATC

TCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAAC

GAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCT

TCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAG

TATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAG

GCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGAC

TCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCC

CAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTA

TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTG

CAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAG

AGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCT

ACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCT

CCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAA

AAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTG

GCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTA

CTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAAC

CAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCG

GCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGC

TCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACC

GCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCT

TCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAA

GGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAAT

ACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTAT

TGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA

TAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC

Bold and underline = compound sequence

Example 2: In Vitro Transcription of RNA Constructs and Data Analysis

The pMA-RQ vectors encoding Compounds A1-A8 and a homologous primer pair (Table 4) were used for PCR based in vitro transcription mRNA production. A transcription template was generated by PCR using forward and reverse primers in Table 4. The poly(A) tail was encoded in the template; the resulting PCR product encoded a 120 bp poly(A) tail (SEQ ID NO: 193). A few optimizations were made due to the repetitive sequence of siRNA flanking regions (see Tables 2 and 3) to achieve a specific amplification. These optimizations included: 1) low amount of plasmid DNA of vector; 2) use of special DNA polymerase (Q5 hot start polymerase, New England Biolabs); 3) reduced time for denaturation (30 seconds to 10 seconds) and extension (45 seconds/kb to 10 seconds/kb) for each cycle of PCR; 4) increased time for annealing (10 seconds to 30 seconds) for each cycle of PCR, and; 5) increased time for final extension (up to 15 minutes) for each cycle of PCR. In addition, to avoid non-specific primer binding, the PCR reaction mixture was prepared on ice, including thawing reagents, and the number of PCR cycles was reduced to 25.

For in vitro transcription, T7 RNA polymerase (MEGAscript kit, Thermo Fisher Scientific) was used at 37° C. for 2 hours and synthesized RNAs were chemically modified with 100% N1-methylpseudo-UTP and co-transcriptionally capped with an anti-reverse CAP analog (ARCA; [m₂^7,3′-OG(5′) ppp(5′)G]) at the 5′ end (Jena Bioscience). After in vitro transcription, the mRNAs were column-purified using MEGAclear kit (Thermo Fisher Scientific) and quantified using Nanophotometer-N60 (Implen).

TABLE 4

Primers for Template Generation

SEQ
Primer

ID NO
Direction
Sequence (5′ to 3′)

17
Forward
GCTGCAAGGCGATTAAGTTG

18
Reverse
U(2′OMe)U(2′OMe)U(2′OMe)TTTTTTTTTTTTTTTTTTTTTT

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

TTTCAGCTATGACCATGTTAATGCAG

Using in vitro transcription, Compounds A1-A5 were generated at 50-200 μg range and were tested for IL-8 down regulation and IGF-1 expression in overexpression models of HEK-293 (Example 3) and THP-1 cells (Example 4) where IL-8 was overexpressed using respective mRNA. In addition, Compounds A6 and A7 were generated at 50-200 μg range and were tested for endogenous IL-1 beta down regulation and IGF-1 expression in THP-1 cells which were stimulated by LPS and dsDNA for endogenous secretion of IL-1 beta (Example 4). Compound A8 was generated at 50-200 μg range and was tested for endogenous TNF-α down regulation and IL-4 expression in THP-1 cells where endogenous TNF-α expression was stimulated by the treatment with LPS and R848 (Example 4). Likewise, Compound A8 was tested for TNF-α down regulation and IL-4 expression in overexpression models of HEK-293 cells where TNF-α was overexpressed using TNF-α encoding mRNA (Example 3).

Data were analyzed using GraphPad Prism 8 (San Diego, USA). For the estimation of the protein (IGF-1, IL-4, IL-8, IL-1 beta or TNF-α) levels using ELISA in the standard or the sample, the mean absorbance value of the blank was subtracted from the mean absorbance of the standards or the samples. A standard curve was generated and plotted using a four parameters nonlinear regression according to manufacturer's protocol. To determine the concentration of proteins (IGF-1, IL-4, IL-8, IL-1 beta or TNF-α) in each sample, the concentration of the protein was interpolated from the standard curve. The final protein concentration of the sample was calculated by multiplication with the dilution factor. Statistical analyses were made using a Student's t-test.

Example 3: In Vitro Transfection of HEK-293 and IL-8 Overexpression Model in HEK-293 Cells

In Vitro Transfection of HEK-293

Human embryonic kidney cells 293 (HEK-293; ATCC CRL-1573) were maintained in Dulbecco's Modified Eagle's medium (DMEM, Biochrom) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) and Penicillin-Streptomycin-Amphotericin B mixture (882087, Biozym Scientific). Cells were seeded at 20,000 cell/well in a 96 well culture plate and incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours prior to transfection. Cells were grown in DMEM growth medium containing 10% of FBS without antibiotics to reach confluency <60% before transfection. Thereafter, HEK-293 cells were transfected with specific mRNA constructs with varying concentrations (100-900 ng) using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions with the mRNA to Lipofectamine ratio of 1:1 w/v. 100 μl of DMEM was removed and replaced with 50 μl of Opti-MEM and 50 μl mRNA and Lipofectamine 2000 complex in Opti-MEM (Thermo Fisher Scientific). After 5 hours, the medium was replaced by fresh medium and the plates were incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours.

IL-8 Overexpression Model in HEK-293 Cells

To assess the simultaneous effect of IL-8 RNA interference (RNAi) and IGF-1 expression of RNA constructs (Compounds A1-A5) in HEK-293 cells, the IL-8 overexpression model was established using IL-8 mRNA transfection (300 ng/well). To assess the capability of mRNA constructs containing IL-8-targeting siRNA (Compounds A1-A5) in interfering with IL-8 expression and at the same time expressing IGF-1, the mRNA constructs (Compounds A1-A5; 300-900 ng/well) were co-transfected with IL-8 mRNA (300 ng/well). Post transfection, the cells were incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours followed by quantification of IL-8 (target gene to downregulate) and IGF-1 (Gene of Interest to overexpress) by ELISA in the cell culture supernatant.

TNF-α Overexpression Model in HEK-293 Cells

To assess the simultaneous effect of TNF-α RNA interference (RNAi) and IL-4 expression of Compound A8 in HEK-293 cells, the TNF-α overexpression model was established using TNF-α mRNA transfection (600 ng/well). To assess the capability of Compound A8 containing TNF-α targeting siRNA in TNF-α downregulation and simultaneous IL-4 expression, the cells were co-transfected with Compound A8 (600 ng/well) and TNF-α mRNA (600 ng/well). Post transfection, the cells were incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours followed by quantification of TNF-α (target gene to downregulate) and IL-4 (Gene of Interest to overexpress) by ELISA in the same cell culture supernatant.

Results

Compound A1 comprising IL-8-targeting siRNA and IGF-1 protein coding sequence was tested for IL-8 downregulation and simultaneous IGF-1 expression in HEK-293 cells (100-900 ng/well). The data demonstrate that Compound A1 expresses IGF-1 protein to the same level or above the level expressed by the control IGF-1 mRNA as shown in FIG. 2A (open circles—expression of IGF-1 from control IGF-1 mRNA; closed circles—Compound A1 IGF-1 expression). In the same experiment, the RNA interference of Compound A1 (300 ng/well) against IL-8 expression was assessed with IL-8 overexpression construct (300 ng/well) followed by IL-8 ELISA. As shown in FIG. 2B, Compound A1 (right bar) downregulated the IL-8 level compared to untreated control (left bar) (P<0.01). These assays showed that Compound A1 downregulated IL-8 by at least approximately 3-fold (65%), without reducing the expression of IGF-1.

To assess the dose-dependent capability of Compound A1 in interfering with IL-8 expression in HEK-293 IL-8 overexpression model, HEK-293 cells were co-transfected with an increasing dose of Compound A1 (300-900 ng of Compound A1/well) and constant IL-8 mRNA (300 ng/well) and assessed for IL-8 expression by ELISA. As demonstrated in FIG. 3, Compound A1 mRNA constructs comprising IL-8-targeting siRNA and IGF-1 protein coding sequence inhibited IL-8 expression in HEK-293 cells in a dose-dependent manner. FIG. 3 shows that at 300 ng/well Compound A1 reduced IL-8 expression by at least approximately 3.5-fold (70%) and at 600 or 900 ng/well, Compound A1 reduced IL-8 expression by at least approximately 4.25-fold (75%).

Compound A2 and Compound A3, which comprise 1× and 3×siRNA targeting IL-8, respectively, and IGF-1 protein coding sequence were tested to assess whether the presence of siRNA sequence in the same construct affect the IGF-1 expression. The HEK-293 cells were transfected with IGF-1 mRNA (600 ng/well). The results, in FIG. 4B (Compound A2) and 5B (Compound A3), show that IGF-1 is expressed from Compounds A2 and A3.

Compound A6 and Compound A7, which comprise 1× and 3×siRNA targeting IL-1 beta, respectively, and IGF-1 protein coding sequence were tested to assess whether the presence of siRNA in the same construct affect the IGF-1 expression. The HEK-293 cells were transfected with IGF-1 mRNA (600 ng/well). The results, in FIG. 8C (Compound A6) and 9C (Compound A7), show that IGF-1 is expressed from Compounds A6 and A7.

Compound A8, comprising TNF-α-targeting siRNA and IL-4 protein coding sequence was tested for TNF-α downregulation and IL-4 expression at the same time in HEK-293 cells (600 ng/well) with exogenously delivered TNF-α mRNA (600 ng/well). The data demonstrate that Compound A8 expresses IL-4 as shown in FIG. 10C. In the same experiment with the same cell culture supernatant, the RNA interference of Compound A8 (600 ng/well) against TNF-α expression from a TNF-α overexpression construct (600 ng/well) was assessed by TNF-α ELISA. As shown in FIG. 10A, Compound A8 (right bar) downregulated the TNF-α level compared to untreated control (left bar) (P<0.05). In this assay, Compound A8 downregulated TNF-α level by at least approximately 50%. These data demonstrate that Compound A8 downregulated TNF-α without affecting the IL-4 expression.

Next, Compound A4 and Compound A5, which comprise 1× and 3×siRNA targeting IL-8, respectively, but do not comprise IGF-1 coding sequence, were assessed for dose-dependent capability in interfering with IL-8 expression in HEK-293 cells. HEK-293 cells overexpressing IL-8 (600 ng of IL-8 mRNA) were transfected with various concentrations (300-900 ng/well) of Compound A4 (1×siRNA) and Compound A5 (3×siRNA). As demonstrated in FIG. 7, Compound A4 and Compound A5 inhibited IL-8 expression in HEK-293 cells in a dose-dependent manner.

Example 4: In Vitro Transfection of THP-1 Cells, Endogenous IL-1 Beta/TNF-α Expression Model in THP-1 Cells and IL-8 Overexpression Model in THP-1 Cells

In Vitro Transfection of THP-1 Cells

Human monocyte leukemia cell line THP-1 (Sigma-Aldrich, Cat. #88081201) was maintained in growth medium (RPMI 1640 supplemented with 10% FBS and 2 mM glutamine). The cells were seeded at 30,000 THP-1 cells in a 96 well cell culture plate 72 hours before transfection and activated with 50 nM of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Cat. #P8139) diluted in growth medium. The cells were transfected with specific mRNA as mono transfection or co-transfection (300-1200 ng/well) using Lipofectamine 2000 (Thermo Fisher Scientific). 100 μl of DMEM was removed from each well and replaced with 50 μl of Opti-MEM (Thermo Fisher Scientific) and 50 μl mRNA and Lipofectamine 2000 complex in Opti-MEM. After 5 hours, the medium was replaced with fresh growth medium supplemented with 50 nM PMA and the plates were incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours.

Endogenous IL-1 Beta Expression Model in THP-1 Cells

For the endogenous secretion of IL-1 beta in THP-1 cells, THP-1 cells were stimulated with E. coli-derived lipopolysaccharide (LPS-L4391; Sigma Aldrich) at 10 μg/mL final concentration with dsDNA (a specific PCR amplicon; 50 ng/well) and incubated for 90 minutes. The induced production of IL-1 beta corresponds to the physiological conditions observed in Osteoarthritis and IVDD. Post stimulation, 50 μl of media was removed and replaced with the transfection complex containing specific mRNA constructs (Compounds A6 and A7) complexed with Lipofectamine 2000 in Opti-MEM and incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours followed by IL-1 beta quantification by ELISA.

Endogenous TNF-α Expression Model in THP-1 Cells

For the endogenous secretion of TNF-α in THP-1 cells, THP-1 cells were stimulated with E. coli-derived lipopolysaccharide (LPS-L4391; Sigma Aldrich) at 10 μg/mL final concentration with R848 (TLR7/8 agonist; Invivogen) at 1 μg/mL final concentration and incubated for 90 minutes. The induced production of TNF-α corresponds to the physiological conditions observed in psoriasis. Post stimulation, 50 μl of media was removed and replaced with the transfection complex containing specific mRNA constructs (Compound A8) complexed with Lipofectamine 2000 in Opti-MEM and incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours. Post transfection, the cell culture supernatant was collected and quantified for TNF-α (target gene to downregulate) and IL-4 (Gene of Interest to overexpress) by ELISA.

IL-8 Overexpression Model in THP-1 Cells

To assess the RNA interference (RNAi) of mRNA constructs in THP-1 cells, the IL-8 overexpression model was established using IL-8 mRNA transfection (300 ng/well). To assess the capability of mRNA constructs containing IL-8-targeting siRNA (Compounds A1-A5) in interfering with IL-8 expression, the mRNA constructs (300-900 ng/well) were co-transfected with IL-8 mRNA (300 ng/well). Post transfection, the cells were incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours followed by quantification of IL-8 and IGF-1 by ELISA.

Results

Compound A2 and Compound A3 were designed to have 1× and 3×siRNA targeting IL-8, respectively, and IGF-1 coding sequence (Tables 1 and 2) and were tested to assess whether having more than one siRNA can maximize the effect of the targeted RNAi. Compound A4 and Compound A5 were designed as internal controls, which comprise only 1× and 3×siRNA targeting IL-8, respectively, without IGF-1 coding sequence (Tables 1 and 2). As demonstrated in FIGS. 4A, 5A, 6A, and 6B, Compounds A2-A5 inhibit IL-8 expression in THP-1 cells regardless of whether the compound has IGF-1 coding sequence. Compound A2 inhibited IL-8 expression by at least approximately 30% (FIG. 4A). Compound A3 inhibited IL-8 expression by at least approximately 45% (FIG. 6B). Compound A4 inhibited IL-8 expression by approximately 40% (FIG. 6A). Compound A5 inhibited IL-8 expression by at least approximately 70% (FIGS. 6A and 6B). Therefore, the compounds having three siRNA (Compounds A3 and A5) inhibited IL-8 expression by at least approximately 45% to at least approximately 70%, whereas the compounds having one siRNA (Compounds A2 and A4) inhibited IL-8 expression by at least approximately 30% to at least approximately 40%.

Next, the effect of Compound A6 (1×siRNA targeting IL-1 beta+IGF-1 coding sequence) and Compound A7 (3×siRNA targeting IL-1 beta+IGF-1 coding sequence) in interfering with IL-1 beta expression was evaluated in THP-1 cells stimulated with 10 μg/mL LPS and 50 ng/well dsDNA to induce endogenous IL-1 beta secretion. The established THP-1 model mimics the physiological immune condition of osteoarthritis and IVDD. As demonstrated in FIGS. 8A, 8B, 9A, and 9B, Compound A6 and Compound A7 downregulated the expression of endogenous IL-1 beta expression in THP-1 cells (P<0.001). Compound A6 downregulated IL-1 beta expression by at least approximately 40% (FIGS. 8A and 8B). Compound A7 downregulated IL-1 beta expression by at least approximately 45% to at least approximately 50% (FIGS. 9A and 9B, respectively).

The effect of Compound A8 (comprising siRNA targeting TNF-α and IL-4 coding sequence) in downregulation of TNF-α was evaluated in THP-1 cells stimulated with 10 μg/mL LPS and 1 μg/mL R848 to induce endogenous TNF-α secretion. The established THP-1 model mimics the physiological immune condition of psoriasis. As demonstrated in FIG. 10B, Compound A8 downregulated the expression of endogenous TNF-α expression in THP-1 cells (P<0.05). In this assay, Compound A8 downregulated TNF-α expression by at least approximately 20%. The same cell culture supernatant was measured for IL-4 expression and it was confirmed that IL-4 expression was not impaired (FIG. 10D).

Example 5: Anti-Viral Construct Design, Sequence, and Synthesis

Anti-Viral Construct Design

Both siRNAs and proteins of interest are simultaneously expressed from a single transcript generated by in vitro transcription. Polynucleotide or RNA constructs are engineered to include siRNA designs as described in Cheng, et al. (2018) J. Mater. Chem. B., 6, 4638-4644, and further comprise one or more gene of interest downstream or upstream of the siRNA sequence (schematic in FIG. 1). The construct may encode or comprise more than one siRNA sequence targeting the same or different target mRNA. Likewise, the construct may comprise nucleic acid sequences of two or more genes of interest. A linker sequence may be present between any two elements of the construct (e.g., 2A peptide linker or tRNA linker).

As presented in FIG. 1, a polynucleic acid construct may comprise a T7 promoter sequence (5′ TAATACGACTCACTATA 3′; SEQ ID NO: 25) upstream of the gene of interest sequence, for RNA polymerase binding and successful in vitro transcription of both the gene of interest and siRNA in a single transcript. An alternative promoter, e.g., SP6, T3, P60, Syn5, and KP34 may be used. A transcription template is generated by PCR to produce mRNA, using primers designed to flank the T7 promoter, IFN-beta and siRNA sequences. The reverse primer includes a stretch of T(120) (SEQ ID NO: 197) to add the 120 bp length of poly(A) tail (SEQ ID NO: 193) to the mRNA.

Anti-Viral Construct Synthesis

The constructs as shown in Table 5 are synthesized by GeneArt, Germany (Thermo Fisher Scientific) as vectors containing a T7 RNA polymerase promoter (pMX, e.g., pMA-T or pMA-RQ), with codon optimization (GeneOptimizer algorithm). Table 5 shows, for each compound, the protein to be downregulated through siRNA binding to the corresponding mRNA, the number of siRNAs of the construct (e.g., either multiple siRNA targeting the same mRNA, or multiple siRNA each targeting a different mRNA), and the protein target for upregulation, i.e., the product of the gene of interest. All uridines in Compounds B1-B19 used in the examples described herein were modified to N¹-methylpseudouridine. The sequences of each construct are shown in Table 6 and annotated as indicated below the table.

TABLE 5

Summary of Compounds B1-B19

Compound

siRNA
#of
Protein Target

ID
siRNA Target
Position
siRNAs
(gene of interest)
Mechanism

B1
IL-6
3’
3
IFN-β
Cytokine storm, anti-

inflammation

B2
IL-6
3’
1
IFN-β
Cytokine storm, anti-

inflammation

B3
IL-6R
3’
3
IFN-β
Cytokine storm, anti-

inflammation

B4
IL-6R alpha
3’
1
IFN-β
Cytokine storm, anti-

inflammation

B5
IL-6R beta
3’
1
IFN-β
Cytokine storm, anti-

inflammation

B6
ACE2
3’
3
IFN-β
Viral entry, anti-

inflammation

B7
ACE2
3’
1
IFN-β
Viral entry, anti-

inflammation

B8
SARS CoV-2
3’
3
IFN-β
Anti-viral, anti-inflammation

(ORF1ab, S, N)

B9
SARS CoV-2 (S)
3’
1
IFN-β
Anti-viral, anti-inflammation

B10
SARS CoV-2 (N)
3’
1
IFN-β
Anti-viral, anti-inflammation

B11
SARS CoV-2 (S)
3’
3
IFN-β
Anti-viral, anti-inflammation

B12
SARS CoV-2
3’
3
IFN-β
Anti-viral, anti-inflammation

(ORF1ab)

B13
SARS CoV-2
3’
1
IFN-β
Anti-viral, anti-inflammation

(ORF1ab)

B14
SARS CoV-2
3’
1
IFN-β
Anti-viral, anti-inflammation

(ORF1ab)

B15
IL6/ACE2/SARS
3’
3
IFN-β
Cytokine storm, viral entry,

CoV-2 (S)

anti-viral, anti-inflammation

B16
IL6/ACE2/SARS
3’
3
IFN-β (1)*
Cytokine storm, viral entry,

CoV-2 (S)

anti-viral, anti-inflammation

B17
IL6/ACE2/SARS
3’
3
IFN-β (2)*
Cytokine storm, viral entry,

CoV-2 (S)

anti-viral, anti-inflammation

B18
SARS CoV-2
3’
3
ACE2 soluble
Anti-viral, viral

(ORF1ab, S, N)

receptor
neutralization

B19
SARS CoV-2 (S)
3’
3
ACE2 soluble
Anti-viral, viral

receptor
neutralization

*IFN-β (1) and IFN-β (2) represent the modified signal peptide (SP) to enhance secretion

TABLE 6

Sequences of Compounds B1-B19

SEQ ID NO
Compound
Sequence (5′ to 3′)

29
Compound B1

GCCACC
ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

5′ to 3′:

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

B1-1 sense

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

strand siRNA

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

93, antisense

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

123;

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

B1-2 sense

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

strand siRNA

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

94, antisense

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

124;

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

B1-3 sense

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

strand siRNA

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

95, antisense

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

125

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGCCC

TGAGAAAGGAGACATGTACTTG custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGAGGAGACT

TGCCTGGTGAAAACTTG custom-character

TTTAT

CTTAGAGGCATATCCCTACGTACCAACAAGAGGGCTCTTCGGC

AAATGTAACTTG custom-character

TTTATCTTAG

AGGCATATCCCTTTTATCTTAGAGGCATATCCCT

30
Compound B2

GCCACC
ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

B2 sense

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

strand siRNA

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

94, antisense

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

124

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGAGG

AGACTTGCCTGGTGAAAACTTG custom-character

TTTATCTTAGAGGCATATCCCTTTTATCTTAGAGGCATATCCC

T

31
Compound B3

GCCACC
ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

5′ to 3′:

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

B3-1 sense

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

strand siRNA

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

96, antisense

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

126;

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

B3-2 sense

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

strand siRNA

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

97, antisense

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

127;

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

B3-3 sense

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

strand siRNA

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

98, antisense

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

128

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGTGA

GGAAGTTTCAGAACAGTACTTG custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGAACGGTCA

AAGACATTCACAACTTG custom-character

TTTAT

CTTAGAGGCATATCCCTACGTACCAACAAGGGAAGGTTACATC

AGATCATACTTG custom-character

TTTATCTTAG

AGGCATATCCCTTTTATCTTAGAGGCATATCCCT

32
Compound B4

GCCACC
ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

B2 sense

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

strand siRNA

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

96, antisense

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

126

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGTGA

GGAAGTTTCAGAACAGTACTTG custom-character

TTTATCTTAGAGGCATATCCCTTTTATCTTAGAGGCATATCCC

T

33
Compound B5

GCCACC
ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

B5 sense

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

strand siRNA

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

98, antisense

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

128

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGGGA

AGGTTACATCAGATCATACTTG custom-character

TTTATCTTAGAGGCATATCCCTTTTATCTTAGAGGCATATCCC

T

34
Compound B6

GCCACC
ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

5′ to 3′:

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

B6-1 sense

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

strand siRNA

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

99, antisense

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

129;

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

B6-2 sense

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

strand siRNA

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

100,

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

antisense

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

130;

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

B6-3 sense

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

strand siRNA

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

101,

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGCAG

antisense 131

CTGAGGCCATTATATGAACTTG custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGGACCCAGG

AAATGTTCAGAAACTTG custom-character

TTTAT

CTTAGAGGCATATCCCTACGTACCAACAAGGCTGAAAGACCAG

AACAAGAACTTG custom-character

TTTATCTTAG

AGGCATATCCCTTTTATCTTAGAGGCATATCCCT

35
Compound B7

GCCACC
ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

B6 sense

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

strand siRNA

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

99, antisense

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

129

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGCAG

CTGAGGCCATTATATGAACTTG custom-character

TTTATCTTAGAGGCATATCCCTTTTATCTTAGAGGCATATCCC

T

36
Compound B8

GCCACC
ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

5′ to 3′:

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

B8-1 sense

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

strand siRNA

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

102,

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

antisense

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

132;

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

B8-2 sense

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

strand siRNA

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

107,

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

antisense

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

137;

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

B8-3 sense

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

strand siRNA

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGTGT

109,

GACCGAAAGGTAAGATGACTTG custom-character

antisense 139

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGAGGTGATG

AAGTCAGACAAAACTTG custom-character

TTTAT

CTTAGAGGCATATCCCTACGTACCAACAAGCAACTGAGGGAGC

CTTGAATACTTG custom-character

TTTATCTTAG

AGGCATATCCCTTTTATCTTAGAGGCATATCCCT

37
Compound B9

GCCACC
ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

B9 sense

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

strand siRNA

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

107,

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

antisense 137

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGAGG

TGATGAAGTCAGACAAAACTTGT custom-character

TTTATCTTAGAGGCATATCCCTTTTATCTTAGAGGCATATCCC

T

38
Compound B10

GCCACC
ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

B10 sense

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

strand siRNA

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

109,

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

antisense 139

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGCAA

CTGAGGGAGCCTTGAATACTTG custom-character

TTTATCTTAGAGGCATATCCCTTTTATCTTAGAGGCATATCCC

T

39
Compound B11

GCCACC
ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

5′ to 3′:

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

B11-1 sense

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

strand siRNA

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

106,

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

antisense

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

136;

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

B11-2 sense

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

strand siRNA

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

107,

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

antisense

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

137;

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

B11-3 sense

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

strand siRNA

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGTTG

108,

CTGATTATTCTGTCCTAACTTG custom-character

antisense 138

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGAGGTGATG

AAGTCAGACAAAACTTG custom-character

TTTAT

CTTAGAGGCATATCCCTACGTACCAACAAGCCGGTAGCACACC

TTGTAATACTTG custom-character

TTTATCTTAG

AGGCATATCCCTTTTATCTTAGAGGCATATCCCT

40
Compound B12

GCCACC
ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

5′ to 3′:

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

B12-1 sense

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

strand siRNA

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

103,

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

antisense

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

133;

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

B12-2 sense

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

strand siRNA

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

104,

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

antisense

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

134;

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

B12-3 sense

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

strand siRNA

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAATTTA

105,

AATATTGGGATCAGACACTTG custom-character

TT

antisense 135

TATCTTAGAGGCATATCCCTACGTACCAACAAAAGAATAGAGC

TCGCACACTTG custom-character

TTTATCTTAGAGGCA

TATCCCTACGTACCAACAAACTGTTGATTCATCACAGGGACTT

GCCC custom-character

TTTATCTTAGAGGCATATCCCT

TTTATCTTAGAGGCATATCCCT

41
Compound B13

GCCACC
ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

B13 sense

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

strand siRNA

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

104,

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

antisense 134

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAAAGA

ATAGAGCTCGCACACTTG custom-character

TTTATCTT

AGAGGCATATCCCTTTTATCTTAGAGGCATATCCCT

42
Compound B14

GCCACC

ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

B14 sense

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

strand siRNA

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

102,

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

antisense 132

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGTGT

GACCGAAAGGTAAGATGACTTG custom-character

TTTATCTTAGAGGCATATCCCTTTTATCTTAGAGGCATATCCC

T

43
Compound B15

GCCACC
ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGC

5′ to 3′:

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

B15-1 sense

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

strand siRNA

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

94, antisense

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

124;

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

B15-2 sense

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

strand siRNA

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

99, antisense

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

129;

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

B15-3 sense

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

strand siRNA

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

109,

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

antisense 139

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGAGG

AGACTTGCCTGGTGAAAACTTG custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGCAGCTGAG

GCCATTATATGAACTTG custom-character

TTTAT

CTTAGAGGCATATCCCTACGTACCAACAAGCAACTGAGGGAGC

CTTGAATACTTG custom-character

TTTATCTTAG

AGGCATATCCCTTTTATCTTAGAGGCATATCCCT

44
Compound

GCCACC
ATGCTCCTGATCTGCCTGCTGGTGATTGCCCTGCTGC

5′ to 3′:
B16*

TGTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

B16-1 sense

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

strand siRNA

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

94, antisense

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

124;

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

B16-2 sense

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

strand siRNA

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

99, antisense

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

129;

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

B16-3 sense

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

strand siRNA

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

109,

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

antisense 139

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGAGG

AGACTTGCCTGGTGAAAACTTG custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGCAGCTGAG

GCCATTATATGAACTTG custom-character

TTTAT

CTTAGAGGCATATCCCTACGTACCAACAAGCAACTGAGGGAGC

CTTGAATACTTG custom-character

TTTATCTTAG

AGGCATATCCCTTTTATCTTAGAGGCATATCCCT

45
Compound

GCCACC
ATGCTCCTGAAGCTCCTGCTGGTGATTGCCCTGCTGG

5′ to 3′:
B17*

CCTGCTTCAGCACAACAGCCCTGAGCATGAGCTACAACCTGCT

B17-1 sense

GGGCTTCCTGCAGCGGAGCAGCAACTTCCAGTGCCAGAAACTG

strand siRNA

CTGTGGCAGCTGAACGGCCGGCTGGAATACTGCCTGAAGGACC

94, antisense

GGATGAACTTCGACATCCCCGAGGAAATCAAGCAGCTGCAGCA

124;

GTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGCTG

B17-2 sense

CAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAG

strand siRNA

GCTGGAACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTA

99, antisense

CCACCAGATCAACCACCTGAAAACCGTGCTGGAAGAGAAGCTG

129;

GAAAAAGAGGACTTCACCCGGGGCAAGCTGATGAGCAGCCTGC

B17-3 sense

ACCTGAAGCGGTACTACGGCAGAATCCTGCACTACCTGAAGGC

strand siRNA

CAAAGAGTACAGCCACTGCGCCTGGACCATCGTGCGCGTGGAA

109,

ATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCTACC

antisense 139

TGAGAAACTGAATAGTGAGTCGTATTAACGTACCAACAAGAGG

AGACTTGCCTGGTGAAAACTTG custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGCAGCTGAG

GCCATTATATGAACTTG custom-character

TTTAT

CTTAGAGGCATATCCCTACGTACCAACAAGCAACTGAGGGAGC

CTTGAATACTTG custom-character

TTTATCTTAG

AGGCATATCCCTTTTATCTTAGAGGCATATCCCT

46
Compound B18

GCCACC
ATGTCTAGCAGCTCTTGGCTGCTGCTGTCTCTGGTGG

5′ to 3′:

CTGTGACAGCCGCTCAGAGCACCATTGAGGAACAGGCCAAGAC

B18-1 sense

CTTCCTGGACAAGTTCAACCACGAGGCCGAGGACCTGTTCTAC

strand siRNA

CAGTCTAGCCTGGCCAGCTGGAACTACAACACCAACATCACCG

102,

AAGAGAACGTGCAGAACATGAACAACGCCGGCGACAAGTGGAG

antisense

CGCCTTCCTGAAAGAGCAGAGCACACTGGCCCAGATGTACCCT

132;

CTGCAAGAGATCCAGAACCTGACCGTGAAGCTCCAGCTGCAGG

B18-2 sense

CCCTCCAGCAGAATGGAAGCTCTGTGCTGAGCGAGGACAAGAG

strand siRNA

CAAGCGGCTGAACACCATCCTGAATACCATGAGCACCATCTAC

107,

AGCACCGGCAAAGTGTGCAACCCCGACAATCCCCAAGAGTGCC

antisense

TGCTGCTGGAACCCGGCCTGAATGAGATCATGGCCAACAGCCT

137;

GGACTACAACGAGAGACTGTGGGCCTGGGAGTCTTGGAGAAGC

B18-3 sense

GAAGTGGGAAAGCAGCTGCGGCCCCTGTACGAGGAATACGTGG

strand siRNA

TGCTGAAGAACGAGATGGCCAGAGCCAACCACTACGAGGACTA

109,

CGGCGACTATTGGAGAGGCGACTACGAAGTGAATGGCGTGGAC

antisense 139

GGCTACGACTACAGCAGAGGCCAGCTGATCGAGGACGTGGAAC

ACACCTTCGAGGAAATCAAGCCTCTGTACGAGCATCTGCACGC

CTACGTGCGGGCCAAGCTGATGAATGCTTACCCCAGCTACATC

AGCCCCATCGGCTGTCTGCCTGCTCATCTGCTGGGAGACATGT

GGGGCAGATTCTGGACCAACCTGTACAGCCTGACAGTGCCCTT

CGGCCAGAAACCTAACATCGACGTGACCGACGCCATGGTGGAT

CAGGCTTGGGATGCCCAGCGGATCTTCAAAGAGGCCGAGAAGT

TCTTCGTGTCCGTGGGCCTGCCTAATATGACCCAAGGCTTCTG

GGAGAACTCCATGCTGACAGACCCCGGCAATGTGCAGAAAGCC

GTGTGTCATCCTACCGCCTGGGATCTCGGCAAGGGCGACTTCA

GAATCCTGATGTGCACCAAAGTGACGATGGACGACTTCCTGAC

AGCCCACCACGAGATGGGCCACATCCAGTACGATATGGCCTAC

GCCGCTCAGCCCTTCCTGCTGAGAAATGGCGCCAATGAGGGCT

TCCACGAAGCCGTGGGAGAGATCATGAGCCTGTCTGCCGCCAC

ACCTAAGCACCTGAAGTCTATCGGACTGCTGAGCCCCGACTTC

CAAGAGGACAACGAGACAGAGATCAACTTCCTGCTCAAGCAGG

CCCTGACCATCGTGGGCACACTGCCCTTTACCTACATGCTGGA

AAAGTGGCGGTGGATGGTCTTTAAGGGCGAGATCCCCAAGGAC

CAGTGGATGAAGAAATGGTGGGAGATGAAGCGCGAGATCGTGG

GCGTTGTGGAACCTGTGCCTCACGACGAGACATACTGCGATCC

TGCCAGCCTGTTTCACGTGTCCAACGACTACTCCTTCATCCGG

TACTACACCCGGACACTGTACCAGTTCCAGTTTCAAGAGGCTC

TGTGCCAGGCCGCCAAGCACGAAGGACCTCTGCACAAGTGCGA

CATCAGCAACTCTACAGAGGCCGGACAGAAACTGTTCAACATG

CTGCGGCTGGGCAAGAGCGAGCCTTGGACACTGGCTCTGGAAA

ATGTCGTGGGCGCCAAGAATATGAACGTGCGGCCACTGCTGAA

CTACTTCGAGCCCCTGTTCACCTGGCTGAAGGACCAGAACAAG

AACAGCTTCGTCGGCTGGTCCACCGATTGGAGCCCTTACGCCG

ACCAGAGCATCAAAGTGCGGATCAGCCTGAAAAGCGCCCTGGG

CGATAAGGCCTATGAGTGGAACGACAATGAGATGTACCTGTTC

CGGTCCAGCGTGGCCTATGCTATGCGGCAGTACTTTCTGAAAG

TCAAGAACCAGATGATCCTGTTCGGCGAAGAGGATGTGCGCGT

GGCCAACCTGAAGCCTCGGATCAGCTTCAACTTCTTCGTGACT

GCCCCTAAGAACGTGTCCGACATCATCCCCAGAACCGAGGTGG

AAAAGGCCATCAGAATGAGCAGAAGCCGGATCAACGACGCCTT

CCGGCTGAACGACAACTCCCTGGAATTCCTGGGCATTCAGCCC

ACACTGGGCCCTCCAAATCAGCCTCCTGTGTCCTAAATAGTGA

GTCGTATTAACGTACCAACAAGTGTGACCGAAAGGTAAGATGA

CTTG custom-character

TTTATCTTAGAGGCATAT

CCCTACGTACCAACAAGAGGTGATGAAGTCAGACAAAACTTG custom-character

TTTATCTTAGAGGCATATCCCTA

CGTACCAACAAGCAACTGAGGGAGCCTTGAATACTTG custom-character

TTTATCTTAGAGGCATATCCCTTTTATC

TTAGAGGCATATCCCT

47
Compound B19

GCCACC
ATGTCTAGCAGCTCTTGGCTGCTGCTGTCTCTGGTGG

5′ to 3′:

CTGTGACAGCCGCTCAGAGCACCATTGAGGAACAGGCCAAGAC

B19-1 sense

CTTCCTGGACAAGTTCAACCACGAGGCCGAGGACCTGTTCTAC

strand siRNA

CAGTCTAGCCTGGCCAGCTGGAACTACAACACCAACATCACCG

106,

AAGAGAACGTGCAGAACATGAACAACGCCGGCGACAAGTGGAG

antisense

CGCCTTCCTGAAAGAGCAGAGCACACTGGCCCAGATGTACCCT

136;

CTGCAAGAGATCCAGAACCTGACCGTGAAGCTCCAGCTGCAGG

B19-2 sense

CCCTCCAGCAGAATGGAAGCTCTGTGCTGAGCGAGGACAAGAG

strand siRNA

CAAGCGGCTGAACACCATCCTGAATACCATGAGCACCATCTAC

107,

AGCACCGGCAAAGTGTGCAACCCCGACAATCCCCAAGAGTGCC

antisense

TGCTGCTGGAACCCGGCCTGAATGAGATCATGGCCAACAGCCT

137;

GGACTACAACGAGAGACTGTGGGCCTGGGAGTCTTGGAGAAGC

B19-3 sense

GAAGTGGGAAAGCAGCTGCGGCCCCTGTACGAGGAATACGTGG

strand siRNA

TGCTGAAGAACGAGATGGCCAGAGCCAACCACTACGAGGACTA

108,

CGGCGACTATTGGAGAGGCGACTACGAAGTGAATGGCGTGGAC

antisense 138

GGCTACGACTACAGCAGAGGCCAGCTGATCGAGGACGTGGAAC

ACACCTTCGAGGAAATCAAGCCTCTGTACGAGCATCTGCACGC

CTACGTGCGGGCCAAGCTGATGAATGCTTACCCCAGCTACATC

AGCCCCATCGGCTGTCTGCCTGCTCATCTGCTGGGAGACATGT

GGGGCAGATTCTGGACCAACCTGTACAGCCTGACAGTGCCCTT

CGGCCAGAAACCTAACATCGACGTGACCGACGCCATGGTGGAT

CAGGCTTGGGATGCCCAGCGGATCTTCAAAGAGGCCGAGAAGT

TCTTCGTGTCCGTGGGCCTGCCTAATATGACCCAAGGCTTCTG

GGAGAACTCCATGCTGACAGACCCCGGCAATGTGCAGAAAGCC

GTGTGTCATCCTACCGCCTGGGATCTCGGCAAGGGCGACTTCA

GAATCCTGATGTGCACCAAAGTGACGATGGACGACTTCCTGAC

AGCCCACCACGAGATGGGCCACATCCAGTACGATATGGCCTAC

GCCGCTCAGCCCTTCCTGCTGAGAAATGGCGCCAATGAGGGCT

TCCACGAAGCCGTGGGAGAGATCATGAGCCTGTCTGCCGCCAC

ACCTAAGCACCTGAAGTCTATCGGACTGCTGAGCCCCGACTTC

CAAGAGGACAACGAGACAGAGATCAACTTCCTGCTCAAGCAGG

CCCTGACCATCGTGGGCACACTGCCCTTTACCTACATGCTGGA

AAAGTGGCGGTGGATGGTCTTTAAGGGCGAGATCCCCAAGGAC

CAGTGGATGAAGAAATGGTGGGAGATGAAGCGCGAGATCGTGG

GCGTTGTGGAACCTGTGCCTCACGACGAGACATACTGCGATCC

TGCCAGCCTGTTTCACGTGTCCAACGACTACTCCTTCATCCGG

TACTACACCCGGACACTGTACCAGTTCCAGTTTCAAGAGGCTC

TGTGCCAGGCCGCCAAGCACGAAGGACCTCTGCACAAGTGCGA

CATCAGCAACTCTACAGAGGCCGGACAGAAACTGTTCAACATG

CTGCGGCTGGGCAAGAGCGAGCCTTGGACACTGGCTCTGGAAA

ATGTCGTGGGCGCCAAGAATATGAACGTGCGGCCACTGCTGAA

CTACTTCGAGCCCCTGTTCACCTGGCTGAAGGACCAGAACAAG

AACAGCTTCGTCGGCTGGTCCACCGATTGGAGCCCTTACGCCG

ACCAGAGCATCAAAGTGCGGATCAGCCTGAAAAGCGCCCTGGG

CGATAAGGCCTATGAGTGGAACGACAATGAGATGTACCTGTTC

CGGTCCAGCGTGGCCTATGCTATGCGGCAGTACTTTCTGAAAG

TCAAGAACCAGATGATCCTGTTCGGCGAAGAGGATGTGCGCGT

GGCCAACCTGAAGCCTCGGATCAGCTTCAACTTCTTCGTGACT

GCCCCTAAGAACGTGTCCGACATCATCCCCAGAACCGAGGTGG

AAAAGGCCATGAGAATGAGCAGAAGCCGGATCAAGGACGCCTT

CCGGCTGAACGACAACTCCCTGGAATTCCTGGGCATTCAGCCC

ACACTGGGCCCTCCAAATCAGCCTCCTGTGTCCTAAATAGTGA

GTCGTATTAACGTACCAACAAGTTGCTGATTATTCTGTCCTAA

CTTG custom-character

TTTATCTTAGAGGCATAT

CCCTACGTACCAACAAGAGGTGATGAAGTCAGACAAAACTTG custom-character

TTTATCTTAGAGGCATATCCCTA

CGTACCAACAAGCCGGTAGCACACCTTGTAATACTTG custom-character

TTTATCTTAGAGGCATATCCCTTTTATC

TTAGAGGCATATCCCT

Bold = Sense siRNA strand

Bold and Italics = Anti-sense siRNA strand

Underline = Signal peptide

Italics = Kozak sequence

*Bolding within the underlined sequence indicates the modified IFN-β signal peptide.

Example 6: In Vitro Transcription of Anti-Viral RNA Constructs and Data Analysis

PCR-based in vitro transcription is carried out using the pMX vectors encoding Compounds B1-B19 to produce mRNA. A transcription template is generated by PCR using the forward and reverse primers in Table 4. The poly(A) tail is encoded in the template resulting in a 120 bp poly(A) tail (SEQ ID NO: 193). Optimizations are made as needed due to achieve specific amplification given the repetitive sequences of siRNA flanking regions. Optimizations include: 1) decreasing the amount of vector DNA, 2) changing the DNA polymerase (Q5 hot start polymerase, New England Biolabs), 3) reducing denaturation time (30 seconds to 10 seconds) and extension time (45 seconds/kb to 10 seconds/kb) for each cycle of PCR, 4) increasing the annealing time (10 seconds to 30 seconds) for each cycle of PCR, and 5) increasing the final extension time (up to 15 minutes) for each cycle of PCR. In addition, to avoid non-specific primer binding, the PCR reaction mixture is prepared on ice, including thawing reagents, and the number of PCR cycles is reduced to 25.

For in vitro transcription, T7 RNA polymerase (MEGAscript kit, Thermo Fisher Scientific) is used at 37° C. for 2 hours. Synthesized RNAs are chemically modified with 100% N1-methylpseudo-UTP and co-transcriptionally capped with an anti-reverse CAP analog (ARCA; [m₂^7,3′-OG(5)ppp(5′)G]) at the 5′ end (Jena Bioscience). After in vitro transcription, the mRNAs are column-purified using MEGAclear kit (Thermo Fisher Scientific) and quantified using Nanophotometer-N60 (Implen).

Using in vitro transcription, Compounds B1-B17 are generated and tested for target mRNA/protein down regulation and gene of interest/protein of interest expression and compared with overexpression models wherein the gene of interest/protein of interest is overexpressed.

Data are analyzed using GraphPad Prism 8 (San Diego, USA). For the estimation of protein levels using ELISA in the standard or the sample, the mean absorbance value of the blank is subtracted from the mean absorbance of the standards or the samples. A standard curve is generated and plotted using a four parameters nonlinear regression according to manufacturer's protocol. To determine the concentration of a protein in each sample, the concentration of each protein is interpolated from the standard curve. The final protein concentration of the sample is calculated by multiplication with the dilution factor. Statistical analyses are carried out using a Student's t-test. The percent of GFP positive cells is calculated using SoftMax Pro tool. Relative quantification of viral RNA by qPCR are analyzed by pair-wise fixed reallocation randomization tests with REST 2009 software.

Example 7: A549 Cell IFN-Beta Overexpression Model

In Vitro Transfection of A549 Cells with IFN-Beta Overexpression Compounds

A549 cells are typical alveolar type II (ATII) cells derived from human lung carcinoma. Since COVID-19 mortality primarily is associated with respiratory illness due to the high viral entry receptor (ACE2) expression in host ATII cells, A549 cells are used to mimic the clinical situation. The A549 cells (Sigma-Aldrich, Buchs Switzerland Cat. #6012804) will be maintained on Dulbecco's Modified Eagle's medium-high glucose (DMEM, Sigma-Aldrich, Buchs Switzerland cat #D0822) supplemented with 10% FBS (Thermofischer, Basel, Switzerland cat #10500-064). To assess the IFN-beta expression the A549 cells are plated at a density of 10,000 cells/well in a regular growth medium 24 hours prior to transfection. Thereafter, cells are transfected with Compounds B1-19 (0.3-0.6 micrograms) using Lipofectamine 2000 (www.invitrogen.com) following the manufacturer's instructions. 100 μl of DMEM are removed and 50 μl of Opti-MEM (www.thermofisher.com) are added to each well followed by 50 μl mRNA and Lipofectamine 2000 complex in Opti-MEM. After 5 hours of incubation, the medium is replaced by fresh growth medium and the plates are incubated for 24 hours at 37° C. in a humidified atmosphere containing 5% CO₂followed by IFN-beta quantification by ELISA (Human IFN-beta bioluminescent ELISA kit 2.0, Cat. Code: luex-hifnbv2, Invivogen).

Example 8: Endogenous IL-6 Stimulation Model in A549 Cells

In Vitro Transfection of A549 Cells with IL-6 Suppressing Compounds

For the endogenous secretion of IL-6 in A549 cells, A549 cells are stimulated with recombinant human IL1-beta (20 ng/mL; Cat. Code: rcyec-hil1b; Invivogen) and recombinant human TNF-alpha (20 ng/mL; Cat. Code: rcyc-htnfa; Invivogen) and incubated for 120 minutes. The induced production of IL-6 corresponds to the physiological conditions observed in COVID-19. Post stimulation, 50 μl of media are removed and replaced with the transfection complex containing specific mRNA constructs (Compounds B1, B2, B15, B16 and B17) complexed with Lipofectamine 2000 in Opti-MEM and incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours followed by IL-6 quantification by ELISA (ThermoFisher Scientific, cat #88-7066-22). A reduction in IL-6 compared to untreated samples is confirmed. To verify the functional suppression of IL-6, HEK-Blue™ IL-6 reporter cells stably transfected with IL-6R and a STAT3-inducible SEAP reporter gene (cat. Code: hkb-hil6, Invivogen) are used. The cell culture supernatant of the IL-6 stimulated samples with or without treatment is measured for bioactive human IL-6 to determine that due to the siRNA mediated interference, the cell culture supernatant with the treatment of Compounds B1, B2, B15, B16 and B17 leads to reduced bioactive human IL-6 compared to untreated control. The cell supernatant is used to quantitatively measure IFN-beta by ELISA (Human IFN-beta bioluminescent ELISA kit 2.0, Cat. Code: luex-hifnbv2, Invivogen).

