Compositions and methods for treating COVID-19

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in an ASCII text file, named as 38534Z_SequenceListing.txt of 114 KB, created on Oct. 25, 2021, and submitted to the United States Patent and Trademark Office via EFS-Web, is incorporated herein by reference.

BACKGROUND

The novel coronavirus disease (COVID-19) is caused by the SARS-Cov-2 virus and is known for inducing multisystem organ dysfunction associated with significant morbidity and mortality. Despite several available vaccines, effective therapeutics targeted specifically to the virus are still lacking. Specifically, effective prophylactics with a few side-effects and therapeutics targeted specifically towards SARS-CoV-2 are needed since some of the current vaccines have been found some serious side-effects, e.g., blood clotting or increasing the heart myocarditis. It has also been observed that IgG antibodies, while abundantly present in the vasculature, are present at a much lesser extent in mucosal tissues, such as epithelial cells of nasal and lung, where most ACE2-expressing cells (i.e., targets of SARS-CoV-2). This means that IgG antibodies against SARS-CoV-2, either induced by vaccination or exogenously provided, may not effectively protect ACE2-expressing cells on the mucosal tissues from a SARS-CoV2 infection.

SUMMARY OF THE DISCLOSURE

An aspect of the disclosure is directed to a composition comprising a plurality of inhibitory oligonucleotides, wherein the plurality of inhibitory oligonucleotides targets at least two SARS-CoV-2 genes selected from the group consisting of ORF1ab, RdRp, the S-protein gene, the N-protein gene, and the E protein gene.

In some embodiments, the plurality of inhibitory oligonucleotides targets all of the ORF1ab, RdRp, S-protein, N-protein and E protein genes. In some embodiments, a selected SARS-CoV-2 gene is targeted by at least two inhibitory oligonucleotides.

In some embodiments, the inhibitory oligonucleotides are selected from an antisense oligonucleotide, a small interfering RNA (siRNA), a Dicer-substrate RNA (DsiRNA), and a microRNA.

In some embodiments, the plurality of inhibitory oligonucleotides comprises at least two oligonucleotides which comprise a nucleotide sequence selected from the group consisting of SEQ ID NOS: 9-16 and modified forms of SEQ ID NOS: 9-16.

In some embodiments, the plurality of inhibitory oligonucleotides comprises eight oligonucleotides as shown in SEQ ID NOS: 9-16 or modified forms of SEQ ID NOS: 9-16.

In some embodiments, the plurality of inhibitory oligonucleotides comprises at least two pairs of Dicer-substrate RNAs (DsiRNAs) selected from the group consisting of DsiRNA pair 1 (SEQ ID NOs: 17 & 18), DsiRNA pair 2 (SEQ ID NOs: 19 & 20), DsiRNA pair 3 (SEQ ID NOs: 21 & 22), DsiRNA pair 4 (SEQ ID NOs: 23 & 24), DsiRNA pair 5 (SEQ ID NOs: 25 & 26), DsiRNA pair 6 (SEQ ID NOs: 27 & 28), DsiRNA pair 7 (SEQ ID NOs: 29 & 30), and DsiRNA pair 8 (SEQ ID NOs: 31 & 32).

In some embodiments, the plurality of inhibitory oligonucleotides comprises Dicer-substrate RNA (DsiRNA) pair 1 (SEQ ID NOs: 17 & 18), DsiRNA pair 2 (SEQ ID NOs: 19 & 20), DsiRNA pair 3 (SEQ ID NOs: 21 & 22), DsiRNA pair 4 (SEQ ID NOs: 23 & 24), DsiRNA pair 5 (SEQ ID NOs: 25 & 26), DsiRNA pair 6 (SEQ ID NOs: 27 & 28), DsiRNA pair 7 (SEQ ID NOs: 29 & 30), and DsiRNA pair 8 (SEQ ID NOs: 31 & 32).

In some embodiments, the inhibitory oligonucleotides are modified oligonucleotides.

In some embodiments, the modified inhibitory oligonucleotides are 2′-Deoxy, 2′-Fluoroarabino Nucleic Acid (FANA)-modified antisense oligonucleotides.

In some embodiments, the modified inhibitory oligonucleotides are 2′ O-Methyl RNA modified antisense oligonucleotides selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.

In some embodiments, at least one inhibitory oligonucleotide within the plurality of oligonucleotides comprises a detectable label.

In some embodiments, the label is a fluorescent label.

In some embodiments, the plurality of inhibitory oligonucleotides is provided in one or more nucleic acid vectors.

In some embodiments, the nucleic acid vectors are selected from a viral vector, a non-viral vector, an integrative vector, or a non-integrative vector.

In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises nanoparticles or other delivery vehicles to which the plurality of inhibitory oligonucleotides is conjugated.

Another aspect of the specification is directed to a composition comprising at least one peptide mimicking a portion of the ligand binding domain (LBD) of human ACE2 protein, wherein the at least one peptide prevents binding of the S-protein of SARS-CoV-2 to the human ACE2 protein.

In some embodiments, the LBD of human ACE2 comprises the amino acid sequence of SEQ ID NO: 56.

In some embodiments, the composition comprises a plurality of peptides, each mimicking a different portion of the ligand binding domain (LBD) of human ACE2 protein.

In some embodiments, the at least one peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-44, 54, and 63-82.

In some embodiments, the composition comprises a plurality of peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-44, 54, and 63-82.

In some embodiments, the composition comprises at least five peptides, wherein the at least five peptides are selected from peptides comprising an amino acid sequence as shown in SEQ ID NOS: 41-44, 54, and 63-82.

Another aspect of the disclosure is directed to a composition comprising a peptide mimicking a portion of the receptor binding domain (RBD) of the S-Protein of SARS-CoV-2, wherein the peptide prevents binding of the S-protein of SARS-CoV-2 to a human ACE2 protein.

In some embodiments, the RBD of the S protein of SARS-CoV-2 comprises SEQ ID NO: 62.

In some embodiments, the peptide comprises an amino acid sequence as shown in SEQ ID NO: 45.

In some embodiments, the at least one peptide comprises a label or is conjugated with a probe, a nucleic acid or a chemical molecule. In some embodiments, the label is a fluorescent label.

Another aspect of the disclosure is directed to a dietary supplement comprising a composition as described herein. In some embodiments, the dietary supplement further comprises at least one additional nutrient selected from Vitamin C, Vitamin B6, Vitamin B12, Vitamin D, Zinc, polypeptides, nucleotide, L-arginine or peppermint oil. In some embodiments, the dietary supplement is formulated for oral, nasal, eye, ear, or topical application.

Another aspect of the disclosure is directed to a method comprising expressing a plurality of inhibitory oligonucleotides in a target cell, wherein the plurality of inhibitory oligonucleotides targets at least two SARS-CoV-2 genes selected from the group consisting of ORF1ab, RdRp, the S-protein gene, the N-protein gene and the E protein gene (aka. the “viral infective functional group”). In some embodiments, a selected SARS-CoV-2 gene is targeted by at least two inhibitory oligonucleotides. In some embodiments, the plurality of inhibitory oligonucleotides targets all of ORF1ab, RdRp, S-protein, N-proteins and E protein genes. In some embodiments, the inhibitory oligonucleotides are selected from an antisense oligonucleotide, a small interfering RNA (siRNA), a Dicer-substrate RNA (DsiRNA), or a microRNA.

In some embodiments, the plurality of inhibitory oligonucleotides comprises at least two oligonucleotides which comprise a nucleotide sequence selected from the group consisting of SEQ ID NOS: 9-16.

In some embodiments, the plurality of inhibitory oligonucleotides comprises eight oligonucleotides as shown in SEQ ID NOS: 9-16.

In some embodiments, the target cell is a human cell. In some embodiments, the target cell is a lung epithelial cell. In some embodiments, the target cell is selected from the group consisting of a small airway epithelial cell, a bronchial/tracheal epithelial cell, and a nasal epithelial cell.

In some embodiments, the plurality of inhibitory oligonucleotides are expressed from at least one vector. In some embodiments, the at least one vector is selected from a viral vector, or a non-viral vector, an integrative vector, or a non-integrative vector.

In some embodiments, the at least one vector is delivered to a subject in need via oral, nasal, intravenous (i.v.) injection or topical administration routes.

Another aspect of the disclosure is directed to a nucleic acid vector encoding a plurality of inhibitory oligonucleotides that targets at least two SARS-CoV-2 genes selected from the group consisting of ORF1ab, RdRp, the S-protein gene, the N-protein gene, and the E protein gene.

Another aspect of the disclosure is directed to a combination of nucleic acid vectors, wherein each nucleic acid vector encodes an inhibitory oligonucleotide that targets at least one SARS-CoV-2 genes selected from the group consisting of ORF1ab, RdRp, the S-protein gene, the N-protein gene, and the E protein gene, and wherein the combination of nucleic acid vectors target at least two SARS-CoV-2 genes.

In some embodiments, the nucleic acid vector is a viral vector.

In some embodiments, the combination of nucleic acid vectors comprises an AAV-based vector selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAV13 and AAV14.

In some embodiments, wherein the nucleic acid vector is a non-viral vector.

Another aspect of the disclosure is directed to a method of treating a SARS-CoV-2 infection in a subject in need thereof, comprising administering a subject an effective amount of a nucleic acid vector or a combination of nucleic acid vectors disclosed herein.

Another aspect of the disclosure is directed to a method for treating a SARS-CoV-2 infection comprising administering to a subject an effective amount of a composition described herein.

Another aspect of the disclosure is directed to a method for treating a SARS-CoV-2 infection comprising administering to a subject an effective amount of a first composition as described herein and a second (different) composition as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1. Experiments designed for investigating cell penetration and therapeutic effects of ASO(s) and siRNA on human primary small airway epithelial cells transfected with viral protein of SARS-CoV-2. The human lung small airway epithelial cells were cultured in the 24 well-dish, and the cells were transfected with the genes encoding the viral proteins of SARS-CoV-2. The VS_ASO_1-FANA-FITC, VS_DsiRNA-Cy5 and VS_ASO_2-Cy3 were into the cells for 24-48 hours before analysis with fluorescent microscope. The VS_ASO_1-FANA-FITC designed with FITC labeled shown in the Table 1, and VS_ASO_2-Cy3 with Cy3 label shown in the Table 2; and VS_DsiRNA-Cy5 with Cy5 label shown in Table 3. A1&A2: No treatment as control; A3&A4: Overexpression of both COVID-19 N-protein and the VS_ASO_1-FANA using lipofectamine reagent; A5&A6: Overexpression of both COVID-19 N-protein and the VS_ASO_1-FANA without any regents; B1&B2: No treatment as control; B3&B4: Overexpression of both COVID-19 N-protein and the VS_DsiRNA-Cy5 using lipofectamine reagent; B5&B6: Overexpression of both COVID-19 N-protein and the VS_DsiRNA-Cy5 using Poly-arginine (5 μl/well) only; C1&C2: No treatment as control; C3&C4: Overexpression of both COVID-19 N-protein and the VS_ASO_2-Cy3 using lipofectamine reagent; C5&C6: Overexpression of both COVID-19 N-protein and the VS_ASO_2-Cy3 using Poly-arginine (5 μl/well) only.

FIGS. 2A-2F. Microscopic analysis showing entry of VS_ASO_1-FANA-FITC into the primary human lung small airway epithermal cells (20×). FIGS. 2A-2C were captured under the FITC florescent filter, and FIGS. 2D-2F were captured in the same view of bright fields (20×). FIGS. 2A and 2D were taken in well A3 & A4 (as shown in FIG. 1), FIGS. 2B and 2E were taken in well A5 & A6 (as shown in FIG. 1), and FIGS. 2C and 2F were taken in well A1 & A2 (as shown in FIG. 1).

FIGS. 3A-3F. Microscopic analysis showing entry of VS_ASO_1-FANA-FITC into primary human lung small airway epithermal cells (10×). FIGS. 3A-3C were captured under the FITC florescent filter, and FIGS. 3D-3F were captured in the same view of bright fields (20×). FIGS. 3A and 3D were taken in well A3 & A4 (as shown in FIG. 1), FIGS. 3B and 3E were taken in well A5 & A6 (as shown in FIG. 1), and FIGS. 3C and 3F were taken in well A1 & A2 (as shown in FIG. 1).

FIGS. 4A-4F. Microscopic analysis showing entry of VS_DsiRNA-Cy5 into primary human lung small airway epithermal cells (20×). FIGS. 4A-4C were captured under the Cy5 florescent filter, and FIGS. 4D-4F were captured in the same view of bright fields (20×). FIGS. 4A and 4D were taken in well B3 & B4 (as shown in FIG. 1), FIGS. 4B and 4E were taken in well B5 & B6 (as shown in FIG. 1), and FIGS. 4C and 4F were taken in well B1 & B2 (as shown in FIG. 1).

FIGS. 5A-5F. Microscopic analysis showing entry of VS_DsiRNA-Cy5 into primary human lung small airway epithermal cells (10×). FIGS. 5A-5C were captured under the Cy5 florescent filter, and FIGS. 5D-5F were captured in the same view of bright field images (10×). FIGS. 5A and 5D were taken in well B3 & B4 (as shown in FIG. 1), FIGS. 5B and 5E were taken in well B5 & B6 (as shown in FIG. 1), and FIGS. 5C and 5F were taken in well B1 & B2 (as shown in FIG. 1).

FIGS. 6A-6F. Microscopic analysis showing entry of VS_ASO_2-Cy3 into primary human lung small airway epithermal cells (20×). FIGS. 6A-6C were captured under the Cy3 florescent filter, and FIGS. 6D-6F were captured in the same view of bright fields (20×). FIGS. 6A and 6D were taken in well C3 & C4 (as shown in FIG. 1), FIGS. 6B and 6E were taken in well C5 & C6 (as shown in FIG. 1), and FIGS. 6C and 6F were taken in well C1 & C2 (as shown in FIG. 1).

FIGS. 7A-7F. Microscopic analysis showing entry of VS_ASO_2-Cy3 into primary human lung small airway epithermal cells (10×). The FIGS. 7A-7C were captured under the Cy3 florescent filter, and FIGS. 7D-7F were captured in the same view of bright fields (10×). FIGS. 7A and 7D were taken in well C3 & C4 (as shown in FIG. 1), FIGS. 7B and 7E were taken in well C5 & C6 (as shown in FIG. 1), and FIGS. 7C and 7F were taken in well C1 & C2 (as shown in FIG. 1).

FIG. 8. Experimental design for FACS detection of intercellular delivery of oligos in the human primary lung small airway epithelial cells (HSAEC). The human lung small airway epithelial cells were cultured in the 6-well dish, and the genes encoding the viral proteins of SARS-CoV-2 were delivered by transfection or arginine delivery. The siRNA or ASO were added into the cells for 24-48 hours before analysis with FACS. The VS_ASO_1-FANA-FITC designed with labeled with FITC shown in the Table 1, and VS_ASO_2-Cy3 with modification shown in the Table 2; and VS_DsiRNA-Cy5 shown in Table 3. A1: No treatment as control; A2: Overexpression of N-protein+VS_ASO_1-FANA-FITC without lipofectamine or arginine; A3: Overexpression of N-protein+VS_DsiRNA-Cy5 with lipofectamine; B1: Overexpression of N-protein+VS_DsiRNA-Cy5 with Arginine (10p/well); B2: Overexpression of N-protein+VS_ASO_2-Cy3 with lipofectamine; B3: Overexpression of N-protein+VS_ASO_2-Cy3 with Arginine (10 μl/well).

FIGS. 9A-9C. FACS analysis of in vitro treatment with VS_ASO_1-FANA-FITC without lipofectamine or Arginine in human primary lung small airway epithelial cells (HSAEC). FACS analysis of HSAEC treated by VS_ASO_1-FANA-FITC (excitation: 488 nm, emission band pass filter: 530/30, Total event: 20,000). FIG. 9A no-treatment control, FIG. 9B VS_ASO_1-FANA-FITC and FIG. 9C Merge. The FACS data indicate that the intensities of FITC signals were significantly stronger with shifting to the right (B: FL1-H:FITC) when compared with the control FIG. 9A in the cells after treated with the VS_ASO_1-FANA-FITC without lipofectamine or Arginine reagents FIG. 9B. The FIG. 9C is the merged figures of FIGS. 9A and 9B.

FIGS. 10A-10B. FACS analysis of in vitro treatment with VS_DsiRNA-Cy5 with lipofectamine FIG. 10A or Arginine only FIG. 10B in human primary lung small airway epithelial cells (HSAEC). FACS analysis of HSAEC treated by VS_DsiRNA-Cy5 (excitation: 635 nm, emission band pass filter: 661/16, Total event: 20,000). Left to right panel: no-treatment control, VS_DsiRNA-Cy5 and merge. The FACS data indicates that the intensities of Cy5 signals were significantly higher with shifting to the right (middle panel: FL4-H:Cy5) in both of panel FIGS. 10A and 10B, it also shown that there are more cells with intercellular signals of the oligos in the presence of 10 μl/well Arginine (panel FIG. 10B) when compared with the lipofectamine (panel FIG. 10A).

FIGS. 11A-11B. FACS analysis of in vitro treatment with VS_ASO_2-Cy3 with lipofectamine FIG. 11A or Arginine only FIG. 11B in human primary lung small airway epithelial cells (HSAEC). FACS analysis of HSAEC treated by VS_VS_ASO-Cy3 (excitation: 488 nm, emission band pass filter: 585/42, Total event: 20,000). Left to right panel: no-treatment control, VS_ASO_2-Cy3 and merge. The FACS data indicates that the intensities of Cy3 signals were significantly higher with shifting to the right (middle panel: FL2-H:Cy3) in both of FIGS. 11A and 11B, it also shown that there are more cells with intercellular signals of the oligos in the presence of 10 μl/well Arginine (panel FIG. 11B) when compared with the lipofectamine (panel FIG. 11A).

FIG. 12. Experimental design for detection of SARS-CoV-2 N-protein expressed in the human primary lung small airway epithelial cells (HSAEC) by qRT-PCR after treatment. The human lung small airway epithelial cells were cultured in a 24-well dish, and the cells were transfected with the genes encoding the viral protein (N-protein) of SARS-CoV-2. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with RT-PCR. The VS_ASO_1-FANA-FITC designed with FITC labeled shown in the Table 1, and VS_ASO_2-Cy3 with Cy3 modification shown in the Table 2; and VS_DsiRNA-Cy5 with Cy5 modification shown in Table 3. A1&A2: No treatment as control, A3&A4: Overexpression of COVID-19 N-protein, A5&A6: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA_Cy5 with lipofectamine, B1&B2: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2-Cy3 with lipofectamine, B3&B4: Overexpression of COVID-19 N-protein+/treated by VS_ASO_1-FANA without any reagents.

FIG. 13. Detection of SARS-CoV-2 N-protein expressed in the human primary lung small airway epithelial cells (HSAEC) by qRT-PCR after treatment with VS-Nucleotides. Significant down-regulation was observed: about 5-fold in group treated by VS_DsiRNA-Cy5 oligos (p<0.005); about 1.5 fold in the group treated by VS_ASO_2-cy3 oligo (p<0.01), and about 6 fold in the group treated by the VS_ASO_1-FANA-FITC oligo (p<0.005); when compared with the group with SARS-CoV-2 N-protein overexpression only. The cycle threshold of no-treatment is non-detectable, but in this case for calculation purposes, the number “40” was used as the cycle threshold for a base-line control.

FIG. 14. Experimental design for detection of SARS-CoV-2 S-protein expressed in the primary human lung small airway epithelial cells (HSAEC) by qRT-PCR after treatment. The human primary lung small airway epithelial cells (HSAEC) were cultured in the 24 well-dish, and the cells were transfected with the genes encoding the viral protein (S-protein) of SARS-CoV-2. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with RT-PCR. The VS_ASO_1-FANA (oligo 3) designed shown in the Table 1, and VS_ASO_2 (oligo 3) shown in the Table 2; and VS_DsiRNA (oligo 3) shown in Table 3. A1&A2: No treatment as control, A3&A4: Overexpression of COVID-19 S-protein, A5&A6: Overexpression of COVID-19 S-protein+/treated by VS_DsiRNA (oligo 3) with lipofectamine, B1&B2: Overexpression of COVID-19 S-protein+/treated by VS_DsiRNA (oligo 3) with Arginine (5 μl/well), B3&B4: Overexpression of COVID-19 S-protein+/treated by VS_ASO_2 (oligo 3) with lipofectamine, B5&B6: Overexpression of COVID-19 S-protein+/treated by VS_ASO_2 (oligo 3) with Arginine (5 μl/well), C1&C2: Overexpression of COVID-19 S-protein+/treated by VS_ASO_1-FANA (oligo 3) without any reagents.

FIG. 15. Detection of SARS-CoV-2 S-protein expressed in the human primary lung small airway epithelial cells (HSAEC) by qRT-PCR after treatment with VS-Nucleotides. Significant down-regulation was observed: about 4 fold in group treated by VS_DsiRNA oligo (purple/L: p<0.01) and about 2.8 fold in the presence of poly-Arginine only (yellow/A: p<0.001); about 4 fold in the group treated by VS_ASO_2 oligo (red/L: p<0.001) and about 4 fold in the in the presence of poly-Arginine only (orange/A: p<0.001); and about 11.5 fold in the group treated by the VS_ASO_1-FANA oligo (green/p<0.001); when compared with the group with SARS-CoV-2 S-protein overexpression only. The cycle threshold of no-treatment is non-detectable, but in this case for calculation purposes, the number “40” was used as the cycle threshold for a base-line control.

FIG. 16. Experimental design for detection of both of SARS-CoV-2 ORF1ab and RdRp expressed in the primary human lung small airway epithelial cells (HSAEC) by qRT-PCR after treatment. The human primary lung small airway epithelial cells (HSAEC) were cultured in the 24 well-dish, and the cells were transfected with the genes encoding both ORF1ab and RdRp of SARS-CoV-2 viral protein. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with RT-PCR. The VS_ASO_1-FANA (oligo 1, 2, 5 and 6) designed shown in the Table 1, and VS_ASO_2 (oligo 1, 2, 5 and 6) shown in the Table 2; and the VS_DsiRNA (oligo 1, 2, 5 and 6) shown in Table 4. A1&A2: No treatment as control, A3&A4: Overexpression of COVID-19 viral genes encoding both ORF1ab and RdRp, A5&A6: Overexpression of both ORF1ab and RdRp+/treated by VS_DsiRNA (oligo 1, 2, 5 and 6) with lipofectamine, B1&B2: Overexpression of both ORF1ab and RdRp+/treated by VS_DsiRNA (oligo 1, 2, 5 and 6) with Arginine (5 μl/well), B3&B4: Overexpression of both ORF1ab and RdRp+/treated by VS_ASO_2 (oligo 1, 2, 5 and 6) with lipofectamine, B5&B6: Overexpression of both ORF1ab and RdRp+/treated by VS_ASO_2 (oligo 1, 2, 5 and 6) with Arginine (5 μl/well), C1&C2: Overexpression of both ORF1ab and RdRp+/treated by VS_ASO_1-FANA (oligo 1, 2, 5 and 6) without any reagents.

FIG. 17. Detection of SARS-CoV-2 ORF1ab and RdRp expressed in the human primary lung small airway epithelial cells (HSAEC) by qRT-PCR after treatment with inhibitory oligonucleotides. Significant down-regulation was observed: about 3.5 fold in group treated by VS_DsiRNA oligo (purple/L: p<0.01) and about 2.2 fold in the presence of poly-Arginine only (yellow/A: p<0.001); about 4.5 fold in the group treated by VS_ASO_2 oligo (red/L: p<0.001) and about 2.1 fold in the in the presence of poly-Arginine only (orange/A: p<0.001); and about 11.8 fold in the group treated by the VS_ASO_1-FANA oligo (green/p<0.001); when compared with the group with SARS-CoV-2 ORF1ab and RdRp overexpression only. The cycle threshold of no-treatment is non-detectable, but in this case for calculation purposes, the number “40” was used as the cycle threshold for a base-line control.

FIG. 18. Experimental design for detection of SARS-CoV-2 N-protein expressed in the primary human lung small airway epithelial cells (HSAEC) by Western Blot after treatment. The human primary lung small airway epithelial cells (HSAEC) were cultured in the 6 well-dish, and the cells were transfected with the genes encoding the viral protein (N-protein) of SARS-CoV-2. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with Western Blot. The VS_ASO_2 (oligo 4 & 8) shown in the Table 2; and VS_DsiRNA (oligo 4 & 8) shown in Table 4. A1: No treatment as control, A2: Overexpression of COVID-19 N-protein, A3: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA (oligo 4 and 8) with lipofectamine, B1: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2 (oligo 4 and 8) with lipofectamine.

FIG. 19. Detection of SARS-CoV-2 N-protein expressed in the human primary lung small airway epithelial cells (HSAEC) by Western Blot after treatment with inhibitory oligonucleotides: Lane-1: no treatment; Lane-2: SARS-CoV-2 N-protein overexpression (OE); Lane-3: SARS-CoV-2 N-protein OE+/treated by VS_DsiRNA (oligo 4 & 8); Lane-4: SARS-CoV-2 N-protein OE+/treated by the VS_ASO_2 (oligo 4 & 8). The 10 ug total cell-lysis were added into each well. primary antibody: 1 μg/mL anti-SARS-CoV-2-N-protein antibody (ProSci, 3857) and anti-GAPDH antibody (Novus Biologicals, NBP2-27103) with 1:1000 dilution. The secondary antibody: goat-anti-rabbit HRP-conjugated Antibody (R&D System, HAF008) with 1:1000 dilution and goat-anti-mouse IgG HRP-conjugated Antibody (R&D System, HAF007) with 1:1000 dilution. The detection was done using horseradish peroxidase-labeled secondary antibodies and enhanced chemiluminescence detection reagent.

