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WO2018183808A1 - Thérapeutique antivirale - Google Patents

Thérapeutique antivirale Download PDF

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Publication number
WO2018183808A1
WO2018183808A1 PCT/US2018/025350 US2018025350W WO2018183808A1 WO 2018183808 A1 WO2018183808 A1 WO 2018183808A1 US 2018025350 W US2018025350 W US 2018025350W WO 2018183808 A1 WO2018183808 A1 WO 2018183808A1
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WIPO (PCT)
Prior art keywords
guide rna
seq
composition according
nos
mrna
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Ceased
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PCT/US2018/025350
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WO2018183808A9 (fr
Inventor
Derek D. Sloan
Sarah LEDOUX
Xin Cindy XIONG
Michael Patrick HOUSLEY
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Agenovir Corp
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Agenovir Corp
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Priority to JP2019553095A priority Critical patent/JP2020515258A/ja
Priority to EP18721530.6A priority patent/EP3601568A1/fr
Priority to CN201880035095.7A priority patent/CN110678548A/zh
Publication of WO2018183808A1 publication Critical patent/WO2018183808A1/fr
Publication of WO2018183808A9 publication Critical patent/WO2018183808A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/335—Modified T or U
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/53—Methods for regulating/modulating their activity reducing unwanted side-effects

Definitions

  • the disclosure relates to viral treatment compositions and methods.
  • Viral infections are a significant cause of disease and death worldwide.
  • cervical cancer is caused by infection with certain types of human papillomavirus (HPV).
  • HPV human papillomavirus
  • Two HPV types (16 and 18) cause 70% of cervical cancers and precancerous cervical lesions.
  • HPV is also linked to cancers of the head and neck (oropharynx), anus, vulva, vagina, and penis. According to the World Health Organization, approximately
  • cervical cancer is the fourth most frequent cancer in women, with an estimated 530,000 new cases in 2012 representing 7.5% of all female cancer deaths.
  • HPV is a member of the Papillomaviridae, a family of DNA viruses collectively known as papillomaviruses.
  • Papillomaviruses replicate in the basal layer of the body surface tissues.
  • Papillomaviruses are non-enveloped, meaning that the outer shell or capsid of the virus is not covered by a lipid membrane.
  • a single viral protein, known as LI forms a 55-60 nanometer capsid.
  • the capsid is geometrically regular and presents icosahedral symmetry.
  • the HPV genome is a double- stranded circular DNA molecule about 8,000 base pairs in length. It is packaged within the LI shell along with cellular histone proteins, which package the genomic viral DNA.
  • HBV Hepatitis B virus
  • cccDNA covalently closed circular DNA
  • the present disclosure provides guide RNAs that target viral nucleic acids, such as HPV16, HPV18, or HBV nucleic acids.
  • the present disclosure provides a composition comprising a Cas endonuclease and one or more guide RNAs that target a viral nucleic acid, or a nucleic acid encoding such a Cas
  • the present disclosure provides a composition that comprises (a) an mRNA encoding a Cas endonuclease and (b) one or more guide RNAs, for the treatment of a viral infection, such as an HBV or HPV infection.
  • a viral infection such as an HBV or HPV infection.
  • the mRNAs and guide RNAs may include particular sequences and modifications, and may be encapsulated by nanoparticles.
  • the composition includes one or more guide RNAs that are specific to a viral target such as Epstein-Barr virus (EB V), human papillomavirus (HPV), Kaposi sarcoma virus (KSHV), hepatitis B virus (HBV), herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), varicella zoster virus (VZV), cytomegalovirus (CMV), human herpesvirus-6 (HHV-6), or human herpesvirus-7 (HHV-7).
  • EB V Epstein-Barr virus
  • HPV human papillomavirus
  • KSHV Kaposi sarcoma virus
  • HBV hepatitis B virus
  • HSV-1 herpes simplex virus-1
  • HSV-2 herpes simplex virus-2
  • VZV varicella zoster virus
  • CMV cytomegalovirus
  • HHV-6 human herpesvirus-6
  • HHV-7 human herpesvirus-7
  • the composition includes mRNA encoding a modified
  • Cas endonuclease as well as one or more guide RNAs that target the Cas endonuclease to the HPV genome, including full-length and partial fragments that exist in episomal form and/or as integrated into the host genomic DNA.
  • guide RNAs comprise targeting regions that are complementary to sites within the genomes of HPV16 or HPV18, which sites do not also appear in a human genome.
  • the composition includes mRNA encoding a modified Cas endonuclease as well as one or more guide RNAs that target the Cas endonuclease to the HBV genome.
  • the guide RNAs may include features such as modified nucleotides that promote the delivery of the RNAs to, and retention within, infected cells.
  • modified nucleotides in RNAs may function to improve RNA stability, reduce immunogenicity, or improve specificity of the endonuclease activity.
  • the guide RNAs can include features such as one or more 2'-0-methyl groups on a ribose ring, one or more phosphorothioate bonds between nucleotides, or both, and particularly located proximal to the 5' and 3 ' termini of the guide RNAs, which may protect against exonuclease digestion.
  • the guide RNAs may optionally include one or more locked nucleic acid, bridged nucleic acid, or conformationally restricted nucleic acid— e.g., within the targeting region— which may stabilize binding to the viral target, reduce binding to non-viral targets (e.g., human DNA), or enhance specificity of endonuclease activity.
  • the Cas endonuclease can include mutations, relative to wild-type Cas9, that may enhance specificity and decrease off-target activity (e.g., by destabilizing interactions with target DNA at locations outside of the guide RNA targets).
  • the RNAs may include modifications such as pseudouridine or 5- methyl-cytosine that may minimize an immune response by the patient.
  • the mRNA preferably encodes a programmable nuclease such as a Cas endonuclease or a modified Cas endonuclease.
  • a programmable nuclease such as a Cas endonuclease or a modified Cas endonuclease.
  • the mRNA encodes a modified Cas endonuclease that is modified, relative to a wild-type version, for decreased immunogenicity.
  • the encoded, modified nuclease may include, relative to wild type, one or a plurality of substitutions within T-cell epitopes.
  • a therapeutic protein is delivered as an mRNA
  • the resulting protein can have, relative to wild-type, alternative or modified epitopes that do not trigger an anti-drug antibody (ADA) response.
  • ADA anti-drug antibody
  • the present disclosure includes modified Cas endonuclease protein sequences in which one or more predicted T-cell epitopes have substitutions that avoid or decrease an immune response relative to wild-type Cas endonuclease.
  • the modified Cas endonuclease, with the modified T-cell epitopes may be delivered to cells encoded in a DNA vector, in protein form (e.g., as an active ribonucleoprotein (R P) with the modified Cas endonuclease complexed with an antiviral guide RNA), or as an mRNA to be translated within the target cells.
  • the nuclease is delivered in mRNA form, e.g., along with one or more guide RNAs.
  • the Cas9-encoding mRNA and guide RNAs are packaged in a lipid nanoparticle (LNP), solid nanoparticle, or liposome.
  • LNP lipid nanoparticle
  • the Cas9-encoding mRNA and guide RNAs are packaged in a lipid nanoparticle (LNP), solid nanoparticle, or liposome.
  • RNA-encapsulating nanoparticles can be formulated for topical, mucosal, or local delivery to infected tissue, which avoids systemic delivery and circulation, thus minimizing drug exposure, off-target activity, and immunogenicity of the Cas endonuclease.
  • the RNAs are packaged in lipid nanoparticles that include, for example, cationic lipids, which balance the charge of the phosphate backbone and promote penetration through tissue and into cells and release of RNA within the cell.
  • Lipid nanoparticles encapsulating a Cas-9 encoding mRNA and guide RNA may further be provided in a topical formulation that contains a suitable gel or suspension, such as an aqueous suspension, which may include a tissue retention-enhancing or thickening agent such as, for example, hydroxy ethyl cellulose or carboxymethyl cellulose.