Example 9: Endogenous IL-6R Suppression Model in THP-1 Cells

In Vitro Transfection of THP-1 Cells with IL-6R Suppressing Compounds

A549 cells do not express IL-6R endogenously, therefore THP-1 cells are used due to their high endogenous expression of the receptor (54×, www.proteinatlas.org). Human monocyte leukemia cell line THP-1 (Sigma-Aldrich, Cat. #88081201) is maintained in growth medium (RPMI 1640 supplemented with 10% FBS and 2 mM glutamine). The cells are seeded at 30,000 THP-1 cells in a 96-well cell culture plate 72 hours before transfection, and activated with 50 nM of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Cat. #P8139) diluted in growth medium. The cells are transfected with Compounds B3-B5 (300-1200 ng/well) using Lipofectamine 2000 (Thermo Fisher Scientific). 100 μl of DMEM is removed from each well and replaced with 50 μl of Opti-MEM (Thermo Fisher Scientific) and 50 μl mRNA and Lipofectamine 2000 complex in Opti-MEM. After 5 hours, the medium is replaced with fresh growth medium supplemented with 50 nM PMA and the plates are incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours. After infection, cell culture supernatant (ThermoFisher Scientific, cat #BMS214) and cell lysate are processed (LSBio, cat #LS-F1001) to quantitatively detect IL-6R by ELISA. To verify the functional suppression of IL-6R, HEK-Blue™ IL-6 reporter cells stably transfected with IL-6R and a STAT3-inducible SEAP reporter gene (cat. Code: hkb-hil6, Invivogen) are used. Since transfection of Compounds B3-B5 leads to siRNA mediated suppression of IL-6R in HEK-Blue™ cells, the addition of recombinant human IL-6 (cat. Code:rcyec-hil6, Invivogen) does not activate the STAT-3 inducible SEAP reporter gene. This is an effective functional assay to validate the blockade of IL-6R signalling pathway. The cell supernatant is used to quantitatively measure IFN-beta by ELISA (Human IFN-beta bioluminescent ELISA kit 2.0, Cat. Code: luex-hifnbv2, Invivogen).

Example 10: ACE2 Overexpression Model in A549 Cells

In Vitro Transfection of A549 Cells with ACE2 mRNA and ACE2 Suppressing/IFN-Beta Overexpression Compounds

An ACE2 overexpression model is used to evaluate simultaneous ACE2 RNA interference (RNAi) and IFN-beta overexpression by mRNA Compounds B6, B7, B15, B16 and B17 in A549 cells. The model is established by transfection with ACE2 mRNA (from SEQ ID NO: 57). Each sample of cells is co-transfected with one of the mRNA Compounds B6, B7, B15, B16 and B17 (300-900 ng/well), and ACE2 mRNA (300 ng/well). Post transfection, the cells are incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours, followed by quantification of ACE2 (target mRNA to downregulate) and IFN-beta (gene of interest to overexpress) by ELISA in the cell culture supernatant (Aviva Systems Biology, cat #OKBB00649).

Example 11: SARS CoV-2 Spike Protein Overexpression Model in A549 Cells

In Vitro Transfection of A549 Cells with SARS CoV-2 Spike Protein mRNA and SARS CoV-2 Spike Protein Suppressing/IFN-Beta Overexpression Compounds

A SARS CoV-2 Spike (S) protein overexpression model is used to evaluate simultaneous SARS CoV-2 Spike protein RNA interference (RNAi) and IFN-beta overexpression by mRNA Compounds B8, B9, B11, B15, B16 and B17 in A549 cells. The model is established by transfection with mRNA encoding the receptor binding domain (RBD) of SARS CoV-2 spike protein (S-RBD, SEQ ID NO: 60). Each sample of cells is co-transfected with one of the mRNA Compounds B8, B9, B11, B15, B16 and B17 (300-900 ng/well), and S-RBD mRNA (300 ng/well). Post transfection, the cells are incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours, followed by quantification of S-RBD by ELISA (Sino biological, cat #KIT40591). Simultaneously, the IFN-beta expression is measured by ELISA in the cell culture supernatant (Human IFN-beta bioluminescent ELISA kit 2.0, Cat. Code: luex-hifnbv2, Invivogen).

Example 12: SARS CoV-2 Nucleocapsid Protein Overexpression Model in A549 Cells

In Vitro Transfection of A549 Cells with SARS CoV-2 Nucleocapsid Protein mRNA and SARS CoV-2 Nucleocapsid Protein Suppressing/IFN-Beta Overexpression Compounds

A SARS CoV-2 Spike protein overexpression model is used to evaluate simultaneous SARS CoV-2 Nucleocapsid (N) protein RNAi suppression and IFN-beta overexpression by mRNA Compounds B8 and B10 in A549 cells. The model is established by transfection with mRNA encoding the complete coding domain of SARS CoV-2 N protein (SEQ ID NO: 62) tagged with 3′ eGFP. In a separate, additional, approach, the SARS CoV-2 N protein is overexpressed from a plasmid (pcDNA3+vector) thereby providing two independent systems to evaluate the effect of RNAi suppression by Compounds B8 and B10. The RNAi of Compounds B8 and B10 targeting SARS CoV-2 N protein disrupt the eGFP translation and expression.

Each sample of cells (mRNA-transfected cells or cells carrying the plasmid) is co-transfected with one of the mRNA Compounds B8 and B10 (300-900 ng/well), and SARS CoV-2 N mRNA (300 ng/well).

Post transfection, the cells are incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours, followed by quantification of SARS CoV-2 N protein by ELISA (Sino biological, cat #KIT40588). Simultaneously, the IFN-beta expression is measured by ELISA in the cell culture supernatant (Human IFN-beta bioluminescent ELISA kit 2.0, Cat. Code: luex-hifnbv2, Invivogen). To determine whether RNAi suppression by Compounds B8 and B10 leads to the disruption of eGFP translation, the SARS CoV-2 Nucleocapsid proteins tagged with eGFP (from expression of both plasmid and mRNA), are microscopically examined for eGFP expression using SpectraMax i3X multi-mode microplate reader (Molecular Devices). The percentage of eGFP positive cells is calculated in treated and control untreated samples.

Example 13: SARS CoV-2 Nsp1 Overexpression Model in A549 Cells

In Vitro Transfection of A549 Cells with SARS CoV-2 Nonstructural Protein mRNA and SARS CoV-2 Nonstructural Protein Suppressing/IFN-Beta Overexpression Compounds

A genome sequence alignment of SARS CoV-2 with SARS CoV and MERS-CoV at the RNA level showed less conservation than an amino acid comparison. Phylogenetic tree analysis (Genetic distance model: Tamura-Nei; Tree build method: UPGMA) showed that MERS-CoV has high level of dissimilar RNA sequence (>45%) whereas SARS CoV and SARS CoV-2 exhibited low level of dissimilarity (up to 21%) (See FIG. 11). We aligned SARS CoV with SARS CoV-2 separately and searched for conserved minimum 20 bp loci for siRNA design. We identified a 47 bp homology near the beginning of viral genome (235-281 bp) which we used to design siRNA (Compounds B8 and B14). The siRNA is located at the first codon (ATG) of the non-structural protein 1 (Nsp1). Targeting the first codon (methionine; AUG) of viral genome ideally lead to huge impact on viral replication as next methionine (AUG) base located 84 amino acids distant to initiate alternative translation.

A SARS CoV-2 Nsp1 overexpression model is used to evaluate simultaneous SARS CoV-2 Nsp1 RNAi suppression and IFN-beta overexpression by mRNA Compounds B8 and B14 in A549 cells.

The model is established by transfection with mRNA encoding the partial domain (first 100 amino acids) of SARS CoV-2 Nsp1 (SEQ ID NO: 64) tagged with 3′ eGFP. In a separate, additional, approach, SARS CoV-2 Nsp1 is overexpressed from a plasmid (pcDNA3+vector) thereby providing two independent systems to evaluate the effect of RNAi suppression by Compounds B8 and B14. The RNAi of Compounds B8 and B14 targeting SARS CoV-2 Nsp1 disrupt the eGFP translation and expression.

Each sample of cells (mRNA-transfected cells or cells carrying the plasmid) is co-transfected with one of the mRNA Compounds B8 and B14 (300-900 ng/well), and SARS CoV-2 Nsp1 mRNA (300 ng/well).

Post transfection, the cells are incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours. To determine whether RNAi suppression by Compounds B8 and B14 leads to the disruption of eGFP translation, the SARS CoV-2 Nsp1 tagged with eGFP (from expression of both plasmid and mRNA), are microscopically examined for eGFP expression using SpectraMax i3X multi-mode microplate reader (Molecular Devices). The percentage of eGFP positive cells is calculated in treated and control untreated samples. Simultaneously, the IFN-beta expression is measured by ELISA in the cell culture supernatant (Human IFN-beta bioluminescent ELISA kit 2.0, Cat. Code: luex-hifnbv2, Invivogen). To determine whether RNAi suppression by Compounds B8 and B14 leads to the disruption of eGFP translation, the SARS CoV-2 Nucleocapsid proteins tagged with eGFP (from expression of both plasmid and mRNA), are microscopically examined for eGFP expression. The percentage of eGFP positive cells is calculated in treated and control untreated samples.

Example 14: Design of Nsp12-Nsp13 siRNA Targeting SARS CoV-2, SARS-CoV and MERS-CoV mRNA, and Nsp12-Nsp13 Overexpression Model in A549 Cells

Design of Nsp12-Nsp13 siRNA Targeting SARS CoV-2, SARS-CoV and MERS-CoV mRNA

To design siRNAs that target all three of SARS CoV-2, SARS-CoV and MERS-CoV, we identified siRNA of as short as 17 bp, tolerating up to 1 mismatch among the sequences. Using this relaxed approach we designed one siRNA of 17 bp in length (between 14299-14318, referenced to SARS CoV-2 genome) and two additional siRNAs each having one bp mismatch tolerance among the three genomic sequences (15091-15107 and 17830-17849, referenced to SARS CoV-2 genome), combining them in a construct with IFN-beta overexpression.

A SARS CoV-2 Nsp12-13 overexpression model is used to evaluate simultaneous SARS CoV-2 Nsp12-13 RNAi suppression and IFN-beta overexpression by mRNA Compounds B12 and B13 in A549 cells. The model is established by transfection with mRNA encoding a non-coding domain of NSP12 and NSP13 (14202-17951 bp; 3749 bp) of SARS CoV-2 genome (SEQ ID NO: 67) tagged with 3′ eGFP. Each sample of cells (mRNA-transfected cells or cells carrying the plasmid) is co-transfected with one of the mRNA Compounds B12 and B13 (300-900 ng/well), and SARS CoV-2 NSP12 and NSP-13 partial genomic RNA (300 ng/well).

Post transfection, the cells are incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours, followed by Taqman-qPCR based assays to assess the viral RNA degradation, as compared to untransfected control. Simultaneously, the IFN-beta expression is measured by ELISA in the cell culture supernatant (Human IFN-beta bioluminescent ELISA kit 2.0, Cat. Code: luex-hifnbv2, Invivogen).

Example 15: A549 Cell ACE2 Soluble Receptor Overexpression Model

In Vitro Transfection of A549 Cells with ACE2 Soluble Receptor Overexpression Compounds

A549 cells are typical alveolar type II (ATII) cells derived from human lung carcinoma. Since COVID-19 mortality primarily is associated with respiratory illness due to the high viral entry receptor (ACE2) expression in host ATII cells, A549 cells are used to mimic the clinical situation. The A549 cells (Sigma-Aldrich, Buchs Switzerland Cat. #6012804) are maintained on Dulbecco's Modified Eagle's medium-high glucose (DMEM, Sigma-Aldrich, Buchs Switzerland cat #D0822) supplemented with 10% FBS (Thermofischer, Basel, Switzerland cat #10500-064). To assess the ACE2 soluble receptor expression the A549 cells are plated at a density of 10,000 cells/well in a regular growth medium 24 hours prior to transfection. Thereafter, cells are transfected with Compounds B18 and B19 (0.3-0.6 micrograms) using Lipofectamine 2000 (www.invitrogen.com) following the manufacturer's instructions. 100 μl of DMEM are removed and 50 μl of Opti-MEM (www.thermofisher.com) are added to each well followed by 50 μl mRNA and Lipofectamine 2000 complex in Opti-MEM. After 5 hours of incubation, the medium is replaced by fresh growth medium and the plates are incubated for 24 hours at 37° C. in a humidified atmosphere containing 5% CO₂followed by ACE2 quantification by ELISA (Aviva Systems Biology, cat #OKBB00649). The anti-viral activity of Compound B18 and Compound B19 are investigated in Examples 11-13.

Example 16: Additional Constructs

Construct Design, Sequence, and Synthesis

Details of construct design and synthesis are described in Example 1. Table 8 summarizes additional compounds used in the examples in the present disclosure with their respective siRNA target to downregulate protein expression, and protein target for upregulated protein expression. The sequences of the constructs of A9-A15 are shown in Table 9 and annotated as indicated in the table below. All uridines in Compounds A9-A15 used in the examples described herein were modified to N¹-methylpseudouridine. For each compound, the position of siRNA sequence is indicated in regard to the gene of interest. For example, “5′ siRNA position” indicates that siRNA sequences are upstream of or 5′ to the gene of interest in the compound. Conversely, “3′ siRNA position” indicates that siRNA sequences are downstream of or 3′ to the gene of interest in the compound. The plasmid sequences of the constructs of A9-A15 are shown in Table 10.

TABLE 8

Summary of Compounds A9-A15

Compound
siRNA
siRNA
# of
Protein Target

ID
Target
Position
siRNAs
(gene of interest)
Indication

A9
TNF-alpha
5’
3
IL-4
Psoriasis

A10
TNF-alpha
3’
3
IL-4
Psoriasis

A11
ALK2
3’
3
IGF-1
FOP

A12
SOD1
5’
3
IGF-1
ALS

A13
SOD1
5’
3
EPO
ALS

A14
IL-1 beta
5’
3
IGF-1
OA, IVDD

A15
IL-1 beta
3’
3
IGF-1
OA, IVDD

FOP: Fibrodysplasia ossificans progressiva;

ALS: Amyotrophic lateral sclerosis;

OA: Osteoarthritis;

IVDD: Intervertebral disc disease

TABLE 9

Sequences of Compounds A9-A15

SEQ ID NO:
Compound #
Sequence (5′→3′ direction)

152
Compound A9
ATAGTGAGTCGTATTAACGTACCAACAAGGCGTGGAGCTGAGAGATAAA

5′ to 3′:

CTTG custom-character

TTTATCTTAGAGGCATATCCCTACG

A9-1 sense

TACCAACAAGGGCCTGTACCTCATCTACTACTTG custom-character

strand siRNA

custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGGTATGAGCC

87, antisense

CATCTATCTACTTG custom-character

TTTATCTTAGAGGCAT

117;

ATCCCTGCCACCATGGGACTGACATCTCAACTGCTGCCTCCACTGTTCT

A9-2 sense

TTCTGCTGGCCTGCGCCGGCAATTTTGTGCACGGCCACAAGTGCGACAT

strand siRNA

CACCCTGCAAGAGATCATCAAGACCCTGAACAGCCTGACCGAGCAGAAA

88, antisense

ACCCTGTGCACCGAGCTGACCGTGACCGATATCTTTGCCGCCAGCAAGA

118;

ACACAACCGAGAAAGAGACATTCTGCAGAGCCGCCACCGTGCTGAGACA

A9-3 sense

GTTCTACAGCCACCACGAGAAGGACACCAGATGCCTGGGAGCTACAGCC

strand siRNA

CAGCAGTTCCACAGACACAAGCAGCTGATCCGGTTCCTGAAGCGGCTGG

89, antisense

ACAGAAATCTGTGGGGACTCGCCGGCCTGAATAGCTGCCCTGTGAAAGA

119

GGCCAACCAGTCTACCCTGGAAAACTTCCTGGAACGGCTGAAAACCATC

ATGCGCGAGAAGTACAGCAAGTGCAGCAGCTGATTTATCTTAGAGGCAT

ATCCCT

153
Compound A10

GCCACC
ATGGGACTGACATCTCAACTGCTGCCTCCACTGTTCTTTCTGC

5′ to 3′:

TGGCCTGCGCCGGCAATTTTGTGCACGGCCACAAGTGCGACATCACCCT

A10-1 sense

GCAAGAGATCATCAAGACCCTGAACAGCCTGACCGAGCAGAAAACCCTG

strand siRNA

TGCACCGAGCTGACCGTGACCGATATCTTTGCCGCCAGCAAGAACACAA

87, antisense

CCGAGAAAGAGACATTCTGCAGAGCCGCCACCGTGCTGAGACAGTTCTA

117;

CAGCCACCACGAGAAGGACACCAGATGCCTGGGAGCTACAGCCCAGCAG

A10-2 sense

TTCCACAGACACAAGCAGCTGATCCGGTTCCTGAAGCGGCTGGACAGAA

strand siRNA

ATCTGTGGGGACTCGCCGGCCTGAATAGCTGCCCTGTGAAAGAGGCCAA

88, antisense

CCAGTCTACCCTGGAAAACTTCCTGGAACGGCTGAAAACCATCATGCGC

118;

GAGAAGTACAGCAAGTGCAGCAGCTGAATAGTGAGTCGTATTAACGTAC

A10-3 sense

CAACAAGGCGTGGAGCTGAGAGATAAACTTG custom-character

strand siRNA

custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGGGCCTGTACCTC

89, antisense

ATCTACTACTTG custom-character

TTTATCTTAGAGGCATA

119

TCCCTACGTACCAACAAGGTATGAGCCCATCTATCTACTTG custom-character

TTTATCTTAGAGGCATATCCCTTTTATCTTAGAGGCAT

ATCCCT

154
Compound A11

GCCACC
ATGACCATCCTGTTTCTGACAATGGTCATCAGCTACTTCGGCT

5′ to 3′:

GCATGAAGGCCGTGAAGATGCACACCATGAGCAGCAGCCACCTGTTCTA

A11-1 sense

TCTGGCCCTGTGCCTGCTGACCTTTACCAGCTCTGCTACCGCCGGACCT

strand siRNA

GAGACACTTTGTGGCGCTGAACTGGTGGACGCCCTGCAGTTTGTGTGTG

140,

GCGACAGAGGCTTCTACTTCAACAAGCCCACAGGCTACGGCAGCAGCTC

antisense

TAGAAGGGCTCCTCAGACCGGAATCGTGGACGAGTGCTGCTTCAGAAGC

146; A11-2

TGCGACCTGCGGCGGCTGGAAATGTATTGTGCCCCTCTGAAGCCTGCCA

sense strand

AGAGCGCCTAAATAGTGAGTCGTATTAACGTACCAACAAGGCCTCATTA

siRNA 141,

TTCTCTCTACTTG custom-character

TTTATCTTAGAGGCATAT

antisense

CCCTACGTACCAACAAGTGTTCGCAGTATGTCTTACTTG custom-character

147; A11-3

custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGCCTGCC

sense strand

TGCTGGGAGTTACTTG custom-character

TTTATCTTAGAGGCA

siRNA 142,

TATCCCTTTTATCTTAGAGGCATATCCCT

antisense 148

155
Compound A12
ATAGTGAGTCGTATTAACGTACCAACAAGAAGGAAAGTAATGGACCAGT

5′ to 3′:

ACTTG custom-character

TTTATCTTAGAGGCATATCCCTA

A12-1 sense

CGTACCAACAAGGTCCTCACTTTAATCCTCTAACTTG custom-character

strand siRNA

custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGGAGAC

143,

TTGGGCAATGTGACTACTTG custom-character

TTTATCTT

antisense

AGAGGCATATCCCTGCCACCATGGGCAAGATTAGCAGCCTGCCTACACA

149; A12-2

GCTGTTCAAGTGCTGCTTCTGCGACTTCCTGAAAGTGAAGATGCACACC

sense strand

ATGAGCAGCAGCCACCTGTTCTATCTGGCCCTGTGCCTGCTGACCTTTA

siRNA 144,

CCAGCTCTGCTACCGCCGGACCTGAGACACTTTGTGGCGCTGAACTGGT

antisense

GGACGCCCTGCAGTTTGTGTGTGGCGACAGAGGCTTCTACTTCAACAAG

150; A12-3

CCCACAGGCTACGGCAGCAGCTCTAGAAGGGCTCCTCAGACCGGAATCG

sense strand

TGGACGAGTGCTGTTTCAGAAGCTGCGACCTGCGGCGGCTGGAAATGTA

siRNA 145,

TTGTGCCCCTCTGAAGCCTGCCAAGAGCGCCTAATTTATCTTAGAGGCA

antisense 151

TATCCCT

156
Compound A13
ATAGTGAGTCGTATTAACGTACCAACAAGAAGGAAAGTAATGGACCAGT

5′ to 3′:

ACTTG custom-character

TTTATCTTAGAGGCATATCCCTA

A13-1 sense

CGTACCAACAAGGTCCTCACTTTAATCCTCTAACTTG custom-character

strand siRNA

custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGGAGAC

143,

TTGGGCAATGTGACTACTTG custom-character

TTTATCTT

antisense

AGAGGCATATCCCTGCCACCATGGGAGTGCATGAATGTCCTGCTTGGCT

149; A13-2

GTGGCTGCTGCTGAGCCTGCTGTCTCTGCCTCTGGGACTGCCTGTTCTT

sense strand

GGAGCCCCTCCTAGACTGATCTGCGACAGCAGAGTGCTGGAAAGATACC

siRNA 144,

TGCTGGAAGCCAAAGAGGCCGAGAACATCACCACAGGCTGTGCCGAGCA

antisense

CTGCAGCCTGAACGAGAATATCACCGTGCCTGAGACCAAAGTGAACTTC

150; A13-3

TACGCCTGGAAGCGGATGGAAGTGGGCCAGCAGGCTGTGGAAGTTTGGC

sense strand

AAGGACTGGCCCTGCTGAGCGAAGCTGTTCTGAGAGGACAGGCTCTGCT

siRNA 145,

GGTCAACAGCTCTCAGCCTTGGGAACCTCTGCAACTGCACGTGGACAAG

antisense 151

GCCGTGTCTGGCCTGAGAAGCCTGACCACACTGCTGAGAGCACTGGGAG

CCCAGAAAGAGGCCATCTCTCCACCTGATGCTGCCTCTGCTGCCCCTCT

GAGAACCATCACCGCCGACACCTTCAGAAAGCTGTTCCGGGTGTACAGC

AACTTCCTGCGGGGCAAGCTGAAGCTGTACACAGGCGAGGCTTGCAGAA

CCGGCGACAGATAATTTATCTTAGAGGCATATCCCT

157
Compound A14
ATAGTGAGTCGTATTAACGTACCAACAAGAAAGATGATAAGCCCACTCT

5′ to 3′:

ACTTG custom-character

TTTATCTTAGAGGCATATCCCTA

A14-1 sense

CGTACCAACAAGGTGATGTCTGGTCCATATGAACTTG custom-character

strand siRNA

custom-character

TTTATCTTAGAGGCATATCCCTACGTACCAACAAGATGAT

84, antisense

AAGCCCACTCTAACTTG custom-character

TTTATCTTAGAGGC

114; A14-2

ATATCCCTGCCACCATGGGCAAGATTAGCAGCCTGCCTACACAGCTGTT

sense strand

CAAGTGCTGCTTCTGCGACTTCCTGAAAGTGAAGATGCACACCATGAGC

siRNA 85,

AGCAGCCACCTGTTCTATCTGGCCCTGTGCCTGCTGACCTTTACCAGCT

antisense

CTGCTACCGCCGGACCTGAGACACTTTGTGGCGCTGAACTGGTGGACGC

115; A14-3

CCTGCAGTTTGTGTGTGGCGACAGAGGCTTCTACTTCAACAAGCCCACA

sense strand

GGCTACGGCAGCAGCTCTAGAAGGGCTCCTCAGACCGGAATCGTGGACG

siRNA 86,

AGTGCTGTTTCAGAAGCTGCGACCTGCGGCGGCTGGAAATGTATTGTGC

antisense 116

CCCTCTGAAGCCTGCCAAGAGCGCCTAATTTATCTTAGAGGCATATCCC

T

158
Compound A15

GCCACC
ATGGGCAAGATTAGCAGCCTGCCTACACAGCTGTTCAAGTGCT

5′ to 3′:

GCTTCTGCGACTTCCTGAAAGTGAAGATGCACACCATGAGCAGCAGCCA

A15-1 sense

CCTGTTCTATCTGGCCCTGTGCCTGCTGACCTTTACCAGCTCTGCTACC

strand siRNA

GCCGGACCTGAGACACTTTGTGGCGCTGAACTGGTGGACGCCCTGCAGT

84, antisense

TTGTGTGTGGCGACAGAGGCTTCTACTTCAACAAGCCCACAGGCTACGG

114; A15-2

CAGCAGCTCTAGAAGGGCTCCTCAGACCGGAATCGTGGACGAGTGCTGT

sense strand

TTCAGAAGCTGCGACCTGCGGCGGCTGGAAATGTATTGTGCCCCTCTGA

siRNA 85,

AGCCTGCCAAGAGCGCCTAAATAGTGAGTCGTATTAACGTACCAACAAG

antisense

AAAGATGATAAGCCCACTCTACTTG custom-character

TTT

115; A15-3

ATCTTAGAGGCATATCCCTACGTACCAACAAGGTGATGTCTGGTCCATA

sense strand

TGAACTTG custom-character

TTTATCTTAGAGGCATATCC

siRNA 86,

CTACGTACCAACAAGATGATAAGCCCACTCTAACTTG custom-character

antisense 116

custom-character

TTTATCTTAGAGGCATATCCCTTTTATCTTAGAGGCATATCCC

T

Bold = Sense siRNA strand

Bold and Italics = anti-Sense siRNA strand

Underline = Signal peptide

Italics = Kozak sequence

TABLE 10

Plasmid Sequences for Compounds A9-A15

SEQ ID NO
Compound #
Sequence (5′→3′ direction)

160
Compound A9 in
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATATAGTGAGTCGTATTAACGTACC

AACAAGGCGTGGAGCTGAGAGATAAACTTGTTATCTCTCAGCTCCACGC

CTTTATCTTAGAGGCATATCCCTACGTACCAACAAGGGCCTGTACCTCA

TCTACTACTTGAGTAGATGAGGTACAGGCCCTTTATCTTAGAGGCATAT

CCCTACGTACCAACAAGGTATGAGCCCATCTATCTACTTGAGATAGATG

GGCTCATACCTTTATCTTAGAGGCATATCCCTGCCACCATGGGACTGAC

ATCTCAACTGCTGCCTCCACTGTTCTTTCTGCTGGCCTGCGCCGGCAAT

TTTGTGCACGGCCACAAGTGCGACATCACCCTGCAAGAGATCATCAAGA

CCCTGAACAGCCTGACCGAGCAGAAAACCCTGTGCACCGAGCTGACCGT

GACCGATATCTTTGCCGCCAGCAAGAACACAACCGAGAAAGAGACATTC

TGCAGAGCCGCCACCGTGCTGAGACAGTTCTACAGCCACCACGAGAAGG

ACACCAGATGCCTGGGAGCTACAGCCCAGCAGTTCCACAGACACAAGCA

GCTGATCCGGTTCCTGAAGCGGCTGGACAGAAATCTGTGGGGACTCGCC

GGCCTGAATAGCTGCCCTGTGAAAGAGGCCAACCAGTCTACCCTGGAAA

ACTTCCTGGAACGGCTGAAAACCATCATGCGCGAGAAGTACAGCAAGTG

CAGCAGCTGATTTATCTTAGAGGCATATCCCT
CTGGGCCTCATGGGCCT

TCCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCA

TTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCC

TCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGC

CTAATGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCG

TTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAA

ATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATA

CCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACC

CTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGG

CGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGT

TCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGC

TGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACG

ACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG

GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGC

TACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTA

CCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGC

TGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAA

AAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTC

AGTGGAACGAAAACTGAGGTTAAGGGATTTTGGTCATGAGATTATCAAA

AAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCA

ATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAA

TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGT

TGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCA

TCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTC

CAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAG

TGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGG

GAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTG

CCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTC

ATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATG

TTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAA

GTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAA

TTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAG

TACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCT

CTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTT

AAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGG

ATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCA

ACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAA

AACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAA

TGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC

AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAA

TAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC

161
Compound A10
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

in pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATGCCACCATGGGACTGACATCTCA

ACTGCTGCCTCCACTGTTCTTTCTGCTGGCCTGCGCCGGCAATTTTGTG

CACGGCCACAAGTGCGACATCACCCTGCAAGAGATCATCAAGACCCTGA

ACAGCCTGACCGAGCAGAAAACCCTGTGCACCGAGCTGACCGTGACCGA

TATCTTTGCCGCCAGCAAGAACACAACCGAGAAAGAGACATTCTGCAGA

GCCGCCACCGTGCTGAGACAGTTCTACAGCCACCACGAGAAGGACACCA

GATGCCTGGGAGCTACAGCCCAGCAGTTCCACAGACACAAGCAGCTGAT

CCGGTTCCTGAAGCGGCTGGACAGAAATCTGTGGGGACTCGCCGGCCTG

AATAGCTGCCCTGTGAAAGAGGCCAACCAGTCTACCCTGGAAAACTTCC

TGGAACGGCTGAAAACCATCATGCGCGAGAAGTACAGCAAGTGCAGCAG

CTGAATAGTGAGTCGTATTAACGTACCAACAAGGCGTGGAGCTGAGAGA

TAAACTTGTTATCTCTCAGCTCCACGCCTTTATCTTAGAGGCATATCCC

TACGTACCAACAAGGGCCTGTACCTCATCTACTACTTGAGTAGATGAGG

TACAGGCCCTTTATCTTAGAGGCATATCCCTACGTACCAACAAGGTATG

AGCCCATCTATCTACTTGAGATAGATGGGCTCA
TACCTTTATCTTAGAG

GCATATCCCTTTTATCTTAGAGGCATATCCCTCTGGGCCTCATGGGCCT

TCCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCA

TTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCC

TCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGC

CTAATGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCG

TTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAA

ATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATA

CCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACC

CTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGG

CGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGT

TCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGC

TGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACG

ACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG

GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGC

TACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTA

CCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGC

TGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAA

AAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTC

AGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAA

AAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCA

ATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAA

TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGT

TGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCA

TCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTC

CAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAG

TGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGG

GAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTG

CCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTC

ATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATG

TTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAA

GTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAA

TTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAG

TACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCT

CTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTT

AAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGG

ATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCA

ACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAA

AACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAA

TGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC

AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAA

TAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC

162
Compound A11
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

in pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATGCCACCATGACCATCCTGTTTCT

GACAATGGTCATCAGCTACTTCGGCTGCATGAAGGCCGTGAAGATGCAC

ACCATGAGCAGCAGCCACCTGTTCTATCTGGCCCTGTGCCTGCTGACCT

TTACCAGCTCTGCTACCGCCGGACCTGAGACACTTTGTGGCGCTGAACT

GGTGGACGCCCTGCAGTTTGTGTGTGGCGACAGAGGCTTCTACTTCAAC

AAGCCCACAGGCTACGGCAGCAGCTCTAGAAGGGCTCCTCAGACCGGAA

TCGTGGACGAGTGCTGCTTCAGAAGCTGCGACCTGCGGCGGCTGGAAAT

GTATTGTGCCCCTCTGAAGCCTGCCAAGAGCGCCTAAATAGTGAGTCGT

ATTAACGTACCAACAAGGCCTCATTATTCTCTCTACTTGAGAGAGAATA

ATGAGGCCTTTATCTTAGAGGCATATCCCTACGTACCAACAAGTGTTCG

CAGTATGTCTTACTTGAAGACATACTGCGAACACTTTATCTTAGAGGCA

TATCCCTACGTACCAACAAGCCTGCCTGCTG
GGAGTTACTTGAACTCCC

AGCAGGCAGGCTTTATCTTAGAGGCATATCCCTTTTATCTTAGAGGCAT

ATCCCTCTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAGTCG

GGAAACCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTG

CGTATTGGGCGCTCTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGT

CGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAAAGG

CCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCG

CCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGA

AACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCC

TCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGC

CTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGG

TATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACG

AACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCT

TGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACT

GGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCT

TGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTAT

CTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCT

TGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCA

AGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGAT

CTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG

ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAA

ATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTG

GTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATC

TGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAA

CTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACC

GCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCA

GCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCA

TCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGT

TAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCA

CGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAA

GGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTT

CGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTC

ATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAA

GATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATA

GTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAAT

ACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTT

CTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTC

GATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTC

ACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAA

AGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTT

TCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATAC

ATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACAT

TTCCCCGAAAAGTGCCAC

163
Compound A12
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

in pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATATAGTGAGTCGTATTAACGTACC

AACAAGAAGGAAAGTAATGGACCAGTACTTGACTGGTCCATTACTTTCC

TTCTTTATCTTAGAGGCATATCCCTACGTACCAACAAGGTCCTCACTTT

AATCCTCTAACTTGTAGAGGATTAAAGTGAGGACCTTTATCTTAGAGGC

ATATCCCTACGTACCAACAAGGAGACTTGGGCAATGTGACTACTTGAGT

CACATTGCCCAAGTCTCCTTTATCTTAGAGGCATATCCCTGCCACCATG

GGCAAGATTAGCAGCCTGCCTACACAGCTGTTCAAGTGCTGCTTCTGCG

ACTTCCTGAAAGTGAAGATGCACACCATGAGCAGCAGCCACCTGTTCTA

TCTGGCCCTGTGCCTGCTGACCTTTACCAGCTCTGCTACCGCCGGACCT

GAGACACTTTGTGGCGCTGAACTGGTGGACGCCCTGCAGTTTGTGTGTG

GCGACAGAGGCTTCTACTTCAACAAGCCCACAGGCTACGGCAGCAGCTC

TAGAAGGGCTCCTCAGACCGGAATCGTGGACGAGTGCTGTTTCAGAAGC

TGCGACCTGCGGCGGCTGGAAATGTATTGTGCCCCTCTGAAGCCTGCCA

AGAGCGCCTAATTTATCTTAGAGGCATATCCCT
CTGGGCCTCATGGGCC

TTCCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGC

ATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTC

CTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTG

CCTAATGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGC

GTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAA

AATCGAGGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGAT

ACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGAC

CCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG

GCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCG

TTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCG

CTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACAC

GACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGA

GGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGG

CTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTT

ACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCG

CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA

AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCT

CAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAA

AAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATC

AATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA

ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAG

TTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACC

ATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCT

CCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAA

GTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCG

GGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT

GCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTT

CATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCAT

GTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGA

AGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATA

ATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGA

GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGC

TCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTT

TAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAG

GATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCC

AACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAA

AAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA

ATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTAT

CAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAA

ATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC

164
Compound A13
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

in pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATATAGTGAGTCGTATTAACGTACC

AACAAGAAGGAAAGTAATGGACCAGTACTTGACTGGTCCATTACTTTCC

TTCTTTATCTTAGAGGCATATCCCTACGTACCAACAAGGTCCTCACTTT

AATCCTCTAACTTGTAGAGGATTAAAGTGAGGACCTTTATCTTAGAGGC

ATATCCCTACGTACCAACAAGGAGACTTGGGCAATGTGACTACTTGAGT

CACATTGCCCAAGTCTCCTTTATCTTAGAGGCATATCCCTGCCACCATG

GGAGTGCATGAATGTCCTGCTTGGCTGTGGCTGCTGCTGAGCCTGCTGT

CTCTGCCTCTGGGACTGCCTGTTCTTGGAGCCCCTCCTAGACTGATCTG

CGACAGCAGAGTGCTGGAAAGATACCTGCTGGAAGCCAAAGAGGCCGAG

AACATCACCACAGGCTGTGCCGAGCACTGCAGCCTGAACGAGAATATCA

CCGTGCCTGACACCAAAGTGAACTTCTACGCCTGGAAGCGGATGGAAGT

GGGCCAGCAGGCTGTGGAAGTTTGGCAAGGACTGGCCCTGCTGAGCGAA

GCTGTTCTGAGAGGACAGGCTCTGCTGGTCAACAGCTCTCAGCCTTGGG

AACCTCTGCAACTGCACGTGGACAAGGCCGTGTCTGGCCTGAGAAGCCT

GACCACACTGCTGAGAGCACTGGGAGCCCAGAAAGAGGCCATCTCTCCA

CCTGATGCTGCCTCTGCTGCCCCTCTGAGAACCATCACCGCCGACACCT

TCAGAAAGCTGTTCCGGGTGTACAGCAACTTCCTGCGGGGCAAGCTGAA

GCTGTACACAGGCGAGGCTTGCAG
AACCGGCGACAGATAATTTATCTTA

GAGGCATATCCCTCTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTT

CCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGT

TTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCGCTCACTGACTCGCTGC

GCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAG

CAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATA

GGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAG

GTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA

AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACC

TGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACG

CTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT

GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACT

ATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGC

AGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACA

GAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTAT

TTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGG

TAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTT

GTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATC

CTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACG

TTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATC

CTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGT

AAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTC

AGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTG

TAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAA

TGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAA

CCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCC

GCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTT

CGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGT

GGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAA

CGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTA

GCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTT

ATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCA

TCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCT

GAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACG

GGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGA

AAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGAT

CCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTT

TACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCC

GCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCT

TCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAG

CGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCG

CGCACATTTCCCCGAAAAGTGCCAC

165
Compound A14
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

in pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATAATAGTGAGTCGTATTAACGTAC

CAACAAGAAAGATGATAAGCCCACTCTACTTGAGAGTGGGCTTATCATC

TTTCTTTATCTTAGAGGCATATCCCTACGTACCAACAAGGTGATGTCTG

GTCCATATGAACTTGTCATATGGACCAGACATCACCTTTATCTTAGAGG

CATATCCCTACGTACCAACAAGATGATAAGCCCACTCTAACTTGTAGAG

TGGGCTTATCATCTTTATCTTAGAGGCATATCCCTGCCACCATGGGCAA

GATTAGCAGCCTGCCTACACAGCTGTTCAAGTGCTGCTTCTGCGACTTC

CTGAAAGTGAAGATGCACACCATGAGCAGCAGCCACCTGTTCTATCTGG

CCCTGTGCCTGCTGACCTTTACCAGCTCTGCTACCGCCGGACCTGAGAC

ACTTTGTGGCGCTGAACTGGTGGACGCCCTGCAGTTTGTGTGTGGCGAC

AGAGGCTTCTACTTCAACAAGCCCACAGGCTACGGCAGCAGCTCTAGAA

GGGCTCCTCAGACCGGAATCGTGGACGAGTGCTGTTTCAGAAGCTGCGA

CCTGCGGCGGCTGGAAATGTATTGTGCCCCTCTGAAGCCTGCCAAGAGC

GCCTAATTTATCTTAGAGGCATATCCCT
CTGGGCCTCATGGGCCTTCCG

CTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAA

CATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCGC

TCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAA

TGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGC

TGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCG

ACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAG

GCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGC

CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCT

TTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGC

TCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCG

CCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTT

ATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTAT

GTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACA

CTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT

CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGT

AGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAG

GATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTG

GAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG

ATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCT

AAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAG

TGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCC

TGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTG

GCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGA

TTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGT

CCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAG

CTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCAT

TGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC

AGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGT

GCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAA

GTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCT

CTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACT

CAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTG

CCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAA

GTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCT

TACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTG

ATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACA

GGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTT

GAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGG

TTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAA

CAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC

166
Compound A15
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

in pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATGCCACCATGGGCAAGATTAGCAG

CCTGCCTACACAGCTGTTCAAGTGCTGCTTCTGCGACTTCCTGAAAGTG

AAGATGCACACCATGAGCAGCAGCCACCTGTTCTATCTGGCCCTGTGCC

TGCTGACCTTTACCAGCTCTGCTACCGCCGGACCTGAGACACTTTGTGG

CGCTGAACTGGTGGACGCCCTGCAGTTTGTGTGTGGCGACAGAGGCTTC

TACTTCAACAAGCCCACAGGCTACGGCAGCAGCTCT

AGAAGGGCTCCTC

AGACCGGAATCGTGGACGAGTGCTGTTTCAGAAGCTGCGACCTGCGGCG

GCTGGAAATGTATTGTGCCCCTCTGAAGCCTGCCAAGAGCGCCTAAATA

GTGAGTCGTATTAACGTACCAACAAGAAAGATGATAAGCCCACTCTACT

TGAGAGTGGGCTTATCATCTTTCTTTATCTTAGAGGCATATCCCTACGT

ACCAACAAGGTGATGTCTGGTCCATATGAACTTGTCATATGGACCAGAC

ATCACCTTTATCTTAGAGGCATATCCCTACGTACCAACAAGATGATAAG

CCCACTCTAACTTGTAGAGTGGGCTTATCATCTTTATCTTAGAGGCATA

TCCCTTTTATCTTAGAGGCATATCCCT
CTGGGCCTCATGGGCCTTCCGC

TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAAC

ATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCGCT

CACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAAT

GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCT

GGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA

CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGG

CGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCC

GCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTT

TCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCT

CCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGC

CTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTA

TCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATG

TAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACAC

TAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTC

GGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTA

GCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGG

ATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGG

AACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGA

TCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTA

AAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGT

GAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCT

GACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGG

CCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGAT

TTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTC

CTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGC

TAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATT

GCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCA

GCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTG

CAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAG

TTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTC

TTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTC

AACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGC

CCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAG

TGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTT

ACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGA

TCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAG

GAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTG

AATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGT

TATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAAC

AAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC

159
Compound B18
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGT

in pMA-RQ
TAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT

ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGC

GCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC

AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT

AAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGCGAA

TTGGCGGAAGGCCGTCAAGGCCGCATGCCACCATGTCTAGCAGCTCTTG

GCTGCTGCTGTCTCTGGTGGCTGTGACAGCCGCTCAGAGCACCATTGAG

GAACAGGCCAAGACCTTCCTGGACAAGTTCAACCACGAGGCCGAGGACC

TGTTCTACCAGTCTAGCCTGGCCAGCTGGAACTACAACACCAACATCAC

CGAAGAGAACGTGCAGAACATGAACAACGCCGGCGACAAGTGGAGCGCC

TTCCTGAAAGAGCAGAGCACACTGGCCGAGATGTACCCTCTGCAAGAGA

TCCAGAACCTGACCGTGAAGCTCCAGCTGCAGGCCCTCCAGCAGAATGG

AAGCTCTGTGCTGAGCGAGGACAAGAGCAAGCGGCTGAACACCATCCTG

AATACCATGAGCACCATCTACAGCACCGGCAAAGTGTGCAACCCCGACA

ATCCCCAAGAGTGCCTGCTGCTGGAACCCGGCCTGAATGAGATCATGGC

CAACAGCCTGGACTACAACGAGAGACTGTGGGCCTGGGAGTCTTGGAGA

AGCGAAGTGGGAAAGCAGCTGCGGCCCCTGTACGAGGAATACGTGGTGC

TGAAGAACGAGATGGCCAGAGCCAACCACTACGAGGACTACGGCGACTA

TTGGAGAGGCGACTACGAAGTGAATGGCGTGGACGGCTACGACTACAGC

AGAGGCCAGCTGATCGAGGAGGTGGAACACACCTTCGAGGAAATCAAGC

CTCTGTACGAGCATCTGCACGCCTACGTGCGGGCCAAGCTGATGAATGC

TTACCCCAGCTACATCAGCCCCATCGGCTGTCTGCCTGCTCATCTGCTG

GGAGACATGTGGGGCAGATTCTGGACCAACCTGTACAGCCTGACAGTGC

CCTTCGGCCAGAAACCTAACATCGACGTGACCGACGCCATGGTGGATCA

GGCTTGGGATGCCCAGCGGATCTTCAAAGAGGCCGAGAAGTTCTTCGTG

TCCGTGGGCCTGCCTAATATGACCCAAGGCTTCTGGGAGAACTCCATGC

TGACAGACCCCGGCAATGTGCAGAAAGCCGTGTGTCATCCTACCGCCTG

GGATCTCGGCAAGGGCGACTTCAGAATCCTGATGTGCACCAAAGTGACG

ATGGACGACTTCCTGACAGCCCACCACGAGATGGGCCACATCCAGTACG

ATATGGCCTACGCCGCTCAGCCCTTCCTGCTGAGAAATGGCGCCAATGA

GGGCTTCCACGAAGCCGTGGGAGAGATCATGAGCCTGTCTGCCGCCACA

CCTAAGCACCTGAAGTCTATCGGACTGCTGAGCCCCGACTTCCAAGAGG

ACAACGAGACAGAGATCAACTTCCTGCTCAAGCAGGCCCTGACCATCGT

GGGCACACTGCCCTTTACCTACATGCTGGAAAAGTGGCGGTGGATGGTC

TTTAAGGGCGAGATCCCCAAGGACCAGTGGATGAAGAAATGGTGGGAGA

TGAAGCGCGAGATCGTGGGCGTTGTGGAACCTGTGCCTCACGACGAGAC

ATACTGCGATCCTGCCAGCCTGTTTCACGTGTCCAACGACTACTCCTTC

ATCCGGTACTACACCCGGACACTGTACCAGTTCCAGTTTCAAGAGGCTC

TGTGCCAGGCCGCCAAGCACGAAGGACCTCTGCACAAGTGCGACATCAG

CAACTCTACAGAGGCCGGACAGAAACTGTTCAACATGCTGCGGCTGGGC

AAGAGCGAGCCTTGGACACTGGCTCTGGAAAATGTCGTGGGCGCCAAGA

ATATGAACGTGCGGCCACTGCTGAACTACTTCGAGCCCCTGTTCACCTG

GCTGAAGGACCAGAACAAGAACAGCTTCGTCGGCTGGTCCACCGATTGG

AGCCCTTACGCCGACCAGAGCATCAAAGTGCGGATCAGCCTGAAAAGCG

CCCTGGGCGATAAGGCCTATGAGTGGAACGACAATGAGATGTACCTGTT

CCGGTCCAGCGTGGCCTATGCTATGCGGCAGTACTTTCTGAAAGTCAAG

AACCAGATGATCCTGTTCGGCGAAGAGGATGTGCGCGTGGCCAACCTGA

AGCCTCGGATCAGCTTCAACTTCTTCGTGACTGCCCCTAAGAACGTGTC

CGACATCATCCCCAGAACCGAGGTGGAAAAGGCCATCAGAATGAGCAGA

AGCCGGATCAACGACGCCTTCCGGCTGAACGACAACTCCCTGGAATTCC

TGGGCATTCAGCCCACACTGGGCCCTCCAAATCAGCCTCCTGTGTCCTA

AATAGTGAGTCGTATTAACGTACCAACAAGTGTGACCGAAAGGTAAGAT

GACTTGCATCTTACCTTTCGGTCACACTTTATCTTAGAGGCATATCCCT

ACGTACCAACAAGAGGTGATGAAGTCAGACAAAACTTGTTTGTCTGACT

TCATCACCTCTTTATCTTAGAGGCATATCCCTACGTACCAACAAGCAAC

TGAGGGAGCCTTGAATACTTGATTCAAGGCTCCCTCAGTTGCTTTATCT

TAGAGGCATATCCCTTTTATCTTAGAGGCATATCCCTCTGGGCCTCATG

GGCCTTCCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAG

CTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCG

CTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGG

GGTGCCTAATGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGG

CCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCA

CAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA

AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTC

CGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAG

CGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAG

GTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCG

ACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAG

ACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGA

GCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT

ACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCC

AGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACC

ACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA

GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA

CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTA

TCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTA

AATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATG

CTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCC

ATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCT

TACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACC

GGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGC

AGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTT

GCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGT

TGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATG

GCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCC

CCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGT

CAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTG

CATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTG

GTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAG

TTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGA

ACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCT

CAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGC

ACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA

GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGAGAC

GGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCAT

TTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAG

AAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC

Bold and underline = compound sequence

In Vitro Transcription of RNA Constructs and Data Analysis

Details of in vitro transcription are provided in Example 2. Using in vitro transcription, Compound A9 and Compound A10 were generated at 50-200 μg range and were tested for endogenous TNF-α downregulation and IL-4 expression in THP-1 cells where endogenous TNF-α expression was stimulated by the treatment with LPS and R848 (Example 17). Likewise, Compound A9 and Compound A10 were tested for TNF-α downregulation and IL-4 expression in overexpression models of HEK-293 cells where TNF-α was overexpressed using TNF-α encoding mRNA (Example 18).

