WO2021226019A2 - Compositions et méthodes de traitement d'infections virales - Google Patents

Compositions et méthodes de traitement d'infections virales Download PDF

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WO2021226019A2
WO2021226019A2 PCT/US2021/030570 US2021030570W WO2021226019A2 WO 2021226019 A2 WO2021226019 A2 WO 2021226019A2 US 2021030570 W US2021030570 W US 2021030570W WO 2021226019 A2 WO2021226019 A2 WO 2021226019A2
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cov
sars
aso
gene
grna
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PCT/US2021/030570
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WO2021226019A3 (fr
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Dennis J. Hartigan-O'connor
David J. Segal
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The Regents Of The University Of California
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • RNA-targeted therapies represent a platform for drug discovery that is inherently more specific than traditional small-molecule drugs and infinitely customizable.
  • ASO antisense oligonucleotide
  • the present disclosure provides a guide RNA (gRNA) that is either identical to or complementary to an equal length portion of a sequence of a gene in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome.
  • SARS-CoV-2 genome has the sequence of GenBank Accession No. MN908947.
  • the gRNA targets a Cas nuclease to the sequence of the gene or to its reverse complement in the SARS-CoV-2 genome.
  • the Cas nuclease cleaves the sequence of the gene or its reverse complement in the SARS-CoV-2 genome.
  • the sequence of the gene or its reverse complement is in a SARS-CoV-2 gene selected from the group consisting of orflab, S gene, ORF3a, E gene, M gene, ORF6, ORF7a, ORF8, N gene, and ORFIO.
  • the gRNA comprises between 15 and 45 nucleotides in length. In some embodiments, the gRNA forms a double-stranded RNA duplex with a scaffold RNA. In some embodiments, the gRNA is a portion of a single-guide RNA. [0005] In some embodiments, the Cas nuclease is selected from the group consisting of Casl3a, Casl3b, or Casl3d.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a gRNA described herein and a Cas nuclease and one or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical composition comprises two or more gRNAs.
  • the two or more gRNAs are cloned in a tandem array from which individual crRNAs can be cleaved.
  • the disclosure provides a method for modifying a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gene in a SARS-CoV-2 genome in a cell, comprising introducing into the cell a Cas nuclease and the gRNA described herein, or the pharmaceutical composition comprising the gRNA, wherein the Cas nuclease cleaves the sequence of the SARS-CoV-2 gene or its reverse complement in the SARS-CoV-2 genome.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the gRNA is introduced into the cell in an adeno-associated viral (AAV) vector.
  • the Cas nuclease and the gRNA are introduced into the cell in an adeno-associated viral (AAV) vector.
  • the disclosure provides a method of treating a subject having a coronavirus disease 2019 (COVID-19) caused by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising administering to the subject a therapeutically effective amount of a Cas nuclease and the gRNA described herein, or the pharmaceutical composition described herein, wherein the Cas nuclease cleaves the sequence of a SARS- CoV-2 gene or its reverse complement in the SARS-CoV-2 genome.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the disclosure provides a method for identifying a gRNA that is either identical to or complementary to an equal length portion of a sequence of a gene in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and targets a Cas nuclease to the sequence of the gene, comprising: (a) designing a library of gRNAs that hybridize to a plurality of different portions in the gene or its reverse complement; (b) synthesizing a library of DNA templates encoding the gRNAs; (c) introducing the library in step (b) and a Cas nuclease to a plurality of cells, wherein each cell comprises at least one gRNA from the library and the Cas nuclease; (d) contacting the plurality of cells with SARS- CoV-2; (e) selecting cells that do not undergo a cytopathic effect (CPE); and (f) isolating and sequencing the gRNAs from the cells selected in step (e).
  • SARS-CoV-2
  • the Cas nuclease is Casl3b.
  • the disclosure provides an antisense oligonucleotide (ASO), or a portion thereof, that is either identical to or complementary to an equal length portion of a sequence of a gene in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome.
  • SARS-CoV-2 genome has the sequence of GenBank Accession No. MN908947.
  • the ASO comprises between 10 and 30 nucleotides in length.
  • the ASO comprises at least one modified nucleobase.
  • the ASO can comprise at least one modified internucleoside linkage.
  • the modified internucleoside linkage can be a phosphorothioate internucleoside linkage.
  • the ASO can comprises at least one modified sugar.
  • the modified sugar can be selected from the group consisting of a 2’-0-methoxyethyl modified sugar, a bicyclic sugar, a 2’-methoxy modified sugar, a 2’-0-alkyl modified sugar, and an unlocked sugar.
  • the ASO comprises a sequence having at least 90% (e.g ., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of any one of SEQ ID NOS: 1-16 and 21-24 (e.g., SEQ ID NOS:2, 10, 15, and 23).
  • one or more nucleotides in the ASO e.g, ASO having a sequence of any one of SEQ ID NOS: 1-16 and 21-24 (e.g, SEQ ID NO:2, 10, 15, and 23)
  • the modified nucleotide comprises 5-methyl cytosine or a modified sugar comprising 2'-0-methoxy-ethyl.
  • the ASO comprises one or more modified internucleoside linkages.
  • the ASO comprises a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of any one of SEQ ID NOS:25-40 and 45-48 (e.g, SEQ ID NOS:26, 34, 39, and 47), including the modifications on the nucleotides and intemucleoside linkages.
  • the disclosure provides an ASO described herein and one or more pharmaceutically acceptable carriers or excipients.
  • the disclosure provides a method of inhibiting the expression or replication of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gene in a SARS-CoV-2 genome in a subject, comprising administering to the subject a therapeutically effective amount of an ASO described herein or a pharmaceutical composition comprising thereof.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the disclosure provides a method of treating a subject having a coronavirus disease 2019 (COVID-19) caused by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising administering to the subject a therapeutically effective amount of an ASO described herein or a pharmaceutical composition comprising thereof, wherein the ASO inhibits the expression or replication of the SARS-CoV-2 gene.
  • SARS-CoV-2 genome has the sequence of GenBank Accession No. MN908947.
  • the disclosure provides a method for identifying an antisense oligonucleotide (ASO) that is either identical to or complementary to an equal length portion of a sequence in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gene in a SARS-CoV-2 genome, comprising: (a) designing a library of ASOs that hybridize to a portion of the SARS-CoV-2 gene; (b) synthesizing the library of ASOs; (c) introducing the library of ASOs to cells individually or in a pool of ASOs, wherein each cell comprises at least one ASO; (d) contacting the cell(s) with SARS-CoV-2; (e) selecting the ASO or the pool of ASOs if it confers resistance in the cell to cytopathic effect (CPE), and/or inhibits the expression or replication of the gene.
  • ASO antisense oligonucleotide
  • FIG. 1 Scheme of CRISPRa library screen.
  • Target cells expressing the dCas9 activation system are transduced with a lentivirus library to express one gRNA/cell.
  • the resulting cell library with regulators for 18,885 specific genes can be incubated with or without drug.
  • gRNAs are recovered by PCR and analyzed by sequencing. gRNAs causing cell death will be underrepresented, while those that improve growth and survival will be overrepresented.
  • FIG. 2 Stable expression of dCas9-VP64 activator.
  • FIGS. 3 A and 3B Analysis of CRISPRa screen hits.
  • FIGS. 4A-4C AAV-PHP.eB/Casl3b vector for the treatment of Angelman syndrome.
  • FIG. 5 Unbiased Casl3 nuclease screen for potent inhibitors of SARS-CoV-2- induced CPE.
  • FIG. 6 Combiantion of an unbiased screen of ASOs “tiling” the SARS-CoV-2 genome (200 phosporothioate 18-mers, PS-ASOs) and a smaller screen leveraging the accessible regions of the genome identified by Casl3b screening (100 PS-ASOs).
  • FIG. 7 A uniform challenge and monitoring protocol is established so that consistent sets of virologic, immunologic, and pathologic data may be leveraged across experiments.
  • FIG. 8 Scatterplot showing ASOs of various tiers identified in the ORFlab, S region, and N region.
  • FIG. 9 Scatterplot showing ASOs of various tiers and the position and targeted strand of the viruses they targeted. It appears that efficacious ASOs can target either strand.
  • FIGS. 10A and 10B Photomicrographs showing infected samples with many dead cells that were treated with control ASOs.