  • a suitable gel or suspension such as an aqueous suspension
  • tissue retention-enhancing or thickening agent such as, for example, hydroxy ethyl cellulose or carboxymethyl cellulose.
  • An LNP formulation as contemplated herein may include one or more excipients to enhance LNP stability such as, for example, sucrose or mannitol.
  • an LNP formulation as described herein may include excipients to enhance tissue penetration such as, for example, sodium lauryl sulfate, ethanol, diethylene glycol monoethyl ether (Transcutol), propylene glycol, polyethylene glycol (PEG) esters, sucrose esters, or N-methyl pyrrolidone.
  • excipients to enhance tissue penetration such as, for example, sodium lauryl sulfate, ethanol, diethylene glycol monoethyl ether (Transcutol), propylene glycol, polyethylene glycol (PEG) esters, sucrose esters, or N-methyl pyrrolidone.
  • an LNP formulation according to the present description may be administered with a device to enhance RNA delivery to the basal epithelium, such as, for example, a microneedle array.
  • a LNP formulation as described herein is applied topically or locally to a site of infection such as a high-grade pre-cancerous lesion associated with an HPV infection.
  • the guide RNA(s) encapsulated by the LNPs is are released within the cells, and the mRNAs encapsulated by the L Ps are released and translated by the cell's ribosomes to produce a Cas endonuclease.
  • Cas9 endonucleases for use in the context of the present compositions and formulations may include linker sequences and one or more nuclear localization sequences ( LS) at the N-terminus and/or C-terminus designed for optimal nuclear localization.
  • the Cas endonuclease complexes with the provided guide RNA or guide RNAs to form active RNP.
  • the RNP traffics to the nucleus and binds to the viral genome by virtue of sequence-specific interaction between the complementary portions of the guide RNA and the target within the viral genome.
  • the Cas endonuclease cleaves the viral genome. Resultant viral DNA fragments may be degraded or repaired by cellular pathways, thereby clearing or disrupting the infection.
  • the present disclosure provides a composition that includes an mRNA encoding a Cas endonuclease; a guide RNA comprising a nucleic acid sequence as set forth in any one of SEQ ID NOs. : 1-38, 71-76, and 79-83, or versions thereof with modified bases; a plurality of nanoparticles comprising a cationic lipid (i.e., "lipid nanoparticles" or "LNPs”) and encapsulating the mRNA and the guide RNA; and a carrier formulation.
  • the carrier formulation may include one or more constituents that stabilize the LNPs encapsulating the mRNA and the guide RNA.
  • the carrier formulation may include one or more constituents that stabilize the LNPs
  • the cationic lipid used in the lipid nanoparticles may include l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) or N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP).
  • DOPE dioleoyl-sn-glycero-3- phosphoethanolamine
  • DOTAP N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate
  • the plurality of LNPs are solid lipid nanoparticles dispersed within the carrier formulation, and the carrier formulation comprises a carrier liquid, oil, or gel.
  • the LNPs included in the compositions described herein may be PEG-ylated.
  • the mRNA encapsulated in the LNPs comprises SEQ ID NO. : 55, 56, 57, or 58;
  • the guide RNA encapsulated in the LNPs comprises SEQ ID NO.: 1, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37;
  • the plurality of LNPs are dispersed within a carrier liquid, oil, or gel provided by the carrier formulation.
  • the Cas endonuclease may be Cas9, optionally including at least one variation, relative to wild type Cas9, to an amino acid selected from the group consisting of R780, K810, K848, K855, H982, K1003, and R1060.
  • the Cas9 may include K848A, K1003A, and R1060A.
  • an mRNA encoding a Cas9 comprises SEQ ID NO. : 55, 56, 57, 58, 59, 60, 67, 68, 69, 70, 77, or 78.
  • a coding sequence of the mRNA comprises a plurality of 5- methylcytidine.
  • the coding sequence of the mRNA may include a plurality of pseudouridine or 5 methoxy-uridine.
  • the compositions include a Cas endonuclease, or a polynucleotide encoding the Cas endonuclease, with one or more substitutions (e.g., between 1 and 25) within, relative to SEQ ID NO. : 63 : amino acids 149-165; amino acids 235-249; amino acids 566-580; amino acids 721 -735; amino acids 880-894; amino acids 952-966; or amino acids 1312-1326.
  • the one or more substitutions are at locations, relative to SEQ ID NO.
  • 63 selected from R152, 1154, A157, F238, N240, A243, F569, K571, C574, W883, Q885, N888, V955, L1315, and N1317, and each is substituted to one of K, V, S, L, Q, R, E, T, or N.
  • the Cas endonuclease may include at least one nuclear localization signal.
  • the guide RNA comprises a plurality of (e.g., between about 20 and 100%) modified bases each selected from the group consisting of 2'-0- methyladenosine (am); 2'-0-methylcytidine (cm); 2'-0-methylguanosine (gm); 2'-0- methyluridine (um); and 2'-0-methylpseudouridine (fm).
  • the guide RNA comprises between one and nine phosphorothioate bonds between the ten terminal nucleotides at each of a 5' end and at a 3 ' end of the guide RNA.
  • the guide RNA may include at least one bridged nucleic acid (BNA), locked nucleic acid (LNA), and/or conformationally -restricted nucleotide (CRN).
  • the guide RNA comprises a targeting region comprising a nucleic acid sequence as set forth in SEQ ID NO. : 39 or SEQ ID NO : 40.
  • the guide RNA is one of SEQ ID NOs. : 1, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37 and optionally includes a plurality of modified bases each selected from the group consisting of 2'-0-methyladenosine (am); 2'-0-methylcytidine (cm); 2'-0-methylguanosine (gm); 2'-0-methyluridine (um); and 2'-0-methylpseudouridine (fm). In some embodiments about 20 to 100% of the nucleotides of the guide RNA comprise a 2'-0-methyl group on a ribose sugar.
  • the guide RNA is one of SEQ ID NOs. : 41, 43, and 45 optionally with about 20 to 100% of the nucleotides having a modification such as a methyl or a 2'-0-methyl group on a ribose sugar, and preferably include a plurality (e.g., about 20 to 100%)) of modified bases each selected from the group consisting of 2'-0-methyladenosine (am); 2'-0-methylcytidine (cm); 2'-0-methylguanosine (gm); 2'-0-methyluridine (um); and 2'-0-methylpseudouridine (fm).
  • the guide RNA comprises one selected from the group consisting of SEQ ID NO.
  • the guide RNA comprises a plurality of modified bases each selected from the group consisting of 2'-0- methylcytidine (cm); 2'-0-methylguanosine (gm); 2'-0-methyluridine (um); and 2'-0- methylpseudouridine (fm).
  • Embodiments of such methods include administering to a site of infection in a patient in need thereof an effective amount of a composition described herein.
  • the patient may have HPV infection or has been diagnosed with HPV-positive pre-cancerous low-grade or high-grade lesions, HPV-positive squamous cell carcinoma in situ, or HPV- positive invasive cancer.
  • the invention provides a composition as disclosed herein for treating a viral infection.
  • the composition preferably includes one or more guide RNAs that are specific to a viral target from a virus selected from the group consisting of Epstein-Barr virus (EBV), human papillomavirus (HPV), Kaposi sarcoma virus (KSHV), hepatitis B virus (HBV), herpes simplex virus- 1 (HSV-1), herpes simplex virus-2 (HSV-2), varicella zoster virus (VZV), cytomegalovirus (CMV), human herpesvirus-6 (HHV-6), human herpesvirus-7 (HHV-7), and human polyomavirus (BK, JC, or Merkel cell, MCV).