Further, Compound A11 was generated at 50-200 μg range and were tested for endogenous ALK2 downregulation and IGF-1 expression in A549 cells (Example 19). In addition, Compound A12 and Compound A13 were generated at 50-200 μg range and were tested for endogenous SOD1 downregulation along with expression of IGF-1 and Erythropoietin (EPO), respectively, in IMR32 cells (Example 20). Compounds A15 and A16 were generated at 50-200 μg range and were tested for the expression of IGF-1 and IL-1 beta downregulation in an overexpression model using HEK293 cells. IL-1-beta protein was overexpressed using IL-1 beta encoding mRNA (Example 21).

Compound B18 was generated at 50-200 μg range and was tested for the expression of soluble ACE2 receptor and downregulation of eGFP tagged SARS CoV-2 Nucleocapsid protein in an overexpression model using A549 cells where eGFP tag-SARS CoV-2 Nucleocapsid protein was overexpressed from a pCDNA3⁺ vector (Example 22).

Data were analyzed using GraphPad Prism 8 (San Diego, USA). For the estimation of the protein (IGF-1, IL-4, IL-1 beta, ALK2, SOD1, EPO, and TNF-α) levels using ELISA in the standard or the sample, the mean absorbance value of the blank was subtracted from the mean absorbance of the standards or the samples. A standard curve was generated and plotted using a four parameters nonlinear regression according to manufacturer's protocol. To determine the concentration of proteins (IGF-1, IL-4, IL-1 beta, ALK2, SOD1, EPO, and TNF-α) in each sample, the concentration of the protein was interpolated from the standard curve. The final protein concentration of the sample was calculated by multiplication with the dilution factor.

Statistical analyses were made using a Student's t-test or one way ANOVA followed by Dunnet's multiple comparing test related to control. The percent of GFP positive cells was calculated using SoftMax Pro tool in Example 22. Relative quantification of remaining target mRNA post treatment with compounds was carried out using the 2^−ΔΔctmethod between study groups. The level of significance was set to a P-value of <0.05. Determination of the molecular weight of Compound A11 was performed as below. The molecular weight of Compound A11 was calculated based on its mRNA sequence by multiplying the number of each base by the molecular weight of the base (e.g., A: 347.2 g/mol; C 323.2 g/mol; G 363.2 g/mol; N1-UTP:338.2 g/mol). The compound molecular weight was determined by adding the obtained weight totals for each base to the ARCA molecular weight of 817.4 g/mol. The molecular weight of the construct was used to convert the amount of transfected mRNA in the well to nM concentration.

Example 17: Endogenous TNF-α Expression Model in THP-1 Cells

Compound A9 and Compound A10 were assayed for their ability to downregulate TNF-α expression, and overexpress IL-4, in THP-1 cells. For the endogenous secretion of TNF-α in THP-1 cells, THP-1 cells were stimulated with E. coli-derived lipopolysaccharide (LPS-L4391; Sigma Aldrich) at 10 μg/mL final concentration with R848 (TLR7/8 agonist; Invivogen) at 1 μg/mL final concentration and incubated for 90 minutes. The induced production of TNF-α corresponds to the physiological conditions observed in psoriasis. Post stimulation, 50 μl of media was removed and replaced with the transfection complex containing specific mRNA constructs (Compounds A9 and A10) or scrambled siRNA (sc-siRNA) complexed with Lipofectamine 2000 in Opti-MEM and incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours. The sc-siRNA were used to rule out transfection related cell death (Universal siRNA, Sigma; Cat. SIC002). Post transfection, the cell culture supernatant was collected and quantified for of TNF-α (target gene to downregulate) and IL-4 (Gene of Interest to overexpress) by ELISA. The TNF-α levels in samples transfected only with TNF-α mRNA were used as controls and set to 100% and percent of TNF-α knock down was calculated.

Results

The effect of Compound A9 (comprising siRNA targeting TNF-α 5′ to the IL-4 coding sequence) and Compound A10 (comprising siRNA targeting TNF-α 3′ to the IL-4 coding sequence) on downregulation of TNF-α was evaluated in THP-1 cells stimulated with 10 μg/mL LPS and 1 μg/mL R848 to induce endogenous TNF-α secretion. The established THP-1 model mimics the physiological immune condition of psoriasis. As demonstrated in FIG. 12A, Compound A9 and Compound A10 downregulated the expression of endogenous TNF-α expression in THP-1 cells by at least approximately 80% relative to control (P<0.001). Interestingly, Compound A10 induced significantly stronger TNF-α downregulation compared to Compound A9 which has siRNA positioned upstream of (or 5′ to) IL-4 ORF (FIG. 12A; P<0.05). Compound A10 induced TNF-α downregulation of at least approximately 85% relative to control, and at approximately 5-10% greater than Compound A9. The same cell culture supernatant was measured for IL-4 expression and the data show that the expression of IL-4 by Compound A10 is 2.5-fold higher than the expression of IL-4 by Compound A9 as shown in FIG. 12B (P<0.01). This assay demonstrates that Compound A10 (TNF-α-targeting siRNA positioned at 3′ of IL-4 gene), when compared to Compound A9 (TNF-α-targeting siRNA positioned 5′ of IL-4 gene), has 5-10% greater TNF-α-targeting (downregulating) siRNA activity and 2.5-fold greater IL-4 expression (a 70% increase).

Example 18: TNF-α Overexpression Model in HEK-293 Cells

Compound A9 and Compound A10 were assayed for their ability to downregulate TNF-α expression, and overexpress IL-4, in HEK-293 cells. To assess the simultaneous effect of TNF-α RNA interference (RNAi) and IL-4 expression, the TNF-α overexpression model was established using TNF-α mRNA transfection (600 ng/well). As described, Compound A9 comprises TNF-α-targeting siRNA 5′ of the IL-4 coding sequence (upstream of IL-4 gene) while Compound A10 comprises TNF-α-targeting siRNA 3′ of the IL-4 coding sequence (downstream of IL-4 gene). To assess the capability of Compound A9 and Compound A10 containing TNF-α targeting siRNA in TNF-α downregulation and simultaneous IL-4 expression, the cells were co-transfected with Compound A9 or Compound A10 (900 ng/well) and TNF-α mRNA (600 ng/well). Post transfection, the cells were incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours followed by quantification of TNF-α (target gene to downregulate) and IL-4 (Gene of Interest to overexpress) by ELISA in the same cell culture supernatant. The TNF-α levels in samples transfected only with TNF-α mRNA were used as controls and set to 100% and percent of TNF-α knock down was calculated.

Results

Compound A9 and Compound A10 were tested for TNF-α downregulation and IL-4 expression at the same time in HEK-293 cells (900 ng/well) with exogenously delivered TNF-α mRNA (600 ng/well). The data show 20-fold higher IL-4 expression from Compound A10 than from Compound A9, as shown in FIG. 13B (P<0.001). In the same experiment with the same cell culture supernatant, the RNA interference of Compound A9 and Compound A10 (900 ng/well) against TNF-α expression was assessed using a TNF-α overexpression construct (600 ng/well), followed by TNF-α ELISA. Both Compound A9 and Compound A10 downregulated the TNF-α level compared to untreated control up to 80% (P<0.01) as shown in FIG. 13A. The assay data shown in FIGS. 13A and 13B demonstrate that Compound A10 (which comprises TNF-α-targeting siRNA 3′ of the IL-4 coding sequence) downregulated TNF-α at least as well as Compound A9 (which comprises TNF-α-targeting siRNA 5′ of the IL-4 coding sequence), by approximately 80%. Additionally, Compound A10 induced at least a 20-fold increase in IL-4 expression relative to Compound A9.

Example 19: Endogenous ALK2 Expression Model in A549 Cells

In Vitro Transfection of A549 Cells with Compound A11

A549 cells are typical alveolar type II (ATII) cells derived from human lung carcinoma. Since A549 cells express endogenous ALK2 RNA transcripts at a moderate level, A549 cells were used to study the effect of Compound A11 in degrading the ALK2 mRNA in parallel to measuring IGF-1 expression. The A549 cells (Sigma-Aldrich, Buchs Switzerland Cat. #6012804) were maintained on Dulbecco's Modified Eagle's medium-high glucose (DMEM, Sigma-Aldrich, Buchs Switzerland cat #D0822) supplemented with 10% FBS (Thermo Fisher Scientific, Basel, Switzerland; cat. #10500-064). To assess Compound A11 activity, the A549 cells were plated at a density of 10,000 cells/well in a regular growth medium 24 hours prior to transfection. Thereafter, cells were transfected with increasing concentration of Compound A11 (0, 0.61, 1.25, 2.54, 5.08, 10.16 and 20.33 nM, corresponding to 0, 19, 38, 75, 150, 300 or 600 ng/well, respectively) using Lipofectamine 2000 (www.invitrogen.com) following the manufacturer's instructions. 100 μl of DMEM were removed and 50 μl of Opti-MEM (www.thermofisher.com) was added to each well followed by 50 μl mRNA and Lipofectamine 2000 complex in Opti-MEM. After 5 hours of incubation, the medium was replaced by fresh growth medium and the plates were incubated for 24 hours at 37° C. in a humidified atmosphere containing 5% CO₂, followed by IGF-1 quantification by ELISA and ALK2 mRNA by relative quantification using qPCR with primers targeting human ALK2 mRNA (Forward primer: 5′-GACGTGGAGTATGGCACTATCG-3′ and Reverse primer: 5′-CACTCCAACAGTGTAATCTGGCG-3′; SEQ ID NOs: 171 and 172, respectively) using SYBR 1-Step Cells to CT kit (Thermo Fisher Scientific, Basel, Switzerland; cat. #A25599). The human 18S rRNA was used as a reference control (Forward primer: 5′-ACCCGTTGAACCCCATTCGTGA-3′ and Reverse primer: 5′-GCCTCACTAAACCATCCAATCGG-3′; SEQ ID NOs: 173 and 174, respectively).

Results

The effect of Compound A11 (comprising 3×ALK2-targeting siRNA 3′ to an IGF-1 protein coding sequence) was evaluated for ALK-2 downregulation and simultaneous IGF-1 expression in A549 cells with dose response (0.6 nM to 20.33 nM). The data demonstrate that Compound A11 expresses IGF-1 protein dose dependently, reaching a level above 150 ng/ml as shown in FIG. 14. In the same cell culture supernatant, the RNA interference of Compound A11 against remaining ALK-2 expression was assessed. As demonstrated in FIG. 14, Compound A11 downregulated the endogenous ALK2 RNA transcripts expression up to approximately 75%. This assay demonstrated that Compound A11 downregulated ALK2 expression by 75% and simultaneously expressed IGF-1 in a dose-dependent manner up to at least 150 ng/ml.

Example 20: Endogenous SOD1 Expression Model in IMR32 Cells

Compound A12 and Compound A13 were assayed for their ability to downregulate SOD-1 expression, and overexpress IGF-1 (Compound A12) or EPO (Compound A13) in Human Caucasian Neuroblastoma (IMR32) cells. IMR32 cells (Cat #86041809, ECACC, UK) were plated at a density of 20,000 cells per well in a 96 pre-coated BRAND microtiter plate (Cat #782082) in Minimum Essential Medium Eagle (EMEM, Bioconcept Cat #1-31501-I, www.bioconcept.ch) supplemented with 10% (v/v) heat-inactivated Fetal Bovine Serum (FBS), L-Glutamine (2 mM) and Non-essential Amino acids (NEAA, 1×). Cells were grown overnight at 37° C. in a humidified atmosphere containing 5% CO₂. Cells were transfected with three doses of Compound A12 or Compound A13 (150, 300 or 900 ng/well,) constructs using JetMessenger (www.polyplus-transfection.com) following manufacturer's instructions. The scrambled siRNA (sc-siRNA) was used to rule out transfection-related cell death (Universal siRNA, Sigma; Cat. SIC002). Briefly, mRNA/JetMessenger complex was formed by mixing 0.25 μl JetMessenger reagent per 0.1 μg mRNA construct. After incubating 15 minutes at room temperature the JetMessenger complex was added as 10 μl and 5 hours after transfection medium/mRNA/JetMessenger was removed from the wells and replaced with fresh 100 μl growth medium and the plates were incubated 24 hours at 37° C. in a humidified atmosphere containing 5% CO₂. The measurement of remaining SOD1 mRNA was measured by qPCR in cell lysates 24 hours after transfection with Compound A12 and Compound A13 by relative quantification using qPCR with primers targeting human SOD1 mRNA (Forward primer: 5′-CTCACTCTCAGGAGACCATTGC-3′ and Reverse primer: 5′-CCACAAGCCAAACGACTTCCAG-3′; SEQ ID NOs: 175 and 176, respectively) using SYBR 1-Step Cells to CT kit (Thermo Fischer Scientific, Basel, Switzerland; cat. #A25599). The human 18S rRNA used as a reference control using the same primers specified in Example 19. The same cell culture supernatant was used to measure IGF-1 and EPO (Thermo Fisher Scientific, Basel, Switzerland; cat. #BMS2035) by ELISA.

Results

The effect on SOD1 downregulation in IMR32 cells of an escalating series of three doses of Compound A12 (comprising 3×SOD1-targeting siRNA and IGF-1 protein coding sequence) and Compound A13 (comprising 3×SOD1-targeting siRNA and EPO protein coding sequence) was evaluated (150, 300 and 900 ng/well). The assay showed that Compound A12 and Compound A13 reduced the SOD1 transcripts in a dose-dependent manner (up to at least approximately 70%) (FIG. 15A, open circles and closed circles, respectively). The scrambled siRNA did not show an effect (FIG. 15A, shaded circles). In the same cell culture supernatant (IMR32 cells), the expression of EPO protein of Compound A13 was assessed. As demonstrated in FIG. 15B, Compound A13 induced EPO expression in a dose-dependent manner. Likewise, the expression of IGF-1 protein from Compound A12 in the same IMR32 cell culture supernatant was assessed. As shown in FIG. 15C, Compound A12 simultaneously expressed IGF-1.

Example 21: IL-1 Beta Overexpression Model in HEK-293 Cells

Compound A14 and Compound A15 were assayed for their ability to downregulate IL-1 beta expression, and overexpress IGF-1 in HEK-293 cells. An IL-1 beta overexpression model was established in HEK-293 cells using IL-1 beta mRNA transfection (300 ng/well). Compound A14 comprises siRNA targeting IL-1 beta 5′ to the IGF-1 coding sequence (upstream of the IGF-1 gene) while Compound A15 comprises siRNA targeting IL-1 beta 3′ to the IGF-1 coding sequence (downstream of the IGF-1 gene). To assess the capability of Compound A14 and Compound A15 containing siRNAs targeting IL-1 beta in IL-1 beta downregulation and simultaneous IGF-1 expression, the HEK-293 cells were co-transfected with Compound A14 or Compound A15 (900 ng/well) and IL-1 beta mRNA (300 ng/well). Post transfection, the cells were incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours followed by quantification of IL-1 beta (target gene to downregulate) and IGF-1 (Gene of Interest to overexpress) by ELISA in the same cell culture supernatant.

Results

Compound A14 and Compound A15 comprise IL-1 beta-targeting siRNA either 5′ or 3′ of IGF-1 coding sequence, respectively. The constructs were tested for IL-1 beta downregulation and IGF-1 expression at the same time in HEK-293 cells (900 ng/well) with exogenously delivered IL-1 beta mRNA (300 ng/well). The data demonstrate that Compound A15 expresses approximately 13-fold higher IGF-1 than Compound A14 as shown in FIG. 16B (P<0.001). In the same experiment with the same cell culture supernatant, the RNA interference of Compound A14 and Compound A15 (900 ng/well) against IL-1 beta expression from the IL-1 beta overexpression construct (300 ng/well) was assessed, as measured by IL-1 beta ELISA. Compound A14 and Compound A15 downregulated the IL-1 beta levels by more than approximately 150-fold and 290-fold, respectively, compared to untreated control (P<0.001) as shown in FIG. 16A. Compound A15 induced at least approximately 2-fold IL-1 beta downregulation as compared to Compound A14 in which the siRNA is positioned upstream of (5′ to) the IGF-1 ORF (FIG. 16A; P<0.05). These data demonstrated that Compound A15 (having IL-1 beta-targeting siRNA positioned 3′ to the IGF-1 gene) downregulated IL-1 beta by 290-fold, and increased IGF-1 expression while significantly increasing IGF-1 expression. Compound A14 (having IL-1 beta-targeting siRNA positioned 5′ to IGF-1 gene) downregulated IL-1 beta by 150-fold, and increased IGF-1 expression while significantly increasing IGF-1 expression. Thus, Compound A15 downregulation of IL-1 beta was 2-fold greater than that observed for Compound A14. Additionally, Compound A15 expression of IGF-1 was 13 fold greater than that observed for Compound A14.

Example 22: SARS CoV-2 Nucleocapsid Protein Overexpression Model in A549 Cells

In Vitro Transfection of A549 Cells with SARS CoV-2 Nucleocapsid Protein with eGFP Tag pCDNA3⁺ Vector and SARS CoV-2 Nucleocapsid Protein Suppressing/Soluble ACE2 Overexpression Compounds

A SARS CoV-2 Nucleocapsid protein overexpression model was used to evaluate simultaneous SARS CoV-2 Nucleocapsid (N) protein RNAi suppression and soluble ACE2 overexpression by Compound B18 in A549 cells. The model was established by transfection of a plasmid pcDNA3⁺ vector (300 ng/well) containing a SARS CoV-2 N protein with eGFP tag. The RNAi of Compound B18 targeting SARS CoV-2 N protein disrupts the downstream eGFP translation and expression. Compound B18 contains a soluble ACE2 encoding ORF and 3×SARS CoV-2-targeting siRNA (lx target ORF1ab region, lx target Spike protein and 1× target nucleocapsid protein) 3′ to (downstream of) the ACE2 ORF. The cells were co-transfected with Compound B18 (600 ng/well) and a SARS CoV-2 Nucleocapsid protein overexpressing plasmid construct (300 ng/well). Post transfection, the cells were incubated at 37° C. in a humidified atmosphere containing 5% CO₂for 24 hours, followed by determination of whether RNAi suppression by Compound B18 leads to the disruption of eGFP translation. The SARS CoV-2 Nucleocapsid proteins tagged with eGFP (from expression of plasmid) were microscopically examined for eGFP expression using SpectraMax i3X multi-mode microplate reader (Molecular Devices). The percentage of eGFP positive cells was calculated in treated and control untreated samples.

Results

The effect of Compound B18 (comprising 3×SARS CoV-2 targeting siRNA 3′ to a soluble ACE2 protein coding sequence) was evaluated for SARS CoV-2 N-Protein downregulation in A549 cells. A reduced number of eGFP positive cells was observed, showing the targeting effect of Compound B18 against SARS CoV-2 N-Protein encoding mRNA (FIGS. 17A and 17B). The cumulative analysis from different samples showed an approximately 8-fold reduction in eGFP positive cells by Compound B18 compared to untreated control (FIG. 17C).

The examples and embodiments described herein are for illustrative purposes only and various modifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.

TABLE 7

Table of Sequences Listed

Protein or

SEQ ID

Nucleic Acid
Sequence (protein: N-term to C-term; nucleic acid: 5′ to 3)
NO:

Compounds A1-
See Table 2
1-8

A8

Compounds A1-
See Table 3
9-16

A8 (plasmid

sequences)

Forward primer
GCTGCAAGGCGATTAAGTTG
17

for template

generation

Reverse primer
U(2′OMe)U(2′OMe)U(2′OMe)TTTTTTTTTTTTTTTTTTTTTTTTTT
18

for template
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

generation
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCAGCTATGA

CCATGTTAATGCAG

A mature human
GGACCTGAGACACTTTGTGGCGCTGAACTGGTGGACGCCCTGCAGTTTGT
19

IGF-1 coding
GTGTGGCGACAGAGGCTTCTACTTCAACAAGCCCACAGGCTACGGCAGCA

sequence
GCTCTAGAAGGGCTCCTCAGACCGGAATCGTGGACGAGTGCTGTTTCAGA

AGCTGCGACCTGCGGCGGCTGGAAATGTATTGTGCCCCTCTGAAGCCTGC

CAAGAGCGCC

A modified
MLILLLPLLLFKCFCDFLK
20

signal peptide of

IGF-1

A modified
ATGCTGATTCTGCTGCTGCCCCTGCTGCTGTTCAAGTGCTTCTGCGACTT
21

signal peptide of
CCTGAAA

IGF-1-coding

sequence

A modified
MLFYLALCLLTFTSSATA
22

IGF-1 pro

domain

A modified
ATGCTGTTCTATCTGGCCCTGTGCCTGCTGACCTTTACCAGCTCTGCTAC
23

IGF-1 pro
CGCC

domain-coding

sequence

tRNA linker
AACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAG
24

ACCCGGGTTCGATTCCCGGCTGGTGCA

T7 promoter
TAATACGACTCACTATA
25

Kozak sequence
GCCACC
26

Flexible linker
GGGGS
27

amino acid

Flexible linker
GGGGGTGGAGGCTCT
28

nucleic acid

Compounds B1-
See Table 5 and 6
29-47

B19 anti-viral

nucleic acid

sequences

Human IFN-

MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLE
48

beta amino acid
YCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGW

(Genbank
NETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI

NM_002176.3)
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN

Underlined:

signal sequence

Human IFN-
CCATACCCATGGAGAAAGGACATTCTAACTGCAACCTTTCGAAGCCTTTG
49

beta nucleic acid
CTCTGGCACAACAGGTAGTAGGCGACACTGTTCGTGTTGTCAACATGACC

(Genbank
AACAAGTGTCTCCTCCAAATTGCTCTCCTGTTGTGCTTCTCCACTACAGC

NM_002176.3)
TCTTTCCATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGCAGCAATT

TTCAGTGTCAGAAGCTCCTGTGGCAATTGAATGGGAGGCTTGAATACTGC

CTCAAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA

GCAGTTCCAGAAGGAGGACGCCGCATTGACCATCTATGAGATGCTCCAGA

ACATCTTTGCTATTTTCAGACAAGATTCATCTAGCACTGGCTGGAATGAG

ACTATTGTTGAGAACCTCCTGGCTAATGTCTATCATCAGATAAACCATCT

GAAGACAGTCCTGGAAGAAAAACTGGAGAAAGAAGATTTCACCAGGGGAA

AACTCATGAGCAGTCTGCACCTGAAAAGATATTATGGGAGGATTCTGCAT

TACCTGAAGGCCAAGGAGTACAGTCACTGTGCCTGGACCATAGTCAGAGT

GGAAATCCTAAGGAACTTTTACTTCATTAACAGACTTACAGGTTACCTCC

GAAACTGAAGATCTCCTAGCCTGTGCCTCTGGGACTGGACAATTGCTTCA

AGCATTCTTCAACCAGCAGATGCTGTTTAAGTGACTGATGGCTAATGTAC

TGCATATGAAAGGACACTAGAAGATTTTGAAATTTTTATTAAATTATGAG

TTATTTTTATTTATTTAAATTTTATTTTGGAAAATAAATTATTTTTGGTG

CAAAAGTCA

Optimized

ATGACCAACAAGTGCCTGCTGCAGATTGCCCTGCTGCTGTGCTTCAGCAC

50

Human IFN-

AACAGCCCTGAGCATGAGCTACAACCTGCTGGGCTTCCTGCAGCGGAGCA

beta nucleic acid
GCAACTTCCAGTGCCAGAAACTGCTGTGGCAGCTGAACGGCCGGCTGGAA

sequence
TACTGCCTGAAGGACCGGATGAACTTCGACATCCCCGAGGAAATCAAGCA

encoding SEQ
GCTGCAGCAGTTCCAGAAAGAGGACGCCGCTCTGACCATCTACGAGATGC

ID NO: 48
TGCAGAACATCTTCGCCATCTTCCGGCAGGACAGCAGCTCCACAGGCTGG

Underlined:
AACGAGACAATCGTGGAAAATCTGCTGGCCAACGTGTACCACCAGATCAA

signal sequence
CCACCTGAAAACCGTGCTGGAAGAGAAGCTGGAAAAAGAGGACTTCACCC

GGGGCAAGCTGATGAGCAGCCTGCACCTGAAGCGGTACTACGGCAGAATC

CTGCACTACCTGAAGGCCAAAGAGTACAGCCACTGCGCCTGGACCATCGT

GCGCGTGGAAATCCTGCGGAACTTCTACTTCATCAACCGGCTGACCGGCT

ACCTGAGAAACTGA

IFN-beta signal
MTNKCLLQIALLLCFSTTALS
51

peptide

(Genbank

NM_002176.3)

Modified IFN-
MLLICLLVIALLLCFSTTALS
52

beta signal

peptide (SP1)

amino acid

(T2L/N3L/K4I

and Q8V)

Modified IFN-
ATGCTCCTGATCTGCCTGCTGGTGATTGCCCTGCTGCTGTGCTTCAGCAC
53

beta signal
AACAGCCCTGAGC

peptide (SP1)

nucleic acid

Modified IFN-
MLLKLLLVIALLACFSTTALS
54

beta signal

peptide (SP2)

amino acid

(T2L/N3L/C5L/

Q8V and L13A)

Modified IFN-
ATGCTCCTGAAGCTCCTGCTGGTGATTGCCCTGCTGGCCTGCTTCAGCAC
55

beta signal
AACAGCCCTGAGC

peptide (SP2)

nucleic acid

ACE2 amino
MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNY
56

acid (Genbank
NTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQAL

NM_021804.2)
QQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNE

Bold: ACE2
IMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYG

transmembrane
DYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMN

domain and
AYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQ

intracellular
AWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWD

domain
LGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGF

(residues 741-
HEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTL

805)
PFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDP

ASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEA

GQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNK

NSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYA

MRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEV

EKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSIWLIVFGVVM

GVIVVGIVILIFTGIRDRKKKNKARSGENPYASIDISKGENNPGFQNTDD

VQTSF

ACE2 nucleic
ATGTCAAGCTCTTCCTGGCTCCTTCTCAGCCTTGTTGCTGTAACTGCTGC
57

acid encoding
TCAGTCCACCATTGAGGAACAGGCCAAGACATTTTTGGACAAGTTTAACC

SEQ ID NO: 56
ACGAAGCCGAAGACCTGTTCTATCAAAGTTCACTTGCTTCTTGGAATTAT

(from Genbank
AACACCAATATTACTGAAGAGAATGTCCAAAACATGAATAATGCTGGGGA

NM_021804.2)
CAAATGGTCTGCCTTTTTAAAGGAACAGTCCACACTTGCCCAAATGTATC

Bold and
CACTACAAGAAATTCAGAATCTCACAGTCAAGCTTCAGCTGCAGGCTCTT

italicized:
CAGCAAAATGGGTCTTCAGTGCTCTCAGAAGACAAGAGCAAACGGTTGAA

siRNA binding
CACAATTCTAAATACAATGAGCACCATCTACAGTACTGGAAAAGTTTGTA

regions
ACCCAGATAATCCACAAGAATGCTTATTACTTGAACCAGGTTTGAATGAA

Bold: ACE2
ATAATGGCAAACAGTTTAGACTACAATGAGAGGCTCTGGGCTTGGGAAAG

transmembrane
CTGGAGATCTGAGGTCGGCAA custom-character

AGAGTATG

domain and
TGGTCTTGAAAAATGAGATGGCAAGAGCAAATCATTATGAGGACTATGGG

intracellular
GATTATTGGAGAGGAGACTATGAAGTAAATGGGGTAGATGGCTATGACTA

domain coding
CAGCCGCGGCCAGTTGATTGAAGATGTGGAACATACCTTTGAAGAGATTA

sequence
AACCATTATATGAACATCTTCATGCCTATGTGAGGGCAAAGTTGATGAAT

GCCTATCCTTCCTATATCAGTCCAATTGGATGCCTCCCTGCTCATTTGCT

TGGTGATATGTGGGGTAGATTTTGGACAAATCTGTACTCTTTGACAGTTC

CCTTTGGACAGAAACCAAACATAGATGTTACTGATGCAATGGTGGACCAG

GCCTGGGATGCACAGAGAATATTCAAGGAGGCCGAGAAGTTCTTTGTATC

TGTTGGTCTTCCTAATATGACTCAAGGATTCTGGGAAAATTCCATGCTAA

C custom-character

AGCAGTCTGCCATCCCACAGCTTGGGAC

CTGGGGAAGGGCGACTTCAGGATCCTTATGTGCACAAAGGTGACAATGGA

CGACTTCCTGACAGCTCATCATGAGATGGGGCATATCCAGTATGATATGG

CATATGCTGCACAACCTTTTCTGCTAAGAAATGGAGCTAATGAAGGATTC

CATGAAGCTGTTGGGGAAATCATGTCACTTTCTGCAGCCACACCTAAGCA

TTTAAAATCCATTGGTCTTCTGTCACCCGATTTTCAAGAAGACAATGAAA

CAGAAATAAACTTCCTGCTCAAACAAGCACTCACGATTGTTGGGACTCTG

CCATTTACTTACATGTTAGAGAAGTGGAGGTGGATGGTCTTTAAAGGGGA

AATTCCCAAAGACCAGTGGATGAAAAAGTGGTGGGAGATGAAGCGAGAGA

TAGTTGGGGTGGTGGAACCTGTGCCCCATGATGAAACATACTGTGACCCC

GCATCTCTGTTCCATGTTTCTAATGATTACTCATTCATTCGATATTACAC

AAGGACCCTTTACCAATTCCAGTTTCAAGAAGCACTTTGTCAAGCAGCTA

AACATGAAGGCCCTCTGCACAAATGTGACATCTCAAACTCTACAGAAGCT

GGACAGAAACTGTTCAATATGCTGAGGCTTGGAAAATCAGAACCCTGGAC

CCTAGCATTGGAAAATGTTGTAGGAGCAAAGAACATGAATGTAAGGCCAC

TGCTCAACTACTTTGAGCCCTTATTTACCT custom-character

ATTCTTTTGTGGGATGGAGTACCGACTGGAGTCCATATGCAGACCAAAG

CATCAAAGTGAGGATAAGCCTAAAATCAGCTCTTGGAGATAAAGCATATG

AATGGAACGACAATGAAATGTACCTGTTCCGATCATCTGTTGCATATGCT

ATGAGGCAGTACTTTTTAAAAGTAAAAAATCAGATGATTCTTTTTGGGGA

GGAGGATGTGCGAGTGGCTAATTTGAAACCAAGAATCTCCTTTAATTTCT

TTGTCACTGCACCTAAAAATGTGTCTGATATCATTCCTAGAACTGAAGTT

GAAAAGGCCATCAGGATGTCCCGGAGCCGTATCAATGATGCTTTCCGTCT

GAATGACAACAGCCTAGAGTTTCTGGGGATACAGCCAACACTTGGACCTC

CTAACCAGCCCCCTGTTTCCATATGGCTGATTGTTTTTGGAGTTGTGATG

GGAGTGATAGTGGTTGGCATTGTCATCCTGATCTTCACTGGGATCAGAGA

TCGGAAGAAGAAAAATAAAGCAAGAAGTGGAGAAAATCCTTATGCCTCCA

TCGATATTAGCAAAGGAGAAAATAATCCAGGATTCCAAAACACTGATGAT

GTTCAGACCTCCTTTTAG

ACE2 Soluble
MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNY
58

Receptor-
NTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQAL

Ectodomain
QQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNE

amino acid
IMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYG

sequence
DYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMN

(derived from
AYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQ

Genbank
AWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWD

NM_021804.2;
LGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGF

does not include
HEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTL

transmembrane
PFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDP

domain and
ASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEA

intracellular
GQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNK

domain)
NSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYA

MRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEV

EKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS

ACE2 Soluble

ATGTCAAGCTCTTCCTGGCTCCTTCTCAGCCTTGTTGCTGTAACTGCTGC

59

Receptor-

TCAGTCCACCATTGAGGAACAGGCCAAGACATTTTTGGACAAGTTTAACC

Ectodomain
ACGAAGCCGAAGACCTGTTCTATCAAAGTTCACTTGCTTCTTGGAATTAT

nucleic acid
AACACCAATATTACTGAAGAGAATGTCCAAAACATGAATAATGCTGGGGA

sequence
CAAATGGTCTGCCTTTTTAAAGGAACAGTCCACACTTGCCCAAATGTATC

encoding SEQ
CACTACAAGAAATTCAGAATCTCACAGTCAAGCTTCAGCTGCAGGCTCTT

ID NO: 58
CAGCAAAATGGGTCTTCAGTGCTCTCAGAAGACAAGAGCAAACGGTTGAA

Underlined:
CACAATTCTAAATACAATGAGCACCATCTACAGTACTGGAAAAGTTTGTA

signal sequence
ACCCAGATAATCCACAAGAATGCTTATTACTTGAACCAGGTTTGAATGAA

(derived from
ATAATGGCAAACAGTTTAGACTACAATGAGAGGCTCTGGGCTTGGGAAAG

Genbank
CTGGAGATCTGAGGTCGGCAAGCAGCTGAGGCCATTATATGAAGAGTATG

NM_021804.2;
TGGTCTTGAAAAATGAGATGGCAAGAGCAAATCATTATGAGGACTATGGG

does not include
GATTATTGGAGAGGAGACTATGAAGTAAATGGGGTAGATGGCTATGACTA

transmembrane
CAGCCGCGGCCAGTTGATTGAAGATGTGGAACATACCTTTGAAGAGATTA

domain and
AACCATTATATGAACATCTTCATGCCTATGTGAGGGCAAAGTTGATGAAT

intracellular
GCCTATCCTTCCTATATCAGTCCAATTGGATGCCTCCCTGCTCATTTGCT

domain coding
TGGTGATATGTGGGGTAGATTTTGGACAAATCTGTACTCTTTGACAGTTC

sequence)
CCTTTGGACAGAAACCAAACATAGATGTTACTGATGCAATGGTGGACCAG

GCCTGGGATGCACAGAGAATATTCAAGGAGGCCGAGAAGTTCTTTGTATC

TGTTGGTCTTCCTAATATGACTCAAGGATTCTGGGAAAATTCCATGCTAA

CGGACCCAGGAAATGTTCAGAAAGCAGTCTGCCATCCCACAGCTTGGGAC

CTGGGGAAGGGCGACTTCAGGATCCTTATGTGCACAAAGGTGACAATGGA

CGACTTCCTGACAGCTCATCATGAGATGGGGCATATCCAGTATGATATGG

CATATGCTGCACAACCTTTTCTGCTAAGAAATGGAGCTAATGAAGGATTC

CATGAAGCTGTTGGGGAAATCATGTCACTTTCTGCAGCCACACCTAAGCA

TTTAAAATCCATTGGTCTTCTGTCACCCGATTTTCAAGAAGACAATGAAA

CAGAAATAAACTTCCTGCTCAAACAAGCACTCACGATTGTTGGGACTCTG

CCATTTACTTACATGTTAGAGAAGTGGAGGTGGATGGTCTTTAAAGGGGA

AATTCCCAAAGACCAGTGGATGAAAAAGTGGTGGGAGATGAAGCGAGAGA

TAGTTGGGGTGGTGGAACCTGTGCCCCATGATGAAACATACTGTGACCCC

GCATCTCTGTTCCATGTTTCTAATGATTACTCATTCATTCGATATTACAC

AAGGACCCTTTACCAATTCCAGTTTCAAGAAGCACTTTGTCAAGCAGCTA

AACATGAAGGCCCTCTGCACAAATGTGACATCTCAAACTCTACAGAAGCT

GGACAGAAACTGTTCAATATGCTGAGGCTTGGAAAATCAGAACCCTGGAC

CCTAGCATTGGAAAATGTTGTAGGAGCAAAGAACATGAATGTAAGGCCAC

TGCTCAACTACTTTGAGCCCTTATTTACCTGGCTGAAAGACCAGAACAAG

AATTCTTTTGTGGGATGGAGTACCGACTGGAGTCCATATGCAGACCAAAG

CATCAAAGTGAGGATAAGCCTAAAATCAGCTCTTGGAGATAAAGCATATG

AATGGAACGACAATGAAATGTACCTGTTCCGATCATCTGTTGCATATGCT

ATGAGGCAGTACTTTTTAAAAGTAAAAAATCAGATGATTCTTTTTGGGGA

GGAGGATGTGCGAGTGGCTAATTTGAAACCAAGAATCTCCTTTAATTTCT

TTGTCACTGCACCTAAAAATGTGTCTGATATCATTCCTAGAACTGAAGTT

GAAAAGGCCATCAGGATGTCCCGGAGCCGTATCAATGATGCTTTCCGTCT

GAATGACAACAGCCTAGAGTTTCTGGGGATACAGCCAACACTTGGACCTC

CTAACCAGCCCCCTGTTTCCTAA

SARS CoV-2
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVL
60

Spike RBD
YNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKI

amino acid
ADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDI

sequence
STEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL

HAPATVCGPKKSTNLVKNKCVNF

SARS CoV-2
AGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTT
61

Spike RBD
GTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATG

nucleic acid
CTTGGAACAGGAAGAGAATCAGCAACTGT custom-character

sequence
TATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTAC

(encoding SEQ
TAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAA

ID NO: 36)
TTA custom-character

TCGCTCCAGGGCAAACTGGAAAGATT

Bold and
GCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGC

italicized:
TTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACC

siRNA binding
TGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATT

regions
TCAACTGAAATCTATCAG custom-character

GGTGTTGAAGG

TTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATG

GTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTA

CATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAA

AAACAAATGTGTCAATTTC

SARS CoV-2
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTA
62

Nucleocapsid
SWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGK

protein (N)
MKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRN

amino acid
PANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPG

sequence (NCBI
SSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKS

YP_009724397.2)
AAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKH

WPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQV

ILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADL

DDFSKQLQQSMSSADSTQA

SARS CoV-2

GCCACC

ATGTCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCGCAT

63

Nucleocapsid

TACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGAATGGAGAAC

protein (N)

GCAGTGGGGCGCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAAT

nucleic acid

ACTGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTTAA

sequence

ATTCCCTCGAGGACAAGGCGTTCCAATTAACACCAATAGCAGTCCAGATG

encoding SEQ

ACCAAATTGGCTACTACCGAAGAGCTACCAGACGAATTCGTGGTGGTGAC

ID NO: 38

GGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAAC

Bold and

TGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATAT

underlined:

GGGTT
custom-character

ACACCAAAAGATCACATTGGCACC

Kozak sequence

CGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAAC

Italicized: ORF

AACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAG

of SARS CoV-2

CCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACT

Nucleocapsid

CCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGG

(N) protein

TGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGA

Bold and

GCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAG

italicized:

AAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCAC

siRNA binding

TAAAGCATACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGAACAAA

region

CCCAAGGAAATTTTGGGGACCAGGAACTAATCAGACAAGGAACTGATTAC

Bold: Flexible

AAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTT

Linker

CGGAATGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGA

Underlined:

CCTACACAGGTGCCATCAAATTGGATGACAAAGATCCAAATTTCAAAGAT

ORF of eGFP

CAAGTCATTTTGCTGAATAAGCATATTGACGCATACAAAACATTCCCACC

reporter protein

AACAGAGCCTAAAAAGGACAAAAAGAAGAAGGCTGATGAAACTCAAGCCT

TACCGCAGAGACAGAAGAAACAGCAAACTGTGACTCTTCTTCCTGCTGCA

GATTTGGATGATTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGA

CTCAACTCAGGCC
GGGGGTGGAGGCTCT
GTGTCCAAGGGCGAAGAACTGT

TCACCGGCGTGGTGCCCATTCTGGTGGAACTGACGGGGATGTGAACGGC

CACAAGTTTAGCGTTAGCGGCGAAGGCGAAGGGGATGCCACATACGGAAA

GCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCTGTGCCTTGGC

CTACACTGGTCACCACACTGACATACGGCGTGCAGTGCTTCAGCAGATAC

CCCGACCATATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCTGAGGG

CTACGTGCAAGAGCGGACCATCTTCTTTAAGGACGACGGCAACTACAAGA

CCAGGGCCGAAGTGAAGTTCGAGGGCGACACCCTGGTCAACCGGATCGAG

CTGAAGGGCATCGACTTCAAAGAGGACGGCAACATCCTGGGCCACAAGCT

CGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGA

AAAACGGCATCAAAGTGAACTTCAAGATCCGGCACAACATCGAGGACGGC

TCTGTGCAGCTGGCCGATCACTACCAGCAGAACACACCCATCGGAGATGG

CCCTGTGCTGCTGCCCGATAACCACTACCTGAGCACACAGAGCGCCCTGA

GCAAGGACCCCAACGAGAAGAGGGATCACATGGTGCTGCTGGAATTCGTG

ACCGCCGCTGGCATCACACTCGGCATGGATGAGCTGTACAAGTGA

SARS CoV-2
MESLVPGFNEKTHVQLSLPVLQVRDVLVRGFGDSVEEVLSEARQHLKDGT
64

NSP1 protein
CGLVEVEKGVLPQLEQPYVFIKRSDARTAPHGHVMVELVAELEGIQYGRS

(NCBI
GETLGVLVPHVGEIPVAYRKVLLRKNGNKGAGGHSYGADLKSFDLGDELG

YP_009725297.1)
TDPYEDFQENWNTKHSSGVTRELMRELNGG

SARS CoV-2
MESLVPGFNEKTHVQLSLPVLQVRDVLVRGFGDSVEEVLSEARQHLKDGT
65

NSP1 protein
CGLVEVEKGVLPQLEQPYVFIKRSDARTAPHGHVMVELVAELEGIQYGRS

(first 100 amino

acids of SEQ ID

NO: 40)

SARS CoV-2
GACACGAGTAACTCGTCTATCTTCTGCAGGCTGCTTACGGTTTCGTCCGT
66

NSP1 protein
GTTGCAGCCGATCATCAGCACATCTAGGTTTCGTCCGG custom-character

nucleic acid

custom-character

GAGAGCCTTGTCCCTGGTTTCAACGAGAAAACACACGTCCA

sequence

ACTCAGTTTGCCTGTTTTACAGGTTCGCGACGTGCTCGTACGTGGCTTTG

(encoding SEQ

GAGACTCCGTGGAGGAGGTCTTATCAGAGGCACGTCAACATCTTAAAGAT

ID NO: 40 at

GGCACTTGTGGCTTAGTAGAAGTTGAAAAAGGCGTTTTGCCTCAACTTGA

positions 107 to

ACAGCCCTATGTGTTCATCAAACGTTCGGATGCTCGAACTGCACCTCATG

406, ORF

GTCATGTTATGGTTGAGCTGGTAGCAGAACTCGAAGGCATTCAGTACGGT

italicized)

CGTAGT
GGGGGTGGAGGCTCT
GTGTCCAAGGGCGAAGAACTGTTCACCGG

Bold and

CGTGGTGCCCATTCTGGTGGAACTGGACGGGGATGTGAACGGCCACAAGT

italicized:

TTAGCGTTAGCGGCGAAGGCGAAGGGGATGCCACATACGGAAAGCTGACC

siRNA binding

CTGAAGTTCATCTGCACCACCGGCAAGCTGCCTGTGCCTTGGCCTACACT

region

GGTCACCACACTGACATACGGCGTGCAGTGCTTCAGCAGATACCCCGACC

Bold: Flexible

ATATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCTGAGGGCTACGTG

Linker

CAAGAGCGGACCATCTTCTTTAAGGACGACGGCAACTACAAGACCAGGGC

Underlined:

CGAAGTGAAGTTCGAGGGCGACACCCTGGTCAACCGGATCGAGCTGAAGG

ORF of eGFP

GCATCGACTTCAAAGAGGACGGCAACATCCTGGGCCACAAGCTCGAGTAC

reporter protein

AACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAAAACGG

(The 5′ UTR of

CATCAAAGTGAACTTCAAGATCCGGCACAACATCGAGGACGGCTCTGTGC

SARS CoV-2 is

AGCTGGCCGATCACTACCAGCAGAACACACCCATCGGAGATGGCCCTGTG

shown upstream

CTGCTGCCCGATAACCACTACCTGAGCACACAGAGCGCCCTGAGCAAGGA

of the first ATG

CCCCAACGAGAAGAGGGATCACATGTGCTGCTGGAATTCGTGACCGCCG

codon at

CTGGCATCACACTCGGCATGGATGAGCTGTACAAGTGA

position 107)