  • FIGS. lOC-lOF Photomicrographs showing successful inhibition of replication using the ASOs A_APL-PR07n (tier 2) (FIG. IOC), A_PL-PR07p (tier 2) (FIG. 10D), A NUClp (tier 1) (FIG. 10E), or A_MOE_SPIKE2p.JPG (tier 1) (FIG. 10F).
  • Coronavirus disease 2019 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Some symptoms of the disease include, for example, fever, cough, shortness of breath, muscle pain, sputum production, diarrhea, sore throat, loss of smell, and abdominal pain. While the majority of cases result in mild symptoms, some progress to viral pneumonia and multi-organ failure.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the virus is spread mainly through close contact and via respiratory droplets produced when people cough or sneeze. People may also contract COVID-19 by touching a contaminated surface and then their face. The infection is most contagious when people are symptomatic, although spread may be possible before symptoms appear.
  • the standard method of diagnosis is by reverse transcription polymerase chain reaction (rRT-PCR) of a subject’s biological sample, such as a nasopharyngeal swab.
  • rRT-PCR reverse transcription polymerase chain reaction
  • compositions and methods for treating COVID-19 that involve the use of guide RNAs (gRNAs) and antisense oligonucleotides (ASOs).
  • gRNAs and ASOs can be either identical to or complementary to an equal length portion of a sequence of a SAR-CoV-2 gene in a SARS-CoV-2 genome.
  • the gRNAs can target a Cas nuclease to the sequence of the SARS-CoV-2 gene or to its reverse complement in the SARS-CoV-2 genome and the Cas nuclease can cleave the gene or its reverse complement.
  • the ASOs can hybridize to a SARS-CoV-2 gene or to its reverse complement and inhibit the expression or replication of the gene.
  • any reference to “about X” specifically indicates at least the values X, 0.8X, 0.8 IX, 0.82X, 0.83X, 0.84X, 0.85X, 0.86X, 0.87X, 0.88X, 0.89X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.11X, 1.12X, 1.13X, 1.14X, 1.15X, 1.16X, 1.17X, 1.18X, 1.19X, and 1.2X.
  • gene refers to a combination of polynucleotide elements, that when operatively linked in either a native or recombinant manner, provide some product or function.
  • gene is to be interpreted broadly, and can encompass mRNA, cDNA, cRNA and genomic DNA or RNA forms of a gene.
  • a guide RNA refers to a targeting RNA that can guide a Cas nuclease to a target nucleic acid by hybridizing to the target nucleic acid.
  • a guide RNA can be a portion of a “single-guide RNA” or “sgRNA,” which contains the guide RNA ( i.e crRNA equivalent portion of the single-guide RNA) that targets the Cas nuclease to the target nucleic acid as well as a scaffold sequence (i.e., tracrRNA equivalent portion of the single-guide RNA) that interacts with the Cas nuclease.
  • a guide RNA can be a part of a two-component system that includes the guide RNA and a scaffold sequence that interacts with the Cas nuclease, in which the guide RNA, or a portion thereof, and the scaffold sequence, or a portion thereof, can hybridize to each other to form a double-stranded RNA duplex.
  • Cas nuclease refers to a Clustered Regularly Interspaced Short Palindromic Repeats-associated polypeptide or nuclease that cleaves single- or double- stranded nucleic acids at sites specified by a guide sequence (which may vary in length from about 20 to about 90 nucelotides) contained within a crRNA transcript.
  • Some Cas nucleases e.g., Cas9, require both a crRNA and a tracrRNA for site-specific nucleic acid recognition and cleavage. In these cases the crRNA associates, through a region of partial complementarity, with the tracrRNA to guide the Cas nuclease to a region homologous to the crRNA in the target nucleic acid.
  • antisense oligonucleotide refers to an oligomer or polymer of nucleotides.
  • This oligomer or polymer of nucleotides includes naturally-occurring nucleosides (i.e., adenosine, guanosine, cytidine, 5-methyluridine, or uridine) or modified forms thereof, that are covalently linked to each other though internucleoside linkages.
  • An ASO is complementary to a target nucleic acid, such that the ASO hybridizes to the target nucleic acid sequence or to its reverse complement.
  • An ASO can include one or more modified nucleotides, which are nucleotides that have at least one change that is structurally distinguishable from a naturally-occurring nucleotide.
  • a modified nucleotide includes a modified nucleobase and/or a modified sugar.
  • the term “nucleotide” refers a nucleobase covalently linked to a sugar and a 5’ functional moiety (e.g ., a phosphorous moiety).
  • a nucleotide includes a nucleoside and a 5’ functional moiety (e.g., a phosphorous moiety) covalently linked to the 5’ carbon of the sugar portion of the nucleoside.
  • a 5’ functional moiety in a nucleotide refers to a functional group that is covalently attached to the 5’ carbon of the sugar and generally serves to connect neighboring nucleotides (i.e., the functional moiety joined to the 5’ carbon of the sugar of one nucleoside is covalently linked to the 3’ carbon of the sugar of the adjacent nucleoside).
  • An example of a 5’ functional moiety is a phosphorous moiety, which refers to a phosphorous-containing functional moiety that is covalently linked to the 5’ carbon of the sugar and functions to connect neighboring nucleotides.
  • phosphorous moieties include, but are not limited to, a phosphate, a phosphorothioate, a phosphorodithioate, a phosphoramidate, a phosphorodiamidate, a thiophosphoramidate, and a thiophosphorodiamidate.
  • the 5’ functional moiety (e.g, a phosphorous moiety) of a nucleotide forms part of the internucleoside linkage, which is defined further herein.
  • a nucleotide may be a naturally-occurring nucleotide or a modified nucleotide.
  • a naturally-occurring nucleotide has a naturally-occurring nucleoside (e.g, adenosine, guanosine, cytidine, 5-methyluridine, or uridine) covalently linked to a phosphate at the 5’ carbon of the sugar.
  • a “modified nucleotide” refers to a nucleotide having at least one change that is structurally distinguishable from a naturally-occurring nucleotide.
  • a modified nucleotide may include a modified nucleobase and/or a modified sugar. Examples of modified nucleobases and modified sugars are described in detail further herein.
  • modified nucleobase refers to a nucleobase having at least one change from a naturally-occurring nucleobase (e.g, adenine, guanine, cytosine, thymine, or uracil).
  • a naturally-occurring nucleobase e.g, adenine, guanine, cytosine, thymine, or uracil.
  • modified sugar refers to a sugar having at least one change from a naturally-occurring sugar (e.g, 2’-deoxyribose in DNA or ribose in RNA).
  • a modified sugar is a pentofuranosyl sugar.
  • a modified sugar is a locked sugar.
  • a modified sugar is an unlocked sugar.
  • a modified sugar is a 2'-0-methoxy-ethyl (2 -MOE) substituted sugar.
  • internucleoside linkage refers to the backbone linkage of the oligonucleotide that connects the neighboring nucleosides.
  • An internucleoside linkage may be a naturally-occurring internucleoside linkage (e.g, a phosphate linkage, also referred to as a 3’ to 5’ phosphodiester linkage) or a modified internucleoside linkage.
  • modified intemucleoside linkage refers to an internucleoside linkage having at least one change from a naturally-occurring intemucleoside linkage.
  • modified intemucleoside linkages include, but are not limited to, a phosphorothioate linkage, a phosphorodithioate linkage, a phosphoramidate linkage, a phosphorodiamidate linkage, a thiophosphoramidate linkage, a thiophosphorodiamidate linkage, a phosphoramidate morpholino linkage, and a thiophosphoramidate morpholino linkage, and a thiophosphorodiamidate morpholino linkage, which are known in the art and described in, e.g., Bennett and Swayz e, Annu Rev Pharmacol Toxicol. 50:259-293, 2010.
  • hybridize refers to the annealing of complementary nucleic acids (i.e., a gRNA or ASO and its target nucleic acid) through hydrogen bonding interactions that occur between complementary nucleobases, nucleosides, or nucleotides.
  • the hydrogen bonding interactions may be Watson-Crick hydrogen bonding or Hoogsteen or reverse Hoogsteen hydrogen bonding.
  • Examples of complementary nucleobase pairs include, but are not limited to, adenine and thymine, cytosine and guanine, and adenine and uracil, which all pair through the formation of hydrogen bonds.
  • the term “complementary” or “complementarity” refers to the capacity for base pairing between nucleobases, nucleosides, or nucleotides, as well as the capacity for base pairing between one polynucleotide to another polynucleotide.