  • the one or more guide RNAs are specific to EBV, HPV, or HBV.
  • the Cas endonuclease comprises an amino acid sequence as set forth in SEQ ID NO. : 62, or the polynucleotide encoding the Cas
  • the composition includes the polynucleotide encoding the Cas endonuclease in the form of a mRNA.
  • the guide RNA may target HPV and include, e.g., one selected from the group consisting of SEQ ID NOs. : 1-38 and 71-76.
  • the guide RNA comprises one selected from the group consisting of SEQ ID NOs.
  • the guide RNA includes comprises SEQ ID NO.: 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 71, 72, 73, 74, 75, or 76.
  • the Cas endonuclease, or the polynucleotide encoding the Cas endonuclease is packaged in a liposome or a lipid nanoparticle.
  • the composition comprises the Cas endonuclease as an active RNP enveloped in a LNP or a liposome or the composition includes the polynucleotide as an mRNA encapsulated in a solid lipid nanoparticle.
  • the one or more substitutions are, relative to SEQ ID NO.: 63, selected from R152K, I154V, A157S, F238L, N240Q, A243S, F569L, K571R, C574E, W883T, Q885N, N888Q, V955I, L13151, and N1317Q.
  • the substitutions include, relative to SEQ ID NO. : 63, six to fifteen ofR152K, I154V, A157S, F238L, N240Q, A243S, F569L, K571R, C574E, W883T, Q885N, N888Q, V955I, L1315I, and N1317Q.
  • composition according to the present description includes an mRNA encoding a Cas endonuclease; a guide RNA with a portion
  • the guide RNA comprising one selected from the list consisting of SEQ ID NOs. : 1-38, 71-76, and 79-83 optionally with modified bases; a plurality of solid lipid nanoparticles (LNP) comprising a cationic lipid and encapsulating the mRNA and the guide RNA; and a carrier formulation.
  • the carrier formulation may include one or more constituents that stabilizes the lipid nanoparticle, enhances topical or local delivery by promoting tissue retention, and/or enhances topical or local delivery by promoting tissue penetration.
  • the guide RNA may be one of SEQ ID NOs.
  • the Cas endonuclease is Cas9.
  • the guide RNA includes a plurality of modified bases each selected from the group consisting of 2'-0- methylcytidine (cm); 2'-0-methylguanosine (gm); 2'-0-methyluridine (um); and 2'-0- methylpseudouridine (fm).
  • the coding sequence of the mRNA may include a plurality of pseudouridine or 5 methoxy -uridine.
  • the guide RNA may include one or more
  • the carrier includes one or more excipients selected from hydroxyethyl cellulose, carboxymethyl cellulose, sucrose, mannitol, sodium lauryl sulfate, ethanol, diethylene glycol monoethyl ether, propylene glycol, polyethylene glycol (PEG) esters, sucrose esters, or N-methyl pyrrolidone.
  • excipients selected from hydroxyethyl cellulose, carboxymethyl cellulose, sucrose, mannitol, sodium lauryl sulfate, ethanol, diethylene glycol monoethyl ether, propylene glycol, polyethylene glycol (PEG) esters, sucrose esters, or N-methyl pyrrolidone.
  • the Cas9 includes between one and seven variations, relative to wild type Cas9, to an amino acid selected from the group consisting of R780, K810, K848, K855, H982, K1003, R1060.
  • the guide RNA comprises at least one bridged nucleic acid (BNA), locked nucleic acid (LNA), and/or conformationally -restricted nucleotide (CRN).
  • BNA bridged nucleic acid
  • LNA locked nucleic acid
  • CNN conformationally -restricted nucleotide
  • at least about 20% of the nucleotides of the guide RNA comprise a 2'-0-methyl group on a ribose sugar.
  • the LNP may be PEG-ylated.
  • the Cas9 may include K848A, K1003A, and R1060A.
  • the cationic lipid may include, for example, l,2-dioleoyl-sn-glycero-3 phosphoethanolamine (DOPE) or N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP).
  • DOPE dioleoyl-sn-glycero-3 phosphoethanolamine
  • DOTAP N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate
  • the mRNA comprises one selected from the group consisting of SEQ ID NOs. : 55, 56, 57, and 58 and the guide RNA comprises one selected from the group consisting of SEQ ID NOs. : 1, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 74, 75, and 76, preferably with a plurality (e.g., 20 to 100%) of modified bases.
  • the compositions described herein are useful for treating a viral infection.
  • the composition includes a Cas endonuclease, or a polynucleotide encoding the Cas endonuclease, with between one and twenty -five substitutions within, relative to SEQ ID NO. : 63, one selected from the group consisting of: amino acids 149-165; amino acids 235-249; amino acids 566-580; amino acids 721-735; amino acids 880-894; amino acids 952-966; and amino acids 1312-1326; and one or more guide RNAs that are specific to a viral target form a virus selected from the group consisting of EBV, HPV, and HBV.
  • the composition comprises the polynucleotide as an mRNA encapsulated in a lipid nanoparticle.
  • the substitutions may be at locations, relative to SEQ ID NO. : 63, selected from R152, 1154, A157, F238, N240, A243, F569, K571, C574, W883, Q885, N888, V955, L1315, and N1317, with each optionally substituted to one of K, V, S, L, Q, R, E, T, and N.
  • the Cas endonuclease comprises at least one nuclear localization signal.
  • the Cas endonuclease may include SEQ ID NO.
  • the polynucleotide encoding the Cas endonuclease may include one selected from the group consisting of SEQ ID NOs. : 55, 56, 57, 58, and 61.
  • the composition includes the polynucleotide encoding the Cas endonuclease in the form of a mRNA.
  • the guide RNA targets UPV.
  • the guide RNA may include one selected from the group consisting of SEQ ID NOs. : 1-38, 71-76, and 79-83, optionally with about 20 to 100% of the bases modified.
  • the guide RNA comprises one selected from the group consisting of SEQ ID NOs.
  • the guide RNA includes one selected from the group consisting of SEQ ID NOs. : 2, 3, 4, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 71-73, and 79-83.
  • the substitutions may include, relative to SEQ ID NO. : 63, R152K, I154V, A157S, F238L, N240Q, A243 S, F569L, K571R, C574E, W883T, Q885N, N888Q, V955I, L1315I, and N1317Q.
  • the substitutions include, relative to SEQ ID NO. : 63, at least six of R152K, I154V, A157S, F238L, N240Q, A243 S, F569L, K571R, C574E, W883T, Q885N, N888Q, V955I, L1315I, and N1317Q.
  • FIG. 1 shows a composition 101 that includes an mRNA encoding a Cas endonuclease 1 13 and a guide RNA 121.
  • the guide RNA 121 comprises a targeting region 127, which is complementary to a target nucleic acid in viral genome.
  • One or a plurality of nanoparticles 105 (which include a cationic lipid 107) encapsulate the mRNA 1 13 and the guide RNA 121.
  • the nanoparticles 105 are optionally carried by a carrier formulation 135, such as water, an aqueous solution, or a gel.
  • the carrier formulation 135 optionally includes one or more of an excipient 136.
  • FIG. 2 shows an mRNA 113 according to certain embodiments.
  • the mRNA 113 includes a 5' cap 205, a 5' untranslated region (UTR) 211, an open reading frame (ORF) 215 encoding a Cas endonuclease, a 3' UTR 221, and a poly- A tail 227 (e.g., 120 adenosine nucleotides).
  • UTR 5' untranslated region
  • ORF open reading frame
  • FIG. 3 shows a ribonucleoprotein ("RNP”), comprising a Cas endonuclease
  • FIG. 4 shows a liposome 401 that may be used to encapsulate nucleic acid molecules of the present disclosure, e.g., the mRNA and the guide RNA.
  • FIG. 5 diagrams a method 501 of treating an UPV infection.