FIG. 20. Experiments designed for investigating cell penetration and therapeutic effects of VS-Nucleotides (inhibitory oligonucleotides) on human primary bronchial/tracheal epithelial cells (HBTEC) transfected with viral protein of SARS-CoV-2 after treatment. The primary human bronchial/tracheal epithelial cells (HBTEC) were cultured in a 24-well dish, and the cells were transfected with the genes encoding the viral proteins of SARS-CoV-2. The VS_ASO_1-FANA-FITC, VS_DsiRNA-Cy5 and VS_ASO_2-Cy3 were added into the cells for 24-48 hours before analysis with fluorescent microscope. VS_ASO_1-FANA-FITC designed with FITC labeled shown in the Table 1, and VS_ASO_2-Cy3 with Cy3 label shown in the Table 2; and VS_DsiRNA-Cy5 with Cy5 label shown in Table 3. A1&A2: No treatment as control, A3&A4: Overexpression of COVID-19 N-protein+/treated by VS_ASO_1-FANA-FITC without lipofectamine or/and Poly-arginine, B1&B2: No treatment as control, B3&B4: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA-Cy5 with lipofectamine, B5&B6: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNAi-Cy5 with arginine (5 μl/well), C1&C2: No treatment as control, C3&C4: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2-Cy3 with lipofectamine, C5&C6: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2-Cy3 with arginine (5 μl/well).

FIGS. 21A-21D. Microscopic analysis showing entry of VS_ASO_1-FANA-FITC into primary human lung bronchial/tracheal epithelial cells (20×). FIGS. 21A-21B were captured under FITC florescent filter, and FIGS. 21C and 21D were captured in the same view of bright fields (20×). FIGS. 21A and 21C were taken in well A1 & A2 (as shown in FIG. 20), and FIGS. 21B and 21D were taken in well A3 & A4 (as shown in FIG. 20).

FIGS. 22A-22D. Microscopic analysis showing entry of VS_ASO_1-FANA-FITC into primary human lung bronchial/tracheal epithelial cells (10×). FIGS. 22A-22B were captured under FITC florescent filter, and FIGS. 22C and 22D were captured in the same view of bright fields (10×). FIGS. 22A and 22C were taken in well A1 & A2 (as shown in FIG. 20), and FIGS. 22B and 22D were taken in well A3 & A4 (as shown in FIG. 20).

FIGS. 23A-23F. Microscopic analysis showing entry of VS_DsiRNA-Cy5 into primary human lung bronchial/tracheal epithermal cells (20×). FIGS. 23A-23C were captured under the Cy5 florescent filter, and FIGS. 23D-23F were captured in the same view of bright fields (20×). FIGS. 23A and 23D were taken in well B3 & B4 (as shown in FIG. 20), FIGS. 23B and 23E were taken in well B5 & B6 (as shown in FIG. 20), and FIGS. 23C and 23F were taken in well B1 & B2 (as shown in FIG. 20).

FIGS. 24A-24F. Microscopic analysis showing entry of VS_DsiRNA-Cy5 into primary human lung bronchial/tracheal epithermal cells (10×). FIGS. 24A-24C were captured under the Cy5 florescent filter, and FIGS. 24D-24F were captured in the same view of bright fields (20×). FIGS. 24A and 24D were taken in well B3 & B4 (as shown in FIG. 20), FIGS. 24B and 24E were taken in well B5 & B6 (as shown in FIG. 20), and FIGS. 24C and 24F were taken in well B1 & B2 (as shown in FIG. 20).

FIGS. 25A-25F. Microscopic analysis showing entry of VS_ASO_2-Cy3 into primary human lung bronchial/tracheal epithermal cells (20×). FIGS. 25A-25C were captured under the Cy3 florescent filter, and FIGS. 25D-25F were captured in the same view of bright fields (20×). FIGS. 25A and 25D were taken in well B3 & B4 (as shown in FIG. 20), FIGS. 25B and 25E were taken in well B5 & B6 (as shown in FIG. 20), and FIGS. 25C and 25F were taken in well B1 & B2 (as shown in FIG. 20).

FIGS. 26A-26F. Microscopic analysis showing entry of VS_ASO_2-Cy3 into primary human lung bronchial/tracheal epithermal cells (10×). FIGS. 26A-26C were captured under the Cy3 florescent filter, and FIGS. 26D-26F were captured in the same view of bright fields (20×). FIGS. 26A and 26D were taken in well B3 & B4 (as shown in FIG. 20), FIGS. 26B and 26E were taken in well B5 & B6 (as shown in FIG. 20), and FIGS. 26C and 26F were taken in well B1 & B2 (as shown in FIG. 20).

FIG. 27. Experimental design for detection of SARS-CoV-2 N-protein expressed on human primary bronchial/tracheal epithelial cells (HBTEC) by qRT-PCR. The human primary bronchial/tracheal epithelial cells (HBTEC) were cultured in a 24-well dish, and the cells were transfected with the genes encoding the viral protein (N-protein) of SARS-CoV-2. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with RT-PCR. The VS_ASO_1-FANA-FITC designed with FITC labeled shown in the Table 1, and VS_ASO_2-Cy3 with Cy3 modification shown in the Table 2; and VS_DsiRNA-Cy5 with Cy5 modification shown in Table 3. A1&A2: No treatment as control, A3&A4: Overexpression of COVID-19 N-protein, A5&A6: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA-Cy5 with lipofectamine, B1&B2: Overexpression of COVID-19 N-protein+/treated by VS_Dsi RNA-Cy3 with lipofectamine, B3&B4: Overexpression of COVID-19 N-protein+/treated by VS_ASO_1-FANA without any reagents.

FIG. 28. Detection of SARS-CoV-2 N-protein expressed in the human primary bronchial/tracheal epithelial cells (HBTEC) by qRT-PCR after treatment with inhibitory oligonucleotides. Significant down-regulation was observed: about 4 fold in the group treated by VS_DsiRNA-Cy5 oligos (p<0.005); about 6 fold in the group treated by VS_ASO_2-cy3 oligo (p<0.01), and about 8 fold in the group treated by the VS_ASO_1-FANA-FITC oligo (p<0.005); when compared with the group with SARS-CoV-2 N-protein overexpression only. The cycle threshold of no-treatment is non-detectable, but in this case for calculation purposes, the number “40” was used as the cycle threshold for a base-line control.

FIG. 29. Experimental design for detection of SARS-CoV-2 S-protein expressed in human primary bronchial/tracheal epithelial cells (HBTEC) by qRT-PCR. The human primary bronchial/tracheal epithelial cells (HBTEC) were cultured in a 24-well dish, and the cells were transfected with the genes encoding the viral protein (S-protein) of SARS-CoV-2. The siRNA or ASO were added into the cells for 24-48 hours before analysis with RT-PCR. The VS_ASO_1-FANA (oligo 3) designed is shown in the Table 1, VS_ASO_2 (oligo 3) is shown in the Table 2; and VS_DsiRNA (oligo 3) is shown in Table 3. A1&A2: No treatment, A3&A4: Overexpression of COVID-19 S-protein, A5&A6: Overexpression of COVID-19 S-protein+/treated by DsiRNA-Cy5 with lipofectamine, B1&B2: Overexpression of COVID-19 S-protein+/treated by VS_DsiRNA-Cy5 with Arginine (5 μl/well), B3&B4: Overexpression of COVID-19 S-protein+/treated by the VS_ASO_2-Cy3 with lipofectamine, B5&B6: Overexpression of COVID-19 S-protein+/treated by VS_ASO_2-Cy3 with Arginine (5 μl/well), C1&C2: Overexpression of COVID-19 S-protein+/treated by VS_ASO_1-FANA without any reagents.

FIG. 30. Detection of SARS-CoV-2 S-protein expressed in the human bronchial/tracheal epithelial cells (HBTEC) by qRT-PCR after treatment with inhibitory oligonucleotides. Significant down-regulation was observed: about 8 fold in the group treated by VS-DsiRNA oligo (purple/L: p<0.01), but about 16.3 fold in presence of Poly-arginine only (yellow/p<0.001); about 15.8 fold in the group treated by VS_ASO_2 oligo (red/L: p<0.001), but about 16.6 fold in presence of Poly-arginine only (orange/A: p<0.001); about 11.7 fold in the group treated by the VS_ASO_1 oligo (green/p<0.001); when compared with the group with SARS-CoV-2 S-protein overexpression only (1). The cycle threshold of no-treatment is non-detectable, but in this case for calculation purposes, the number “40” was used as the cycle threshold for a base-line control.

FIG. 31. Experimental design for detection of SARS-CoV-2 ORF1ab and RdRp expressed in the human primary bronchial/tracheal epithelial cells (HBTEC) detected by qRT-PCR after treatment. The human primary bronchial/tracheal epithelial cells (HBTEC) were cultured in the 24 well-dish, and the cells were transfected with the genes encoding the viral protein (ORF1ab and RdRp) of SARS-CoV-2. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with RT-PCR. VS_ASO_1-FANA (oligo 1, 2, 5 and 6) designed is shown in the Table 1, VS_ASO_2 (oligo 1, 2, 5 and 6) is shown in the Table 2; and the VS_DsiRNA (oligo 1, 2, 5 and 6) is shown in Table 4. A1&A2: No treatment as control, A3&A4: Overexpression of COVID-19 viral genes encoding both ORF1ab and RdRp, A5&A6: Overexpression of COVID-19 ORF1ab and RdRp+/treated by VS_DsiRNA (oligo 1, 2, 5 and 6) with lipofectamine, B1&B2: Overexpression of COVID-19 ORF1ab and RdRp+/treated by VS_DsiRNA (oligo 1, 2, 5 and 6) with Arginine (51/well), B3&B4: Overexpression of COVID-19 ORF1ab and RdRp+/treated by VS_ASO_2 (oligo 1, 2, 5 and 6) with lipofectamine, B5&B6: Overexpression of COVID-19 ORF1ab and RdRp+/treated by VS_ASO_2 (oligo 1, 2, 5 and 6) with Arginine (5p l/well), C1&C2: Overexpression of COVID-19 ORF1ab and RdRp+/treated by VS_ASO_1-FANA (oligo 1, 2, 5 and 6) without any reagents.

FIG. 32. Detection of SARS-CoV-2 ORF1ab and RdRp expressed in the human primary bronchial/tracheal epithelial cells (HBTEC) detected by qRT-PCR after treatment with inhibitory oligonucleotides. Significant down-regulation was observed: about 3 fold in the group treated by VS_DsiRNA oligo (purple/L: p<0.01) and about 2.6 fold in the presence of poly-Arginine only (yellow/A: p<0.001); about 6.4 fold in the group treated by VS_ASO_2 oligo (red/L: p<0.001) and about 4.5 fold in the presence of poly-Arginine only (orange/A: p<0.001); and about 11.9 fold in the group treated by the VS_ASO_1-FANA oligo (green/p<0.001); when compared with the group with SARS-CoV-2 ORF1ab and RdRp overexpression only. The cycle threshold of no-treatment is non-detectable, but in this case for calculation purposes, the number “40” was used as the cycle threshold for a base-line control.

FIG. 33. Experimental design for detection of SARS-CoV-2 N-protein expressed in the human primary bronchial/tracheal epithelial cells (HBTEC) by Western Blot after treatment. The human primary bronchial/tracheal epithelial cells (HBTEC) were cultured in a 6-well dish, and the cells were transfected with the genes encoding the viral protein (N-protein) of SARS-CoV-2. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with Western Blot. The VS_ASO_2 (oligo 4 & 8) is shown in the Table 2; and VS_DsiRNA (oligo 4 & 8) is shown in Table 4. A1: No treatment as control, A2: Overexpression of COVID-19 N-protein, A3: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA (oligo 4 and 8) with lipofectamine, B1: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2 (oligo 4 and 8) with lipofectamine.

FIG. 34. Detection of SARS-CoV-2 N-protein expressed in the human primary bronchial/tracheal epithelial cells (HBTEC) by Western Blot after treatment with inhibitory oligonucleotides. Lane 1: no treatment, Lane 2: SARS-CoV-2 N-protein overexpression (OE); Lane 3: SARS-CoV-2 N-protein OE+VS_DsiRNA (oligo 4 & 8); Lane 4: SARS-CoV-2 N-protein OE+VS_ASO_2 (oligo 4 & 8). 10 μg total cell lysis were added into each well and blotted with primary antibody (1 μg/mL anti-SARS-CoV-2-N-protein antibody (ProSci, 3857)) or anti-GAPDH antibody (Novus Biologicals, NBP2-27103) (1:1000 dilution). The secondary antibodies were goat-anti-rabbit HRP-conjugated Antibody (R&D System, HAF008) (1:1000) dilution and goat-anti-mouse IgG HRP-conjugated Antibody (R&D System, HAF007) (1:1000) dilution. The detection was done using horseradish peroxidase-labeled secondary antibodies and an enhanced chemiluminescence detection reagent.

FIG. 35. Experiments designed for investigating cell penetration and therapeutic effects of ASO(s) and DsiRNA on human primary nasal epithelial cells (HNEpC) transfected with viral protein of SARS-CoV-2 after treatment. The human primary nasal epithelial cells (HNEpC) were cultured in a 24-well dish, and the cells were transfected with the genes encoding the viral proteins of SARS-CoV-2. The VS_ASO_1-FANA-FITC, VS_DsiRNA-Cy5 and VS_ASO_2-Cy3 were delivered into the cells for 24-48 hours before analysis with fluorescent microscope. The VS_ASO_1-FANA-FITC was FITC labeled (see Table 1), and VS_ASO_2-Cy3 was Cy3 labeled (See Table 2); and VS_DsiRNA-Cy5 was Cy5 labeled (see Table 3). A1&A2: No treatment as control, A3&A4: Overexpression of COVID-19 N-protein+/treated by VS_ASO_1-FANA without lipofectamine or arginine, B1&B2: No treatment as control, B3&B4: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA-Cy5 with lipofectamine, B5&B6: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA-Cy5 with poly-arginine (5 μl/well), C1&C2: No treatment as control, C3&C4: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2-Cy3 with lipofectamine, C5&C6: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2-Cy3 with poly-arginine (5 μl/well).

FIGS. 36A-36D. Microscopic analysis of Human Primary Nasal Epithelial Cells at 20×. This analysis showed that VS_ASO_1-FANA-FITC can enter epithelial cells (20×). FIGS. 36A and 36B were captured under FITC florescent filter, and FIGS. 36C and 36D were captured in the same view of bright fields (20×). FIGS. 36A and 36C were taken in well A3 & A4 (as shown in FIG. 35), FIGS. 36B and 36D were taken in well A1 & A2 (as shown in FIG. 35).

FIGS. 37A-37D. Microscopic analysis of Human Primary Nasal Epithelial Cells at 10×. This analysis showed that VS_ASO_1-FANA-FITC can enter epithelial cells (10×). FIGS. 37A and 37B were captured under FITC florescent filter, and FIGS. 37C and 37D were captured in the same view of bright fields (20×). FIGS. 37A and 37C were taken in well A3 & A4 (as shown in FIG. 35), FIGS. 37B and 37D were taken in well A1 & A2 (as shown in FIG. 35).

FIGS. 38A-38F. Microscopic analysis of Human Primary Nasal Epithelial Cells at 20×. This analysis showed that VS_DsiRNA-Cy5 can enter epithelial cells (20×). FIGS. 38A-38C were captured under the Cy5 florescent filter, and FIGS. 38D-38F were captured in the same view of bright fields (20×). FIGS. 38A and 38D were taken in well B3 & B4 (as shown in FIG. 35), FIGS. 38B and 38E were taken in well B5 & B6 (as shown in FIG. 35), FIGS. 38C and 38F were taken in well B1 & B2 (as shown in FIG. 35).

FIGS. 39A-39F. Microscopic analysis of Human Primary Nasal Epithelial Cells at 10×. This analysis showed that VS_DsiRNA-Cy5 can enter epithelial cells (10×). FIGS. 39A-39C were captured under the Cy5 florescent filter, and FIGS. 39D-39F were captured in the same view of bright fields (20×). FIGS. 39A and 39D were taken in well B3 & B4 (as shown in FIG. 35), FIGS. 39B and 39E were taken in well B5 & B6 (as shown in FIG. 35), FIGS. 39C and 39F were taken in well B1 & B2 (as shown in FIG. 35).

FIGS. 40A-40F. Microscopic analysis of Human Primary Nasal Epithelial Cells at 20×. This analysis showed that VS_ASO_2-Cy3 can enter epithelial cells (20×). FIGS. 40A-40C were captured under the Cy3 florescent filter, and FIGS. 40D-40F were captured in the same view of bright fields (20×). FIGS. 40A and 40D were taken in well B3 & B4 (as shown in FIG. 35), FIGS. 40B and 40E were taken in well B5 & B6 (as shown in FIG. 35), FIGS. 40C and 40F were taken in well B1 & B2 (as shown in FIG. 35).

FIGS. 41A-41F. Microscopic analysis of Human Primary Nasal Epithelial Cells at 10×. This analysis showed that VS_ASO_2-Cy3 can enter epithelial cells (10×). FIGS. 41A-41C were captured under the Cy3 florescent filter, and FIGS. 41D-41F were captured in the same view of bright fields (10×). FIGS. 41A and 41D were taken in well B3 & B4 (as hown in FIG. 35), FIGS. 41B and 41E were taken in well B5 & B6 (as shown in FIG. 35), FIGS. 41C and 41F were taken in well B1 & B2 (as shown in FIG. 35).

FIG. 42. Experimental design for detection of SARS-CoV-2 N-protein expressed on human primary nasal epithelial cells (HNEpC) by qRT-PCR. The human primary nasal epithelial cells (HNEpC) were cultured in a 24-well dish, and the cells were transfected with the genes encoding the viral protein (N-protein) of SARS-CoV-2. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with RT-PCR. VS_ASO_1-FANA-FITC was labeled with FITC as shown in the Table 1, and VS_ASO_2-Cy3 was labeled with Cy3 as shown in Table 2; and VS_DsiRNA-Cy5 was labeled with Cy5 as shown in Table 3. A1&A2: No treatment, A3&A4: Overexpression of COVID-19 N-protein, A5&A6: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA-Cy5 with lipofectamine, B1&B2: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA-Cy5 with Arginine (5 μl/well), B3&B4: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2-Cy3 with lipofectamine, B5&B6: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2-Cy3 with Arginine (5 μl/well), C1&C2: Overexpression of COVID-19 N-protein+/treated by VS_ASO_1-FANA-FITC without any reagents.

FIG. 43. Detection of SARS-CoV-2 N-protein expressed in the human primary nasal epithelial cells (HNEpC) by qRT-PCR after treatment with siRNA or ASO. Significant down-regulation was observed: about 90 fold in the group treated by VS_DsiRNA-Cy5 oligo (2: p<0.01); about 15 fold in the group treated by VS_ASO_2-Cy3 oligo (p<0.01), and about 350 fold of down-regulation in the group treated by the VS_ASO_1-FANA-FITC oligo (3: p<0.001); when compared with the group with COIVD-19 N-protein overexpression only. The cycle threshold of no-treatment is non-detectable, but in this case for calculation purposes, the number “40” was used as the cycle threshold for a base-line control.

FIG. 44. Experimental design for inhibiting viral infections using inhibitory nucleotides. WV=Wild-type of pseud-COVID-19 virus, 5 μl (titer: 10⁵TU/ml) of the virus added into each well (C1 to C9). MV=Mutant form of pseud-COVID-19 virus, 5 μl (titer: 105 TU/ml) of the virus added into each well (D1 to D9). N 1=VS_ASO_3 oligo (targeting on S-protein of COVID-19), N 2=VS_siRNA/RNAi_3 oligo (targeting on S-protein of COVID-19, Control-1=Scramble nucleotide oligo (SN) only.

FIGS. 45A-45F. Experimental data of inhibitions of wild-type viral infections by inhibitory nucleotides. FIG. 45A Brightfield image of VS_ASO_3-treated cells. FIG. 45B Brightfield image of VS_RNAi_3-treated cells. FIG. 45C Brightfield image of scramble-treated cells. FIG. 45D Fluorescence image of VS_ASO_3-treated cells. FIG. 45E Fluorescence image of VS_RNAi_3-treated cells. FIG. 45F Fluorescence image of scramble-treated cells. There were no significant GFP expressions found in those cells treated by VS_ASO_3 and VS_RNAi_3 oligos, detected under the confocal microscope; but the inventors were able to see the GFP expressions in the control group of the cells treated with the scramble nucleotide only. This data thus indicated that the VS_ASO_3 and VS_RNAi_3 have inhibited the wild-type of the viruses coupled with eGFP (WV) inside the cells; but not in the group treated with the scramble nucleotide.

FIGS. 46A-46F. Experimental data of inhibitions of mutant viral infections by inhibitory nucleotides. FIG. 46A Brightfield image of VS_ASO_3-treated cells. FIG. 46B Brightfield image of VS_RNAi_3-treated cells. FIG. 46C Brightfield image of scramble-treated cells. FIG. 46D Fluorescence image of VS_ASO_3-treated cells. FIG. 46E Fluorescence image of VS_RNAi_3-treated cells. FIG. 46F Fluorescence image of scramble-treated cells. There was no significant GFP expressions found in those cells treated by VS_ASO_3 and VS_RNAi_3, detected under the confocal microscope; but the inventors were able to see the GFP expressions in the control group of the cells treated with the scramble nucleotide only. This data thus indicated that the VS_ASO_3 and VS_RNAi_3 have also inhibited the mutant viruses coupled with eGFP (MV) inside the cells; but not in the group treated with the scramble nucleotide.

FIG. 47. Analysis of the amino acid sequence of SARS-CoV-2 Spike protein (S-protein) (GenBank ID: QHD43416.1, SEQ ID NO: 61). The region of the sequence highlighted in red represents the predicted sequences of ACE2 binding sequences/motifs (aka. the Ligand binding Domain).

FIGS. 48A-48B. Analysis of the amino acid sequence of the BD motifs. FIG. 48A 3D interaction between the SARS-CoV-2 Spike protein and human ACE2. FIG. 48B Analysis of the amino acids of the RBD motifs in 3D structure between the SARS-CoV-2 Spike protein (B: K417 to Y505) and human ACE2 (B: Q24 to R393) was used to order to locate which regions of the sequences contribute to the protein-protein interaction, and to design peptides that mimic the RBD sequences (mimics act like a human ACE 2 and prevent or block the binding activities for the SARS-CoV-2 on the real ACE2 in the cells).

FIG. 49. Experimental design. A1&A2: No treatment as control, A3&A4: peptide 5-FITC, A5&A6: peptide 5-FITC+/treated by the peptide 1 (low dosage), B1&B2: peptide 5-FITC+/treated by the peptide 1 (high dosage), B3&B4: peptide 5-FITC+/treated by the peptide 2 (low dosage), B5&B6: peptide 5-FITC+/treated by the peptide 2 (high dosage), 1&C2: peptide 5-FITC+/treated by the peptide 3 (low dosage), C3&C4: peptide 5-FITC+/treated by the peptide 3 (high dosage), C5&C6: peptide 5-FITC+/treated by the peptide 4 (low dosage), D1&D2: peptide 5-FITC+/treated by the peptide 4 (high dosage), D3&D4: peptide 5-FITC+/treated by the peptide 1 (high dosage)+peptide 2 (high dosage)+peptide 3 (high dosage)+peptide 4 (high dosage), The dosage-1=1 μg per 10⁵cells; and the dosage-2=10 μg per 10⁵cells.

FIGS. 50A-50H. Cells infected with wild-type SARS-COV-2 virus (WV) in the presence of inhibitory peptides. FIG. 50A Brightfield image of cells treated with Peptide 1 (P1). FIG. 50B Brightfield image of cells treated with Peptide 2 (P2). FIG. 50C Brightfield image of cells treated with Peptide 3 (P3). FIG. 50D Brightfield image of cells treated with normal human serum (NHS). FIG. 50E Fluorescence (GFP) image cells treated with Peptide 1 (P1). FIG. 50F Fluorescence (GFP) image of cells treated with Peptide 2 (P2). FIG. 50G Fluorescence (GFP) image of cells treated with Peptide 3 (P3). FIG. 50H Fluorescence (GFP) image of cells treated with normal human serum (NHS).

FIGS. 51A-51H. Cells infected with mutant SARS-COV-2 virus (MV) in the presence of inhibitory peptides. FIG. 51A Brightfield image of cells treated with Peptide 1 (P1). FIG. 51B Brightfield image of cells treated with Peptide 2 (P2). FIG. 51C Brightfield image of cells treated with Peptide 3 (P3). FIG. 51D Brightfield image of cells treated with normal human serum (NHS). FIG. 51E Fluorescence (GFP) image cells treated with Peptide 1 (P1). FIG. 51F Fluorescence (GFP) image of cells treated with Peptide 2 (P2). FIG. 51G Fluorescence (GFP) image of cells treated with Peptide 3 (P3). FIG. 51H Fluorescence (GFP) image of cells treated with normal human serum (NHS).