  • one polynucleotide can have “complete complementarity,” or be “completely complementary,” to another polynucleotide, which means that when the two polynucleotides are optimally aligned, each nucleotide in one polynucleotide can engage in Watson-Crick base pairing with its corresponding nucleotide in the other polynucleotide.
  • one polynucleotide can have “partial complementarity,” or be “partially complementary,” to another polynucleotide, which means that when the two polynucleotides are optimally aligned, at least 60% (e.g, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97%) but less than 100% of the nucleotides in one polynucleotide can engage in Watson-Crick base pairing with their corresponding nucleotides in the other polynucleotide.
  • mismatched nucleotide base pair that does not engage in Watson-Crick base pairing when the two partially complementary polynucleotides are hybridized.
  • Pairs of nucleotides that engage in Watson-Crick base pairing includes, e.g, adenine and thymine, cytosine and guanine, and adenine and uracil, which all pair through the formation of hydrogen bonds.
  • mismatched bases include a guanine and uracil, guanine and thymine, and adenine and cytosine pairing.
  • CPE cytopathic effect
  • the infecting virus causes cell death via lysis of the host cell or inhibition of cell growth and reproduction. Both of these effects can occur due to CPE. If a virus causes these morphological changes in the host cell, it is said to be cytopathogenic or cytopathic.
  • Common examples of CPEs caused by viral infections include, but are not limited to, rounding of the infected cell, detachment of the infected cell from a substrate, fusion of the infected cell with adjacent cells to form syncytia, and the appearance of nuclear or cytoplasmic inclusion bodies.
  • polypeptide “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • composition refers to a composition that is physiologically acceptable and pharmacologically acceptable.
  • the composition includes an agent for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration.
  • pharmaceutically acceptable carrier refers to a substance that aids the administration of an agent (e.g gRNA or ASO) to a cell, an organism, or a subject.
  • “Pharmaceutically acceptable carrier” refers to a carrier or excipient that can be included in a composition or formulation and that causes no significant adverse toxicological effect on the patient.
  • Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors and colors, and the like.
  • pharmaceutically acceptable carriers include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors and colors, and the like.
  • subject refers to a vertebrate, preferably a mammal, more preferably a human.
  • Mammals include, but are not limited to, murines, mice, rats, simians, humans, farm animals, sport animals, and pets.
  • Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • administering includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intratumoral, intrathecal, intranasal, intraosseous, or subcutaneous administration to a subject. Administration is by any route, including parenteral and transmucosal (e.g ., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arterial, intradermal, subcutaneous, intraperitoneal, intraventricular, intraosseous, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, depot formulations, etc.
  • treating refers to an approach for obtaining beneficial or desired results including, but not limited to, a therapeutic benefit and/or a prophylactic benefit.
  • Therapeutic benefit means any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
  • Therapeutic benefit can also mean to effect a cure of one or more diseases, conditions, or symptoms under treatment.
  • therapeutic benefit can also mean to increase survival and/or to alleviate symptoms and/or suffering of the subject.
  • the present disclosure provides a guide RNA (gRNA) that is either identical to or complementary to an equal length portion of a sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gene in the SARS-CoV-2 genome.
  • the gRNA can target a Cas nuclease (e.g, Casl3b) to the sequence of the gene or to its reverse complement in the SARS-CoV-2 genome. Once targeted to the SARS-CoV-2 gene, the Cas nuclease can cleave the sequence of the gene or its reverse complement.
  • the SARS-CoV-2 genome has the sequence of GenBank Accession No.
  • the gRNA is identical to or complementary to an equal length portion of a sequence of a SARS-CoV-2 gene selected from the group consisting of orflab, S gene (encoding a surface glycoprotein), ORF3a, E gene (encoding an envelope protein), M gene (encoding a membrane glycoprotein), ORF6, ORF7a, ORF8, N gene (encoding a nucleocapsid phosphoprotein), and ORFIO.
  • the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated protein) nuclease system is an engineered nuclease system based on a bacterial system that can be used for genome engineering. It is based on part of the adaptive immune response of many bacteria and archaea. When a virus or plasmid invades a bacterium, segments of the invader’s gene are converted into CRISPR RNAs (crRNA) by the “immune” response.
  • crRNA CRISPR RNAs
  • the crRNA then associates, through a region of partial complementarity, with another type of RNA called tracrRNA to guide the Cas nuclease to a region homologous to the crRNA in the target nucleic acid called a “protospacer.”
  • CRISPR/Cas systems There are several main types of CRISPR/Cas systems (type I, type II, type III, type IV, and type V).
  • Type I, II, and V are DNA-targeting Cas nucleases.
  • Type III and type IV are RNA-targeting Cas nucleases.
  • the Cas nuclease cleaves the nucleic acid to generate blunt ends at sites specified by an about 20- to about 90-nucleotide guide sequence contained within the crRNA transcript.
  • the Cas nuclease can require both the crRNA and the tracrRNA for site-specific nucleic acid recognition and cleavage.
  • This system has now been engineered such that the crRNA and tracrRNA can be combined into one molecule (the “single guide RNA” or “sgRNA”), and the crRNA equivalent portion of the single guide RNA can be engineered to guide the Cas nuclease to target any desired sequence (see, e.g., Jinek et al. (2012) Science 337:816-821; Jinek et al.
  • the CRISPR/Cas system can be engineered to create a strand break at a desired target nucleic acid in a genome of a cell, and harness the cell’s endogenous mechanisms to repair the induced break by homology-directed repair (HDR) or nonhomologous end-joining (NHEJ).
  • HDR homology-directed repair
  • NHEJ nonhomologous end-joining
  • the Cas nuclease has RNA cleavage activity.
  • RNA-targeting Cas nucleases include type III and type VI Cas nucleases.
  • type III Cas nucleases include, but are not limited to, CaslO, Csm2, Cmr5, CaslO, Csxll, and CsxlO.
  • type VI Cas nucleases include, but are not limited to, Casl3a, Casl3b, and Cas 13c.
  • a Cas nuclease that can cleave a SARS-CoV-2 gene is Casl3b.
  • Cas nucleases can be derived from a variety of bacterial species including, but not limited to, Veillonella atypical , Fusobacterium nucleatum , Filifactor alocis , Solobacterium moorei , Coprococcus catus, Treponema denticola , Peptoniphilus duerdenii , Catenibacterium mitsuokai , Streptococcus mutans , Listeria innocua , Staphylococcus pseudintermedius , Acidaminococcus intestine , Olsenella uli , Oenococcus kitaharae , Bifidobacterium bifidum , Lactobacillus rhamnosus ,
  • Torque ns Ilyobacter polytropus, Ruminococcus albus , Akkermansia muciniphila , Acidothermus cellulolyticus , Bifidobacterium longum , Bifidobacterium dentium , Corynebacterium diphtheria , Elusimicrobium minutum , Nitratifractor salsuginis , Sphaerochaeta globus , Fibrobacter succinogenes subsp.
  • a Cas nuclease (e.g., Casl3b) can be guided to a SARS-CoV-2 gene by a gRNA described herein.
  • the gRNA comprises between 15 and 45 nucleotides in length (e. ., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides).
  • the gRNA can form a double-stranded RNA duplex with a scaffold RNA.
  • the gRNA can form a portion of a single-guide RNA (sgRNA).
  • An sgRNA is a version of the naturally occurring two-piece guide RNA (crRNA and tracrRNA) engineered into a single, continuous sequence.
  • An sgRNA may contain a gRNA as described herein (e. ., the crRNA equivalent portion of the sgRNA) that targets the Cas protein (e. ., Casl3b) to the target nucleic acid and a scaffold sequence that interacts with the Cas protein (e. ., the tracrRNAs equivalent portion of the sgRNA).
  • a gRNA or sgRNA may be selected using a software.
  • a gRNA can contain natural nucleotides, as well as non-natural or modified nucleotides.
  • a modified nucleotide can contain a modified nucleobase, a modified internucleoside linkage, and/or a modified sugar. Descriptions and examples of modified nucleobases, modified internucleoside linkages, and modified sugars are provided further herein.
  • the present disclosure also provides methods for modifying a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gene in a SARS-CoV-2 genome in a cell, comprising introducing into the cell a Cas nuclease (e.g ., Casl3b) and a gRNA described herein, or a pharmaceutical composition that comprises the Cas nuclease and the gRNA, in which the Cas nuclease (e.g., Cas 13b) cleaves the sequence of the SARS-CoV-2 gene or its reverse complement in the SARS-CoV-2 genome.
