Patent Application
20240043851
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file, created on Aug. 31, 2023, is named 55190-701_302_SL.xml and is 245,571 bytes in size.
Targeted editing of nucleic acids is a highly promising approach for studying genetic functions and for treating and ameliorating symptoms of genetic disorders and diseases. Most notable target-specific genetic modification methods involve engineering and using of zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and RNA-guided DNA endonuclease Cas. Frequency of introducing mutations such as deletions and insertions at the targeted nucleic acids through the non-homologous end joining (NHEJ) repair mechanism limits the applications of genetic targeting and editing in the development of therapeutics.
The disclosure is summarized here in part in the claims disclosed herein. Disclosed herein is a method comprising introducing a first vector into a plurality of cells wherein said first vector encodes a fusion protein complex comprising a Cas9 nuclease fused to an exonuclease; wherein a viability of said plurality of cells comprising said vector is at least 1.5 times that of a second plurality of cells comprising a second vector encoding a Cas9 nuclease; wherein said second plurality of cells are K562 cells transfected with said second vector. The first vector can encode the Cas9 fused to an exonuclease and a gRNA. The exonuclease can be selected from the group consisting of MRE11, EXO1, EXOIII, EXOVII, EXOT, DNA2, CtIP, TREX1, TREX2, Apollo, RecE, RecJ, T5, Lexo, RecBCD, and Mungbean. A donor polynucleotide can be introduced into the first plurality of cells. The method can comprise making an edit to an abnormal locus of a gene by said Cas9-fused to an exonuclease. The donor polynucleotide can comprise an integration cassette further comprising a functional locus of said gene. The viability can be measured by resazurin assay. The exonuclease can be ExoI. The abnormal locus can be an abnormal locus of a HBB gene. The donor polynucleotide can encode a functional locus of said HBB gene. The fusion protein complex can encode at least one nuclear localization signal (NLS). The first vector encoding the fusion protein complex can have at least 80% sequence identity with any one of SEQ ID NO: 2-18. The first vector can be delivered by electroporation. The donor polynucleotide can comprise a mutated protospacer adjacent motif (PAM) sequence located at the immediate 3′ end of a cleavage site, wherein said mutated PAM sequence comprises 5′-NCG-3′ or 5′-NGC-3′. The fusion protein complex can be unable to cleave said mutated PAM sequence. The donor polynucleotide can be single-stranded DNA. The donor polynucleotide can be double-stranded DNA.
Disclosed herein is a polypeptide, comprising a first functional fragment, a second functional fragment comprising a Cas nuclease, and a linker peptide, wherein said first functional fragment is coupled to a first end of the linker peptide and the second functional fragment is coupled to a second end of said linker peptide; and when a first complex comprising said polypeptide and a ribonucleic acid (RNA) molecule is administered to a first plurality of cells, a reduced toxicity is observed in said first plurality of cells compared to said toxicity observed in a second plurality of cells when a second complex comprising a Cas9 nuclease and said RNA molecule is administered to said second plurality of cells. The first functional fragment can comprise an exonuclease wherein the exonuclease is selected from the group consisting of MRE11, EXO1, EXOIII, EXOVII, EXOT, DNA2, CtIP, TREX1, TREX2, Apollo, RecE, RecJ, T5, Lexo, RecBCD, and Mungbean. The RNA molecule can be a guide RNA. The exonuclease can be a human Exo1 enzyme. The N-terminal of the human Exo1 enzyme can be coupled to said C-terminal of said linker which is coupled to said C-terminal of said Cas nuclease. The human Exo1 enzyme can comprise SEQ ID NO: 1. The human Exo1 enzyme can comprise a fragment that has a 80% sequence identity of SEQ ID NO:1. The human Exo1 enzyme can comprise a fragment that has a 90% sequence identity of SEQ ID NO:1. The human Exo1 enzyme can comprise a fragment that has a 95% sequence identity of SEQ ID NO:1. The second functional fragment can comprise a Cas9 enzyme. The Cas9 enzyme can comprise a N-terminal nuclear localizing sequence (NLS) and a C-terminal NLS. The Cas9 enzyme can comprise a N-terminal nuclear localizing sequence (NLS). The Cas9 enzyme can comprise a C-terminal nuclear localizing sequence (NLS). The linker peptide can be selected from a group consisting of FL2X, SLA2X, AP5X, FL1X, SLA1X. The linker peptide can be SLA2X. The peptide can comprise 5 to 200 amino acids. The reduced toxicity can be quantified by measuring resorufin accumulation. After administration of said first complex, the first plurality of cells can have at least two times a number of viable cells compared to said second plurality of cells after administration of said second complex wherein the number of viable cells is quantified by a resorufin assay. After administration of the first complex, the first plurality of cells has at least two times said amount of HDR edited cells when compared to the second plurality of cells after administration of the second complex as quantified by a cellular HDR assay. The cellular HDR assay can comprise IHC, qPCR or deep sequencing.
Disclosed herein is a polynucleotide encoding the aforementioned polypeptide and the RNA molecule. The first end of the linker peptide can be a 3′ end and the second end of the linker peptide can be a 5′ end. The first end of said linker peptide can be a 5′end and the second end of said linker peptide can be a 3′ end. The RNA molecule can be a guide RNA (gRNA). The polynucleotide can comprise a homology directed repair (HDR) template. The gRNA can be selected from sequences listed in Table 2. The HDR template can be single-strand DNA. The HDR template can be double-strand DNA. The polynucleotide can be formulated in a liposome. The liposome can comprise a polyethylene glycol (PEG), a cell-penetrating peptide, a ligand, an aptamer, an antibody, or a combination thereof.
Disclosed herein is a vector comprising a nucleotide sequence of the aforementioned polypeptide. The vector can comprise a promoter. The promoter can be a CMV or a CAG promoter. The vector can be selected from a group consisting of retroviral vectors, adenoviral vectors, lentiviral vectors, herpesvirus vectors, and adeno-associated viral vectors. The vector can be an adeno-associated viral vector. Disclosed herein is a virus-like particle (VLP) comprising the aforementioned vector. Disclosed herein is a kit comprising the aforementioned polypeptide formulated in a compatible pharmaceutical excipient, an insert with administering instructions, reagents.
Disclosed herein is a kit comprising the aforementioned polynucleotide formulated in a compatible pharmaceutical excipient, an insert with administering instructions, reagents.
Disclosed herein is a kit comprising the aforementioned vector formulated in a compatible pharmaceutical excipient, an insert with administering instructions, reagents.
Disclosed herein is a method for inducing homologous recombination of DNA in a cell, comprising contacting the DNA with the aforementioned polypeptide.
Disclosed herein is a method for inducing HDR in a cell in vitro or ex vivo, comprising delivering the aforementioned polynucleotide into a cell. The cell can be a human cell, a non-human mammalian cell, a stem cell, a non-mammalian cell, an invertebrate cell, a plant cell, or a single-eukaryotic organism.
Disclosed herein is a method, comprising: contacting a first of plurality of cells with an aforementioned polynucleotide and a second plurality of cells with a second polynucleotide encoding a wild-type Cas9 enzyme; and inducing a site-specific cleavage at an intended locus followed by HDR in the first plurality of cells and the second plurality of cells; and recovering at least 30-90% more cells in the first plurality of cells compared to the second plurality of cells. The method can further comprise measuring cell viability by measuring an amount of resorufin produced in the first plurality of cells and the second plurality of cells. The first plurality of cells can have 2-5 times an amount of viable cells as quantified by a resorufin assay when compared to the second plurality of cells. The first plurality of cells and the second plurality of cells can comprise a human cell, a non-human mammalian cell, a stem cell, a non-mammalian cell, a invertebrate cell, a plant cell, or a single-eukaryotic organism. The human cell can be a T cell, a B cell, a dendritic cell, a natural killer cell, a macrophage, a neutrophil, an eosinophil, a basophil, a mast cell, a hematopoietic progenitor cell, a hematopoietic stem cell (HSC), a red blood cell, a blood stem cell, an endoderm stem cell, an endoderm progenitor cell, an endoderm precursor cell, a differentiated endoderm cell, a mesenchymal stem cell (MSC), a mesenchymal progenitor cell, a mesenchymal precursor cell, or a differentiated mesenchymal cell. The differentiated endoderm cell can be a hepatocytes progenitor cell, a pancreatic progenitor cell, a lung progenitor cell, or a tracheae progenitor cell. The differentiated mesenchymal cell can be a bone cell, a cartilage cell, a muscle cell, an adipose cell, a stromal cell, a fibroblast, or a dermal cell.