FIG. 52A-52H. Microscope analysis of human primary small airway epithelial cells treated with inhibitory peptides (VS-peptides). FIGS. 52A, and 52E were captured under the FITC florescent filter, FIGS. 52B and 52E were captured in brightfield (20×). FIG. 52C shows the merge of FIGS. 52A and 52B. FIG. 52G is a merge photo of FIGS. 52E and 52F. The white dots indicate the box that was enlarged as shown in FIG. 52D. The yellow dots indicate the box that was enlarged as shown in FIG. 52H. White arrows suggested peptide 5-FITC internalized into cells cytoplasm and nucleus, while the yellow arrows suggested the VS-peptides combination can block the peptide 5-FITC from internalization, staying outside of cells.

FIG. 53. Gene therapy vector AAV-U6-A1-H1-A2-SV40-eGFP. This AAV vector expresses two transgenes (namely ASO1 (A1) and ASO2 (A2)) simultaneously in one cell. U6=The 1st promoter that controls the expression of A1 gene in the mammalian cells, H1=The 2nd promoter that controls the expression of A2 gene in the mammalian cells, SV=The 3rd promoter that controls the expression of GFP gene in the mammalian cells. Full sequence of AAV-U6-A1-H1-A2-SV40-GFP is shown by SEQ ID NO: 46.

FIG. 54. Gene therapy vector AAV-U6-A3-H1-A4-SV40-eGFP. This AAV vector expresses two transgenes (namely ASO3 (A3) and ASO4 (A4)) simultaneously in one cell. U6=The 1st promoter that controls the expression of A3 gene in the mammalian cells, H1=The 2nd promoter that controls the expression of A4 gene in the mammalian cells SV40=The 3rd promoter that controls the expression of GFP gene in the mammalian cells. Full sequence of AAV-U6-A1-H1-A2-SV40-eGFP is shown by SEQ ID NO: 47.

FIG. 55. Gene therapy vector AAV-U6-shRNA1-CMV-eGFP. This AAV vector expresses the transgene shRNA1. U6=The 1st promoter that controls the expression of shRNA1 gene in the mammalian cells, CMV=the 2nd promoter that controls the expression of eGFP gene in the mammalian cells. The full DNA sequence is shown by SEQ ID NO: 48 of AAV-U6-shRNA1-eGFP.

FIG. 56. Gene therapy vector AAV-U6-shRNA2-CMV-eGFP. This AAV vector expresses the transgene shRNA1 in one cell: U6=The 1st promoter that controls the expression of shRNA2 gene in the mammalian cells, CMV=the 2^ndpromoter that controls the expression of eGFP gene in the mammalian cells. The sequence of AAV-U6-shRNA2-eGFP is shown by SEQ ID NO: 49.

FIG. 57. Gene therapy vector AAV-U6-shRNA3-CMV-eGFP. A. This AAV vector expresses the transgene shRNA1. U6=The 1st promoter that controls the expression of shRNA3 gene in the mammalian cells, CMV=The 2nd promoter that controls the expression of eGFP gene in the mammalian cells. The full DNA sequence of AAV-U6-shRNA3-eGFP is shown by SEQ ID NO: 50.

FIG. 58. Gene therapy vector AAV-U6-shRNA4-CMV-eGFP. A. This AAV vector expresses the transgene shRNA1. U6=The 1st promoter that controls the expression of shRNA4 gene in the mammalian cells, CMV=The 2nd promoter that controls the expression of eGFP gene in the mammalian cells. The full DNA sequence of AAV-U6-shRNA4-eGFP is shown by SEQ ID NO: 51.

FIGS. 59A-59B. These experiments were designed as plates A and B as follows: FIG. 59A Plate A: #1 vector: AAV-U6-shRNA1-GFP (ORF1ab), #2 vector: AAV-U6-shRNA2-GFP (RdRp), #3 vector: AAV-U6-shRNA3-GFP (S), #4 vector: AAV-U6-shRNA4-GFP (N), #5 vector: AAV-U6-ASO1-H1-ASO2-GFP (ORF1ab&RdRp), #6 vector: AAV-U6-ASO3-H1-ASO4-GFP (S & N). FIG. 59B Plate B: No treatment: Mock transfections, and Positive control: Cells transfected with COVID19 plasmids, but no gene therapy vectors.

FIGS. 60A-60D. Data analysis by qRT-PCR. FIG. 60A Nasal-epithelial cells transfected with designed COVID-19 plasmid encoding N protein. FIG. 60B Nasal-epithelial cells transfected with designed COVID-19 plasmid encoding ORF1ab. FIG. 60C Nasal-epithelial cells transfected with designed COVID-19 plasmid encoding S protein. FIG. 60D Nasal-epithelial cells transfected with designed COVID-19 plasmid encoding RdRp. The cells were transfected with designed vectors with COVID-19 plasmids (S, N, RdRp and ORF1ab), and also the gene therapy vectors (#1 to #6) after 24 hours post-seeding. At the 48 hours post-transfection, the cells were then harvested, and their total RNA were extracted with including the DNase digestion before PCR assays. S proteins were conducted with TaqMan-probe assay kit (Thermofisher, A47532). The N protein, ORF1ab and RdRp proteins were determined by the GenScript kits (SARS-CoV-2PCR detection assay kit). The data indicated that the treatments by using the gene vectors have significant therapeutic effects to inhibit the expressions of the viral proteins. Synergic enhanced effects were observed when more than one peptide was used (see, #1 to 4 and #5 to 6).

FIGS. 61A-61B. FIG. 61A Western blot analysis of S-protein expression. FIG. 61B Quantification of (A). The cells were seed onto 6 well-plates. At 24 hours post-seeding, cells were transfected by the gene therapy vectors (#1 to #6) with including the plasmids encoding the COVID-19 S-proteins. After 48 hours post-transfection, cells were harvested and lysed. The primary antibody is SARS-CoV-2 Spike with 1 μg/mL (ProSc, Inc), and the secondary antibody is Goat-anti-Rabbit HRP conjugated antibody by 1:1000 dilution (R&D System). The data indicated that the treatments by using the gene vectors have significant therapeutic effects to block the expression of viral proteins. Synergic enhanced effects were observed when more than one peptide was used (see, #1 to 4 and #5 to 6).

FIG. 62. Experimental design of in vitro gene therapy on inhibitions of the viral infections. WV=Wild-type of pseud-COVID-19 virus, 5 ul (titer: 10⁵TU/ml) of the virus added into each well (E1 to E9). MV=Mutant form of pseud-COVID-19 virus, 5 ul (titer: 10⁵TU/ml) of the virus added into each well (F1 to F9). V1=AAV-U6-A3_H1-A4-GFP, V2=AAV-U6-shRNA3-GFP, Control-1=irrelevant vector plasmid, Control-2=AAV-U6-A3_H1-A4-GFP (without virus added), Control-3=AAV-U6-shRNA3-GFP (without virus added).

FIGS. 63A-63F. In vitro gene therapy inhibits WV viral infections. ASO (V1) and RNAi (V2) were delivered by gene vectors into mammalian cells that express ACE2 proteins, in order to inhibit the WV viral infections (WV=wild-type pseudo-virus of COVID-19). FIG. 63A Brightfield image of cells treated with ASO (V1). FIG. 63B Brightfield image of cells treated with RNAi (V2). FIG. 63C Brightfield image of cells treated with control. FIG. 63D Fluorescence image of cells treated with ASO (V1). FIG. 63E Fluorescence image of cells treated with RNAi (V2). FIG. 63F Fluorescence image of cells treated with control.

FIGS. 64A-64F. In vitro gene therapy inhibits MV viral infections. ASO (V1) and RNAi (V2) were delivered by gene vectors into mammalian cells that express ACE2 proteins, in order to inhibit the MV viral infections (MV=mutant pseudo-virus of COVID-19). FIG. 64A Brightfield image of cells treated with ASO (V1). FIG. 64B Brightfield image of cells treated with RNAi (V2). FIG. 64C Brightfield image of cells treated with control. FIG. 64D Fluorescence image of cells treated with ASO (V1). FIG. 64E Fluorescence image of cells treated with RNAi (V2). FIG. 64F Fluorescence image of cells treated with control.

FIGS. 65A-65D. In vitro delivery of gene vectors into living cells. ASO (V1) and RNAi (V2) were delivered by using the gene vectors into mammalian cells that express ACE2 proteins, the cells were not incubated with any viruses, which were served as background controls. The concentrations of the vectors encoding the ASO (control-2) or/and RNAi (control-3) were the same used in the FIGS. 61A-61B and 62. FIG. 65A Brightfield image of cells treated with ASO control. FIG. 65B Brightfield image of cells treated with RNAi control. FIG. 65C Fluorescence image of cells treated with ASO control. FIG. 65D Fluorescence image of cells treated with RNAi control.

FIGS. 66A-66B. FIG. 66A Wild-type pseudo-virus experiment results and FIG. 66B mutant pseudo-virus experiment results. Since both gene vectors, encoding ASO and shRNA, also contain marker gene of GFP, normalized data was calculated based on control-2 and control-3 constructs (also see FIG. 62). The data analysis confirmed that ASO or shRNA vector expressing cells showed very little GFP signal, when compared with the control group-1. This data indicates that the gene vectors carrying either ASO or shRNA (inhibitory oligonucleotides) suppress viral infection and propagation in both wild-type and mutant viruses of COVID-19, pseudo-typed by lentiviruses.

FIG. 67. Experimental design for detection of apoptosis/cytotoxicity of VS-nutrition in human bronchial/tracheal epithelial cells (HBTEC) by qRT-PCR. The human primary bronchial/tracheal epithelial cells (HBTEC) were cultured in the 24 well-dish, and the cells were treated with VS-nutrition with designated dilution (1:1, 1:300 and 1:500) for 5 days (in every day, refresh cell culture medium and added new VS-nutrition with same composition and ratio) before analysis by qRT-PCR.

FIG. 68. Detection of on apoptosis/cytotoxicity of VS-nutrition in the human bronchial/tracheal epithelial cells (HBTEC) by qRT-PCR after treatment. Detection of apoptosis/cytotoxicity of VS-nutrition in the human primary bronchial/tracheal epithelial cells (HBTEC) by qRT-PCR after treatment with VS-nutrition. There are no significant up or down-regulation of BAX/BCL2 ratio in group treated by VS-nutrition when compared with the normal cells with no-treatment (p>0.05).

FIG. 69. Experimental design for detection of apoptosis/cytotoxicity of VS-nutrition in Human Primary Nasal Epithelial Cells (HNEpC) by qRT-PCR. The human primary nasal epithelial cells (HNEpC) were cultured in the 24 well-dish, and the cells were treated with VS-nutrition with designated dilution (1:1, 1:300 and 1:500) for 5 days (in every day, refresh cell culture medium and added new VS-nutrition with same composition and ratio) before analysis by qRT-PCR.

FIG. 70. Detection of on apoptosis/cytotoxicity of VS-nutrition in the human primary nasal epithelial cells (HNEpC) by qRT-PCR after treatment. Detection of apoptosis/cytotoxicity of VS-nutrition in the human primary nasal epithelial cells by RT-PCR after treatment with VS-nutrition. There are no significant up or down-regulation of BAX/BCL2 ratio in group treated by VS-nutrition when compared with the normal cells with no-treatment (p>0.05).

FIGS. 71A-71B. Oral intake formulations of VS product (nutritional supplement). Bottle product (10-15 ml) with FIG. 71A 1.5 ml spoon or FIG. 71B 1.0 ml drop.

FIGS. 72A-72C. Nasal (liquid) spray. Spray product with 10-15 ml bottle nasal spray. FIG. 72A composition and size of the product. FIGS. 72B & 72C usage example.

FIGS. 73A-73B. FIG. 73A The alignment of all ASO (ASO_1 and ASO_2) and all oligos in Tables 1, 2 and 4 showed that the designed inhibitory oligonucleotides specifically target the SARS-COV-2 virus genes. The alignment did not show any significant match to any human genes (thereby, avoiding potential side-effects when applied in human). FIG. 73B: The analysis of all DsiRNA indicated all oligos (in Tables 1, 2 and 4) specifically target the SARS-COV-2 virus genes. The alignment did not show any significant match to any human genes (thereby, avoiding potential side-effects when applied in human)

FIGS. 74A-74C. Peptide ELISA assays. FIG. 74A Schematic of ELISA assays. FIG. 74B Analysis of inhibitions of COVID-19 Spike Protein Receptor Binding Domain (S-RBD)-ACE2 binding by inhibitory peptides. FIG. 74C Table of p-values of the results in FIG. 74A and the number of amino acids participating in S-RBD/ACE2 interaction. These results indicated that the peptides could compete with ACE2 proteins and prevent S-RBD binding to ACE2. When the designed inhibitory peptides contained more amino acids interacting with S-RBD, stronger affinities were measured.

FIGS. 75A-75B. FIG. 75A Inhibition rate of S-RBD binding to ACE2 using VS peptides. FIG. 75B S-RBD signal rate. The data were converted and calculated as inhibition/suppression rates of the peptides based on the intensities of the S-RBD signals after treatment with the peptides compared with the control groups FIG. 75B. All peptides have shown their dosage-responses in the ELISA reactions, and indicated their strong biological affinities to bind with the S-RBD.

FIGS. 76A-76J. VS-peptides (VS-peptides 1, 2, 3, 4 and 5) block COVID-19 S-RBD-FITC from entering living mammalian cells expressing ACE2 receptors, as determined by FACS analysis. FIG. 76A Negative control (no peptide or FITC). FIG. 76B Positive control (FITC added, no peptide). FIG. 76C Human normal serum control (FITC added, no peptide). FIG. 76D VS-Peptide 1. FIG. 76E VS-Peptide 1. FIG. 76F VS-Peptide 1. FIG. 76G VS-Peptide 1. FIG. 76H VS-Peptide 1. FIG. 76I VS-Peptide 1. FIG. 76J Schematic of the experiment. Without a VS peptide, S-RBD (which is conjugated to FITC) binds to ACE2 and results the HeLa-ACE2 cells giving a FITC signal. When incubated with a VS peptide, S-RBD (conjugated to FITC) binds to VSB peptide instead of HeLa-ACE2 cells. In this case, HeLa-ACE2 cells have no FITC signal.

FIG. 77. ELISA results of S-RBD inhibition by inhibitory peptides derived from VS-Peptides 1-5 (P1 to P20—see Table 6 for sequences shown by SEQ ID NOS: 63-82). The competitive ELISA was performed as depicted in FIG. 74A. PL: VS-Peptide 1-a, P2: VS-Peptide 1-b, P3: VS-Peptide 1-c, P4: VS-Peptide 1-d, P5: VS-Peptide 2-a, P6: VS-Peptide 2-b, P7: VS-Peptide 2-c, P8: VS-Peptide 2-d, P9: VS-Peptide 3-a, P10: VS-Peptide 3-b, P11: VS-Peptide 3-c, P12: VS-Peptide 3-d, P13: VS-Peptide 4-a, P14: VS-Peptide 4-b, P15: VS-Peptide 4-c, P16: VS-Peptide 4-d, P17: VS-Peptide 5-a, P18: VS-Peptide 5-b, P19: VS-Peptide 5-c, P20: VS-Peptide 5-d. The data indicated that the derivative inhibitory peptides are capable of targeting the S-RBD of SARS-CoV-2 significantly to prevent viral binding on the human ACE2 receptors (p<0.05).

DETAILED DESCRIPTION

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

This disclosure is directed to compositions and methods for treating Coronavirus disease 2019 (COVID-19).

The term “COVID-19 treatment” (or “treating COVID-19”), refers to reduction, alleviation, or elimination of one or more of the COVID-19 disease symptoms, or prevention or inhibition of the onset of one or more COVID-19 disease or disease symptoms. Documented symptoms of include, for example, fever, dry cough, tiredness, aches and pains, sore throat, diarrhea, conjunctivitis, headache, loss of taste, loss of smell, a rash on skin, discoloration of fingers or toes, difficulty breathing, shortness of breath, chest pain or pressure, loss of speech, and loss of movement.

The compositions and methodologies described herein are effective to treat COVID-19 caused by infection of SARS-CoV2, including the originally or earlier isolated viral strains of SARS-CoV2, as well as variants of the original or earlier SARS-CoV2 viral isolates. In some embodiments, a SARS-CoV2 variant has a mutation in the spike protein. In some embodiments, the mutations comprise at least one amino acid deletion or substitution. In a specific embodiment, the SARS-CoV2 variant is the viral isolate known as B.1.1.7. In a specific embodiment, the B.1.1.7 has deletions of H69, V70 and Y144 of the spike protein as shown by SEQ ID NO: 61, and also has the following amino acid substitutions N501Y, A570D, D614G, P681H, T7161, S982A, and D118H of the spike protein as shown by SEQ ID NO: 61. All known variants of SARS-CoV2 cause similar and overlapping disease symptoms, as described above.

In one aspect, the compositions and the methods disclosed herein are directed towards targeting a plurality of selected target genes in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) genome by employing a plurality of inhibitory oligonucleotides. In some embodiments, a selected target gene is targeted with a plurality of inhibitory oligonucleotides. The inhibitory oligonucleotides can be used directly in a composition formulated as a dietary supplement or a pharmaceutical composition (e.g., in the form of nanoparticles or liposomes) for administration to a subject; or alternatively, can be placed in one or more nucleic acid vectors which are administered to a subject.

This approach developed by the inventors has several advantages. RNA viruses have a tendency to mutate and a recent study suggests that mutations could make coronavirus more infectious. The genes selected to be targeted herein are essential to the function of the SARS-CoV-2 virus. Therefore, by providing a plurality of inhibitory oligonucleotides targeting multiple genes, inhibition of the viral function is accomplished even if one of the target genes has mutated. Further, the inhibitory oligonucleotides are small in size, which permits effective cell penetration including penetration of infected cells, an advantage not provided by vaccines or antibodies against SARS-CoV-2 proteins which are not effective once the virus has entered into host cells.

In another aspect, the compositions and the methods disclosed herein are directed towards inhibiting the interactions between the SARS-CoV-2 virus and the Angiotensin-converting enzyme 2 (ACE2) receptor by one or more inhibitory peptides that mimic either the binding surface of ACE2 ligand binding domain (LBD) or the binding surface of the SARS-CoV-2 S-protein receptor binding domain (RBD). The inhibitory peptides can be included in a composition formulated as a dietary supplement or a pharmaceutical composition (e.g., in the form of nanoparticles or liposomes) for administration to a subject.

In a further aspect, the compositions and the methods disclosed herein are based on a combination of a plurality of inhibitory oligonucleotides and inhibitory peptides.

In some embodiments, the gene vectors described herein, encoding the inhibitory oligonucleotides and peptides described herein, can target viral infective functional group genes that have been integrated into the host cell (e.g., a human cell) genome (see, Zhang, L., et al., PNAS, 118.21 (2021), incorporated herein in its entirety).

SARS-CoV-2 Genes

In some embodiments, the entire SARS-CoV-2 cDNA sequence is shown under GenBank Accession No: NC_045512.2 (SEQ ID NO: 52). The individual SARS-CoV-2 genes are as follows:

ORF1ab: The open reading frame for starting transcriptional genes of SARS-CoV-2, 1ab, and is between nucleotide numbers 266 and 21555 of SEQ ID NO: 52;

N-protein gene: encodes the Nucleocapsid Protein of SARS-CoV-2 (which is a structural protein that binds to the coronavirus RNA genome, thus creating a shell), and is between nucleotide numbers 28274 and 29533 of SEQ ID NO: 52;

S-protein gene: encodes the spike protein of SARS-CoV-2 (which binds to the host cell receptors, i.e., ACE2 to enter the host cells, and is between nucleotide numbers 21563 and 25384 of SEQ ID NO: 52;

E-protein gene: encodes envelope protein of SARS-CoV-2 (which is a small membrane protein that has an important role in the assembly of virions), and is between nucleotide numbers 26245 and 26472 of SEQ ID NO: 52;

RdRp: encodes the RNA-dependent RNA polymerase of SARS-CoV-2 (an enzyme that catalyzes the replication of RNA from a viral RNA template) and is between nucleotide numbers 13442 and 16236 of SEQ ID NO: 52.

Targeting/Target Site

As used herein, the term “targeting” refers to the action of an inhibitory oligonucleotide binding or hybridizing to a target site in a nucleic acid that results in inhibition of the expression of the nucleic acid.

As used herein, a “target site” refers to a stretch of nucleotides on an mRNA of a target gene to which an inhibitory oligonucleotide binds, which ultimately leads to inhibition of the function of the mRNA and thus the expression of the gene. In some embodiments, a target site comprises at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, a target site comprises not more than 50, 45, 40 or 35 nucleotides. In some embodiments, a target site comprises between 15-30, 18-28, 20-25, or 30-35 nucleotides. Selected target sites are unique to the virus with minimal or no overlap with mRNA sequences found in human, so that the oligonucleotides are specific in their inhibition of the viral mRNA, with minimal side effects/off-target effects. In specific embodiments, a target site comprises the nucleotide sequence of one of SEQ ID NOS: 1-8; for example, a target site may have a nucleotide sequence that includes one of SEQ ID NOS: 1-8 and additional nucleotide(s) (e.g., 1, 2, 3, 4, or 5 nucleotides) on either 5′ or 3′ of the selected sequence.

Inhibitory Oligonucleotides

As used herein, the phrase “inhibitory oligonucleotide” refers to an oligonucleotide that can inhibit expression of a target SARS-CoV-2 gene. In some embodiments, an inhibitory oligonucleotide binds to a target site in a nucleic acid (e.g., a selected SARS-CoV-2 mRNA). In some embodiments, an inhibitory oligonucleotide comprises the reverse complementary sequence of a target site. In some embodiments, an inhibitory oligonucleotide comprises a nucleotide sequence that is substantially complementary to the sequence of a target site and capable of binding to the target site. “Substantially complementary” means that the oligonucleotide may be identical to the reverse complementary sequence of a target site, or may differ from the reverse complementary sequence of a target site in one or more (e.g., 1, 2, or 3) nucleotide positions via substitution, addition or deletion of one or more nucleotides.

The inhibitory oligonucleotides disclosed herein have the following features:

- (i) The inhibitory oligonucleotides can specifically target multiple target sites of SARS-CoV-2 RNA directly.
- (ii) The inhibitory oligonucleotides can cross the cell membrane and interact with the viral RNA inside cellular cytoplasm.
- (iii) Since the oligonucleotide compositions of the disclosure can target the multiple-target sites in the viral RNA, this approach is able to circumvent mutations which often occur in RNA viruses.
- (iv) The inhibitory oligonucleotides can be easily, quickly and economically manipulated, and different kinds of viral infections (including, but not limited to, different strains of SARS-CoV-2) can be treated.
- (v) Nucleotides provide nutrition for supporting on metabolism and health of the human body.

In some embodiments, the inhibitory oligonucleotides of this disclosure comprise at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the inhibitory oligonucleotides comprise not more than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, or 31 nucleotides. In some embodiments, the inhibitory oligonucleotides comprise between 15-20, 15-25, 15-30, 16-20, 16-25, 16-27, 16-30, 17-20, 17-25, 17-27, 17-30, 18-20, 18-25, 18-28, 18-30, 19-20, 19-25, 19-28, 19-30, 20-22, 20-25, 20-28, 20-30, 21-22, 21-25, 21-28, 21-30, 21-25, 21-28, 21-30, or 25-30 nucleotides.

In some embodiments, the inhibitory oligonucleotides are selected from the group consisting of an antisense oligonucleotide (ASO), a small interfering RNA (siRNA), a Dicer-substrate RNA (DsiRNA), and a microRNA.

In some embodiments, the inhibitory oligonucleotide is an ASO selected from SEQ ID NOS: 9-16, and 33-40. In some embodiments, the inhibitory oligonucleotide comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to a nucleotide sequence selected from SEQ ID NOS: 9-16 and 33-40.

In some embodiments, the inhibitory oligonucleotide is an ASO targeting SARS-CoV2 ORF1ab gene. In a specific embodiment, the ASO comprises a sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 33. In a specific embodiment, the ASO comprises a sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 37.

In some embodiments, the inhibitory oligonucleotide is an ASO targeting SARS-CoV2 RdRp gene. In a specific embodiment, the ASO comprises a sequence as shown in SEQ ID NO: 10 or SEQ ID NO: 34. In a specific embodiment, the ASO comprises a sequence as shown in SEQ ID NO: 14 or SEQ ID NO: 38.

In some embodiments, the inhibitory oligonucleotide is an ASO targeting SARS-CoV2 S-protein gene. In a specific embodiment, the ASO comprises a sequence as shown in SEQ ID NO: 11 or SEQ ID NO: 35.

In some embodiments, the inhibitory oligonucleotide is an ASO targeting SARS-CoV2 N-protein gene. In a specific embodiment, the ASO comprises a sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 36. In a specific embodiment, the ASO comprises a sequence as shown in SEQ ID NO: 16 or SEQ ID NO: 40.

In some embodiments, the inhibitory oligonucleotide is an ASO targeting SARS-CoV2 E-protein gene. In a specific embodiment, the ASO comprises a sequence as shown in SEQ ID NO: 15 or SEQ ID NO: 39.