  • a Cas nuclease e.g ., Casl3b
  • a gRNA described herein or a pharmaceutical composition that comprises the Cas nuclease and the gRNA, in which the Cas nuclease (e.g., Cas 13b) cleaves the sequence of the SARS-CoV-2 gene or its reverse complement in the SARS-CoV-2
  • the methods described herein also include methods for treating a subject having a coronavirus disease 2019 (COVID-19) caused by a SARS-CoV-2, comprising administering to the subject a therapeutically effective amount of a Cas nuclease (e.g, Casl3b) and a gRNA described herein, or a pharmaceutical composition comprising a Cas nuclease (e.g, Cas 13b) and a gRNA described herein, in which the Cas nuclease cleaves the sequence of a SARS-CoV-2 gene or its reverse complement in the SARS-CoV-2 genome.
  • a Cas nuclease e.g, Casl3b
  • a gRNA described herein or a pharmaceutical composition comprising a Cas nuclease (e.g, Cas 13b) and a gRNA described herein, in which the Cas nuclease cleaves the sequence of a SARS-CoV-2 gene or its reverse complement
  • the gRNA is introduced into the cell in an adeno-associated viral (AAV) vector.
  • AAV adeno-associated viral
  • a nucleic acid encoding the Cas nuclease (e.g, Cas 13b) and the guide RNA are introduced into the cell in an AAV vector.
  • AAV serotype e.g, human AAV serotype
  • AAV serotype 1 AAV1
  • AAV2 AAV serotype 2
  • AAV3 AAV 3
  • AAV serotype 4 AAV4
  • AAV serotype 5 AAV5
  • AAV serotype 6 AAV6
  • AAV serotype 7 AAV7
  • AAV serotype 8 AAV8
  • AAV serotype 9 AAV9
  • AAV serotype 10 AAV10)
  • AAV serotype 11 AAV11
  • AAV serotype 11 AAV11
  • a variant thereof or a shuffled variant thereof (e.g, a chimeric variant thereof).
  • an AAV variant has at least 90%, e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to a wild-type AAV.
  • one or more regions of at least two different AAV serotype viruses are shuffled and reassembled to generate an AAV chimera virus.
  • a chimeric AAV can comprise inverted terminal repeats (ITRs) that are of a heterologous serotype compared to the serotype of the capsid.
  • ITRs inverted terminal repeats
  • the resulting chimeric AAV virus can have a different antigenic reactivity or recognition, compared to its parental serotypes.
  • a chimeric variant of an AAV includes amino acid sequences from 2, 3, 4, 5, or more different AAV serotypes.
  • AAV variants and methods for generating thereof are found, e.g., in Weitzman and Linden. Chapter 1-Adeno- Associated Virus Biology in Adeno- Associated Virus: Methods and Protocols Methods in Molecular Biology, vol. 807. Snyder and Moullier, eds., Springer, 2011; Potter et al., Molecular Therapy Methods & Clinical Development , 2014, 1, 14034; Bartel et al ., Gene Therapy , 2012, 19, 694-700; Ward and Walsh, Virology , 2009, 386(2):237-248; and Li et al. , Mol Ther , 2008, 16(7): 1252-1260.
  • AAV virions e.g, viral vectors or viral particle
  • AAV virions can be transduced into cells to introduce the gRNA and/or the nucleic acid encoding the Cas nuclease into the cell.
  • the gRNA and/or the nucleic acid encoding the Cas nuclease can be packaged into an AAV viral vector according to any method known to those skilled in the art. Examples of useful methods are described in McClure et al., J Vis Exp, 2001, 57:3378.
  • the present disclosure also provides methods for identifying a gRNA that is either identical to or complementary to an equal length portion of a sequence of a SARS-CoV-2 gene in a SARS-CoV-2 genome and targets a Cas nuclease to the sequence of the gene, resulting in reduced SARS-CoV-2 replication.
  • the SARS-CoV-2 genome has the sequence of GenBank Accession No. MN908947, which provides the sequence of each gene in the SARS-CoV-2 genome.
  • the methods can identify gRNAs that are either identical to or complementary to an equal length portion of a sequence of a SARS-CoV-2 gene selected from the group consisting of orflab, S gene (encoding a surface glycoprotein), ORF3a, E gene (encoding an envelope protein), M gene (encoding a membrane glycoprotein), ORF6, ORF7a, ORF8, N gene (encoding a nucleocapsid phosphoprotein), and ORF10.
  • the methods comprise: (a) designing a library of gRNAs that hybridize to a plurality of different portions in a SARS-CoV-2 gene or its reverse complement; (b) synthesizing a library of DNA templates encoding the gRNAs; (c) introducing the library in step (b) and a Cas nuclease (e.g, Casl3b) to a plurality of cells, wherein each cell comprises at least one gRNA from the library and the Cas nuclease; (d) contacting the plurality of cells with SARS-CoV-2; (e) selecting cells that do not show cytopathic effect (CPE); and (f) isolating and sequencing the gRNAs from the cells selected in step (e).
  • a Cas nuclease e.g, Casl3b
  • the introducing can comprise introducing the library and the Cas nuclease (e.g ., Cas 13b) by packaging a DNA template encoding a gRNA and a nucleic acid encoding a Cas nuclease (e.g., Cas 13b) into a vector (e.g, a lentiviral vector or AAV).
  • a vector e.g, a lentiviral vector or AAV.
  • each cell can contain between one and five (e.g, one, two, three, four, or five) gRNAs.
  • the present disclosure provides an antisense oligonucleotide (ASO), or a portion thereof, that is either identical to or complementary to an equal length portion of a sequence of a SARS-CoV-2 gene in a SARS-CoV-2 genome.
  • ASO antisense oligonucleotide
  • the ASO can hybridize to a SARS-CoV-2 gene or its reverse complement and activate endonuclease cleavage, i.e., RNaseH cleavage, of the gene.
  • the SARS-CoV-2 genome has the sequence of GenBank Accession No. MN908947, which provides the sequence of each gene in the SARS-CoV-2 genome.
  • An ASO can be identical to or complementary to an equal length portion of a sequence of a SARS-CoV-2 gene selected from the group consisting of orflab, S gene (encoding a surface glycoprotein), ORF3a, E gene (encoding an envelope protein), M gene (encoding a membrane glycoprotein), ORF6, ORF7a, ORF8, N gene (encoding a nucleocapsid phosphoprotein), and ORF10.
  • the ASO comprises between 10 and 30 nucleotides in length (e.g, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides).
  • an ASO can contain natural nucleotides, as well as non-natural or modified nucleotides.
  • a modified nucleotide can contain a modified nucleobase, a modified intemucleoside linkage, and/or a modified sugar. Descriptions and examples of modified nucleobases, modified intemucleoside linkages, and modified sugars are provided further herein.
  • the ASO comprises a sequence having at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of any one of SEQ ID NOS:l-16 and 21-24 (e.g, SEQ ID NOS:2, 10, 15, and 23).
  • SEQ ID NOS: 1-24 indicate the ASO sequences without any modifications on the nucleotides or intemucleoside linkages.
  • SEQ ID NOS:25-48 indicate the ASO sequences with modifications on the nucleotides and intemucleoside linkages as described in Table 1.
  • the ASO comprises a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of any one of SEQ ID NOS:25-40 and 45-48 (e.g., SEQ ID NOS:26, 34, 39, and 47), including the modifications on the nucleotides and internucleoside linkages.
  • the present disclosure also provides methods of inhibiting the expression or replication of a SARS-CoV-2 gene in a SARS-CoV-2 genome in a subject and/or treating a subject having a coronavirus disease 2019 (COVID-19) caused by a SARS-CoV-2, comprising administering to the subject a therapeutically effective amount of an ASO described herein or a pharmaceutical composition containing an ASO described herein.
  • the SARS-CoV-2 genome can have a sequence of GenBank Accession No. MN908947, which provides the sequence of each gene in the SARS-CoV-2 genome.
  • An ASO described herein, or a portion thereof, can be either identical to or complementary to an equal length portion of a sequence of a SARS-CoV-2 gene selected from the group consisting of orflab, S gene (encoding a surface glycoprotein), ORF3a, E gene (encoding an envelope protein), M gene (encoding a membrane glycoprotein), ORF6, ORF7a, ORF8, N gene (encoding a nucleocapsid phosphoprotein), and ORF10.