Disclosed herein is a method for treating a single gene disorder in a subject, comprising: culturing a plurality of primary cells obtained from said subject; administering the aforementioned polynucleotide to a plurality of primary cells, wherein the gRNA is configured to recognize a locus of the gene that causes said disorder and the HDR template is configured to provide a functioning sequence of the gene; and inducing a site-specific cleavage at the locus followed by HDR, wherein the functioning sequence of said gene is inserted at the locus. The method can further comprise selecting primary cells in which said functioning sequence of the gene is inserted at the locus; and reintroducing the selected primary cells back into the subject. The subject can be a mammal. The mammal can be a human. The plurality of primary cells can be selected from a group comprising T cells, B cells, dendritic cells, natural killer cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, mast cells, hematopoietic progenitor cells, hematopoietic stem cells (HSCs), red blood cells, blood stem cells, endoderm stem cells, endoderm progenitor cells, endoderm precursor cells, differentiated endoderm cells, mesenchymal stem cells (MSCs), mesenchymal progenitor cells, mesenchymal precursor cells, differentiated mesenchymal cells, hepatocytes progenitor cells, pancreatic progenitor cells, lung progenitor cells, tracheae progenitor cells, bone cells, cartilage cells, muscle cells, adipose cells, stromal cells, fibroblasts, and dermal cells. The gene that causes said single gene disorder can be selected from Table 3.
Disclosed herein is a method for treating sickle cell anemia caused by an abnormal HBB gene in a subject, comprising: culturing a plurality of primary cells obtained from said subject; administering the aforementioned polynucleotide to the plurality of primary cells, wherein the gRNA is configured to recognize a locus of said HBB gene that causes the disorder and the HDR template is configured to provide a functioning sequence of said HBB gene; and inducing a site-specific cleavage at said locus followed by HDR, wherein the functioning sequence of said HBB gene is inserted at the locus. The method can further comprise selecting primary cells in which said functioning sequence of said HBB gene is inserted at said locus; and reintroducing said selected primary cells back into said subject. The subject can be a mammal. The mammal can be a human. The primary cell can be a hematopoietic stem cell. The primary cell can be a CD34+ hematopoietic stem cell. The primary cell can be a CD34+ hematopoietic stem cell. The vector can comprise plasmid PX330. The cell can be a CD34+ hematopoietic stem cell.
Disclosed herein is a method for treating sickle cell anemia caused by an abnormal HBB gene in a subject, comprising: culturing a plurality of primary cells obtained from the subject; administering the aforementioned polynucleotide to the plurality of primary cells, wherein the gRNA is configured to recognize a locus of the HBB gene that causes the disorder and the HDR template is configured to provide a functioning sequence of the HBB gene; and inducing a site-specific cleavage at the locus followed by HDR, wherein the functioning sequence of the HBB gene is inserted at the locus. The method can further comprise selecting primary cells in which the functioning sequence of the HBB gene is inserted at the locus; and reintroducing the selected primary cells back into the subject. The subject can be a mammal. The mammal can be a human. The primary cell can be a CD34+ hematopoietic stem cell.
Disclosed herein is a method, comprising: contacting a first of plurality of cells with a first complex comprising the aforementioned polynucleotide and a RNA molecule; inducing a site-specific cleavage followed by HDR in the first plurality of cells, wherein a percentage of cells of the first plurality of cells edited by HDR quantified by a cellular HDR assay is at least two times higher compared to a percentage of cells of a second plurality of cells contacted with a second complex comprising a polynucleotide encoding a wild-type Cas9 enzyme and the RNA molecule. The cellular HDR assay can comprise IHC. The cellular HDR assay can comprise qPCR. The cellular HDR assay can comprise nucleic acid sequencing.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
Some understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:
A brief description about the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system is included. The CRISPR/Cas enzyme system first found in bacteria and archaea is an immune defense against viral infection. During viral infection, segments of viral DNA are integrated into CRISPR locus. These segments of integrated viral DNA are transcribed into guide RNA (gRNA), which is sequentially complementary to the viral genome. gRNA directs the Cas enzymes to the gRNA targeted viral genome, where Cas proteins cleave the viral genome, thus defending against viral infection.
The CRISPR system typically comprises a gRNA that is specific to the target DNA sequence and a non-specific Cas 9 protein. Generally, the gRNA includes two distinct segments-CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA). The crRNA is complementary to the target DNA sequences, and thus recognize the sequence to be cleaved. And the tracrRNA functions as a scaffold for the crRNA-Cas9 interaction. Guide RNA naturally form a duplex molecule, with the crRNA and tracrRNA fragments annealed together. Cas proteins have been investigated and engineered as a tool for genetic editing by generating site-specific double strand breaks (DSBs). Custom designed gRNA directs the Cas proteins to generate DSB at any nucleic acid loci that are complementary to the sequence of gRNA. Cas proteins have been shown to successfully introduce nucleotide changes, deletions, insertions, and substitutions in eukaryotic cells.
The use CRISPR and Cas9 proteins for editing nucleic acids are limited by the endogenous repair mechanism of the cell. DSBs are preferentially repaired by NHEJ. Unintended insertions and deletions at sites of repair associated with NHEJ render development of genetic-based therapy undesirable. Alternatively, if the generated DSBs are resected so that long (<200 bp) 3′ overhangs are generated, the endogenous repair pathway is forced to use HR. Targeted error-free insertions and deletions of anywhere from 1-1000s of bp of DNA can be achieved by addition of a polynucleotide (template sequence) comprising homology arms flanking the desired insertion or deletion.
Homology directed repair is error free, and results in the ability to insert or delete specific sequences of DNA in a given genome.
Further, the HDR reduces cellular toxicity, which is caused by DSBs introduced by CRISPR and Cas9 enzyme system. The cellular toxicity is dependent on the p53 tumor suppressor pathway, as inhibition or loss of p53 function greatly reduces cellular toxicity in both Human Pluripotent Stem Cells (hPSCs) and in immortalized Retinal Pigment Epithelium (RPE) cells. Since permanent loss of p53 functionality has some severe effects on cells including genomic instability, altered cellular homeostasis, and increased rates of cancer in-vivo, one solution is transient inhibition of p53 by either small molecule or overexpression of dominant negative inhibitors. However, the transient inhibition of p53 in vivo is challenging and could produce undesirable side effects. Therefore, generating a non-toxic Cas9 enzyme is desirable for in vivo applications.
Disclosed herein are compositions and methods related to the selective targeting and editing endogenous nucleic acid segment in both normal cell and in cell associated with genetic diseases with reduced cellular toxicity. Targeted endogenous nucleic acids are cleaved, digested, and edited through HDR. gRNA directs a protein fusion complex comprising of the Cas protein moiety and a human Exo1 enzyme to a specific endogenous nucleic acid segment, where the protein fusion complex introduces cleavage and digestion, leaving 3′ or 5′ overhangs on the targeted endogenous nucleic acid segment. The overhangs allow for increased rates of HDR when the cell is further presented with a polynucleotide fragment that shares some degrees of sequence homology as the targeted and digested endogenous nucleic acid segment.
Disclosed herein are compositions wherein the targeted endogenous nucleic acids are located in known disease loci. Targeted known disease loci are cleaved, digested, and edited through HDR. gRNA directs a protein fusion complex comprising the Cas protein moiety and a human Exo1 enzyme to a specific known disease locus where the protein fusion complex introduces cleavage and digestion, leaving 3′ or 5′ overhangs on the targeted endogenous nucleic acid segment. The overhangs allow for increased rates of HDR when the cell is further presented with a polynucleotide fragment that shares some degrees of sequence homology as the targeted and digested endogenous nucleic acid segment.
Some aspects of the compositions and methods disclosed herein involve at least one modified polypeptide comprising a programmable endonuclease such as a Cas9 or other CRISPR-related programmable endonucleases coupled to a fragment of an exonuclease such as human Exo1 exonuclease or other exonucleases, such as MRE11, EXO1, EXOIII, EXOVII, EXOT, DNA2, CtIP, TREX1, TREX2, Apollo, RecE, RecJ, T5, Lexo, RecBCD, and Mungbean, to reduce cellular toxicity relative to that of an unmodified programmable endonuclease such as Cas9 enzyme in the CRISPR-Cas9 system.