SARS CoV-2
GTCACATGTTGACACTGACTTAACAAAGCCTTACATTAAGTGGGATTTGT
67

NSP12 and
TAAAATATGACTTCACGGAAGAGAGGTTAAAACTCTTTGACCGTTAT custom-character

NSP13 nucleic

custom-character

ATACCACCCAAATTGTGTTAACTGTTTGGATGA

acid sequence
CAGATGCATTCTGCATTGTGCAAACTTTAATGTTTTATTCTCTACAGTGT

Bold and
TCCCACCTACAAGTTTTGGACCACTAGTGAGAAAAATATTTGTTGATGGT

italicized:
GTTCCATTTGTAGTTTCAACTGGATACCACTTCAGAGAGCTAGGTGTTGT

siRNA binding
ACATAATCAGGATGTAAACTTACATAGCTCTAGACTTAGTTTTAAGGAAT

regions
TACTTGTGTATGCTGCTGACCCTGCTATGCACGCTGCTTCTGGTAATCTA

TTACTAGATAAACGCACTACGTGCTTTTCAGTAGCTGCACTTACTAACAA

TGTTGCTTTTCAAACTGTCAAACCCGGTAATTTTAACAAAGACTTCTATG

ACTTTGCTGTGTCTAAGGGTTTCTTTAAGGAAGGAAGTTCTGTTGAATTA

AAACACTTCTTCTTTGCTCAGGATGGTAATGCTGCTATCAGCGATTATGA

CTACTATCGTTATAATCTACCAACAATGTGTGATATCAGACAACTACTAT

TTGTAGTTGAAGTTGTTGATAAGTACTTTGATTGTTACGATGGTGGCTGT

ATTAATGCTAACCAAGTCATCGTCAACAACCTAGACAAATCAGCTGGTTT

TCCATTTAATAAATGGGGTAAGGCTAGACTTTATTATGATTCAATGAGTT

ATGAGGATCAAGATGCACTTTTCGCATATACAAAACGTAATGTCATCCCT

ACTATAACTCAAATGAATCTTAAGTATGCCATTAGTGC custom-character

CGTAGCTGGTGTCTCTATCTGTAGTACTATGACCAATAGACAGT

TTCATCAAAAATTATTGAAATCAATAGCCGCCACTAGAGGAGCTACTGTA

GTAATTGGAACAAGCAAATTCTATGGTGGTTGGCACAACATGTTAAAAAC

TGTTTATAGTGATGTAGAAAACCCTCACCTTATGGGTTGGGATTATCCTA

AATGTGATAGAGCCATGCCTAACATGCTTAGAATTATGGCCTCACTTGTT

CTTGCTCGCAAACATACAACGTGTTGTAGCTTGTCACACCGTTTCTATAG

ATTAGCTAATGAGTGTGCTCAAGTATTGAGTGAAATGGTCATGTGTGGCG

GTTCACTATATGTTAAACCAGGTGGAACCTCATCAGGAGATGCCACAACT

GCTTATGCTAATAGTGTTTTTAACATTTGTCAAGCTGTCACGGCCAATGT

TAATGCACTTTTATCTACTGATGGTAACAAAATTGCCGATAAGTATGTCC

GCAATTTACAACACAGACTTTATGAGTGTCTCTATAGAAATAGAGATGTT

GACACAGACTTTGTGAATGAGTTTTACGCATATTTGCGTAAACATTTCTC

AATGATGATACTCTCTGACGATGCTGTTGTGTGTTTCAATAGCACTTATG

CATCTCAAGGTCTAGTGGCTAGCATAAAGAACTTTAAGTCAGTTCTTTAT

TATCAAAACAATGTTTTTATGTCTGAAGCAAAATGTTGGACTGAGACTGA

CCTTACTAAAGGACCTCATGAATTTTGCTCTCAACATACAATGCTAGTTA

AACAGGGTGATGATTATGTGTACCTTCCTTACCCAGATCCATCAAGAATC

CTAGGGGCCGGCTGTTTTGTAGATGATATCGTAAAAACAGATGGTACACT

TATGATTGAACGGTTCGTGTCTTTAGCTATAGATGCTTACCCACTTACTA

AACATCCTAATCAGGAGTATGCTGATGTCTTTCATTTGTACTTACAATAC

ATAAGAAAGCTACATGATGAGTTAACAGGACACATGTTAGACATGTATTC

TGTTATGCTTACTAATGATAACACTTCAAGGTATTGGGAACCTGAGTTTT

ATGAGGCTATGTACACACCGCATACAGTCTTACAGGCTGTTGGGGCTTGT

GTTCTTTGCAATTCACAGACTTCATTAAGATGTGGTGCTTGCATACGTAG

ACCATTCTTATGTTGTAAATGCTGTTACGACCATGTCATATCAACATCAC

ATAAATTAGTCTTGTCTGTTAATCCGTATGTTTGCAATGCTCCAGGTTGT

GATGTCACAGATGTGACTCAACTTTACTTAGGAGGTATGAGCTATTATTG

TAAATCACATAAACCACCCATTAGTTTTCCATTGTGTGCTAATGGACAAG

TTTTTGGTTTATATAAAAATACATGTGTTGGTAGCGATAATGTTACTGAC

TTTAATGCAATTGCAACATGTGACTGGACAAATGCTGGTGATTACATTTT

AGCTAACACCTGTACTGAAAGACTCAAGCTTTTTGCAGCAGAAACGCTCA

AAGCTACTGAGGAGACATTTAAACTGTCTTATGGTATTGCTACTGTACGT

GAAGTGCTGTCTGACAGAGAATTACATCTTTCATGGGAAGTTGGTAAACC

TAGACCACCACTTAACCGAAATTATGTCTTTACTGGTTATCGTGTAACTA

AAAACAGTAAAGTACAAATAGGAGAGTACACCTTTGAAAAAGGTGACTAT

GGTGATGCTGTTGTTTACCGAGGTACAACAACTTACAAATTAAATGTTGG

TGATTATTTTGTGCTGACATCACATACAGTAATGCCATTAAGTGCACCTA

CACTAGTGCCACAAGAGCACTATGTTAGAATTACTGGCTTATACCCAACA

CTCAATATCTCAGATGAGTTTTCTAGCAATGTTGCAAATTATCAAAAGGT

TGGTATGCAAAAGTATTCTACACTCCAGGGACCACCTGGTACTGGTAAGA

GTCATTTTGCTATTGGCCTAGCTCTCTACTACCCTTCTGCTCGCATAGTG

TATACAGCTTGCTCTCATGCCGCTGTTGATGCACTATGTGAGAAGGCATT

AAAATATTTGCCTATAGATAAATGTAGTAGAATTATACCTGCACGTGCTC

GTGTAGAGTGTTTTGATAAATTCAAAGTGAATTCAACATTAGAACAGTAT

GTCTTTTGTACTGTAAATGCATTGCCTGAGACGACAGCAGATATAGTTGT

CTTTGATGAAATTTCAATGGCCACAAATTATGATTTGAGTGTTGTCAATG

CCAGATTACGTGCTAAGCACTATGTGTACATTGGCGACCCTGCTCAATTA

CCTGCACCACGCACATTGCTAACTAAGGGCACACTAGAACCAGAATATTT

CAATTCAGTGTGTAGACTTATGAAAACTATAGGTCCAGACATGTTCCTCG

GAACTTGTCGGCGTTGTCCTGCTGAAATTGTTGACACTGTGAGTGCTTTG

GTTTATGATAATAAGCTTAAAGCACATAAAGACAAATCAGCTCAATGCTT

TAAAATGTTTTATAAGGGTGTTATCACGCATGATGTTTCATCTGCAATTA

ACAGGCCACAAATAGGCGTGGTAAGAGAATTCCTTACACGTAACCCTGCT

TGGAGAAAAGCTGTCTTTATTTCACCTTATAATTCACAGAATGCTGTAGC

CTCAAAGATTTTGGGACTACCAACTCAA custom-character

CT

CAGAATATGACTATGTCATATTCACTCAAACCACTGAAACAGCTCACTCT

TGTAATGTAAACAGATTTAATGTTGCTATTACCAGAGCAAAAGTAGGCA

qPCR Set 1
GATGTGGTGCTTGCATACGT
68

Primer

Forward-1

qPCR Set 1
TGCTGTTACGACCATGTCAT
69

Probe-1

qPCR Set 1
TCACAACCTGGAGCATTGCA
70

Primer

Reverse-1

qPCR Set 2
AATAGAGCTCGCACCGTAGC
71

Primer

Forward-2

qPCR Set 2
GGTGTCTCTATCTGTAGTACTATGACC
72

Probe-2

qPCR Set 2
AGTGGCGGCTATTGATTTCA
73

Primer

Reverse-2

IL-6 nucleic
ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGG
74

acid sequence
GCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGTACCCCCAGGAG

(protein coding
AAGATTCCAAAGATGTAGCCGCCCCACACAGACAGCCACTCACCTCTTCA

sequence)
GAACGAATTGACAAACAAATTCGGTACATCCTCGACGGCATCTCA custom-character

Bold and

AACAAGAGTAACATGTGTGAAAGCAGCAAAGAGG

italicized:
GAGTGGCAGAAAACAACCTGAACCTTCGAAAGATGGCTGAAAAAGATGGA

siRNA binding
TGCTTCCAATCTGGATTCAAT custom-character

ATCATGAG

regions
TGGTCTTTTGGAGTTTGAGGTATACCTAGAGTACCTCCAGAACAGATTTG

AGAGTAGTGAGGAACAAGCCAGAGCTGTGCAGATGAGTACAAAAGTCCTG

ATCCAGTTCCTGCAGAAAAAGGCAAAGAATCTAGATGCAATAACCACCCC

TGACCCAACCACAAATGCCAGCCTGCTGACGAAGCTGCAGGCACAGAACC

AGTGGCTGCAGGACATGACAACTCATCTCATTCTGCGCAGCTTTAAGGAG

TTCCTGCAGTCCAGCCT custom-character

G

IL-6R-alpha
ATGCTGGCCGTCGGCTGCGCGCTGCTGGCTGCCCTGCTGGCCGCGCCGGG
75

nucleic acid
AGCGGCGCTGGCCCCAAGGCGCTGCCCTGCGCAGGAGGTGGCGAGAGGCG

sequence
TGCTGACCAGTCTGCCAGGAGACAGCGTGACTCTGACCTGCCCGGGGGTA

(protein coding
GAGCCGGAAGACAATGCCACTGTTCACTGGGTGCTCAGGAAGCCGGCTGC

sequence)
AGGCTCCCACCCCAGCAGATGGGCTGGCATGGGAAGGAGGCTGCTGCTGA

Bold and
GGTCGGTGCAGCTCCACGACTCTGGAAACTATTCATGCTACCGGGCCGGC

italicized:
CGCCCAGCTGGGACTGTGCACTTGCTGGTGGATGTTCCCCCCGAGGAGCC

siRNA binding
CCAGCTCTCCTGCTTCCGGAAGAGCCCCCTCAGCAATGTTGTTTGTGAGT

regions
GGGGTCCTCGGAGCACCCCATCCCTGACGACAAAGGCTGTGCTCTTG custom-character

GCGGCCGAAGACTTCCAGGAGCCGTGCCAGTA

TTCCCAGGAGTCCCAGAAGTTCTCCTGCCAGTTAGCAGTCCCGGAGGGAG

ACAGCTCTTTCTACATAGTGTCCATGTGCGTCGCCAGTAGTGTCGGGAGC

AAGTTCAGCAAAACTCAAACCTTTCAGGGTTGTGGAATCTTGCAGCCTGA

TCCGCCTGCCAACATCACAGTCACTGCCGTGGCCAGAAACCCCCGCTGGC

TCAGTGTCACCTGGCAAGACCCCCACTCCTGGAACTCATCTTTCTACAGA

CTACGGTTTGAGCTCAGATATCGGGCT custom-character

AC

ATGGATGGTCAAGGACCTCCAGCATCACTGTGTCATCCACGACGCCTGGA

GCGGCCTGAGGCACGTGGTGCAGCTTCGTGCCCAGGAGGAGTTCGGGCAA

GGCGAGTGGAGCGAGTGGAGCCCGGAGGCCATGGGCACGCCTTGGACAGA

CAGGCTTTCTCCTCGTTGCCCAGGATGGAGTACAGCAGTGCAATCACAGC

TCACGGCAACTTCTGCCTCCTGGGTTCAAGCAATCCTCCCGCCTCAGCCT

CCTAAGTAG

IL-6R-beta
ATGTTGACGTTGCAGACTTGGCTAGTGCAAGCCTTGTTTATTTTCCTCAC
76

nucleic acid
CACTGAATCTACAGGTGAACTTCTAGATCCATGTGGTTATATCAGTCCTG

sequence
AATCTCCAGTTGTACAACTTCATTCTAATTTCACTGCAGTTTGTGTGCTA

(protein coding
AAGGAAAAATGTATGGATTATTTTCATGTAAATGCTAATTACATTGTCTG

sequence)
GAAAACAAACCATTTTACTATTCCTAAGGAGCAATATACTATCATAAACA

Bold and
GAACAGCATCCAGTGTCACCTTTACAGATATAGCTTCATTAAATATTCAG

italicized:
CTCACTTGCAACATTCTTACATTCGGACAGCTTGAACAGAATGTTTATGG

siRNA binding
AATCACAATAATTTCAGGCTTGCCTCCAGAAAAACCTAAAAATTTGAGTT

regions
GCATTGTGAACGAGGGGAAGAAAATGAGGTGTGAGTGGGATGGTGGAAGG

GAAACACACTTGGAGACAAACTTCACTTTAAAATCTGAATGGGCAACACA

CAAGTTTGCTGATTGCAAAGCAAAACGTGACACCCCCACCTCATGCACTG

TTGATTATTCTACTGTGTATTTTGTCAACATTGAAGTCTGGGTAGAAGCA

GAGAATGCCCTT custom-character

ATCAATTTTGATCCTGT

ATATAAAGTGAAGCCCAATCCGCCACATAATTTATCAGTGATCAACTCAG

AGGAACTGTCTAGTATCTTAAAATTGACATGGACCAACCCAAGTATTAAG

AGTGTTATAATACTAAAATATAACATTCAATATAGGACCAAAGATGCCTC

AACTTGGAGCCAGATTCCTCCTGAAGACACAGCATCCACCCGATCTTCAT

TCACTGTCCAAGACCTTAAACCTTTTACAGAATATGTGTTTAGGATTCGC

TGTATGAAGGAAGATGGTAAGGGATACTGGAGTGACTGGAGTGAAGAAGC

AAGTGGGATCACCTATGAAGATAACATTGCCTCCTTTTGA

SARS CoV-
ATTAAAGGTTTATACCTTCCCAGGTAACAAACCAACCAACTTTCGATCTC
77

2_Refseq
TTGTAGATCTGTTCTCTAAACGAACTTTAAAATCTGTGTGGCTGTCACTC

GGCTGCATGCTTAGTGCACTCACGCAGTATAATTAATAACTAATTACTGT

CGTTGACAGGACACGAGTAACTCGTCTATCTTCTGCAGGCTGCTTACGGT

TTCGTCCGTGTTGCAGCCGATCATCAGCACATCTAGGTTTCGTCCGGGTG

TGACCGAAAGGTAAGATGGAGAGCCTTGTCCCTGGTTTCAACGAGAAAAC

ACACGTCCAACTCAGTTTGCCTGTTTTACAGGTTCGCGACGTGCTCGTAC

GTGGCTTTGGAGACTCCGTGGAGGAGGTCTTATCAGAGGCACGTCAACAT

CTTAAAGATGGCACTTGTGGCTTAGTAGAAGTTGAAAAAGGCGTTTTGCC

TCAACTTGAACAGCCCTATGTGTTCATCAAACGTTCGGATGCTCGAACTG

CACCTCATGGTCATGTTATGGTTGAGCTGGTAGCAGAACTCGAAGGCATT

CAGTACGGTCGTAGTGGTGAGACACTTGGTGTCCTTGTCCCTCATGTGGG

CGAAATACCAGTGGCTTACCGCAAGGTTCTTCTTCGTAAGAACGGTAATA

AAGGAGCTGGTGGCCATAGTTACGGCGCCGATCTAAAGTCATTTGACTTA

GGCGACGAGCTTGGCACTGATCCTTATGAAGATTTTCAAGAAAACTGGAA

CACTAAACATAGCAGTGGTGTTACCCGTGAACTCATGCGTGAGCTTAACG

GAGGGGCATACACTCGCTATGTCGATAACAACTTCTGTGGCCCTGATGGC

TACCCTCTTGAGTGCATTAAAGACCTTCTAGCACGTGCTGGTAAAGCTTC

ATGCACTTTGTCCGAACAACTGGACTTTATTGACACTAAGAGGGGTGTAT

ACTGCTGCCGTGAACATGAGCATGAAATTGCTTGGTACACGGAACGTTCT

GAAAAGAGCTATGAATTGCAGACACCTTTTGAAATTAAATTGGCAAAGAA

ATTTGACACCTTCAATGGGGAATGTCCAAATTTTGTATTTCCCTTAAATT

CCATAATCAAGACTATTCAACCAAGGGTTGAAAAGAAAAAGCTTGATGGC

TTTATGGGTAGAATTCGATCTGTCTATCCAGTTGCGTCACCAAATGAATG

CAACCAAATGTGCCTTTCAACTCTCATGAAGTGTGATCATTGTGGTGAAA

CTTCATGGCAGACGGGCGATTTTGTTAAAGCCACTTGCGAATTTTGTGGC

ACTGAGAATTTGACTAAAGAAGGTGCCACTACTTGTGGTTACTTACCCCA

AAATGCTGTTGTTAAAATTTATTGTCCAGCATGTCACAATTCAGAAGTAG

GACCTGAGCATAGTCTTGCCGAATACCATAATGAATCTGGCTTGAAAACC

ATTCTTCGTAAGGGTGGTCGCACTATTGCCTTTGGAGGCTGTGTGTTCTC

TTATGTTGGTTGCCATAACAAGTGTGCCTATTGGGTTCCACGTGCTAGCG

CTAACATAGGTTGTAACCATACAGGTGTTGTTGGAGAAGGTTCCGAAGGT

CTTAATGACAACCTTCTTGAAATACTCCAAAAAGAGAAAGTCAACATCAA

TATTGTTGGTGACTTTAAACTTAATGAAGAGATCGCCATTATTTTGGCAT

CTTTTTCTGCTTCCACAAGTGCTTTTGTGGAAACTGTGAAAGGTTTGGAT

TATAAAGCATTCAAACAAATTGTTGAATCCTGTGGTAATTTTAAAGTTAC

AAAAGGAAAAGCTAAAAAAGGTGCCTGGAATATTGGTGAACAGAAATCAA

TACTGAGTCCTCTTTATGCATTTGCATCAGAGGCTGCTCGTGTTGTACGA

TCAATTTTCTCCCGCACTCTTGAAACTGCTCAAAATTCTGTGCGTGTTTT

ACAGAAGGCCGCTATAACAATACTAGATGGAATTTCACAGTATTCACTGA

GACTCATTGATGCTATGATGTTCACATCTGATTTGGCTACTAACAATCTA

GTTGTAATGGCCTACATTACAGGTGGTGTTGTTCAGTTGACTTCGCAGTG

GCTAACTAACATCTTTGGCACTGTTTATGAAAAACTCAAACCCGTCCTTG

ATTGGCTTGAAGAGAAGTTTAAGGAAGGTGTAGAGTTTCTTAGAGACGGT

TGGGAAATTGTTAAATTTATCTCAACCTGTGCTTGTGAAATTGTCGGTGG

ACAAATTGTCACCTGTGCAAAGGAAATTAAGGAGAGTGTTCAGACATTCT

TTAAGCTTGTAAATAAATTTTTGGCTTTGTGTGCTGACTCTATCATTATT

GGTGGAGCTAAACTTAAAGCCTTGAATTTAGGTGAAACATTTGTCACGCA

CTCAAAGGGATTGTACAGAAAGTGTGTTAAATCCAGAGAAGAAACTGGCC

TACTCATGCCTCTAAAAGCCCCAAAAGAAATTATCTTCTTAGAGGGAGAA

ACACTTCCCACAGAAGTGTTAACAGAGGAAGTTGTCTTGAAAACTGGTGA

TTTACAACCATTAGAACAACCTACTAGTGAAGCTGTTGAAGCTCCATTGG

TTGGTACACCAGTTTGTATTAACGGGCTTATGTTGCTCGAAATCAAAGAC

ACAGAAAAGTACTGTGCCCTTGCACCTAATATGATGGTAACAAACAATAC

CTTCACACTCAAAGGCGGTGCACCAACAAAGGTTACTTTTGGTGATGACA

CTGTGATAGAAGTGCAAGGTTACAAGAGTGTGAATATCACTTTTGAACTT

GATGAAAGGATTGATAAAGTACTTAATGAGAAGTGCTCTGCCTATACAGT

TGAACTCGGTACAGAAGTAAATGAGTTCGCCTGTGTTGTGGCAGATGCTG

TCATAAAAACTTTGCAACCAGTATCTGAATTACTTACACCACTGGGCATT

GATTTAGATGAGTGGAGTATGGCTACATACTACTTATTTGATGAGTCTGG

TGAGTTTAAATTGGCTTCACATATGTATTGTTCTTTCTACCCTCCAGATG

AGGATGAAGAAGAAGGTGATTGTGAAGAAGAAGAGTTTGAGCCATCAACT

CAATATGAGTATGGTACTGAAGATGATTACCAAGGTAAACCTTTGGAATT

TGGTGCCACTTCTGCTGCTCTTCAACCTGAAGAAGAGCAAGAAGAAGATT

GGTTAGATGATGATAGTCAACAAACTGTTGGTCAACAAGACGGCAGTGAG

GACAATCAGACAACTACTATTCAAACAATTGTTGAGGTTCAACCTCAATT

AGAGATGGAACTTACACCAGTTGTTCAGACTATTGAAGTGAATAGTTTTA

GTGGTTATTTAAAACTTACTGACAATGTATACATTAAAAATGCAGACATT

GTGGAAGAAGCTAAAAAGGTAAAACCAACAGTGGTTGTTAATGCAGCCAA

TGTTTACCTTAAACATGGAGGAGGTGTTGCAGGAGCCTTAAATAAGGCTA

CTAACAATGCCATGCAAGTTGAATCTGATGATTACATAGCTACTAATGGA

CCACTTAAAGTGGGTGGTAGTTGTGTTTTAAGCGGACACAATCTTGCTAA

ACACTGTCTTCATGTTGTCGGCCCAAATGTTAACAAAGGTGAAGACATTC

AACTTCTTAAGAGTGCTTATGAAAATTTTAATCAGCACGAAGTTCTACTT

GCACCATTATTATCAGCTGGTATTTTTGGTGCTGACCCTATACATTCTTT

AAGAGTTTGTGTAGATACTGTTCGCACAAATGTCTACTTAGCTGTCTTTG

ATAAAAATCTCTATGACAAACTTGTTTCAAGCTTTTTGGAAATGAAGAGT

GAAAAGCAAGTTGAACAAAAGATCGCTGAGATTCCTAAAGAGGAAGTTAA

GCCATTTATAACTGAAAGTAAACCTTCAGTTGAACAGAGAAAACAAGATG

ATAAGAAAATCAAAGCTTGTGTTGAAGAAGTTACAACAACTCTGGAAGAA

ACTAAGTTCCTCACAGAAAACTTGTTACTTTATATTGACATTAATGGCAA

TCTTCATCCAGATTCTGCCACTCTTGTTAGTGACATTGACATCACTTTCT

TAAAGAAAGATGCTCCATATATAGTGGGTGATGTTGTTCAAGAGGGTGTT

TTAACTGCTGTGGTTATACCTACTAAAAAGGCTGGTGGCACTACTGAAAT

GCTAGCGAAAGCTTTGAGAAAAGTGCCAACAGACAATTATATAACCACTT

ACCCGGGTCAGGGTTTAAATGGTTACACTGTAGAGGAGGCAAAGACAGTG

CTTAAAAAGTGTAAAAGTGCCTTTTACATTCTACCATCTATTATCTCTAA

TGAGAAGCAAGAAATTCTTGGAACTGTTTCTTGGAATTTGCGAGAAATGC

TTGCACATGCAGAAGAAACACGCAAATTAATGCCTGTCTGTGTGGAAACT

AAAGCCATAGTTTCAACTATACAGCGTAAATATAAGGGTATTAAAATACA

AGAGGGTGTGGTTGATTATGGTGCTAGATTTTACTTTTACACCAGTAAAA

CAACTGTAGCGTCACTTATCAACACACTTAACGATCTAAATGAAACTCTT

GTTACAATGCCACTTGGCTATGTAACACATGGCTTAAATTTGGAAGAAGC

TGCTCGGTATATGAGATCTCTCAAAGTGCCAGCTACAGTTTCTGTTTCTT

CACCTGATGCTGTTACAGCGTATAATGGTTATCTTACTTCTTCTTCTAAA

ACACCTGAAGAACATTTTATTGAAACCATCTCACTTGCTGGTTCCTATAA

AGATTGGTCCTATTCTGGACAATCTACACAACTAGGTATAGAATTTCTTA

AGAGAGGTGATAAAAGTGTATATTACACTAGTAATCCTACCACATTCCAC

CTAGATGGTGAAGTTATCACCTTTGACAATCTTAAGACACTTCTTTCTTT

GAGAGAAGTGAGGACTATTAAGGTGTTTACAACAGTAGACAACATTAACC

TCCACACGCAAGTTGTGGACATGTCAATGACATATGGACAACAGTTTGGT

CCAACTTATTTGGATGGAGCTGATGTTACTAAAATAAAACCTCATAATTC

ACATGAAGGTAAAACATTTTATGTTTTACCTAATGATGACACTCTACGTG

TTGAGGCTTTTGAGTACTACCACACAACTGATCCTAGTTTTCTGGGTAGG

TACATGTCAGCATTAAATCACACTAAAAAGTGGAAATACCCACAAGTTAA

TGGTTTAACTTCTATTAAATGGGCAGATAACAACTGTTATCTTGCCACTG

CATTGTTAACACTCCAACAAATAGAGTTGAAGTTTAATCCACCTGCTCTA

CAAGATGCTTATTACAGAGCAAGGGCTGGTGAAGCTGCTAACTTTTGTGC

ACTTATCTTAGCCTACTGTAATAAGACAGTAGGTGAGTTAGGTGATGTTA

GAGAAACAATGAGTTACTTGTTTCAACATGCCAATTTAGATTCTTGCAAA

AGAGTCTTGAACGTGGTGTGTAAAACTTGTGGACAACAGCAGACAACCCT

TAAGGGTGTAGAAGCTGTTATGTACATGGGCACACTTTCTTATGAACAAT

TTAAGAAAGGTGTTCAGATACCTTGTACGTGTGGTAAACAAGCTACAAAA

TATCTAGTACAACAGGAGTCACCTTTTGTTATGATGTCAGCACCACCTGC

TCAGTATGAACTTAAGCATGGTACATTTACTTGTGCTAGTGAGTACACTG

GTAATTACCAGTGTGGTCACTATAAACATATAACTTCTAAAGAAACTTTG

TATTGCATAGACGGTGCTTTACTTACAAAGTCCTCAGAATACAAAGGTCC

TATTACGGATGTTTTCTACAAAGAAAACAGTTACACAACAACCATAAAAC

CAGTTACTTATAAATTGGATGGTGTTGTTTGTACAGAAATTGACCCTAAG

TTGGACAATTATTATAAGAAAGACAATTCTTATTTCACAGAGCAACCAAT

TGATCTTGTACCAAACCAACCATATCCAAACGCAAGCTTCGATAATTTTA

AGTTTGTATGTGATAATATCAAATTTGCTGATGATTTAAACCAGTTAACT

GGTTATAAGAAACCTGCTTCAAGAGAGCTTAAAGTTACATTTTTCCCTGA

CTTAAATGGTGATGTGGTGGCTATTGATTATAAACACTACACACCCTCTT

TTAAGAAAGGAGCTAAATTGTTACATAAACCTATTGTTTGGCATGTTAAC

AATGCAACTAATAAAGCCACGTATAAACCAAATACCTGGTGTATACGTTG

TCTTTGGAGCACAAAACCAGTTGAAACATCAAATTCGTTTGATGTACTGA

AGTCAGAGGACGCGCAGGGAATGGATAATCTTGCCTGCGAAGATCTAAAA

CCAGTCTCTGAAGAAGTAGTGGAAAATCCTACCATACAGAAAGACGTTCT

TGAGTGTAATGTGAAAACTACCGAAGTTGTAGGAGACATTATACTTAAAC

CAGCAAATAATAGTTTAAAAATTACAGAAGAGGTTGGCCACACAGATCTA

ATGGCTGCTTATGTAGACAATTCTAGTCTTACTATTAAGAAACCTAATGA

ATTATCTAGAGTATTAGGTTTGAAAACCCTTGCTACTCATGGTTTAGCTG

CTGTTAATAGTGTCCCTTGGGATACTATAGCTAATTATGCTAAGCCTTTT

CTTAACAAAGTTGTTAGTACAACTACTAACATAGTTACACGGTGTTTAAA

CCGTGTTTGTACTAATTATATGCCTTATTTCTTTACTTTATTGCTACAAT

TGTGTACTTTTACTAGAAGTACAAATTCTAGAATTAAAGCATCTATGCCG

ACTACTATAGCAAAGAATACTGTTAAGAGTGTCGGTAAATTTTGTCTAGA

GGCTTCATTTAATTATTTGAAGTCACCTAATTTTTCTAAACTGATAAATA

TTATAATTTGGTTTTTACTATTAAGTGTTTGCCTAGGTTCTTTAATCTAC

TCAACCGCTGCTTTAGGTGTTTTAATGTCTAATTTAGGCATGCCTTCTTA

CTGTACTGGTTACAGAGAAGGCTATTTGAACTCTACTAATGTCACTATTG

CAACCTACTGTACTGGTTCTATACCTTGTAGTGTTTGTCTTAGTGGTTTA

GATTCTTTAGACACCTATCCTTCTTTAGAAACTATACAAATTACCATTTC

ATCTTTTAAATGGGATTTAACTGCTTTTGGCTTAGTTGCAGAGTGGTTTT

TGGCATATATTCTTTTCACTAGGTTTTTCTATGTACTTGGATTGGCTGCA

ATCATGCAATTGTTTTTCAGCTATTTTGCAGTACATTTTATTAGTAATTC

TTGGCTTATGTGGTTAATAATTAATCTTGTACAAATGGCCCCGATTTCAG

CTATGGTTAGAATGTACATCTTCTTTGCATCATTTTATTATGTATGGAAA

AGTTATGTGCATGTTGTAGACGGTTGTAATTCATCAACTTGTATGATGTG

TTACAAACGTAATAGAGCAACAAGAGTCGAATGTACAACTATTGTTAATG

GTGTTAGAAGGTCCTTTTATGTCTATGCTAATGGAGGTAAAGGCTTTTGC

AAACTACACAATTGGAATTGTGTTAATTGTGATACATTCTGTGCTGGTAG

TAGATTTATTAGTGATGAAGTTGCGAGAGACTTGTCACTACAGTTTAAAA

GACCAATAAATCCTACTGACCAGTCTTCTTACATCGTTGATAGTGTTACA

GTGAAGAATGGTTCCATCCATCTTTACTTTGATAAAGCTGGTCAAAAGAC

TTATGAAAGACATTCTCTCTCTCATTTTGTTAACTTAGACAACCTGAGAG

CTAATAACACTAAAGGTTCATTGCCTATTAATGTTATAGTTTTTGATGGT

AAATCAAAATGTGAAGAATCATCTGCAAAATCAGCGTCTGTTTACTACAG

TCAGCTTATGTGTCAACCTATACTGTTACTAGATCAGGCATTAGTGTCTG

ATGTTGGTGATAGTGCGGAAGTTGCAGTTAAAATGTTTGATGCTTACGTT

AATACGTTTTCATCAACTTTTAACGTACCAATGGAAAAACTCAAAACACT

AGTTGCAACTGCAGAAGCTGAACTTGCAAAGAATGTGTCCTTAGACAATG

TCTTATCTACTTTTATTTCAGCAGCTCGGCAAGGGTTTGTTGATTCAGAT

GTAGAAACTAAAGATGTTGTTGAATGTCTTAAATTGTCACATCAATCTGA

CATAGAAGTTACTGGCGATAGTTGTAATAACTATATGCTCACCTATAACA

AAGTTGAAAACATGACACCCCGTGACCTTGGTGCTTGTATTGACTGTAGT

GCGCGTCATATTAATGCGCAGGTAGCAAAAAGTCACAACATTGCTTTGAT

ATGGAACGTTAAAGATTTCATGTCATTGTCTGAACAACTACGAAAACAAA

TACGTAGTGCTGCTAAAAAGAATAACTTACCTTTTAAGTTGACATGTGCA

ACTACTAGACAAGTTGTTAATGTTGTAACAACAAAGATAGCACTTAAGGG

TGGTAAAATTGTTAATAATTGGTTGAAGCAGTTAATTAAAGTTACACTTG

TGTTCCTTTTTGTTGCTGCTATTTTCTATTTAATAACACCTGTTCATGTC

ATGTCTAAACATACTGACTTTTCAAGTGAAATCATAGGATACAAGGCTAT

TGATGGTGGTGTCACTCGTGACATAGCATCTACAGATACTTGTTTTGCTA

ACAAACATGCTGATTTTGACACATGGTTTAGCCAGCGTGGTGGTAGTTAT

ACTAATGACAAAGCTTGCCCATTGATTGCTGCAGTCATAACAAGAGAAGT

GGGTTTTGTCGTGCCTGGTTTGCCTGGCACGATATTACGCACAACTAATG

GTGACTTTTTGCATTTCTTACCTAGAGTTTTTAGTGCAGTTGGTAACATC

TGTTAGACACCATCAAAACTTATAGAGTACACTGACTTTGCAACATCAGC

TTGTGTTTTGGCTGCTGAATGTACAATTTTTAAAGATGCTTCTGGTAAGC

CAGTACCATATTGTTATGATACCAATGTACTAGAAGGTTCTGTTGCTTAT

GAAAGTTTACGCCCTGACACACGTTATGTGCTCATGGATGGCTCTATTAT

TCAATTTCCTAACACCTACCTTGAAGGTTCTGTTAGAGTGGTAACAACTT

TTGATTCTGAGTACTGTAGGCACGGCACTTGTGAAAGATCAGAAGCTGGT

GTTTGTGTATCTACTAGTGGTAGATGGGTACTTAACAATGATTATTACAG

ATCTTTACCAGGAGTTTTCTGTGGTGTAGATGCTGTAAATTTACTTACTA

ATATGTTTACACCACTAATTCAACCTATTGGTGCTTTGGACATATCAGCA

TCTATAGTAGCTGGTGGTATTGTAGCTATCGTAGTAACATGCCTTGCCTA

CTATTTTATGAGGTTTAGAAGAGCTTTTGGTGAATACAGTCATGTAGTTG

CCTTTAATACTTTACTATTCCTTATGTCATTCACTGTACTCTGTTTAACA

CCAGTTTACTCATTCTTACCTGGTGTTTATTCTGTTATTTACTTGTACTT

GACATTTTATCTTACTAATGATGTTTCTTTTTTAGCACATATTCAGTGGA

TGGTTATGTTCACACCTTTAGTACCTTTCTGGATAACAATTGCTTATATC

ATTTGTATTTCCACAAAGCATTTCTATTGGTTCTTTAGTAATTACCTAAA

GAGACGTGTAGTCTTTAATGGTGTTTCCTTTAGTACTTTTGAAGAAGCTG

CGCTGTGCACCTTTTTGTTAAATAAAGAAATGTATCTAAAGTTGCGTAGT

GATGTGCTATTACCTCTTACGCAATATAATAGATACTTAGCTCTTTATAA

TAAGTACAAGTATTTTAGTGGAGCAATGGATACAACTAGCTACAGAGAAG

CTGCTTGTTGTCATCTCGCAAAGGCTCTCAATGACTTCAGTAACTCAGGT

TCTGATGTTCTTTACCAACCACCACAAACCTCTATCACCTCAGCTGTTTT

GCAGAGTGGTTTTAGAAAAATGGCATTCCCATCTGGTAAAGTTGAGGGTT

GTATGGTACAAGTAACTTGTGGTACAACTACACTTAACGGTCTTTGGCTT

GATGACGTAGTTTACTGTCCAAGACATGTGATCTGCACCTCTGAAGACAT

GCTTAACCCTAATTATGAAGATTTACTCATTCGTAAGTCTAATCATAATT

TCTTGGTACAGGCTGGTAATGTTCAACTCAGGGTTATTGGACATTCTATG

CAAAATTGTGTACTTAAGCTTAAGGTTGATACAGCCAATCCTAAGACACC

TAAGTATAAGTTTGTTCGCATTCAACCAGGACAGACTTTTTCAGTGTTAG

CTTGTTACAATGGTTCACCATCTGGTGTTTACCAATGTGCTATGAGGCCC

AATTTCACTATTAAGGGTTCATTCCTTAATGGTTCATGTGGTAGTGTTGG

TTTTAACATAGATTATGACTGTGTCTCTTTTTGTTACATGCACCATATGG

AATTACCAACTGGAGTTCATGCTGGCACAGACTTAGAAGGTAACTTTTAT

GGACCTTTTGTTGACAGGCAAACAGCACAAGCAGCTGGTACGGACACAAC

TATTACAGTTAATGTTTTAGCTTGGTTGTACGCTGCTGTTATAAATGGAG

ACAGGTGGTTTCTCAATCGATTTACCACAACTCTTAATGACTTTAACCTT

GTGGCTATGAAGTACAATTATGAACCTCTAACACAAGACCATGTTGACAT

ACTAGGACCTCTTTCTGCTCAAACTGGAATTGCCGTTTTAGATATGTGTG

CTTCATTAAAAGAATTACTGCAAAATGGTATGAATGGACGTACCATATTG

GGTAGTGCTTTATTAGAAGATGAATTTACACCTTTTGATGTTGTTAGACA

ATGCTCAGGTGTTACTTTCCAAAGTGCAGTGAAAAGAACAATCAAGGGTA

CACACCACTGGTTGTTACTCACAATTTTGACTTCACTTTTAGTTTTAGTC

CAGAGTACTCAATGGTCTTTGTTCTTTTTTTTGTATGAAAATGCCTTTTT

ACCTTTTGCTATGGGTATTATTGCTATGTCTGCTTTTGCAATGATGTTTG

TCAAACATAAGCATGCATTTCTCTGTTTGTTTTTGTTACCTTCTCTTGCC

ACTGTAGCTTATTTTAATATGGTCTATATGCCTGCTAGTTGGGTGATGCG

TATTATGACATGGTTGGATATGGTTGATACTAGTTTGTCTGGTTTTAAGC

TAAAAGACTGTGTTATGTATGCATCAGCTGTAGTGTTACTAATCCTTATG

ACAGCAAGAACTGTGTATGATGATGGTGCTAGGAGAGTGTGGACACTTAT

GAATGTCTTGACACTCGTTTATAAAGTTTATTATGGTAATGCTTTAGATC

AAGCCATTTCCATGTGGGCTCTTATAATCTCTGTTACTTCTAACTACTCA

GGTGTAGTTACAACTGTCATGTTTTTGGCCAGAGGTATTGTTTTTATGTG

TGTTGAGTATTGCCCTATTTTCTTCATAACTGGTAATACACTTCAGTGTA

TAATGCTAGTTTATTGTTTCTTAGGCTATTTTTGTACTTGTTACTTTGGC

CTCTTTTGTTTACTCAACCGCTACTTTAGACTGACTCTTGGTGTTTATGA

TTACTTAGTTTCTACACAGGAGTTTAGATATATGAATTCACAGGGACTAC

TCCCACCCAAGAATAGCATAGATGCCTTCAAACTCAACATTAAATTGTTG

GGTGTTGGTGGCAAACCTTGTATCAAAGTAGCCACTGTACAGTCTAAAAT

GTCAGATGTAAAGTGCACATCAGTAGTCTTACTCTCAGTTTTGCAACAAC

TCAGAGTAGAATCATCATCTAAATTGTGGGCTCAATGTGTCCAGTTACAC

AATGACATTCTCTTAGCTAAAGATACTACTGAAGCCTTTGAAAAAATGGT

TTCACTACTTTCTGTTTTGCTTTCCATGCAGGGTGCTGTAGACATAAACA

AGCTTTGTGAAGAAATGCTGGACAACAGGGCAACCTTACAAGCTATAGCC

TCAGAGTTTAGTTCCCTTCCATCATATGCAGCTTTTGCTACTGCTCAAGA

AGCTTATGAGCAGGCTGTTGCTAATGGTGATTCTGAAGTTGTTCTTAAAA

AGTTGAAGAAGTCTTTGAATGTGGCTAAATCTGAATTTGACCGTGATGCA

GCCATGCAACGTAAGTTGGAAAAGATGGCTGATCAAGCTATGACCCAAAT

GTATAAACAGGCTAGATCTGAGGACAAGAGGGCAAAAGTTACTAGTGCTA

TGCAGACAATGCTTTTCACTATGCTTAGAAAGTTGGATAATGATGCACTC

AACAACATTATCAACAATGCAAGAGATGGTTGTGTTCCCTTGAACATAAT

ACCTCTTACAACAGCAGCCAAACTAATGGTTGTCATACCAGACTATAACA

CATATAAAAATACGTGTGATGGTACAACATTTACTTATGCATCAGCATTG

TGGGAAATCCAACAGGTTGTAGATGCAGATAGTAAAATTGTTCAACTTAG

TGAAATTAGTATGGACAATTCACCTAATTTAGCATGGCCTCTTATTGTAA

CAGCTTTAAGGGCCAATTCTGCTGTCAAATTACAGAATAATGAGCTTAGT

CCTGTTGCACTACGACAGATGTCTTGTGCTGCCGGTACTACACAAACTGC

TTGCACTGATGACAATGCGTTAGCTTACTACAACACAACAAAGGGAGGTA

GGTTTGTACTTGCACTGTTATCCGATTTACAGGATTTGAAATGGGCTAGA

TTCCCTAAGAGTGATGGAACTGGTACTATCTATACAGAACTGGAACCACC

TTGTAGGTTTGTTACAGACACACCTAAAGGTCCTAAAGTGAAGTATTTAT

ACTTTATTAAAGGATTAAACAACCTAAATAGAGGTATGGTACTTGGTAGT

TTAGCTGCCACAGTACGTCTACAAGCTGGTAATGCAACAGAAGTGCCTGC

CAATTCAACTGTATTATCTTTCTGTGCTTTTGCTGTAGATGCTGCTAAAG

CTTACAAAGATTATCTAGCTAGTGGGGGACAACCAATCACTAATTGTGTT

AAGATGTTGTGTACACACACTGGTACTGGTCAGGCAATAACAGTTACACC

GGAAGCCAATATGGATCAAGAATCCTTTGGTGGTGCATCGTGTTGTCTGT

ACTGCCGTTGCCACATAGATCATCCAAATCCTAAAGGATTTTGTGACTTA

AAAGGTAAGTATGTACAAATACCTACAACTTGTGCTAATGACCCTGTGGG

TTTTACACTTAAAAACACAGTCTGTACCGTCTGCGGTATGTGGAAAGGTT

ATGGCTGTAGTTGTGATCAACTCCGCGAACCCATGCTTCAGTCAGCTGAT

GCACAATCGTTTTTAAACGGGTTTGCGGTGTAAGTGCAGCCCGTCTTACA

CCGTGCGGCACAGGCACTAGTACTGATGTCGTATACAGGGCTTTTGACAT

CTACAATGATAAAGTAGCTGGTTTTGCTAAATTCCTAAAAACTAATTGTT

GTCGCTTCCAAGAAAAGGACGAAGATGACAATTTAATTGATTCTTACTTT

GTAGTTAAGAGACACACTTTCTCTAACTAGCAACATGAAGAAACAATTTA

TAATTTACTTAAGGATTGTCCAGCTGTTGCTAAACATGACTTCTTTAAGT

TTAGAATAGACGGTGACATGGTACCACATATATCACGTCAACGTCTTACT

AAATACACAATGGCAGACCTCGTCTATGCTTTAAGGCATTTTGATGAAGG

TAATTGTGACACATTAAAAGAAATACTTGTCACATACAATTGTTGTGATG

ATGATTATTTCAATAAAAAGGACTGGTATGATTTTGTAGAAAACCCAGAT

ATATTACGCGTATACGCCAACTTAGGTGAACGTGTACGCCAAGCTTTGTT

AAAAACAGTACAATTCTGTGATGCCATGCGAAATGCTGGTATTGTTGGTG

TACTGACATTAGATAATCAAGATCTCAATGGTAACTGGTATGATTTCGGT

GATTTCATACAAACCACGCCAGGTAGTGGAGTTCCTGTTGTAGATTCTTA

TTATTCATTGTTAATGCCTATATTAACCTTGACCAGGGCTTTAACTGCAG

AGTCACATGTTGACACTGACTTAACAAAGCCTTACATTAAGTGGGATTTG

TTAAAATATGACTTCACGGAAGAGAGGTTAAAACTCTTTGACCGTTATTT

TAAATATTGGGATCAGACATACCACCCAAATTGTGTTAACTGTTTGGATG

ACAGATGCATTCTGCATTGTGCAAACTTTAATGTTTTATTCTCTACAGTG

TTCCCACCTACAAGTTTTGGACCACTAGTGAGAAAAATATTTGTTGATGG

TGTTCCATTTGTAGTTTCAACTGGATACCACTTCAGAGAGCTAGGTGTTG

TAGATAATCAGGATGTAAACTTACATAGCTCTAGACTTAGTTTTAAGGAA

TTACTTGTGTATGCTGCTGACCCTGCTATGCACGCTGCTTCTGGTAATCT

ATTACTAGATAAACGCACTACGTGCTTTTCAGTAGCTGCACTTACTAACA

ATGTTGCTTTTCAAACTGTCAAACCCGGTAATTTTAACAAAGACTTCTAT

GACTTTGCTGTGTCTAAGGGTTTCTTTAAGGAAGGAAGTTCTGTTGAATT

AAAACACTTCTTCTTTGCTCAGGATGGTAATGCTGCTATCAGCGATTATG

ACTACTATCGTTATAATCTACCAACAATGTGTGATATCAGACAACTACTA

TTTGTAGTTGAAGTTGTTGATAAGTACTTTGATTGTTACGATGGTGGCTG

TATTAATGCTAACCAAGTCATCGTCAACAACCTAGACAAATCAGCTGGTT

TTCCATTTAATAAATGGGGTAAGGCTAGACTTTATTATGATTCAATGAGT

TATGAGGATCAAGATGCACTTTTCGCATATACAAAACGTAATGTCATCCC

TAGTATAACTCAAATGAATCTTAAGTATGCCATTAGTGCAAAGAATAGAG

CTCGCACCGTAGCTGGTGTCTCTATCTGTAGTACTATGACCAATAGACAG

TTTCATCAAAAATTATTGAAATCAATAGCCGCCACTAGAGGAGCTACTGT

AGTAATTGGAACAAGCAAATTCTATGGTGGTTGGCACAACATGTTAAAAA

CTGTTTATAGTGATGTAGAAAACCCTCACCTTATGGGTTGGGATTATCCT

AAATGTGATAGAGCCATGCCTAACATGCTTAGAATTATGGCCTCACTTGT

TCTTGCTCGCAAACATACAACGTGTTGTAGCTTGTCACACCGTTTCTATA

GATTAGCTAATGAGTGTGCTCAAGTATTGAGTGAAATGGTCATGTGTGGC

GGTTCACTATATGTTAAACCAGGTGGAACCTCATCAGGAGATGCCACAAC

TGCTTATGCTAATAGTGTTTTTAACATTTGTCAAGCTGTCACGGCCAATG

TTAATGCACTTTTATCTACTGATGGTAACAAAATTGCCGATAAGTATGTC

CGCAATTTACAACACAGACTTTATGAGTGTCTCTATAGAAATAGAGATGT

TGACACAGACTTTGTGAATGAGTTTTACGCATATTTGCGTAAACATTTCT

CAATGATGATACTCTCTGACGATGCTGTTGTGTGTTTCAATAGCACTTAT

GCATCTCAAGGTCTAGTGGCTAGCATAAAGAACTTTAAGTCAGTTCTTTA

TTATCAAAACAATGTTTTTATGTCTGAAGCAAAATGTTGGACTGAGACTG

ACCTTACTAAAGGACCTCATGAATTTTGCTCTCAACATACAATGCTAGTT

AAACAGGGTGATGATTATGTGTACCTTCCTTACCCAGATCCATCAAGAAT

CCTAGGGGCCGGCTGTTTTGTAGATGATATCGTAAAAACAGATGGTACAC

TTATGATTGAACGGTTCGTGTCTTTAGCTATAGATGCTTACCCACTTACT

AAACATCCTAATCAGGAGTATGCTGATGTCTTTCATTTGTACTTACAATA

CATAAGAAAGCTACATGATGAGTTAACAGGACACATGTTAGAGATGTATT

CTGTTATGCTTACTAATGATAACACTTCAAGGTATTGGGAACCTGAGTTT

TATGAGGCTATGTACACACCGCATACAGTCTTACAGGCTGTTGGGGCTTG

TGTTCTTTGCAATTCACAGACTTCATTAAGATGTGGTGCTTGCATACGTA

GACCATTCTTATGTTGTAAATGCTGTTACGACCATGTCATATCAACATCA

CATAAATTAGTCTTGTCTGTTAATCCGTATGTTTGCAATGCTCCAGGTTG

TGATGTCACAGATGTGACTCAACTTTACTTAGGAGGTATGAGCTATTATT

GTAAATCACATAAACCACCCATTAGTTTTCCATTGTGTGCTAATGGACAA

GTTTTTGGTTTATATAAAAATACATGTGTTGGTAGCGATAATGTTACTGA

CTTTAATGCAATTGCAACATGTGACTGGACAAATGCTGGTGATTACATTT

TAGCTAACACCTGTACTGAAAGACTCAAGCTTTTTGCAGCAGAAACGCTC

AAAGCTACTGAGGAGACATTTAAACTGTCTTATGGTATTGCTACTGTACG

TGAAGTGCTGTCTGACAGAGAATTACATCTTTCATGGGAAGTTGGTAAAC

CTAGACCACCACTTAACCGAAATTATGTCTTTACTGGTTATCGTGTAACT

AAAAACAGTAAAGTACAAATAGGAGAGTACACCTTTGAAAAAGGTGACTA

TGGTGATGCTGTTGTTTACCGAGGTACAACAACTTACAAATTAAATGTTG

GTGATTATTTTGTGCTGACATCACATACAGTAATGCCATTAAGTGCACCT

ACACTAGTGCCACAAGAGCACTATGTTAGAATTACTGGCTTATACCCAAC

ACTCAATATCTCAGATGAGTTTTCTAGCAATGTTGCAAATTATCAAAAGG

TTGGTATGCAAAAGTATTCTACACTCCAGGGACCACCTGGTACTGGTAAG

AGTCATTTTGCTATTGGCCTAGCTCTCTACTACCCTTCTGCTCGCATAGT

GTATACAGCTTGCTCTCATGCCGCTGTTGATGCACTATGTGAGAAGGCAT

TAAAATATTTGCCTATAGATAAATGTAGTAGAATTATACCTGCACGTGCT

CGTGTAGAGTGTTTTGATAAATTCAAAGTGAATTCAACATTAGAACAGTA

TGTCTTTTGTACTGTAAATGCATTGCCTGAGACGACAGCAGATATAGTTG

TCTTTGATGAAATTTCAATGGCCACAAATTATGATTTGAGTGTTGTCAAT

GCCAGATTACGTGCTAAGCACTATGTGTACATTGGCGACCCTGCTCAATT

ACCTGCACCACGCACATTGCTAACTAAGGGCACACTAGAACCAGAATATT

TCAATTCAGTGTGTAGACTTATGAAAACTATAGGTCCAGACATGTTCCTC

GGAACTTGTCGGCGTTGTCCTGCTGAAATTGTTGACACTGTGAGTGCTTT

GGTTTATGATAATAAGCTTAAAGCACATAAAGACAAATCAGCTCAATGCT

TTAAAATGTTTTATAAGGGTGTTATCACGCATGATGTTTCATCTGCAATT

AACAGGCCACAAATAGGCGTGGTAAGAGAATTCCTTACACGTAACCCTGC

TTGGAGAAAAGCTGTCTTTATTTCACCTTATAATTCACAGAATGCTGTAG

CCTCAAAGATTTTGGGACTACCAACTCAAACTGTTGATTCATCACAGGGC

TCAGAATATGACTATGTCATATTCACTCAAACCACTGAAACAGCTCACTC

TTGTAATGTAAACAGATTTAATGTTGCTATTACCAGAGCAAAAGTAGGCA

TACTTTGCATAATGTCTGATAGAGACCTTTATGACAAGTTGCAATTTACA

AGTCTTGAAATTCCACGTAGGAATGTGGCAACTTTACAAGCTGAAAATGT

AACAGGACTCTTTAAAGATTGTAGTAAGGTAATCACTGGGTTACATCCTA

CACAGGCACCTACACACCTCAGTGTTGACACTAAATTCAAAACTGAAGGT

TTATGTGTTGACATACCTGGCATACCTAAGGACATGACCTATAGAAGACT

CATCTCTATGATGGGTTTTAAAATGAATTATCAAGTTAATGGTTACCCTA

ACATGTTTATCACCCGCGAAGAAGCTATAAGACATGTACGTGCATGGATT

GGCTTCGATGTCGAGGGGTGTCATGCTACTAGAGAAGCTGTTGGTACCAA

TTTACCTTTACAGCTAGGTTTTTCTACAGGTGTTAACCTAGTTGCTGTAC

CTACAGGTTATGTTGATACACCTAATAATACAGATTTTTCCAGAGTTAGT

GCTAAACCACCGCCTGGAGATCAATTTAAACACCTCATACCACTTATGTA

CAAAGGACTTCCTTGGAATGTAGTGCGTATAAAGATTGTACAAATGTTAA

GTGACACACTTAAAAATCTCTCTGACAGAGTCGTATTTGTCTTATGGGCA

CATGGCTTTGAGTTGACATCTATGAAGTATTTTGTGAAAATAGGACCTGA

GCGCACCTGTTGTCTATGTGATAGACGTGCCACATGCTTTTCCACTGCTT

CAGACACTTATGCCTGTTGGCATCATTCTATTGGATTTGATTACGTCTAT

AATCCGTTTATGATTGATGTTCAACAATGGGGTTTTACAGGTAACCTACA

AAGCAACCATGATCTGTATTGTCAAGTCCATGGTAATGCACATGTAGCTA

GTTGTGATGCAATCATGACTAGGTGTCTAGCTGTCCACGAGTGCTTTGTT

AAGCGTGTTGACTGGACTATTGAATATCCTATAATTGGTGATGAACTGAA

GATTAATGCGGCTTGTAGAAAGGTTCAACACATGGTTGTTAAAGCTGCAT

TATTAGCAGACAAATTCCCAGTTCTTCACGACATTGGTAACCCTAAAGCT

ATTAAGTGTGTACCTCAAGCTGATGTAGAATGGAAGTTCTATGATGCACA

GCCTTGTAGTGACAAAGCTTATAAAATAGAAGAATTATTCTATTCTTATG

CCACACATTCTGACAAATTCACAGATGGTGTATGCCTATTTTGGAATTGC

AATGTCGATAGATATCCTGCTAATTCCATTGTTTGTAGATTTGACACTAG

AGTGCTATCTAACCTTAACTTGCCTGGTTGTGATGGTGGCAGTTTGTATG

TAAATAAACATGCATTCCACACACCAGCTTTTGATAAAAGTGCTTTTGTT

AATTTAAAACAATTACCATTTTTCTATTACTCTGACAGTCCATGTGAGTC

TCATGGAAAACAAGTAGTGTCAGATATAGATTATGTACCACTAAAGTCTG

CTACGTGTATAACACGTTGCAATTTAGGTGGTGCTGTCTGTAGACATCAT

GCTAATGAGTACAGATTGTATCTCGATGCTTATAACATGATGATCTCAGC

TGGCTTTAGCTTGTGGGTTTACAAACAATTTGATACTTATAACCTCTGGA

ACACTTTTACAAGACTTCAGAGTTTAGAAAATGTGGCTTTTAATGTTGTA

AATAAGGGACACTTTGATGGACAACAGGGTGAAGTACCAGTTTCTATCAT

TAATAACACTGTTTACACAAAAGTTGATGGTGTTGATGTAGAATTGTTTG

AAAATAAAACAACATTACCTGTTAATGTAGCATTTGAGCTTTGGGCTAAG

CGCAACATTAAACCAGTACCAGAGGTGAAAATACTCAATAATTTGGGTGT

GGACATTGCTGCTAATACTGTGATCTGGGACTACAAAAGAGATGCTCCAG

CACATATATCTACTATTGGTGTTTGTTCTATGACTGACATAGCCAAGAAA

CCAACTGAAACGATTTGTGCACCACTCACTGTCTTTTTTGATGGTAGAGT

TGATGGTCAAGTAGACTTATTTAGAAATGCCCGTAATGGTGTTCTTATTA

CAGAAGGTAGTGTTAAAGGTTTACAACCATCTGTAGGTCCCAAACAAGCT

AGTCTTAATGGAGTCACATTAATTGGAGAAGCCGTAAAAACACAGTTCAA

TTATTATAAGAAAGTTGATGGTGTTGTCCAACAATTACCTGAAACTTACT

TTACTCAGAGTAGAAATTTACAAGAATTTAAACCCAGGAGTCAAATGGAA

ATTGATTTCTTAGAATTAGCTATGGATGAATTCATTGAACGGTATAAATT

AGAAGGCTATGCCTTCGAACATATCGTTTATGGAGATTTTAGTCATAGTC

AGTTAGGTGGTTTACATCTACTGATTGGACTAGCTAAACGTTTTAAGGAA

TCACCTTTTGAATTAGAAGATTTTATTCCTATGGACAGTACAGTTAAAAA

CTATTTCATAACAGATGCGCAAACAGGTTCATCTAAGTGTGTGTGTTCTG

TTATTGATTTATTACTTGATGATTTTGTTGAAATAATAAAATCCCAAGAT

TTATCTGTAGTTTCTAAGGTTGTCAAAGTGACTATTGACTATACAGAAAT

TTCATTTATGCTTTGGTGTAAAGATGGCCATGTAGAAACATTTTACCCAA

AATTACAATCTAGTCAAGCGTGGCAACCGGGTGTTGCTATGCCTAATCTT

TACAAAATGCAAAGAATGCTATTAGAAAAGTGTGACCTTCAAAATTATGG

TGATAGTGCAACATTACCTAAAGGCATAATGATGAATGTCGCAAAATATA

CTCAACTGTGTCAATATTTAAACACATTAACATTAGCTGTACCCTATAAT

ATGAGAGTTATACATTTTGGTGCTGGTTCTGATAAAGGAGTTGCACCAGG

TACAGCTGTTTTAAGACAGTGGTTGCCTACGGGTACGCTGCTTGTCGATT

CAGATCTTAATGACTTTGTCTCTGATGCAGATTCAACTTTGATTGGTGAT

TGTGCAACTGTACATACAGCTAATAAATGGGATCTCATTATTAGTGATAT

GTACGACCCTAAGACTAAAAATGTTACAAAAGAAAATGACTCTAAAGAGG

GTTTTTTCACTTACATTTGTGGGTTTATACAACAAAAGCTAGCTCTTGGA

GGTTCCGTGGCTATAAAGATAACAGAACATTCTTGGAATGCTGATCTTTA

TAAGCTCATGGGACACTTCGCATGGTGGACAGCCTTTGTTACTAATGTGA

ATGCGTCATCATCTGAAGCATTTTTAATTGGATGTAATTATCTTGGCAAA

CCACGCGAACAAATAGATGGTTATGTCATGCATGCAAATTACATATTTTG

GAGGAATACAAATCCAATTCAGTTGTCTTCCTATTCTTTATTTGACATGA

GTAAATTTCCCCTTAAATTAAGGGGTACTGCTGTTATGTCTTTAAAAGAA

GGTCAAATCAATGATATGATTTTATCTCTTCTTAGTAAAGGTAGACTTAT

AATTAGAGAAAACAACAGAGTTGTTATTTCTAGTGATGTTCTTGTTAACA

ACTAAACGAACAATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAG

TCAGTGTGTTAATCTTACAACCAGAACTCAATTACCCCCTGCATACACTA

ATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCA

GTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTAC

TTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTG

ATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAG

AAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAA

GACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAG

TCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCAC

AAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGC

GAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTG

AAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAAT

ATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGT

GCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGC

CAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGA

AGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGC

AGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATA

ATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTC

TCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTA

TCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTC

CTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGA

TTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGC

TGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTT

ATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTAT

GCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGG

GCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTA

CAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGT

GGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACC

TTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTT

GTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGT

TTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACT

TTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGT

CTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTA

ACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCA

ACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCAC

AGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGT

GTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCA

GGATGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTA

CTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGT

GCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGA

CATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATT

CTCCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACT

ATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGC

CATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGT

CTATGACCAAGACATCAGTAGATTGTACAATGTACATTTGTGGTGATTCA

ACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATT

AAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAG

AAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGAT

TTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAG

CAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAG

ATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCT

AGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACC

TTTGCTCACAGATGAAATGATTGCTCAATACACTTCTGCACTGTTAGCGG

GTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATA

CCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACA

GAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTG

CTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGA

AAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCTTGT

TAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATA

TCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTG

ATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAAT

TAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGT

CAGAGTGTGTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGC

TATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTT

GCATGTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTG

CCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTT

TCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACA

AATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGATGTTGTAA

TAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGAC

TCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGA

TGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTC

AAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCT

CTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCC

ATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGG

TGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGC

TGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCC

AGTGCTCAAAGGAGTCAAATTAGATTACACATAAACGAACTTATGGATTT

GTTTATGAGAATCTTCACAATTGGAACTGTAACTTTGAAGCAAGGTGAAA

TCAAGGATGCTACTCCTTCAGATTTTGTTCGCGCTACTGCAACGATACCG

ATACAAGCCTCACTCCCTTTCGGATGGCTTATTGTTGGCGTTGCACTTCT

TGCTGTTTTTCAGAGCGCTTCCAAAATCATAACCCTCAAAAAGAGATGGC

AACTAGCACTCTCCAAGGGTGTTCACTTTGTTTGCAACTTGCTGTTGTTG

TTTGTAACAGTTTACTCACACCTTTTGCTCGTTGCTGCTGGCCTTGAAGC

CCCTTTTCTCTATCTTTATGCTTTAGTCTACTTCTTGCAGAGTATAAACT

TTGTAAGAATAATAATGAGGCTTTGGCTTTGCTGGAAATGCCGTTCCAAA

AACCCATTACTTTATGATGCCAACTATTTTCTTTGCTGGCATACTAATTG

TTACGACTATTGTATACCTTACAATAGTGTAACTTCTTCAATTGTCATTA

CTTCAGGTGATGGCACAACAAGTCCTATTTCTGAACATGACTACCAGATT

GGTGGTTATACTGAAAAATGGGAATCTGGAGTAAAAGACTGTGTTGTATT

ACACAGTTACTTCACTTCAGACTATTACCAGCTGTACTCAACTCAATTGA

GTACAGACACTGGTGTTGAACATGTTACCTTCTTCATCTACAATAAAATT

GTTGATGAGCCTGAAGAACATGTCCAAATTCACACAATCGACGGTTCATC

CGGAGTTGTTAATCCAGTAATGGAACCAATTTATGATGAACCGACGACGA

CTACTAGCGTGCCTTTGTAAGCACAAGCTGATGAGTACGAACTTATGTAC

TCATTCGTTTCGGAAGAGACAGGTACGTTAATAGTTAATAGCGTACTTCT

TTTTCTTGCTTTCGTGGTATTCTTGCTAGTTACACTAGCCATCCTTACTG

CGCTTCGATTGTGTGCGTACTGCTGCAATATTGTTAACGTGAGTCTTGTA

AAACCTTCTTTTTACGTTTACTCTCGTGTTAAAAATCTGAATTCTTCTAG

AGTTCCTGATCTTCTGGTCTAAACGAACTAAATATTATATTAGTTTTTCT

GTTTGGAACTTTAATTTTAGCCATGGCAGATTCCAACGGTACTATTACCG

TTGAAGAGCTTAAAAAGCTCCTTGAACAATGGAACCTAGTAATAGGTTTC

CTATTCCTTACATGGATTTGTCTTCTACAATTTGCCTATGCCAACAGGAA

TAGGTTTTTGTATATAATTAAGTTAATTTTCCTCTGGCTGTTATGGCCAG

TAACTTTAGCTTGTTTTGTGCTTGCTGCTGTTTACAGAATAAATTGGATC

ACCGGTGGAATTGCTATCGCAATGGCTTGTCTTGTAGGCTTGATGTGGCT

CAGCTACTTCATTGCTTCTTTCAGACTGTTTGCGCGTACGCGTTCCATGT

GGTCATTCAATCCAGAAACTAACATTCTTCTCAACGTGCCACTCCATGGC

ACTATTCTGACCAGACCGCTTCTAGAAAGTGAACTCGTAATCGGAGCTGT

GATCCTTCGTGGACATCTTCGTATTGCTGGACACCATCTAGGACGCTGTG

ACATCAAGGACCTGCCTAAAGAAATCACTGTTGCTACATCACGAACGCTT

TCTTATTACAAATTGGGAGCTTCGCAGCGTGTAGCAGGTGACTCAGGTTT

TGCTGCATACAGTCGCTACAGGATTGGCAACTATAAATTAAACACAGACC

ATTCCAGTAGCAGTGACAATATTGCTTTGCTTGTACAGTAAGTGACAACA

GATGTTTCATCTCGTTGACTTTCAGGTTACTATAGCAGAGATATTACTAA

TTATTATGAGGACTTTTAAAGTTTCCATTTGGAATCTTGATTACATCATA

AACCTCATAATTAAAAATTTATCTAAGTCACTAACTGAGAATAAATATTC

TCAATTAGATGAAGAGCAACCAATGGAGATTGATTAAACGAACATGAAAA

TTATTCTTTTCTTGGCACTGATAACACTCGCTACTTGTGAGCTTTATCAC

TACCAAGAGTGTGTTAGAGGTACAACAGTACTTTTAAAAGAACCTTGCTC

TTCTGGAACATACGAGGGCAATTCACCATTTCATCCTCTAGCTGATAACA

AATTTGCACTGACTTGCTTTAGCACTCAATTTGCTTTTGCTTGTCCTGAC

GGCGTAAAACACGTCTATCAGTTACGTGCCAGATCAGTTTCACCTAAACT

GTTCATCAGACAAGAGGAAGTTCAAGAACTTTACTCTCCAATTTTTCTTA

TTGTTGCGGCAATAGTGTTTATAACACTTTGCTTCACACTCAAAAGAAAG