  • the methods described herein also include methods for identifying an ASO that is either identical to or complementary to an equal length portion of a sequence in a SARS- CoV-2 gene in a SARS-CoV-2 genome and can reduce SARS-CoV-2 replication.
  • the methods include: (a) designing a library of ASOs that hybridize to a portion of the SARS-CoV-2 gene; b) synthesizing the library of ASOs; (c) introducing the library of ASOs to cells individually or in a pool of ASOs, wherein each cell comprises at least one ASO; (d) contacting the cells with SARS-CoV-2; (e) selecting the ASO or the pool of ASOs if it confers resistance in the cell to cytopathic effect (CPE), and/or inhibits the expression or replication of the gene.
  • CPE cytopathic effect
  • each cell can contain between 1 and 100 (e.g, between 1 and 90, between 1 and 80, between 1 and 70, between 1 and 60, between 1 and 50, between 1 and 40, between 1 and 30, between 1 and 20, between 1 and 10, between 10 and 100, between 20 and 100, between 30 and 100, between 40 and 100, between 50 and 100, between 60 and 100, between 70 and 100, between 80 and 100, between 90 and 100) ASOs.
  • a pool of ASOs can contain between 2 and 10 (e.g ., 2, 3, 4, 5, 6, 7, 8, 9, or 10) different ASOs.
  • ASOs described herein can be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution, and/or cellular uptake of the resulting ASOs.
  • Typical conjugate groups include, but are not limited to, cholesterol moieties and lipid moieties.
  • Other conjugate groups include, but are not limited to, carbohydrates, phospholipids, peptides, antibodies, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, dyes, and other small molecules.
  • ASOs described herein can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of ASOs to enhance properties such as, for example, nuclease stability.
  • Stabilizing groups include, e.g., cap structures. These terminal modifications can protect the terminal nucleic acids in the ASO from exonuclease degradation, and can help in delivery and/or localization of the ASO within a cell.
  • the cap can be present at the 5 '-terminus (5 '-cap), or at the 3'- terminus (3 '-cap), or can be present on both termini.
  • Cap structures are well-known in the art and include, for example, inverted deoxy abasic caps.
  • a gRNA or an ASO can contain natural nucleotides, as well as non-natural or modified nucleotides.
  • a modified nucleotide can contain a modified nucleobase, a modified internucleoside linkage, and/or a modified sugar.
  • a modified nucleobase refers to a nucleobase having at least one change that is structurally distinguishable from a naturally-occurring nucleobase (e.g, adenine, guanine, cytosine, thymine, or uracil).
  • a modified nucleobase is functionally interchangeable with its naturally-occurring counterpart. Both naturally- occurring and modified nucleobases are capable of hydrogen bonding. Modifications on modified nucleobases may help to improve the stability of the gRNAs and ASOs to nucleases, increase binding affinity of the gRNAs and ASOs to their target nucleic acids, and decrease off-target binding of the gRNAs and ASOs.
  • a gRNA or an ASO described herein may include at least one modified nucleobase.
  • modified nucleobases include, but are not limited to, 5-methyl cytosine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2- propyladenine, 2-propylguanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5- halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6- azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-haloadenine, 8-aminoadenine, 8- thioladenine, 8-thioalkyladenine, 8-hydroxyladenine, 8-haloguanine, 8-aminoguanine
  • a modified sugar refers to a sugar having at least one change that is structurally distinguishable from a naturally-occurring sugar (e.g., 2’-deoxyribose in DNA or ribose in RNA). Modifications on modified sugars may help to improve the stability of the gRNAs and ASOs to nucleases, increase binding affinity of the gRNAs and ASOs to their target nucleic acids, and decrease off-target binding of the gRNAs and ASOs.
  • the sugar is a pentofuranosyl sugar.
  • the pentofuranosyl sugar ring of a nucleoside may be modified in various ways including, but not limited to, addition of a substituent group, particularly, at the T position of the ring; bridging two non-geminal ring atoms to form a bicyclic sugar (i.e., a locked sugar); and substitution of an atom or group such as — S — , -N(R)- or -C(RI)(R2) for the ring oxygen.
  • modified sugars include, but are not limited to, substituted sugars, especially 2 '-substituted sugars having a 2'-F, 2'-OCH3 (2'-OMe), or a 2'-0(CH 2 )2-0CH3 (2'-0-methoxy-ethyl or 2'-MOE) substituent group; and bicyclic sugars.
  • a bicyclic sugar refers to a modified pentofuranosyl sugar containing two fused rings.
  • a bicyclic sugar may have the T ring carbon of the pentofuranose linked to the 4’ ring carbon by way of one or more carbons (e.g, a methylene) and/or heteroatoms (e.g, sulfur, oxygen, or nitrogen).
  • the second ring in the sugar limits the flexibility of the sugar ring and thus, constrains the oligonucleotide in a conformation that is favorable for base pairing interactions with its target nucleic acids.
  • An example of a bicyclic sugar is a locked sugar, which is a pentofuranosyl sugar having the T- oxygen linked to the 4’ ring carbon by way of a carbon (e.g, a methylene) or a heteroatom (e.g, sulfur, oxygen, or nitrogen).
  • a locked sugar has the 2’-oxygen linked to the 4’ ring carbon by way of a carbon (e.g, a methylene).
  • a locked sugar has a 4'-(CH2)-0-2' bridge, such as a-L-methyleneoxy (4'-CH2-0-2') and b-D- methyleneoxy (4 -CH2-0-2').
  • a nucleoside having a lock sugar is referred to as a locked nucleoside.
  • bicyclic sugars include, but are not limited to, (6'S)— 6' methyl bicyclic sugar, aminooxy (4'-CH2-0-N(R)-2') bicyclic sugar, oxyamino (4'-CH2-N(R)-0- 2') bicyclic sugar, wherein R is, independently, H, a protecting group or C1-C12 alkyl.
  • a modified sugar is an unlocked sugar.
  • An unlocked sugar refers to an acyclic sugar that has a 2’, 3’-seco acyclic structure, where the bond between the T carbon and the 3’ carbon in a pentofuranosyl ring is absent.
  • An intemucleoside linkage refers to the backbone linkage that connects the nucleosides.
  • An intemucleoside linkage may be a naturally-occurring intemucleoside linkage (e.g, a phosphate linkage, also referred to as a 3’ to 5’ phosphodiester linkage, which is found in DNA and RNA) or a modified intemucleoside linkage.
  • a modified intemucleoside linkage refers to an intemucleoside linkage having at least one change that is structurally distinguishable from a naturally-occurring intemucleoside linkage. Modified intemucleoside linkages may help to improve the stability of the gRNAs and ASOs to nucleases and enhance cellular uptake.
  • modified intemucleoside linkages include, but are not limited to, a phosphorothioate linkage, a phosphorodithioate linkage, a phosphoramidate linkage, a phosphorodiamidate linkage, a thiophosphoramidate linkage, a thiophosphorodiamidate linkage, a phosphoramidate morpholino linkage, and a thiophosphoramidate morpholino linkage, and a thiophosphorodiamidate morpholino linkage, which are known in the art and described in, e.g., Bennett and Swayze, Annu Rev Pharmacol Toxicol. 50:259-293, 2010.
  • a phosphorothioate linkage is a 3’ to 5’ phosphodiester linkage that has a sulfur atom for a non bridging oxygen in the phosphate backbone of an oligonucleotide.
  • a phosphorodithioate linkage is a 3’ to 5’ phosphodiester linkage that has two sulfur atoms for non-bridging oxygens in the phosphate backbone of an oligonucleotide.
  • a thiophosphoramidate linkage refers to a 3’ to 5’ phospho-linkage that has a sulfur atom for a non-bridging oxygen and a NH group as the 3’-bridging oxygen in the phosphate backbone of an oligonucleotide.
  • an antisense oligonucleotide described herein has at least one phosphorothioate linkage. In some embodiments, all of the intemucleoside linkages in an antisense oligonucleotide described herein are phosphorothioate linkages.
  • the present disclosure features pharmaceutical compositions that include a gRNA described herein and a Cas nuclease and one or more pharmaceutically acceptable carriers or excipients.
  • the present disclosure also features pharmaceutical compositions that include an ASO described herein and one or more pharmaceutically acceptable carriers or excipients.