The polypeptide (fusion protein) comprises a programmable endonuclease such as Cas9, other CRISPR-related programmable endonucleases, other site-specific endonucleases, or a fragment thereof and an exonuclease such as human Exo1 exonuclease or a fragment thereof covalently connected by a peptidyl linker. As used herein, the “Cas9,” “Cas9 domain,” or “Cas9 fragment” refers to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof, e.g., a protein comprising an active DNA cleavage domain of Cas9. A Cas9 nuclease is sometimes referred to as a casn1 nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. Cas9 nuclease sequences and structures are well known to those of ordinary skill in the art. Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Wild type (unmodified) Cas9 can be from any of the sequences listed below in Table 1. The Cas9 protein sequences listed in Table 1 is not meant to be limiting. Additional suitable Cas9 nucleases and protein sequences will be apparent to a person of ordinary skill in the art.
GNTDRHSIKKNLIGALLFGSGETAEATRLKRTARR
pyogenes)
GILQTVKIVDELVKVMGHKPENIVIEMARENQTTQ
QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI
VPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVV
KKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEL
DKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN
DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY
HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY
DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT
LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKV
LSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA
Streptococcus
thermophilus
Francisella
tularensis subsp.
novicida (strain
Staphylococcus
aureus
Streptococcus
thermophilus
Actinomyces
naeslundii
Neisseria
meningitidis
Listeria innocua
serovar 6a
Pasteurella
multocida
Corynebacterium
diphtheriae
Campylobacter
jejuni subsp.
jejuni serotype O:
Rhodobacteraceae
bacterium
Campylobacter
coli
Ignavibacteria
bacterium
Fructobacillus sp.
Pedobacter
glucosidilyticus
Geobacillus
thermodenitrificans
Further, in some embodiments, fragments of Cas9 or other programmable nuclease that retain DNA cleaving function can be used to generate the fusion proteins. For example, a Cas9 or other programmable nuclease polypeptide fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a wild type Cas9. In some embodiments, the Cas9 fragment may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a wild type Cas9.
The Cas9 enzymes or other programmable nuclease disclosed herein also comprises at least one nuclear localization signal (NLS), which is an amino acid sequence that attaches to a protein for import into the cell nucleus by nuclear transport. Generally, the NLS comprises one or more short sequences of positively charged lysines or arginines exposed on the protein surface. These types of classical NLSs can be further classified as either monopartite or bipartite. The major structural difference between the two is that the two basic amino acid clusters in bipartite NLSs are separated by a relatively short spacer sequence (hence bipartite—2 parts), while monopartite NLSs are not. In some embodiments, the NLS comprises sequence PKKKRKV (SEQ ID NO: 19) of the SV40 Large T-antigen (a monopartite NLS). In other embodiments, the NLS of nucleoplasmin comprises sequence KR[PAATKKAGQA]KKKK (SEQ ID NO: 20). There are also many other types of non-classical NLSs. Different types of NLSs disclosed herein are not meant to be limiting and a person of ordinary skill in the art is able to select a NLS to attach to a Cas9 protein. In some embodiments, the Cas9 protein comprises an N-terminal NLS. In other embodiments, the Cas9 protein comprises a C-terminal NLS. In yet other embodiments, the Cas9 protein comprises both N-terminal and C-terminal NLSs.
In some embodiments, the other CRISPR-related programmable endonucleases often includes CRISPR-associated (Cas) polypeptides or Cas nucleases including Class 1 Cas polypeptides, Class 2 Cas polypeptides, type I Cas polypeptides, type II Cas polypeptides, type III Cas polypeptides, type IV Cas polypeptides, type V Cas polypeptides, and type VI CRISPR-associated (Cas) polypeptides, CRISPR-associated RNA binding proteins, or a functional fragment thereof. Further, Cas polypeptides suitable for use with the present disclosure often include Cpf1 (or Cas12a), c2c1, C2c2 (or Cas13a), Cas13, Cas13a, Cas13b, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Csn1, Csx12, Cas10, Cas10d, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cul966; any derivative thereof; any variant thereof; and any fragment thereof.
Additionally, other site-specific endonucleases that are suitable for the fusion protein composition disclosed herein often comprise zinc finger nucleases (ZFN); transcription activator-like effector nucleases (TALEN); meganucleases; RNA-binding proteins (RBP); recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), and eukaryotic Argonaute (eAgo)); or any functional fragment thereof.
hExo1 Protein
A programmable nuclease is often tethered to an exonuclease domain so as to effect the results disclosed herein. A number of exonuclease/programmable exonuclease combinations are consistent with the disclosure herein. With respect to the exonuclease, certain exemplary exonucleases suitable for use as part of the fusion protein in present application include MRE11, EXO1, EXOIII, EXOVII, EXOT, DNA2, CtIP, TREX1, TREX2, Apollo, RecE, RecJ, T5, Lexo, RecBCD, and Mungbean. Additional suitable exonucleases are also contemplated. In certain embodiments, human Exo1 (hExo1) is used herein as a part of the fusion protein. Full length hExo1 can be divided into roughly two regions: the N-terminal nuclease region (1-392) (SEQ ID NO: 1) MGIQGLLQFI KEASEPIHVR KYKGQVVAVD TYCWLHKGAI ACAEKLAKGE PTDRYVGFCM KFVNMLLSHG IKPILVFDGC TLPSKKEVER SRRERRQANL LKGKQLLREG KVSEARECFT RSINITHAMA HKVIKAARSQ GVDCLVAPYE ADAQLAYLNK AGIVQAIITE DSDLLAFGCK KVILKMDQFG NGLEIDQARL GMCRQLGDVF TEEKFRYMCI LSGCDYLSSL RGIGLAKACK VLRLANNPDI VKVIKKIGHY LKMNITVPED YINGFIRANN TFLYQLVFDP IKRKLIPLNA YEDDVDPETL SYAGQYVDDS IALQIALGNK DINTFEQIDD YNPDTAMPAH SRSHSWDDKT CQKSANVSSI WHRNYSPRPE SGTVSDAPQL KE), and the C-terminal MLH2/MSH1 interaction region (393-846). In some embodiments, the N-terminal nuclease region of hExo1 (SEQ ID NO: 1) is used to covalently link to a Cas9 with at least one NLS via a peptidyl linker. In other embodiments, a fragment of SEQ ID NO: 1 or other exonuclease domain that retains the nuclease function is used herein. For example, the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to SEQ ID NO: 1. In some embodiments, the fragment may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to SEQ ID NO: 1 or other untruncated or unmutated domain. The N-terminal nuclease region of the hExo1 is exemplary, and additionally suitable Exo1 or other exonuclease sequences can be utilized for the purpose disclosed herein by a person of ordinary skill in the art.
An exonuclease such as a hExo1 peptide is connected to a programmable endonuclease such as a Cas9 peptide and at least one NLS in some cases using a linker. In some embodiments, the linker is a linker peptide. The linker peptides not only serves to connect the protein moieties, but in some cases also provides many other functions, such as maintaining cooperative inter-domain interactions or preserving biological activity (Gokhale R S, Khosla C. Role of linkers in communication between protein modules. Curr Opin Chem Biol. 2000; 4: 22-27; Ikebe M, Kambara T, Stafford W F, Sata M, Katayama E, Ikebe R. A hinge at the central helix of the regulatory light chain of myosin is critical for phosphorylation-dependent regulation of smooth muscle myosin motor activity. J Biol Chem. 1998; 273: 17702-17707; and Chen X Y, Zaro J, and Shen W C. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 2014; 65, 1357-1369 are incorporated herein). The linker peptides can be grouped into small, medium, and large linkers with average length of less than or up to 4.5±0.7, 9.1±2.4, and 21.0±7.6 residues or greater, respectively, although examples anywhere within the set defined by these three ranges are also contemplated. In some embodiments, the linker peptide comprises 5 to 200 amino acids. In other embodiments, the linker peptide comprises 5 to 25 amino acids. In certain embodiments, the linker peptide is selected from the group consisting of FL2X (encoded by SEQ ID NO: 122 (ggtctccttaaacctgtcttgt)), SLA2X (encoded by SEQ ID NO: 123 (GGAGGTGGAGGCTCTGGTGGAGGCGGATCA)), APSX (encoded by SEQ ID NO: 124 (GCAGAGGCTGCAGCCGCTAAGGCC)), FL1X (encoded by SEQ ID NO: 125 (GCAGAGGCTGCAGCCGCTAAGGAGGCAGCTGCCGCTAAGGCC)), SLA1X, (encoded by SEQ ID NO: 126 (GCACCTGCTCCAGCGCCCGCACCAGCTCCC)) and any combinations thereof. In some embodiments, the linker peptide is SLA2X. Again, these disclosed linker peptides are not meant to be limiting. A person of ordinary skill in the art would be able to select an appropriate linker peptide.