  • FIG. 6 is a map of the UPV genome.
  • FIG. 7 shows the results of an in vitro CRISPR endonuclease assay.
  • the upper panel shows a conceptual diagram of how CRISPR-mediated cleavage can be used to insert a DNA fragment.
  • the lower panel shows the detection of CRISPR-mediated cleavage by PCR detection of the inserted DNA fragment.
  • A HPV guide RNA 1.1.1 (SEQ ID NO. : 2);
  • B HPV guide RNA E6-2 (SEQ ID NO. : 81);
  • C HPV guide RNA E7-1 (SEQ ID NO.: 82).
  • FIG. 8A and 8B shows results of an in vitro assay showing that CRISPR/Cas9 compositions can reduce viral DNA and kill viral cells.
  • Viral DNA levels are lower in cells provided a Cas9 mRNA and an HPV16-targeting guide RNA compared to cells provided with non-specific guide RNA.
  • B Cell death was greater in HPV16+ cells provided a Cas9 mRNA and an HPV16-targeting guide RNA compared to HPV16+ cells provided with non-specific guide RNA (left). Cell death was much lower in HPV16- 293 cells, and was more similar between those that were provided an HPV16-targeting guide RNA and those provided with non-specific guide RNA (right).
  • FIG. 9 shows the results of an in vitro assay showing that CRISPR/Cas9 may be used to kill HPV16+ cancer cells. Cytoxicity was greater for cells receiving Cas9 mRNA and HPV16-targeting guide RNAs, compared to cells receiving non-specific guide RNA. Cell killing tended to increase in days following treatment (left) and with increasing amounts of Cas9 mRNA (right).
  • FIG. 10 diagrams a method 1001 of making a medicament for treatment of an
  • FIG. 11 diagrams a strategy for verifying specificity of the composition based on GUIDE-Seq technology (see Tsai et al., Nature Biotechnology 33 : 187-197, 2015).
  • FIG. 12 shows the detected specificity of HPV16-targeting guide RNAs at different doses in targeting portions of the HPV16 genome (SEQ ID NOs.: 64 and 65).
  • FIG. 13 show the results of measurement of in vitro DNA cleavage efficiency of a Cas endonuclease administered with HPV16-targeting guide RNAs corresponding to HPV16 guide RNAs 1.1.1 (A, circles) (SEQ ID NO.: 2), 1.1.3 (B, squares) (SEQ ID NO.: 4), and E6-1 BNA/LNA (C, diamonds) (SEQ ID NO.: 83).
  • FIG. 14 shows in vitro cleavage specificity for UPV guide RNAs corresponding to HPV16 guide RNAs 1.1.1 (A) (SEQ ID NO. : 2), 1.1.3 (B) (SEQ ID NO. : 4), and E6-1 BNA/LNA (C) (SEQ ID NO. : 83).
  • FIG. 15 shows successful tissue penetration of mRNA-LNP, wherein the mRNA encodes green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • FIG. 16 shows exemplary Cas9 nucleotide (mRNA) sequences.
  • mRNA Cas9 nucleotide sequences.
  • cm 2'-0-methylcytidine
  • gm 2'-0-methylguanosine
  • um 2'-0-methyluridine
  • am 2'-0- methyladenosine
  • m5c 5-methylcytidine
  • p pseudouridine
  • n any nucleotide
  • n* phosphorothioate bond
  • +n Locked Nucleic Acid or Bridged Nucleic Acid
  • (mo5u) 5methoxyuridine.
  • FIG. 17 shows exemplary Cas9 protein sequences.
  • cm 2'-0-methylcytidine
  • gm 2'-0- methylguanosine
  • um 2'-0-methyluridine
  • am 2'-0-methyladenosine
  • m5c 5- methylcytidine
  • p pseudouridine
  • n any nucleotide
  • n* phosphorothioate bond
  • +n Locked Nucleic Acid or Bridged Nucleic Acid
  • (mo5u) 5methoxyuridine.
  • FIG. 18 shows exemplary guide RNA sequences for targeting HPV16.
  • nucleotide denotes 2'-0-methyl RNA base modification
  • n* phosphorothioate bond
  • +n Locked Nucleic Acid or Bridged Nucleic Acid.
  • FIG. 19 shows exemplary guide RNA sequences for targeting HPV18.
  • cm 2'-0-methylcytidine
  • gm 2'-0-methylguanosine
  • um 2'-0-methyluridine
  • am 2'-0-methyladenosine
  • m5c 5- methylcytidine
  • p pseudouridine
  • n any nucleotide
  • n* phosphorothioate bond
  • +n Locked Nucleic Acid or Bridged Nucleic Acid
  • (mo5u) 5methoxyuridine.
  • FIG. 20 shows exemplary guide RNA sequences for targeting HBV.
  • cm 2'-0-methylcytidine
  • gm 2'-0-methylguanosine
  • um 2'-0-methyluridine
  • am 2'-0-methyladenosine
  • m5c 5- methylcytidine
  • p pseudouridine
  • n any nucleotide
  • n* phosphorothioate bond
  • +n Locked Nucleic Acid or Bridged Nucleic Acid
  • (mo5u) 5methoxyuridine.
  • FIG. 21 shows exemplary target sequences within the EBV genome for guide RNA targeting EBV.
  • FIG. 23 shows the specificity of Cas9 mRNA + HPV16 E7 guide RNA compositions.
  • FIG. 24A-24C shows enhanced Cas9 specificity with a Conformationally Restricted Nucleotides (CRN)-modified HPV16 E7 gRNA (SEQ ID NO. : 79).
  • CRN Conformationally Restricted Nucleotides
  • the present disclosure provides guide RNA sequences that are complementary to a target in a viral nucleic acid, such as an HPV or HBV nucleic acid.
  • the guide RNA comprises various modifications, e.g., a 2'-0-methyl modified nucleotide, a locked or bridged nucleotide, or a phosphorothioate bond between two nucleotides.
  • the guide RNAs can be used with a Cas endonuclease (e.g., a Cas9) to cleave the target nucleic acid.
  • the guide RNAs are delivered to a cell or a tissue in compositions that further comprise an mRNA molecule that encodes a Cas endonuclease. In some embodiments, the guide RNA is delivered to a cell or a tissue as part of a ribonucleoprotein (RNP) complex.
  • RNP ribonucleoprotein
  • the present disclosure provides a composition comprising a Cas endonuclease and one or more guide RNAs that target a viral nucleic acid, or a nucleic acid encoding such a Cas endonuclease and/or guide RNAs.
  • the present disclosure provides a composition that comprises (a) an mRNA encoding a Cas endonuclease and (b) one or more guide RNAs, for the treatment of a viral infection, such as an HPV or HB V infection.
  • the present disclosure provides methods of preventing or treating viral infections comprising administering a composition comprising the guide RNAs as described herein.
  • a protein consists essentially of a particular amino acid sequence when the protein includes additional amino acids that contribute to at most 20% of the length of the protein and do not substantially affect the activity of the protein (e.g., alters the activity of the protein by no more than 50%).
  • a cell includes a plurality of cells, including mixtures thereof.
  • a composition for treatment of preparation of medicaments as described herein contemplates using one or more compositions of the invention for such treatment or preparation unless the context clearly dictates otherwise.
  • nucleic acid molecule or peptide refers to either the nucleic acid molecule or the peptide, or both of them.
  • SEQ ID NO. : 1 is 102 nucleotides long and includes 18 cytosines.
  • a plurality of bases in SEQ ID NO. : 1 are modified means that between 2 and 102 bases are modified.
  • SEQ ID NO. : 1 contains a plurality of 5- methyl-cytosine modifications means that between 2 and 18 of the cytosines have 5' methyl groups.
  • a value of 0.1 is understood to mean from 0.05 to 0.14.