ACAGAATGATTGAACTTTCATTAATTGACTTCTATTTGTGCTTTTTAGCC

TTTCTGCTATTCCTTGTTTTAATTATGCTTATTATCTTTTGGTTCTCACT

TGAACTGCAAGATCATAATGAAACTTGTCACGCCTAAACGAACATGAAAT

TTCTTGTTTTCTTAGGAATCATCACAACTGTAGCTGCATTTCACCAAGAA

TGTAGTTTACAGTCATGTACTCAACATCAACCATATGTAGTTGATGACCC

GTGTCCTATTCACTTCTATTCTAAATGGTATATTAGAGTAGGAGCTAGAA

AATCAGCACCTTTAATTGAATTGTGCGTGGATGAGGCTGGTTCTAAATCA

CCCATTCAGTACATCGATATCGGTAATTATACAGTTTCCTGTTTACCTTT

TACAATTAATTGCCAGGAACCTAAATTGGGTAGTCTTGTAGTGCGTTGTT

CGTTCTATGAAGACTTTTTAGAGTATCATGACGTTCGTGTTGTTTTAGAT

TTCATCTAAACGAACAAACTAAAATGTCTGATAATGGACCCCAAAATCAG

CGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAG

TAACCAGAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCC

AAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACAT

GGCAAGGAAGACCTTAAATTCCCTCGAGGACAAGGCGTTCCAATTAACAC

CAATAGCAGTCCAGATGACCAAATTGGCTACTACCGAAGAGCTACCAGAC

GAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTAT

TTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAA

CAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAA

AAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTA

CAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAG

CAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACA

GTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGA

ATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAG

ATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAG

GCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGG

CAAAAACGTACTGCCACTAAAGCATACAATGTAACACAAGCTTTCGGCAG

ACGTGGTCCAGAACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATCA

GACAAGGAACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCCCCC

AGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAGTCACACC

TTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGACAAAG

ATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATATTGACGCA

TACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAAGAAGGC

TGATGAAACTCAAGCCTTACCGCAGAGACAGAAGAAACAGCAAACTGTGA

CTCTTCTTCCTGCTGCAGATTTGGATGATTTCTCCAAACAATTGCAACAA

TCCATGAGCAGTGCTGACTCAACTCAGGCCTAAACTCATGCAGACCACAC

AAGGCAGATGGGCTATATAAACGTTTTCGCTTTTCCGTTTACGATATATA

GTCTACTCTTGTGCAGAATGAATTCTCGTAACTACATAGCACAAGTAGAT

GTAGTTAACTTTAATCTCACATAGCAATCTTTAATCAGTGTGTAACATTA

GGGAGGACTTGAAAGAGCCACCACATTTTCACCGAGGCCACGCGGAGTAC

GATCGAGTGTACAGTGAACAATGCTAGGGAGAGCTGCCTATATGGAAGAG

CCCTAATGTGTAAAATTAATTTTAGTAGTGCTATCCCCATGTGATTTTAA

TAGCTTCTTAGGAGAATGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAA

SARS
ATATTAGGTTTTTACCTACCCAGGAAAAGCCAACCAACCTCGATCTCTTG
78

CoV_Refseq
TAGATCTGTTCTCTAAACGAACTTTAAAATCTGTGTAGCTGTCGCTCGGC

TGCATGCCTAGTGCACCTACGCAGTATAAACAATAATAAATTTTACTGTC

GTTGACAAGAAACGAGTAACTCGTCCCTCTTCTGCAGACTGCTTACGGTT

TCGTCCGTGTTGCAGTCGATCATCAGCATACCTAGGTTTCGTCCGGGTGT

GACCGAAAGGTAAGATGGAGAGCCTTGTTCTTGGTGTCAACGAGAAAACA

CACGTCCAACTCAGTTTGCCTGTCCTTCAGGTTAGAGACGTGCTAGTGCG

TGGCTTCGGGGACTCTGTGGAAGAGGCCCTATCGGAGGCACGTGAACACC

TCAAAAATGGCACTTGTGGTCTAGTAGAGCTGGAAAAAGGCGTACTGCCC

CAGCTTGAACAGCCCTATGTGTTCATTAAACGTTCTGATGCCTTAAGCAC

CAATCACGGCCACAAGGTCGTTGAGCTGGTTGCAGAAATGGACGGCATTC

AGTACGGTCGTAGCGGTATAACACTGGGAGTACTCGTGCCACATGTGGGC

GAAACCCCAATTGCATACCGCAATGTTCTTCTTCGTAAGAACGGTAATAA

GGGAGCCGGTGGTCATAGCTATGGCATCGATCTAAAGTCTTATGACTTAG

GTGACGAGCTTGGCACTGATCCCATTGAAGATTATGAACAAAACTGGAAC

ACTAAGCATGGCAGTGGTGCACTCCGTGAACTCACTCGTGAGCTCAATGG

AGGTGCAGTCACTCGCTATGTCGACAACAATTTCTGTGGCCCAGATGGGT

ACCCTCTTGATTGCATCAAAGATTTTCTCGCACGCGCGGGCAAGTCAATG

TGCACTCTTTCCGAACAACTTGATTACATCGAGTCGAAGAGAGGTGTCTA

CTGCTGCCGTGACCATGAGCATGAAATTGCCTGGTTCACTGAGCGCTCTG

ATAAGAGCTACGAGCACCAGACACCCTTCGAAATTAAGAGTGCCAAGAAA

TTTGACACTTTCAAAGGGGAATGCCCAAAGTTTGTGTTTCCTCTTAACTC

AAAAGTCAAAGTCATTCAACCACGTGTTGAAAAGAAAAAGACTGAGGGTT

TCATGGGGCGTATACGCTCTGTGTACCCTGTTGCATCTCCACAGGAGTGT

AACAATATGCACTTGTCTACCTTGATGAAATGTAATCATTGCGATGAAGT

TTCATGGCAGACGTGCGACTTTCTGAAAGCCACTTGTGAACATTGTGGCA

CTGAAAATTTAGTTATTGAAGGACCTACTACATGTGGGTACCTACCTACT

AATGCTGTAGTGAAAATGCCATGTCCTGCCTGTCAAGACCCAGAGATTGG

ACCTGAGCATAGTGTTGCAGATTATCACAACCACTCAAACATTGAAACTC

GACTCCGCAAGGGAGGTAGGACTAGATGTTTTGGAGGCTGTGTGTTTGCC

TATGTTGGCTGCTATAATAAGCGTGCCTACTGGGTTCCTCGTGCTAGTGC

TGATATTGGCTCAGGCCATACTGGCATTACTGGTGACAATGTGGAGACCT

TGAATGAGGATCTCCTTGAGATACTGAGTCGTGAACGTGTTAACATTAAC

ATTGTTGGCGATTTTCATTTGAATGAAGAGGTTGCCATCATTTTGGCATC

TTTCTCTGCTTCTACAAGTGCCTTTATTGACACTATAAAGAGTCTTGATT

ACAAGTCTTTCAAAACCATTGTTGAGTCCTGCGGTAACTATAAAGTTACC

AAGGGAAAGCCCGTAAAAGGTGCTTGGAACATTGGACAACAGAGATCAGT

TTTAACACCACTGTGTGGTTTTCCCTCACAGGCTGCTGGTGTTATCAGAT

CAATTTTTGCGCGCACACTTGATGCAGCAAACCACTCAATTCCTGATTTG

CAAAGAGCAGCTGTCACCATACTTGATGGTATTTCTGAACAGTCATTACG

TCTTGTCGACGCCATGGTTTATACTTCAGACCTGCTCACCAACAGTGTCA

TTATTATGGCATATGTAACTGGTGGTCTTGTACAACAGACTTCTCAGTGG

TTGTCTAATCTTTTGGGCACTACTGTTGAAAAACTCAGGCCTATCTTTGA

ATGGATTGAGGCGAAACTTAGTGCAGGAGTTGAATTTCTCAAGGATGCTT

GGGAGATTCTCAAATTTCTCATTACAGGTGTTTTTGACATCGTCAAGGGT

CAAATACAGGTTGCTTCAGATAACATCAAGGATTGTGTAAAATGCTTCAT

TGATGTTGTTAACAAGGCACTCGAAATGTGCATTGATCAAGTCACTATCG

CTGGCGCAAAGTTGCGATCACTCAACTTAGGTGAAGTCTTCATCGCTCAA

AGCAAGGGACTTTACCGTCAGTGTATACGTGGCAAGGAGCAGCTGCAACT

ACTCATGCCTCTTAAGGCACCAAAAGAAGTAACCTTTCTTGAAGGTGATT

CACATGACACAGTACTTACCTCTGAGGAGGTTGTTCTCAAGAACGGTGAA

CTCGAAGCACTCGAGACGCCCGTTGATAGCTTCACAAATGGAGCTATCGT

TGGCACACCAGTCTGTGTAAATGGCCTCATGCTCTTAGAGATTAAGGACA

AAGAACAATACTGCGCATTGTCTCCTGGTTTACTGGCTACAAACAATGTC

TTTCGCTTAAAAGGGGGTGCACCAATTAAAGGTGTAACCTTTGGAGAAGA

TACTGTTTGGGAAGTTCAAGGTTACAAGAATGTGAGAATCACATTTGAGC

TTGATGAACGTGTTGACAAAGTGCTTAATGAAAAGTGCTCTGTCTACACT

GTTGAATCCGGTACCGAAGTTACTGAGTTTGCATGTGTTGTAGCAGAGGC

TGTTGTGAAGACTTTACAACCAGTTTCTGATCTCCTTACCAACATGGGTA

TTGATCTTGATGAGTGGAGTGTAGCTACATTCTACTTATTTGATGATGCT

GGTGAAGAAAACTTTTCATCACGTATGTATTGTTCCTTTTACCCTCCAGA

TGAGGAAGAAGAGGACGATGCAGAGTGTGAGGAAGAAGAAATTGATGAAA

CCTGTGAACATGAGTACGGTACAGAGGATGATTATCAAGGTCTCCCTCTG

GAATTTGGTGCCTCAGCTGAAACAGTTCGAGTTGAGGAAGAAGAAGAGGA

AGACTGGCTGGATGATACTACTGAGCAATCAGAGATTGAGCCAGAACCAG

AACCTACACCTGAAGAACCAGTTAATCAGTTTACTGGTTATTTAAAACTT

ACTGACAATGTTGCCATTAAATGTGTTGACATCGTTAAGGAGGCACAAAG

TGCTAATCCTATGGTGATTGTAAATGCTGCTAACATACACCTGAAACATG

GTGGTGGTGTAGCAGGTGCACTCAACAAGGCAACCAATGGTGCCATGCAA

AAGGAGAGTGATGATTACATTAAGCTAAATGGCCCTCTTACAGTAGGAGG

GTCTTGTTTGCTTTCTGGACATAATCTTGCTAAGAAGTGTCTGCATGTTG

TTGGACCTAACCTAAATGCAGGTGAGGACATCCAGCTTCTTAAGGCAGCA

TATGAAAATTTCAATTCACAGGACATCTTACTTGCACCATTGTTGTCAGC

AGGCATATTTGGTGCTAAACCACTTCAGTCTTTACAAGTGTGCGTGCAGA

CGGTTCGTACACAGGTTTATATTGCAGTCAATGACAAAGCTCTTTATGAG

CAGGTTGTCATGGATTATCTTGATAACCTGAAGCCTAGAGTGGAAGCACC

TAAACAAGAGGAGCCACCAAACACAGAAGATTCCAAAACTGAGGAGAAAT

CTGTCGTACAGAAGCCTGTCGATGTGAAGCCAAAAATTAAGGCCTGCATT

GATGAGGTTACCACAACACTGGAAGAAACTAAGTTTCTTACCAATAAGTT

ACTCTTGTTTGCTGATATCAATGGTAAGCTTTACCATGATTCTCAGAACA

TGCTTAGAGGTGAAGATATGTCTTTCCTTGAGAAGGATGCACCTTACATG

GTAGGTGATGTTATCACTAGTGGTGATATCACTTGTGTTGTAATACCCTC

CAAAAAGGCTGGTGGCACTACTGAGATGCTCTCAAGAGCTTTGAAGAAAG

TGCCAGTTGATGAGTATATAACCACGTACCCTGGACAAGGATGTGCTGGT

TATACACTTGAGGAAGCTAAGACTGCTCTTAAGAAATGCAAATCTGCATT

TTATGTACTACCTTCAGAAGCACCTAATGCTAAGGAAGAGATTCTAGGAA

CTGTATCCTGGAATTTGAGAGAAATGCTTGCTCATGCTGAAGAGACAAGA

AAATTAATGCCTATATGCATGGATGTTAGAGCCATAATGGCAACCATCCA

ACGTAAGTATAAAGGAATTAAAATTCAAGAGGGCATCGTTGACTATGGTG

TCCGATTCTTCTTTTATACTAGTAAAGAGCCTGTAGCTTCTATTATTACG

AAGCTGAACTCTCTAAATGAGCCGCTTGTCACAATGCCAATTGGTTATGT

GACACATGGTTTTAATCTTGAAGAGGCTGCGCGCTGTATGCGTTCTCTTA

AAGCTCCTGCCGTAGTGTCAGTATCATCACCAGATGCTGTTACTACATAT

AATGGATACCTCACTTCGTCATCAAAGACATCTGAGGAGCACTTTGTAGA

AACAGTTTCTTTGGCTGGCTCTTACAGAGATTGGTCCTATTCAGGACAGC

GTACAGAGTTAGGTGTTGAATTTCTTAAGCGTGGTGACAAAATTGTGTAC

CACACTCTGGAGAGCCCCGTCGAGTTTCATCTTGACGGTGAGGTTCTTTC

ACTTGACAAACTAAAGAGTCTCTTATCCCTGCGGGAGGTTAAGACTATAA

AAGTGTTCACAACTGTGGACAACACTAATCTCCACACACAGCTTGTGGAT

ATGTCTATGACATATGGACAGCAGTTTGGTCCAACATACTTGGATGGTGC

TGATGTTACAAAAATTAAACCTCATGTAAATCATGAGGGTAAGACTTTCT

TTGTACTACCTAGTGATGACACACTACGTAGTGAAGCTTTCGAGTACTAC

CATACTCTTGATGAGAGTTTTCTTGGTAGGTACATGTCTGCTTTAAACCA

CACAAAGAAATGGAAATTTCCTCAAGTTGGTGGTTTAACTTCAATTAAAT

GGGCTGATAACAATTGTTATTTGTCTAGTGTTTTATTAGCACTTCAACAG

CTTGAAGTCAAATTCAATGCACCAGCACTTCAAGAGGCTTATTATAGAGC

CCGTGCTGGTGATGCTGCTAACTTTTGTGCACTCATACTCGCTTACAGTA

ATAAAACTGTTGGCGAGCTTGGTGATGTCAGAGAAACTATGACCCATCTT

CTACAGCATGCTAATTTGGAATCTGCAAAGCGAGTTCTTAATGTGGTGTG

TAAACATTGTGGTCAGAAAACTACTACCTTAACGGGTGTAGAAGCTGTGA

TGTATATGGGTACTCTATCTTATGATAATCTTAAGACAGGTGTTTCCATT

CCATGTGTGTGTGGTCGTGATGCTACACAATATCTAGTACAACAAGAGTC

TTCTTTTGTTATGATGTCTGCACCACCTGCTGAGTATAAATTACAGCAAG

GTACATTCTTATGTGCGAATGAGTACACTGGTAACTATCAGTGTGGTCAT

TACACTCATATAACTGCTAAGGAGACCCTCTATCGTATTGACGGAGCTCA

CCTTACAAAGATGTCAGAGTACAAAGGACCAGTGACTGATGTTTTCTACA

AGGAAACATCTTACACTACAACCATCAAGCCTGTGTCGTATAAACTCGAT

GGAGTTACTTACACAGAGATTGAACCAAAATTGGATGGGTATTATAAAAA

GGATAATGCTTACTATACAGAGCAGCCTATAGACCTTGTACCAACTCAAC

CATTACCAAATGCGAGTTTTGATAATTTCAAACTCACATGTTCTAACACA

AAATTTGCTGATGATTTAAATCAAATGACAGGCTTCACAAAGCCAGCTTC

ACGAGAGCTATCTGTCACATTCTTCCCAGACTTGAATGGCGATGTAGTGG

CTATTGACTATAGACACTATTCAGCGAGTTTCAAGAAAGGTGCTAAATTA

CTGCATAAGCCAATTGTTTGGCACATTAACCAGGCTACAACCAAGACAAC

GTTCAAACCAAACACTTGGTGTTTACGTTGTCTTTGGAGTACAAAGCCAG

TAGATACTTCAAATTCATTTGAAGTTCTGGCAGTAGAAGACACACAAGGA

ATGGACAATCTTGCTTGTGAAAGTCAACAACCCACCTCTGAAGAAGTAGT

GGAAAATCCTACCATACAGAAGGAAGTCATAGAGTGTGACGTGAAAACTA

CCGAAGTTGTAGGCAATGTCATACTTAAACCATCAGATGAAGGTGTTAAA

GTAACACAAGAGTTAGGTCATGAGGATCTTATGGCTGCTTATGTGGAAAA

CACAAGCATTACCATTAAGAAACCTAATGAGCTTTCACTAGCCTTAGGTT

TAAAAACAATTGCCACTCATGGTATTGCTGCAATTAATAGTGTTCCTTGG

AGTAAAATTTTGGCTTATGTCAAACCATTCTTAGGACAAGCAGCAATTAC

AACATCAAATTGCGCTAAGAGATTAGCACAACGTGTGTTTAACAATTATA

TGCCTTATGTGTTTACATTATTGTTCCAATTGTGTACTTTTACTAAAAGT

ACCAATTCTAGAATTAGAGCTTCACTACCTACAACTATTGCTAAAAATAG

TGTTAAGAGTGTTGCTAAATTATGTTTGGATGCCGGCATTAATTATGTGA

AGTCACCCAAATTTTCTAAATTGTTCACAATCGCTATGTGGCTATTGTTG

TTAAGTATTTGCTTAGGTTCTCTAATCTGTGTAACTGCTGCTTTTGGTGT

ACTCTTATCTAATTTTGGTGCTCCTTCTTATTGTAATGGCGTTAGAGAAT

TGTATCTTAATTCGTCTAACGTTACTACTATGGATTTCTGTGAAGGTTCT

TTTCCTTGCAGCATTTGTTTAAGTGGATTAGACTCCCTTGATTCTTATCC

AGCTCTTGAAACCATTCAGGTGACGATTTCATCGTACAAGCTAGACTTGA

CAATTTTAGGTCTGGCCGCTGAGTGGGTTTTGGCATATATGTTGTTCACA

AAATTCTTTTATTTATTAGGTCTTTCAGCTATAATGCAGGTGTTCTTTGG

CTATTTTGCTAGTCATTTCATCAGCAATTCTTGGCTCATGTGGTTTATCA

TTAGTATTGTACAAATGGCACCCGTTTCTGCAATGGTTAGGATGTACATC

TTCTTTGCTTCTTTCTACTACATATGGAAGAGCTATGTTCATATCATGGA

TGGTTGCACCTCTTCGACTTGCATGATGTGCTATAAGCGCAATCGTGCCA

CACGCGTTGAGTGTACAACTATTGTTAATGGCATGAAGAGATCTTTCTAT

GTCTATGCAAATGGAGGCCGTGGCTTCTGCAAGACTCACAATTGGAATTG

TCTCAATTGTGACACATTTTGCACTGGTAGTACATTCATTAGTGATGAAG

TTGCTCGTGATTTGTCACTCCAGTTTAAAAGACCAATCAACCCTACTGAC

CAGTCATCGTATATTGTTGATAGTGTTGCTGTGAAAAATGGCGCGCTTCA

CCTCTACTTTGACAAGGCTGGTCAAAAGACCTATGAGAGACATCCGCTCT

CCCATTTTGTCAATTTAGACAATTTGAGAGCTAACAACACTAAAGGTTCA

CTGCCTATTAATGTCATAGTTTTTGATGGCAAGTCCAAATGCGACGAGTC

TGCTTCTAAGTCTGCTTCTGTGTACTACAGTCAGCTGATGTGCCAACCTA

TTCTGTTGCTTGACCAAGCTCTTGTATCAGACGTTGGAGATAGTACTGAA

GTTTCCGTTAAGATGTTTGATGCTTATGTCGACACCTTTTCAGCAACTTT

TAGTGTTCCTATGGAAAAACTTAAGGCACTTGTTGCTACAGCTCACAGCG

AGTTAGCAAAGGGTGTAGCTTTAGATGGTGTCCTTTCTACATTCGTGTCA

GCTGCCCGACAAGGTGTTGTTGATACCGATGTTGACACAAAGGATGTTAT

TGAATGTCTCAAACTTTCACATCACTCTGACTTAGAAGTGACAGGTGACA

GTTGTAACAATTTCATGCTCACCTATAATAAGGTTGAAAACATGACGCCC

AGAGATCTTGGCGCATGTATTGACTGTAATGCAAGGCATATCAATGCCCA

AGTAGCAAAAAGTCACAATGTTTCACTCATCTGGAATGTAAAAGACTACA

TGTCTTTATCTGAACAGCTGCGTAAACAAATTCGTAGTGCTGCCAAGAAG

AACAACATACCTTTTAGACTAACTTGTGCTACAACTAGACAGGTTGTCAA

TGTCATAACTACTAAAATCTCACTCAAGGGTGGTAAGATTGTTAGTACTT

GTTTTAAACTTATGCTTAAGGCCACATTATTGTGCGTTCTTGCTGCATTG

GTTTGTTATATCGTTATGCCAGTACATACATTGTCAATCCATGATGGTTA

CACAAATGAAATCATTGGTTACAAAGCCATTCAGGATGGTGTCACTCGTG

ACATCATTTCTACTGATGATTGTTTTGCAAATAAACATGCTGGTTTTGAC

GCATGGTTTAGCCAGCGTGGTGGTTCATACAAAAATGACAAAAGCTGCCC

TGTAGTAGCTGCTATCATTACAAGAGAGATTGGTTTCATAGTGCCTGGCT

TACCGGGTACTGTGCTGAGAGCAATCAATGGTGACTTCTTGCATTTTCTA

CCTCGTGTTTTTAGTGCTGTTGGCAACATTTGCTACACACCTTCCAAACT

CATTGAGTATAGTGATTTTGCTACCTCTGCTTGCGTTCTTGCTGCTGAGT

GTACAATTTTTAAGGATGCTATGGGCAAACCTGTGCCATATTGTTATGAC

ACTAATTTGCTAGAGGGTTCTATTTCTTATAGTGAGCTTCGTCCAGACAC

TCGTTATGTGCTTATGGATGGTTCCATCATACAGTTTCCTAACACTTACC

TGGAGGGTTCTGTTAGAGTAGTAACAACTTTTGATGCTGAGTACTGTAGA

CATGGTACATGCGAAAGGTCAGAAGTAGGTATTTGCCTATCTACCAGTGG

TAGATGGGTTCTTAATAATGAGCATTACAGAGCTCTATCAGGAGTTTTCT

GTGGTGTTGATGCGATGAATCTCATAGCTAACATCTTTACTCCTCTTGTG

CAACCTGTGGGTGCTTTAGATGTGTCTGCTTCAGTAGTGGCTGGTGGTAT

TATTGCCATATTGGTGACTTGTGCTGCCTACTACTTTATGAAATTCAGAC

GTGTTTTTGGTGAGTACAACCATGTTGTTGCTGCTAATGCACTTTTGTTT

TTGATGTCTTTCACTATACTCTGTCTGGTACCAGCTTACAGCTTTCTGCC

GGGAGTCTACTCAGTCTTTTACTTGTACTTGACATTCTATTTCACCAATG

ATGTTTCATTCTTGGCTCACCTTCAATGGTTTGCCATGTTTTCTCCTATT

GTGCCTTTTTGGATAACAGCAATCTATGTATTCTGTATTTCTCTGAAGCA

CTGCCATTGGTTCTTTAACAACTATCTTAGGAAAAGAGTCATGTTTAATG

GAGTTACATTTAGTACCTTCGAGGAGGCTGCTTTGTGTACCTTTTTGCTC

AACAAGGAAATGTACCTAAAATTGCGTAGCGAGACACTGTTGCCACTTAC

ACAGTATAACAGGTATCTTGCTCTATATAACAAGTACAAGTATTTCAGTG

GAGCCTTAGATACTACCAGCTATCGTGAAGCAGCTTGCTGCCACTTAGCA

AAGGCTCTAAATGACTTTAGCAACTCAGGTGCTGATGTTCTCTACCAACC

ACCACAGACATCAATCACTTCTGCTGTTCTGCAGAGTGGTTTTAGGAAAA

TGGCATTCCCGTCAGGCAAAGTTGAAGGGTGCATGGTACAAGTAACCTGT

GGAACTACAACTCTTAATGGATTGTGGTTGGATGACACAGTATACTGTCC

AAGACATGTCATTTGCACAGCAGAAGACATGCTTAATCCTAACTATGAAG

ATCTGCTCATTCGCAAATCCAACCATAGCTTTCTTGTTCAGGCTGGCAAT

GTTCAACTTCGTGTTATTGGCCATTCTATGCAAAATTGTCTGCTTAGGCT

TAAAGTTGATACTTCTAACCCTAAGACACCCAAGTATAAATTTGTCCGTA

TCCAACCTGGTCAAACATTTTCAGTTCTAGCATGCTACAATGGTTCACCA

TCTGGTGTTTATCAGTGTGCCATGAGACCTAATCATACCATTAAAGGTTC

TTTCCTTAATGGATCATGTGGTAGTGTTGGTTTTAACATTGATTATGATT

GCGTGTCTTTCTGCTATATGCATCATATGGAGCTTCCAACAGGAGTACAC

GCTGGTACTGACTTAGAAGGTAAATTCTATGGTCCATTTGTTGACAGACA

AACTGCACAGGCTGCAGGTACAGACACAACCATAACATTAAATGTTTTGG

CATGGCTGTATGCTGCTGTTATCAATGGTGATAGGTGGTTTCTTAATAGA

TTCACCACTACTTTGAATGACTTTAACCTTGTGGCAATGAAGTACAACTA

TGAACCTTTGACACAAGATCATGTTGACATATTGGGACCTCTTTCTGCTC

AAACAGGAATTGCCGTCTTAGATATGTGTGCTGCTTTGAAAGAGCTGCTG

CAGAATGGTATGAATGGTCGTACTATCCTTGGTAGCACTATTTTAGAAGA

TGAGTTTACACCATTTGATGTTGTTAGACAATGCTCTGGTGTTACCTTCC

AAGGTAAGTTCAAGAAAATTGTTAAGGGCACTCATCATTGGATGCTTTTA

ACTTTCTTGACATCACTATTGATTCTTGTTCAAAGTACACAGTGGTCACT

GTTTTTCTTTGTTTACGAGAATGCTTTCTTGCCATTTACTCTTGGTATTA

TGGCAATTGCTGCATGTGCTATGCTGCTTGTTAAGCATAAGCACGCATTC

TTGTGCTTGTTTCTGTTACCTTCTCTTGCAACAGTTGCTTACTTTAATAT

GGTCTACATGCCTGCTAGCTGGGTGATGCGTATCATGACATGGCTTGAAT

TGGCTGACACTAGCTTGTCTGGTTATAGGCTTAAGGATTGTGTTATGTAT

GCTTCAGCTTTAGTTTTGCTTATTCTCATGACAGCTCGCACTGTTTATGA

TGATGCTGCTAGACGTGTTTGGACACTGATGAATGTCATTACACTTGTTT

ACAAAGTCTACTATGGTAATGCTTTAGATCAAGCTATTTCCATGTGGGCC

TTAGTTATTTCTGTAACCTCTAACTATTCTGGTGTCGTTACGACTATCAT

GTTTTTAGCTAGAGCTATAGTGTTTGTGTGTGTTGAGTATTACCCATTGT

TATTTATTACTGGCAACACCTTACAGTGTATCATGCTTGTTTATTGTTTC

TTAGGCTATTGTTGCTGCTGCTACTTTGGCCTTTTCTGTTTACTCAACCG

TTACTTCAGGCTTACTCTTGGTGTTTATGACTACTTGGTCTCTACACAAG

AATTTAGGTATATGAACTCCCAGGGGCTTTTGCCTCCTAAGAGTAGTATT

GATGCTTTCAAGCTTAACATTAAGTTGTTGGGTATTGGAGGTAAACCATG

TATCAAGGTTGCTACTGTACAGTCTAAAATGTCTGACGTAAAGTGCACAT

CTGTGGTACTGCTCTCGGTTCTTCAACAACTTAGAGTAGAGTCATCTTCT

AAATTGTGGGCACAATGTGTACAACTCCACAATGATATTCTTCTTGCAAA

AGACACAACTGAAGCTTTCGAGAAGATGGTTTCTCTTTTGTCTGTTTTGC

TATCCATGCAGGGTGCTGTAGACATTAATAGGTTGTGCGAGGAAATGCTC

GATAACCGTGCTACTCTTCAGGCTATTGCTTCAGAATTTAGTTCTTTACC

ATCATATGCCGCTTATGCCACTGCCCAGGAGGCCTATGAGCAGGCTGTAG

CTAATGGTGATTCTGAAGTCGTTCTCAAAAAGTTAAAGAAATCTTTGAAT

GTGGCTAAATCTGAGTTTGACCGTGATGCTGCCATGCAACGCAAGTTGGA

AAAGATGGCAGATCAGGCTATGACCCAAATGTACAAACAGGCAAGATCTG

AGGACAAGAGGGCAAAAGTAACTAGTGCTATGCAAACAATGCTCTTCACT

ATGCTTAGGAAGCTTGATAATGATGCACTTAACAACATTATCAACAATGC

GCGTGATGGTTGTGTTCCACTCAACATCATACCATTGACTACAGCAGCCA

AACTCATGGTTGTTGTCCCTGATTATGGTACCTACAAGAACACTTGTGAT

GGTAACACCTTTACATATGCATCTGCACTCTGGGAAATCCAGCAAGTTGT

TGATGCGGATAGCAAGATTGTTCAACTTAGTGAAATTAACATGGACAATT

CACCAAATTTGGCTTGGCCTCTTATTGTTACAGCTCTAAGAGCCAACTCA

GCTGTTAAACTACAGAATAATGAACTGAGTCCAGTAGCACTACGACAGAT

GTCCTGTGCGGCTGGTACCACACAAACAGCTTGTACTGATGACAATGCAC

TTGCCTACTATAACAATTCGAAGGGAGGTAGGTTTGTGCTGGCATTACTA

TCAGACCACCAAGATCTCAAATGGGCTAGATTCCCTAAGAGTGATGGTAC

AGGTACAATTTACACAGAACTGGAACCACCTTGTAGGTTTGTTACAGACA

CACCAAAAGGGCCTAAAGTGAAATACTTGTACTTCATCAAAGGCTTAAAC

AACCTAAATAGAGGTATGGTGCTGGGCAGTTTAGCTGCTACAGTACGTCT

TCAGGCTGGAAATGCTACAGAAGTACCTGCCAATTCAACTGTGCTTTCCT

TCTGTGCTTTTGCAGTAGACCCTGCTAAAGCATATAAGGATTACCTAGCA

AGTGGAGGACAACCAATCACCAACTGTGTGAAGATGTTGTGTACACACAC

TGGTACAGGACAGGCAATTACTGTAACACCAGAAGCTAACATGGACCAAG

AGTCCTTTGGTGGTGCTTCATGTTGTCTGTATTGTAGATGCCACATTGAC

CATCCAAATCCTAAAGGATTCTGTGACTTGAAAGGTAAGTACGTCCAAAT

ACCTACCACTTGTGCTAATGACCCAGTGGGTTTTACACTTAGAAACACAG

TCTGTACCGTCTGCGGAATGTGGAAAGGTTATGGCTGTAGTTGTGACCAA

CTCCGCGAACCCTTGATGCAGTCTGCGGATGCATCAACGTTTTTAAACGG

GTTTGCGGTGTAAGTGCAGCCCGTCTTACACCGTGCGGCACAGGCACTAG

TACTGATGTCGTCTACAGGGCTTTTGATATTTACAACGAAAAAGTTGCTG

GTTTTGCAAAGTTCCTAAAAACTAATTGCTGTCGCTTCCAGGAGAAGGAT

GAGGAAGGCAATTTATTAGACTCTTACTTTGTAGTTAAGAGGCATACTAT

GTCTAACTACGAAGATGAAGAGACTATTTATAACTTGGTTAAAGATTGTC

CAGCGGTTGCTGTCCATGACTTTTTCAAGTTTAGAGTAGATGGTGACATG

GTACCACATATATCACGTCAGCGTCTAACTAAATACACAATGGCTGATTT

AGTCTATGCTCTACGTCATTTTGATGAGGGTAATTGTGATACATTAAAAG

AAATAGTCGTCACATACAATTGCTGTGATGATGATTATTTCAATAAGAAG

GATTGGTATGACTTCGTAGAGAATCCTGACATCTTACGCGTATATGCTAA

CTTAGGTGAGCGTGTACGCCAATCATTATTAAAGACTGTACAATTCTGCG

ATGCTATGCGTGATGCAGGCATTGTAGGCGTACTGACATTAGATAATCAG

GATCTTAATGGGAACTGGTACGATTTCGGTGATTTCGTACAAGTAGCACC

AGGCTGCGGAGTTCCTATTGTGGATTCATATTACTCATTGCTGATGCCCA

TCCTCACTTTGACTAGGGCATTGGCTGCTGAGTCCCATATGGATGCTGAT

CTCGCAAAACCACTTATTAAGTGGGATTTGCTGAAATATGATTTTACGGA

AGAGAGACTTTGTCTCTTCGACCGTTATTTTAAATATTGGGACCAGACAT

ACCATCCCAATTGTATTAACTGTTTGGATGATAGGTGTATCCTTCATTGT

GCAAACTTTAATGTGTTATTTTCTACTGTGTTTCCACCTACAAGTTTTGG

ACCACTAGTAAGAAAAATATTTGTAGATGGTGTTCCTTTTGTTGTTTCAA

CTGGATACCATTTTCGTGAGTTAGGAGTCGTACATAATCAGGATGTAAAC

TTACATAGCTCGCGTCTCAGTTTCAAGGAACTTTTAGTGTATGCTGCTGA

TCCAGCTATGCATGCAGCTTCTGGCAATTTATTGCTAGATAAACGCACTA

CATGCTTTTCAGTAGCTGCACTAACAAACAATGTTGCTTTTCAAACTGTC

AAACCCGGTAATTTTAATAAAGACTTTTATGACTTTGCTGTGTCTAAAGG

TTTCTTTAAGGAAGGAAGTTCTGTTGAACTAAAACACTTCTTCTTTGCTC

AGGATGGCAACGCTGCTATCAGTGATTATGACTATTATCGTTATAATCTG

CCAACAATGTGTGATATCAGACAACTCCTATTCGTAGTTGAAGTTGTTGA

TAAATACTTTGATTGTTACGATGGTGGCTGTATTAATGCCAACCAAGTAA

TCGTTAACAATCTGGATAAATCAGCTGGTTTCCCATTTAATAAATGGGGT

AAGGCTAGACTTTATTATGACTCAATGAGTTATGAGGATCAAGATGCACT

TTTCGCGTATACTAAGCGTAATGTCATCCCTACTATAACTCAAATGAATC

TTAAGTATGCCATTAGTGCAAAGAATAGAGCTCGCACCGTAGCTGGTGTC

TCTATCTGTAGTACTATGACAAATAGACAGTTTCATCAGAAATTATTGAA

GTCAATAGCCGCCACTAGAGGAGCTACTGTGGTAATTGGAACAAGCAAGT

TTTACGGTGGCTGGCATAATATGTTAAAAACTGTTTACAGTGATGTAGAA

ACTCCACACCTTATGGGTTGGGATTATCCAAAATGTGACAGAGCCATGCC

TAACATGCTTAGGATAATGGCCTCTCTTGTTCTTGCTCGCAAACATAACA

CTTGCTGTAACTTATCACACCGTTTCTACAGGTTAGCTAACGAGTGTGCG

CAAGTATTAAGTGAGATGGTCATGTGTGGCGGCTCACTATATGTTAAACC

AGGTGGAACATCATCCGGTGATGCTACAACTGCTTATGCTAATAGTGTCT

TTAACATTTGTCAAGCTGTTACAGCCAATGTAAATGCACTTCTTTCAACT

GATGGTAATAAGATAGCTGACAAGTATGTCCGCAATCTACAACACAGGCT

CTATGAGTGTCTCTATAGAAATAGGGATGTTGATCATGAATTCGTGGATG

AGTTTTACGCTTACCTGCGTAAACATTTCTCCATGATGATTCTTTCTGAT

GATGCCGTTGTGTGCTATAACAGTAACTATGCGGCTCAAGGTTTAGTAGC

TAGCATTAAGAACTTTAAGGCAGTTCTTTATTATCAAAATAATGTGTTCA

TGTCTGAGGCAAAATGTTGGACTGAGACTGACCTTACTAAAGGACCTCAC

GAATTTTGCTCACAGCATACAATGCTAGTTAAACAAGGAGATGATTACGT

GTACCTGCCTTACCCAGATCCATCAAGAATATTAGGCGCAGGCTGTTTTG

TCGATGATATTGTCAAAACAGATGGTACACTTATGATTGAAAGGTTCGTG

TCACTGGCTATTGATGCTTACCCACTTACAAAACATCCTAATCAGGAGTA

TGCTGATGTCTTTCACTTGTATTTACAATACATTAGAAAGTTACATGATG

AGCTTACTGGCCACATGTTGGACATGTATTCCGTAATGCTAACTAATGAT

AACACCTCACGGTACTGGGAACCTGAGTTTTATGAGGCTATGTACACACC

ACATACAGTCTTGCAGGCTGTAGGTGCTTGTGTATTGTGCAATTCACAGA

CTTCACTTCGTTGCGGTGCCTGTATTAGGAGACCATTCCTATGTTGCAAG

TGCTGCTATGACCATGTCATTTCAACATCACACAAATTAGTGTTGTCTGT

TAATCCCTATGTTTGCAATGCCCCAGGTTGTGATGTCACTGATGTGACAC

AACTGTATCTAGGAGGTATGAGCTATTATTGCAAGTCACATAAGCCTCCC

ATTAGTTTTCCATTATGTGCTAATGGTCAGGTTTTTGGTTTATACAAAAA

CACATGTGTAGGCAGTGACAATGTCACTGACTTCAATGCGATAGCAACAT

GTGATTGGACTAATGCTGGCGATTACATACTTGCCAACACTTGTACTGAG

AGACTCAAGCTTTTCGCAGCAGAAACGCTCAAAGCCACTGAGGAAACATT

TAAGCTGTCATATGGTATTGCCACTGTACGCGAAGTACTCTCTGACAGAG

AATTGCATCTTTCATGGGAGGTTGGAAAACCTAGACCACCATTGAACAGA

AACTATGTCTTTACTGGTTACCGTGTAACTAAAAATAGTAAAGTACAGAT

TGGAGAGTACACCTTTGAAAAAGGTGACTATGGTGATGCTGTTGTGTACA

GAGGTACTACGACATACAAGTTGAATGTTGGTGATTACTTTGTGTTGACA

TCTCACACTGTAATGCCACTTAGTGCACCTACTCTAGTGCCACAAGAGCA

CTATGTGAGAATTACTGGCTTGTACCCAACACTCAACATCTCAGATGAGT

TTTCTAGCAATGTTGCAAATTATCAAAAGGTCGGCATGCAAAAGTACTCT

ACACTCCAAGGACCACCTGGTACTGGTAAGAGTCATTTTGCCATCGGACT

TGCTCTCTATTACCCATCTGCTCGCATAGTGTATACGGCATGCTCTCATG

CAGCTGTTGATGCCCTATGTGAAAAGGCATTAAAATATTTGCCCATAGAT

AAATGTAGTAGAATCATACCTGCGCGTGCGCGCGTAGAGTGTTTTGATAA

ATTCAAAGTGAATTCAACACTAGAACAGTATGTTTTCTGCACTGTAAATG

CATTGCCAGAAACAACTGCTGACATTGTAGTCTTTGATGAAATCTCTATG

GCTACTAATTATGACTTGAGTGTTGTCAATGCTAGACTTCGTGCAAAACA

CTACGTCTATATTGGCGATCCTGCTCAATTACCAGCCCCCCGCACATTGC

TGACTAAAGGCACACTAGAACCAGAATATTTTAATTCAGTGTGCAGACTT

ATGAAAACAATAGGTCCAGACATGTTCCTTGGAACTTGTCGCCGTTGTCC

TGCTGAAATTGTTGACACTGTGAGTGCTTTAGTTTATGACAATAAGCTAA

AAGCACACAAGGATAAGTCAGCTCAATGCTTCAAAATGTTCTACAAAGGT

GTTATTACACATGATGTTTCATCTGCAATCAACAGACCTCAAATAGGCGT

TGTAAGAGAATTTCTTACACGCAATCCTGCTTGGAGAAAAGCTGTTTTTA

TCTCACCTTATAATTCACAGAACGCTGTAGCTTCAAAAATCTTAGGATTG

CCTACGCAGACTGTTGATTCATCACAGGGTTCTGAATATGACTATGTCAT

ATTCACACAAACTACTGAAACAGCACACTCTTGTAATGTCAACCGCTTCA

ATGTGGCTATCACAAGGGCAAAAATTGGCATTTTGTGCATAATGTCTGAT

AGAGATCTTTATGACAAACTGCAATTTACAAGTCTAGAAATACCACGTCG

CAATGTGGCTACATTACAAGCAGAAAATGTAACTGGACTTTTTAAGGACT

GTAGTAAGATCATTACTGGTCTTCATCCTACACAGGCACCTACACACCTC

AGCGTTGATATAAAGTTCAAGACTGAAGGATTATGTGTTGACATACCAGG

CATACCAAAGGACATGACCTACCGTAGACTCATCTCTATGATGGGTTTCA

AAATGAATTACCAAGTCAATGGTTACCCTAATATGTTTATCACCCGCGAA

GAAGCTATTCGTCACGTTCGTGCGTGGATTGGCTTTGATGTAGAGGGCTG

TCATGCAACTAGAGATGCTGTGGGTACTAACCTACCTCTCCAGCTAGGAT

TTTCTACAGGTGTTAACTTAGTAGCTGTACCGACTGGTTATGTTGACACT

GAAAATAACACAGAATTCACCAGAGTTAATGCAAAACCTCCACCAGGTGA

CCAGTTTAAACATCTTATACCACTCATGTATAAAGGCTTGCCCTGGAATG

TAGTGCGTATTAAGATAGTACAAATGCTCAGTGATACACTGAAAGGATTG

TCAGACAGAGTCGTGTTCGTCCTTTGGGCGCATGGCTTTGAGCTTACATC

AATGAAGTACTTTGTCAAGATTGGACCTGAAAGAACGTGTTGTCTGTGTG

ACAAACGTGCAACTTGCTTTTCTACTTCATCAGATACTTATGCCTGCTGG

AATCATTCTGTGGGTTTTGACTATGTCTATAACCCATTTATGATTGATGT

TCAGCAGTGGGGCTTTACGGGTAACCTTCAGAGTAACCATGACCAACATT

GCCAGGTACATGGAAATGCACATGTGGCTAGTTGTGATGCTATCATGACT

AGATGTTTAGCAGTCCATGAGTGCTTTGTTAAGCGCGTTGATTGGTCTGT

TGAATACCCTATTATAGGAGATGAACTGAGGGTTAATTCTGCTTGCAGAA

AAGTACAACACATGGTTGTGAAGTCTGCATTGCTTGCTGATAAGTTTCCA

GTTCTTCATGACATTGGAAATCCAAAGGCTATCAAGTGTGTGCCTCAGGC

TGAAGTAGAATGGAAGTTCTACGATGCTCAGCCATGTAGTGACAAAGCTT

ACAAAATAGAGGAACTCTTCTATTCTTATGCTACACATCACGATAAATTC

ACTGATGGTGTTTGTTTGTTTTGGAATTGTAACGTTGATCGTTACCCAGC

CAATGCAATTGTGTGTAGGTTTGACACAAGAGTCTTGTCAAACTTGAACT

TACCAGGCTGTGATGGTGGTAGTTTGTATGTGAATAAGCATGCATTCCAC

ACTCCAGCTTTCGATAAAAGTGCATTTACTAATTTAAAGCAATTGCCTTT

CTTTTACTATTCTGATAGTCCTTGTGAGTCTCATGGCAAACAAGTAGTGT

CGGATATTGATTATGTTCCACTCAAATCTGCTACGTGTATTACACGATGC

AATTTAGGTGGTGCTGTTTGCAGACACCATGCAAATGAGTACCGACAGTA

CTTGGATGCATATAATATGATGATTTCTGCTGGATTTAGCCTATGGATTT

ACAAACAATTTGATACTTATAACCTGTGGAATACATTTACCAGGTTACAG

AGTTTAGAAAATGTGGCTTATAATGTTGTTAATAAAGGACACTTTGATGG

ACACGCCGGCGAAGCACCTGTTTCCATCATTAATAATGCTGTTTACACAA

AGGTAGATGGTATTGATGTGGAGATCTTTGAAAATAAGACAACACTTCCT

GTTAATGTTGCATTTGAGCTTTGGGCTAAGCGTAACATTAAACCAGTGCC

AGAGATTAAGATACTCAATAATTTGGGTGTTGATATCGCTGCTAATACTG

TAATCTGGGACTACAAAAGAGAAGCCCCAGCACATGTATCTACAATAGGT

GTCTGCACAATGACTGACATTGCCAAGAAACCTACTGAGAGTGCTTGTTC

TTCACTTACTGTCTTGTTTGATGGTAGAGTGGAAGGACAGGTAGACCTTT

TTAGAAACGCCCGTAATGGTGTTTTAATAACAGAAGGTTCAGTCAAAGGT

CTAACACCTTCAAAGGGACCAGCACAAGCTAGCGTCAATGGAGTCACATT

AATTGGAGAATCAGTAAAAACACAGTTTAACTACTTTAAGAAAGTAGACG

GCATTATTCAACAGTTGCCTGAAACCTACTTTACTCAGAGCAGAGACTTA

GAGGATTTTAAGCCCAGATCACAAATGGAAACTGACTTTCTCGAGCTCGC

TATGGATGAATTCATACAGCGATATAAGCTCGAGGGCTATGCCTTCGAAC

ACATCGTTTATGGAGATTTCAGTCATGGACAACTTGGCGGTCTTCATTTA

ATGATAGGCTTAGCCAAGCGCTCACAAGATTCACCACTTAAATTAGAGGA

TTTTATCCCTATGGACAGCACAGTGAAAAATTACTTCATAACAGATGCGC

AAACAGGTTCATCAAAATGTGTGTGTTCTGTGATTGATCTTTTACTTGAT

GACTTTGTCGAGATAATAAAGTCACAAGATTTGTCAGTGATTTCAAAAGT

GGTCAAGGTTACAATTGACTATGCTGAAATTTCATTCATGCTTTGGTGTA

AGGATGGACATGTTGAAACCTTCTACCCAAAACTACAAGCAAGTCAAGCG

TGGCAACCAGGTGTTGCGATGCCTAACTTGTACAAGATGCAAAGAATGCT

TCTTGAAAAGTGTGACCTTCAGAATTATGGTGAAAATGCTGTTATACCAA

AAGGAATAATGATGAATGTCGCAAAGTATACTCAACTGTGTCAATACTTA

AATACACTTACTTTAGCTGTACCCTACAACATGAGAGTTATTCACTTTGG

TGCTGGCTCTGATAAAGGAGTTGCACCAGGTACAGCTGTGCTCAGACAAT

GGTTGCCAACTGGCACACTACTTGTCGATTCAGATCTTAATGACTTCGTC

TCCGACGCAGATTCTACTTTAATTGGAGACTGTGCAACAGTACATACGGC

TAATAAATGGGACCTTATTATTAGCGATATGTATGACCCTAGGACCAAAC

ATGTGACAAAAGAGAATGACTCTAAAGAAGGGTTTTTCACTTATCTGTGT

GGATTTATAAAGCAAAAACTAGCCCTGGGTGGTTCTATAGCTGTAAAGAT

AACAGAGCATTCTTGGAATGCTGACCTTTACAAGCTTATGGGCCATTTCT

CATGGTGGACAGCTTTTGTTACAAATGTAAATGCATCATCATCGGAAGCA

TTTTTAATTGGGGCTAACTATCTTGGCAAGCCGAAGGAACAAATTGATGG

CTATACCATGCATGCTAACTACATTTTCTGGAGGAACACAAATCCTATCC

AGTTGTCTTCCTATTCACTCTTTGACATGAGCAAATTTCCTCTTAAATTA

AGAGGAACTGCTGTAATGTCTCTTAAGGAGAATCAAATCAATGATATGAT

TTATTCTCTTCTGGAAAAAGGTAGGCTTATCATTAGAGAAAACAACAGAG

TTGTGGTTTCAAGTGATATTCTTGTTAACAACTAAACGAACATGTTTATT

TTCTTATTATTTCTTACTCTCACTAGTGGTAGTGACCTTGACCGGTGCAC

CACTTTTGATGATGTTCAAGCTCCTAATTACACTCAACATACTTCATCTA

TGAGGGGGGTTTACTATCCTGATGAAATTTTTAGATCAGACACTCTTTAT

TTAACTCAGGATTTATTTCTTCCATTTTATTCTAATGTTACAGGGTTTCA

TACTATTAATCATACGTTTGGCAACCCTGTCATACCTTTTAAGGATGGTA

TTTATTTTGCTGCCACAGAGAAATCAAATGTTGTCCGTGGTTGGGTTTTT

GGTTCTACCATGAACAACAAGTCACAGTCGGTGATTATTATTAACAATTC

TACTAATGTTGTTATACGAGCATGTAACTTTGAATTGTGTGACAACCCTT

TCTTTGCTGTTTCTAAACCCATGGGTACACAGACACATACTATGATATTC

GATAATGCATTTAATTGCACTTTCGAGTACATATCTGATGCCTTTTCGCT

TGATGTTTCAGAAAAGTCAGGTAATTTTAAACACTTACGAGAGTTTGTGT

TTAAAAATAAAGATGGGTTTCTCTATGTTTATAAGGGCTATCAACCTATA

GATGTAGTTCGTGATCTACCTTCTGGTTTTAACACTTTGAAACCTATTTT

TAAGTTGCCTCTTGGTATTAACATTACAAATTTTAGAGCCATTCTTACAG

CCTTTTCACCTGCTCAAGACATTTGGGGCACGTCAGCTGCAGCCTATTTT

GTTGGCTATTTAAAGCCAACTACATTTATGCTCAAGTATGATGAAAATGG

TACAATCACAGATGCTGTTGATTGTTCTCAAAATCCACTTGCTGAACTCA

AATGCTCTGTTAAGAGCTTTGAGATTGACAAAGGAATTTACCAGACCTCT

AATTTCAGGGTTGTTCCCTCAGGAGATGTTGTGAGATTCCCTAATATTAC

AAACTTGTGTCCTTTTGGAGAGGTTTTTAATGCTACTAAATTCCCTTCTG

TCTATGCATGGGAGAGAAAAAAAATTTCTAATTGTGTTGCTGATTACTCT

GTGCTCTACAACTCAACATTTTTTTCAACCTTTAAGTGCTATGGCGTTTC

TGCCACTAAGTTGAATGATCTTTGCTTCTCCAATGTCTATGCAGATTCTT

TTGTAGTCAAGGGAGATGATGTAAGACAAATAGCGCCAGGACAAACTGGT

GTTATTGCTGATTATAATTATAAATTGCCAGATGATTTCATGGGTTGTGT

CCTTGCTTGGAATACTAGGAACATTGATGCTACTTCAACTGGTAATTATA

ATTATAAATATAGGTATCTTAGACATGGCAAGCTTAGGCCCTTTGAGAGA

GACATATCTAATGTGCCTTTCTCCCCTGATGGCAAACCTTGCACCCCACC

TGCTCTTAATTGTTATTGGCCATTAAATGATTATGGTTTTTACACCACTA

CTGGCATTGGCTACCAACCTTACAGAGTTGTAGTACTTTCTTTTGAACTT

TTAAATGCACCGGCCACGGTTTGTGGACCAAAATTATCCACTGACCTTAT

TAAGAACCAGTGTGTCAATTTTAATTTTAATGGACTCACTGGTACTGGTG

TGTTAACTCCTTCTTCAAAGAGATTTCAACCATTTCAACAATTTGGCCGT

GATGTTTCTGATTTCACTGATTCCGTTCGAGATCCTAAAACATCTGAAAT

ATTAGACATTTCACCTTGCGCTTTTGGGGGTGTAAGTGTAATTACACCTG

GAACAAATGCTTCATCTGAAGTTGCTGTTCTATATCAAGATGTTAACTGC

ACTGATGTTTCTACAGCAATTCATGCAGATCAACTCACACCAGCTTGGCG

CATATATTCTACTGGAAACAATGTATTCCAGACTCAAGCAGGCTGTCTTA

TAGGAGCTGAGCATGTCGACACTTCTTATGAGTGCGACATTCCTATTGGA

GCTGGCATTTGTGCTAGTTACCATACAGTTTCTTTATTACGTAGTACTAG

CCAAAAATCTATTGTGGCTTATACTATGTCTTTAGGTGCTGATAGTTCAA

TTGCTTACTCTAATAACACCATTGCTATACCTACTAACTTTTCAATTAGC

ATTACTACAGAAGTAATGCCTGTTTCTATGGCTAAAACCTCCGTAGATTG

TAATATGTACATCTGCGGAGATTCTACTGAATGTGCTAATTTGCTTCTCC

AATATGGTAGCTTTTGCACACAACTAAATCGTGCACTCTCAGGTATTGCT

GCTGAACAGGATCGCAACACACGTGAAGTGTTCGCTCAAGTCAAACAAAT

GTACAAAACCCCAACTTTGAAATATTTTGGTGGTTTTAATTTTTCACAAA

TATTACCTGACCCTCTAAAGCCAACTAAGAGGTCTTTTATTGAGGACTTG

CTCTTTAATAAGGTGACACTCGCTGATGCTGGCTTCATGAAGCAATATGG

CGAATGCCTAGGTGATATTAATGCTAGAGATCTCATTTGTGCGCAGAAGT

TCAATGGACTTACAGTGTTGCCACCTCTGCTCACTGATGATATGATTGCT

GCCTACACTGCTGCTCTAGTTAGTGGTACTGCCACTGCTGGATGGACATT

TGGTGCTGGCGCTGCTCTTCAAATACCTTTTGCTATGCAAATGGCATATA

GGTTCAATGGCATTGGAGTTACCCAAAATGTTCTCTATGAGAACCAAAAA

CAAATCGCCAACCAATTTAACAAGGCGATTAGTCAAATTCAAGAATCACT

TACAACAACATCAACTGCATTGGGCAAGCTGCAAGACGTTGTTAACCAGA

ATGCTCAAGCATTAAACACACTTGTTAAACAACTTAGCTCTAATTTTGGT

GCAATTTCAAGTGTGCTAAATGATATCCTTTCGCGACTTGATAAAGTCGA

GGCGGAGGTACAAATTGACAGGTTAATTACAGGCAGACTTCAAAGCCTTC

AAACCTATGTAACACAACAACTAATCAGGGCTGCTGAAATCAGGGCTTCT

GCTAATCTTGCTGCTACTAAAATGTCTGAGTGTGTTCTTGGACAATCAAA

AAGAGTTGACTTTTGTGGAAAGGGCTACCACCTTATGTCCTTCCCACAAG

CAGCCCCGCATGGTGTTGTCTTCCTACATGTCACGTATGTGCCATCCCAG

GAGAGGAACTTCACCACAGCGCCAGCAATTTGTCATGAAGGCAAAGCATA

CTTCCCTCGTGAAGGTGTTTTTGTGTTTAATGGCACTTCTTGGTTTATTA

CACAGAGGAACTTCTTTTCTCCACAAATAATTACTACAGACAATACATTT

GTCTCAGGAAATTGTGATGTCGTTATTGGCATCATTAACAACACAGTTTA

TGATCCTCTGCAACCTGAGCTTGACTCATTCAAAGAAGAGCTGGACAAGT

ACTTCAAAAATCATACATCACCAGATGTTGATCTTGGCGACATTTCAGGC

ATTAACGCTTCTGTCGTCAACATTCAAAAAGAAATTGACCGCCTCAATGA

GGTCGCTAAAAATTTAAATGAATCACTCATTGACCTTCAAGAATTGGGAA

AATATGAGCAATATATTAAATGGCCTTGGTATGTTTGGCTCGGCTTCATT

GCTGGACTAATTGCCATCGTCATGGTTACAATCTTGCTTTGTTGCATGAC

TAGTTGTTGCAGTTGCCTCAAGGGTGCATGCTCTTGTGGTTCTTGCTGCA

AGTTTGATGAGGATGACTCTGAGCCAGTTCTCAAGGGTGTCAAATTACAT

TACACATAAACGAACTTATGGATTTGTTTATGAGATTTTTTACTCTTAGA

TCAATTACTGCACAGCCAGTAAAAATTGACAATGCTTCTCCTGCAAGTAC

TGTTCATGCTACAGCAACGATACCGCTACAAGCCTCACTCCCTTTCGGAT

GGCTTGTTATTGGCGTTGCATTTCTTGCTGTTTTTCAGAGCGCTACCAAA

ATAATTGCGCTCAATAAAAGATGGCAGCTAGCCCTTTATAAGGGCTTCCA

GTTCATTTGCAATTTACTGCTGCTATTTGTTACCATCTATTCACATCTTT

TGCTTGTCGCTGCAGGTATGGAGGCGCAATTTTTGTACCTCTATGCCTTG

ATATATTTTCTACAATGCATCAACGCATGTAGAATTATTATGAGATGTTG

GCTTTGTTGGAAGTGCAAATCCAAGAACCCATTACTTTATGATGCCAACT

ACTTTGTTTGCTGGCACACACATAACTATGACTACTGTATACCATATAAC

AGTGTCACAGATACAATTGTCGTTACTGAAGGTGACGGCATTTCAACACC

AAAACTCAAAGAAGACTACCAAATTGGTGGTTATTCTGAGGATAGGCACT

CAGGTGTTAAAGACTATGTCGTTGTACATGGCTATTTCACCGAAGTTTAC

TACCAGCTTGAGTCTACACAAATTACTACAGACACTGGTATTGAAAATGC

TACATTCTTCATCTTTAACAAGCTTGTTAAAGACCCACCGAATGTGCAAA

TACACACAATCGACGGCTCTTCAGGAGTTGCTAATCCAGCAATGGATCCA

ATTTATGATGAGCCGACGACGACTACTAGCGTGCCTTTGTAAGCACAAGA

AAGTGAGTACGAACTTATGTACTCATTCGTTTCGGAAGAAACAGGTACGT

TAATAGTTAATAGCGTACTTCTTTTTCTTGCTTTCGTGGTATTCTTGCTA

GTCACACTAGCCATCCTTACTGCGCTTCGATTGTGTGCGTACTGCTGCAA

TATTGTTAACGTGAGTTTAGTAAAACCAACGGTTTACGTCTACTCGCGTG

TTAAAAATCTGAACTCTTCTGAAGGAGTTCCTGATCTTCTGGTCTAAACG

AACTAACTATTATTATTATTCTGTTTGGAACTTTAACATTGCTTATCATG

GCAGACAACGGTACTATTACCGTTGAGGAGCTTAAACAACTCCTGGAACA

ATGGAACCTAGTAATAGGTTTCCTATTCCTAGCCTGGATTATGTTACTAC

AATTTGCCTATTCTAATCGGAACAGGTTTTTGTACATAATAAAGCTTGTT

TTCCTCTGGCTCTTGTGGCCAGTAACACTTGCTTGTTTTGTGCTTGCTGC

TGTCTACAGAATTAATTGGGTGACTGGCGGGATTGCGATTGCAATGGCTT

GTATTGTAGGCTTGATGTGGCTTAGCTACTTCGTTGCTTCCTTCAGGCTG

TTTGCTCGTACCCGCTCAATGTGGTCATTCAACCCAGAAACAAACATTCT

TCTCAATGTGCCTCTCCGGGGGACAATTGTGACCAGACCGCTCATGGAAA

GTGAACTTGTCATTGGTGCTGTGATCATTCGTGGTCACTTGCGAATGGCC

GGACACTCCCTAGGGCGCTGTGACATTAAGGACCTGCCAAAAGAGATCAC

TGTGGCTACATCACGAACGCTTTCTTATTACAAATTAGGAGCGTCGCAGC

GTGTAGGCACTGATTCAGGTTTTGCTGCATACAACCGCTACCGTATTGGA

AACTATAAATTAAATACAGACCACGCCGGTAGCAACGACAATATTGCTTT

GCTAGTACAGTAAGTGACAACAGATGTTTCATCTTGTTGACTTCCAGGTT

ACAATAGCAGAGATATTGATTATCATTATGAGGACTTTCAGGATTGCTAT

TTGGAATCTTGACGTTATAATAAGTTCAATAGTGAGACAATTATTTAAGC

CTCTAACTAAGAAGAATTATTCGGAGTTAGATGATGAAGAACCTATGGAG

TTAGATTATCCATAAAACGAACATGAAAATTATTCTCTTCCTGACATTGA

TTGTATTTACATCTTGCGAGCTATATCACTATCAGGAGTGTGTTAGAGGT

ACGACTGTACTACTAAAAGAACCTTGCCCATCAGGAACATACGAGGGCAA

TTCACCATTTCACCCTCTTGCTGACAATAAATTTGCACTAACTTGCACTA

GCACACACTTTGCTTTTGCTTGTGCTGACGGTACTCGACATACCTATCAG

CTGCGTGCAAGATCAGTTTCACCAAAACTTTTCATCAGACAAGAGGAGGT

TCAACAAGAGCTCTACTCGCCACTTTTTCTCATTGTTGCTGCTCTAGTAT

TTTTAATACTTTGCTTCACCATTAAGAGAAAGACAGAATGAATGAGCTCA

CTTTAATTGACTTCTATTTGTGCTTTTTAGCCTTTCTGCTATTCCTTGTT

TTAATAATGCTTATTATATTTTGGTTTTCACTCGAAATCCAGGATCTAGA

AGAACCTTGTACCAAAGTCTAAACGAACATGAAACTTCTCATTGTTTTGA

CTTGTATTTCTCTATGCAGTTGCATATGCACTGTAGTACAGCGCTGTGCA

TCTAATAAACCTCATGTGCTTGAAGATCCTTGTAAGGTACAACACTAGGG

GTAATACTTATAGCACTGCTTGGCTTTGTGCTCTAGGAAAGGTTTTACCT

TTTCATAGATGGCACACTATGGTTCAAACATGCACACCTAATGTTACTAT

CAACTGTCAAGATCCAGCTGGTGGTGCGCTTATAGCTAGGTGTTGGTACC

TTCATGAAGGTCACCAAACTGCTGCATTTAGAGACGTACTTGTTGTTTTA

AATAAACGAACAAATTAAAATGTCTGATAATGGACCCCAATCAAACCAAC

GTAGTGCCCCCCGCATTACATTTGGTGGACCCACAGATTCAACTGACAAT

AACCAGAATGGAGGACGCAATGGGGCAAGGCCAAAACAGCGCCGACCCCA

AGGTTTACCCAATAATACTGCGTCTTGGTTCACAGCTCTCACTCAGCATG

GCAAGGAGGAACTTAGATTCCCTCGAGGCCAGGGCGTTCCAATCAACACC

AATAGTGGTCCAGATGACCAAATTGGCTACTACCGAAGAGCTACCCGACG

AGTTCGTGGTGGTGACGGCAAAATGAAAGAGCTCAGCCCCAGATGGTACT

TCTATTACCTAGGAACTGGCCCAGAAGCTTCACTTCCCTACGGCGCTAAC

AAAGAAGGCATCGTATGGGTTGCAACTGAGGGAGCCTTGAATACACCCAA

AGACCACATTGGCACCCGCAATCCTAATAACAATGCTGCCACCGTGCTAC

AACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAGGGAAGC

AGAGGCGGCAGTCAAGCCTCTTCTCGCTCCTCATCACGTAGTCGCGGTAA

TTCAAGAAATTCAACTCCTGGCAGCAGTAGGGGAAATTCTCCTGCTCGAA

TGGCTAGCGGAGGTGGTGAAACTGCCCTCGCGCTATTGCTGCTAGACAGA