In some embodiments, the inhibitory oligonucleotide comprises a pair of Dicer-substrate RNAs (DsiRNAs) selected from the group consisting of DsiRNA pair 1 (SEQ ID NOs: 17 & 18), DsiRNA pair 2 (SEQ ID NOs: 19 & 20), DsiRNA pair 3 (SEQ ID NOs: 21 & 22), DsiRNA pair 4 (SEQ ID NOs: 23 & 24), DsiRNA pair 5 (SEQ ID NOs: 25 & 26), DsiRNA pair 6 (SEQ ID NOs: 27 & 28), DsiRNA pair 7 (SEQ ID NOs: 29 & 30), and DsiRNA pair 8 (SEQ ID NOs: 31 & 32. In some embodiments, the plurality of inhibitory oligonucleotides comprise Dicer-substrate RNA (DsiRNA) pair 1 (SEQ ID NOs: 17 & 18), DsiRNA pair 2 (SEQ ID NOs: 19 & 20), DsiRNA pair 3 (SEQ ID NOs: 21 & 22), DsiRNA pair 4 (SEQ ID NOs: 23 & 24), DsiRNA pair 5 (SEQ ID NOs: 25 & 26), DsiRNA pair 6 (SEQ ID NOs: 27 & 28), DsiRNA pair 7 (SEQ ID NOs: 29 & 30), and DsiRNA pair 8 (SEQ ID NOs: 31 & 32).

In some embodiments, the inhibitory oligonucleotide is a pair of DsiRNAs targeting SARS-CoV2 ORF1ab gene. In a specific embodiment, the pair of DsiRNAs comprises SEQ ID NOs: 17 & 18 or SEQ ID NOs: 25 & 26.

In some embodiments, the inhibitory oligonucleotide is a pair of DsiRNAs targeting SARS-CoV2 RdRb gene. In some embodiments, the pair of DsiRNAs comprises a pair of nucleotide sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NOs: 19 & 20 or SEQ ID NOs: 27 & 28. In a specific embodiment, the pair of DsiRNAs comprises SEQ ID NOs: 19 & 20 or SEQ ID NOs: 27 & 28.

In some embodiments, the inhibitory oligonucleotide is a pair of DsiRNAs targeting SARS-CoV2 S-protein gene. In some embodiments, the pair of DsiRNAs comprises a pair of nucleotide sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NOs: 21 & 22. In a specific embodiment, the pair of DsiRNAs comprises SEQ ID NOs: 21 & 22.

In some embodiments, the inhibitory oligonucleotide is a pair of DsiRNAs targeting SARS-CoV2 N-protein gene. In some embodiments, the pair of DsiRNAs comprises a pair of nucleotide sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NOs: 23 & 24 or SEQ ID NOs: 31 & 32. In a specific embodiment, the pair of DsiRNAs comprises SEQ ID NOs: 23 & 24 or SEQ ID NOs: 31 & 32.

In some embodiments, the inhibitory oligonucleotide is a pair of DsiRNAs targeting SARS-CoV2 E-protein gene. In some embodiments, the pair of DsiRNAs comprises a pair of nucleotide sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NOs: 29 & 30. In a specific embodiment, the pair of DsiRNAs comprises SEQ ID NOs: 29 & 30.

In some embodiments, the inhibitory oligonucleotides are modified. As used herein the term “modified” or “modification” refers to a change in chemical structure of one or more nucleotides of an oligonucleotide, while leaving the sequence of the oligonucleotide unchanged as compared to the sequence before the modification. In some embodiments, the modification results in improved in vivo stability of the inhibitory oligonucleotides (e.g., by preventing degradation by cellular enzymes). In some embodiments, the modification results in improved entry of the inhibitory oligonucleotides into a cell (e.g., by improving cell membrane crossing properties). In a specific embodiment, the inhibitory oligonucleotides are 2′-Deoxy, 2′-Fluoroarabino Nucleic Acid (FANA)-modified antisense oligonucleotides. In a specific embodiment, the inhibitory oligonucleotides are 2′ O-Methyl RNA modified antisense oligonucleotides.

In some embodiments, the inhibitory oligonucleotides of the disclosure comprise at least one detectable label. Non-limiting examples of detectable labels include, but are not limited to, Alexa 405, Pacific Blue, Pacific Green, Alexa 488, Alexa 532, Alexa 546, Rhodamine Red X, Alexa 610, Alexa 647, DyLight-510-LS, Hydroxycoumarin, methoxycoumarin, Cy2, FAM, Flourescein FITC, Alexa 430, R-phycoerythrin (PE), Tamara, Cy3.5 581, Rox, Alexa fluor 568, Red 613, Texas Red, Alexa fluor 594, Alexa fluor 633, Alexa fluor 660, Alexa fluor 680, Cy5, Cy 5.5, Cy 7, and Allophycocyanin.

Compositions Comprising Inhibitory Oligonucleotides

One aspect of the disclosure is directed to a composition comprising at least one inhibitory oligonucleotide as described herein, wherein the at least one inhibitory oligonucleotide targets a SARS-CoV-2 gene selected from the group consisting of ORF1ab, RdRp, the S-protein gene, the N-protein gene, and the E protein gene.

In some embodiments, the composition comprises at least one inhibitory oligonucleotide, wherein the at least one inhibitory oligonucleotide is selected from the group consisting of an antisense oligonucleotide (ASO), a small interfering RNA (siRNA), a Dicer-substrate RNA (DsiRNA), and a microRNA.

In some embodiments, the composition comprises at least one ASO selected from SEQ ID NOS: 9-16, and 33-40.

In some embodiments, the composition comprises at least one pair of Dicer-substrate RNAs (DsiRNAs) selected from the group consisting of DsiRNA pair 1 (SEQ ID NOs: 17 & 18), DsiRNA pair 2 (SEQ ID NOs: 19 & 20), DsiRNA pair 3 (SEQ ID NOs: 21 & 22), DsiRNA pair 4 (SEQ ID NOs: 23 & 24), DsiRNA pair 5 (SEQ ID NOs: 25 & 26), DsiRNA pair 6 (SEQ ID NOs: 27 & 28), DsiRNA pair 7 (SEQ ID NOs: 29 & 30), and DsiRNA pair 8 (SEQ ID NOs: 31 & 32. In some embodiments, the plurality of inhibitory oligonucleotides comprise Dicer-substrate RNA (DsiRNA) pair 1 (SEQ ID NOs: 17 & 18), DsiRNA pair 2 (SEQ ID NOs: 19 & 20), DsiRNA pair 3 (SEQ ID NOs: 21 & 22), DsiRNA pair 4 (SEQ ID NOs: 23 & 24), DsiRNA pair 5 (SEQ ID NOs: 25 & 26), DsiRNA pair 6 (SEQ ID NOs: 27 & 28), DsiRNA pair 7 (SEQ ID NOs: 29 & 30), and DsiRNA pair 8 (SEQ ID NOs: 31 & 32).

In one aspect, the disclosure is directed to a composition comprising a plurality of inhibitory oligonucleotides, wherein the plurality of inhibitory oligonucleotides targets at least two SARS-CoV-2 genes selected from the group consisting of ORF1ab, RdRp, the S-protein gene, the N-protein gene, and the E protein gene. In some embodiments, the plurality of inhibitory oligonucleotides targets all of the ORF1ab, RdRp, S-protein, N-protein and E protein genes.

In some embodiments, a selected SARS-CoV-2 gene is targeted by at least two inhibitory oligonucleotides. In some embodiments, a selected SARS-CoV-2 gene is targeted by two, three, four, five, or six different, e.g., non-overlapping, inhibitory oligonucleotides. In some embodiments, the at least two inhibitory oligonucleotides simultaneously target at least two different sites on at least two SARS-CoV-2 genes.

In some embodiments, the plurality of inhibitory oligonucleotides comprises at least two, at least three, at least four, at least five, at least six, at least seven, or more oligonucleotides which comprise a nucleotide sequence selected from the group consisting of SEQ ID NOS: 9-16 and modified forms of SEQ ID NOS: 9-16 (e.g., SEQ ID NOS: 33-40). In some embodiments, the plurality of inhibitory oligonucleotides comprises all eight oligonucleotides as shown in SEQ ID NOS: 9-16 or modified forms of SEQ ID NOS: 9-16 (e.g., SEQ ID NOS: 33-40), respectively.

In some embodiments, the plurality of inhibitory oligonucleotides comprise at least two, at least three, at least four, at least five, at least six, at least seven, or more pairs of Dicer-substrate RNAs (DsiRNAs) selected from the group consisting of DsiRNA pair 1 (SEQ ID NOs: 17 & 18), DsiRNA pair 2 (SEQ ID NOs: 19 & 20), DsiRNA pair 3 (SEQ ID NOs: 21 & 22), DsiRNA pair 4 (SEQ ID NOs: 23 & 24), DsiRNA pair 5 (SEQ ID NOs: 25 & 26), DsiRNA pair 6 (SEQ ID NOs: 27 & 28), DsiRNA pair 7 (SEQ ID NOs: 29 & 30), and DsiRNA pair 8 (SEQ ID NOs: 31 & 32). In some embodiments, the plurality of inhibitory oligonucleotides comprise Dicer-substrate RNA (DsiRNA) pair 1 (SEQ ID NOs: 17 & 18), DsiRNA pair 2 (SEQ ID NOs: 19 & 20), DsiRNA pair 3 (SEQ ID NOs: 21 & 22), DsiRNA pair 4 (SEQ ID NOs: 23 & 24), DsiRNA pair 5 (SEQ ID NOs: 25 & 26), DsiRNA pair 6 (SEQ ID NOs: 27 & 28), DsiRNA pair 7 (SEQ ID NOs: 29 & 30), and DsiRNA pair 8 (SEQ ID NOs: 31 & 32).

In some embodiments, the plurality of inhibitory oligonucleotides are expressed from at least one nucleic acid vector (i.e., one or more vectors). In some embodiments, the at least one nucleic acid vector is selected from a viral vector, a non-viral vector, an integrative vector, or a non-integrative vector. In some embodiments, the plurality of inhibitory oligonucleotides are expressed from one nucleic acid vector.

In some embodiments, the inhibitory oligonucleotides are 2′ O-Methyl RNA modified antisense oligonucleotides and have a nucleotide sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.

In some embodiments, the inhibitory oligonucleotides are in modified forms that comprise phosphorothioate bonds that render them resistant to nucleases.

In some embodiment, the inhibitory oligonucleotides comprise a 5-methyl dC modification at the in 5′ ends.

In some embodiments, the composition comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises nanoparticles or other delivery vehicles (e.g., lipid-based delivery vehicles such as lipofectamine and oligofectamine) to which the plurality of inhibitory oligonucleotides is conjugated.

Compositions Comprising Inhibitory Peptides

ACE2 is an enzyme that plays a critical role in human biology and metabolism. ACE2 also functions as the receptor for the SARS-CoV-2 for its cellular entry. Disclosed herein are peptides designed to block SARS-CoV-2 S-protein receptor binding domain (RBD) from interacting with ACE2 ligand binding domain (LBD), thereby preventing viral entry.

The inventors recognized that if a peptide that is too long is used to treat the disease, that peptide's effect would be limited because of folding of the 3D structure during the binding on SARS-CoV-2. Therefore, the inventors designed peptides that are long enough to prevent ACE2 LBD and SARS-CoV-2 RBD interaction, but short enough to not require secondary structures to work. In some embodiments, the length of a peptide is about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 23 amino acids, about 24 amino acids, about 25 amino acids, about 26 amino acids, about 27 amino acids, about 28 amino acids, about 29 amino acids, about 30 amino acids, about 31 amino acids, about 32 amino acids, about 33 amino acids, about 34 amino acids, or about 35 amino acids. In some embodiments, the length of a peptide is not more than 50 amino acids, not more than 49 amino acids, not more than 48 amino acids, not more than 47 amino acids, not more than 46 amino acids, not more than 45 amino acids, not more than 44 amino acids, not more than 43 amino acids, not more than 42 amino acids, not more than 41 amino acids, not more than 40 amino acids, not more than 39 amino acids, not more than 38 amino acids, not more than 37 amino acids, not more than 36 amino acids, not more than 37 amino acids, not more than 36 amino acids, not more than 35 amino acids, not more than 34 amino acids, not more than 33 amino acids, not more than 32 amino acids, not more than 31 amino acids, or not more than 30 amino acids in length. As used herein, the term “about” refers to ±10% of any given value. The inhibitory peptides bind to ACE2 LBD or SARS-CoV-2 RBD by mimicking a portion of ACE2 LBD or a portion of SARS-CoV-2 RBD. A “portion” means a contiguous peptide sequence of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acids.

In some embodiments, the inhibitory peptides of the instant disclosure comprise modifications to a peptide of a naturally occurring protein, e.g., by adding, substituting or removing one or more amino acids in a peptide of a naturally occurring protein (e.g., at the N-terminus, the C-terminus or within the peptide) such that the modified peptide differs in sequence from the peptide of a naturally occurring protein; by including one or more non-natural amino acids in the peptide, by making a modification (e.g., a label or a tag) to the side chain of an amino acid in the peptide.

The inhibitory peptides disclosed herein can also serve as antigens for generation of antibodies against these peptides. The generated antibodies can bind to the RBD domain of the S protein of SARS-CoV-2, or to the LBD of human ACE2, thereby blocking the interaction between the S protein and human ACE receptor. Thus, the inhibitory peptides can be administered to a subject and antibodies can be generated in the subject; or alternatively, the inhibitory peptides, especially peptides that mimic the binding surface of the RBD domain of the S protein of SARS-CoV-2 (such as peptide 6 in Table 5, SEQ ID NO: 45), can be used to produce antibodies in vitro or in a host animal, which antibodies are then administered to a subject.

In one aspect, the disclosure is directed to a composition comprising at least one peptide mimicking the ligand binding domain (LBD) of human Angiotensin-converting Enzyme 2 (ACE2) protein, wherein the at least one peptide prevents binding of the S-protein of SARS-CoV-2 to the human ACE2 protein. The amino acid sequence of the human ACE2 protein is shown in SEQ ID NO: 55. The nucleotide sequence of the human ACE2 gene is shown in GenBank Accession Number: AB046569.1. The amino acids in the ACE2 protein that directly interact with SARS-CoV-2 S-protein are as follows: Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L79, M82, Y83, N330, K353, G354, D355, R357 and R393 of SEQ ID NO: 55.

In some embodiments, the LBD of human ACE2 is as defined in Lan, Jun et al. (Nature, vol. 581, 7807 (2020): 215-220, Extended Data Table 2|Contact residues of the SARS-CoV-2 RBD-ACE2 and SARS-CoV RBD-ACE2 interfaces), which is incorporated herein in its entirety. In some embodiments, the LBD of human ACE2 comprises the amino acid sequence of any one of SEQ ID NOS: 41-44, 54, and 63-82. In some embodiments, the LBD of human ACE2 comprises the amino acid sequence shown by SEQ ID NO: 56, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 56.

In some embodiments, the composition comprises at least one peptide. In some embodiments, the at least one peptide is between 15 and 30 amino acids in length (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length).

In some embodiments, the peptide is an LBD mimic peptide (e.g., a peptide that correspond to a region/segment of the LBD).

In some embodiments, the LBD mimic peptide comprises a core amino acid sequence as shown in SEQ ID NO: 63, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 63. In some embodiments, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 63 and has a length of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 64, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 64. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 41, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 41. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 65, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 65. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 66, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 66.

In some embodiments, the LBD mimic peptide comprises a core amino acid sequence as shown in SEQ ID NO: 67, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 67. In some embodiments, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 67 and has a length of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 42, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 42. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 68, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 68. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 69, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 69. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 70, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 70.

In some embodiments, the LBD mimic peptide comprises a core amino acid sequence as shown in SEQ ID NO: 71, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 71. In some embodiments, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 71 and has a length of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 43, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 43. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 72, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 72. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 73, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 73. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 74, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 74.

In some embodiments, the LBD mimic peptide comprises a core amino acid sequence as shown in SEQ ID NO: 75, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 75. In some embodiments, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 75 and has a length of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 44, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 44. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 76, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 76. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 77, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 77. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 78, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 78.

In some embodiments, the LBD mimic peptide comprises a core amino acid sequence as shown in SEQ ID NO: 79, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 79. In some embodiments, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 79 and has a length of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 54, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 54. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 80, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 80. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 81, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 81. In a specific embodiment, the LBD mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 82, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 82.

In a specific embodiment, the composition comprises a plurality of LBD mimic peptides (e.g., peptides that correspond to different regions/segments of the LBD). In some embodiments, the composition comprises LBD mimic peptides that comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-44, 54, and 63-82, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-44, 54, and 63-82. In some embodiments, the composition comprises a plurality of peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-44, 54, and 63-82. In some embodiments, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, 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 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24 or 25 peptides, and each peptide in the composition is selected from the group of peptides comprising an amino acid sequence as shown in SEQ ID NOS: 41-44, 54, and 63-82, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-44, 54, and 63-82.

Another aspect of the disclosure is directed to a composition comprising a peptide mimicking the receptor binding domain (RBD) of the S-Protein of SARS-CoV-2 (an RBD mimic peptide), wherein the peptide prevents binding of the S-protein of SARS-CoV-2 to the human ACE2 protein. In some embodiments, the full-length S-protein of SARS-CoV-2 comprises an amino acid sequence as shown in SEQ ID NO: 62 (GenBank Accession No: QHD43416.1), or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 62.

In some embodiments, the RBD-mimic peptide comprises a core amino acid sequence as shown in SEQ ID NO: 45, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 45. In a specific embodiment, the RBD-mimic peptide comprises an amino acid sequence as shown in SEQ ID NO: 45 and has a length of at least 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, wherein the additional amino acids are selected from the amino acids immediate N or C terminal of the SEQ ID NO: 45 core sequence in the full length S-protein as shown by SEQ ID NO: 62. SEQ ID NO: 45 corresponds to amino acids 17-39 of SEQ ID NO: 69. In some embodiments, the RBD-mimic peptide comprises a sequence between amino acids 10-39 and 17-46 of SEQ ID NO: 62, and has a length of at least 23, 24, 25, 26, 27, 28, 29, or 30 amino acids (e.g., amino acids 10-39, 11-39, 11-40, 12-39, 12-40, 12-41, 13-39, 13-40, 13-41, 13-42, 14-39, 14-40, 14-41, 14-42, 14-43, 15-39, 15-40, 15-41, 15-42, 15-43, 15-44, 16-39, 16-40, 16-41, 16-42, 16-43, 16-44, 16-45, 17-39 (SEQ ID NO:42), 17-40, 17-41, 17-42, 17-43, 17-44, 17-45, or 17-46 of SEQ ID NO: 62).

In some embodiments, the composition comprises a plurality of LBD and/or RBD mimic peptides (e.g., peptides that correspond to different regions/segments of the LBD and/or at least one peptide that corresponds to different regions/segments of the RBD).

In some embodiments, the at least one peptide is labeled. In some embodiment the label is a fluorescent label. In some exemplary embodiments, the fluorescent label is selected from the group consisting of Alexa 405, Pacific Blue, Pacific Green, Alexa 488, Alexa 532, Alexa 546, Rhodamine Red X, Alexa 610, Alexa 647, DyLight-510-LS, Hydroxycoumarin, methoxycoumarin, Cy2, FAM, Flourescein FITC, Alexa 430, R-phycoerythrin (PE), Tamara, Cy3.5 581, Rox, Alexa fluor 568, Red 613, Texas Red, Alexa fluor 594, Alexa fluor 633, Alexa fluor 660, Alexa fluor 680, FAM (6-carboxyfluorescein), Cy 5.5, Cy 7, and Allophycocyanin

In some embodiments, the composition comprises a pharmaceutically acceptable carrier as described herein. In some embodiments, the pharmaceutically acceptable carrier comprises nanoparticles or other delivery vehicles (e.g., lipid-based carriers such as lipofectamine, oligofectamine, etc.) to which the at least one peptide is conjugated.

Dietary Supplements

The instant disclosure is also directed to dietary supplements compositions that are capable of supporting the immune system and helping with viral infection, such as SARS-CoV-2.

Inventors of the instant disclosure have formulated dietary supplements that comprise at least one of the compositions (comprising inhibitory oligonucleotides, inhibitory peptides, or a combination thereof) described above.

In some embodiments, a dietary supplement comprises inhibitory oligonucleotides at an amount between about 0.1 microgram (mcg) and about 1 milligram (mg) per serving of the dietary supplement. In some embodiments, a dietary supplement comprises about 0.1 mcg, 0.5 meg, 1 mcg, 1.5 mcg, 2 mcg, 2.5 mcg, 5 mcg, 8 mcg, 9 mcg, 10 mcg, 15 mcg, 20 mcg, 25 mcg, 30 mcg, 50, mcg, 100 mcg, 150 mcg, 200 mcg, 250 mcg, 300 mcg, 350 mcg, 400 mcg, 450 mcg, 500 mcg, 550 mcg, 600 mcg, 650 mcg, 700 mcg, 750 mcg, 800 mcg, 850 mcg, 900 mcg, 950 meg or 1000 mcg (1 mg) of inhibitory oligonucleotides per serving of the dietary supplement. As used herein, a “serving of a dietary supplement” refers to the maximum amount recommended, as appropriate, for consumption per eating occasion, or in the absence of recommendations, 1 unit (e.g., tablet, capsule, packet, teaspoonful, etc.). For example, if the directions on the label say to take 1-3 tablets with breakfast, the serving size would be 3 tablets. If the dietary supplement is a liquid, a serving may be measured in milliliters (ml) (e.g., 0.5 ml, 1 ml, 2 ml, etc.) or teaspoons.

In some embodiments, a dietary supplement comprises inhibitory peptides at an amount between about 0.1 microgram (mcg) and about 10 milligram (mg) per serving of the dietary supplement. In some embodiments, a dietary supplement comprises about 0.1 mcg, 0.5 mcg, 1 mcg, 1.5 mcg, 2 mcg, 2.5 mcg, 5 mcg, 8 mcg, 9 mcg, 10 mcg, 15 mcg, 20 mcg, 25 mcg, 30 mcg, 50, mcg, 100 mcg, 150 mcg, 200 mcg, 250 mcg, 300 mcg, 350 mcg, 400 mcg, 450 mcg, 500 meg, 550 mcg, 600 mcg, 650 mcg, 700 mcg, 750 mcg, 800 mcg, 850 mcg, 900 mcg, 950 mcg, 1000 mcg (1 mg), 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg or 10 mg of inhibitory peptides per serving of the dietary supplement.

In some embodiments, the dietary supplement comprises a composition comprising a plurality of inhibitory oligonucleotides as described herein, wherein the plurality of inhibitory oligonucleotides targets at least two SARS-CoV-2 genes selected from the group consisting of ORF1ab, RdRp, the S-protein gene, the N-protein gene, and the E protein gene.

In some embodiments, the dietary supplement comprises a composition as described herein comprising at least one peptide mimicking the ligand binding domain (LBD) of human ACE2 protein, wherein the at least one peptide prevents binding of the S-protein of SARS-CoV-2 to the human ACE2 protein.

In some embodiments, the dietary supplement comprises both a composition comprising a plurality of inhibitory oligonucleotides and a composition comprising at least one peptide, as described herein. In some embodiments, the dietary supplement comprises a composition comprising a plurality of inhibitory oligonucleotides and at least one peptide, as described herein.

In some embodiments, the dietary supplement the disclosure further comprises at least one additional nutrient selected from Vitamin C, Vitamin B6, Vitamin B12, Vitamin D, Zinc, polypeptides, nucleotide, L-arginine or peppermint oil.

In some embodiments, the dietary supplement of the disclosure is formulated for oral (e.g., pills, tablets, capsules, inhalers, liquid formulations), nasal (e.g., nasal sprays), ocular (e.g. eye drops), ear (e.g. ear drops), or topical (e.g., cream, lotion, shampoo, paper towels, wet wipes) application.

In some embodiments, in addition to at least one of the compositions (comprising inhibitory oligonucleotides, inhibitory peptides, or a combination thereof) described herein, an oral tablet comprises:

1) Vitamin C, 1-1000 mg

2) Vitamin B-mix (B6 and B12), 0.1-0.6 mg B6/1-2.4 mcg B12

3) Vitamin D, 1-800 IU

4) Zinc supplement, 1-50 mg

5) Polypeptides, 1-1000 mcg

6) L-Arginine, 1-10 mg

7) Peppermint oil, 1-2 mg.

In some embodiments, an oral drop/spray/tablet formula comprises Inhibitory Polypeptides (0.1 mcg-10 mg) and/or Inhibitory Nucleotides (0.1 mcg-1 mg) and one or more of: N-Acetyl Cysteine (1-100 mg), L-Arginine (1-10 mg), Glutathione (0.1-10 mg), Vitamin D3 (1-5000 IU), Vitamin C (1-1000 mg), Zinc (1 mcg-50 mg), Vitamin B6 (1 mcg-800 mcg), Vitamin B12 (0.1-10 mcg), Peppermint (leaf powder or oil) (10 mcg-5 mg), DHA (Docosahexaenoic acid) or EPA (Eicosapentaenoic acid) (5 mg) and other ingredients such as sweeteners (e.g., sugar, stevia), preservatives (e.g., potassium sorbate), glycerin and/or sorbic acid (amounts in parentheses are per serving (e.g., 0.5 ml Droplet for liquid)).

In a specific embodiment, an oral drop/spray/tablet formula comprises Inhibitory Polypeptides (50 mcg) and/or Inhibitory Nucleotides (2.5 mcg), and one or more of: N-Acetyl Cysteine (25 mg), L-Arginine (2.5 mg), Glutathione (2.5 mg), Vitamin D3 (500 IU), Vitamin C (50 mg), Zinc (65 mcg), Vitamin B6 (30 mcg), Vitamin B12 (0.5 mcg), Peppermint (leaf powder or oil) (0.1 mg), DHA (Docosahexaenoic acid) or EPA (Eicosapentaenoic acid) (5 mg) and other ingredients such as sweeteners (e.g., sugar, stevia), preservatives (e.g., potassium sorbate), glycerin and/or sorbic acid (amounts in parentheses are per serving (e.g., 0.5 ml droplet)).