The fusion protein disclosed herein can be fused together directly post-translationally or translated from a polynucleotide (fusion nucleotide) that encodes the disclosed fusion protein in a common open reading frame. In some embodiments, a first nucleic acid sequence encoding hExo1 or the N-terminal nuclease region thereof is ligated to one end of a second nucleic acid sequence encoding a selected linker peptide. Further, the other end of the second nucleic acid sequence is ligated with a third nucleic acid sequence encoding Cas9 enzyme with at least one NLS. Generally, stop codons of the first, second, and third nucleic acid sequences are removed. In some embodiments, the first, second and third nucleic acid sequences are codon optimized or engineered for more efficient transfection or expression in a target cell. Similarly, in some instances, intronic sequences are removed.
In some embodiments, hExo1-Cas9-DN1s (or reverse orientation DN1s-Cas9-HR) can be a fusion of hExo1(1-352) via linker 1(FL1X, AP5X or other) to Cas9 possessing or lacking an N-terminal FLAG+NLS (noted as NLS in
In some instances, hExo1-Cas9-DN1s-Geminin(1-110) (or DN1s-Cas9-HR-Geminin) can be fusion of hExo1(1-352) via linker 1(FL1X, AP5X or other) to Cas9 possessing or lacking an N-terminal FLAG+NLS subsequently fused via linker 2 (either TGS or other) to a fragment of human p53 (1231-1644). DN1s can either have an NLS added to its C-Terminus, which can then be fused to Geminin(1-110) via L3 (any sequence), or fused to Geminin with an NLS sequence at its C-Terminus, which can be fused to DN1s via L3. In some embodiments, the cellular toxicity of the hExo1-Cas9-DN1s-Geminin(1-110) (or DN1s-Cas9-HR-Geminin) can be reduced compared to Cas9. In some embodiments, error free editing efficiency of hExo1-Cas9-DN1s-Geminin(1-110) (or DN1s-Cas9-HR-Geminin) can be increased compared to Cas9. In some embodiments, the error free editing efficiency of hExo1-Cas9-DN1s-Geminin(1-110) (or DN1s-Cas9-HR-Geminin) can be increased compared to Cas9 due to post-translational regulation via geminin of hExo1-Cas9-DN1s-Geminin restricting nuclease activity to S/G2 phase, when endogenous HR is highest in the cell.
In some embodiments, hExo1-Cas9-Geminin(1-110) (or Cas9-hExo1-Geminin) can be a fusion of hExo1(1-352) via linker 1(FL1X, AP5X or other) to Cas9 possessing or lacking an N-terminal FLAG+NLS, possessing or lacking a C-terminal NLS sequence subsequently fused via linker 2 (either TGS or other) to a fragment of Geminin (1-110) either possessing or lacking a C-terminal NLS sequence. In some embodiments, hExo1-Cas9-Geminin(1-110) (or Cas9-hExo1-Geminin) comprises reduced cellular toxicity and increased error free editing efficiency compared to Cas9.
In some embodiments, hExo1-Cas9-PCV (or PCV-Cas9-hExo1 can be a fusion of hExo1(1-352) via linker 1 (FL1X, AP5X or other) to Cas9 possessing or lacking an N-terminal FLAG+NLS, possessing or lacking a C-terminal NLS sequence subsequently fused via linker 2 (either TGS or other) to PCV. In some embodiments, PCV can bind to a specific ssDNA sequence thereby tethering the repair template to the Cas9 complex. In some embodiments, hExo1-Cas9-PCV comprises increased error free editing efficiency compared to Cas9. In some embodiments, hExo1-Cas9-PCV comprises reduced cellular toxicity compared to Cas9.
In some embodiments, hExo1-Cas9-PCV-Geminin(1-110) (or PCV-Cas9-hExo1-Geminin) can be a fusion of hExo1(1-352) via linker 1(FL1X, APSX or other) to Cas9 possessing or lacking an N-terminal FLAG+NLS, possessing or lacking a C-terminal NLS sequence subsequently fused via linker 2 (either TGS or other) to PCV, which can then be fused to a fragment of Geminin (1-110). In some embodiments, hExo1-Cas9-PCV-Geminin(1-110) (or PCV-Cas9-hExo1-Geminin) comprises higher error free editing efficiency compared to Cas9. In some embodiments, hExo1-Cas9-PCV-Geminin(1-110) (or PCV-Cas9-hExo1-Geminin) comprises higher error free editing efficiency compared to Cas9 due to restriction of nuclease activity to S/G2 phase.
In some embodiments, hExo1-Cas9-CtIP(1-296) (or CtIP-Cas9-hExo1) can be a fusion of hExo1(1-352) via linker 1(FL1X, APSX or other) to Cas9 possessing or lacking an N-terminal FLAG+NLS, possessing or lacking a C-terminal NLS sequence subsequently fused via linker 2 (either TGS or other) to CtIP. In some embodiments, CtIP can improve error free editing efficiency compared to Cas9 without CtIP. In some embodiments, CtIP can improve error free editing efficiency compared to Cas9 via binding downstream of blocked DSBs (double-strand breaks) and resecting back towards the break using 3′-5′ exonuclease activity.
Escherichia coli (E. coli) version of Exo I
In certain embodiments, the Escherichia coli (E. coli) version of Exo I (E. coli Exo1) is used herein as a part of the fusion protein. E. coli Exo1 possesses 3′ to 5′ exonuclease activity as opposed to the 5′ to 3′ exonuclease activity of hExo1. The E. coli Exo1 Cas9 fusion can generate much longer deletions than traditional Cas9.
Some nucleotide constructs consistent with the disclosure comprise nucleic acid encoding an exonuclease such as hExo1. Further, some nucleotide constructs consistent with the disclosure comprise nucleic acid encoding a programmable endonuclease such as a Cas9 or other CRISPR-related programmable endonucleases. In some embodiments, the nucleic acid sequence encoding hExo1 or the N-terminal nuclease region thereof is non-naturally occurring, but the hExo1 or the N-terminal nuclease region thereof encoded by it has an amino acid sequence that is naturally occurring. In some instances, the nucleic acid sequence is different from a naturally occurring hExo1 or the N-terminal nuclease region thereof nucleic acid sequence but encodes a polypeptide identical to hExo1 or the N-terminal nuclease region thereof owning to codon degeneracy. Similarly, the third nucleic acid sequence encoding Cas9 enzyme with at least one NLS is non-naturally occurring, but the Cas9 protein encoded by it has an amino acid sequence that is naturally occurring. In some instances, the nucleic acid sequence is different from a naturally occurring Cas9 nucleic acid sequence but encodes a polypeptide identical to Cas9 owning to codon degeneracy.
A ribonucleoprotein (RNP) typically comprises at least two parts: one part comprises a programmable endonuclease such as a Cas9 or other CRISPR-related programmable endonucleases; and the other part comprises a gRNA or other specificity-conveying nucleic acid. Often, a wild type Cas9 enzyme or other Cas or non-Cas programmable endonuclease can be one part of the CRISPR-Cas9 system. The modified Cas9 protein coupled to a fragment of hExo1 via a linker peptide can also be one part of the CRISPR-Cas9 system. Further, the modified Cas9 protein and a gRNA can form a ribonucleoprotein (RNP).
gRNA
A ribonucleic acid that comprises a sequence for guiding the ribonucleic acid to a target site on a gene and another sequence for binding to an endonuclease such as Cas9 enzyme is used herein. Often, the ribonucleic acid is a gRNA. In some embodiments, the gRNA is a synthetic gRNA (sgRNA). The gRNA directs the fusion protein complex to a targeted nucleotide sequence of the DNA molecule. The gRNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined about 20 nucleotide spacer that defines the genomic target to be modified. In certain embodiments, a spacer of a gRNA can be designed to recognize the exon 1 of HBB gene. Thus, one can change the genomic target of the Cas protein by simply changing the target sequence present in the gRNA.
There are several ways to deliver gRNA into cells. One is to deliver gRNA into the cells as plasmid DNA. In some embodiments, the nucleic acids encoding the fusion proteins can be cloned into one plasmid or other suitable vectors with a nucleic acid sequence encoding a designed gRNA targeting a gene of interest.
A list of representative gRNA constituents is provided below.