  • the interval of values 0.1 to 0.2 includes the range from 0.05 to 0.24.
  • a concentration of from 0.1 mg/mL to 2 mg/mL means a concentration range of from 0.05 mg/mL to 2.4 mg/mL.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer generally to a polymer of amino acids linked by peptide (amide) bonds. It may be of any length and may be linear, branched, or cyclic.
  • the amino acid may be naturally-occurring, non-naturally-occurring, or may be an altered amino acid. This term can also include an assembly of a plurality of polypeptide chains into a complex. This term also includes natural or artificially altered amino acid polymers. Such alteration includes disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or alteration.
  • This definition also includes, for example, polypeptides including one or two or more analogs of amino acids (e.g., including non-naturally-occurring amino acids), peptide-like compounds (e.g., peptoids) and other alterations known in the art.
  • amino acids e.g., including non-naturally-occurring amino acids
  • peptide-like compounds e.g., peptoids
  • a functionally equivalent peptide refers to a peptide that may vary in terms of structure (sequence) but is the same or similar to the original peptide.
  • Functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Those skilled in the art may introduce designed changes through the application mutagenesis techniques.
  • a “signal peptide”, also referred to as “signal sequence”, “leader sequence”, “leader peptide”, “localization signal” or “localization sequence”, is a short peptide (usually 15-30 amino acids in length) present at the N-terminus of newly synthesized proteins that are destined for the secretory pathway.
  • a signal peptide typically comprises a short stretch of hydrophilic, positively charged amino acids at the N-terminus, a central hydrophobic domain of 5-15 residues, and a C-terminal region with a cleavage site for a signal peptidase.
  • a signal peptide prompts translocation of the newly synthesized protein to the endoplasmic reticulum where it is cleaved by the signal peptidase, creating a mature protein that then proceeds to its appropriate destination.
  • nucleic acid or “nucleic acid molecule” refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments generated, for example, by the polymerase chain reaction (PCR) or by in vitro translation, and fragments generated by any of ligation, scission, endonuclease action, or exonuclease action.
  • the nucleic acids of the present disclosure are produced by PCR.
  • Nucleic acids may be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides ⁇ e.g., a-enantiomeric forms of naturally-occurring nucleotides), or a combination of both.
  • naturally occurring nucleotides such as deoxyribonucleotides and ribonucleotides
  • analogs of naturally occurring nucleotides e.g., a-enantiomeric forms of naturally-occurring nucleotides
  • Modified nucleotides can have "modifications" or be “modified,” wherein the nucleotide differs from the wild-type or original or comparator nucleotide molecule in, for example, replacement of or modification of sugar moieties ⁇ e.g., 2'-0-methylation), pyrimidine or purine base moieties ⁇ e.g., methylation), or linkages between nucleotides ⁇ e.g., a phosphorothioate linkage). Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages.
  • Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. Nucleic acid molecules can be either single stranded or double stranded.
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region "leader and trailer” as well as intervening sequences (introns) between individual coding segments (exons).
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated.
  • nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide.
  • a composition e.g., a cell lysate
  • the term "recombinant" refers to a cell, microorganism, nucleic acid molecule, or vector that has been modified by introduction of an exogenous nucleic acid molecule, or refers to a cell or microorganism that has been altered such that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated or constitutive, where such alterations or modifications may be introduced by genetic engineering. Genetic alterations may include, for example, modifications introducing nucleic acid molecules (which may include an expression control element, such as a promoter) encoding one or more proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of or addition to a cell's genetic material.
  • nucleic acid molecules which may include an expression control element, such as a promoter
  • mutation refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively.
  • a mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).
  • a “conservative mutation” or “conservative substitution” refers to a substitution of one amino acid for another amino acid that has similar properties.
  • the percentage sequence identity values can be generated using the NCBI BLAST2.0 software as defined by Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402, with the parameters set to default values.
  • a "guide RNA” refers to the inclusion of both trans-activating RNA (tracrRNA) and crispr RNA (crRNA), which function together to form the active ribonucleoprotein (RNP) with an endonuclease, or to the single-molecule version known as single guide RNA, or sgRNA.
  • a "ribonucleoprotein” or RNP” refers to a CRISPR/Cas protein complex formed by the association of a guide RNA with a Cas endonuclease. RNPs can form in vivo as a result of the natural association of the guide RNA and endonuclease, or they can be assembled in vitro, and delivered directly to cells using electroporation or transfection techniques.
  • the guide RNA is present in the single guide RNA form. In one embodiment, the guide RNA includes one or more modifications in the form of modified nucleotides or modifications to the canonical phosphate backbone bonds.
  • guide RNAs comprise targeting regions or targeting portions that are complementary to sites within the genome of a virus, which sites do not also appear in a human genome.
  • the guide RNA includes a targeting region that hybridizes specifically to a target within an HPV genome.
  • the guide RNAs may include features such as modified nucleotides that promote the delivery of the RNAs to, and retention within, infected cells.
  • modified nucleotides in RNAs may function to improve RNA stability, reduce immunogenicity, or improve specificity of the endonuclease activity.
  • the guide RNAs can include features such as one or more 2'-0-methyl groups on a ribose ring, one or more phosphorothioate bonds between nucleotides, or both. Such modifications may, in some embodiments, be located proximal to the 5' and 3' termini of the guide RNAs, which may protect against exonuclease digestion.
  • the guide RNAs may optionally include one or more locked nucleic acid, bridged nucleic acid, or
  • the guide RNAs may include modifications such as pseudouridine or 5-methyl-cytosine that may minimize an immune response by the patient.
  • the guide RNA comprises a plurality of (e.g., between about 20 and 100%) modified bases each selected from the group consisting of 2'-0- methylcytidine ("cm”); 2'-0-methylguanosine ("gm”); 2'-0-methyluridine (“urn”); 2'-0- methylpseudouridine (“fm”); and 2'-0-methyladenosine ("am”).
  • the guide RNA comprises between one and nine phosphorothioate bonds between the ten terminal nucleotides at each of a 5' end and at a 3' end of the guide RNA.
  • the guide RNA may include at least one bridged nucleic acid (BNA), locked nucleic acid (LNA), and/or conformationally-restricted nucleotide (CRN).
  • FIG. 3 shows an RNP 301 that includes a guide RNA 121, showing the targeting region 127.
  • the guide RNA 121 includes one of SEQ ID NO. : 1-30, 71- 76, and 79-83, optionally with one or a plurality of modifications.
  • the guide RNA 121 includes one of SEQ ID NO.: 31-38, optionally with one or a plurality of modifications. Modifications may include, for example, a 2'-0-methyl group on a ribose ring of a nucleotide, a phosphorothioate bond between nucleotides, or a Locked Nucleic Acid or Bridged Nucleic Acid. Except where otherwise specified, RNA preferably includes standard ribonucleotides.
  • the guide RNA 121 may include 2'-0-methylated nucleotides.
  • the 2'-0- Methyl oligo modification may be characterized as a RNA analog protecting against general base hydrolysis and nucleases, as well as increased Tm of duplexes by 1-4 °C per addition. It may be found that the configuration of a 2'-0-Methyl oligonucleotide is in the A-form like RNA, and not the B-form like DNA. It may only require a couple of those modifications in a row to effect the transition from one form to the other. Another benefit of 2' O-methyl groups includes the stabilization of RNA molecules from nuclease activity.
  • 2'-0-methylated nucleotides are available from TriLink BioTechnologies, LLC (San Diego, CA). In one embodiment, between 1 and about 10, preferably about 5, 2'-0-methyl groups are included within the first 5 or 6 or 7 bases at both the 5' and the 3' end of the guide RNA 121 to protect from exonuclease activity when the composition 101 is delivered to cells.