In some embodiments, a nasal spray formula comprises: N-Acetyl Cysteine (1-100 mg), Glutathione (0.1-10 mg), Vitamin C (1-1000 mg), Vitamin B6 (1 mcg-800 mcg), Vitamin B12 (0.1-10 mcg), Inhibitory Polypeptides (0.1 mcg-10 mg), Inhibitory Nucleotides (0.1 mcg-1 mg), Xylitol (0.1 mg-50 mg) and saline (amounts in parentheses are per serving (e.g., 0.5 ml Droplet for liquid)).

In a specific embodiment, a nasal spray formula comprises: N-Acetyl Cysteine (2.5 mg), Glutathione (1 mg), Vitamin C (5 mg), Vitamin B6 (5 mcg), Vitamin B12 (0.5 mcg), Inhibitory Polypeptides (20 mcg), Inhibitory Nucleotides (5 mcg), Xylitol (0.25 mg) and saline (amounts in parentheses are per serving (e.g., 0.5 ml Droplet for liquid)).

In some embodiments, a nasal spray formula comprises:

1) Vitamin C, 1-1000 mg

2) Vitamin B-mix (B6 and B12), 0.1-0.6 mg/1-2.4 ug

3) Vitamin D, 1-800 IU

4) Zinc supplement, 1-50 mg

5) Polypeptides, 1-1000 ug

6) L-Arginine, 1-10 mg

7) Peppermint oil, 1-2 mg

In some embodiments, a dietary supplement is formulated for kids (ages between 5-12) or teens (ages between 13-19). In some embodiments, a kid/teen formula comprises Inhibitory Polypeptides (0.1 mcg-10 mg) and/or Inhibitory Nucleotides (0.1 mcg-1 mg) and one or more of Vitamin A (0.1-10 mg), Vitamin C (1-1000 mg), Vitamin D (0.1 mcg, 1 mg), Vitamin E (1 mg-100 mg), Vitamin K (0.1 mcg-1 mg), Vitamin B6 (1 mcg-5 mg), Vitamin B12 (0.1 mcg-10 meg), Zinc (0.1 mg-50 mg) and other ingredients (calcium (1 mg-500 mg), Iron (0.1 mg-15 mg), sweetener (sugar or stevia—0.1 g-3 g) (amounts in parentheses are per serving (e.g., 1 pellet or gummy per day)). It is understood that

In a specific embodiment, a teen formula comprises: Vitamin A (1.5 mg), Vitamin C (80 mg), Vitamin D (20 mcg), Vitamin E (27 mg), Vitamin K (20 mg), Vitamin B6 (1.4 mg), Vitamin B12 (3 mcg), Inhibitory Polypeptides (200 mcg), Inhibitory Nucleotides (5 mcg), Zinc (0.5 mg) and other ingredients (calcium (120 mg), Iron (9 mg), sweetener (sugar or stevia—0.5 g) (amounts in parentheses are per serving (e.g., 1 pellet or gummy per day)).

In a specific embodiment, a kid formula comprises: Vitamin A (0.3 mg), Vitamin C (40 mg), Vitamin D (10 mcg), Vitamin E (6 mg), Vitamin K (10 mg), Vitamin B6 (0.7 mg), Vitamin B112 (2 mcg), Inhibitory Polypeptides (50 mcg), Inhibitory Nucleotides (2.5 mcg), Zinc (0.1 mg) and other ingredients (calcium (25 mg), Iron (1 mg), sweetener (sugar or stevia—0.5 g) (amounts in parentheses are per serving (e.g., 1 pellet or gummy per day)).

In some embodiments, the dietary supplement is formulated for oral (e.g., pills, tablets, capsules, inhalers, liquid formulations), nasal (e.g., nasal sprays), eye (eye drop or ointment), ear (ear drop), or topical (e.g., cream, lotion, shampoo, paper towels, wet wipes) application.

Nucleic Acid Vectors

Another aspect of the disclosure is directed to a nucleic acid vector encoding at least one inhibitory oligonucleotides disclosed herein. In some embodiments, the disclosure is directed to a nucleic acid vector encoding a plurality of inhibitory oligonucleotides disclosed herein.

In some embodiments, a nucleic acid vector encodes at least two inhibitory oligonucleotides. In some embodiments, a nucleic acid vector encodes for at least two inhibitory oligonucleotides of different types (e.g., at least two inhibitory oligonucleotides selected from an antisense oligonucleotide, a small interfering RNA (siRNA), a Dicer-substrate RNA (DsiRNA), and a microRNA).

In some embodiments, each nucleic acid vector encodes only one inhibitory oligonucleotide, and a combination of such nucleic acid vectors are provided.

In some embodiments, the nucleic acid vectors are suitable for delivery to a subject and capable of expression of the encoded inhibitory oligonucleotides in the subject.

In some embodiments, the nucleic acid vector is an integrative vector, i.e., a vector that integrates into the genome of a host cell. In some embodiments, the nucleic acid vector is a non-integrative vector. In some embodiments, the nucleic acid vector is viral vector, e.g., an Adeno-Associated Virus (AAV)-based vector, or a lentiviral vector. In some embodiments, the nucleic acid vector is a non-viral vector.

In some embodiments, the AAV-based vector is selected from AAV1, AAV2, AAV3, VAAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, and AAV13 and AAV14. In some embodiments, the AAV-derived vector is one of the vectors described in Lykken, Erik Allen, et al., Journal of Neurodevelopmental Disorders 10.1 (2018): 16, incorporated herein in its entirety.

In some embodiments, the nucleic acid vector has the ability to integrate into the genome of a cell (i.e., is an integrative vector). In some embodiments, the nucleic acid vector does not have the ability to integrate into the genome of a cell (i.e., is a non-integrative vector).

In some embodiments, the nucleic acid vector is a lentiviral vector. In some embodiments, lentiviral vector has the ability to integrate into the genome of a cell. In some embodiments, the lentiviral vector does not have the ability to integrate into the genome of a cell (e.g., as described in Philippe, Stéphanie, et al. PNAS, 103.47 (2006): 17684-17689, and Lai et al., PNAS 97 (21), (2000): 11297-11302, both of which are incorporated herein in their entirety). In some embodiments, the lentiviral vector has a defective (i.e., nonfunctional) integrase (which prevents its genome integration).

Methods for Gene Therapy

Another aspect of the disclosure is directed at a method of treating or preventing a SARS-CoV-2 infection comprising expressing a plurality of inhibitory oligonucleotides in a target cell, wherein the plurality of inhibitory oligonucleotides are those disclosed herein.

In some embodiments, the target cell is a mammalian cell expressing an ACE2 receptor. In some embodiments, the target cell is a human cell. In some embodiments, the target cell is a lung epithelial cell. In some embodiments, the target cell is selected from the group consisting of a small airway epithelial cell, a bronchial/tracheal epithelial cell, and a nasal epithelial cell.

In some embodiments, the plurality of inhibitory oligonucleotides are expressed from at least one nucleic acid vector (i.e., one or more vectors) disclosed herein.

In some embodiments, the at least one vector is administered to a subject in need via oral (e.g., pills, tablets, capsules, inhalers, liquid formulations), nasal (e.g., nasal sprays), ocular (e.g., eye drops), ear (e.g., ear drops), intravenous (i.v.) injection or topical (e.g., cream, lotion, shampoo) routes.

In some embodiments, the at least one vector is selected from a viral vector, (e.g., an Adeno-Associated Virus (AAV)-based vector, or a lentiviral vector), or a non-viral vector (e.g., lipid, carbon, metal, or polymer nanoparticles), an integrative vector, or a non-integrative vector (e.g., a lentiviral vector with a defective integrase).

In some embodiments, combinations of gene therapy application include the following gene therapy vectors described herein:

1) AAV-ASO (A1 to A8): 1) AAV-U6-A1_H1-A2_CMV/EF-A3-E2A-A4; 2) AAV-U6-A5_H1-A6 CMV/EF-A7-E2A-A8.

2) AAV-shRNA (siRNA1 to siRNA8): 1) AAV-US-shRNA1_H1-shRNA2 CMV-shRNA3_EF-shRNA4; 2) AAV-US-shRNA5_H1-shRNA6_CMV-shRNA7_EF-shRNA8.

Methods for Treatment

Another aspect of the disclosure is directed to a method for treating a SARS-CoV-2 infection comprising administering to a subject an effective amount of; 1) a composition; 2) a nucleic acid vector; 3) combination of nucleic acid vectors; or 4) a combination thereof. The compositions (comprising inhibitory oligonucleotides, peptides, or a combination thereof), nucleic acid vectors, and combination of nucleic acid vectors are described above.

Pharmaceutical Carriers and Administration

A “pharmaceutically-acceptable carrier” includes any of the standard pharmaceutical carriers. Examples of suitable carriers are well known in the art and may include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution and various wetting agents. Other carriers may include additives used in tablets, granules and capsules, and the like. Typically, such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gum, glycols or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well-known conventional methods.

In some embodiments, the pharmaceutically acceptable carrier of the present disclosure comprises non-viral delivery vehicles such as nanoparticles (as described in Jin, Sha, and Kaiming Ye., (2007), Biotechnology Progress, 23.1: 3241; Z. Liu, et al., Advanced Drug Delivery Reviews, vol. 60, no. 15, 2008, pp. 1650-1662; and Saravanakumar G. et al., Curr. Med. Chem., 19(19), 2012, pp. 3212-3229, all of which are herein incorporated by reference in their entirety). In some embodiments, the nanoparticles are lipid nanoparticles (as described in WO2017218704A1, which is incorporated by reference in its entirety). In some embodiments, the nanoparticles are lipid nanoparticles, carbon nanoparticles, metal nanoparticles (e.g., iron nanoparticles), or polymer nanoparticles.

In some embodiments, the pharmaceutically acceptable carrier of the present disclosure comprises lipid-based delivery vehicles such as liposomes (as described in U.S. Ser. No. 10/258,629B2; Gabizon, A. et al. J Control Release 1998, 53 (1-3), 275-9; Bomgaars, L. et al., J Clin. Oncol. 2004, 22 (19), 3916-21; Drummond, D. C. et al, Pharmacol. Rev. 1999, 51 (4), 691-743; Allen, T. M.; Cullis, P. R., Science 2004, 303 (5665), 1818-22, which are incorporated by reference in their entirety).

The pharmaceutical preparations of the present disclosure can be made up in any conventional form including, inter alia, (a) a solid form for oral administration such as tablets, capsules (e.g., hard or soft gelatin capsules), pills, cachets, powders, granules, and the like; (b) preparations for topical administrations such as solutions, suspensions, ointments, creams, gels, micronized powders, sprays, aerosols and the like. The pharmaceutical preparations may be sterilized and/or may contain adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, salts for varying the osmotic pressure and/or buffers.

The pharmaceutical compositions of the present disclosure can be used in liquid, solid, tablet, capsule, pill, ointment, cream, nebulized or other forms as explained below. In some embodiments, the composition of the present disclosure can be administered by different routes of administration such as oral, oronasal, parenteral or topical.

“Oral” or “peroral” administration refers to the introduction of a substance into a subject's body through or by way of the mouth and involves swallowing or transport through the oral mucosa (e.g., sublingual or buccal absorption) or both.

“Oronasal” administration refers to the introduction of a substance into a subject's body through or by way of the nose and the mouth, as would occur, for example, by placing one or more droplets in the nose. Oronasal administration involves transport processes associated with oral and intranasal administration.

“Parenteral administration” refers to the introduction of a substance into a subject's body through or by way of a route that does not include the digestive tract. Parenteral administration includes subcutaneous administration, intramuscular administration, transcutaneous administration, intradermal administration, intraperitoneal administration, intraocular administration, and intravenous administration. For the purposes of this disclosure, parenteral administration excludes administration routes that primarily involve transport of the substance through mucosal tissue in the mouth, nose, trachea, and lungs.

Abbreviations Used in this Disclosure

ASO/Antisense Oligos: A sequence of nucleotides complementary to (and hence capable of binding to) a coding sequence of a messenger RNA molecule.

FANA: 2′-deoxy-2′-fluoroarabinonucleotide (FANA) modified, a technology to induce oligos self delivery

siRNA: Small interfering RNA/Short interfering RNA or Silencing RNA,

siRNA therapy: The siRNA interferes with specific genes. This may be used to turn off overactive genes within the human body or turn off genes from foreign invaders, such as virtues in the body to cure disease.

Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to cause a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate.

VS_ASO_1-FANA: ASO designed by the inventors with 2′-deoxy-2′-fluoroarabinonucleotide (FANA) modified, including Table 1: oligos 1, 2, 3, 4, 5, 6, 7 and 8

VS_ASO_1-FANA-FITC: VS_ASO_1-FANA oligo 9 (FITC labeled) (Table 1)

VS_ASO_2: ASO designed by the inventors including Table 2: oligo 1, 2, 3, 4, 5, 6, 7, 8 and 9 1, 2, 3, 4, 5, 6, 7 and 8

VS_ASO_2-Cy3: VS_ASO_2 oligo 9 (Cy3 labeled) (Table 2)

VS_DsiRNA: siRNA designed by the inventors with DsiRNA technology including table 3: oligo 1, 2, 3, 4, 5, 6, 7 and 8

VS_DsiRNA-Cy5: VS_DsiRNA oligo 9 (Cy5 labeled) (Table 3)

N-Protein: Nucleocapsid Protein of SARS-CoV-2, which is a structural protein that binds to the coronavirus RNA genome, thus creating a capsid.

S-Protein: The spike protein of SARS-CoV-2, which bind to the host cell receptors, i.e., ACE2, to enter the host cells.

RdRp: The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, which is an enzyme that catalyzes the replication of RNA from a viral RNA template.

E-Protein: The envelope protein of SARS-CoV-2, which is a small membrane protein that has an important role in the assembly of virions.

ORF1ab: The open reading frame for starting transcriptional genes of SARS-CoV-2, 1ab.

VS-nutrition: The inventors designed nutrition with Inhibitory oligonucleotides and poly-peptides.

FACS: Fluorescence-activated cell sorting (a cell-based fluorescent analysis technique used in biological experiment to detect and analyze fluorescent signal in every single cell).

Cycle threshold: Real-Time PCR calculation parameter used to quantify specific gene expression level. Cycle threshold refers to the cycle number in an RT-PCR reaction when the specific amplification signal rises above a predetermined level (e.g., above the background noise). As used herein, cycle threshold is a value that ranges from 0 to 40. Genes with high expression have lower cycle thresholds (e.g., between 0 and 20), and genes with low expression have high cycle thresholds (e.g., between 21 and 40).

Inhibitory oligonucleotides: ASO (VS_ASO_1-FANA and VS_ASO_2), and siRNA (VS_DsiRNA) designed and produced by VS

qRT-PCR: Real-Time Quantitative Reverse Transcription PCR

AAV: Adeno-associated viral vector

U6: Human U6 promoter

H1: Human H1 promoter

SV40: Simian Virus 40 promoter

A1: The gene encoding the Antisense 1 (ASO1) which binds to a target sequence on the SARS-CoV-2 ORF1ab protein.

A2: The gene encoding the Antisense 2 (ASO2) which binds to a target sequence on the SARS-CoV-2 RdRp protein

A3: The gene encoding the Antisense 3 (ASO3) which binds to a target sequence on the SARS-CoV-2 S-protein

A4: The gene encoding the Antisense 4 (ASO4) which binds to a target sequence on the SARS-CoV-2 N-protein

A5: The gene encoding the Antisense 5 (ASO5) which binds to a target sequence on the SARS-CoV-2 ORF1ab protein (but in a different region from the region that ASO1 binds)

A6: The gene encoding the Antisense 6 (ASO6) which binds to a target sequence on the SARS-CoV-2 RdRp protein (but in a different region from the region that ASO2 binds)

A7: The gene encoding the Antisense 7 (ASO7) which binds to a target sequence on the SARS-CoV-2 E-protein

A8: The gene encoding the Antisense 8 (ASO8) which binds to a target sequence on the SARS-CoV-2 N-proteins (but in a different region from the region that ASO4 binds)

eGFP: Enhanced green fluorescent protein

hGHpA: Human grown hormone poly-A

ITR: The inverted terminal repeat (ITR)

CMV promoter: Human cytomegalovirus (CMV) promoter

EF promoter: Human elongation factor promoter

E2A: 2A peptide with 19 amino acids derived from equine rhinitis A virus

TABLE 1

The VS_ASO_1-FANA designed, and all the ASO sequences are included

in the Table; but the sequence of oligo 4 is same as olgo 9 with

its fluorescent probe labeled for studying on its intercellular

delivering capacity. The FANA: 2′-deoxy-2′-fluoroarabinonucleotide

(FANA) modified, a technology to make the oligos with self-delivery

into the mammalian cells.

VS_ASO_1-

SEQ

FANA oligos
ASO_1 Sequences
Target on
ID

numbers
(from 5′ to 3′)
SARS-COV-2
NO
Fluorescence

1
AAGAACCTTGCGGTAAGCCAC
ORFlab
9
None

2
ATACGACATCAGTACTAGTGC
RdRp
10
None

3
ATAAGTAGGGACTGGGTCTTC
S-protein
11
None

4
TGTTAATTGGAACGCCTTGTC
N-protein
12
None

5
AGTTGTGCGTAATATCGTGCC
ORFlab
13
None

6
AAGTCTAGAGCTATGTAAGTT
RdRp
14
None

7
TATTAACGTACCTGTCTCTTC
E-protein
15
None

8
TGTCTGATTAGTTCCTGGTCC
N-protein
16
None

9
TGTTAATTGGAACGCCTTGTC
N-protein
12
FITC

TABLE 2

The VS_ASO_2 designed, and all the ASO sequences are included in the

Table, which are the same DNA sequences as VS_ASO_1, but their

structures were modified by another different technique (see Table 3).

The sequence of oligo 4 is same as oligo 9 with its fluorescent probe

labeled for studying on its intercellular delivering capacity.

VS_ASO_2
ASO_2 sequences
Target on
SEQ ID

oligos numbers
(from 5′ to 3′)
SARS-COV-2
NO
Fluorescence

1
AAGAACCTTGCGGTAAGCCAC
ORFlab
9
None

2
ATACGACATCAGTACTAGTGC
RdRp
10
None

3
ATAAGTAGGGACTGGGTCTTC
S-protein
11
None

4
TGTTAATTGGAACGCCTTGTC
N-protein
12
None

5
AGTTGTGCGTAATATCGTGCC
ORFlab
13
None

6
AAGTCTAGAGCTATGTAAGTT
RdRp
14
None

7
TATTAACGTACCTGTCTCTTC
E-protein
15
None

8
TGTCTGATTAGTTCCTGGTCC
N-protein
16
None

9
TGTTAATTGGAACGCCTTGTC
N-protein
12
Cy3

TABLE 3

The structure of VS_ASO_2 molecules modified: The ASO

molecules modified as shown: (1) *: Phosphorothioate-bond

in all base pairs to provide resistance to exonuclease

degradation; (2) m: 2′ O-Methyl RNA modified in those pb,

to increases both 5′ end nuclease stability and affinity

of the antisense oligo to the target RNA; (3) 5-methyl dC

in 5′ end of the oligos (ASO), it can also reduce the

chance of adverse immune response to Toll-like receptor 9

(TLR9). The middle region is a ″gapmer″ designed.

/5Me-dC/mA*mA*mG*mA*mA*C*C*T*T*G*C*G*G*T*A*mA*mG*mC*mC*mA*C

(SEQ ID NO: 33) Oligo-1 (Table 2)

/5Me-dC/mA*mU*mA*mC*mG*A*C*A*T*C*A*G*T*A*C*mU*mA*mG*mU*mG*C

(SEQ ID NO: 34) Oligo-2 (Table 2)

/5Me-dC/mA*mU*mA*mA*mG*T*A*G*G*G*A*C*T*G*G*mG*mU*mC*mU*mU*C

(SEQ ID NO: 35) Oligo-3 (Table 2)

/5Me-dC/mU*mG*mU*mU*mA*A*T*T*G*G*A*A*C*G*C*mC*mU*mU*mG*T*C

(SEQ ID NO: 36) Oligo-4 (Table 2)

/5Me-dC/mA*mG*mU*mU*mG*T*G*C*G*T*A*A*T*A*T*mC*mG*mU*mG*mC*C

(SEQ ID NO: 37) Oligo-5 (Table 2)

/5Me-dC/mA*mA*mG*mU*mC*T*A*G*A*G*C*T*A*T*G*mU*mA*mA*mG*mU*T

(SEQ ID NO: 38) Oligo-6 (Table 2)

/5Me-dC/ mU*mA*mU*mU*mA*A*C*G*T*A*C*C*T*G*T*mC*mU*mC*mU*mU*C

(SEQ ID NO: 39) Oligo-7 (Table 2)

/5Me-dC/ mU*mG*mU*mC*mU*G*A*T*T*A*G*T*T*C*C*mU*mG*mG*mU*mC*C

(SEQ ID NO: 40) Oligo-8 (Table 2)

TABLE 4

The VS_DsiRNA- Cy5 designed, all their sequences shown in the table. The sequence of oligo

4 is same as oligo 9 with its fluorescent probe labeled for studying on its intercellular

delivering capacity. The ′r′s in SEQ ID NOS: 17-32 in the table below denote “ribonucleic

acid” nucleotides in the sequences. If there is no ″r″ before a nucleotide, that nucleotide

is a deoxyribonucleic acid.

VS_DsiRNA

SEQ ID

oligos #
Sequences (from 5′ to 3′)
Target
NO:
Tag

1
5′ rGrCrCrUrUrGrUrCrCrCrUrGrGrUrUrUrCrArArCrGrArGAA 3′
ORFlab
17
None

5′rUrUrCrUrCrGrUrUrGrArArArCrCrArGrGrGrArCrArArGrGrCrUrC3′

18

2
5′ rCrArGrCrUrGrArUrGrCrArCrArArUrCrGrUrUrUrUrUrAAA 3′
RdRp
19
None

5′rUrUrUrArArArArArCrGrArUrUrGrUrGrCrArUrCrArGrCrUrGrArC3′

20

3
5′ rCrUrArGrUrCrArGrUrGrUrGrUrUrArArUrCrUrUrArCrAAC 3′
S-protein
21
None

5′rGrUrUrGrUrArArGrArUrUrArArCrArCrArCrUrGrArCrUrArGrArG3′

22

4
5′ rArArArCrUrArArArArUrGrUrCrUrGrArUrArArUrGrGrACC 3′
N-protein
23
None

5′rGrGrUrCrCrArUrUrArUrCrArGrArCrArUrUrUrUrArGrUrUrUrGrU3′

24

5
5′ rGrGrUrArGrUrUrArUrArCrUrArArUrGrArCrArArArGrCTT 3′
ORFlab
25
None

5′rArArGrCrUrUrUrGrUrCrArUrUrArGrUrArUrArArCrUrArCrCrArC3′

26

6
5′ rCrUrUrCrUrGrGrUrArArUrCrUrArUrUrArCrUrArGrArUAA 3′
RdRp
27
None

5′rUrUrArUrCrUrArGrUrArArUrArGrArUrUrArCrCrArGrArArGrCrA3′

28

7
5′ rGrGrArArGrArGrArCrArGrGrUrArCrGrUrUrArArUrArGTT 3′
E-protein
29
None

5′rArArCrUrArUrUrArArCrGrUrArCrCrUrGrUrCrUrCrUrUrCrCrGrA3′

30

8
5′ rGrGrCrCrArArArCrUrGrUrCrArCrUrArArGrArArArUrCTG 3′
N-protein
31
None

5′rCrArGrArUrUrUrCrUrUrArGrUrGrArCrArGrUrUrUrGrGrCrCrUrU3′

32

9
5′ rArArArCrUrArArArArUrGrUrCrUrGrArUrArArUrGrGrACC 3′
N-protein
23
Cy5

5′rGrGrUrCrCrArUrUrArUrCrArGrArCrArUrUrUrUrArGrUrUrUrGrU3′

24

TABLE 5

Sequences of VS-Peptides 1-6.

Name
Sequence
SEQ ID NO

Peptide 1
EEQAKTFLDKFNHEAEDLFYQSS
41

Peptide 2
FLKEQSTLAQMYPLQEIQNL
42

Peptide 3
LPNMTQGFWENSMLTDPGNVQ
43

Peptide 4
HPTAWDLGKGDFRILMCTKV
44

Peptide 5
MAYAAQPFLLRNGANEGFHEA
54

Peptide 6
GNYNYLYRLFRKSNLKPFERDIS
45

The sequences for the peptides of 1, 2, 3, 4 and 5 were designed based on the RBD amino acids of human ACE2, a receptor for COVID-19 entering the cells (see FIGS. 47 through FIGS. 52A-52H); which are capable to bind on the S-protein of COVID-19, in order to protect the human ACE 2 receptors by blocking the binding sites of the S-protein to elicit their therapeutic effects. However, the AA sequences of peptide 6 was designed to mimic the BD of S-Protein (see, FIG. 49), which was labelled by FITC; this peptide 5 could also be served as a pre-blocker on the RBD of ACE2 to prevent the COVID-19 infection as well.