Genome stability necessitates the correct and efficient repair of DSBs. In eukaryotic cells, mechanistic repair of DSBs occurs primarily by two pathways: Non-Homologous End-Joining (NHEJ) and Homology Directed Repair (HDR). NHEJ is the canonical homology-independent pathway as it involves the alignment of only one to a few complementary bases at most for the re-ligation of two ends, whereas HDR uses longer stretches of sequence homology to repair DNA lesions. HDR is the more accurate mechanism for DSB repair due to the requirement of higher sequence homology between the damaged and intact donor strands of DNA. The process is error-free if the DNA template used for repair is identical to the original DNA sequence at the DSB, or it can introduce very specific mutations into the damaged DNA.
As addressed above, HDR methods provide the great freedom in genomic engineering, allowing for as little as single base mutations and up to insertions or deletions of kilo-bases (kb) of DNA. In eukaryotes, HDR rate is governed by the competition between two different pathways: Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ). The competition between these two pathways begins by competitive binding by either MRN/CtIP complex or Ku 70/80 heterodimer. If MRN/CtIP bind first, they recruit other proteins, including Exonuclease I (Exo1), which possess 5′->3′ exonuclease activity 20. 5′ end resection of double strand DNA breaks by either Exo1 or Dna2 at each side of the break commits the DSB to be repaired by the HR pathway. Alternatively, if the Ku 70/80 heterodimer binds, it can then recruit other NHEJ pathway members, including DNA Ligase IV, and eventually repairs the double strand break via NHEJ.
HDR template sequences are needed to be delivered into cells when delivering the CRISPR-Cas9 system to the cells. HDR templates used to create specific mutations or insert new elements into a gene require a certain amount of homology surrounding the target sequence that will be modified. In some embodiments, the 5′ and 3′ homology arms start at the CRISPR-induced DSB. In general, the insertion sites of the modification can be very close to the DSB, ideally less than 10 bp away if possible. In some embodiments, the 5′ and 3′ homology arm of the HDR template sequences are at least 80% identical to the targeted sequence. Further, in some embodiments, single stranded donor oligonucleotide (ssDON) is utilized for smaller insertions. Each homology arm of the ssDON may comprise about 30-80 bp nucleotide sequence. The length of the homology arm is not meant to be limiting and the length can be adjusted by a person of ordinary skill in the art according to a locus of gene interest and experimental system. For larger insertions such as fluorescent proteins or selection cassettes, double stranded donor oligonucleotide (dsDON) can be utilized as HDR template sequence. In some embodiments, each homology arm of the ssDON may comprise about 800-1500 bp nucleotide sequence. To prevent Cas9 enzyme cleaving the HDR template, in some embodiments, a single base mutation can be introduced in the Protospacer Adjacent Motif (PAM) sequence of the HDR template.
Several different methods are used to deliver ribonucleoproteins and ssDON or other nucleic acids to a cleavage site, such as transfection. Transfection methods can be used to deliver CRISPR-Cas9 or other programmable endonuclease components to cells. Some of exemplary methods can be used to deliver the disclosed modified CRISPR-Cas9 system to cells and additional methods consistent with the disclosure known to a person of ordinary skill in the art can choose a particular method depending on the type of cells and the format of the CRISPR-Cas9 components.
Delivery can be broken into two major categories: cargo and delivery vehicle. Regarding CRISPR/Cas9 cargoes, three approaches are commonly available: (1) DNA plasmid encoding both the Cas9 protein or other programmable endonuclease and the guide RNA, (2) mRNA for Cas9 or other programmable endonuclease translation alongside a separate guide RNA, and (3) Cas9 protein or other programmable endonuclease with guide RNA (ribonucleoprotein complex). The delivery vehicle used will often dictate which of these three cargos can be packaged, and whether the system is usable in vitro and/or in vivo.
Vehicles used to deliver the gene editing system cargo can be classified into three general groups: physical delivery, viral vectors, and non-viral vectors. The most common physical delivery methods are microinjection, electroporation, and nucleofection. Electroporation enables delivery of the CRISPR machinery in cell types that are difficult to transform using lipid-based delivery systems. Application of a controlled, short electric pulse to the cells forms pores in the cell membrane, allowing entry of foreign material. Nucleofection is a variant of electroporation, in which the electric pulse is optimized such that the nuclear membrane of the cells also forms pores. The CRISPR components are thus directly delivered inside the nucleus. Microinjection is commonly used to inject the Cas9 or other programmable endonuclease and gRNA ribonucleoprotein complex in embryos, although it can also be used in cells. Zebrafish, mouse, and most recently human embryos have been manipulated using this technique.
Viral delivery vectors include specifically engineered adeno-associated virus (AAV), and full sized adenovirus and lentivirus vehicles. Especially for in vivo work, viral vectors have found favor and are the most common CRISPR/Cas9 delivery vectors. AAV, of the Dependovirus genus and Parvoviridae family, is a single stranded DNA virus that has been extensively utilized for gene therapy. While LVs and AdVs are clearly distinct, the way they are utilized for delivery of CRISPR/Cas9 components is quite similar. In the case of LV delivery, the backbone virus is a provirus of HIV; for AdV delivery, the backbone virus is one of the many different serotypes of known AdVs. Both LV and AdV can infect dividing and non-dividing cells; however, unlike LV, AdV does not integrate into the genome. This is advantageous in the case of CRISPR/Cas9-based editing for limiting off-target effects. As is the case with AAV particles, both LV and AdV can be used in in vitro, ex vivo, and in vivo applications, which eases both efficacy and safety testing. In terms of mechanism, this class of CRISPR/Cas9 delivery is like AAV delivery described above. Full viral particles containing the desired Cas9 and sgRNA are created via transformation of HEK 293 T cells. These viral particles are then used to infect the target cell type. The biggest difference between LV/AdV delivery and AAV delivery is the size of the particle; both LVs and AdVs are roughly 80-100 nm in diameter. Compared with the 20 nm diameter of AAV, larger insertions are better tolerated in these systems. When considering CRISPR/Cas9, additional packaging space for differently-sized Cas9 constructs or several sgRNAs for multiplex genome editing is a significant advantage over the AAV delivery system.
A viral vector can be a modified viral vector, alternatively, it can be an unmodified vector. Often, the modified viral vector is a genetically modified vector. The modified viral vector can show reduced immunogenicity, an increase in the persistence of the vector in the blood stream, or impaired uptake of the vector by macrophages and antigen presenting cells.
The modified viral vector can further comprise a polymer, a lipid, a peptide, a magnetic nanoparticle (MNP), an additional compound, or a combination thereof. The polymer, lipid, or magnetic nanoparticle can be attached to a capsid of the viral vector. The polymer can be a polyethylene glycol (PEG). The polymer can be N-[2-hydroxypropyl] methacrylamide (HPMA), poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA), or arginine-grafted bioreducible polymers (ABPs). The peptide can be a cell-penetrating peptide, a cell adhesion peptide, or a peptide which binds to a receptor on a cell. The cell can be a tumor cell. Any suitable cell-penetrating peptide can be used. Examples of cell-penetrating peptides include, but are not limited to a polylysine peptide and a polyarginine peptide. The cell adhesion peptide can be an arginylglycylaspartic acid (RGD) peptide. An additional compound can be a compound which binds to a receptor on a cell, such as folic acid.
In some instances, the modified viral vector is a genetically modified vector. The genetically modified vector can have reduced immunogenicity, reduced genotoxicity, increased loading capacity, increased transgene expression, or a combination thereof. In some instances, the genetically modified viral vector is a pseudotyped viral vector. The pseudotyped viral vector can have at least one foreign viral envelope protein. The foreign viral envelope protein can be an envelope protein from a lyssavirus, an arenavirus, a hepadnavirus, a flavivirus, a paramyxovirus, a baculovirus, a filovirus, or an alphavirus. The foreign viral envelope protein can be the glycoprotein G of a vesicular stomatitis virus (VSV). In some instances, the foreign viral envelope protein is a genetically modified viral envelope protein. The genetically modified viral envelope protein can be a non-naturally occurring viral envelope protein.
In some embodiments, the viral vectors are virus-like particles (VLPs). VLPs resemble viruses but are non-infectious because they do not contain viral genetic materials. VLPs have been produced from components of a wide variety of virus families including Parvoviridae (e.g. adeno-associated virus), Retroviridae (e.g. HIV), Flaviviridae (e.g. Hepatitis C virus) and bacteriophages. VLPs can be produced in multiple cell culture systems including bacteria, mammalian cell lines, insect cell lines, yeast and plant cells.