  • the guide RNA 121 may also include conformationally-restricted nucleic acids (CRN), which includes both locked nucleic acids and bridged nucleic acids.
  • CRN conformationally-restricted nucleic acids
  • a locked nucleic acid is a modified RNA nucleotide.
  • the ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the 3'- endo (North) conformation, which is often found in the A-form duplexes.
  • LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired and hybridize with DNA or RNA according to Watson-Crick base-pairing rules. Such oligomers are synthesized chemically and are commercially available from Exiqon, a Qiagen company (Venlo, Netherlands).
  • Bridged nucleic acids are modified RNA nucleotides. They are sometimes also referred to as constrained or inaccessible RNA molecules. BNA monomers can contain a five-membered, six-membered or even a seven-membered bridged structure with a "fixed" C3'-endo sugar puckering. The bridge is synthetically incorporated at the 2', 4'-position of the ribose to afford a 2', 4'-BNA monomer. The monomers can be incorporated into oligonucleotide polymeric structures using standard phosphoramide chemistry. BNAs are structurally rigid oligonucleotides with increased binding affinities and stability. BNAs are available from Bio-Synthesis, Inc. (Lewisville, TX).
  • the guide RNA targets an HPV genome, such as, for example an HPV16 genome, an HPV18 genome, an HPV6 genome, or an HPV11 genome.
  • the guide RNA comprises a nucleic acid sequence as set forth in SEQ ID NOs.: 1, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 74, 75, or 76, and optionally wherein one or more of the nucleotides of the guide RNA are further substituted with a plurality of modified bases each selected from the group consisting of 2'-0-methylcytidine (cm); 2'-0- methylguanosine (gm); 2'-0-methyluridine (um); 2'-0-methylpseudouridine (fm); and 2'-0- methyladenosine (am).
  • the guide RNA comprises a nucleic acid sequence as set forth in SEQ ID NO. : 2, 3, 4, 6-16, 18, 20, 22, 24, 26, 28, 32, 34, 36, 38, or 71-73.
  • the guide RNA is one of SEQ ID NO. : 1, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 4, 37, 41, 43, and 45, and optionally with about 20 to 100% of the nucleotides having a modification such as a methyl or a 2'-0-methyl group on a ribose sugar, and preferably include a plurality (e.g., about 20 to 100%) of modified bases each selected from the group consisting of 2'-0-methylcytidine (cm); 2'-0-methylguanosine (gm); 2'-0- methyluridine (um); and 2'-0-methylpseudouridine (fm), and 2'-0-methyladenosine (am).
  • the guide RNA comprises one selected from the group consisting of SEQ ID NO. : 41, 43, 45, and optionally wherein one or more of the nucleotides of the guide RNA are further substituted with a plurality of modified bases each selected from the group consisting of 2'-0-methylcytidine (cm); 2'-0-methylguanosine (gm); 2'-0-methyluridine (um); 2'-0- methylpseudouridine (fm); and 2'-0-methyladenosine (am).
  • cm 2'-0-methylcytidine
  • gm 2'-0-methylguanosine
  • gm 2'-0-methyluridine
  • fm 2'-0-methylpseudouridine
  • am 2'-0-methyladenosine
  • the composition 101 includes (a) a mRNA polynucleotide encoding the Cas endonuclease, or the active Cas endonuclease, and (b) one or more guide RNAs that are specific to a viral target such as Epstein-Barr virus (EB V), human
  • EB V Epstein-Barr virus
  • HPV Kaposi sarcoma virus
  • KSHV Kaposi sarcoma virus
  • HBV hepatitis B virus
  • HSV-1 herpes simplex virus-1
  • HSV-2 herpes simplex virus-2
  • VZV varicella zoster virus
  • CMV cytomegalovirus
  • HHV-6 human herpesvirus-6
  • HHV-7 human herpesvirus-7
  • Embodiments of a composition 101 as described herein may be provided for the treatment of an HPV infection.
  • the guide RNA 121 may include a targeting region 127 that includes SEQ ID NO. : 39 or SEQ ID NO. : 40.
  • the guide RNA 121 included in compositions provided for the treatment of an HPV infection may include one or more of SEQ ID NO. : 1, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 4, or 37, and optionally further include ten or more 2'-0-methyluridine.
  • at least about 20% of the nucleotides of the guide RNA 121 include a 2'-0-methyl group on a ribose sugar.
  • compositions of the invention may include a first guide RNA for use against HPV16.
  • the guide RNA comprises one of SEQ ID NO. : 1, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 4, 37, 41, 43, 45, and optionally includes a plurality of modified bases each selected from the group consisting of 2'-0-methylcytidine (cm); 2'-0- methylguanosine (gm); 2'-0-methyluridine (um); and 2'-0-methylpseudouridine (fm).
  • the guide RNA may be one of SEQ ID NOs. : 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42, 44, and 46.
  • the guide RNA targets UPV, and includes one of SEQ ID NOs.: 1, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37 and optionally includes one or a plurality of modified bases each selected from the group consisting of 2'-0-methylcytidine (cm); 2'-0-methylguanosine (gm); 2'-0-methyluridine (um); and 2'-0-methylpseudouridine (fm).
  • the guide RNA may be one of SEQ ID NOs.: 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38.
  • the guide RNA targets HBV and comprises one of SEQ ID NOs.: 41, 43, or 45, and further optionally includes one or a plurality of modified bases each selected from the group consisting of 2'-0-methylcytidine (cm); 2'-0- methylguanosine (gm); 2'-0-methyluridine (um); 2'-0-methylpseudouridine (fm); and 2'-0- methyladenosine (am)
  • the guide RNA may be one of SEQ ID NOs. : 42, 44, or 46.
  • the guide RNA targets EB V and includes a targeting region substantially complementary to one of SEQ ID NO. : 47, 48, 49, 50, 51, 52, and 53, and preferably includes a plurality of modified bases each selected from the group consisting of 2'-0-methylcytidine (cm); 2'-0-methylguanosine (gm); 2'-0-methyluridine (um); and 2'- O-methylpseudouridine (fm).
  • SEQ ID NO. : 47 is a first target in an EBV genome.
  • SEQ ID NO. : 48 is a second target in an EBV genome.
  • SEQ ID NO. : 49 is a third target in an EBV genome.
  • SEQ ID NO. : 50 is a fourth target in an EBV genome.
  • SEQ ID NO. : 51 is a fifth target in an EBV genome.
  • SEQ ID NO. : 52 is a sixth target in an EBV genome.
  • SEQ ID NO. : 53 is a seventh target in an EBV genome.
  • the guide RNA(s) that target any of SEQ ID NOs.
  • : 47-53 may include any of the modifications described in connection with guide RNAs herein, and preferably include at least three modified bases each selected from the group consisting of 2'-0-methylcytidine (cm); 2'-0-methylguanosine (gm); 2'-0- methyluridine (um); and 2'-0-methylpseudouridine (fm) within the terminal 7 to 10 nucleotides of the guide RNA.
  • the present disclosure provides a synthetic guide RNA, comprising a guide RNA nucleic acid molecule having a targeting region complementary to a target in a viral nucleic acid.
  • the viral nucleic acid is a human papillomavirus (HPV) nucleic acid.
  • the viral nucleic acid is a hepatitis B virus (HBV) nucleic acid.
  • the present disclosure provides a synthetic guide RNA, comprising a guide RNA nucleic acid molecule having a targeting region complementary to a target in a an HPV16 nucleic acid, and comprising (a) any one of SEQ ID NOs. : 1-30, 71-76, and 79-83; or (b) a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs.: 1-30, 71-76, and 79-83.
  • the guide RNA comprises SEQ ID NO. : 1, 5, 17, 19, 21, 23, 25, 27, 29, 74, 75, or 76.