TABLE 6

Alternative peptides based on VS Peptide 1-5

Name
Length
Sequence
SEQ ID NO

VS-Peptide 1
23
EEQAKTFLDKFNHEAEDLFYQSS
41

VS-Peptide 1-a
15
TFLDKFNHEAEDLFY
63

VS-Peptide 1-b
20
QAKTFLDKFNHEAEDLFYQS
64

VS-Peptide 1-c
25
TIEEQAKTFLDKFNHEAEDLFYQSSL
65

VS-Peptide 1-d
30
QSTIEEQAKTFLDKFNHEAEDLFYQSSLAS
66

VS-Peptide 2
20
FLKEQSTLAQMYPLQEIQNL
42

VS-Peptide 2-a
15
EQSTLAQMYPLQEIQ
67

VS-Peptide 2-b
20
LKEQSTLAQMYPLQEIQNLT
68

VS-Peptide 2-c
25
WSAFLKEQSTLAQMYPLQEIQNLTV
69

VS-Peptide 2-d
30
DKWSAFLKEQSTLAQMYPLQEIQNLTVKLQ
70

VS-Peptide 3
21
LPNMTQGFWENSMLTDPGNVQ
43

VS-Peptide 3-a
15
TQGFW ENSML TDPGN
71

VS-Peptide 3-b
20
LPNMT QGFWE NSMLT DPGNV
72

VS-Peptide 3-c
25
VGLPN MTQGF WENSM LTDPG NVQKA
73

VS-Peptide 3-d
30
VSVGL PNMTQ GFWEN SMLTD PGNVQ
74

KAVCH

VS-Peptide 4
20
HPTAWDLGKGDFRILMCTKV
44

VS-Peptide 4-a
15
WDLGKGDFRILMCTK
75

VS-Peptide 4-b
20
PTAWDLGKGDFRILMCTKVT
76

VS-Peptide 4-c
25
VCHPTAWDLGKGDFRILMCTKVTMD
77

VS-Peptide 4-d
30
KAVCHPTAWDLGKGDFRILMCTKVTMDDFL
78

VS-Peptide 5
21
MAYAAQPFLLRNGANEGFHEA
54

VS-Peptide 5-a
15
AQPFLLRNGANEGFH
79

VS-Peptide 5-b
20
AYAAQPFLLRNGANEGFHEA
80

VS-Peptide 5-c
25
YDMAYAAQPFLLRNGANEGFHEAVG
81

VS-Peptide 5-d
30
IQYDMAYAAQPFLLRNGANEGFHEAVGEIMS
82

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

The present disclosure is further illustrated by the following non-limiting examples.

EXAMPLES
Example 1: Materials and Methods

Tissue Culture

Human Normal Primary Small Airway Epithelial Cells (HSAEC) (ATCC Number: PCS-301-010) and Human Normal Primary Bronchial/Tracheal Epithelial Cells (HBTEC) (ATCC Number: PCS-300-010) were purchased from American Type Culture Collection (ATCC) (Manassas, Va.). Human Nasal Epithelial Cells (HNEpC) were purchased from PromoCell (Catalog Number: C-12621) (Heidelberg, Germany). HSAEC and HBTEC were grown Airway Epithelial Cell Basal Medium supplement with Bronchial Epithelial Cell Growth Kit (ATCC, PCS-300-030 and PCS-300-040). HNEpC were grown in Airway/Nasal Epithelial Cell Grow Medium (PromoCell, C-21060)). Cells were maintained in a humidified atmosphere with 5% CO₂at 37° C.

RNAi, ASO Delivery

Transfection

HSAEC, HBTEC and HNEpC were seeded into 6-well tissue-culture plate (VWR-USA, 10062-892), and transfected with lipofectamine 3000 (Thermo Fisher Scientific, L3000001) per manufacturer's protocol.

“Untreated”: medium

“Treated”: SARS-CoV-2 viral protein expression vectors (S-protein, E-protein, N-protein, RdRp and ORF1ab) or SARS-CoV-2 viral protein expression vectors (S-protein, E-protein, N-protein, RdRp and ORF1ab)+VS-vector.

“Overexpression vector”: N-protein: MC_0101137, GenScript, S-protein: MC_0101080, GenScript

E-protein: MC_0101135, GenScript

ORF1ab: MC_0101079, GenScript

RdRP: MC_0101076, GenScript”

Arginine Delivery

HSAEC, HBTEC and HNEpC were seeded into tissue-culture plate (VWR-USA, 10062-892). The cells were either left untreated, or treated with the following mixture: 10 μL arginine (200 mg/mL), SARS-CoV-2 N, S and E protein overexpression vector, and VS-vector (VS-RNAi or VS-ASO).

“Untreated”: medium

“Treated”: COVID-19 viral protein expression vectors (S-protein, E-protein, N-protein, RdRp and ORF1ab) or COVID-19 viral protein expression vectors (S-protein, E-protein, N-protein, RdRp and ORF1ab)+VS-vector.

“Overexpression vector”: N-protein: MC_0101137, GenScript; S-protein: MC_0101080, GenScript; E-protein: MC_0101135, GenScript; ORF1ab: MC_0101079, GenScript; RdRP: MC_0101076, GenScript.

Confocal Microscopy

Untreated or VS-vector treated HSAEC, HBTEC and HNEpC were washed twice in phosphate buffered solution (PBS, pH 7.4). The cells were then observed under confocal microscope (BD pathway 855) with four channels: transmitted, FITC (excitation filter: 488/10, emission: 515LP), Cy3 (excitation filter: 548/20, emission filter: 84101) and Cy5 (excitation filter: 635/20, emission filter: 84101) respectively.

Western Blot Analysis

Untreated or VS-vector treated HSAEC, HBTEC and HNEpC were washed twice in phosphate buffered solution (PBS, pH 7.4), scraped into 15-mL conical tubes, and centrifuged at 1,000×g at 4° C. for 5 minutes. Cell extracts were prepared by lysis in NET buffer [50 mmol/L Tris-HCl, (pH 7.4), 150 mmol/L NaCl, 0.1% NP40, 1 mmol/L EDTA, 0.25% gelatin, 0.02% sodium azide, 1 mmol/L phenylmethylsulfonyl fluoride, and 1% aprotinin]. The lysates were centrifuged at 15,000×g for 30 minutes at 4° C. The protein concentrations in the supernatant fractions were determined by Bicinchoninic Acid assay (Thermo Fisher Scientific, 23255).

Ten micrograms of total HSAEC protein extracts were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in PBS/Tween 20 (0.05%) for 1 hour, followed by overnight incubation with 1p g/mL anti-COVID19-Spike-protein antibody (ProSci, 3223) in 10% milk/PBS-T and 1 μg/mL anti-COVID19-N-protein antibody (ProSci, 3857) in 10% milk/PBS-T. Loading control antibody used rabbit anti-GAPDH antibody (Novus Biologicals, NB100-56875) with 1:1000 dilution or mouse anti-GAPDH antibody (Novus Biologicals, NBP2-27103). Secondary antibody: rabbit IgG HRP-conjugated Antibody (R&D System, HAF008) with 1:1000 dilution and mouse IgG HRP-conjugated Antibody (R&D System, HAF007) with 1:1000 dilution. The detection was done using horseradish peroxidase-labeled secondary antibodies and enhanced chemiluminescence detection reagent.

S-Protein, N-Protein, E-Protein, ORF1ab and RdRP Detection by Quantitative Real-Time PCR (qRT-PCR)

Untreated or treated HSAEC, HBTEC and HNEpC were washed twice in PBS. RNA was extracted from cells per manufacturer's protocol using Qiagen RNA extraction kit (Qiagen, 74104). One-step RT-PCR was conducted by StepOne Real-Time PCR System (Thermo Fisher Scientific, CA): 5 μl extracted RNA (300 ng/μl) samples were added to a mixture of 6.25 μl Taqman-Master-Mix (Thermo Fisher Scientific, 4444432), 11.25 μl PCR-grade water (Thermo Fisher Scientific, AM9916) and 1.25 FAM Probe (information as following):

For SARS-CoV-2 S-protein: Thermo Fisher Scientific, A47532

For SARS-CoV-2 N-protein: Thermo Fisher Scientific, A47532

For SARS-CoV-2 ORF1ab: Thermo Fisher Scientific, A47532

For RNAse P (internal control): Thermo Fisher Scientific, A47532

For SARS-CoV-2 E protein: GenScript, E_Sarbeco_P1

(SEQ ID NO: 53)

(ACACTAGCCATCCTTACTGCGCTTCG).

Apoptosis Analysis by qRT-PCR

Untreated or treated HSAEC, HBTEC and HNEpC were washed twice in PBS. RNA was extracted from cells per manufacturer's protocol using Qiagen RNA extraction kit (Qiagen, 74104). Total RNA was reverse-transcribed to cDNA according to manufacturer's protocol (Thermo Fisher Scientific, 18091050). The cDNA concentration was determined by NanoDrop 1000 (Thermo Scientific). Real-Time PCR was conducted by StepOne Real-Time PCR System (Thermo Fisher Scientific, CA): 3 μl cDNA (300 ng/ul) samples were added to a mixture of 10 μl Taqman-Master-Mix (Thermo Fisher Scientific, 4444557), 5 μl of PCR-grade water (Thermo Fisher Scientific, AM9916) and 2 μl FAM Probe (information as following):

For BCL2: Thermo Fisher Scientific, Hs04986394_s1

For BAX: Thermo Fisher Scientific, Hs00180269_m1

For GAPDH (internal control): Hs02786624_g1

Fluorescence Signal Detection by Flow Cytometry

Untreated or treated HSAEC, HBTEC and HNEpC were washed twice in PBS (pH 7.4), scraped into 15-mL conical tubes, and centrifuged at 1,000×g at 4° C. for 5 minutes. Cells were resuspended into 0.5 mL PBS/BSA (0.5%). Fluorescence signal detection was carried out on BD FacsCalibur (BD Biosciences) with excitation laser (485 nm, 635 nm) and emission channel (530/30, 585/42, 670/LP and 661/16). 10,000 cells (or event) were collected for analysis.

Cytotoxicity by MTT Assay

Cells were seeded in 96-well plates (100 μL per well of 1×10⁵cells/mL). The cells were left undisturbed until they adhered to the plate. The cells were then treated with culture medium (control) or VS-vectors. After the cells were incubated with MTT dye (Sigma) for another 4 h at 37° C., the medium was removed and the crystal formazan dye was solubilized in 150 μL dimethyl sulphoxide (DMSO; Sigma). Absorbance was measured at 570 nm by using the Bio-Tek Powerwave X microplate reader (BioTek Instruments).

Cell Cycle and Cell Apoptosis Rate Analysis

HSAEC, HTBEC and HNEpC were seeded in 25-mL culture flasks at a density of 5×10⁵cell/mL and cultured in recommended medium until the cells adhered to the flasks. The cells were then treated with culture medium (control) or VS-vector for 24 h. Cells were harvested by scrapping, and aliquots of 1×10⁶cell/mL were prepared for analysis. The cells were washed with PBS twice (centrifuged at 300 g with 5 min and resuspended in PBS), fixed with ice-chilled 70% ethanol for 24 h, and then treated with 20 mg/L RNase for 30 min. Propidium iodide (Sigma) was added to a final concentration of 20 mg/L. DNA contents of the samples were analyzed on a BD FacsCalibur (BD Biosciences), and the number of cells in every phase was calculated using FlowJo.

VS-Peptide In Vitro Fluorescent Observation and Detection

HSAEC, HBTEC and HNEpC were seeded into 24-well tissue-culture plate (VWR-USA, 10062-896), and transfected (Thermo Fisher Scientific, L3000001) with CMV-human ACE2-vector by lipofectamine 3000 (Thermo Fisher Scientific, L3000001) per manufacturer's protocol. After 24 hours, peptides were added into 24-well tissue-culture plate as follows:

Untreated: SARS-CoV-2-S-Protein-peptide-FITC (final concentration: 1 ug per 1×10⁵cells)

VS-peptide-1-dosage-1: SARS-CoV-2-S-Protein-peptide-FITC (final concentration: 1 ug per 1×10⁵cells)+VS-peptide-1 (final concentration: 1 ug per 1×10⁵cells)

VS-peptide-1-dosage-2: SARS-CoV-2-S-Protein-peptide-FITC (final concentration: 1 ug per 1×10⁵cells)+VS-peptide-1 (final concentration: 1 ug per 1×10⁵cells)

VS-peptide-2-dosage-1: SARS-CoV-2-S-Protein-peptide-FITC (final concentration: 1 ug per 1×10⁵cells)+VS-peptide-2 (final concentration: 1 ug per 1×10⁵cells)

VS-peptide-2-dosage-2: SARS-CoV-2-S-Protein-peptide-FITC (final concentration: 1 ug per 1×10⁵cells)+VS-peptide-2 (final concentration: 1 ug per 1×10⁵cells)

VS-peptide-3-dosage-1: SARS-CoV-2-S-Protein-peptide-FITC (final concentration: 1 ug per 1×10⁵cells)+VS-peptide-3 (final concentration: 1 ug per 1×10⁵cells)

VS-peptide-3-dosage-2: SARS-CoV-2-S-Protein-peptide-FITC (final concentration: 1 ug per 1×10⁵cells)+VS-peptide-3 (final concentration: 1 ug per 1×10⁵cells)

VS-peptide-4-dosage-1: SARS-CoV-2-S-Protein-peptide-FITC (final concentration: 1 ug per 1×10⁵cells)+VS-peptide-4 (final concentration: 1 ug per 1×10⁵cells)

VS-peptide-4-dosage-2: SARS-CoV-2-S-Protein-peptide-FITC (final concentration: 1 ug per 1×10⁵cells)+VS-peptide-4 (final concentration: 1 ug per 1×10⁵cells)

VS-peptide-combination: SARS-CoV-2-S-Protein-peptide-FITC (final concentration: 1 ug per 1×10⁵cells)+VS-peptide-1+VS-peptide-2+VS-peptide-3+VS-peptide-4 (final concentration: 1 ug per 1×10⁵cells)

Observed under confocal microscope (BD pathway 855) with channel: transmitted, FITC (excitation: 488/10, emission: 515LP) and detected FITC intensity via fluorescent microplate reader (BIO-TEK Synergy HT) in each well.

Inhibitions of Nucleotides on the Viral Infections from Both Pseudo-Viruses of Wild-Type and Mutant Forms of the COVID-19

5000 HELA cells were seeded per well in 96-well-plate. Co-transfections of ACE-2 (50 ng) and nucleosides (500 nM). For control-1, transfection of ACE-2 (50 ng) in the cells, and the scramble nucleotides (500 nM) was added.

24 hr-post transfection, add 4 ul of the wild-type virus of SARS-CoV-2 Spike pseudo-typed in the lentivirus, (WV: titer 10⁵TU/ml, eGFP reporter with catalog: 79981, BPS Bioscience) or/and 4 ul of the mutant virus of SARS-CoV-2 Spike (MV: B.1.1.7 mutant variant virus from UK/England) pseudo-typed in the lentivirus (titer 10⁵TU/ml, eGFP reporter with catalog: 78158, BPS Bioscience), and the polybrene was added into each well, as its final concentration of 5 ug/ml.

Observation on the GFP expression in the transduced cells under the confocal microscope in 48-72 hrs after viruses added into the wells.

ELISA Assays

A 96 well dish was coated with the ACE2 protein (Cat #: 10-014, ProSci, Inc) at the concentration of 10 ng/well at 4° C. overnight. After overnight, washed 3 times (400 ul/well/time) by 1× washing buffer (Cat #: DY008, R&D System). Later, it was blocked by 3% BSA for 1 hr RT, and washing 3 times (400 ul/well/time) by 1× washing buffer (Cat #: DY008, R&D System). VS Peptides of 1 to 4 (see Table 1) were diluted into two concentrations of 100 ug/well and 50 ug/well, and then mixed with the Receptor-Binding Domain (RBD) of Covid-19 viral Spike recombinant protein (S-RBD) (Cat #: 10-303, ProSci, Inc) at the concentration of 3000 ng/well for 30 min at 37° C. before adding onto each well. After adding the mixture solution of peptides and S-RBD, the specific anti-S-RBD antibody (Cat #: 9087, ProSci, Inc) was added onto each well at a concentration of 1000 ng/well at 37° C. for 30 min at a tissue-culture incubator. After the incubation, wells were washed by 1× washing buffer for 3 times (300 ul/well/time). The secondary HRP (Horseradish Peroxidase) antibody (Cat #: HAF008, R&D System), that binds to the primary antibody, was then added onto each well (1:30000) 100 ul/well for 30 min in RT. After the incubation, the well was washed by 1× washing buffer for 3 times (300 ul/well/time) (Cat #: DY008, R&D System). 100 ul color substrate (Cat #: DY008, R&D System) was added to each well for 20 min at RT to show the color. Finally, 50 ul of stop solution was added to each well (Cat #: DY008, R&D System). The 96-well plate was placed inside the Microplate Reader (Model: Bio-TEK Synergy HT) and read the wavelength signal autumnally by the computer program. The signals of intensities from the “yellow-like” color, called as TMB signals, were scanned and read at a wave-length of 450 nm by the Microplate Reader, and recorded and calculated by its computer software automatedly (see the FIG. 1S: the table at the left-bottom), the 570 nm was measured as the background signals that was subtracted out from the final data analysis.

Example 2: VS-Nucleotide Treatment of Human Primary Small Airway Epithelial Cells (HSAEC) Transfected with COVID-19 Viral Proteins

FIG. 1 shows experimental designs of investigating on delivery capable and therapeutic effects of ASO(s) and siRNA on human primary small airway epithelial cells transfected with viral protein of SARS-CoV-2. The human lung small airway epithelial cells were cultured in the 24 well-dish, and the cells were transfected with the genes encoding the viral proteins of SARS-CoV-2. The VS_ASO_1-FANA-FITC, VS_DsiRNA-Cy5 and VS_ASO_2-Cy3 were into the cells for 24-48 hours before analysis with fluorescent microscope. The VS_ASO_1-FANA-FITC designed with FITC labeled shown in the Table 1, and VS_ASO_2-Cy3 with Cy3 label shown in the Table 2; and VS_DsiRNA-Cy5 with Cy5 label shown in Table 3. A1&A2: No treatment as control, A3&A4: Overexpression of both COVID-19 N-protein and the VS_ASO_1-FANA using lipofectamine reagent, A5&A6: Overexpression of both COVID-19 N-protein and the VS_ASO_1-FANA without any regents; B1&B2: No treatment as control, B3&B4: Overexpression of both COVID-19 N-protein and the VS_DsiRNA-Cy5 using lipofectamine reagent, B5&B6: Overexpression of both COVID-19 N-protein and the VS_DsiRNA-Cy5 using Poly-arginine (5 μl/well) only; C1&C2: No treatment as control, C3&C4: Overexpression of both COVID-19 N-protein and the VS_ASO_2-Cy3 using lipofectamine reagent, C5&C6: Overexpression of both COVID-19 N-protein and the VS_ASO_2-Cy3 using Poly-arginine (5 μl/well) only.

FIGS. 2A-2F show microscopic analysis showing entry of VS_ASO_1-FANA-FITC into the primary human lung small airway epithermal cells (20×). FIGS. 2A-2C were captured under the FITC florescent filter, and FIGS. 2D-2F were captured in the same view of bright fields (20×). FIGS. 2A and 2D were taken in well A3 & A4 (as shown in FIG. 1), FIGS. 2B and 2E were taken in well A5 & A6 (as shown in FIG. 1), and FIGS. 2C and 2F were taken in well A 1 & A2 (as shown in FIG. 1).

FIGS. 3A-3F show microscopic analysis showing entry of VS_ASO_1-FANA-FITC into primary human lung small airway epithermal cells (10×). FIGS. 3A-3C were captured under the FITC florescent filter, and FIGS. 3D-3F were captured in the same view of bright fields (20×). FIGS. 3A and 3D were taken in well A3 & A4 (as shown in FIG. 1), FIGS. 3B and 3E were taken in well A5 & A6 (as shown in FIG. 1), and FIGS. 3C and 3F were taken in well A1 & A2 (as shown in FIG. 1).

FIGS. 4A-4F show microscopic analysis showing entry of VS_DsiRNA-Cy5 into primary human lung small airway epithermal cells (20×). FIGS. 4A-4C were captured under the Cy5 florescent filter, and FIGS. 4D-4F were captured in the same view of bright fields (20×). FIGS. 4A and 4D were taken in well B3 & B4 (as shown in FIG. 1), FIGS. 4B and 4E were taken in well B5 & B6 (as shown in FIG. 1), and FIGS. 4C and 4F were taken in well B1 & B2 (as shown in FIG. 1).

FIGS. 5A-5F show microscopic analysis entry of VS_DsiRNA-Cy5 into primary human lung small airway epithermal cells (10×). FIGS. 5A-5C were captured under the Cy5 florescent filter, and FIGS. 5D-5F were captured in the same view of bright field images (10×). FIGS. 5A and 5D were taken in well B3 & B4 (as shown in FIG. 1), FIGS. 5B and 5E were taken in well B5 & B6 (as shown in FIG. 1), and FIGS. 5C and SF were taken in well B1 & B2 (as shown in FIG. 1).

FIGS. 6A-6F show microscopic analysis entry of VS_ASO_2-Cy3 into primary human lung small airway epithermal cells (20×). FIGS. 6A-6C were captured under the Cy3 florescent filter, and FIGS. 6D-6F were captured in the same view of bright fields (20×). FIGS. 6A and 6D were taken in well C3 & C4 (as shown in FIG. 1), FIGS. 6B and 6E were taken in well C5 & C6 (as shown in FIG. 1), and FIGS. 6C and 6F were taken in well C1 & C2 (as shown in FIG. 1).

FIGS. 7A-7F show microscopic analysis showing entry of VS_ASO_2-Cy3 into primary human lung small airway epithermal cells (10×). The FIGS. 7A-7C were captured under the Cy3 florescent filter, and FIGS. 7D-7F were captured in the same view of bright fields (10×). FIGS. 7A and 7D were taken in well C3 & C4 (as shown in FIG. 1), FIGS. 7B and 7E were taken in well C5 & C6 (as shown in FIG. 1), and FIGS. 7C and 7F were taken in well C1 & C2 (as shown in FIG. 1).

Example 3: Intercellular Delivery of Inhibitory Oligonucleotides in Human Primary Lung Small Airway Epithelial Cells (HSAEC)

FIG. 8 shows experimental design for FACS detection of intercellular delivery of oligos in the human primary lung small airway epithelial cells (HSAEC). The human lung small airway epithelial cells were cultured in the 6-well dish, and the genes encoding the viral proteins of SARS-CoV-2 were delivered by transfection or arginine delivery. The siRNA or ASO were added into the cells for 24-48 hours before analysis with FACS. The VS_ASO_1-FANA-FITC designed with labeled with FITC shown in the Table 1, and VS_ASO_2-Cy3 with modification shown in the Table 2; and VS_DsiRNA-Cy5 shown in Table 3. A1: No treatment as control; A2: Overexpression of N-protein+VS_ASO_1-FANA-FITC without lipofectamine or arginine; A3: Overexpression of N-protein+VS_DsiRNA-Cy5 with lipofectamine; B1: Overexpression of N-protein+VS_DsiRNA-Cy5 with Arginine (10 μl/well); B2: Overexpression of N-protein+VS_ASO_2-Cy3 with lipofectamine; B3: Overexpression of N-protein+VS_ASO_2-Cy3 with Arginine (10 μl/well).

FIGS. 9A-9C show FACS analysis of in vitro treatment with VS_ASO_1-FANA-FITC without lipofectamine or Arginine in human primary lung small airway epithelial cells (HSAEC). FACS analysis of HSAEC treated by VS_ASO_1-FANA-FITC (excitation: 488 nm, emission band pass filter: 530/30, Total event: 20,000). FIG. 9A no-treatment control, FIG. 9B VS_ASO_1-FANA-FITC and FIG. 9C Merge. The FACS data indicate that the intensities of FITC signals were significantly stronger with shifting to the right (B: FL1-H:FITC) when compared with the control FIG. 9A in the cells after treated with the VS_ASO_1-FANA-FITC without lipofectamine or Arginine reagents FIG. 9B. The FIG. 9C is the merged figures of FIGS. 9A and 9B.

FIGS. 10A-10B show FACS analysis of in vitro treatment with VS_DsiRNA-Cy5 with lipofectamine FIG. 10A or Arginine only FIG. 10B in human primary lung small airway epithelial cells (HSAEC). FACS analysis of HSAEC treated by VS_DsiRNA-Cy5 (excitation: 635 nm, emission band pass filter: 661/16, Total event: 20,000). Left to right panel: no-treatment control, VS_DsiRNA-Cy5 and merge. The FACS data indicates that the intensities of Cy5 signals were significantly higher with shifting to the right (middle panel: FL4-H:Cy5) in both of panel (FIG. 10A) and (FIG. 10B), it also shown that there are more cells with intercellular signals of the oligos in the presence of 10 μl/well Arginine (panel FIG. 10B) when compared with the lipofectamine (panel FIG. 10A).

FIGS. 11A-11B show FACS analysis of in vitro treatment with VS_ASO_2-Cy3 with lipofectamine FIG. 11A or Arginine only FIG. 11B in human primary lung small airway epithelial cells (HSAEC). FACS analysis of HSAEC treated by VS_VS_ASO-Cy3 (excitation: 488 nm, emission band pass filter: 585/42, Total event: 20,000). Left to right panel: no-treatment control, VS_ASO_2-Cy3 and merge. The FACS data indicates that the intensities of Cy3 signals were significantly higher with shifting to the right (middle panel: FL2-H:Cy3) in both of (FIG. 11A) and (FIG. 11B), it also shown that there are more cells with intercellular signals of the oligos in the presence of 10 μl/well Arginine (panel FIG. 11B) when compared with the lipofectamine (panel FIG. 11A).