  • a pharmaceutical composition includes a gRNA and a Cas nuclease ( e.g ., Cas 13b), or an ASO in a therapeutically effective amount.
  • the therapeutically effective amount can be sufficient to alleviate or ameliorate symptoms of the COVID-19 caused by a SARS-CoV-2 or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the capability of those skilled in the art.
  • compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • a gRNA or an ASO described herein can be utilized in pharmaceutical compositions by combining the gRNA or ASO with a suitable pharmaceutically acceptable diluent or carrier.
  • a pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS).
  • PBS is a diluent suitable for use in compositions to be delivered parenterally.
  • compositions can include any pharmaceutically acceptable salts or esters thereof, which, upon administration to a subject (e.g., a human), is capable of providing (directly or indirectly) the biologically active form of the gRNA or ASO.
  • a subject e.g., a human
  • the disclosure is also drawn to pharmaceutically acceptable salts of gRNAs and ASOs, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
  • Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • pharmaceutical compositions include one or more pharmaceutically acceptable carriers or excipients. Acceptable carriers and excipients in the pharmaceutical compositions are nontoxic to recipients at the dosages and concentrations employed.
  • Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES, and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran, and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, histidine, and lysine, and carbohydrates such as glucose, mannose, sucrose, and sorbitol.
  • buffers such as phosphate, citrate, HEPES, and TAE
  • antioxidants such as ascorbic acid and methionine
  • preservatives such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, re
  • carriers and excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulosem, and polyvinylpyrrolidone.
  • a pharmaceutical composition includes a delivery system.
  • delivery systems include, but are not limited to, exosomes, liposomes, and emulsions.
  • gRNAs or ASOs described herein may be loaded or packaged in exosomes that specifically target a cell type, tissue, or organ to be treated.
  • Exosomes are small membrane-bound vesicles of endocytic origin that are released into the extracellular environment following fusion of mutivesicular bodies with the plasma membrane. Exosome production has been described for many immune cells including B cells, T cells, and dendritic cells. Techniques used to load a therapeutic compound into exosomes are known in the art and described in, e.g., U.S.
  • therapeutic compounds may be loaded into exosomes by electroporation or the use of a transfection reagent (i.e cationic liposomes).
  • an exosome- producing cell can be engineered to produce the exosome and load it with the therapeutic compound.
  • exosomes may be loaded by transforming or transfecting an exosome-producing host cell with a genetic construct that expresses the therapeutic compound, such that the therapeutic compound is taken up into the exosomes as the exosomes are produced by the host cell.
  • a pharmaceutical composition is prepared for gene therapy.
  • the pharmaceutical composition for gene therapy is in an acceptable diluent.
  • Vectors that may be used as in vivo gene delivery vehicle include, but are not limited to, adeno-associated viral vectors (AAVs), adenoviral vectors, retroviral vectors, poxviral vectors (e.g, vaccinia viral vectors, such as Modified Vaccinia Ankara), cytomegalovirus vectors, and alphaviral vectors.
  • Example 1 Test molecular drugs targeting SARS-CoV-2 RNA in a non-human primate model of COVID-19
  • the goal of this work is to test molecular drugs targeting SARS-CoV-2 RNA in a non-human primate model of COVID-19.
  • ASOs and Casl3b guide molecules that prevent virus growth in vitro and interrupt pathogenesis in vivo are identified.
  • the preliminary results demonstrate the use of CRISPR screening methods, which can be applied to identify useful Casl3 crRNAs, as well as the use of AAV-Casl3b in vivo.
  • An unbiased screen of Casl3b and ASO target sites can identify common vulnerable (accessible) regions in the SARS-CoV-2 genome that can serve as targets of effective molecular drugs.
  • Specific Aim 1 Identify effective anti-SARS-CoV-2 Casl3b guide RNAs in an unbiased screen covering both strands of the viral genome.
  • Guide RNAs for 31,835 sites encompassing both the plus and minus strands of the SARS-CoV-2 genome and excluding human sequences are introduced to Casl3b-expressing Vero E6 cells by retroviral transduction.
  • the library-containing cells are then exposed to SARS-CoV-2 in the BSL-3, before being passaged to a new plate permitting attachment of cytopathic effect (CPE)- resistant cells.
  • CPE cytopathic effect
  • the virus challenge can be graded by multiplicity of infection and time point of passage.
  • Candidate guide RNAs for COVID-19 treatment are identified by enrichment in the CPE-resistant vs. unselected library cells.
  • Ten guides are selected for further study and eventually four for packaging with a Casl3b expression cassette into AAV serotype 6.
  • Specific Aim 2 Identify effective anti-SARS-CoV-2 ASOs chosen either (i) in an unbiased manner or (ii) for reactivity with Casl3b-accessible regions of the viral genome.
  • 200 phosophorothioate-containing ASOs are synthesized that randomly tile both strands of SARS-CoV-2; an additional 100 ASOs are synthesized within Casl3b guides that confer resistance to CPE in Aim 1.
  • SARS-CoV-2 genomic regions targeted by 20 ASOs that confer resistance to coronavirus replication in vitro are selected for more detailed study by synthesis of additional ASOs in those regions.
  • three ASOs are selected for synthesis as T - MOE derivatives (i.e., 2'-0-(2-methoxyethyl)-modified ASOs) and evaluated in modified and unmodified form.
  • Casl3a is an RNA-guided RNA-targeting nuclease.
  • This class-2, type- VI CRISPR protein is activated upon recognition of ssRNA targets. Scanning of bacterial genome sequences subsequently led to the identification of a class-2, type VT-D CRISPR effector, termed Casl3d.
  • Casl3d-mediated target recognition and cleavage promotes collateral RNA cleavage in bacteria but not when expressed in mammalian cells.
  • RNA recognition by Casl3d is PAM independent, permitting great flexibility in targert-site selection.
  • Casl3d’s smaller size makes packaging into vectors such as AAV possible for in vivo applications.
  • ASOs Antisense oligonucleotides
  • CRISPRa CRISPR activation
  • TNBC triple-negative breast cancer
  • Approximately lxlO 7 cells expressing the activator (“Test” cells) or not expressing activator (“Control” cells) were then transduced with a lentiviral gRNA library (Human CRISPRa sgRNA library Calabrese [P65 HSF] Set A [3 gRNA/gene]) at low multiplicity of infection (0.1-0.5 MOI) such that each transduced cell expressed a single member of the gRNA library. After 8 days of incubation, gRNAs were recovered from Test and Control cells by PCR and submitted for next-gen sequencing. Reads were analyzed using MAGeCK-VISPR pipeline.
  • gRNAs able to activate a gene that inhibited cell growth or resulted in death would be underrepresented in Test cells compared to Control cells.
  • gRNAs for the genes GAS1, SAMD9L, HCRTR1, and VWA2 were identified as significantly depleted with a false discovery rate (FDR) ⁇ 20% (FIGS. 3A and 3B).
  • FDR false discovery rate
  • FIGS. 3A and 3B The depletion of GAS1 was further evidenced by a closer examination of read counts for each gRNA.
  • Angelman syndrome is caused by loss of expression of the maternal allele of UBE3A in the brain.
  • a long antisense RNA silences expression of the paternal copy of UBE3A; therefore, inactivation of the antisense RNA by Casl3b cleavage could restore normal UBE3A expression from the paternal allele.
  • a Casl3b crRNA to the antisense transcript just before it enters the UBE3A open reading frame in mice was designed (FIG. 4A).
  • Example 4 Identify effective anti-SARS-CoV-2 Casl3b guide RNAs in an unbiased screen covering both strands of the viral genome
  • SARS-CoV-2 replication can be sensitive to targeting of rare negative-strand intermediates.
  • Coronavirus RNA synthesis produces both genomic and sub-genomic RNAs.
  • Sub-genomic RNAs serve as mRNAs for the structural and accessory genes which reside downstream of the replicase polyproteins. Both genomic and sub-genomic RNAs are produced through negative-strand intermediates that are only about 1% as abundant as their positive-sense counterparts, suggesting that targeting the negative-sense RNA strands may be a particularly potent strategy.
  • a CRISPR screen for virus-targeted guide sequences that permit cell survival despite SARS-CoV-2 infection is performed (FIG. 5).
  • the starting point for the screen is the collection of densely tiled Casl3b guide sequences in the SARS-CoV-2 genome. Because Casl3 enzymes do not require a PAM sequence (having instead a single-base preference), there are 31,835 plausible guide sequences on both strands. All of these guide sequences are synthesized on an Agilent array, converted to a lentivirus library, and used to transduce Vero E6 cells that express cytoplasmic Casl3b.