  • the guide RNA comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO. : 1, 5, 17, 19, 21, 23, 25, 27, 29, 74, 75, or 76.
  • the guide RNA comprises a sequence having at least 95% identity to SEQ ID NO. : 1, 5, 17, 19, 21, 23, 25, 27, 29, 74, 75, or 76.
  • the targeting region of the guide RNA comprises SEQ ID NO. : 39 or SEQ ID NO. : 40.
  • the target in the viral nucleic acid comprises SEQ ID NO. : 64 or SEQ ID NO. : 65.
  • the guide RNA comprises SEQ ID NO. : 80.
  • the guide RNA may comprise one or more modified bases.
  • the guide RNA may comprise 2'-0-methylcytidine; 2'-0-methylguanosine; 2'-0-methyluridine; 2'-0- methylpseudouridine; or 2'-0-methyladenosine.
  • the guide RNA may comprise a plurality of modified bases each selected from the group consisting of 2'-0-methylcytidine; 2'-0-methylguanosine; 2'-0-methyluridine; 2'-0- methylpseudouridine; and 2'-0-methyladenosine.
  • the guide RNA comprises SEQ ID NO.
  • nucleotides of the guide RNA are further substituted with nucleotides having a modified base, and each modified base is 2'-0-methylcytidine; 2'-0-methylguanosine; 2'-0- methyluridine; 2'-0-methylpseudouridine; or 2'-0-methyladenosine.
  • at least about 20% of the nucleotides of the guide RNA may comprise a 2'-0-methyl group on a ribose sugar.
  • the guide RNA may comprise a phosphorothioate bond between two nucleosides.
  • the guide RNA may comprise one or more
  • the guide RNA comprises SEQ ID NO.: 1, 5, 17, 19, 21, 23, 25, 27, 29, 74, 75, or 76, wherein one or more of the internucleoside bonds in the guide RNA is further substituted with a phosphorothioate bond.
  • the guide RNA comprises SEQ ID NO.: 1, 5, 17, 19, 21, 23, 25, 27, 29, 74, 75, or 76, wherein one or more of the internucleoside bonds within the ten terminal nucleotides at each of a 5' end and at a 3' end of the guide RNA is further substituted with a phosphorothioate bond.
  • the guide RNA may comprise a bridged nucleic acid (BNA), a locked nucleic acid (LNA), or a conformationally-restricted nucleotide (CRN).
  • the guide RNA comprises SEQ ID NO.
  • nucleotides of the guide RNA are further substituted with nucleotides having a conformationally-restricted nucleotide (CRN).
  • CRN conformationally-restricted nucleotide
  • one or more of the nucleotides of the guide RNA are further substituted with nucleotides having (a) a modified base, and each modified base is 2'-0-methylcytidine, 2'-0- methylguanosine; 2'-0-methyluridine, 2'-0-methylpseudouridine, or 2'-0-methyladenosine; (b) a 2'-0-methyl group on a ribose sugar; (c) a phosphorothioate bond between two nucleosides; (d) an LNA, BNA, or CRN; or (e) any combination of (a)-(d).
  • the present disclosure provides a synthetic guide RNA, comprising a guide RNA nucleic acid molecule having a targeting region complementary to a target in a an HPV18 nucleic acid, and comprising (a) any one of SEQ ID NOs. : 31-38; or (b) a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs.: 31-38.
  • the guide RNA comprises any one of SEQ ID NOs. : 31-38.
  • the guide RNA comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs.: 31-38.
  • the guide RNA comprises a sequence having at least 95% identity to any one of SEQ ID NOs. : 31-38.
  • the guide RNA comprises SEQ ID NO. : 31, 33, 35, or 37.
  • the guide RNA comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO. : 31, 33, 35, or 37.
  • the guide RNA may comprise one or more modified bases.
  • the guide RNA may comprise 2'-0-methylcytidine; 2'-0-methylguanosine; 2'-0-methyluridine; 2'-0-methylpseudouridine; or 2'-0- methyladenosine.
  • the guide RNA may comprise a plurality of modified bases each selected from the group consisting of 2'-0-methylcytidine; 2'-0-methylguanosine; 2'-0-methyluridine; 2'-0-methylpseudouridine; and 2'-0- methyladenosine.
  • the guide RNA comprises SEQ ID NO.: 31, 33, 35, or 37, wherein one or more of the nucleotides of the guide RNA are further substituted with nucleotides having a modified base, and each modified base is 2'-0-methylcytidine; 2'-0- methylguanosine; 2'-0-methyluridine; 2'-0-methylpseudouridine; or 2'-0-methyladenosine.
  • at least about 20% of the nucleotides of the guide RNA may comprise a 2'-0-methyl group on a ribose sugar.
  • the guide RNA may comprise a phosphorothioate bond between two nucleosides. In any of the aforementioned embodiments, the guide RNA may comprise one or more phosphorothioate bonds within the ten terminal nucleotides at each of a 5' end and at a 3' end of the guide RNA. In some embodiments, the guide RNA comprises SEQ ID NO. : 31, 33, 35, or 37, wherein one or more of the internucleoside bonds in the guide RNA is further substituted with a phosphorothioate bond. In some embodiments, the guide RNA comprises SEQ ID NO.
  • the guide RNA may comprise a bridged nucleic acid (BNA), a locked nucleic acid (LNA), or a conformationally -restricted nucleotide (CRN).
  • BNA bridged nucleic acid
  • LNA locked nucleic acid
  • CRN conformationally -restricted nucleotide
  • the guide RNA comprises SEQ ID NO.
  • nucleotides of the guide RNA are further substituted with nucleotides having a conformationally-restricted nucleotide (CRN).
  • one or more of the nucleotides of the guide RNA are further substituted with nucleotides having (a) a modified base, and each modified base is 2'-0-methylcytidine, 2'-0-methylguanosine; 2'-0-methyluridine, 2'-0-methylpseudouridine, or 2'-0-methyladenosine; (b) a 2'-0-methyl group on a ribose sugar; (c) a phosphorothioate bond between two nucleosides; (d) an LNA, BNA, or CRN; or (e) any combination of (a)-(d).
  • the present disclosure provides a synthetic guide RNA, comprising a guide RNA nucleic acid molecule having a targeting region complementary to a target in a an HBV nucleic acid, and comprising (a) any one of SEQ ID NOs.: 41-46 and 66; or (b) a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs.: 41-46 and 66.
  • the guide RNA comprises SEQ ID NO. : 41, 43, or 45.
  • the guide RNA comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO. : 41, 43, or 45.
  • the guide RNA may comprise one or more modified bases.
  • the guide RNA may comprise 2'-0-methylcytidine; 2'-0-methylguanosine; 2'- O-methyluridine; 2'-0-methylpseudouridine; or 2'-0-methyladenosine.
  • the guide RNA may comprise a plurality of modified bases each selected from the group consisting of 2'-0-methylcytidine; 2'-0-methylguanosine; 2'-0- methyluridine; 2'-0-methylpseudouridine; and 2'-0-methyladenosine.
  • modified bases each selected from the group consisting of 2'-0-methylcytidine; 2'-0-methylguanosine; 2'-0- methyluridine; 2'-0-methylpseudouridine; and 2'-0-methyladenosine.
  • the guide RNA comprises SEQ ID NO. : 41, 43, or 45, wherein one or more of the nucleotides of the guide RNA are further substituted with nucleotides having a modified base, and each modified base is 2'-0-methylcytidine; 2'-0-methylguanosine; 2'-0- methyluridine; 2'-0-methylpseudouridine; or 2'-0-methyladenosine.
  • at least about 20% of the nucleotides of the guide RNA may comprise a 2'-0-methyl group on a ribose sugar.
  • the guide RNA may comprise a phosphorothioate bond between two nucleosides.