With respect to non-viral vector delivery vehicles, lipid nanoparticles/liposomes can be used herein. A lipid can be a cationic lipid, an anionic lipid, or neutral lipid. The lipid can be a liposome, a small unilamellar vesicle (SUV), a lipidic envelope, a lipidoid, or a lipid nanoparticle (LNP). The lipid can be mixed with the nucleic acid to form a lipoplex (a nucleic acid-liposome complex). The lipid can be conjugated to the nucleic acid. The lipid can be a non-pH sensitive lipid or a pH-sensitive lipid. The lipid can further comprise a polyethylene glycol (PEG).
The cationic lipid can be a monovalent cationic lipid, such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), [1,2-bis(oleoyloxy)-3-(trimethylammonio)propane] (DOTAP), or 3β[N—(N′, N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol). The cationic lipid can be a multivalent cationic lipid, such as Di-octadecyl-amido-glycyl-spermine (DOGS) or {2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate} (DOSPA).
The anionic lipid can be a phospholipid or dioleoylphosphatidylglycerol (DOPG). Examples of phospholipids include, but are not limited to, phosphatidic acid, phosphatidylglycerol, or phosphatidylserine. In some instances, the anionic lipid further comprises a divalent cation, such as Ca2+, Mg2+, Mn2+, and Ba2+.
The cationic lipid or the anionic lipid can further comprise a neutral lipid. The neutral lipid can be dioleoylphosphatidyl ethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC). In some instances, the use of a helper lipid in combination with a charged lipid yields higher transfection efficiencies.
The liposome can further comprise a polymer, a lipid, a peptide, a magnetic nanoparticle (MNP), an additional compound, or a combination thereof. The polymer, lipid, or magnetic nanoparticle can be attached to the liposome or integrated into the liposomal membrane. The polymer can be a polyethylene glycol (PEG). The polymer can be N-[2-hydroxypropyl] methacrylamide (HPMA), poly (2-(dimethylamino)ethyl methacrylate) (pDMAEMA), or arginine-grafted bioreducible polymers (ABPs). The peptide can be a cell-penetrating peptide, a cell adhesion peptide, or a peptide which binds to a receptor on a cell. The cell can be a tumor cell. Any suitable cell-penetrating peptide can be used. Examples of cell-penetrating peptides include, but are not limited to a polylysine peptide and a polyarginine peptide. The cell adhesion peptide can be an arginylglycylaspartic acid (RGD) peptide. An additional compound can be a compound which binds to a receptor on a cell, such as folic acid.
Disclosed herein are kits and articles of manufacture for use with one or more methods and compositions described herein. The kit can comprise a polynucleotide composition described herein formulated in a compatible pharmaceutical excipient and placed in an appropriate container.
The kit can include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. A container can be formed from a variety of materials such as glass or plastic.
The kit can include an identifying description, a label, or a package insert. The label or package insert can list contents of kit or the immunological composition, instructions relating to its use in the methods described herein, or a combination thereof. The label can be on or associated with the container. The label can be on a container when letters, numbers, or other characters forming the label are attached, molded or etched into the container itself. The label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In some instances, the label is used to indicate that the contents are to be used for a specific therapeutic application.
The kit herein can further comprise one or more reagents that used to deliver the polynucleotide sequences to cells, tissues, or organs.
The disclosed RNPs can be introduced into cells using one of the delivery methods disclosed herein to induce homologues recombination of DNA in the cells. Further, the disclosed RNPs can be introduced into cells using one of the delivery methods disclosed herein to induce HDR in cells in vitro or ex vivo. The DNA molecule is contacted with the RNPs. The modified Cas9 protein guided by a gRNA introduces a DSB by cleaving at a location as determined by the hybridization of the gRNA with the DNA molecule. The hExo1 peptide partially digests the cleaved DNA molecule, leaving a 3′ or 5′ overhang. The HDR template sequences comprising some degrees of sequence homology as the digested DNA molecule promotes and serves as the template for HDR. After HDR, the DNA molecule in the cell comprises a sequence that is identical to the HDR template at the region where homologous recombination occurs.
By inducing HDR in cells, the cellular toxicity caused by wild type Cas9 protein along with gRNAs is decreased. Cellular toxicity can be measured by several cell viability assays. In some embodiments, tetrazolium reduction assay is used. A variety of tetrazolium compounds have been used to detect viable cells. The most commonly used compounds include: MTT, MTS, XTT, and WST-1. These compounds fall into two basic categories: 1) MTT which is positively charged and readily penetrates viable eukaryotic cells and 2) those such as MTS, XTT, and WST-1 which are negatively charged and do not readily penetrate cells. The latter class (MTS, XTT, WST-1) are typically used with an intermediate electron acceptor that can transfer electrons from the cytoplasm or plasma membrane to facilitate the reduction of the tetrazolium into the colored formazan product. For example, viable cells with active metabolism convert MTT into a purple colored formazan product with an absorbance maximum near 570 nm. When cells die, they lose the ability to convert MTT into formazan, thus color formation serves as a useful and convenient marker of only the viable cells.
In other embodiments, resazurin reduction assay is used. Resazurin is a cell permeable redox indicator that can be used to monitor viable cell number with protocols similar to those utilizing the tetrazolium compounds. Resazurin can be dissolved in physiological buffers (resulting in a deep blue colored solution) and added directly to cells in culture in a homogeneous format. Viable cells with active metabolism can reduce resazurin into the resorufin product which is pink and fluorescent. The quantity of resorufin produced is proportional to the number of viable cells which can be quantified using a microplate fluorometer equipped with a 530 nm or 560 nm excitation/590 nm emission filter set. The wavelength can be adjusted according to different types of cells and experimental designs. Resorufin also can be quantified by measuring a change in absorbance; however, absorbance detection is not often used because it is far less sensitive than measuring fluorescence.
Further, the disclosed RNPs herein are used to treat diseases where the causes of the diseases are tranced to a locus of chromosomal abnormality. In certain embodiments, a biological sample is obtained from a subject afflicted with a disease. DNA is extracted from the biological sample and sequenced to determine the locus of chromosomal abnormality. Primary cells harboring the chromosomal abnormality are isolated from the subject and cultured ex vivo. The RNPs are delivered into the said cultured primary cells using one of the delivery methods disclosed herein. The HDR template sequences are also delivered into the cultured primary cells. In some embodiments, the gRNA moiety comprises at least 10 nucleotides complementary to the targeted locus of chromosomal abnormality. The HDR template sequences comprise an integration cassette flanked by a 5′ homology region and a 3′ homology region, wherein the 5′ homology region and the 3′ homology region exhibit at least 80% identity to adjacent segments of the targeted locus. The integration cassette of the HDR template comprises a wild type sequence that corresponds to the locus of chromosomal abnormality as detected in the primary cells. Upon delivering of the RNPs, the gRNA directs the protein fusion complex to the targeted locus, where the modified Cas protein moiety creates a DSB by cleaving said targeted locus as recognized by the gRNA. The nuclease moiety partially digests the cleaved locus of chromosomal abnormality, leaving a 3′ overhang. The presence of the HDR template sequences promotes endogenous repair through HDR. Primary cells with wild type sequence replacing chromosomal abnormality are screened and selected for reintroducing back into the subject.
In some embodiments, primary cells are selected from the group comprising T cells, B cells, dendritic cells, natural killer cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, mast cells, hematopoietic progenitor cells, hematopoietic stem cells (HSCs), red blood cells, blood stem cells, endoderm stem cells, endoderm progenitor cells, endoderm precursor cells, differentiated endoderm cells, mesenchymal stem cells (MSCs), mesenchymal progenitor cells, mesenchymal precursor cells, differentiated mesenchymal cells, hepatocytes progenitor cells, pancreatic progenitor cells, lung progenitor cells, tracheae progenitor cells, bone cells, cartilage cells, muscle cells, adipose cells, stromal cells, fibroblasts, and dermal cells.
Further, in some embodiments, the gRNA is configured to recognize exon 1 of the human HBB gene. The HDR template is configured to have 5′ and 3′ arm homology with a functional human HBB gene. In other embodiments, the gRNA is configured to recognize a region of CFTR and the HDR template is designed to have 5′ and 3′ arm homology with a functional CFTR gene.
Please see a list of single gene disorders with the mutated locus of gene respectively listed in Table 3. Examples of human monogenic diseases, modes of inheritance, and associated genes.