  • Cells transduced with each member of the library are then be infected with SARS-CoV-2 as a pool; after a suitable interval, cells not undergoing cytopathic effect (CPE) are selected by re-plating and harvesting adherent cells the following day.
  • CPE cytopathic effect
  • Guide sequences that are enriched in the surviving cells compared to the starting cells are those that are able to prevent SARS-CoV-2 replication.
  • Casl3b crRNAs targeting 28-nt protospacers and a protospacer flanking site (PFS) of [A, C, or U] were designed using CRISPR-RT to the (+) strand and reverse complement of SARS-CoV-2 Accession MN908947 and assessed for self-dimerization and secondary structure. 31,835 crRNAs remained after filtering sites having homology to the human transcriptome (Ensembl GRCh38.86). An additional 500 neutral control crRNAs were designed to not target any site in SARS-CoV-2 nor the human transcriptome.
  • a positive control crRNA targets the mRNA for ACE2, which is the primary receptor for SARS-CoV-2, and should thus be enriched in the CPE-resistant cell populations since cells containing them should be highly resistant to infection.
  • Full-length DNA crRNA oligo pools with appropriate overhang sequences for Gibson cloning are synthesized and amplified by the Agilent SurePrint Oligo Library Synthesis service, cloned, packaged into lentiviral particles, and titered by Cellecta.
  • the target cells for library transduction are Vero cells stably expressing Casl3b.
  • Cells containing the crRNA library are transduced with SARS-CoV-2 at an initial MOI of 10. After four days (an interval that may be adjusted), cells not undergoing cytopathic effect (CPE) are selected by re-plating and harvesting adherent cells. This procedure is repeated and a fraction of surviving cells is collected at every passage until all unprotected negative control cells have been killed by SARS-CoV-2. The lentiviral crRNA insertions are then amplified and barcoded Illumina next-gen sequencing is performed to determine the relative abundance of each gRNA at various time points.
  • CPE cytopathic effect
  • Positive and negative control guide RNAs are included in the library.
  • the ACE2 positive control crRNA should be enriched, while neutral control crRNAs should show minimal variation over time.
  • the stringency of the screening procedure can be adjusted. If a first attempt using an MOI of 10 and cell harvest after four days yields many effective guide sequences, suggesting that the stringency of the screen is too low, then the MOI can be increased and the time for testing CPE resistance extended. Alternatively, if the yield is low then the stringency may be too high and time for testing CPE resistance can be accelerated, e.g., to two days after infection.
  • Positive hits should be enriched at least two standard deviations beyond the variance of the neutral control crRNAs (which should display little positive or negative selection), and should appear in both biological replicates.
  • the first level of validation is to repeat the use of the top 10 crRNAs in individual SARS-CoV-2 challenge assays.
  • the metric for success is the ability of the gRNA to cause the same effect as in the screen (protection from CPE).
  • Cell survival can be further quantitated using a colorimetric live cell assay such as MTT. For cells that survive, the change in viral gene presence and expression can be measured by RT-qPCR.
  • RNA-seq can be performed in biological replicates of i) non-treated Vero cells, ii) cells expressing Casl3/crRNA, iii) cells expressing Casl3/crRNA with SARS-CoV-2 challenge, and iv) non-treated Vero cells with viral challenge.
  • the Casl3b/crRNA treatment is tested to ensure that it is effective when delivered in an AAV6. Because Casl3b possesses a nuclease domain that can cleave individual crRNAs from a tandem array of crRNAs, the top four crRNAs can be cloned into the vector shown in FIG. 4B in tandem, under the same U6 promoter, to target the SARS-CoV-2 genome at four independent positions. This arrangement is expected to increase the likelihood of disruptive cleavage and reduce the possibility of acquired resistance by mutation of the target site.
  • RNA-seq and other assays can be performed in biological triplicates and one-way ANOVA with a Tukey post-hoc test can be used to determine statistical significance.
  • Example 5 Identify effective anti-SARS-CoV-2 ASOs chosen either (i) in an unbiased manner or (ii) for reactivity with Casl3b-accessible regions of the viral genome
  • ASOs and Casl3b guide RNAs that inhibit SARS-CoV-2 replication can both target regions of the genome that are accessible (unimpeded by secondary structure) and relatively scarce, e.g., full-length negative strands. Because the mechanism of ASO action involves base pairing, the molecules are less effective in the presence of a competitor for binding, e.g, sequences that create secondary structure in RNA. As a result, it seems likely that sequences successfully targeted by Casl3b are candidates for targeting with ASOs. However, Casl3b and ASOs approaches may have trade-offs in terms of delivery in vivo , activity, and ease of manufacture. ASOs can be examined as potential candidates for SARS-CoV-2 inactivation.
  • An unbiased screen of ASOs “tiling” the SARS-CoV-2 genome 200 phosporothioate 18-mers, PS-ASOs
  • a smaller screen leveraging the accessible regions of the genome identified by Casl3b screening, above 100 PS-ASOs
  • the PS-ASOs are tested individually for antiviral activity in single wells of 96-well plates.
  • the molecules are administered in solution and allowed to enter the cells by free uptake, an important mechanism for ASO activity.
  • SARS-CoV-2 is subsequently added to the wells at an MOI of 10, allowed to replicate for two days, and finally the cells are harvested for viral RNA extraction.
  • Successful ASOs to be identified in the screen are those that reduce replication of the virus, as reflected by reduction of viral RNA.
  • a second round of screening can test if sequences near to those first identified might have superior activity. For each of the top 20 hits in the first screen, five additional staggered 20-mers can be tested in the same region (100 additional ASOs total)
  • ASOs (10 mM) can be applied to triplicate wells of a 96-well plate (10 4 cells/well).
  • the free uptake mechanism to allow entry of sufficient PS-ASO to cells is chosen for screening for delivery of ASOs intratracheally in vivo.
  • the ASOs can also be delivered in liposomes or nanoparticles.
  • the plate can also contain three wells of cells that receive no ASOs (and thus should not be protected from SARS-CoV-2 infection) as a negative control. All cells can be subsequently transduced with SARS-CoV-2 at an initial MOI of 10. Nucleic acid can be harvested after four days to allow quantitation of SAR.S- CoV-2 genomes by RT-PCR.
  • the stringency of the screen can be adjusted by changing the MOI, the length of incubation before measuring viral RNA levels, or the concentration of ASO added to the culture well for uptake by cells. Such adjustment is likely to be necessary with progression through screening rounds, as progressively more inhibitory ASOs are identified. Presumably, for example, a stringent screen may be needed (high MOI, lower concentration of ASO, longer incubation) to resolve differences between highly effective ASOs and their derivatized counterparts.
  • Phosphothioate bonds are added to antisense oligos to protect them from nuclease degradation.
  • Tm decreases with each phosphothioate bond added.
  • Increased affinity can be achieved via use of modified bases such as 2'-0-methoxy-ethyl (2'-MOE) or locked nucleic acid bases and via substitution of 5-methyl dC for dC.
  • the three top ASOs selected in the first two rounds of screening described above can be synthesized with T- MOE-modifications in a gapped format (ten unmodified bases in the center for RNaseH activity). These derivatized ASOs can be tested head-to-head against their unmodified parental ASOs. The more inhibitory molecule in vitro can be chosen for animal testing.
  • RNA genomes found in ASO-treated and infected cells when treated at two concentrations of ASO. Adjusted p values can be calculated for reduction of viral RNA relative to wells treated with irrelevant controls; ASOs can be prioritized for retesting or further investigation by according to these p values. Linear mixed models may be employed for more sophisticated analyses with batch effects, e.g., comparative testing of oligos across screening days.
  • Example 6 Test efficacy of optimal anti-SARS-CoV-2 ASOs and Casl3b guide RNAs in a COVID-19 rhesus macaque model
  • ASOs and Casl3b guide RNAs that inhibit SARS-CoV-2 replication in vitro are effective treatments for COVID-19 in a rhesus macaque model.
  • Effective delivery strategies for ASOs and Cas enzymes are known and are achievable in macaques.
  • the identified molecules should protect any cell to which they gain access; in that event, the drugs might protect against COVID-19 by direct inhibition of the virus or by preservation or host immune and/or respiratory homeostasis.