Example 4: SARS-CoV-2 N-Protein Expression is Reduced in Human Primary Lung Small Airway Epithelial Cells (HSAEC) after Treatment by VS-Oligonucleotides

FIG. 12 shows the experimental design for detecting SARS-CoV-2 N-protein expressed in the human primary lung small airway epithelial cells (HSAEC) by qRT-PCR after treatment. The human lung small airway epithelial cells were cultured in a 24-well dish, and the cells were transfected with the genes encoding the viral protein (N-protein) of SARS-CoV-2. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with RT-PCR. The VS_ASO_1-FANA-FITC designed with FITC labeled shown in the Table 1, and VS_ASO_2-Cy3 with Cy3 modification shown in the Table 2; and VS_DsiRNA-Cy5 with Cy5 modification shown in Table 3. A1&A2: No treatment as control, A3&A4: Overexpression of COVID-19 N-protein, A5&A6: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA_Cy5 with lipofectamine, B1&B2: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2-Cy3 with lipofectamine, B3&B4: Overexpression of COVID-19 N-protein+/treated by VS_ASO_1-FANA without any reagents.

FIG. 13 shows detection of SARS-CoV-2 N-protein expressed in the human primary lung small airway epithelial cells (HSAEC) by qRT-PCR after treatment with VS-Nucleotides. Significant down-regulation was observed: about 5-fold in group treated by VS_DsiRNA-Cy5 oligos (p<0.005); about 1.5 fold in the group treated by VS_ASO_2-cy3 oligo (p<0.01), and about 6 fold in the group treated by the VS_ASO_1-FANA-FITC oligo (p<0.005); when compared with the group with SARS-CoV-2 N-protein overexpression only. The cycle threshold of no-treatment is non-detectable, but in this case for calculation purposes, the number “40” was used as the cycle threshold for a base-line control.

FIG. 14 shows experimental design for detection of SARS-CoV-2 S-protein expressed in the primary human lung small airway epithelial cells (HSAEC) by qRT-PCR after treatment. The human primary lung small airway epithelial cells (HSAEC) were cultured in the 24 well-dish, and the cells were transfected with the genes encoding the viral protein (S-protein) of SARS-CoV-2. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with RT-PCR. The VS_ASO_1-FANA (oligo 3) designed shown in the Table 1, and VS_ASO_2 (oligo 3) shown in the Table 2; and VS_DsiRNA (oligo 3) shown in Table 3. A1&A2: No treatment as control, A3&A4: Overexpression of COVID-19 S-protein, A5&A6: Overexpression of COVID-19 S-protein+/treated by VS_DsiRNA (oligo 3) with lipofectamine, B1&B2: Overexpression of COVID-19 S-protein+/treated by VS_DsiRNA (oligo 3) with Arginine (5 μl/well), B3&B4: Overexpression of COVID-19 S-protein+/treated by VS_ASO_2 (oligo 3) with lipofectamine, B5&B6: Overexpression of COVID-19 S-protein+/treated by VS ASO_2 (oligo 3) with Arginine (5 μl/well), C1&C2: Overexpression of COVID-19 S-protein+/treated by VS_ASO_1-FANA (oligo 3) without any reagents.

FIG. 15 shows detection of SARS-CoV-2 S-protein expressed in the human primary lung small airway epithelial cells (HSAEC) by qRT-PCR after treatment with VS-Nucleotides. Significant down-regulation was observed: about 4 fold in group treated by VS_DsiRNA oligo (purple/L: p<0.01) and about 2.8 fold in the presence of poly-Arginine only (yellow/A: p<0.001); about 4 fold in the group treated by VS_ASO_2 oligo (red/L: p<0.001) and about 4 fold in the in the presence of poly-Arginine only (orange/A: p<0.001); and about 11.5 fold in the group treated by the VS_ASO_1-FANA oligo (green/p<0.001); when compared with the group with SARS-CoV-2 S-protein overexpression only. The cycle threshold of no-treatment is non-detectable, but in this case for calculation purposes, the number “40” was used as the cycle threshold for a base-line control.

FIG. 16 shows the experimental design for detection of both of SARS-CoV-2 ORF1ab and RdRp expressed in the primary human lung small airway epithelial cells (HSAEC) by qRT-PCR after treatment. The human primary lung small airway epithelial cells (HSAEC) were cultured in the 24-well dish, and the cells were transfected with the genes encoding both ORF1ab and RdRp of SARS-CoV-2 viral protein. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with RT-PCR. The VS_ASO_1-FANA (oligo 1, 2, 5 and 6) designed shown in the Table 1, and VS_ASO_2 (oligo 1, 2, 5 and 6) shown in the Table 2; and the VS_DsiRNA (oligo 1, 2, 5 and 6) shown in Table 4. A1&A2: No treatment as control, A3&A4: Overexpression of COVID-19 viral genes encoding both ORF1ab and RdRp, A5&A6: Overexpression of both ORF1ab and RdRp+/treated by VS_DsiRNA (oligo 1, 2, 5 and 6) with lipofectamine, B1&B2: Overexpression of both ORF1ab and RdRp+/treated by VS_DsiRNA (oligo 1, 2, 5 and 6) with Arginine (5 μl/well), B3&B4: Overexpression of both ORF1ab and RdRp+/treated by VS_ASO_2 (oligo 1, 2, 5 and 6) with lipofectamine, B5&B6: Overexpression of both ORF1ab and RdRp+/treated by VS_ASO_2 (oligo 1, 2, 5 and 6) with Arginine (5 μl/well), C1&C2: Overexpression of both ORF1ab and RdRp+/treated by VS_ASO_1-FANA (oligo 1, 2, 5 and 6) without any reagents.

FIG. 17 shows detection of SARS-CoV-2 ORF1ab and RdRp expressed in the human primary lung small airway epithelial cells (HSAEC) by qRT-PCR after treatment with inhibitory oligonucleotides. Significant down-regulation was observed: about 3.5 fold in group treated by VS_DsiRNA oligo (purple/L: p<0.01) and about 2.2 fold in the presence of poly-Arginine only (yellow/A: p<0.001); about 4.5 fold in the group treated by VS_ASO_2 oligo (red/L: p<0.001) and about 2.1 fold in the in the presence of poly-Arginine only (orange/A: p<0.001); and about 11.8 fold in the group treated by the VS_ASO_1-FANA oligo (green/p<0.001); when compared with the group with SARS-CoV-2 ORF1ab and RdRp overexpression only. The cycle threshold of no-treatment is non-detectable, but in this case for calculation purposes, the number “40” was used as the cycle threshold for a base-line control.

Example 5: SARS-CoV-2 N-Protein Levels are Decreased in Human Primary Lung Small Airway Epithelial Cells (HSAEC) after Treatment by Inhibitory Oligonucleotides

FIG. 18 shows the experimental design for detection of SARS-CoV-2 N-protein expressed in the primary human lung small airway epithelial cells (HSAEC) by Western Blot after treatment. The human primary lung small airway epithelial cells (HSAEC) were cultured in the 6-well dish, and the cells were transfected with the genes encoding the viral protein (N-protein) of SARS-CoV-2. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with Western Blot. The VS_ASO_2 (oligo 4 & 8) shown in the Table 2; and VS_DsiRNA (oligo 4 & 8) shown in Table 4. A1: No treatment as control, A2: Overexpression of COVID-19 N-protein, A3: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA (oligo 4 and 8) with lipofectamine, B1: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2 (oligo 4 and 8) with lipofectamine.

FIG. 19 shows detection of SARS-CoV-2 N-protein expressed in the human primary lung small airway epithelial cells (HSAEC) by Western Blot after treatment with inhibitory oligonucleotides: Lane-1: no treatment; Lane-2: SARS-CoV-2 N-protein overexpression (OE); Lane-3: SARS-CoV-2 N-protein OE+/treated by VS_DsiRNA (oligo 4 & 8); Lane-4: SARS-CoV-2 N-protein OE+/treated by the VS_ASO_2 (oligo 4 & 8). The 10 ug total cell-lysis were added into each well. primary antibody: 1p g/mL anti-SARS-CoV-2-N-protein antibody (ProSci, 3857) and anti-GAPDH antibody (Novus Biologicals, NBP2-27103) with 1:1000 dilution. The secondary antibody: goat-anti-rabbit HRP-conjugated Antibody (R&D System, HAF008) with 1:1000 dilution and goat-anti-mouse IgG HRP-conjugated Antibody (R&D System, HAF007) with 1:1000 dilution. The detection was done using horseradish peroxidase-labeled secondary antibodies and enhanced chemiluminescence detection reagent.

Example 6: Inhibitory Oligonucleotide Treatment of Human Primary Bronchial/Tracheal Epithelial (HBTEC) Cells Transfected with Viral Proteins of SARS-CoV-2

FIG. 20 shows experiments designed for investigating cell penetration and therapeutic effects of VS-Nucleotides (inhibitory oligonucleotides) on human primary bronchial/tracheal epithelial cells (HBTEC) transfected with viral protein of SARS-CoV-2 after treatment. The primary human bronchial/tracheal epithelial cells (HBTEC) were cultured in a 24-well dish, and the cells were transfected with the genes encoding the viral proteins of SARS-CoV-2. The VS_ASO_1-FANA-FITC, VS_DsiRNA-Cy5 and VS_ASO_2-Cy3 were added into the cells for 24-48 hours before analysis with fluorescent microscope. VS_ASO_1-FANA-FITC designed with FITC labeled shown in the Table 1, and VS_ASO_2-Cy3 with Cy3 label shown in the Table 2; and VS_DsiRNA-Cy5 with Cy5 label shown in Table 3. A1&A2: No treatment as control, A3&A4: Overexpression of COVID-19 N-protein+/treated by VS_ASO_1-FANA-FITC without lipofectamine or/and Poly-arginine, B1&B2: No treatment as control, B3&B4: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA-Cy5 with lipofectamine, B5&B6: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNAi-Cy5 with arginine (5 μl/well), C1&C2: No treatment as control, C3&C4: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2-Cy3 with lipofectamine, C5&C6: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2-Cy3 with arginine (5 μl/well).

FIGS. 21A-21D shows microscopic analysis showing entry of VS_ASO_1-FANA-FITC into primary human lung bronchial/tracheal epithelial cells (20×). FIGS. 21A-21B were captured under FITC florescent filter, and FIGS. 21C and 21D were captured in the same view of bright fields (20×). FIGS. 21A and 21C were taken in well A1 & A2 (as shown in FIG. 20), and FIGS. 21B and 21D were taken in well A3 & A4 (as shown in FIG. 20).

FIGS. 22A-22D shows microscopic analysis showing entry of VS_ASO_1-FANA-FITC into primary human lung bronchial/tracheal epithelial cells (10×). FIG. 22A-22B were captured under FITC florescent filter, and FIGS. 22C and 22D were captured in the same view of bright fields (10×). FIGS. 22A and 22C were taken in well A1 & A2 (as shown in FIG. 20), and FIGS. 22B and 22D were taken in well A3 & A4 (as shown in FIG. 20).

FIGS. 23A-23F shows microscopic analysis showing entry of VS_DsiRNA-Cy5 into primary human lung bronchial/tracheal epithermal cells (20×). FIG. 23A-23C were captured under the Cy5 florescent filter, and FIGS. 23D-23F were captured in the same view of bright fields (20×). FIGS. 23A and 23D were taken in well B3 & B4 (as shown in FIG. 20), FIGS. 23B and 23E were taken in well B5 & B6 (as shown in FIG. 20), and FIGS. 23C and 23F were taken in well B1 & B2 (as shown in FIG. 20).

FIGS. 24A-24F shows microscopic analysis showing entry of VS_DsiRNA-Cy5 into primary human lung bronchial/tracheal epithermal cells (10×). FIGS. 24A-24C were captured under the Cy5 florescent filter, and FIGS. 24D-24F were captured in the same view of bright fields (20×). FIGS. 24A and 24D were taken in well B3 & B4 (as shown in FIG. 20), FIGS. 24B and 24E were taken in well B5 & B6 (as shown in FIG. 20), and FIGS. 24C and 24F were taken in well B1 & B2 (as shown in FIG. 20).

FIGS. 25A-25F shows microscopic analysis showing entry of VS_ASO_2-Cy3 into primary human lung bronchial/tracheal epithermal cells (20×). FIGS. 25A-25C were captured under the Cy3 florescent filter, and FIGS. 25D-25F were captured in the same view of bright fields (20×). FIGS. 25A and 25D were taken in well B3 & B4 (as shown in FIG. 20), FIGS. 25B and 25E were taken in well B5 & B6 (as shown in FIG. 20), and FIGS. 25C and 25F were taken in well B1 & B2 (as shown in FIG. 20).

FIGS. 26A-26F shows microscopic analysis showing entry of VS_ASO_2-Cy3 into primary human lung bronchial/tracheal epithermal cells (10×). FIGS. 26A-26C were captured under the Cy3 florescent filter, and FIGS. 26D-26F were captured in the same view of bright fields (20×). FIGS. 26A and 26D were taken in well B3 & B4 (as shown in FIG. 20), FIGS. 26B and 26E were taken in well B5 & B6 (as shown in FIG. 20), and FIGS. 26C and 26F were taken in well B1 & B2 (as shown in FIG. 20).

Example 7: SARS-CoV-2 N-Protein Expression is Decreased Upon Treatment by the Inhibitory Oligonucleotides

FIG. 27 shows the experimental design for detection of SARS-CoV-2 N-protein expressed on human primary bronchial/tracheal epithelial cells (HBTEC) by qRT-PCR. The human primary bronchial/tracheal epithelial cells (HBTEC) were cultured in a 24-well dish, and the cells were transfected with the genes encoding the viral protein (N-protein) of SARS-CoV-2. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with RT-PCR. The VS_ASO_1-FANA-FITC designed with FITC labeled shown in the Table 1, and VS_ASO_2-Cy3 with Cy3 modification shown in the Table 2; and VS_DsiRNA-Cy5 with Cy5 modification shown in Table 3. A1&A2: No treatment as control, A3&A4: Overexpression of COVID-19 N-protein, A5&A6: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA-Cy5 with lipofectamine, B1&B2: Overexpression of COVID-19 N-protein+/treated by VS_Dsi RNA-Cy3 with lipofectamine, B3&B4: Overexpression of COVID-19 N-protein+/treated by VS_ASO_1-FANA without any reagents.

FIG. 28 shows detection of SARS-CoV-2 N-protein expressed in the human primary bronchial/tracheal epithelial cells (HBTEC) by qRT-PCR after treatment with inhibitory oligonucleotides. Significant down-regulation was observed: about 4 fold in the group treated by VS_DsiRNA-Cy5 oligos (p<0.005); about 6 fold in the group treated by VS_ASO_2-cy3 oligo (p<0.01), and about 8 fold in the group treated by the VS_ASO_1-FANA-FITC oligo (p<0.005); when compared with the group with SARS-CoV-2 N-protein overexpression only. The cycle threshold of no-treatment is non-detectable, but in this case for calculation purposes, the number “40” was used as the cycle threshold for a base-line control.

FIG. 29 shows the experimental design for detection of SARS-CoV-2 S-protein expressed in human primary bronchial/tracheal epithelial cells (HBTEC) by qRT-PCR. The human primary bronchial/tracheal epithelial cells (HBTEC) were cultured in a 24-well dish, and the cells were transfected with the genes encoding the viral protein (S-protein) of SARS-CoV-2. The siRNA or ASO were added into the cells for 24-48 hours before analysis with RT-PCR. The VS_ASO_1-FANA (oligo 3) designed is shown in the Table 1, VS_ASO_2 (oligo 3) is shown in the Table 2; and VS_DsiRNA (oligo 3) is shown in Table 3. A1&A2: No treatment, A3&A4: Overexpression of COVID-19 S-protein, A5&A6: Overexpression of COVID-19 S-protein+/treated by DsiRNA-Cy5 with lipofectamine, B1&B2: Overexpression of COVID-19 S-protein+/treated by VS_DsiRNA-Cy5 with Arginine (5 μl/well), B3&B4: Overexpression of COVID-19 S-protein+/treated by the VS_ASO_2-Cy3 with lipofectamine, B5&B6: Overexpression of COVID-19 S-protein+/treated by VS_ASO_2-Cy3 with Arginine (5 μl/well), C1&C2: Overexpression of COVID-19 S-protein+/treated by VS_ASO_1-FANA without any reagents.

FIG. 30 shows detection of SARS-CoV-2 S-protein expression in the human bronchial/tracheal epithelial cells (HBTEC) by qRT-PCR after treatment with inhibitory oligonucleotides. Significant down-regulation was observed: about 8 fold in the group treated by VS-DsiRNA oligo (purple/L: p<0.01), but about 16.3 fold in presence of Poly-arginine only (yellow/p<0.001); about 15.8 fold in the group treated by VS_ASO_2 oligo (red/L: p<0.001), but about 16.6 fold in presence of Poly-arginine only (orange/A: p<0.001); about 11.7 fold in the group treated by the VS_ASO_1 oligo (green/p<0.001); when compared with the group with SARS-CoV-2 S-protein overexpression only (1). The cycle threshold of no-treatment is non-detectable, but in this case for calculation purposes, the number “40” was used as the cycle threshold for a base-line control.

Inhibitory Peptide—ACE2 Protein Competition Assays

Inhibitory peptide (VS-peptide)—ACE2 protein Competition Assays prove the VSB peptides' therapeutic efficiency (blocking/interfere S-RBD binding to ACE2) in live cell condition. HeLa cells were transfected with ACE2 plasmid to express ACE2 (HeLa cells do not have endogenous ACE2 expression). Without VSB peptide, S-RBD (conjugated with FITC) will bind to ACE2 and resulting the HeLa-ACE2 cell with FITC (green) signal. In the presence of a VSB peptide, the S-RBD (conjugated with FITC) binds to VSB peptide instead of HeLa-ACE2 cells. In this case, HeLa-ACE2 cells has no FITC signal.

Example 8: SARS-CoV-2 ORF1ab and RdRp Expression is Decreased Upon Treatment by Inhibitory Oligonucleotides

FIG. 31 shows the experimental design for detection of SARS-CoV-2 ORF1ab and RdRp expressed in the human primary bronchial/tracheal epithelial cells (HBTEC) detected by qRT-PCR after treatment. The human primary bronchial/tracheal epithelial cells (HBTEC) were cultured in the 24-well dish, and the cells were transfected with the genes encoding the viral protein (ORF1ab and RdRp) of SARS-CoV-2. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with RT-PCR. VS_ASO_1-FANA (oligo 1, 2, 5 and 6) designed is shown in the Table 1, VS_ASO_2 (oligo 1, 2, 5 and 6) is shown in the Table 2; and the VS_DsiRNA (oligo 1, 2, 5 and 6) is shown in Table 4. A1&A2: No treatment as control, A3&A4: Overexpression of COVID-19 viral genes encoding both ORF1ab and RdRp, A5&A6: Overexpression of COVID-19 ORF1ab and RdRp+/treated by VS_DsiRNA (oligo 1, 2, 5 and 6) with lipofectamine, B1&B2: Overexpression of COVID-19 ORF1ab and RdRp+/treated by VS_DsiRNA (oligo 1, 2, 5 and 6) with Arginine (5 μl/well), B3&B4: Overexpression of COVID-19 ORF1ab and RdRp+/treated by VS_ASO_2 (oligo 1, 2, 5 and 6) with lipofectamine, B5&B6: Overexpression of COVID-19 ORF1ab and RdRp+/treated by VS_ASO_2 (oligo 1, 2, 5 and 6) with Arginine (5 μl/well), C1&C2: Overexpression of COVID-19 ORF1ab and RdRp+/treated by VS_ASO_1-FANA (oligo 1, 2, 5 and 6) without any reagents.

FIG. 32 shows detection of SARS-CoV-2 ORF1ab and RdRp expressed in the human primary bronchial/tracheal epithelial cells (HBTEC) detected by qRT-PCR after treatment with inhibitory oligonucleotides. Significant down-regulation was observed: about 3 fold in the group treated by VS_DsiRNA oligo (purple/L: p<0.01) and about 2.6 fold in the presence of poly-Arginine only (yellow/A: p<0.001); about 6.4 fold in the group treated by VS_ASO_2 oligo (red/L: p<0.001) and about 4.5 fold in the presence of poly-Arginine only (orange/A: p<0.001); and about 11.9 fold in the group treated by the VS_ASO_1-FANA oligo (green/p<0.001); when compared with the group with SARS-CoV-2 ORF1ab and RdRp overexpression only. The cycle threshold of no-treatment is non-detectable, but in this case for calculation purposes, the number “40” was used as the cycle threshold for a base-line control.

Example 9: SARS-CoV-2 N-Protein Levels are Down after Treatment by the Inhibitory Oligonucleotides

FIG. 33 shows the experimental design for detection of SARS-CoV-2 N-protein expressed in the human primary bronchial/tracheal epithelial cells (HBTEC) by Western Blot after treatment. The human primary bronchial/tracheal epithelial cells (HBTEC) were cultured in a 6-well dish, and the cells were transfected with the genes encoding the viral protein (N-protein) of SARS-CoV-2. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with Western Blot. The VS_ASO_02 (oligo 4 & 8) is shown in the Table 2; and VS_DsiRNA (oligo 4 & 8) is shown in Table 4. A1: No treatment as control, A2: Overexpression of COVID-19 N-protein, A3: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA (oligo 4 and 8) with lipofectamine, B1: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2 (oligo 4 and 8) with lipofectamine.

FIG. 34 shows detection of SARS-CoV-2 N-protein expressed in the human primary bronchial/tracheal epithelial cells (HBTEC) by Western Blot after treatment with inhibitory oligonucleotides. Lane 1: no treatment, Lane 2: SARS-CoV-2 N-protein overexpression (OE); Lane 3: SARS-CoV-2 N-protein OE+VS_DsiRNA (oligo 4 & 8); Lane 4: SARS-CoV-2 N-protein OE+VS_ASO_2 (oligo 4 & 8). 10 μg total cell lysis were added into each well and blotted with primary antibody (1 μg/mL anti-SARS-CoV-2-N-protein antibody (ProSci, 3857)) or anti-GAPDH antibody (Novus Biologicals, NBP2-27103) (1:1000 dilution). The secondary antibodies were goat-anti-rabbit HRP-conjugated Antibody (R&D System, HAF008) (1:1000) dilution and goat-anti-mouse IgG HRP-conjugated Antibody (R&D System, HAF007) (1:1000) dilution. The detection was done using horseradish peroxidase-labeled secondary antibodies and an enhanced chemiluminescence detection reagent.

FIG. 35 shows the experimental designs for investigating cell penetration and therapeutic effects of ASO(s) and DsiRNA on human primary nasal epithelial cells (HNEpC) transfected with viral protein of SARS-CoV-2 after treatment. The human primary nasal epithelial cells (HNEpC) were cultured in a 24-well dish, and the cells were transfected with the genes encoding the viral proteins of SARS-CoV-2. The VS_ASO_1-FANA-FITC, VS_DsiRNA-Cy5 and VS_ASO_2-Cy3 were delivered into the cells for 24-48 hours before analysis with fluorescent microscope. The VS_ASO_1-FANA-FITC was FITC labeled (see Table 1), and VS_ASO_2-Cy3 was Cy3 labeled (See Table 2); and VS_DsiRNA-Cy5 was Cy5 labeled (see Table 3). A1&A2: No treatment as control, A3&A4: Overexpression of COVID-19 N-protein+/treated by VS_ASO_1-FANA without lipofectamine or arginine, B1&B2: No treatment as control, B3&B4: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA-Cy5 with lipofectamine, B5&B6: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA-Cy5 with poly-arginine (5 μl/well), C1&C2: No treatment as control, C3&C4: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2-Cy3 with lipofectamine, C5&C6: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2-Cy3 with poly-arginine (5 μl/well).

FIGS. 36A-36D show the microscopic analysis of Human Primary Nasal Epithelial Cells at 20×. This analysis showed that VS_ASO_1-FANA-FITC can enter epithelial cells (20×). FIGS. 36A and 36B were captured under FITC florescent filter, and FIGS. 36C and 36D were captured in the same view of bright fields (20×). FIGS. 36A and 36C were taken in well A3 & A4 (as shown in FIG. 35), FIGS. 36B and 36D were taken in well A1 & A2 (as shown in FIG. 35).

FIGS. 37A-37D show the microscopic analysis of Human Primary Nasal Epithelial Cells at 20×. This analysis showed that VS_ASO_1-FANA-FITC can enter epithelial cells (10×). FIGS. 37A and 37B were captured under FITC florescent filter, and FIGS. 37C and 37D were captured in the same view of bright fields (20×). FIGS. 37A and 37C were taken in well A3 & A4 (as shown in FIG. 35), FIGS. 37B and 37D were taken in well A1 & A2 (as shown in FIG. 35).

FIGS. 38A-38F show the microscopic analysis of Human Primary Nasal Epithelial Cells at 20×. This analysis showed that VS_DsiRNA-Cy5 can enter epithelial cells (20×). FIGS. 38A-38C were captured under the Cy5 florescent filter, and FIGS. 38D-38F were captured in the same view of bright fields (20×). FIGS. 38A and 38D were taken in well B3 & B4 (as shown in FIG. 35), FIGS. 38B and 38E were taken in well B5 & B6 (as shown in FIG. 35), FIGS. 38C and 38F were taken in well B1 & B2 (as shown in FIG. 35).