TTGAACCAGCTTGAGAGCAAAGTTTCTGGTAAAGGCCAACAACAACAAGG

CCAAACTGTCACTAAGAAATCTGCTGCTGAGGCATCTAAAAAGCCTCGCC

AAAAACGTACTGCCACAAAACAGTACAACGTCACTCAAGCATTTGGGAGA

CGTGGTCCAGAACAAACCCAAGGAAATTTCGGGGACCAAGACCTAATCAG

ACAAGGAACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCTCCAA

GTGCCTCTGCATTCTTTGGAATGTCACGCATTGGCATGGAAGTCACACCT

TCGGGAACATGGCTGACTTATCATGGAGCCATTAAATTGGATGACAAAGA

TCCACAATTCAAAGACAACGTCATACTGCTGAACAAGCACATTGACGCAT

ACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAAAAAGACT

GATGAAGCTCAGCCTTTGCCGCAGAGACAAAAGAAGCAGCCCACTGTGAC

TCTTCTTCCTGCGGCTGACATGGATGATTTCTCCAGACAACTTCAAAATT

CCATGAGTGGAGCTTCTGCTGATTCAACTCAGGCATAAACACTCATGATG

ACCACACAAGGCAGATGGGCTATGTAAACGTTTTCGCAATTCCGTTTACG

ATACATAGTCTACTCTTGTGCAGAATGAATTCTCGTAACTAAACAGCACA

AGTAGGTTTAGTTAACTTTAATCTCACATAGCAATCTTTAATCAATGTGT

AACATTAGGGAGGACTTGAAAGAGCCACCACATTTTCATCGAGGCCACGC

GGAGTACGATCGAGGGTACAGTGAATAATGCTAGGGAGAGCTGCCTATAT

GGAAGAGCCCTAATGTGTAAAATTAATTTTAGTAGTGCTATCCCCATGTG

ATTTTAATAGCTTCTTAGGAGAATGAGAAAAAAAAAAAAAAAAAAAAAAA

A

MERS
GATTTAAGTGAATAGCTTGGCTATCTCACTTCCCCTCGTTCTCTTGCAGA
79

CoV_Refseq
ACTTTGATTTTAACGAACTTAAATAAAAGCCCTGTTGTTTAGCGTATCGT

TGCACTTGTCTGGTGGGATTGTGGCATTAATTTGCCTGCTCATCTAGGCA

GTGGACATATGCTCAACACTGGGTATAATTCTAATTGAATACTATTTTTC

AGTTAGAGCGTCGTGTCTCTTGTACGTCTCGGTCACAATACACGGTTTCG

TCCGGTGCGTGGCAATTCGGGGCACATCATGTCTTTCGTGGCTGGTGTGA

CCGCGCAAGGTGCGCGCGGTACGTATCGAGCAGCGCTCAACTCTGAAAAA

CATCAAGACCATGTGTCTCTAACTGTGCCACTCTGTGGTTCAGGAAACCT

GGTTGAAAAACTTTCACCATGGTTCATGGATGGCGAAAATGCCTATGAAG

TGGTGAAGGCCATGTTACTTAAAAAGGAGCCACTTCTCTATGTGCCCATC

CGGCTGGCTGGACACACTAGACACCTCCCAGGTCCTCGTGTGTACCTGGT

TGAGAGGCTCATTGCTTGTGAAAATCCATTCATGGTTAACCAATTGGCTT

ATAGCTCTAGTGCAAATGGCAGCCTGGTTGGCACAACTTTGCAGGGCAAG

CCTATTGGTATGTTCTTCCCTTATGACATCGAACTTGTCACAGGAAAGCA

AAATATTCTCCTGCGCAAGTATGGCCGTGGTGGTTATCACTACACCCCAT

TCCACTATGAGCGAGACAACACCTCTTGCCCTGAGTGGATGGACGATTTT

GAGGCGGATCCTAAAGGCAAATATGCCCAGAATCTGCTTAAGAAGTTGAT

TGGCGGTGATGTCACTCCAGTTGACCAATACATGTGTGGCGTTGATGGAA

AACCCATTAGTGCCTACGCATTTTTAATGGCCAAGGATGGAATAACCAAA

CTGGCTGATGTTGAAGCGGACGTCGCAGCACGTGCTGATGACGAAGGCTT

CATCACATTAAAGAACAATCTATATAGATTGGTTTGGCATGTTGAGCGTA

AAGACGTTCCATATCCTAAGCAATCTATTTTTACTATTAATAGTGTGGTC

CAAAAGGATGGTGTTGAAAACACTCCTCCTCACTATTTTACTCTTGGATG

CAAAATTTTAACGCTCACCCCACGCAACAAGTGGAGTGGCGTTTCTGACT

TGTCCCTCAAACAAAAACTCCTTTACACCTTCTATGGTAAGGAGTCACTT

GAGAACCCAACCTACATTTACCACTCCGCATTCATTGAGTGTGGAAGTTG

TGGTAATGATTCCTGGCTTACAGGGAATGCTATCCAAGGGTTTGCCTGTG

GATGTGGGGCATCATATACAGCTAATGATGTCGAAGTCCAATCATCTGGC

ATGATTAAGCCAAATGCTCTTCTTTGTGCTACTTGCCCCTTTGCTAAGGG

TGATAGCTGTTCTTCTAATTGCAAACATTCAGTTGCTCAGTTGGTTAGTT

ACCTTTCTGAACGCTGTAATGTTATTGCTGATTCTAAGTCCTTCACACTT

ATCTTTGGTGGCGTAGCTTACGCCTACTTTGGATGTGAGGAAGGTACTAT

GTACTTTGTGCCTAGAGCTAAGTCTGTTGTCTCAAGGATTGGAGACTCCA

TCTTTACAGGCTGTACTGGCTCTTGGAACAAGGTCACTCAAATTGCTAAC

ATGTTCTTGGAACAGACTCAGCATTCCCTTAACTTTGTGGGAGAGTTCGT

TGTCAACGATGTTGTCCTCGCAATTCTCTCTGGAACCACAACTAATGTTG

ACAAAATACGCCAGCTTCTCAAAGGTGTCACCCTTGACAAGTTGCGTGAT

TATTTAGCTGACTATGACGTAGCAGTCACTGCCGGCCCATTCATGGATAA

TGCTATTAATGTTGGTGGTACAGGATTACAGTATGCCGCCATTACTGCAC

CTTATGTAGTTCTCACTGGCTTAGGTGAGTCCTTTAAGAAAGTTGCAACC

ATACCGTATAAGGTTTGCAACTCTGTTAAGGATACTCTGGCTTATTATGC

TCACAGCGTGTTGTACAGAGTTTTTCCTTATGACATGGATTCTGGTGTGT

CATCCTTTAGTGAACTACTTTTTGATTGCGTTGATCTTTCAGTAGCTTCT

ACCTATTTTTTAGTCCGCATCTTGCAAGATAAGACTGGCGACTTTATGTC

TACAATTATTACTTCCTGCCAAACTGCTGTTAGTAAGCTTCTAGATACAT

GTTTTGAAGCTACAGAAGCAACATTTAACTTCTTGTTAGATTTGGCAGGA

TTGTTCAGAATCTTTCTCCGCAATGCCTATGTGTACACTTCACAAGGGTT

TGTGGTGGTCAATGGCAAAGTTTCTACACTTGTCAAACAAGTGTTAGACT

TGCTTAATAAGGGTATGCAACTTTTGCATACAAAGGTCTCCTGGGCTGGT

TCTAAAATCATTGCTGTTATCTACAGCGGCAGGGAGTCTCTAATATTCCC

ATCGGGAACCTATTACTGTGTCACCACTAAGGCTAAGTCCGTTCAACAAG

ATCTTGACGTTATTTTGCCTGGTGAGTTTTCCAAGAAGCAGTTAGGACTG

CTCCAACCTACTGACAATTCTACAACTGTTAGTGTTACTGTATCCAGTAA

CATGGTTGAAACTGTTGTGGGTCAACTTGAGCAAACTAATATGCATAGTC

CTGATGTTATAGTAGGTGACTATGTCATTATTAGTGAAAAATTGTTTGTG

CGTAGTAAGGAAGAAGACGGATTTGCCTTCTACCCTGCTTGCACTAATGG

TCATGCTGTACCGACTCTCTTTAGACTTAAGGGAGGTGCACCTGTAAAAA

AAGTAGCCTTTGGCGGTGATCAAGTACATGAGGTTGCTGCTGTAAGAAGT

GTTACTGTCGAGTACAACATTCATGCTGTATTAGACACACTACTTGCTTC

TTCTAGTCTTAGAACCTTTGTTGTAGATAAGTCTTTGTCAATTGAGGAGT

TTGCTGACGTAGTAAAGGAACAAGTCTCAGACTTGCTTGTTAAATTACTG

CGTGGAATGCCGATTCCAGATTTTGATTTAGACGATTTTATTGACGCACC

ATGCTATTGCTTTAACGCTGAGGGTGATGCATCCTGGTCTTCTACTATGA

TCTTCTCTCTTCACCCCGTCGAGTGTGACGAGGAGTGTTCTGAAGTAGAG

GCTTCAGATTTAGAAGAAGGTGAATCAGAGTGCATTTCTGAGACTTCAAC

TGAACAAGTTGACGTTTCTCATGAGACTTCTGACGACGAGTGGGCTGCTG

CAGTTGATGAAGCGTTCCCTCTCGATGAAGCAGAAGATGTTACTGAATCT

GTGCAAGAAGAAGCACAACCAGTAGAAGTACCTGTTGAAGATATTGCGCA

GGTTGTCATAGCTGACACCTTACAGGAAACTCCTGTTGTGCCTGATACTG

TTGAAGTCCCACCGCAAGTGGTGAAACTTCCGTCTGCACCTCAGACTATC

CAGCCCGAGGTAAAAGAAGTTGCACCTGTCTATGAGGCTGATACCGAACA

GACACAGAATGTTACTGTTAAACCTAAGAGGTTACGCAAAAAGCGTAATG

TTGACCCTTTGTCCAATTTTGAACATAAGGTTATTACAGAGTGCGTTACC

ATAGTTTTAGGTGACGCAATTCAAGTAGCCAAGTGCTATGGGGAGTCTGT

GTTAGTTAATGCTGCTAACACACATCTTAAGCATGGCGGTGGTATCGCTG

GTGCTATTAATGCGGCTTCAAAAGGGGCTGTCCAAAAAGAGTCAGATGAG

TATATTCTGGCTAAAGGGCCGTTACAAGTAGGAGATTCAGTTCTCTTGCA

AGGCCATTCTCTAGCTAAGAATATCCTGCATGTCGTAGGCCCAGATGCCC

GCGCTAAACAGGATGTTTCTCTCCTTAGTAAGTGCTATAAGGCTATGAAT

GCATATCCTCTTGTAGTCACTCCTCTTGTTTCAGCAGGCATATTTGGTGT

AAAACCAGCTGTGTCTTTTGATTATCTTATTAGGGAGGCTAAGACTAGAG

TTTTAGTCGTCGTTAATTCCCAAGATGTCTATAAGAGTCTTACCATAGTT

GACATTCCACAGAGTTTGACTTTTTCATATGATGGGTTACGTGGCGCAAT

ACGTAAAGCTAAAGATTATGGTTTTACTGTTTTTGTGTGCACAGACAACT

CTGCTAACACTAAAGTTCTTAGGAACAAGGGTGTTGATTATACTAAGAAG

TTTCTTACAGTTGACGGTGTGCAATATTATTGCTACACGTCTAAGGACAC

TTTAGATGATATCTTACAACAGGCTAATAAGTCTGTTGGTATTATATCTA

TGCCTTTGGGATATGTGTCTCATGGTTTAGACTTAATGCAAGCAGGGAGT

GTCGTGCGTAGAGTTAACGTGCCCTACGTGTGTCTCCTAGCTAATAAAGA

GCAAGAAGCTATTTTGATGTCTGAAGACGTTAAGTTAAACCCTTCAGAAG

ATTTTATAAAGCACGTCCGCACTAATGGTGGTTACAATTCTTGGCATTTA

GTCGAGGGTGAACTATTGGTGCAAGACTTACGCTTAAATAAGCTCCTGCA

TTGGTCTGATCAAACCATATGCTACAAGGATAGTGTGTTTTATGTTGTAA

AGAATAGTACAGCTTTTCCATTTGAAACACTTTCAGCATGTCGTGCGTAT

TTGGATTCACGCACGACACAGCAGTTAACAATCGAAGTCTTAGTGACTGT

CGATGGTGTAAATTTTAGAACAGTCGTTCTAAATAATAAGAACACTTATA

GATCACAGCTTGGATGCGTTTTCTTTAATGGTGCTGATATTTCTGACACC

ATTCCTGATGAGAAACAGAATGGTCACAGTTTATATCTAGCAGACAATTT

GACTGCTGATGAAACAAAGGCGCTTAAAGAGTTATATGGCCCCGTTGATC

CTACTTTCTTACACAGATTCTATTCACTTAAGGCTGCAGTCCATGGGTGG

AAGATGGTTGTGTGTGATAAGGTACGTTCTCTCAAATTGAGTGATAATAA

TTGTTATCTTAATGCAGTTATTATGACACTTGATTTATTGAAGGACATTA

AATTTGTTATACCTGCTCTACAGCATGCATTTATGAAACATAAGGGCGGT

GATTCAACTGACTTCATAGCCCTCATTATGGCTTATGGCAATTGCACATT

TGGTGCTCCAGATGATGCCTCTCGGTTACTTCATACCGTGCTTGCAAAGG

CTGAGTTATGCTGTTCTGCACGCATGGTTTGGAGAGAGTGGTGCAATGTC

TGTGGCATAAAAGATGTTGTTCTACAAGGCTTAAAAGCTTGTTGTTACGT

GGGTGTGCAAACTGTTGAAGATCTGCGTGCTCGCATGACATATGTATGCC

AGTGTGGTGGTGAACGTCATCGGCAATTAGTCGAACACACCACCCCCTGG

TTGCTGCTCTCAGGCACACCAAATGAAAAATTGGTGACAACCTCCACGGC

GCCTGATTTTGTAGCATTTAATGTCTTTCAGGGCATTGAAACGGCTGTTG

GCCATTATGTTCATGCTCGCCTGAAGGGTGGTCTTATTTTAAAGTTTGAC

TCTGGCACCGTTAGCAAGACTTCAGACTGGAAGTGCAAGGTGACAGATGT

ACTTTTCCCCGGCCAAAAATACAGTAGCGATTGTAATGTCGTACGGTATT

CTTTGGACGGTAATTTCAGAACAGAGGTTGATCCCGACCTATCTGCTTTC

TATGTTAAGGATGGTAAATACTTTACAAGTGAACCACCCGTAACATATTC

ACCAGCTACAATTTTAGCTGGTAGTGTCTACACTAATAGCTGCCTTGTAT

CGTCTGATGGACAACCTGGCGGTGATGCTATTAGTTTGAGTTTTAATAAC

CTTTTAGGGTTTGATTCTAGTAAACCAGTCACTAAGAAATACACTTACTC

CTTCTTGCCTAAAGAAGACGGCGATGTGTTGTTGGCTGAGTTTGACACTT

ATGACCCTATTTATAAGAATGGTGCCATGTATAAAGGCAAACCAATTCTT

TGGGTCAATAAAGCATCTTATGATACTAATCTTAATAAGTTCAATAGAGC

TAGTTTGCGTCAAATTTTTGACGTAGCCCCCATTGAACTCGAAAATAAAT

TCACACCTTTGAGTGTGGAGTCTACACCAGTTGAACCTCCAACTGTAGAT

GTGGTAGCACTTCAACAGGAAATGACAATTGTCAAATGTAAGGGTTTAAA

TAAACCTTTCGTGAAGGACAATGTCAGTTTCGTTGCTGATGATTCAGGTA

CTCCCGTTGTTGAGTATCTGTCTAAAGAAGACCTACATACATTGTATGTA

GACCCTAAGTATCAAGTCATTGTCTTAAAAGACAATGTACTTTCTTCTAT

GCTTAGATTGCACACCGTTGAGTCAGGTGATATTAACGTTGTTGCAGCTT

CCGGATCTTTGACACGTAAAGTGAAGTTACTATTTAGGGCTTCATTTTAT

TTCAAAGAATTTGCTACCCGCACTTTCACTGCTACCACTGCTGTAGGTAG

TTGTATAAAGAGTGTAGTGCGGCATCTAGGTGTTACTAAAGGCATATTGA

CAGGCTGTTTTAGTTTTGCCAAGATGTTATTTATGCTTCCACTAGCTTAC

TTTAGTGATTCAAAACTCGGCACCACAGAGGTTAAAGTGAGTGCTTTGAA

AACAGCCGGCGTTGTGACAGGTAATGTTGTAAAACAGTGTTGCACTGCTG

CTGTTGATTTAAGTATGGATAAGTTGCGCCGTGTGGATTGGAAATCAACC

CTACGGTTGTTACTTATGTTATGCACAACTATGGTATTGTTGTCTTCTGT

GTATCACTTGTATGTCTTCAATCAGGTCTTATCAAGTGATGTTATGTTTG

AAGATGCCCAAGGTTTGAAAAAGTTCTACAAAGAAGTTAGAGCTTACCTA

GGAATCTCTTCTGCTTGTGACGGTCTTGCTTCAGCTTATAGGGCGAATTC

CTTTGATGTACCTACATTCTGCGCAAACCGTTCTGCAATGTGTAATTGGT

GCTTGATTAGCCAAGATTCCATAACTCACTACCCAGCTCTTAAGATGGTT

CAAACACATCTTAGCCACTATGTTCTTAACATAGATTGGTTGTGGTTTGC

ATTTGAGACTGGTTTGGCATACATGCTCTATACCTCGGCCTTCAACTGGT

TGTTGTTGGCAGGTACATTGCATTATTTCTTTGCACAGACTTCCATATTT

GTAGACTGGCGGTCATACAATTATGCTGTGTCTAGTGCCTTCTGGTTATT

CACCCACATTCCAATGGCGGGTTTGGTACGAATGTATAATTTGTTAGCAT

GCCTTTGGCTTTTACGCAAGTTTTATCAGCATGTAATCAATGGTTGCAAA

GATACGGCATGCTTGCTCTGCTATAAGAGGAACCGACTTACTAGAGTTGA

AGCTTCTACCGTTGTCTGTGGTGGAAAACGTACGTTTTATATCACAGCAA

ATGGCGGTATTTCATTCTGTCGTAGGCATAATTGGAATTGTGTGGATTGT

GACACTGCAGGTGTGGGGAATACCTTCATCTGTGAAGAAGTCGCAAATGA

CCTCACTACCGCCCTACGCAGGCCTATTAACGCTACGGATAGATCACATT

ATTATGTGGATTCCGTTACAGTTAAAGAGACTGTTGTTCAGTTTAATTAT

CGTAGAGACGGTCAACCATTCTACGAGCGGTTTCCCCTCTGCGCTTTTAC

AAATCTAGATAAGTTGAAGTTCAAAGAGGTCTGTAAAACTACTACTGGTA

TACCTGAATACAACTTTATCATCTACGACTCATCAGATCGTGGCCAGGAA

AGTTTAGCTAGGTCTGCATGTGTTTATTATTCTCAAGTCTTGTGTAAATC

AATTCTTTTGGTTGACTCAAGTTTGGTTACTTCTGTTGGTGATTCTAGTG

AAATCGCCACTAAAATGTTTGATTCCTTTGTTAATAGTTTCGTCTCGCTG

TATAATGTCACACGCGATAAGTTGGAAAAACTTATCTCTACTGCTCGTGA

TGGCGTAAGGCGAGGCGATAACTTCCATAGTGTCTTAACAACATTCATTG

ACGCAGCACGAGGCCCCGCAGGTGTGGAGTCTGATGTTGAGACCAATGAA

ATTGTTGACTCTGTGCAGTATGCTCATAAACATGACATACAAATTACTAA

TGAGAGCTACAATAATTATGTACCCTCATATGTTAAACCTGATAGTGTGT

CTACCAGCGATTTAGGTAGTCTCATTGATTGTAATGCGGCTTCAGTTAAC

CAAATTGTCTTGCGTAATTCTAATGGTGCTTGCATTTGGAACGCTGCTGC

ATATATGAAACTCTCGGATGCACTTAAACGACAGATTCGCATTGCATGCC

GTAAGTGTAATTTAGCTTTCCGGTTAACCACCTCAAAGCTACGCGCTAAT

GATAATATCTTATCAGTTAGATTCACTGCTAACAAAATTGTTGGTGGTGC

TCCTACATGGTTTAATGCGTTGCGTGACTTTACGTTAAAGGGTTATGTTC

TTGCTACCATTATTGTGTTTCTGTGTGCTGTACTGATGTATTTGTGTTTA

CCTACATTTTCTATGGCACCTGTTGAATTTTATGAAGACCGCATCTTGGA

CTTTAAAGTTCTTGATAATGGTATCATTAGGGATGTAAATCCTGATGATA

AGTGCTTTGCTAATAAGCACCGGTCCTTCACACAATGGTATCATGAGCAT

GTTGGTGGTGTCTATGACAACTCTATCACATGCCCATTGACAGTTGCAGT

AATTGCTGGAGTTGCTGGTGCTCGCATTCCAGACGTACCTACTACATTGG

CTTGGGTGAACAATCAGATAATTTTCTTTGTTTCTCGAGTCTTTGCTAAT

ACAGGCAGTGTTTGCTACACTCCTATAGATGAGATACCCTATAAGAGTTT

CTCTGATAGTGGTTGCATTCTTCCATCTGAGTGCACTATGTTTAGGGATG

CAGAGGGCCGTATGACACCATACTGCCATGATCCTACTGTTTTGCCTGGG

GCTTTTGCGTACAGTCAGATGAGGCCTCATGTTCGTTACGACTTGTATGA

TGGTAACATGTTTATTAAATTTCCTGAAGTAGTATTTGAAAGTACACTTA

GGATTACTAGAACTCTGTCAACTCAGTACTGCCGGTTCGGTAGTTGTGAG

TATGCACAAGAGGGTGTTTGTATTACCACAAATGGCTCGTGGGCCATTTT

TAATGACCACCATCTTAATAGACCTGGTGTCTATTGTGGCTCTGATTTTA

TTGACATTGTCAGGCGGTTAGCAGTATCACTGTTCCAGCCTATTACTTAT

TTCCAATTGACTACCTCATTGGTCTTGGGTATAGGTTTGTGTGCGTTCCT

GACTTTGCTCTTCTATTATATTAATAAAGTAAAACGTGCTTTTGCAGATT

ACACCCAGTGTGCTGTAATTGCTGTTGTTGCTGCTGTTCTTAATAGCTTG

TGCATCTGCTTTGTTACCTCTATACCATTGTGTATAGTACCTTACACTGC

ATTGTACTATTATGCTACATTCTATTTTACTAATGAGCCTGCATTTATTA

TGCATGTTTCTTGGTACATTATGTTCGGGCCTATCGTTCCCATATGGATG

ACCTGCGTCTATACAGTTGCAATGTGCTTTAGACACTTCTTCTGGGTTTT

AGCTTATTTTAGTAAGAAACATGTAGAAGTTTTTACTGATGGTAAGCTTA

ATTGTAGTTTCCAGGACGCTGCCTCTAATATCTTTGTTATTAACAAGGAC

ACTTATGCAGCTCTTAGAAACTCTTTAACTAATGATGCCTATTCACGATT

TTTGGGGTTGTTTAACAAGTATAAGTACTTCTCTGGTGCTATGGAAACAG

CCGCTTATCGTGAAGCTGCAGCATGTCATCTTGCTAAAGCCTTACAAACA

TACAGCGAGACTGGTAGTGATCTTCTTTACCAACCACCCAACTGTAGCAT

AACCTCTGGCGTGTTGCAAAGCGGTTTGGTGAAAATGTCACATCCCAGTG

GAGATGTTGAGGCTTGTATGGTTCAGGTTACCTGCGGTAGCATGACTCTT

AATGGTCTTTGGCTTGACAACACAGTCTGGTGCCCACGACACGTAATGTG

CCCGGCTGACCAGTTGTCTGATCCTAATTATGATGCCTTGTTGATTTCTA

TGACTAATCATAGTTTCAGTGTGCAAAAACACATTGGCGCTCCAGCAAAC

TTGCGTGTTGTTGGTCATGCCATGCAAGGCACTCTTTTGAAGTTGACTGT

CGATGTTGCTAACCCTAGCACTCCAGCCTACACTTTTACAACAGTGAAAC

CTGGCGCAGCATTTAGTGTGTTAGCATGCTATAATGGTCGTCCGACTGGT

ACATTCACTGTTGTAATGCGCCCTAACTACACAATTAAGGGTTCCTTTCT

GTGTGGTTCTTGTGGTAGTGTTGGTTACACCAAGGAGGGTAGTGTGATCA

ATTTCTGTTACATGCATCAAATGGAACTTGCTAATGGTACACATACCGGT

TCAGCATTTGATGGTACTATGTATGGTGCCTTTATGGATAAACAAGTGCA

CCAAGTTCAGTTAACAGACAAATACTGCAGTGTTAATGTAGTAGCTTGGC

TTTACGCAGCAATACTTAATGGTTGCGCTTGGTTTGTAAAACCTAATCGC

ACTAGTGTTGTTTCTTTTAATGAATGGGCTCTTGCCAACCAATTCACTGA

ATTTGTTGGCACTCAATCCGTTGACATGTTAGCTGTCAAAACAGGCGTTG

CTATTGAACAGCTGCTTTATGCGATCCAACAACTGTATACTGGGTTCCAG

GGAAAGCAAATCCTTGGCAGTACCATGTTGGAAGATGAATTCACACCTGA

GGATGTTAATATGCAGATTATGGGTGTGGTTATGCAGAGTGGTGTGAGAA

AAGTTACATATGGTACTGCGCATTGGTTGTTTGCGACCCTTGTCTCAACC

TATGTGATAATCTTACAAGCCACTAAATTTACTTTGTGGAACTACTTGTT

TGAGACTATTCCCACACAGTTGTTCCCACTCTTATTTGTGACTATGGCCT

TCGTTATGTTGTTGGTTAAACACAAACACACCTTTTTGACACTTTTCTTG

TTGCCTGTGGCTATTTGTTTGACTTATGCAAACATAGTCTACGAGCCCAC

TACTCCCATTTCGTCAGCGCTGATTGCAGTTGCAAATTGGCTTGCCCCCA

CTAATGCTTATATGCGCACTACACATACTGATATTGGTGTCTACATTAGT

ATGTCACTTGTATTAGTCATTGTAGTGAAGAGATTGTACAACCCATCACT

TTCTAACTTTGCGTTAGCATTGTGCAGTGGTGTAATGTGGTTGTACACTT

ATAGCATTGGAGAAGCCTCAAGCCCCATTGCCTATCTGGTTTTTGTCACT

ACACTCACTAGTGATTATACGATTACAGTCTTTGTTACTGTCAACCTTGC

AAAAGTTTGCACTTATGCCATCTTTGCTTACTCACCACAGCTTACACTTG

TGTTTCCGGAAGTGAAGATGATACTTTTATTATACACATGTTTAGGTTTC

ATGTGTACTTGCTATTTTGGTGTCTTCTCTCTTTTGAACCTTAAGCTTAG

AGCACCTATGGGTGTCTATGACTTTAAGGTCTCAACACAAGAGTTCAGAT

TCATGACTGCTAACAATCTAACTGCACCTAGAAATTCTTGGGAGGCTATG

GCTCTGAACTTTAAGTTAATAGGTATTGGCGGTACACCTTGTATAAAGGT

TGCTGCTATGCAGTCTAAACTTACAGATCTTAAATGCACATCTGTGGTTC

TCCTCTCTGTGCTCCAACAGTTACACTTAGAGGCTAATAGTAGGGCCTGG

GCTTTCTGTGTTAAATGCCATAATGATATATTGGCAGCAACAGACCCCAG

TGAGGCTTTCGAGAAATTCGTAAGTCTCTTTGCTACTTTAATGACTTTTT

CTGGTAATGTAGATCTTGATGCGTTAGCTAGTGATATTTTTGACACTCCT

AGCGTACTTCAAGCTACTCTTTCTGAGTTTTCACACTTAGCTACCTTTGC

TGAGTTGGAAGCTGCGCAGAAAGCCTATCAGGAAGCTATGGACTCTGGTG

ACACCTCACCACAAGTTCTTAAGGCTTTGCAGAAGGCTGTTAATATAGCT

AAAAACGCCTATGAGAAGGATAAGGCAGTGGCCCGTAAGTTAGAACGTAT

GGCTGATCAGGCTATGACTTCTATGTATAAGCAAGCACGTGCTGAAGACA

AGAAAGCAAAAATTGTCAGTGCTATGCAAACTATGTTGTTTGGTATGATT

AAGAAGCTCGACAACGATGTTCTTAATGGTATCATTTCTAACGCTAGGAA

TGGTTGTATACCTCTTAGTGTCATCCCACTGTGTGCTTCAAATAAACTTC

GCGTTGTAATTCCTGACTTCACCGTCTGGAATCAGGTAGTCACATATCCC

TCGCTTAACTACGCTGGGGCTTTGTGGGACATTACAGTTATAAACAATGT

GGACAATGAAATTGTTAAGTCTTCAGATGTTGTAGACAGCAATGAAAATT

TAACATGGCCACTTGTTTTAGAATGCACTAGGGCATCCACTTCTGCCGTT

AAGTTGCAAAATAATGAGATCAAACCTTCAGGTCTAAAAACCATGGTTGT

GTCTGCGGGTCAAGAGCAAACTAACTGTAATACTAGTTCCTTAGCTTATT

ACGAACCTGTGCAGGGTCGTAAAATGCTGATGGCTCTTCTTTCTGATAAT

GCCTATCTCAAATGGGCGCGTGTTGAAGGTAAGGACGGATTTGTCAGTGT

AGAGCTACAACCTCCTTGCAAATTCTTGATTGCGGGACCAAAAGGACCTG

AAATCCGATATCTCTATTTTGTTAAAAATCTTAACAACCTTCATCGCGGG

CAAGTGTTAGGGCACATTGCTGCGACTGTTAGATTGCAAGCTGGTTCTAA

CACCGAGTTTGCCTCTAATTCCTCGGTGTTGTCACTTGTTAACTTCACCG

TTGATCCTCAAAAAGCTTATCTCGATTTCGTCAATGCGGGAGGTGCCCCA

TTGACAAATTGTGTTAAGATGCTTACTCCTAAAACTGGTACAGGTATAGC

TATATCTGTTAAACCAGAGAGTACAGCTGATCAAGAGACTTATGGTGGAG

CTTCAGTGTGTCTCTATTGCCGTGCGCATATAGAACATCCTGATGTCTCT

GGTGTTTGTAAATATAAGGGTAAGTTTGTCCAAATCCCTGCTCAGTGTGT

CCGTGACCCTGTGGGATTTTGTTTGTCAAATACCCCCTGTAATGTCTGTC

AATATTGGATTGGATATGGGTGCAATTGTGACTCGCTTAGGCAAGCAGCA

CTGCCCCAATCTAAAGATTCCAATTTTTTAAACGAGTCCGGGGTTCTATT

GTAAATGCCCGAATAGAACCCTGTTCAAGTGGTTTGTCCACTGATGTCGT

CTTTAGGGCATTTGACATCTGCAACTATAAGGCTAAGGTTGCTGGTATTG

GAAAATACTACAAGACTAATACTTGTAGGTTTGTAGAATTAGATGACCAA

GGGCATCATTTAGACTCCTATTTTGTCGTTAAGAGGCATACTATGGAGAA

TTATGAACTAGAGAAGCACTGTTACGACTTGTTACGTGACTGTGATGCTG

TAGCTCCCCATGATTTCTTCATCTTTGATGTAGACAAAGTTAAAACACCT

CATATTGTACGTCAGCGTTTAACTGAGTACACTATGATGGATCTTGTATA

TGCCCTGAGGCACTTTGATCAAAATAGCGAAGTGCTTAAGGCTATCTTAG

TGAAGTATGGTTGCTGTGATGTTACCTACTTTGAAAATAAACTCTGGTTT

GATTTTGTTGAAAATCCCAGTGTTATTGGTGTTTATCATAAACTTGGAGA

ACGTGTACGCCAAGCTATCTTAAACACTGTTAAATTTTGTGACCACATGG

TCAAGGCTGGTTTAGTCGGTGTGCTCACACTAGACAACCAGGACCTTAAT

GGCAAGTGGTATGATTTTGGTGACTTCGTAATCACTCAACCTGGTTCAGG

AGTAGCTATAGTTGATAGCTACTATTCTTATTTGATGCCTGTGCTCTCAA

TGACCGATTGTCTGGCCGCTGAGACACATAGGGATTGTGATTTTAATAAA

CCACTCATTGAGTGGCCACTTACTGAGTATGATTTTACTGATTATAAGGT

ACAACTCTTTGAGAAGTACTTTAAATATTGGGATCAGACGTATCACGCAA

ATTGCGTTAATTGTACTGATGACCGTTGTGTGTTACATTGTGCTAATTTC

AATGTATTGTTTGCTATGACCATGCCTAAGACTTGTTTCGGACCCATAGT

CCGAAAGATCTTTGTTGATGGCGTGCCATTTGTAGTATCTTGTGGTTATC

ACTACAAAGAATTAGGTTTAGTCATGAATATGGATGTTAGTCTCCATAGA

CATAGGCTCTCTCTTAAGGAGTTGATGATGTATGCCGCTGATCCAGCCAT

GCACATTGCCTCCTCTAACGCTTTTCTTGATTTGAGGACATCATGTTTTA

GTGTCGCTGCACTTACAACTGGTTTGACTTTTCAAACTGTGCGGCCTGGC

AATTTTAACCAAGACTTCTATGATTTCGTGGTATCTAAAGGTTTCTTTAA

GGAGGGCTCTTCAGTGACGCTCAAACATTTTTTCTTTGCTCAAGATGGTA

ATGCTGCTATTACAGATTATAATTACTATTCTTATAATCTGCCTACTATG

TGTGACATCAAACAAATGTTGTTCTGCATGGAAGTTGTAAACAAGTACTT

CGAAATCTATGACGGTGGTTGTCTTAATGCTTCTGAAGTGGTTGTTAATA

ATTTAGACAAGAGTGCTGGCCATCCTTTTAATAAGTTTGGCAAAGCTCGT

GTCTATTATGAGAGCATGTCTTACCAGGAGCAAGATGAACTTTTTGCCAT

GACAAAGCGTAACGTCATTCCTACCATGACTCAAATGAATCTAAAATATG

CTATTAGTGCTAAGAATAGAGCTCGCACTGTTGCAGGCGTGTCCATACTT

AGCACAATGACTAATCGCCAGTACCATCAGAAAATGCTTAAGTCCATGGC

TGCAACTCGTGGAGCGACTTGCGTCATTGGTACTACAAAGTTCTACGGTG

GCTGGGATTTCATGCTTAAAACATTGTACAAAGATGTTGATAATCCGCAT

CTTATGGGTTGGGATTACCCTAAGTGTGATAGAGCTATGCCTAATATGTG

TAGAATCTTCGCTTCACTCATATTAGCTCGTAAACATGGCACTTGTTGTA

CTACAAGGGACAGATTTTATCGCTTGGCAAATGAGTGTGCTCAGGTGCTA

AGCGAATATGTTCTATGTGGTGGTGGTTACTACGTCAAACCTGGAGGTAC

CAGTAGCGGAGATGCCACCACTGCATATGCCAATAGTGTCTTTAACATTT

TGCAGGCGACAACTGCTAATGTCAGTGCACTTATGGGTGCTAATGGCAAC

AAGATTGTTGACAAAGAAGTTAAAGACATGCAGTTTGATTTGTATGTCAA

TGTTTACAGGAGCACTAGCCCAGACCCCAAATTTGTTGATAAATACTATG

CTTTTCTTAATAAGCACTTTTCTATGATGATACTGTCTGATGACGGTGTC

GTTTGCTATAATAGTGATTATGCAGCTAAGGGTTACATTGCTGGAATACA

GAATTTTAAGGAAACGCTGTATTATCAGAACAATGTCTTTATGTCTGAAG

CTAAATGCTGGGTGGAAACCGATCTGAAGAAAGGGCCACATGAATTCTGT

TCACAGCATACGCTTTATATTAAGGATGGCGACGATGGTTACTTCCTTCC

TTATCCAGACCCTTCAAGAATTTTGTCTGCCGGTTGCTTTGTAGATGATA

TCGTTAAGACTGACGGTACACTCATGGTAGAGCGGTTTGTGTCTTTGGCT

ATAGATGCTTACCCTCTCACAAAGCATGAAGATATAGAATACCAGAATGT

ATTCTGGGTCTACTTACAGTATATAGAAAAACTGTATAAAGACCTTACAG

GACACATGCTTGACAGTTATTCTGTCATGCTATGTGGTGATAATTCTGCT

AAGTTTTGGGAAGAGGCATTCTATAGAGATCTCTATAGTTCGCCTACCAC

TTTGCAGGCTGTCGGTTCATGCGTTGTATGCCATTCACAGACTTCCCTAC

GCTGTGGGACATGCATCCGTAGACCATTTCTCTGCTGTAAATGCTGCTAT

GATCATGTTATAGCAACTCCACATAAGATGGTTTTGTCTGTTTCTCCTTA

CGTTTGTAATGCCCCTGGTTGTGGCGTTTCAGACGTTACTAAGCTATATT

TAGGTGGTATGAGCTACTTTTGTGTAGATCATAGACCTGTGTGTAGTTTT

CCACTTTGCGCTAATGGTCTTGTATTCGGCTTATACAAGAATATGTGCAC

AGGTAGTCCTTCTATAGTTGAATTTAATAGGTTGGCTACCTGTGACTGGA

CTGAAAGTGGTGATTACACCCTTGCCAATACTACAACAGAACCACTCAAA

CTTTTTGCTGCTGAGACTTTACGTGCCACTGAAGAGGCGTCTAAGCAGTC

TTATGCTATTGCCACCATCAAAGAAATTGTTGGTGAGCGCCAACTATTAC

TTGTGTGGGAGGCTGGCAAGTCCAAACCACCACTCAATCGTAATTATGTT

TTTACTGGTTATCATATAACCAAAAATAGTAAAGTGCAGCTCGGTGAGTA

CATTTTCGAGCGCATTGATTATAGTGATGCTGTATCCTACAAGTCTAGTA

CAACGTATAAACTGACTGTAGGTGACATCTTCGTACTTACCTCTCACTCT

GTGGCTACCTTGACGGCGCCCACAATTGTGAATCAAGAGAGGTATGTTAA

AATTACTGGGTTGTACCCAACCATTACGGTACCTGAAGAGTTCGCAAGTC

ATGTTGCCAACTTCCAAAAATCAGGTTATAGTAAATATGTCACTGTTCAG

GGACCACCTGGCACTGGCAAAAGTCATTTTGCTATAGGGTTAGCGATTTA

CTACCCTACAGCACGTGTTGTTTATACAGCATGTTCACACGCAGCTGTTG

ATGCTTTGTGTGAAAAAGCTTTTAAATATTTGAACATTGCTAAATGTTCC

CGTATCATTCCTGCAAAGGCACGTGTTGAGTGCTATGACAGGTTTAAAGT

TAATGAGACAAATTCTCAATATTTGTTTAGTACTATTAATGCTCTACCAG

AAACTTCTGCCGATATTCTGGTGGTTGATGAGGTTAGTATGTGCACTAAT

TATGATCTTTCAATTATTAATGCACGTATTAAAGCTAAGCACATTGTCTA

TGTAGGAGATCCAGCACAGTTGCCAGCTCCTAGGACTTTGTTGACTAGAG

GCACATTGGAACCAGAAAATTTCAATAGTGTCACTAGATTGATGTGTAAC

TTAGGTCCTGACATATTTTTAAGTATGTGCTACAGGTGTCCTAAGGAAAT

AGTAAGCACTGTGAGCGCTCTTGTCTACAATAATAAATTGTTAGCCAAGA

AGGAGCTTTCAGGCCAGTGCTTTAAAATACTCTATAAGGGCAATGTGACG

CATGATGCTAGCTCTGCCATTAATAGACCACAACTCACATTTGTGAAGAA

TTTTATTACTGCCAATCCGGCATGGAGTAAGGCAGTCTTTATTTCGCCTT

ACAATTCACAGAATGCTGTGTCTCGTTCAATGCTGGGTCTTACCACTCAG

ACTGTTGATTCCTCACAGGGTTCAGAATACCAGTACGTTATCTTCTGTCA

AACAGCAGATACGGCACATGCTAACAACATTAACAGATTTAATGTTGCAA

TCACTCGTGCCCAAAAAGGTATTCTTTGTGTTATGACATCTCAGGCACTC

TTTGAGTCCTTAGAGTTTACTGAATTGTCTTTTACTAATTACAAGCTCCA

GTCTCAGATTGTAACTGGCCTTTTTAAAGATTGCTCTAGAGAAACTTCTG

GCCTCTCACCTGCTTATGCACCAACATATGTTAGTGTTGATGACAAGTAT

AAGACGAGTGATGAGCTTTGCGTGAATCTTAATTTACCCGCAAATGTCCC

ATACTCTCGTGTTATTTCCAGGATGGGCTTTAAACTCGATGCAACAGTTC

CTGGATATCCTAAGCTTTTCATTACTCGTGAAGAGGCTGTAAGGCAAGTT

CGAAGCTGGATAGGCTTCGATGTTGAGGGTGCTCATGCTTCCCGTAATGC

ATGTGGCACCAATGTGCCTCTACAATTAGGATTTTCAACTGGTGTGAACT

TTGTTGTTCAGCCAGTTGGTGTTGTAGACACTGAGTGGGGTAACATGTTA

ACGGGCATTGCTGCACGTCCTCCACCAGGTGAACAGTTTAAGCACCTCGT

GCCTCTTATGCATAAGGGGGCTGCGTGGCCTATTGTTAGACGACGTATAG

TGCAAATGTTGTCAGACACTTTAGACAAATTGTCTGATTACTGTACGTTT

GTTTGTTGGGCTCATGGCTTTGAATTAACGTCTGCATCATACTTTTGCAA

GATAGGTAAGGAACAGAAGTGTTGCATGTGCAATAGACGCGCTGCAGCGT

ACTCTTCACCTCTGCAATCTTATGCCTGCTGGACTCATTCCTGCGGTTAT

GATTATGTCTACAACCCTTTCTTTGTCGATGTTCAACAGTGGGGTTATGT

AGGCAATCTTGCTACTAATCACGATCGTTATTGCTCTGTCCATCAAGGAG

CTCATGTGGCTTCTAATGATGCAATAATGACTCGTTGTTTAGCTATTCAT

TCTTGTTTTATAGAACGTGTGGATTGGGATATAGAGTATCCTTATATCTC

ACATGAAAAGAAATTGAATTCCTGTTGTAGAATCGTTGAGCGCAACGTCG

TACGTGCTGCTCTTCTTGCCGGTTCATTTGACAAAGTCTATGATATTGGC

AATCCTAAAGGAATTCCTATTGTTGATGACCCTGTGGTTGATTGGCATTA

TTTTGATGCACAGCCCTTGACCAGGAAGGTACAACAGCTTTTCTATACAG

AGGACATGGCCTCAAGATTTGCTGATGGGCTCTGCTTATTTTGGAACTGT

AATGTACCAAAATATCCTAATAATGCAATTGTATGCAGGTTTGACACACG

TGTGCATTCTGAGTTCAATTTGCCAGGTTGTGATGGCGGTAGTTTGTATG

TTAACAAGCACGCTTTTCATACACCAGCATATGATGTGAGTGCATTCCGT

GATCTGAAACCTTTACCATTCTTTTATTATTCTACTACACCATGTGAAGT

GCATGGTAATGGTAGTATGATAGAGGATATTGATTATGTACCCCTAAAAT

CTGCAGTCTGTATTACAGCTTGTAATTTAGGGGGCGCTGTTTGTAGGAAG

CATGCTACAGAGTACAGAGAGTATATGGAAGCATATAATCTTGTCTCTGC

ATCAGGTTTCCGCCTTTGGTGTTATAAGACCTTTGATATTTATAATCTCT

GGTCTACTTTTACAAAAGTTCAAGGTTTGGAAAACATTGCTTTTAATGTT

GTTAAACAAGGCCATTTTATTGGTGTTGAGGGTGAACTACCTGTAGCTGT

AGTCAATGATAAGATCTTCACCAAGAGTGGCGTTAATGACATTTGTATGT

TTGAGAATAAAACCACTTTGCCTACTAATATAGCTTTTGAACTCTATGCT

AAGCGTGCTGTACGCTCGCATCCCGATTTCAAATTGCTACACAATTTACA

AGCAGACATTTGCTACAAGTTCGTCCTTTGGGATTATGAACGTAGCAATA

TTTATGGTACTGCTACTATTGGTGTATGTAAGTACACTGATATTGATGTT

AATTCAGCTTTGAATATATGTTTTGACATACGCGATAATTGTTCATTGGA

GAAGTTCATGTCTACTCCCAATGCCATCTTTATTTCTGATAGAAAAATCA

AGAAATACCCTTGTATGGTAGGTCCTGATTATGCTTACTTCAATGGTGCT

ATCATCCGTGATAGTGATGTTGTTAAACAACCAGTGAAGTTCTACTTGTA

TAAGAAAGTCAATAATGAGTTTATTGATCCTACTGAGTGTATTTACACTC

AGAGTCGCTCTTGTAGTGACTTCCTACCCCTTTCTGACATGGAGAAAGAC

TTTCTATCTTTTGATAGTGATGTTTTCATTAAGAAGTATGGCTTGGAAAA

CTATGCTTTTGAGCACGTAGTCTATGGAGACTTCTCTCATACTACGTTAG

GCGGTCTTCACTTGCTTATTGGTTTATACAAGAAGCAACAGGAAGGTCAT

ATTATTATGGAAGAAATGCTAAAAGGTAGCTCAACTATTCATAACTATTT

TATTACTGAGACTAACACAGCGGCTTTTAAGGCGGTGTGTTCTGTTATAG

ATTTAAAGCTTGACGACTTTGTTATGATTTTAAAGAGTCAAGACCTTGGC

GTAGTATCCAAGGTTGTCAAGGTTCCTATTGACTTAACAATGATTGAGTT

TATGTTATGGTGTAAGGATGGACAGGTTCAAACCTTCTACCCTCGACTCC

AGGCTTCTGCAGATTGGAAACCTGGTCATGCAATGCCATCCCTCTTTAAA

GTTCAAAATGTAAACCTTGAACGTTGTGAGCTTGCTAATTACAAGCAATC

TATTCCTATGCCTCGCGGTGTGCACATGAACATCGCTAAATATATGCAAT

TGTGCCAGTATTTAAATACTTGCACATTAGCCGTGCCTGCCAATATGCGT

GTTATACATTTTGGCGCTGGTTCTGATAAAGGTATCGCTCCTGGTACCTC

AGTTTTACGACAGTGGCTTCCTACAGATGCCATTATTATAGATAATGATT

TAAATGAGTTCGTGTCAGATGCTGACATAACTTTATTTGGAGATTGTGTA

ACTGTACGTGTCGGCCAACAAGTGGATCTTGTTATTTCCGACATGTATGA

TCCTACTACTAAGAATGTAACAGGTAGTAATGAGTCAAAGGCTTTATTCT

TTACTTACCTGTGTAACCTCATTAATAATAATCTTGCTCTTGGTGGGTCT

GTTGCTATTAAAATAACAGAACACTCTTGGAGCGTTGAACTTTATGAACT

TATGGGAAAATTTGCTTGGTGGACTGTTTTCTGCACCAATGCAAATGCAT

CCTCATCTGAAGGATTCCTCTTAGGTATTAATTACTTGGGTACTATTAAA

GAAAATATAGATGGTGGTGCTATGCACGCCAACTATATATTTTGGAGAAA

TTCCACTCCTATGAATCTGAGTACTTACTCACTTTTTGATTTATCCAAGT

TTCAATTAAAATTAAAAGGAACACCAGTTCTTCAATTAAAGGAGAGTCAA

ATTAACGAACTCGTAATATCTCTCCTGTCGCAGGGTAAGTTACTTATCCG

TGACAATGATACACTCAGTGTTTCTACTGATGTTCTTGTTAACACCTACA

GAAAGTTACGTTGATGTAGGGCCAGATTCTGTTAAGTCTGCTTGTATTGA

GGTTGATATACAACAGACTTTCTTTGATAAAACTTGGCCTAGGCCAATTG

ATGTTTCTAAGGCTGACGGTATTATATACCCTCAAGGCCGTACATATTCT

AACATAACTATCACTTATCAAGGTCTTTTTCCCTATCAGGGAGACCATGG

TGATATGTATGTTTACTCTGCAGGACATGCTACAGGCACAACTCCACAAA

AGTTGTTTGTAGCTAACTATTCTCAGGACGTCAAACAGTTTGCTAATGGG

TTTGTCGTCCGTATAGGAGCAGCTGCCAATTCCACTGGCACTGTTATTAT

TAGCCCATCTACCAGCGCTACTATACGAAAAATTTACCCTGCTTTTATGC

TGGGTTCTTCAGTTGGTAATTTCTCAGATGGTAAAATGGGCCGCTTCTTC

AATCATACTCTAGTTCTTTTGCCCGATGGATGTGGCACTTTACTTAGAGC

TTTTTATTGTATTCTAGAGCCTCGCTCTGGAAATCATTGTCCTGCTGGCA

ATTCCTATACTTCTTTTGCCACTTATCACACTCCTGCAACAGATTGTTCT

GATGGCAATTACAATCGTAATGCCAGTCTGAACTCTTTTAAGGAGTATTT

TAATTTACGTAACTGCACCTTTATGTACACTTATAACATTACCGAAGATG

AGATTTTAGAGTGGTTTGGCATTACACAAACTGCTCAAGGTGTTCACCTC

TTCTCATCTCGGTATGTTGATTTGTACGGCGGCAATATGTTTCAATTTGC

CACCTTGCCTGTTTATGATACTATTAAGTATTATTCTATCATTCCTCACA

GTATTCGTTCTATCCAAAGTGATAGAAAAGCTTGGGCTGCCTTCTACGTA

TATAAACTTCAACCGTTAACTTTCCTGTTGGATTTTTCTGTTGATGGTTA

TATACGCAGAGCTATAGACTGTGGTTTTAATGATTTGTCACAACTCCACT

GCTCATATGAATCCTTCGATGTTGAATCTGGAGTTTATTCAGTTTCGTCT

TTCGAAGCAAAACCTTCTGGCTCAGTTGTGGAACAGGCTGAAGGTGTTGA

ATGTGATTTTTCACCTCTTCTGTCTGGCACACCTCCTCAGGTTTATAATT

TCAAGCGTTTGGTTTTTACCAATTGCAATTATAATCTTACCAAATTGCTT

TCACTTTTTTCTGTGAATGATTTTACTTGTAGTCAAATATCTCCAGCAGC

AATTGCTAGCAACTGTTATTCTTCACTGATTTTGGATTACTTTTCATACC

CACTTAGTATGAAATCCGATCTCAGTGTTAGTTCTGCTGGTCCAATATCC

CAGTTTAATTATAAACAGTCCTTTTCTAATCCCACATGTTTGATTTTAGC

GACTGTTCCTCATAACCTTACTACTATTACTAAGCCTCTTAAGTACAGCT

ATATTAACAAGTGCTCTCGTCTTCTTTCTGATGATCGTACTGAAGTACCT

CAGTTAGTGAACGCTAATCAATACTCACCCTGTGTATCCATTGTCCCATC

CACTGTGTGGGAAGACGGTGATTATTATAGGAAACAACTATCTCCACTTG

AAGGTGGTGGCTGGCTTGTTGCTAGTGGCTCAACTGTTGCCATGACTGAG

CAATTACAGATGGGCTTTGGTATTACAGTTCAATATGGTACAGACACCAA

TAGTGTTTGCCCCAAGCTTGAATTTGCTAATGACACAAAAATTGCCTCTC

AATTAGGCAATTGCGTGGAATATTCCCTCTATGGTGTTTCGGGCCGTGGT

GTTTTTCAGAATTGCACAGCTGTAGGTGTTCGACAGCAGCGCTTTGTTTA

TGATGCGTACCAGAATTTAGTTGGCTATTATTCTGATGATGGCAACTACT

ACTGTTTGCGTGCTTGTGTTAGTGTTCCTGTTTCTGTCATCTATGATAAA

GAAACTAAAACCCACGCTACTCTATTTGGTAGTGTTGCATGTGAACACAT

TTCTTCTACCATGTCTCAATACTCCCGTTCTACGCGATCAATGCTTAAAC

GGCGAGATTCTACATATGGCCCCCTTCAGACACCTGTTGGTTGTGTCCTA

GGACTTGTTAATTCCTCTTTGTTCGTAGAGGACTGCAAGTTGCCTCTTGG

TCAATCTCTCTGTGCTCTTCCTGACACACCTAGTACTCTCACACCTCGCA

GTGTGCGCTCTGTTCCAGGTGAAATGCGCTTGGCATCCATTGCTTTTAAT

CATGCTATTCAGGTTGATCAACTTAATAGTAGTTATTTTAAATTAAGTAT

ACCCACTAATTTTTCCTTTGGTGTGACTCAGGAGTACATTCAGACAACCA

TTCAGAAAGTTACTGTTGATTGTAAACAGTACGTTTGCAATGGTTTCCAG

AAGTGTGAGCAATTACTGCGCGAGTATGGCCAGTTTTGTTCCAAAATAAA

CCAGGCTCTCCATGGTGCCAATTTACGCCAGGATGATTCTGTACGTAATT

TGTTTGCGAGCGTGAAAAGCTCTCAATCATCTCCTATCATACCAGGTTTT

GGAGGTGACTTTAATTTGACACTTCTAGAACCTGTTTCTATATCTACTGG

CAGTCGTAGTGCACGTAGTGCTATTGAGGATTTGCTATTTGACAAAGTCA

CTATAGCTGATCCTGGTTATATGCAAGGTTACGATGATTGCATGCAGCAA

GGTCCAGCATCAGCTCGTGATCTTATTTGTGCTCAATATGTGGCTGGTTA

CAAAGTATTACCTCCTCTTATGGATGTTAATATGGAAGCCGCGTATACTT

CATCTTTGCTTGGCAGCATAGCAGGTGTTGGCTGGACTGCTGGCTTATCC

TCCTTTGCTGCTATTCCATTTGCACAGAGTATCTTTTATAGGTTAAACGG

TGTTGGCATTACTCAACAGGTTCTTTCAGAGAACCAAAAGCTTATTGCCA

ATAAGTTTAATCAGGCTCTGGGAGCTATGCAAACAGGCTTCACTACAACT

AATGAAGCTTTTCAGAAGGTTCAGGATGCTGTGAACAACAATGCACAGGC

TCTATCCAAATTAGCTAGCGAGCTATCTAATACTTTTGGTGCTATTTCCG

CCTCTATTGGAGACATCATACAACGTCTTGATGTTCTCGAACAGGACGCC

CAAATAGACAGACTTATTAATGGCCGTTTGACAACACTAAATGCTTTTGT

TGCACAGCAGCTTGTTCGTTCCGAATCAGCTGCTCTTTCCGCTCAATTGG

CTAAAGATAAAGTCAATGAGTGTGTCAAGGCACAATCCAAGCGTTCTGGA

TTTTGCGGTCAAGGCACACATATAGTGTCCTTTGTTGTAAATGCCCCTAA

TGGCCTTTACTTCATGCATGTTGGTTATTACCCTAGCAACCACATTGAGG

TTGTTTCTGCTTATGGTCTTTGCGATGCAGCTAACCCTACTAATTGTATA

GCCCCTGTTAATGGCTACTTTATTAAAACTAATAACACTAGGATTGTTGA

TGAGTGGTCATATACTGGCTCGTCCTTCTATGCACCTGAGCCCATTACCT

CCCTTAATACTAAGTATGTTGCACCACAGGTGACATACCAAAACATTTCT

ACTAACCTCCCTCCTCCTCTTCTCGGCAATTCCACCGGGATTGACTTCCA

AGATGAGTTGGATGAGTTTTTCAAAAATGTTAGCACCAGTATACCTAATT

TTGGTTCCCTAACACAGATTAATACTACATTACTCGATCTTACCTACGAG

ATGTTGTCTCTTCAACAAGTTGTTAAAGCCCTTAATGAGTCTTACATAGA

CCTTAAAGAGCTTGGCAATTATACTTATTACAACAAATGGCCGTGGTACA

TTTGGCTTGGTTTCATTGCTGGGCTTGTTGCCTTAGCTCTATGCGTCTTC

TTCATACTGTGCTGCACTGGTTGTGGCACAAACTGTATGGGAAAACTTAA

GTGTAATCGTTGTTGTGATAGATACGAGGAATACGACCTCGAGCCGCATA

AGGTTCATGTTCACTAATTAACGAACTATTAATGAGAGTTCAAAGACGAG

CCACTCTCTTGTTAGTGTTTTCACTCTCTCTTTTGGTCACTGCATCCTCA

AAACCTCTCTATGTACCTGAGCATTGTCAGAATTATTCTGGTTGCATGCT

TAGGGCTTGTATTAAAACTGCCCAAGCTGATACAGCTGGTCTTTATACAA

ATTTTCGAATTGACGTCCCATCTGCAGAATCAACTGGTACTCAATCAGTT

TCTGTCGATCTTGAGTCAACTTCAACTCATGATGGTCCTACCGAACATGT

TACTAGTGTGAATCTTTTTGACGTTGGTTACTCAGTTAATTAACGAACTC

TATGGATTACGTGTCTCTGCTTAATCAAATTTGGCAGAAGTACCTTAACT

CACCGTATACTACTTGTTTGTACATCCCTAAACCCACAGCTAAGTATACA

CCTTTAGTTGGCACTTCATTGCACCCTGTGCTGTGGAACTGTCAGCTATC

CTTTGCTGGTTATACTGAATCTGCTGTTAATTCTACAAAAGCTTTGGCCA

AACAGGACGCAGCTCAGCGAATCGCTTGGTTGCTACATAAGGATGGAGGA

ATCCCTGATGGATGTTCCCTCTACCTCCGGCACTCAAGTTTATTCGCGCA

AAGCGAGGAAGAGGAGCCATTCTCCAACTAAGAAACTGCGCTACGTTAAG

CGTAGATTTTCTCTTCTGCGCCATGAAGACCTTAGTGTTATTGTCCAACC

AACACACTATGTCAGGGTTACATTTTCAGACCCCAACATGTGGTATCTAC

GTTCGGGTCATCATTTACACTCAGTTCACAATTGGCTTAAACCTTATGGC

GGCCAACCTGTTTCTGAGTACCATATTACTCTAGCTTTGCTAAATCTCAC

TGATGAAGATTTAGCTAGAGATTTTTCACCCATTGCGCTCTTTTTGCGCA

ATGTCAGATTTGAGCTACATGAGTTCGCCTTGCTGCGCAAAACTCTTGTT

CTTAATGCATCAGAGATCTACTGTGCTAACATACATAGATTTAAGCCTGT

GTATAGAGTTAACACGGCAATCCCTACTATTAAGGATTGGCTTCTCGTTC

AGGGATTTTCCCTTTACCATAGTGGCCTCCCTTTACATATGTCAATCTCT

AAATTGCATGCACTGGATGATGTTACTCGCAATTACATCATTACAATGCC

ATGCTTTAGAACTTACCCTCAACAAATGTTTGTTACTCCTTTGGCCGTAG

ATGTTGTCTCCATACGGTCTTCCAATCAGGGTAATAAACAAATTGTTCAT

TCTTATCCCATTTTACATCATCCAGGATTTTAACGAACTATGGCTTTCTC

GGCGTCTTTATTTAAACCCGTCCAGCTAGTCCCAGTTTCTCCTGCATTTC

ATCGCATTGAGTCTACTGACTCTATTGTTTTCACATACATTCCTGCTAGC

GGCTATGTAGCTGCTTTAGCTGTCAATGTGTGTCTCATTCCCCTATTATT

ACTGCTACGTCAAGATACTTGTCGTCGCAGCATTATCAGAACTATGGTTC

TCTATTTCCTTGTTCTGTATAACTTTTTATTAGCCATTGTACTAGTCAAT

GGTGTACATTATCCAACTGGAAGTTGCCTGATAGCCTTCTTAGTTATCCT

CATAATACTTTGGTTTGTAGATAGAATTCGTTTCTGTCTCATGCTGAATT

CCTACATTCCACTGTTTGACATGCGTTCCCACTTTATTCGTGTTAGTACA

GTTTCTTCTCATGGTATGGTCCCTGTAATACACACCAAACCATTATTTAT

TAGAAACTTCGATCAGCGTTGCAGCTGTTCTCGTTGTTTTTATTTGCACT

CTTCCACTTATATAGAGTGCACTTATATTAGCCGTTTTAGTAAGATTAGC

CTAGTTTCTGTAACTGACTTCTCCTTAAACGGCAATGTTTCCACTGTTTT

CGTGCCTGCAACGCGCGATTCAGTTCCTCTTCACATAATCGCCCCGAGCT

CGCTTATCGTTTAAGCAGCTCTGCGCTACTATGGGTCCCGTGTAGAGGCT

AATCCATTAGTCTCTCTTTGGACATATGGAAAACGAACTATGTTACCCTT

TGTCCAAGAACGAATAGGGTTGTTCATAGTAAACTTTTTCATTTTTACCG

TAGTATGTGCTATAACACTCTTGGTGTGTATGGCTTTCCTTACGGCTACT

AGATTATGTGTGCAATGTATGACAGGCTTCAATACCCTGTTAGTTCAGCC

CGCATTATACTTGTATAATACTGGACGTTCAGTCTATGTAAAATTCCAGG

ATAGTAAACCCCCTCTACCACCTGACGAGTGGGTTTAACGAACTCCTTCA

TAATGTCTAATATGACGCAACTCACTGAGGCGCAGATTATTGCCATTATT

AAAGACTGGAACTTTGCATGGTCCCTGATCTTTCTCTTAATTACTATCGT

ACTACAGTATGGATACCCATCCCGTAGTATGACTGTCTATGTCTTTAAAA

TGTTTGTTTTATGGCTCCTATGGCCATCTTCCATGGCGCTATCAATATTT

AGCGCCGTTTATCCAATTGATCTAGCTTCCCAGATAATCTCTGGCATTGT

AGCAGCTGTTTCAGCTATGATGTGGATTTCCTACTTTGTGCAGAGTATCC

GGCTGTTTATGAGAACTGGATCATGGTGGTCATTCAATCCTGAGACTAAT

TGCCTTTTGAACGTTCCATTTGGTGGTACAACTGTCGTACGTCCACTCGT

AGAGGACTCTACCAGTGTAACTGCTGTTGTAACCAATGGCCACCTCAAAA

TGGCTGGCATGCATTTCGGTGCTTGTGACTACGACAGACTTCCTAATGAA

GTCACCGTGGCCAAACCCAATGTGCTGATTGCTTTAAAAATGGTGAAGCG

GCAAAGCTACGGAACTAATTCCGGCGTTGCCATTTACCATAGATATAAGG

CAGGTAATTACAGGAGTCCGCCTATTACGGCGGATATTGAACTTGCATTG

CTTCGAGCTTAGGCTCTTTAGTAAGAGTATCTTAATTGATTTTAACGAAT

CTCAATTTCATTGTTATGGCATCCCCTGCTGCACCTCGTGCTGTTTCCTT

TGCCGATAACAATGATATAACAAATACAAACCTATCTCGAGGTAGAGGAC

GTAATCCAAAACCACGAGCTGCACCAAATAACACTGTCTCTTGGTACACT

GGGCTTACCCAACACGGGAAAGTCCCTCTTACCTTTCCACCTGGGCAGGG

TGTACCTCTTAATGCCAATTCTACCCCTGCGCAAAATGCTGGGTATTGGC

GGAGACAGGACAGAAAAATTAATACCGGGAATGGAATTAAGCAACTGGCT

CCCAGGTGGTACTTCTACTACACTGGAACTGGACCCGAAGCAGCACTCCC

ATTCCGGGCTGTTAAGGATGGCATCGTTTGGGTCCATGAAGATGGCGCCA

CTGATGCTCCTTCAACTTTTGGGACGCGGAACCCTAACAATGATTCAGCT

ATTGTTACACAATTCGCGCCCGGTACTAAGCTTCCTAAAAACTTCCACAT

TGAGGGGACTGGAGGCAATAGTCAATCATCTTCAAGAGCCTCTAGCTTAA

GCAGAAACTCTTCCAGATCTAGTTCACAAGGTTCAAGATCAGGAAACTCT

ACCCGCGGCACTTCTCCAGGTCCATCTGGAATCGGAGCAGTAGGAGGTGA

TCTACTTTACCTTGATCTTCTGAACAGACTACAAGCCCTTGAGTCTGGCA

AAGTAAAGCAATCGCAGCCAAAAGTAATCACTAAGAAAGATGCTGCTGCT

GCTAAAAATAAGATGCGCCACAAGCGCACTTCCACCAAAAGTTTCAACAT

GGTGCAAGCTTTTGGTCTTCGCGGACCAGGAGACCTCCAGGGAAACTTTG

GTGATCTTCAATTGAATAAACTCGGCACTGAGGACCCACGTTGGCCCCAA

ATTGCTGAGCTTGCTCCTACAGCCAGTGCTTTTATGGGTATGTCGCAATT

TAAACTTACCCATCAGAACAATGATGATCATGGCAACCCTGTGTACTTCC

TTCGGTACAGTGGAGCCATTAAACTTGACCCAAAGAATCCCAACTACAAT

AAGTGGTTGGAGCTTCTTGAGCAAAATATTGATGCCTACAAAACCTTCCC

TAAGAAGGAAAAGAAACAAAAGGCACCAAAAGAAGAATCAACAGACCAAA

TGTCTGAACCTCCAAAGGAGCAGCGTGTGCAAGGTAGCATCACTCAGCGC

ACTCGCACCCGTCCAAGTGTTCAGCCTGGTCCAATGATTGATGTTAACAC

TGATTAGTGTCACTCAAAGTAACAAGATCGCGGCAATCGTTTGTGTTTGG

CAACCCCATCTCACCATCGCTTGTCCACTCTTGCACAGAATGGAATCATG

TTGTAATTACAGTGCAATAAGGTAATTATAACCCATTTAATTGATAGCTA

TGCTTTATTAAAGTGTGTAGCTGTAGAGAGAATGTTAAAGACTGTCACCT

CTGCTTGATTGCAAGTGAACAGTGCCCCCCGGGAAGAGCTCTACAGTGTG

AAATGTAAATAAAAAATAGCTATTATTCAATTAGATTAGGCTAATTAGAT

GATTTGCAAAAAAAAAAAA

IL-8 siRNA
CAAGGAAGTGCTAAAGAA
80

sense strand (A1

siRNA, A4

siRNA)