  • FIG. 7 A uniform challenge and monitoring protocol is established (FIG. 7) so that consistent sets of virologic, immunologic, and pathologic data may be leveraged across experiments.
  • Contemporaneous control animals (group 1) can be infected and followed as part of the work on establishing and evaluating the model.
  • Treatments can be tested in this model when given 7 days after infection, since this is a time point at which humans may become symptomatic and seek medical attention.
  • a virus stock produced by expanding the SARS-CoV-2 isolate obtained from an infected patient can be used for animal inoculations, designated SARS-CoV-2 USA- CA1/2020. If outgrowth of that virus is insufficient, the SARS-CoV-2 isolate USA- WA1/2020 (BEI Resources) can be used. To infect animals, approximately 6 x 10 6 TCID50 in total can be instilled into the conjunctiva, nostrils, and trachea of anesthetized monkeys in 5 ml of 0.9% sterile saline to recapitulate relevant transmission routes of COVID-19.
  • Physiologic parameters can be monitored throughout the experiment. CBCs and serum chemistry can be obtained on all blood samples to monitor host responses and organ function after infection.
  • the sampling schedule is designed to comprehensively characterize viral shedding, cytokine responses, and cellular and humoral immune responses to understand how changes in these parameters reflect pathological changes in the lung.
  • the sampling schedule and procedures have been used successfully to characterize influenza A virus infection in rhesus macaques. Intensive sampling during the first week following infection enables the study of acute virology and host responses in the lung and systemic compartments.
  • ACE2 is expressed in the gastrointestinal and genitourinary tracts of rhesus and humans, in addition to respiratory secretions, virus shedding in saliva, urine, and stool samples can be evaluated.
  • all relevant tissues including the salivary glands, lung, mediastinal lymph nodes, kidney, and gut tissue among others can be collected to evaluate virus localization and immune responses by PCR, quantitative molecular histology (IHC, ISH), and flow cytometry. Tissues can be evaluated for gross pathology, histopathology, and tissue vRNA levels by RT- PCR. Necropsy can be performed by a board-certified pathologist.
  • Thermo’s MagMAX Viral/Pathogen Nucleic Acid Isolation kit (as recommended by the CDC for COVID-19 RNA work) can be used to isolate the total RNA from the respiratory tract samples, prepare cDNA and perform Taqman PCR to amplify a segment of the SARS- CoV-2 nucleoprotein (N) gene.
  • the lung is an important site of SARS-CoV-2 replication and an important target for treatment. It is known that AAV6 vectors delivered intratracheally can reach alveolar cells including type-II pneumocytes, which are the cells with highest expression of ACE2. From the lungs AAV6 is distributed to a limited extent. 10 13 vector genomes/kg are delivered by placement of an endotracheal tube 4-5 cm above the carina followed by 2.5 ml of PBS and air flush to ensure delivery of the inoculum.
  • Intratracheally delivered PS-ASOs are known to be systemically distributed, to some extent, and able to affect gene expression in the liver. Therefore, this delivery method may have some capacity to reach distant SARS-CoV-2-infected tissues.
  • a mixture of three ASOs selected previously can be delivered at a dose of 6 mg/kg for each oligo in 5 ml of PBS.
  • Example 7 Sreening of ASO targets by strand and position in the SARS-CoV-2 genome
  • SEQ ID NOS:25-48 in Table 1 “*” indicates a phosphorothioate internucleoside linkage; “Me-dC” indicates 5-methyl-deoxycytidine; “2MOE” indicates O-methoxy-ethyl at the T position of the ribose moiety of a nucleotide, and lower case “r”, as in “2MOEr,” indicates ribose.
  • SEQ ID NOS: 17-20, as well as their corresponding modified versions SEQ ID NOS:41-44, are negative controls.
  • Vero CCL81 cells (1.3xl0 5 ) were seeded in 24-well plates in 0.9 mL of Dulbecco’s Modified Eagle Media (DMEM) containing 10% FBS and IX Penicillin/Streptomycin (complete DMEM).
  • DMEM Modified Eagle Media
  • ASOs reconstituted in Tris EDTA buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0), were added to each well for a final concentration of 10 mM to bring the volume in each well to 1 mL.
  • the cultures were incubated overnight at 37 °C with 5% CO2 in a humidified incubator. The following day, the cultures were transferred to the Biosafety Level Three (BSL3) laboratory.
  • BSL3 Biosafety Level Three
  • a well-characterized, low-passage (Passage 2) stock of SARS- CoV-2 (isolate SARS-CoV-2/human/USA/CA-CZB-59X002/2020; Genbank: MT394528) was diluted in complete DMEM to generate the viral inocula. Growth media was removed from the cells and replaced with the viral inocula media at a multiplicity of infection of 0.001. ASOs were added the cultures for a final concentration of 10 mM. The cultures were incubated for two hours with rocking every 15 minutes to allow for infection of the cells. The viral inocula media was removed and replaced with complete DMEM growth media. ASOs were added to a final concentration of 10 mM and the cultures were incubated for 48 hours.
  • Passage 2 A well-characterized, low-passage (Passage 2) stock of SARS- CoV-2 (isolate SARS-CoV-2/human/USA/CA-CZB-59X002/2020; Genbank: MT394528) was diluted in
  • CPE cytopathic effect
  • An antisense oligonucleotide or a portion thereof, that is either identical to or complementary to an equal length portion of a sequence of a gene in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • modified sugar is selected from the group consisting of a 2’-0-methoxyethyl modified sugar, a bicyclic sugar, a T - methoxy modified sugar, a 2’-0-alkyl modified sugar, and an unlocked sugar.
  • ASO of embodiment 9 wherein the ASO comprises a sequence having at least 90% identity to the sequence of any one of SEQ ID NOS:2, 10, 15, and 23.
  • modified nucleotide comprises 5-methyl cytosine or a modified sugar comprising 2'-0-methoxy-ethyl.
  • the ASO comprises at least 90% identity to the sequence of any one of SEQ ID NOS:25-40 and 45-48, including the modifications on the nucleotides and intemucleoside linkages.
  • ASO of embodiment 14, wherein the ASO comprises at least 90% identity to the sequence of any one of SEQ ID NOS:26, 34, 39, and 47.
  • a pharmaceutical composition comprising an ASO of any one of embodiments 1 to 15 and one or more pharmaceutically acceptable carriers or excipients.
  • a method of inhibiting the expression or replication of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gene in a SARS-CoV-2 genome in a subject comprising administering to the subject a therapeutically effective amount of an ASO of any one of embodiments 1 to 15 or a pharmaceutical composition of embodiment 16.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • COVID-19 coronavirus disease 2019
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • a method for identifying an antisense oligonucleotide (ASO) that is either identical to or complementary to an equal length portion of a sequence in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gene in a SARS-CoV-2 genome comprising:
  • a guide RNA that is either identical to or complementary to an equal length portion of a sequence of a gene in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome.
  • gRNA of embodiment 24, wherein the sequence of the gene or its reverse complement is in a SARS-CoV-2 gene selected from the group consisting of orflab, S gene, ORF3a, E gene, M gene, ORF6, ORF7a, ORF8, N gene, and ORFIO.
  • a pharmaceutical composition comprising a gRNA of any one of embodiments 21 to 29 and a Cas nuclease and one or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical composition of embodiment 30, comprising two or more gRNAs.
  • a method for modifying a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gene in a SARS-CoV-2 genome in a cell comprising introducing into the cell a Cas nuclease and the gRNA of any one of embodiments 21 to 29, or the pharmaceutical composition of embodiment 30, wherein the Cas nuclease cleaves the sequence of the SARS-CoV-2 gene or its reverse complement in the SARS-CoV-2 genome.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • step (c) introducing the library in step (b) and a Cas nuclease to a plurality of cells, wherein each cell comprises at least one gRNA from the library and the Cas nuclease;
  • step (f) isolating and sequencing the gRNAs from the cells selected in step (e).

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Abstract

L'invention concerne des compositions et des procédés pour traiter une maladie à coronavirus 2019 (COVID-19) provoquée par un coronavirus du syndrome respiratoire aigu sévère 2 (SRAS-CoV-2) chez un sujet. Les compositions et les procédés comprennent l'utilisation d'ARN guides ou d'oligonucléotides antisens (ASO) qui ciblent une partie de la séquence d'un gène du SRAS-CoV-2 dans un génome du SRAS-CoV-2.
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