FIGS. 39A-39F show the microscopic analysis of Human Primary Nasal Epithelial Cells at 10×. This analysis showed that VS_DsiRNA-Cy5 can enter epithelial cells (10×). FIGS. 39A-39C were captured under the Cy5 florescent filter, and FIGS. 39D-39F were captured in the same view of bright fields (20×). FIGS. 39A and 39D were taken in well B3 & B4 (as shown in FIG. 35), FIGS. 39B and 39E were taken in well B5 & B6 (as shown in FIG. 35), FIGS. 39C and 39F were taken in well B1 & B2 (as shown in FIG. 35).

FIGS. 40A-40F show the microscopic analysis of Human Primary Nasal Epithelial Cells at 20×. This analysis showed that VS_ASO_2-Cy3 can enter epithelial cells (20×). FIGS. 40A-40C were captured under the Cy3 florescent filter, and FIGS. 40D-40F were captured in the same view of bright fields (20×). FIGS. 40A and 40D were taken in well B3 & B4 (as shown in FIG. 35), FIGS. 40B and 40E were taken in well B5 & B6 (as shown in FIG. 35), FIGS. 40C and 40F were taken in well B1 & B2 (as shown in FIG. 35).

FIGS. 41A-41F show the microscopic analysis of Human Primary Nasal Epithelial Cells at 10×. This analysis showed that VS_ASO_2-Cy3 can enter epithelial cells (10×). FIGS. 41A-41C were captured under the Cy3 florescent filter, and FIGS. 41D-41F were captured in the same view of bright fields (10×). FIGS. 41A and 41D were taken in well B3 & B4 (as shown in FIG. 35), FIGS. 41B and 41E were taken in well B5 & B6 (as shown in FIG. 35), FIGS. 41C and 41F were taken in well B1 & B2 (as shown in FIG. 35).

Example 10: SARS-CoV-2 N-Gene Expression is Reduced after Treatment by the Inhibitory Oligonucleotides in HNEpCs

FIG. 42. show the experimental design for detection of SARS-CoV-2 N-protein expressed on human primary nasal epithelial cells (HNEpC) by qRT-PCR. The human primary nasal epithelial cells (HNEpC) were cultured in a 24-well dish, and the cells were transfected with the genes encoding the viral protein (N-protein) of SARS-CoV-2. The inhibitory oligonucleotides were added into the cells for 24-48 hours before analysis with RT-PCR. VS_ASO_1-FANA-FITC was labeled with FITC as shown in the Table 1, and VS_ASO_2-Cy3 was labeled with Cy3 as shown in Table 2; and VS_DsiRNA-Cy5 was labeled with Cy5 as shown in Table 3. A1&A2: No treatment, A3&A4: Overexpression of COVID-19 N-protein, A5&A6: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA-Cy5 with lipofectamine, B1&B2: Overexpression of COVID-19 N-protein+/treated by VS_DsiRNA-Cy5 with Arginine (5 μl/well), B3&B4: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2-Cy3 with lipofectamine, B5&B6: Overexpression of COVID-19 N-protein+/treated by VS_ASO_2-Cy3 with Arginine (5 μl/well), C1&C2: Overexpression of COVID-19 N-protein+/treated by VS_ASO_1-FANA-FITC without any reagents.

FIG. 43 shows detection of SARS-CoV-2 N-protein expressed in the human primary nasal epithelial cells (HNEpC) by qRT-PCR after treatment with siRNA or ASO. Significant down-regulation was observed: about 90 fold in the group treated by VS_DsiRNA-Cy5 oligo (2: p<0.01); about 15 fold in the group treated by VS_ASO_2-Cy3 oligo (p<0.01), and about 350 fold of down-regulation in the group treated by the VS_ASO_1-FANA-FITC oligo (3: p<0.001); when compared with the group with COIVD-19 N-protein overexpression only. The cycle threshold of no-treatment is non-detectable, but in this case for calculation purposes, the number “40” was used as the cycle threshold for a base-line control.

FIG. 44 shows experimental design for inhibiting viral infections using inhibitory nucleotides. WV=Wild-type of pseud-COVID-19 virus, 5 μl (titer: 10⁵TU/ml) of the virus added into each well (C1 to C9). MV=Mutant form of pseud-COVID-19 virus, 5 μl (titer: 10⁵TU/ml) of the virus added into each well (D1 to D9). N 1=VS_ASO_3 oligo (targeting on S-protein of COVID-19), N 2=VS_siRNA/RNAi_3 oligo (targeting on S-protein of COVID-19, Control-1=Scramble nucleotide oligo (SN) only.

FIGS. 45A-45F show experimental data of inhibitions of wild-type viral infections by inhibitory nucleotides. FIG. 45A Brightfield image of VS_ASO_3-treated cells. FIG. 45B Brightfield image of VS_RNAi_3-treated cells. FIG. 45C Brightfield image of scramble-treated cells. FIG. 45D Fluorescence image of VS_ASO_3-treated cells. FIG. 45E Fluorescence image of VS_RNAi_3-treated cells. FIG. 45F Fluorescence image of scramble-treated cells. There were no significant GFP expressions found in those cells treated by VS_ASO_3 and VS_RNAi_3 oligos, detected under the confocal microscope; but the inventors were able to see the GFP expressions in the control group of the cells treated with the scramble nucleotide only. This data thus indicated that the VS_ASO_3 and VS_RNAi_3 have inhibited the wild-type pseudoviruses coupled with eGFP (WV) inside the cells; but not in the group treated with the scramble nucleotide. The pseudo-type of COVID-19 virus (both the wild-type virus (WV) and the mutant virus (MV)) used in these experiments is an RNA virus like SARS-CoV-2. Without being bound to a particular theory, the inhibitory nucleotides of the disclosure are believed to inhibit viral infection by blocking the transcription of the spike protein; and also by disrupting the RNA transcription of the pseudo-type of COVID-19 viral genes.

FIGS. 46A-46F show experimental data of inhibitions of mutant viral infections by inhibitory nucleotides. FIG. 46A Brightfield image of VS_ASO_3-treated cells. FIG. 46B Brightfield image of VS_RNAi_3-treated cells. FIG. 46C Brightfield image of scramble-treated cells. FIG. 46D Fluorescence image of VS_ASO_3-treated cells. FIG. 46E Fluorescence image of VS_RNAi_3-treated cells. FIG. 46F Fluorescence image of scramble-treated cells. There was no significant GFP expressions found in those cells treated by VS_ASO_3 and VS_RNAi_3, detected under the confocal microscope; but the inventors were able to see the GFP expressions in the control group of the cells treated with the scramble nucleotide only. This data thus indicated that the VS_ASO_3 and VS_RNAi_3 have also inhibited the mutant pseudo viruses coupled with eGFP (MV) inside the cells; but not in the group treated with the scramble nucleotide. The pseudo-type of COVID-19 virus (both the wild-type virus (WV) and the mutant virus (MV)) used in these experiments is an RNA virus like SARS-CoV-2. Without being bound to a particular theory, the inhibitory nucleotides of the disclosure are believed to inhibit viral infection by blocking the transcription of the spike protein; and also by disrupting the RNA transcription of the pseudo-type of COVID-19 viral genes.

The designed nucleotides of VS_ASO and VS_RNAi (see Table 1) were able to inhibit viral infections and propagations with both the wild-type (WV) and mutant (MV) viruses of the pseudo COVID-19 in the living mammalian cells expressed ACE2 proteins.

Example 11: Inhibitory Peptides Block RBD Derived from the S-Protein of SARS-CoV-2

FIG. 47 shows the analysis of the amino acid sequence of SARS-CoV-2 Spike protein (S-protein) (GenBank ID: QHD43416.1, SEQ ID NO: 61). The region of the sequence highlighted in red represents the predicted sequences of ACE2 binding sequences/motifs (aka. the Ligand binding Domain).

FIGS. 48A-48B show the analysis of the amino acid sequence of the BD motifs. FIG. 48A 3D interaction between the SARS-CoV-2 Spike protein and human ACE2. FIG. 48B Analysis of the amino acids of the RBD motifs in 3D structure between the SARS-CoV-2 Spike protein (B: K417 to Y505) and human ACE2 (B: Q24 to R393) was used to order to locate which regions of the sequences contribute to the protein-protein interaction, and to design peptides that mimic the RBD sequences (mimics act like a human ACE 2 and prevent or block the binding activities for the SARS-CoV-2 on the real ACE2 in the cells).

FIG. 49 shows the experimental design. A1&A2: No treatment as control, A3&A4: peptide 5-FITC, A5&A6: peptide 5-FITC+/treated by the peptide 1 (low dosage), B1&B2: peptide 5-FITC+/treated by the peptide 1 (high dosage), B3&B4: peptide 5-FITC+/treated by the peptide 2 (low dosage), B5&B6: peptide 5-FITC+/treated by the peptide 2 (high dosage), 1&C2: peptide 5-FITC+/treated by the peptide 3 (low dosage), C3&C4: peptide 5-FITC+/treated by the peptide 3 (high dosage), C5&C6: peptide 5-FITC+/treated by the peptide 4 (low dosage), D1&D2: peptide 5-FITC+/treated by the peptide 4 (high dosage), D3&D4: peptide 5-FITC+/treated by the peptide 1 (high dosage)+peptide 2 (high dosage)+peptide 3 (high dosage)+peptide 4 (high dosage), The dosage-1=1 μg per 10⁵cells; and the dosage-2=10 μg per 10⁵cells.

FIGS. 50A-50H show cells infected with wild-type SARS-COV-2 virus (WV) in the presence of inhibitory peptides. FIG. 50A Brightfield image of cells treated with Peptide 1 (P1). FIG. 50B Brightfield image of cells treated with Peptide 2 (P2). FIG. 50C Brightfield image of cells treated with Peptide 3 (P3). FIG. 50D Brightfield image of cells treated with normal human serum (NHS). FIG. 50E Fluorescence (GFP) image cells treated with Peptide 1 (P1). FIG. 50F Fluorescence (GFP) image of cells treated with Peptide 2 (P2). FIG. 50G Fluorescence (GFP) image of cells treated with Peptide 3 (P3). FIG. 50H Fluorescence (GFP) image of cells treated with normal human serum (NHS).

FIGS. 51A-51H show cells infected with mutant SARS-COV-2 virus (MV) in the presence of inhibitory peptides. FIG. 51A Brightfield image of cells treated with Peptide 1 (P1). FIG. 51B Brightfield image of cells treated with Peptide 2 (P2). FIG. 51C Brightfield image of cells treated with Peptide 3 (P3). FIG. 51D Brightfield image of cells treated with normal human serum (NHS). FIG. 51E Fluorescence (GFP) image cells treated with Peptide 1 (P1). FIG. 51F Fluorescence (GFP) image of cells treated with Peptide 2 (P2). FIG. 51G Fluorescence (GFP) image of cells treated with Peptide 3 (P3). FIG. 51H Fluorescence (GFP) image of cells treated with normal human serum (NHS).

FIGS. 52A-52H show microscopic analysis of human primary small airway epithelial cells treated with inhibitory peptides (VS-peptides). FIGS. 52A, and 52E were captured under the FITC florescent filter, FIGS. 52B and 52E were captured in brightfield (20×). FIG. 52C shows the merge of FIGS. 52A and 52B. FIG. 52G is a merge photo of FIGS. 52E and 52F. The white dots indicate the box that was enlarged as shown in (D). The yellow dots indicate the box that was enlarged as shown in FIG. 52H. White arrows suggested peptide 5-FITC internalized into cells cytoplasm and nucleus, while the yellow arrows suggested the VS-peptides combination can block the peptide 5-FITC from internalization, staying outside of cells.

FIGS. 73A-73B. show alignments of inhibitory oligonucleotides. FIG. 73A The alignment of all ASO (ASO_1 and ASO_2) and all oligos in Tables 1, 2 and 4 showed that the designed inhibitory oligonucleotides specifically target the SARS-COV-2 virus genes. The alignment did not show any significant match to any human genes (thereby, avoiding potential side-effects when applied in human). FIG. 73B: The analysis of all DsiRNA indicated all oligos (in Tables 1, 2 and 4) specifically target the SARS-COV-2 virus genes. The alignment did not show any significant match to any human genes (thereby, avoiding potential side-effects when applied in human).

Inhibitory Peptides Block COVID-19 Spike Protein In Vitro

FIGS. 74A-74C show peptide ELISA assays. FIG. 74A Schematic of ELISA assays. FIG. 74B Analysis of inhibitions of COVID-19 Spike Protein Receptor Binding Domain (S-RBD)-ACE2 binding by inhibitory peptides. FIG. 74C Table of p-values of the results in FIG. 74A and the number of amino acids participating in S-RBD/ACE2 interaction. These results indicated that the peptides could compete with ACE2 proteins and prevent S-RBD binding to ACE2. When the designed inhibitory peptides contained more amino acids interacting with S-RBD, stronger affinities were measured.

FIGS. 75A-75B show peptide inhibition data. FIG. 75A Inhibition rate of S-RBD binding to ACE2 using VS peptides. FIG. 75B S-RBD signal rate. The data were converted and calculated as inhibition/suppression rates of the peptides based on the intensities of the S-RBD signals after treatment with the peptides compared with the control groups FIG. 75B. All peptides have shown their dosage-responses in the ELISA reactions, and indicated their strong biological affinities to bind with the S-RBD.

Inhibitory Peptides Block COVID-19 S-RBD-FITC Entry to Living Mammalian Cells

The HeLa-ACE2 cell were harvested after 48 hr post-transfection when ACE2 receptors were expressed on the cell membranes; and suspended in FACS running buffer (0.1% BSA in PBS) with final concentration in 1×106 cells/mL. Divided into 9 tubes (each tube has about 1 million cells). The above cells in each tube were incubated at RT in the dark for 30 min and subsequently subjected to run the FACS analysis BD FACSCalibur (total event: 10 k). The figures were generated and analyzed by a computer program of FlowJo, which were shown as the format of “Scatter-plot.” The data indicated that all peptides could target/bind on the S-RBD-FITC to prevent viral RBD entry the cells expressed the ACE2 receptors. See FIGS. 76A-76I. The peptides 1 (FIG. 76D), 2 (FIG. 76E), 4 (FIG. 76G), 5 (FIG. 76H) and mixture showed significant strongest therapeutic effects, but peptides 3 (FIG. 76F) was weak; when compared with the control groups of “HNS/Tube-9” (FIG. 76C), “Positive control/Tube-8” (FIG. 76B) and “Negative control/Tube-7” (FIG. 76A).

Derivatives of Inhibitory Peptides can Also Inhibit S-RBD as Measured by ELISA Assays

Inventors have developed variants of original inhibitory peptides VS-Peptides 1-5 (see Table 6). FIG. 77 shows ELISA results of S-RBD inhibition by inhibitory peptides derived from VS-Peptides 1-5 (P1 to P20—corresponding to SEQ ID NOS: 63-82, respectively). The data indicated that the derivative inhibitory peptides are also capable of targeting the S-RBD of SARS-CoV-2 significantly to prevent viral binding on the human ACE2 receptors (p<0.05) (FIG. 77).

Example 12: Gene Therapy

FIG. 53 shows the gene therapy vector AAV-U6-A1-H1-A2-SV40-eGFP. This AAV vector expresses two transgenes (namely ASO1 (A1) and ASO2 (A2)) simultaneously in one cell. U6=The 1st promoter that controls the expression of A1 gene in the mammalian cells, H 1=The 2nd promoter that controls the expression of A2 gene in the mammalian cells, SV=The 3rd promoter that controls the expression of GFP gene in the mammalian cells. Full sequence of AAV-U6-A1-H1-A2-SV40-GFP is shown by SEQ ID NO: 46.

FIG. 54 shows the gene therapy vector AAV-U6-A3-H1-A4-SV40-eGFP. This AAV vector expresses two transgenes (namely ASO3 (A3) and ASO4 (A4)) simultaneously in one cell. U6=The 1st promoter that controls the expression of A3 gene in the mammalian cells, H1=The 2nd promoter that controls the expression of A4 gene in the mammalian cells SV40=The 3rd promoter that controls the expression of GFP gene in the mammalian cells. Full sequence of AAV-U6-A1-H1-A2-SV40-eGFP is shown by SEQ ID NO: 47.

FIG. 55 shows the gene therapy vector AAV-U6-shRNA1-CMV-eGFP. This AAV vector expresses the transgene shRNA1. U6=The 1st promoter that controls the expression of shRNA1 gene in the mammalian cells, CMV=the 2nd promoter that controls the expression of eGFP gene in the mammalian cells. The full DNA sequence is shown by SEQ ID NO: 48 of AAV-U6-shRNA1-eGFP.

FIG. 56 shows the gene therapy vector AAV-U6-shRNA2-CMV-eGFP. This AAV vector expresses the transgene shRNA1 in one cell: U6=The 1st promoter that controls the expression of shRNA2 gene in the mammalian cells, CMV=the 2^ndpromoter that controls the expression of eGFP gene in the mammalian cells. The sequence of AAV-U6-shRNA2-eGFP is shown by SEQ ID NO: 49.

FIG. 57 shows the gene therapy vector AAV-U6-shRNA3-CMV-eGFP. A. This AAV vector expresses the transgene shRNA1. U6=The 1st promoter that controls the expression of shRNA3 gene in the mammalian cells, CMV=The 2nd promoter that controls the expression of eGFP gene in the mammalian cells. The full DNA sequence of AAV-U6-shRNA3-eGFP is shown by SEQ ID NO: 50.

FIG. 58 shows the gene therapy vector AAV-U6-shRNA4-CMV-eGFP. A. This AAV vector expresses the transgene shRNA1. U6=The 1st promoter that controls the expression of shRNA4 gene in the mammalian cells, CMV=The 2nd promoter that controls the expression of eGFP gene in the mammalian cells. The full DNA sequence of AAV-U6-shRNA4-eGFP is shown by SEQ ID NO: 51.

FIGS. 59A-59B show the experimental design of plates FIGS. 59A and 59B.

FIGS. 60A-60D show fold change of exogenously-expressed COVID-19 proteins with gene therapy treatment. The cells were transfected with designed vectors with COVID-19 plasmids (S, N, RdRp and ORF1ab), and also the gene therapy vectors (#1 to #6) after 24 hours post-seeding. At the 48 hours post-transfection, the cells were then harvested, and their total RNA were extracted with including the DNase digestion before PCR assays. S proteins were conducted with TaqMan-probe assay kit (Thermofisher, A47532). The N protein, ORF1ab and RdRp proteins were determined by the GenScript kits (SARS-CoV-2PCR detection assay kit). The data indicated that the treatments by using the gene vectors have significant therapeutic effects to inhibit the expressions of the viral proteins. Synergic enhanced effects were observed when more than one peptide was used (see, #1 to 4 and #5 to 6)

FIGS. 61A-61B show western blot analysis of S-protein expression. FIG. 61B shows quantification of FIG. 61A. The cells were seed onto 6 well-plates. At 24 hours post-seeding, cells were transfected by the gene therapy vectors (#1 to #6) with including the plasmids encoding the COVID-19 S-proteins. After 48 hours post-transfection, cells were harvested and lysed. The primary antibody is SARS-CoV-2 Spike with 1 μg/mL (ProSc, Inc), and the secondary antibody is Goat-anti-Rabbit HRP conjugated antibody by 1:1000 dilution (R&D System). The data indicated that the treatments by using the gene vectors have significant therapeutic effects to block the expression of viral proteins. Synergic enhanced effects were observed when more than one peptide was used (see, #1 to 4 and #5 to 6).

FIG. 62 shows the experimental design of in vitro gene therapy on inhibitions of the viral infections.

FIGS. 63A-63F show in vitro gene therapy inhibits WV viral infections. ASO (V1) and RNAi (V2) were delivered by gene vectors into mammalian cells that express ACE2 proteins, in order to inhibit the WV viral infections (WV=wild-type pseudo-virus of COVID-19). FIG. 63A Brightfield image of cells treated with ASO (V1). FIG. 63B Brightfield image of cells treated with RNAi (V2). FIG. 63C Brightfield image of cells treated with control. FIG. 63D Fluorescence image of cells treated with ASO (V1). FIG. 63E Fluorescence image of cells treated with RNAi (V2). FIG. 63F Fluorescence image of cells treated with control.

FIGS. 64A-64F show in vitro gene therapy inhibits MV viral infections. ASO (V1) and RNAi (V2) were delivered by gene vectors into mammalian cells that express ACE2 proteins, in order to inhibit the MV viral infections (MV=mutant pseudo-virus of COVID-19). FIG. 64A Brightfield image of cells treated with ASO (V1). FIG. 64B Brightfield image of cells treated with RNAi (V2). FIG. 64C Brightfield image of cells treated with control. FIG. 64D Fluorescence image of cells treated with ASO (V1). FIG. 64E Fluorescence image of cells treated with RNAi (V2). FIG. 64F Fluorescence image of cells treated with control.

FIG. 65A-65D show in vitro delivery of gene vectors into living cells. ASO (V1) and RNAi (V2) were delivered by using the gene vectors into mammalian cells that express ACE2 proteins, the cells were not incubated with any viruses, which were served as background controls. The concentrations of the vectors encoding the ASO (control-2) or/and RNAi (control-3) were the same used in the FIGS. 61A-61B and 62. FIG. 65A Brightfield image of cells treated with ASO control. FIG. 65B Brightfield image of cells treated with RNAi control. FIG. 65C Fluorescence image of cells treated with ASO control. FIG. 65D Fluorescence image of cells treated with RNAi control.

FIGS. 66A-66B show analysis of FIG. 66A Wild-type pseudovirus experiment results and FIG. 66B mutant pseudovirus experiment results. Since both gene vectors, encoding ASO and shRNA, also contain marker gene of GFP, normalized data was calculated based on control-2 and control-3 constructs (also see FIG. 62). The data analysis confirmed that ASO or shRNA vector expressing cells showed very little GFP signal, when compared with the control group-1. This data indicates that the gene vectors carrying either ASO or shRNA (inhibitory oligonucleotides) suppress viral infection and propagation in both wild-type and mutant pseudoviruses of COVID-19, pseudo-typed by lentiviruses. The pseudo-type of COVID-19 virus (both the wild-type virus (WV) and the mutant virus (MV)) used in these experiments is an RNA virus like SARS-CoV-2. Without being bound to a particular theory, the inhibitory nucleotides expressed by the genes vectors of the instant disclosure are believed to inhibit viral infection by blocking the transcription of the spike protein; and also by disrupting the RNA transcription of the pseudo-type of COVID-19 viral genes.

Example 13: Nutrition/Dietary Supplements

FIG. 67 shows experimental design for detection of apoptosis/cytotoxicity of VS-nutrition in human bronchial/tracheal epithelial cells (HBTEC) by qRT-PCR. The human primary bronchial/tracheal epithelial cells (HBTEC) were cultured in the 24 well-dish, and the cells were treated with VS-nutrition with designated dilution (1:1, 1:300 and 1:500) for 5 days (in every day, refresh cell culture medium and added new VS-nutrition with same composition and ratio) before analysis by qRT-PCR.

FIG. 68 shows detection of on apoptosis/cytotoxicity of VS-nutrition in the human bronchial/tracheal epithelial cells (HBTEC) by qRT-PCR after treatment. Detection of apoptosis/cytotoxicity of VS-nutrition in the human primary bronchial/tracheal epithelial cells (HBTEC) by qRT-PCR after treatment with VS-nutrition. There are no significant up or down-regulation of BAX/BCL2 ratio in group treated by VS-nutrition when compared with the normal cells with no-treatment (p>0.05).

FIG. 69 shows the experimental design for detection of apoptosis/cytotoxicity of VS-nutrition in Human Primary Nasal Epithelial Cells (HNEpC) by qRT-PCR. The human primary nasal epithelial cells (HNEpC) were cultured in the 24 well-dish, and the cells were treated with VS-nutrition with designated dilution (1:1, 1:300 and 1:500) for 5 days (in every day, refresh cell culture medium and added new VS-nutrition with same composition and ratio) before analysis by qRT-PCR.

FIG. 70 shows detection on apoptosis/cytotoxicity of VS-nutrition in the human primary nasal epithelial cells (HNEpC) by qRT-PCR after treatment. Detection of apoptosis/cytotoxicity of VS-nutrition in the human primary nasal epithelial cells by RT-PCR after treatment with VS-nutrition. There are no significant up or down-regulation of BAX/BCL2 ratio in group treated by VS-nutrition when compared with the normal cells with no-treatment (p>0.05).

FIGS. 71A-71B show oral intake formulations of VS product (nutritional supplement). Bottle product (10-15 ml) with FIG. 71A 1.5 ml spoon or FIG. 71B 1.0 ml drop.

FIGS. 72A-72C show an exemplary nasal (liquid) spray. Spray product with 10-15 ml bottle nasal spray. FIG. 72A composition and size of the product. FIGS. 72B & 72C usage example.

Number	Name	Date	Kind
20030138894	Parry et al.	Jul 2003	A1
20060084620	McCray et al.	Apr 2006	A1
20060258611	Kung et al.	Nov 2006	A1
20100261214	Loibner et al.	Oct 2010	A1

Number	Date	Country
WO-2021188966	Sep 2021	WO
WO-2021224918	Nov 2021	WO

	Number	Date	Country
Parent	17350114	Jun 2021	US
Child	17512963		US

Compositions and methods for treating COVID-19

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US Referenced Citations (4)

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Entry
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