Moreover, the disclosed RNPs herein are used to introduce genetic modification to confer immunity against diseases. A biological sample is obtained from a subject. DNA is extracted and the locus for the targeted genetic modification is sequenced. Primary cells the subjected are isolated and cultured ex vivo. RNPs and the HDR template sequences are delivered into said cultured primary cells. The gRNA moiety directs the RNPs to the targeted locus to initiate the formation of DSB and DNA digestion to generate the 3′ overhang. The HDR template comprises an integration cassette flanked by a 5′ homology region and a 3′ homology region, wherein the 5′ homology region and the 3′ homology region exhibit at least 80% identity to adjacent segments of the targeted loci. The integration cassette comprises a wild type sequence that is different from the subject's sequence at the targeted locus. The presence of the polynucleotide promotes endogenous repair through HDR. Primary cells harboring wild type sequence encoded by the polynucleotide are screened and selected for reintroducing back into the subject.
As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
As used herein, the terms “polypeptide,” “peptide” and “protein” are often used interchangeably herein in reference to a polymer of amino acid residues. A protein, generally, refers to a full-length polypeptide as translated from a coding open reading frame, or as processed to its mature form, while a polypeptide or peptide informally refers to a degradation fragment or a processing fragment of a protein that nonetheless uniquely or identifiably maps to a particular protein. A polypeptide can be a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Polypeptides can be modified, for example, by the addition of carbohydrate, phosphorylation, etc. Proteins can comprise one or more polypeptides.
As used herein, the terms “fragment,” “domain,” or equivalent terms refer to a portion of a protein that has less than the full length of the protein and maintains the function of the protein. Further, when the portion of the protein is blasted again the protein, the portion of the protein sequence would align at least with 80% identity to part of the protein sequence.
As used herein, the terms “polynucleotide,” “nucleic acid,” “oligonucleotide,” or equivalent terms, refer to molecules that comprises a polymeric arrangement of nucleotide base monomers, where the sequence of monomers defines the polynucleotide. Polynucleotides can include polymers of deoxyribonucleotides to produce deoxyribonucleic acid (DNA), and polymers of ribonucleotides to produce ribonucleic acid (RNA). A polynucleotide can be single or double stranded. When single stranded, the polynucleotide can correspond to the sense or antisense strand of a gene. A single-stranded polynucleotide can hybridize with a complementary portion of a target polynucleotide to form a duplex, which can be a homoduplex or a heteroduplex. The length of a polynucleotide is not limited in any respect. Linkages between nucleotides can be internucleotide-type phosphodiester linkages, or any other type of linkage. A polynucleotide can be produced by biological means (e.g., enzymatically), either in vivo (in a cell) or in vitro (in a cell-free system). A polynucleotide can be chemically synthesized using enzyme-free systems. A polynucleotide can be enzymatically extendable or enzymatically non-extendable.
As used herein, the terms “vector,” “vehicle,” “construct” and “plasmid” are used in reference to any recombinant polynucleotide molecule that can be propagated and used to transfer nucleic acid segment(s) from one organism to another. Vectors generally comprise parts which mediate vector propagation and manipulation (e.g., one or more origin of replication, genes imparting drug or antibiotic resistance, a multiple cloning site, operably linked promoter/enhancer elements which enable the expression of a cloned gene, etc.). Vectors are generally recombinant nucleic acid molecules, often derived from bacteriophages, or plant or animal viruses. Plasmids and cosmids refer to two such recombinant vectors. A “cloning vector” or “shuttle vector” or “subcloning vector” contain operably linked parts that facilitate subcloning steps (e.g., a multiple cloning site containing multiple restriction endonuclease target sequences). A nucleic acid vector can be a linear molecule, or in circular form, depending on type of vector or type of application. Some circular nucleic acid vectors can be intentionally linearized prior to delivery into a cell.
As used herein, the term “gene” generally refers to a combination of polynucleotide elements, that when operatively linked in either a native or recombinant manner, provide some product or function. The term “gene” is to be interpreted broadly, and can encompass mRNA, cDNA, cRNA and genomic DNA forms of a gene. In some uses, the term “gene” encompasses the transcribed sequences, including 5′ and 3′ untranslated regions (5′-UTR and 3′-UTR), exons and introns. In some genes, the transcribed region will contain “open reading frames” that encode polypeptides. In some uses of the term, a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region”) necessary for encoding a polypeptide. In some aspects, genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some aspects, the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters. The term “gene” encompasses mRNA, cDNA and genomic forms of a gene.
As used herein, the terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. The disease can be cancer. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.
As used herein, the term “in vivo” is used to describe an event that takes place in a subject's body.
As used herein, the term “ex vivo” is used to describe an event that takes place outside of a subject's body. An “ex vivo” assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ‘ex vivo’ assay performed on a sample is an ‘in vitro’ assay.
As used herein, the term “in vitro” is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the living biological source organism from which the material is obtained. In vitro assays can encompass cell-based assays in which cells alive or dead are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed.
“Treating” or “treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder, as well as those prone to have the disorder, or those in whom the disorder is to be prevented. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.
Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes, such as a number that is within 10% of the value of the number that it precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating un-recited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods and compositions described herein are. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods and compositions described herein, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions described herein.
Mention is frequently made to “Cas9” throughout the disclosure. It is understood that, although Cas9 is a particular embodiment, additional programmable endonucleases are also contemplated, such as Cas12 or others. Accordingly, mention of Cas9 should not always be read to exclude alternate or other programmable endonuclease.
Similarly, “hEXO1” is frequently referred to. It is understood that, although hEXO1 is a particular embodiment, additional programmable endonucleases are also contemplated. Accordingly, mention of hEXO1 should not always be read to exclude alternate or other exonuclease.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions described herein belong. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions described herein, representative illustrative methods and materials are now described.
The following examples are given for the purpose of illustrating various embodiments as described in the present disclosure and are not meant to be limiting in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present disclosure. Changes therein and other uses which are encompassed within the spirit of the present disclosure as defined by the scope of the claims will occur to those skilled in the art.
Referring to
Human lung carcinoma A549 cells were cultured and about 2.5×104 cells were plated in 96-well plates, with 8-16 transfection replicates per individual treatment. Each well was then transfected with 62.5 ng of plasmid DNA using a standard Calcium Phosphate transfection technique and incubated overnight for 16-20 hours. Cells were then allowed to recover for one day. Resazurin reduction assay (
Referring to
Similarly to experiments conducted in Example 1, several plasmids containing polynucleotide encoding fusion protein hExo1-Cas9 enzymes (
Similar cell culture and transfection protocols were used as in experiments conducted in Example 1. Referring to
A biological sample is obtained from a subject afflicted with sickle cell anemia. Genomic DNA is extracted from the biological sample and sequenced to verify a single nucleotide substitution (A to T) in the amino acid 6 codon of the (3-globin gene. This mutation converts a glutamic acid codon (GAG) to a valine codon (GTG). Hematopoietic stem cells are isolated from the bone marrow cavity of the patient and cultured ex vivo. Nucleic acid vectors encoding the protein fusion complex of the hExo1-Cas9 and the gRNA moiety are delivered into the cultured hematopoietic stem cells. Further, the DNA template sequences with an integration cassette encoding the wild type sequence of exon 1 of (3-globin gene are delivered to the cultured hematopoietic stem cells. The gRNA moiety comprises at least 10 nucleotides complementary to the GTG locus of exon 1 of the (3-globin gene. The DNA template sequence comprises an integration cassette flanked by a 5′ homology region and a 3′ homology region, wherein the 5′ homology region and the 3′ homology region exhibit at least 80% identity to the segments flanking the GTG locus of exon 1. The integration cassette of the polynucleotide comprises a wild type GAG sequence that corresponds to the locus of chromosomal abnormality as detected in the primary cells. Upon delivering of the nucleic acids encoding the RNPs and DNA template sequences into the cultured hematopoietic stem cells, the gRNA directs the engineered hExo1-Cas9 proteins to the GTG locus, where the Cas9 portion of the engineered hExo1-Cas9 proteins creates a DSB. The hExo1 portion of the engineered hExo1-Cas9 proteins partially digests the cleaved GTG locus of, leaving a 3′ overhang. The presence of the DNA template sequences promotes endogenous repair through HDR, where the integration cassette with the correct wild type sequence, GAG, at amino acid 6 of exon 1 of the (3-globin gene is inserted into the chromosome of the hematopoietic stem cells. Hematopoietic stem cells with corrected GAG sequence is screened for and selected to be transplanted back into the patient.