IL-8 siRNA
CAAGGAGTGCTAAAGAA
81

sense strand (A2

siRNA, A3-1

siRNA, A5-1

siRNA)

IL-8 siRNA
GAGAGTGATTGAGAGTGG
82

sense strand

(A3-2 siRNA,

A5-2 siRNA)

IL-8 siRNA
GAGAGCTCTGTCTGGACC
83

sense strand

(A3-3 siRNA,

A5-3 siRNA)

IL-1beta siRNA
GAAAGATGATAAGCCCACTCT
84

sense strand (A6

siRNA, A7-1

siRNA)

IL-1beta siRNA
GGTGATGTCTGGTCCATATGA
85

sense strand

(A7-2 siRNA)

IL-1beta siRNA
GATGATAAGCCCACTCTA
86

sense strand

(A7-3 siRNA)

TNF-alpha
GGCGTGGAGCTGAGAGATAA
87

sense strand

(A8-1 siRNA)

TNF-alpha
GGGCCTGTACCTCATCTACT
88

sense strand

(A8-2 siRNA)

TNF-alpha
GGTATGAGCCCATCTATCT
89

sense strand

(A8-3 siRNA)

IL-17
GCAATGAGGACCCTGAGAGAT
90

sense strand

(A8-4 siRNA)

IL-17
GCTGATGGGAACGTGGACTA
91

sense strand

(A8-5 siRNA)

IL-17
GGTCCTCAGATTACTACAA
92

sense strand

(A8-6 siRNA)

IL-6
GCCCTGAGAAAGGAGACATGT
93

sense strand

(B1-1 siRNA)

IL-6
GAGGAGACTTGCCTGGTGAAA
94

sense strand

(B1-2, B2, B15-

1, B16-1, B17-1

siRNA)

IL-6
GAGGGCTCTTCGGCAAATGTA
95

sense strand

(B1-3 siRNA)

IL6R-alpha
GTGAGGAAGTTTCAGAACAGT
96

sense strand

(B3-1, B4

siRNA)

IL6R-alpha
GAACGGTCAAAGACATTCACA
97

sense strand

(B3-2 siRNA)

IL6R-Beta
GGGAAGGTTACATCAGATCAT
98

sense strand

(B3-3, B5

siRNA)

ACE2
GCAGCTGAGGCCATTATATGA
99

sense strand

(B6-1, B7, B15-

2, B16-2, Bl7-2

siRNA)

ACE2
GGACCCAGGAAATGTTCAGAA
100

sense strand

(B6-2 siRNA)

ACE2
GGCTGAAAGACCAGAACAAGA
101

sense strand

(B6-3 siRNA)

SARS CoV-
GTGTGACCGAAAGGTAAGATG
102

2_ORF1ab

sense strand

(B8-1, B14,

B18-1 siRNA)

SARS CoV-
TTTAAATATTGGGATCAGAC
103

2_ORF1ab

sense strand

(B12-1 siRNA)

SARS CoV-
AAGAATAGAGCTCGCAC
104

2_ORF1ab

sense strand

(B12-2, B13

siRNA)

SARS CoV-
ACTGTTGATTCATCACAGGG
105

2_ORF1ab

sense strand

(B12-3 siRNA)

SARS CoV-
GTTGCTGATTATTCTGTCCTA
106

2_Spike Protein

sense strand

(B11-1, B19-1

siRNA)

SARS CoV-
GAGGTGATGAAGTCAGACAAA
107

2_Spike Protein

sense strand

(B8-2, B9, B11-

2, B18-2, B19-2

siRNA)

SARS CoV-
GCCGGTAGCACACCTTGTAAT
108

2_Spike Protein

sense strand

(B11-3, B19-3

siRNA)

SARS CoV-
GCAACTGAGGGAGCCTTGAAT
109

2_Nucleocapsid

Protein sense

strand (B8-3,

B10, B15-3,

B16-3, B17-3,

Bl8-3 siRNA)

IL-8 siRNA
TTCTTTAGCACTTCCTTG
110

antisense strand

(A1 siRNA, A4

siRNA)

IL-8 siRNA
TTCTTTAGCACTCCTTG
111

antisense strand

(A2 siRNA, A3-

1 siRNA, A5-1

siRNA)

IL-8 siRNA
CCACTCTCAATCACTCTC
112

antisense strand

(A3-2 siRNA,

A5-2 siRNA)

IL-8 siRNA
GGTCCAGACAGAGCTCTC
113

antisense strand

(A3-3 siRNA,

A5-3 siRNA)

IL-1beta siRNA
AGAGTGGGCTTATCATCTTTC
114

antisense strand

(A6 siRNA, A7-

1 siRNA)

IL-1beta siRNA
TCATATGGACCAGACATCACC
115

antisense strand

(A7-2 siRNA)

IL-1beta siRNA
TAGAGTGGGCTTATCATC
116

antisense strand

(A7-3 siRNA)

TNF-alpha
TTATCTCTCAGCTCCACGCC
117

antisense strand

(A8-1 siRNA)

TNF-alpha
AGTAGATGAGGTACAGGCCC
118

antisense strand

(A8-2 siRNA)

TNF-alpha
AGATAGATGGGCTCATACC
119

antisense strand

(A8-3 siRNA)

IL-17
ATCTCTCAGGGTCCTCATTGC
120

antisense strand

(A8-4 siRNA)

IL-17
TAGTCCACGTTCCCATCAGC
121

antisense strand

(A8-5 siRNA)

IL-17
TTGTAGTAATCTGAGGACC
122

antisense strand

(A8-6 siRNA)

IL-6
ACATGTCTCCTTTCTCAGGGC
123

antisense strand

(Bl-1 siRNA)

IL-6
TTTCACCAGGCAAGTCTCCTC
124

antisense strand

(B1-2, B2, B15-

1, B16-1, B17-1

siRNA)

IL-6
TACATTTGCCGAAGAGCCCTC
125

antisense strand

(B1-3 siRNA)

IL6R-alpha
ACTGTTCTGAAACTTCCTCAC
126

antisense strand

(B3-1, B4

siRNA)

IL6R-alpha
TGTGAATGTCTTTGACCGTTC
127

antisense strand

(B3-2 siRNA)

IL6R-Beta
ATGATCTGATGTAACCTTCCC
128

antisense strand

(B3-3, B5

siRNA)

ACE2
TCATATAATGGCCTCAGCTGC
129

antisense strand

(B6-1, B7, B15-

2, B16-2, B17-2

siRNA)

ACE2
TTCTGAACATTTCCTGGGTCC
130

antisense strand

(B6-2 siRNA)

ACE2
TCTTGTTCTGGTCTTTCAGCC
131

antisense strand

(B6-3 siRNA)

SARS CoV-
CATCTTACCTTTCGGTCACAC
132

2_ORF1ab

antisense strand

(B8-1, B14,

B18-1 siRNA)

SARS CoV-
GTCTGATCCCAATATTTAAA
133

2_ORF1ab

antisense strand

(B12-1 siRNA)

SARS CoV-
GTGCGAGCTCTATTCTT
134

2_ORF1ab

antisense strand

(B12-2, B13

siRNA)

SARS CoV-
CCCTGTGATGAATCAACAGT
135

2_ORF1ab

antisense strand

(B12-3 siRNA)

SARS CoV-
TAGGACAGAATAATCAGCAAC
136

2_Spike Protein

antisense strand

(B11-1, B19-1

siRNA)

SARS CoV-
TTTGTCTGACTTCATCACCTC
137

2_Spike Protein

antisense strand

(B8-2, B9, B11-

2, B18-2, B19-2

siRNA)

SARS CoV-
ATTACAAGGTGTGCTACCGGC
138

2_Spike Protein

antisense strand

(B11-3, B19-3

siRNA)

SARS CoV-
ATTCAAGGCTCCCTCAGTTGC
139

2_Nucleocapsid

Protein

antisense strand

(B8-3, B10,

B15-3, B16-3,

B17-3, B18-3

siRNA)

ALK2 sense
GGCCTCATTATTCTCTCT
140

strand (A11-1

siRNA)

ALK2 sense
GTGTTCGCAGTATGTCTT
141

strand (A11-2

siRNA)

ALK2 sense
GCCTGCCTGCTGGGAGTT
142

strand (A11-3

siRNA)

SOD1 sense
GAAGGAAAGTAATGGACCAGT
143

strand (A12-1,

A13-1 siRNA)

SOD1 sense
GGTCCTCACTTTAATCCTCTA
144

strand (A12-2,

A13-2 siRNA)

SOD1 sense
GGAGACTTGGGCAATGTGACT
145

strand (A12-3,

A13-3 siRNA)

ALK2 antisense
AGAGAGAATAATGAGGCC
146

strand (A11-1

siRNA)

ALK2 antisense
AAGACATACTGCGAACAC
147

strand (A11-2

siRNA)

ALK2 antisense
AACTCCCAGCAGGCAGGC
148

strand (A11-3

siRNA)

SOD1 antisense
ACTGGTCCATTACTTTCCTTC
149

strand (A12-1,

A13-1 siRNA)

SOD1 antisense
TAGAGGATTAAAGTGAGGACC
150

strand (A12-2,

A13-2 siRNA)

SOD1 antisense
AGTCACATTGCCCAAGTCTCC
151

strand (A12-3,

A13-3 siRNA)

Compounds A9-
See Table 9
152-158

A15

Compounds B18
See Table 10
159-166

and A9-A15

(plasmid

sequences)

IL-4
ATCGTTAGCTTCTCCTGATAAACTAATTGCCTCACATTGTCACTGCAAAT
167

Human IL-4
CGACACCTATTAATGGGTCTCACCTCCCAACTGCTTCCCCCTCTGTTCTT

amino acid
CCTGCTAGCATGTGCCGGCAACTTTGTCCACGGACACAAGTGCGATATCA

(Genbank
CCTTACAGGAGATCATCAAAACTTTGAACAGCCTCACAGAGCAGAAGACT

NM_000589.4)
CTGTGCACCGAGTTGACCGTAACAGACATCTTTGCTGCCTCCAAGAACAC

AACTGAGAAGGAAACCTTCTGCAGGGCTGCGACTGTGCTCCGGCAGTTCT

ACAGCCACCATGAGAAGGACACTCGCTGCCTGGGTGCGACTGCACAGCAG

TTCCACAGGCACAAGCAGCTGATCCGATTCCTGAAACGGCTCGACAGGAA

CCTCTGGGGCCTGGCGGGCTTGAATTCCTGTCCTGTGAAGGAAGCCAACC

AGAGTACGTTGGAAAACTTCTTGGAAAGGCTAAAGACGATCATGAGAGAG

AAATATTCAAAGTGTTCGAGCTGAATATTTTAATTTATGAGTTTTTGATA

GCTTTATTTTTTAAGTATTTATATATTTATAACTCATCATAAAATAAAGT

ATATATAGAATCTAA

IL-4

MGLTSQLLPPLFFLLACAGNFVHGHKCDITLQEIIKTLNSLTEQKTLCTE
168

Human IL-4
LTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRH

amino acid
KQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSK

(Genbank
CSS

NP_000580.1)

Underlined:

signal sequence

Erythropoietin
CCTTTCCCAGATAGCACGCTCCGCCAGTCCCAAGGGTGCGCAACCGGCTG
169

(EPO)
CACTCCCCTCCCGCGACCCAGGGCCCGGGAGCAGCCCCCATGACCCACAC

Human EPO
GCACGTCTGCAGCAGCCCCGCTCACGCCCCGGCGAGCCTCAACCCAGGCG

amino acid
TCCTGCCCCTGCTCTGACCCCGGGTGGCCCCTACCCCTGGCGACCCCTCA

(Genbank
CGCACACAGCCTCTCCCCCACCCCCACCCGCGCACGCACACATGCAGATA

NM_000799.4)
ACAGCCCCGACCCCCGGCCAGAGCCGCAGAGTCCCTGGGCCACCCCGGCC

GCTCGCTGCGCTGCGCCGCACCGCGCTGTCCTCCCGGAGCCGGACCGGGG

CCACCGCGCCCGCTCTGCTCCGACACCGCGCCCCCTGGACAGCCGCCCTC

TCCTCCAGGCCCGTGGGGCTGGCCCTGCACCGCCGAGCTTCCCGGGATGA

GGGCCCCCGGTGTGGTCACCCGGCGCGCCCCAGGTCGCTGAGGGACCCCG

GCCAGGCGCGGAGATGGGGGTGCACGAATGTCCTGCCTGGCTGTGGCTTC

TCCTGTCCCTGCTGTCGCTCCCTCTGGGCCTCCCAGTCCTGGGCGCCCCA

CCACGCCTCATCTGTGACAGCCGAGTCCTGGAGAGGTACCTCTTGGAGGC

CAAGGAGGCCGAGAATATCACGACGGGCTGTGCTGAACACTGCAGCTTGA

ATGAGAATATCACTGTCCCAGACACCAAAGTTAATTTCTATGCCTGGAAG

AGGATGGAGGTCGGGCAGCAGGCCGTAGAAGTCTGGCAGGGCCTGGCCCT

GCTGTCGGAAGCTGTCCTGCGGGGCCAGGCCCTGTTGGTCAACTCTTCCC

AGCCGTGGGAGCCCCTGCAGCTGCATGTGGATAAAGCCGTCAGTGGCCTT

CGCAGCCTCACCACTCTGCTTCGGGCTCTGGGAGCCCAGAAGGAAGCCAT

CTCCCCTCCAGATGCGGCCTCAGCTGCTCCACTCCGAACAATCACTGCTG

ACACTTTCCGCAAACTCTTCCGAGTCTACTCCAATTTCCTCCGGGGAAAG

CTGAAGCTGTACACAGGGGAGGCCTGCAGGACAGGGGACAGATGACCAGG

TGTGTCCACCTGGGCATATCCACCACCTCCCTCACCAACATTGCTTGTGC

CACACCCTCCCCCGCCACTCCTGAACCCCGTCGAGGGGCTCTCAGCTCAG

CGCCAGCCTGTCCCATGGACACTCCAGTGCCAGCAATGACATCTCAGGGG

CCAGAGGAACTGTCCAGAGAGCAACTCTGAGATCTAAGGATGTCACAGGG

CCAACTTGAGGGCCCAGAGCAGGAAGCATTCAGAGAGCAGCTTTAAACTC

AGGGACAGAGCCATGCTGGGAAGACGCCTGAGCTCACTCGGCACCCTGCA

AAATTTGATGCCAGGACACGCTTTGGAGGCGATTTACCTGTTTTCGCACC

TACCATCAGGGACAGGATGACCTGGATAACTTAGGTGGCAAGCTGTGACT

TCTCCAGGTCTCACGGGCATGGGCACTCCCTTGGTGGCAAGAGCCCCCTT

GACACCGGGGTGGTGGGAACCATGAAGACAGGATGGGGGCTGGCCTCTGG

CTCTCATGGGGTCCAAGTTTTGTGTATTCTTCAACCTCATTGACAAGAAC

TGAAACCACCAA

Erythropoietin

MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAE
170

(EPO)
NITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEA

Human EPO
VLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPD

amino acid
AASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

(Genbank

NP_000790.2)

Underlined:

signal sequence

ALK2 mRNA
GAGGTGGAGTATGGCACTATCG
171

forward primer

ALK2 mRNA
CACTCCAACAGTGTAATCTGGCG
172

reverse primer

Human 18S
ACCCGTTGAACCCCATTCGTGA
173

rRNA forward

primer

Human 18S
GCCTCACTAAACCATCCAATCGG
174

rRNA reverse

primer

Human SOD1
CTCACTCTCAGGAGACCATTGC
175

mRNA forward

primer

Human SOD1
CCACAAGCCAAACGACTTCCAG
176

mRNA reverse

primer

Compound A1
AUAGUGAGUCGUAUUAACGUACCAACAACAAGGAAGUGCUAAAGAAACUU
177

RNA sequence
GUUCUUUAGCACUUCCUUGUUUAUCUUAGAGGCAUAUGCCUGCCACCAUG

ACCAUCCUGUUUCUGACAAUGGUCAUCAGCUACUUCGGCUGCAUGAAGGC

CGUGAAGAUGCACACCAUGAGCAGCAGCCACCUGUUCUAUCUGGCCCUGU

GCCUGCUGACCUUUACCAGCUCUGCUACCGCCGGACCUGAGACACUUUGU

GGCGCUGAACUGGUGGACGCCCUGCAGUUUGUGUGUGGCGACAGAGGCUU

CUACUUCAACAAGCCCACAGGCUACGGCAGCAGCUCUAGAAGGGCUCCUC

AGACCGGAAUCGUGGACGAGUGCUGCUUCAGAAGCUGCGACCUGCGGCGG

CUGGAAAUGUAUUGUGCCCCUCUGAAGCCUGCCAAGAGCGCCUAAUUUAU

CUUAGAGGCAUAUCCCU

(all Us are modified; N¹-methylpseudouridine)

Compound A2
AUAGUGAGUCGUAUUAACGUACCAACAACAAGGAGUGCUAAAGAAACUUG
178

RNA sequence
UUCUUUAGCACUCCUUGUUUAUCUUAGAGGCAUAUCCCUGCCACCAUGAC

CAUCCUGUUUCUGACAAUGGUCAUCAGCUACUUCGGCUGCAUGAAGGCCG

UGAAGAUGCACACCAUGAGCAGCAGCCACCUGUUCUAUCUGGCCCUGUGC

CUGCUGACCUUUACCAGCUCUGCUACCGCCGGACCUGAGACACUUUGUGG

CGCUGAACUGGUGGACGCCCUGCAGUUUGUGUGUGGCGACAGAGGCUUCU

ACUUCAACAAGCCCACAGGCUACGGCAGCAGCUCUAGAAGGGCUCCUCAG

ACCGGAAUCGUGGACGAGUGCUGCUUCAGAAGCUGCGACCUGCGGCGGCU

GGAAAUGUAUUGUGCCCCUCUGAAGCCUGCCAAGAGCGCCUAAUUUAUCU

UAGAGGCAUAUCCCU

(all Us are modified; N¹-methylpseudouridine)

Compound A3
AUAGUGAGUCGUAUUAACGUACCAACAACAAGGAGUGCUAAAGAAACUUG
179

RNA sequence
UUCUUUAGCACUCCUUGUUUAUCUUAGAGGCAUAUCCCUACGUACCAACA

AGAGAGUGAUUGAGAGUGGACUUGCCACUCUCAAUCACUCUCUUUAUCUU

AGAGGCAUAUCCCUACGUACCAACAAGAGAGCUCUGUCUGGACCACUUGG

GUCCAGACAGAGCUCUCUUUAUCUUAGAGGCAUAUCCCUGCCACCAUGAC

CAUCCUGUUUCUGACAAUGGUCAUCAGCUACUUCGGCUGCAUGAAGGCCG

UGAAGAUGCACACCAUGAGCAGCAGCCACCUGUUCUAUCUGGCCCUGUGC

CUGCUGACCUUUACCAGCUCUGCUACCGCCGGACCUGAGACACUUUGUGG

CGCUGAACUGGUGGACGCCCUGCAGUUUGUGUGUGGCGACAGAGGCUUCU

ACUUCAACAAGCCCACAGGCUACGGCAGCAGCUCUAGAAGGGCUCCUCAG

ACCGGAAUCGUGGACGAGUGCUGCUUCAGAAGCUGCGACCUGCGGCGGCU

GGAAAUGUAUUGUGCCCCUCUGAAGCCUGCCAAGAGCGCCUAAUUUAUCU

UAGAGGCAUAUCCCU

(all Us are modified; N¹-methylpseudouridine)

Compound A6
AUAGUGAGUCGUAUUAACGUACCAACAAGAAAGAUGAUAAGCCCACUCUA
180

RNA sequence
CUUGAGAGUGGGCUUAUCAUCUUUCUUUAUCUUAGAGGCAUAUCCCUGCC

ACCAUGACCAUCCUGUUUCUGACAAUGGUCAUCAGCUACUUCGGCUGCAU

GAAGGCCGUGAAGAUGCACACCAUGAGCAGCAGCCACCUGUUCUAUCUGG

CCCUGUGCCUGCUGACCUUUACCAGCUCUGCUACCGCCGGACCUGAGACA

CUUUGUGGCGCUGAACUGGUGGACGCCCUGCAGUUUGUGUGUGGCGACAG

AGGCUUCUACUUCAACAAGCCCACAGGCUACGGCAGCAGCUCUAGAAGGG

CUCCUCAGACCGGAAUCGUGGACGAGUGCUGCUUCAGAAGCUGCGACCUG

CGGCGGCUGGAAAUGUAUUGUGCCCCUCUGAAGCCUGCCAAGAGCGCCUA

AUUUAUCUUAGAGGCAUAUCCCU

(all Us are modified; N¹-methylpseudouridine)

Compound A7
AUAGUGAGUCGUAUUAACGUACCAACAAGAAAGAUGAUAAGCCCACUCUA
181

RNA sequence
CUUGAGAGUGGGCUUAUCAUCUUUCUUUAUCUUAGAGGCAUAUCCCUACG

UACCAACAAGGUGAUGUCUGGUCCAUAUGAACUUGUCAUAUGGACCAGAC

AUCACCUUUAUCUUAGAGGCAUAUCCCUACGUACCAACAAGAUGAUAAGC

CCACUCUAACUUGUAGAGUGGGCUUAUCAUCUUUAUCUUAGAGGCAUAUC

CCUGCCACCAUGACCAUCCUGUUUCUGACAAUGGUCAUCAGCUACUUCGG

CUGCAUGAAGGCCGUGAAGAUGCACACCAUGAGCAGCAGCCACCUGUUCU

AUCUGGCCCUGUGCCUGCUGACCUUUACCAGCUCUGCUACCGCCGGACCU

GAGACACUUUGUGGCGCUGAACUGGUGGACGCCCUGCAGUUUGUGUGUGG

CGACAGAGGCUUCUACUUCAACAAGCCCACAGGCUACGGCAGCAGCUCUA

GAAGGGCUCCUCAGACCGGAAUCGUGGACGAGUGCUGCUUCAGAAGCUGC

GACCUGCGGCGGCUGGAAAUGUAUUGUGCCCCUCUGAAGCCUGCCAAGAG

CGCCUAAUUUAUCUUAGAGGCAUAUCCCU

(all Us are modified; N¹-methylpseudouridine)

Compound A8
AUAGUGAGUCGUAUUAACGUACCAACAAGGCGUGGAGCUGAGAGAUAAAC
182

RNA sequence
UUGUUAUCUCUCAGCUCCACGCCUUUAUCUUAGAGGCAUAUCCCUACGUA

CCAACAAGGGCCUGUACCUCAUCUACUACUUGAGUAGAUGAGGUACAGGC

CCUUUAUCUUAGAGGCAUAUCCCUACGUACCAACAAGGUAUGAGCCCAUC

UAUCUACUUGAGAUAGAUGGGCUCAUACCUUUAUCUUAGAGGCAUAUCCC

UACGUACCAACAAGCAAUGAGGACCCUGAGAGAUACUUGAUCUCUCAGGG

UCCUCAUUGCUUUAUCUUAGAGGCAUAUCCCUACGUACCAACAAGCUGAU

GGGAACGUGGACUAACUUGUAGUCCACGUUCCCAUCAGCUUUAUCUUAGA

GGCAUAUCCCUACGUACCAACAAGGUCCUCAGAUUACUACAAACUUGUUG

UAGUAAUCUGAGGACCUUUAUCUUAGAGGCAUAUCCCUGCCACCAUGGGA

CUGACAUCUCAACUGCUGCCUCCACUGUUCUUUCUGCUGGCCUGCGCCGG

CAAUUUUGUGCACGGCCACAAGUGCGACAUCACCCUGCAAGAGAUCAUCA

AGACCCUGAACAGCCUGACCGAGCAGAAAACCCUGUGCACCGAGCUGACC

GUGACCGAUAUCUUUGCCGCCAGCAAGAACACAACCGAGAAAGAGACAUU

CUGCAGAGCCGCCACCGUGCUGAGACAGUUCUACAGCCACCACGAGAAGG

ACACCAGAUGCCUGGGAGCUACAGCCCAGCAGUUCCACAGACACAAGCAG

CUGAUCCGGUUCCUGAAGCGGCUGGACAGAAAUCUGUGGGGACUCGCCGG

CCUGAAUAGCUGCCCUGUGAAAGAGGCCAACCAGUCUACCCUGGAAAACU

UCCUGGAACGGCUGAAAACCAUCAUGCGCGAGAAGUACAGCAAGUGCAGC

AGCUGAUUUAUCUUAGAGGCAUAUCCCU

(all Us are modified; N¹-methylpseudouridine)

Compound A9
AUAGUGAGUCGUAUUAACGUACCAACAAGGCGUGGAGCUGAGAGAUAAAC
183

RNA sequence
UUGUUAUCUCUCAGCUCCACGCCUUUAUCUUAGAGGCAUAUCCCUACGUA

CCAACAAGGGCCUGUACCUCAUCUACUACUUGAGUAGAUGAGGUACAGGC

CCUUUAUCUUAGAGGCAUAUCCCUACGUACCAACAAGGUAUGAGCCCAUC

UAUCUACUUGAGAUAGAUGGGCUCAUACCUUUAUCUUAGAGGCAUAUCCC

UGCCACCAUGGGACUGACAUCUCAACUGCUGCCUCCACUGUUCUUUCUGC

UGGCCUGCGCCGGCAAUUUUGUGCACGGCCACAAGUGCGACAUCACCCUG

CAAGAGAUCAUCAAGACCCUGAACAGCCUGACCGAGCAGAAAACCCUGUG

CACCGAGCUGACCGUGACCGAUAUCUUUGCCGCCAGCAAGAACACAACCG

AGAAAGAGACAUUCUGCAGAGCCGCCACCGUGCUGAGACAGUUCUACAGC

CACCACGAGAAGGACACCAGAUGCCUGGGAGCUACAGCCCAGCAGUUCCA

CAGACACAAGCAGCUGAUCCGGUUCCUGAAGCGGCUGGACAGAAAUCUGU

GGGGACUCGCCGGCCUGAAUAGCUGCCCUGUGAAAGAGGCCAACCAGUCU

ACCCUGGAAAACUUCCUGGAACGGCUGAAAACCAUCAUGCGCGAGAAGUA

CAGCAAGUGCAGCAGCUGAUUUAUCUUAGAGGCAUAUCCCU

(all Us are modified; N¹-methylpseudouridine)

Compound A10
GCCACCAUGGGACUGACAUCUCAACUGCUGCCUCCACUGUUCUUUCUGCU
184

RNA sequence
GGCCUGCGCCGGCAAUUUUGUGCACGGCCACAAGUGCGACAUCACCCUGC

AAGAGAUCAUCAAGACCCUGAACAGCCUGACCGAGCAGAAAACCCUGUGC

ACCGAGCUGACCGUGACCGAUAUCUUUGCCGCCAGCAAGAACACAACCGA

GAAAGAGACAUUCUGCAGAGCCGCCACCGUGCUGAGACAGUUCUACAGCC

ACCACGAGAAGGACACCAGAUGCCUGGGAGCUACAGCCCAGCAGUUCCAC

AGACACAAGCAGCUGAUCCGGUUCCUGAAGCGGCUGGACAGAAAUCUGUG

GGGACUCGCCGGCCUGAAUAGCUGCCCUGUGAAAGAGGCCAACCAGUCUA

CCCUGGAAAACUUCCUGGAACGGCUGAAAACCAUCAUGCGCGAGAAGUAC

AGCAAGUGCAGCAGCUGAAUAGUGAGUCGUAUUAACGUACCAACAAGGCG

UGGAGCUGAGAGAUAAACUUGUUAUCUCUCAGCUCCACGCCUUUAUCUUA

GAGGCAUAUCCCUACGUACCAACAAGGGCCUGUACCUCAUCUACUACUUG

AGUAGAUGAGGUACAGGCCCUUUAUCUUAGAGGCAUAUCCCUACGUACCA

ACAAGGUAUGAGCCCAUCUAUCUACUUGAGAUAGAUGGGCUCAUACCUUU

AUCUUAGAGGCAUAUCCCUUUUAUCUUAGAGGCAUAUCCCU

(all Us are modified; N¹-methylpseudouridine)

Compound A11
GCCACCAUGACCAUCCUGUUUCUGACAAUGGUCAUCAGCUACUUCGGCUG
185

RNA sequence
CAUGAAGGCCGUGAAGAUGCACACCAUGAGCAGCAGCCACCUGUUCUAUC

UGGCCCUGUGCCUGCUGACCUUUACCAGCUCUGCUACCGCCGGACCUGAG

ACACUUUGUGGCGCUGAACUGGUGGACGCCCUGCAGUUUGUGUGUGGCGA

CAGAGGCUUCUACUUCAACAAGCCCACAGGCUACGGCAGCAGCUCUAGAA

GGGCUCCUCAGACCGGAAUCGUGGACGAGUGCUGCUUCAGAAGCUGCGAC

CUGCGGCGGCUGGAAAUGUAUUGUGCCCCUCUGAAGCCUGCCAAGAGCGC

CUAAAUAGUGAGUCGUAUUAACGUACCAACAAGGCCUCAUUAUUCUCUCU

ACUUGAGAGAGAAUAAUGAGGCCUUUAUCUUAGAGGCAUAUCCCUACGUA

CCAACAAGUGUUCGCAGUAUGUCUUACUUGAAGACAUACUGCGAACACUU

UAUCUUAGAGGCAUAUCCCUACGUACCAACAAGCCUGCCUGCUGGGAGUU

ACUUGAACUCCCAGCAGGCAGGCUUUAUCUUAGAGGCAUAUCCCUUUUAU

CUUAGAGGCAUAUCCCU

(all Us are modified; N¹-methylpseudouridine)

Compound A12
AUAGUGAGUCGUAUUAACGUACCAACAAGAAGGAAAGUAAUGGACCAGUA
186

RNA sequence
CUUGACUGGUCCAUUACUUUCCUUCUUUAUCUUAGAGGCAUAUCCCUACG

UACCAACAAGGUCCUCACUUUAAUCCUCUAACUUGUAGAGGAUUAAAGUG

AGGACCUUUAUCUUAGAGGCAUAUCCCUACGUACCAACAAGGAGACUUGG

GCAAUGUGACUACUUGAGUCACAUUGCCCAAGUCUCCUUUAUCUUAGAGG

CAUAUCCCUGCCACCAUGGGCAAGAUUAGCAGCCUGCCUACACAGCUGUU

CAAGUGCUGCUUCUGCGACUUCCUGAAAGUGAAGAUGCACACCAUGAGCA

GCAGCCACCUGUUCUAUCUGGCCCUGUGCCUGCUGACCUUUACCAGCUCU

GCUACCGCCGGACCUGAGACACUUUGUGGCGCUGAACUGGUGGACGCCCU

GCAGUUUGUGUGUGGCGACAGAGGCUUCUACUUCAACAAGCCCACAGGCU

ACGGCAGCAGCUCUAGAAGGGCUCCUCAGACCGGAAUCGUGGACGAGUGC

UGUUUCAGAAGCUGCGACCUGCGGCGGCUGGAAAUGUAUUGUGCCCCUCU

GAAGCCUGCCAAGAGCGCCUAAUUUAUCUUAGAGGCAUAUCCCU

(all Us are modified; N¹-methylpseudouridine)

Compound A13
AUAGUGAGUCGUAUUAACGUACCAACAAGAAGGAAAGUAAUGGACCAGUA
187

RNA sequence
CUUGACUGGUCCAUUACUUUCCUUCUUUAUCUUAGAGGCAUAUCCCUACG

UACCAACAAGGUCCUCACUUUAAUCCUCUAACUUGUAGAGGAUUAAAGUG

AGGACCUUUAUCUUAGAGGCAUAUCCCUACGUACCAACAAGGAGACUUGG

GCAAUGUGACUACUUGAGUCACAUUGCCCAAGUCUCCUUUAUCUUAGAGG

CAUAUCCCUGCCACCAUGGGAGUGCAUGAAUGUCCUGCUUGGCUGUGGCU

GCUGCUGAGCCUGCUGUCUCUGCCUCUGGGACUGCCUGUUCUUGGAGCCC

CUCCUAGACUGAUCUGCGACAGCAGAGUGCUGGAAAGAUACCUGCUGGAA

GCCAAAGAGGCCGAGAACAUCACCACAGGCUGUGCCGAGCACUGCAGCCU

GAACGAGAAUAUCACCGUGCCUGACACCAAAGUGAACUUCUACGCCUGGA

AGCGGAUGGAAGUGGGCCAGCAGGCUGUGGAAGUUUGGCAAGGACUGGCC

CUGCUGAGCGAAGCUGUUCUGAGAGGACAGGCUCUGCUGGUCAACAGCUC

UCAGCCUUGGGAACCUCUGCAACUGCACGUGGACAAGGCCGUGUCUGGCC

UGAGAAGCCUGACCACACUGCUGAGAGCACUGGGAGCCCAGAAAGAGGCC

AUCUCUCCACCUGAUGCUGCCUCUGCUGCCCCUCUGAGAACCAUCACCGC

CGACACCUUCAGAAAGCUGUUCCGGGUGUACAGCAACUUCCUGCGGGGCA

AGCUGAAGCUGUACACAGGCGAGGCUUGCAGAACCGGCGACAGAUAAUUU

AUCUUAGAGGCAUAUCCCU

(all Us are modified; N¹-methylpseudouridine)

Compound A14
AUAGUGAGUCGUAUUAACGUACCAACAAGAAAGAUGAUAAGCCCACUCUA
188

RNA sequence
CUUGAGAGUGGGCUUAUCAUCUUUCUUUAUCUUAGAGGCAUAUCCCUACG

UACCAACAAGGUGAUGUCUGGUCCAUAUGAACUUGUCAUAUGGACCAGAC

AUCACCUUUAUCUUAGAGGCAUAUCCCUACGUACCAACAAGAUGAUAAGC

CCACUCUAACUUGUAGAGUGGGCUUAUCAUCUUUAUCUUAGAGGCAUAUC

CCUGCCACCAUGGGCAAGAUUAGCAGCCUGCCUACACAGCUGUUCAAGUG

CUGCUUCUGCGACUUCCUGAAAGUGAAGAUGCACACCAUGAGCAGCAGCC

ACCUGUUCUAUCUGGCCCUGUGCCUGCUGACCUUUACCAGCUCUGCUACC

GCCGGACCUGAGACACUUUGUGGCGCUGAACUGGUGGACGCCCUGCAGUU

UGUGUGUGGCGACAGAGGCUUCUACUUCAACAAGCCCACAGGCUACGGCA

GCAGCUCUAGAAGGGCUCCUCAGACCGGAAUCGUGGACGAGUGCUGUUUC

AGAAGCUGCGACCUGCGGCGGCUGGAAAUGUAUUGUGCCCCUCUGAAGCC

UGGCAAGAGCGCCUAAUUUAUCUUAGAGGCAUAUCCCU

(all Us are modified; N¹-methylpseudouridine)

Compound A15
GCCACCAUGGGCAAGAUUAGCAGCCUGCCUACACAGCUGUUCAAGUGCUG
189

RNA sequence
CUUCUGCGACUUCCUGAAAGUGAAGAUGCACACCAUGAGCAGCAGCCACC

UGUUCUAUCUGGCCCUGUGCCUGCUGACCUUUACCAGCUCUGCUACCGCC

GGACCUGAGACACUUUGUGGCGCUGAACUGGUGGACGCCCUGCAGUUUGU

GUGUGGCGACAGAGGCUUCUACUUCAACAAGCCCACAGGCUACGGCAGCA

GCUCUAGAAGGGCUCCUCAGACCGGAAUCGUGGACGAGUGCUGUUUCAGA

AGCUGCGACCUGCGGCGGCUGGAAAUGUAUUGUGCCCCUCUGAAGCCUGC

CAAGAGCGCCUAAAUAGUGAGUCGUAUUAACGUACCAACAAGAAAGAUGA

UAAGCCCACUCUACUUGAGAGUGGGCUUAUCAUCUUUCUUUAUCUUAGAG

GCAUAUCCCUACGUACCAACAAGGUGAUGUCUGGUCCAUAUGAACUUGUC

AUAUGGACCAGACAUCACCUUUAUCUUAGAGGCAUAUCCCUACGUACCAA

CAAGAUGAUAAGCCCACUCUAACUUGUAGAGUGGGCUUAUCAUCUUUAUC

UUAGAGGCAUAUCCCUUUUAUCUUAGAGGCAUAUCCCU

(all Us are modified; N¹-methylpseudouridine)

Compound B18
GCCACCAUGUCUAGCAGCUCUUGGCUGCUGCUGUCUCUGGUGGCUGUGAC
190

RNA sequence
AGCCGCUCAGAGCACCAUUGAGGAACAGGCCAAGACCUUCCUGGACAAGU

UCAACCACGAGGCCGAGGACCUGUUCUACCAGUCUAGCCUGGCCAGCUGG

AACUACAACACCAACAUCACCGAAGAGAACGUGCAGAACAUGAACAACGC

CGGCGACAAGUGGAGCGCCUUCCUGAAAGAGCAGAGCACACUGGCCCAGA

UGUACCCUCUGCAAGAGAUCCAGAACCUGACCGUGAAGCUCCAGCUGCAG

GCCCUCCAGCAGAAUGGAAGCUCUGUGCUGAGCGAGGACAAGAGCAAGCG

GCUGAACACCAUCCUGAAUACCAUGAGCACCAUCUACAGCACCGGCAAAG

UGUGCAACCCCGACAAUCCCCAAGAGUGCCUGCUGCUGGAACCCGGCCUG

AAUGAGAUCAUGGCCAACAGCCUGGACUACAACGAGAGACUGUGGGCCUG

GGAGUCUUGGAGAAGCGAAGUGGGAAAGCAGCUGCGGCCCCUGUACGAGG

AAUACGUGGUGCUGAAGAACGAGAUGGCCAGAGCCAACCACUACGAGGAC

UACGGCGACUAUUGGAGAGGCGACUACGAAGUGAAUGGCGUGGACGGCUA

CGACUACAGCAGAGGCCAGCUGAUCGAGGACGUGGAACACACCUUCGAGG

AAAUCAAGCCUCUGUACGAGCAUCUGCACGCCUACGUGCGGGCCAAGCUG

AUGAAUGCUUACCCCAGCUACAUCAGCCCCAUCGGCUGUCUGCCUGCUCA

UCUGCUGGGAGACAUGUGGGGCAGAUUCUGGACCAACCUGUACAGCCUGA

CAGUGCCCUUCGGCCAGAAACCUAACAUCGACGUGACCGACGCCAUGGUG

GAUCAGGCUUGGGAUGCCCAGCGGAUCUUCAAAGAGGCCGAGAAGUUCUU

CGUGUCCGUGGGCCUGCCUAAUAUGACCCAAGGCUUCUGGGAGAACUCCA

UGCUGACAGACCCCGGCAAUGUGCAGAAAGCCGUGUGUCAUCCUACCGCC

UGGGAUCUCGGCAAGGGCGACUUCAGAAUCCUGAUGUGCACCAAAGUGAC

GAUGGACGACUUCCUGACAGCCCACCACGAGAUGGGCCACAUCCAGUACG

AUAUGGCCUACGCCGCUCAGCCCUUCCUGCUGAGAAAUGGCGCCAAUGAG

GGCUUCCACGAAGCCGUGGGAGAGAUCAUGAGCCUGUCUGCCGCCACACC

UAAGCACCUGAAGUCUAUCGGACUGCUGAGCCCCGACUUCCAAGAGGACA

ACGAGACAGAGAUCAACUUCCUGCUCAAGCAGGCCCUGACCAUCGUGGGC

ACACUGCCCUUUACCUACAUGCUGGAAAAGUGGCGGUGGAUGGUCUUUAA

GGGCGAGAUCCCCAAGGACCAGUGGAUGAAGAAAUGGUGGGAGAUGAAGC

GCGAGAUCGUGGGCGUUGUGGAACCUGUGCCUCACGACGAGACAUACUGC

GAUCCUGCCAGCCUGUUUCACGUGUCCAACGACUACUCCUUCAUCCGGUA

CUACACCCGGACACUGUACCAGUUCCAGUUUCAAGAGGCUCUGUGCCAGG

CCGCCAAGCACGAAGGACCUCUGCACAAGUGCGACAUCAGCAACUCUACA

GAGGCCGGACAGAAACUGUUCAACAUGCUGCGGCUGGGCAAGAGCGAGCC

UUGGACACUGGCUCUGGAAAAUGUCGUGGGCGCCAAGAAUAUGAACGUGC

GGCCACUGCUGAACUACUUCGAGCCCCUGUUCACCUGGCUGAAGGACCAG

AACAAGAACAGCUUCGUCGGCUGGUCCACCGAUUGGAGCCCUUACGCCGA

CCAGAGCAUCAAAGUGCGGAUCAGCCUGAAAAGCGCCCUGGGCGAUAAGG

CCUAUGAGUGGAACGACAAUGAGAUGUACCUGUUCCGGUCCAGCGUGGCC

UAUGCUAUGCGGCAGUACUUUCUGAAAGUCAAGAACCAGAUGAUCCUGUU

CGGCGAAGAGGAUGUGCGCGUGGCCAACCUGAAGCCUCGGAUCAGCUUCA

ACUUCUUCGUGACUGCCCCUAAGAACGUGUCCGACAUCAUCCCCAGAACC

GAGGUGGAAAAGGCCAUCAGAAUGAGCAGAAGCCGGAUCAACGACGCCUU

CCGGCUGAACGACAACUCCCUGGAAUUCCUGGGCAUUCAGCCCACACUGG

GCCCUCCAAAUCAGCCUCCUGUGUCCUAAAUAGUGAGUCGUAUUAACGUA

CCAACAAGUGUGACCGAAAGGUAAGAUGACUUGCAUCUUACCUUUCGGUC

ACACUUUAUCUUAGAGGCAUAUCCCUACGUACCAACAAGAGGUGAUGAAG

UCAGACAAAACUUGUUUGUCUGACUUCAUCACCUCUUUAUCUUAGAGGCA

UAUCCCUACGUACCAACAAGCAACUGAGGGAGCCUUGAAUACUUGAUUCA

AGGCUCCCUCAGUUGCUUUAUCUUAGAGGCAUAUCCCUUUUAUCUUAGAG

GCAUAUCCCU

(all Us are modified; N¹-methylpseudouridine)

	Number	Date	Country
Parent	PCT/IB2020/001091	Dec 2020	US
Child	17846288		US

Compositions and Methods for Simultaneously Modulating Expression of Genes

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

Priority Claims (1)

CROSS-REFERENCE TO RELATED APPLICATIONS

Provisional Applications (1)

Continuations (1)