Similarly to experiments conducted in Example 2, several plasmids containing polynucleotide encoding fusion protein hExo1-Cas9 enzymes (
Similar cell culture and transfection protocols were used as in experiments conducted in Example 2. Roughly 2.5*10{circumflex over ( )}4 cells were plated in 96 well plates, with 8-16 replicates per individual experiment, as diagramed in
Cas-9 hExo1 fusions were used to integrate an antibiotic resistance cassette into a locus on Chromosome 6 of A549 cells. The Puromycin resistance repair template is diagramed in
Similar cell culture and transfection protocols were used as in experiments conducted in Example 4. Roughly 2.5*10{circumflex over ( )}4 cells were plated in 96 well plates, with 8-16 replicates per individual experiment, as diagramed in
Compared to the experiment in Example 5, K562 cells were used and Neon (Thermo Fisher) electroporation was used. K562 cells lack P53 function. In light of the results of Example 5, it was important to remove the variable of the activation of P53 by the activity of Cas9 as this would differ between fusion Cas9 and wild type Cas9, introducing the possibility of effecting the results of the antibiotic screen.
K562 cells were electroporated with 500 ng of each plasmid and 100 ng of repair template as shown in
Quantification of toxicity was performed as in Example 5, with the addition of fusion construct 9.
Successful amplification using primer specific for the genome and specific for the repair template demonstrates successful integration of the repair template and that the reduction in toxicity of the Cas9-HR series of constructs is not due to lack of nuclease activity. There may be indication that the Cas9-HR series has a higher editing efficiency than Cas9.
As seen in Example 2, different guide RNAs can have radically different cleavage rates and toxicities. Constructs with unmodified Cas9 and guides targeting regions shown in
DNA was extracted from cells transfected with HBB-G1, HBB-G2, and HBB-G3, amplified with the outer primer pair in
Similar to Example 7, K562 cells are used because they lack P53 activity as well as because they share more similarities to hematocytes than A549 cells.
The gRNA of Example 7, HBB-G3, is transfected with Cas9 and Cas9-HR 1-9 respectively to introduce multiple mutations into the HBB locus of K562 cells. The first mutation chosen is Sickle Cell E6V mutation. The Sickle Cell E6V mutation is made along with an additional mutation creating an EcoRI restriction site and two silent mutations designed to prevent re-cutting of the repair template once integrated into the genome, in addition to 60 bp homology arms on each side of the predicted cut-site.
Transfection is achieved with electroporation. Two days after electroporation, toxicity assays with Resazurin are conducted as in Example 6. DNA is also harvested and the HBB locus is amplified to prepare for deep sequencing to measure INDELs and HDR rate. Alternatively, DNA can be digested with EcoRI to measure target efficiency.
The experiments of Example 8 are repeated on CD34+ cells. The gRNA from Example 8, HBB-G3, is transfected with Cas9 and Cas9-HR 1-9 respectively to introduce multiple mutations into the HBB locus of K562 cells. The first mutation chosen is Sickle Cell E6V mutation. The Sickle Cell E6V mutation is made along with an additional mutation creating an EcoRI restriction site and two silent mutations designed to prevent re-cutting of the repair template once integrated into the genome, in addition to 60 bp homology arms on each side of the predicted cut-site.
Transfection is achieved with electroporation. Two days after electroporation, toxicity assays with Resazurin are conducted as in Example 6. DNA is harvested and the HBB locus is amplified to prepare for deep sequencing to measure INDELs and HDR rate. Alternatively, DNA is digested with EcoRI to measure target efficiency.
A 954 bp piece of DNA was amplified from wildtype K562 cells using standard Taq DNA polymerase and HBB-out-4-F (5′-aacgatcctgagacttccaca-3′ (SEQ ID NO: 127)) and HBB-out-5-R (5′-tgcttaccaagctgtgattcc-3′ (SEQ ID NO: 128)), Tm=56 for 35 cycles, and purified using the Qiagen PCR cleanup kit. Next, HBB-G1 (5′-guaacggcagacuucuccuc-3′ (SEQ ID NO: 129),IDT) or HBB-G3 (5′-gaggugaacguggaugaagu-3′ (SEQ ID NO: 130),IDT) were combined with tracrRNA (IDT) at final concentrations of 1 μM each in duplex buffer (IDT). The RNA was heated for 5 minutes at then allowed to cool to room temperature. Cas9 or Cas9-HR3 were then combined with either HBB-G1 or HBB-G3 guide RNA complex and amplified DNA at a 10:10:1 molar ratio (30 nM:30 nM:3 nM) in 1×Cas9 reaction Buffer (50 mM Tris, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, pH7.9) and incubated for 1 hr at 37° C., after which 1 μL of Proteinase K was added and the reaction was incubated for an additional 20 minutes at 50° C. The samples were then electrophoresed on a standard 1% TAE agarose gel and imaged.
Cas9-HR and Cas were utilized to introduce an hH2b fragment into the H2B genomic locus. Primers were designed so that the genomic primer is outside of the H2B-mNeon repair template (RT), while the other is RT specific (within mNeon) as shown in
After transfection of K562 cells, genomic DNA was extracted from cells transfected with repair template (RT) and either Cas9-HR4, Cas9-HR8, Cas9 (NT), or untransfected (Con). Standard Taq polymerase (Bioneer, Tm=56,35 cycles) was used to amplify the fragments flanked by the 5′ primers or the 3′ primers.
Cells from either undifferentiated or mature adipose tissue are isolated from a patient and transfected with either plasmids encoding any one of the versions of Cas9-HR or purified RNPs. The chosen Cas9-HR(s) can be targeted to sites of the human genome which have been already been shown to be amenable to DNA insertion (“safe harbor sites”) or any such novel site identified. Additionally, a repair template containing the cDNA for either Uncoupling Proteins (UCPs) 1, 2, 3 is transfected simultaneously. This transgene contains 5′ Homology Arms (HAs) to the chosen integration site, either a ubiquitous or tissue specific enhancer complexed with a basal promoter, either with or without 5′UTR sequence, an ORF consisting of the aforementioned cDNA from either UCP 1, 2 or 3, with or without a 3′ UTR sequence, a poly-adenylation sequence, and a 3′ HA to the chosen integration site. Integration and subsequent reintroduction of the Adipose Tissue expressing this transgene can increase basal metabolism, leading to overall weight loss and decrease in adipose lipid deposit size. Use of Cas9-HRs can lead to reduction in toxicity and increase the number of cells successfully integrated.
Plasmids encoding Cas9-HR(s) or purified RNPs can be used to transfect either isolated cells or in-situ on the scalp to transfect transgenes expressing either full length or modified Sex Binding Hormone Globulin (SBHG), NRF 2, or SRD5A1,2 or 3. The chosen Cas9-HR(s) can be targeted to sites of the human genome which have been already been shown to be amenable to DNA insertion (“safe harbor sites”) or any such novel site identified. These transgenes contain 5′ Homology Arms (HAs) to the chosen integration site, either a ubiquitous or tissue specific enhancer complexed with a basal promoter, either with or without 5′UTR sequence, an ORF consisting of the aforementioned cDNA from either SBHG, NFR2 or SRD5A1,2, or 3, with or without a 3′ UTR sequence, a poly-adenylation sequence, and a 3′ HA to the chosen integration site. Successful transfection of either in-situ cells or re-introduction of isolated dermal cells can delay or permanently halt hair-loss and result in hair regrowth.
While preferred embodiments of the present disclosure 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 present disclosure. It should be understood that various alternatives to the embodiments described herein may be employed. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Bacterial Immunity. Science 337, 816-821 (2012).
This application is a continuation application of U.S. Non-Provisional application Ser. No. 17/368,369, filed Jul. 6, 2021, which is a continuation application of International Application No. PCT/US2020/012438, filed Jan. 6, 2020, which claims priority to U.S. provisional application 62/789,347, filed on Jan. 7, 2019; U.S. provisional application 62/823,477, filed on Mar. 25, 2019; U.S. provisional application 62/824,164, filed on Mar. 26, 2019, and U.S. provisional application 62/855,612, filed on May 31, 2019, the entirety of which are hereby incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 62789347 | Jan 2019 | US | |
| 62823477 | Mar 2019 | US | |
| 62824164 | Mar 2019 | US | |
| 62855612 | May 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17368369 | Jul 2021 | US |
| Child | 18462921 | US | |
| Parent | PCT/US2020/012438 | Jan 2020 | US |
| Child | 17368369 | US |