  • the guide RNA may comprise one or more
  • the guide RNA comprises SEQ ID NO.: 41, 43, or 45, wherein one or more of the internucleoside bonds in the guide RNA is further substituted with a phosphorothioate bond.
  • the guide RNA comprises SEQ ID NO. : 41, 43, or 45, wherein one or more of the internucleoside bonds within the ten terminal nucleotides at each of a 5' end and at a 3' end of the guide RNA is further substituted with a phosphorothioate bond.
  • the guide RNA may comprise a bridged nucleic acid (BNA), a locked nucleic acid (LNA), or a conformationally-restricted nucleotide (CRN).
  • BNA bridged nucleic acid
  • LNA locked nucleic acid
  • CNN conformationally-restricted nucleotide
  • the guide RNA comprises SEQ ID NO.: 41, 43, or 45, wherein one or more of the nucleotides of the guide RNA are further substituted with nucleotides having a conformationally-restricted nucleotide (CRN).
  • nucleotides of a guide RNA targeting an HB V nucleic acid are further substituted with nucleotides having (a) a modified base, and each modified base is 2'-0- methylcytidine, 2'-0-methylguanosine; 2'-0-methyluridine, 2'-0-methylpseudouridine, or 2'- O-methyladenosine; (b) a 2'-0-methyl group on a ribose sugar; (c) a phosphorothioate bond between two nucleosides; (d) an LNA, BNA, or CRN; or (e) any combination of (a)-(d).
  • a DNA molecule encoding a guide RNA as disclosed herein is provided.
  • a vector comprising such a DNA molecule is also contemplated.
  • Exemplary guide RNA sequences of the present disclosure are shown in FIG. 18 for targeting HPV16, FIG. 19 for targeting HPV18, and FIG. 20 for targeting HBV.
  • FIGs. 21 and 22 show exemplary target sequences.
  • compositions as disclosed herein comprise a Cas endonuclease, e.g., any member of the family of CRISPR associated bacterial endonucleases.
  • the Cas endonuclease is a wild-type Cas9 endonuclease, e.g., one encoded by a nucleic acid molecule as set forth in SEQ ID NO. : 54 and/or comprising an amino acid sequence as set forth in SEQ ID NO.: 63 or 84.
  • the Cas endonuclease is a modified Cas9 endonuclease, wherein the modified Cas9 endonuclease is modified relative to the wild-type molecule.
  • the Cas9 may be modified for deceased immunogenicity (e.g., modified to include, relative to wild type, one or a plurality of substitutions within T-cell epitopes).
  • a therapeutic protein is delivered as an mRNA
  • a modified nucleotide sequence when the mRNA is translated into protein, the resulting protein can have, relative to wild-type, alternative or modified epitopes that do not trigger an anti-drug antibody (ADA) response.
  • ADA anti-drug antibody
  • the present disclosure provides in some embodiments a modified Cas9 endonuclease protein sequence in which one or more predicted T-cell epitopes have substitutions that avoid or decrease an immune response relative to wild-type Cas9 endonuclease.
  • the Cas9 endonuclease comprises at least one amino acid variation, relative to wild-type Cas9, comprising one or more of R780, K810, K848, K855, H982, K1003, and R1060.
  • the Cas9 protein comprises K848A, K1003A, or R1060A.
  • a coding sequence of the mRNA comprises a plurality of 5-methylcytidine. In a further embodiment, the coding sequence of the mRNA comprises a plurality of
  • the mRNA 113 can be manufactured by in vitro transcription by a company such as TriLink BioTechnologies, LLC (San Diego, CA) or AmpTec GmbH (Hamburg, Germany).
  • In vitro transcription manufacturing of mRNA typically uses double stranded DNA template in buffer with an RNA polymerase and a mix of NTPs. The polymerase synthesizes the mRNA 113. The DNA is then enzymatically degraded. The mRNA 113 is purified away from polymerase, free NTPs, and degraded DNA.
  • the Cas9 endonuclease is delivered to cells encoded in a DNA vector, in protein form ⁇ e.g., as an active RNP with the modified Cas9 endonuclease complexed with an antiviral guide RNA), or as an mRNA to be translated within the target cells.
  • the Cas9 endonuclease is delivered in mRNA form along with one or more guide RNAs.
  • FIG. 2 shows an mRNA 113 according to certain embodiments.
  • 113 includes a 5' cap 205, a 5' untranslated region (UTR) 211, ⁇ e.g., such as may be derived from a beta-globin sequence), an open reading frame (ORF) 215 encoding a Cas
  • the ORF 215 begins with start codon (AUG, part of Kozak Sequence) and ends with a stop codon (UAG).
  • the mRNA 113 includes a 3' UTR, which supports translation termination.
  • the mRNA 113 aldo includes a poly(A) tail of about 80 to 120 bases, which supports translation factor binding and stabilizes the mRNA 113, preventing mRNA degradation.
  • a 7-methyl-guanosine is linked to first 5' nucleotide via 5 '-5' triphosphate bridge. Depending on the number of additional methyl groups, this may be referred to as CapO, Capl, or Cap2.
  • mRNA produced as described below is not all capped, i.e., not 100% of the mRNA molecules produced will have a 5' Cap.
  • the 5' UTR 211 provides a site for ribosome binding and translation factor binding. The 5' UTR 211 influences expression level of the mRNA 113. Expression includes the translation of the sequence of the ORF 215 into its corresponding amino acid sequence.
  • the ORF 215 encodes a Cas endonuclease such as Cas9 and is optionally codon-optimized for mammalian expression.
  • the 5' cap 205 may include CapO or Capl, a 7-methylguanosine linked to the first 5' nucleotide via a 5 '-5' triphosphate bridge. Spanning the 3' end of the 5' UTR 211 and a 5' end of the ORF 215 is a Kozak sequence, serving as a translational start region.
  • the ORF comprises one of SEQ ID NOs.: 54, 55, 56, 57, 58, 59, and 60, wherein a plurality of the nucleotides of the guide RNA are further substituted with nucleotides having a modified base, and each modified base is 2'-0-methylcytidine; 2'-0-methylguanosine; 2'-0-methyluridine; and 2'-0- methylpseudouridine; or 2'-0-methyladenosine.
  • the ORF comprises one or more modifications relative to canonical RNA nucleotide sequences.
  • the ORF comprises SEQ ID NO.
  • the ORF 215 encodes a Cas9 variant as described by one of SEQ ID NOs. : 56-60 or a functionally equivalent peptide. In some embodiments, the ORF 215 encodes a Cas9 variant as described by one of SEQ ID NOs.: 67-70, 77, and 78, or a functionally equivalent peptide.
  • the ORF 215 of the mRNA 113 may be codon-optimized for a specific organism.
  • a portion of the ORF may include CAA CCT CAA, which encodes MPQ.
  • proline (P) is incorporated by the ribosome when any base is in the wobble position, so the codon for proline could be represented as CCN.
  • Glutamine (Q) is incorporated when the wobble base is a purine, and thus the Q codon could be represented (again, in DNA sense) as CAR (where one of skill in the art will recognize that N and R are the IUPAC ambiguity codes for any base and a purine, respectively).
  • CAR the IUPAC ambiguity codes for any base and a purine, respectively.
  • a sequence having or approaching 100% identity to SEQ ID NOs.: 54-60 may be most preferable due to codon optimization and, for example, GC content or avoidance of RNA secondary structures.
  • the mRNA 113 encodes a Cas endonuclease such as a version of Cas9.
  • the Cas9 endonuclease is a wild-type S. pyogenes Cas9 (SpCas9), and is encoded by an mRNA comprising SEQ ID NO.: 54.
  • the mRNA 113 encodes a modified Cas9 endonuclease
  • SpCas9-sgRNA complexes cleave target sites composed of an NGG PAM sequence (recognized by SpCas9)21-24 and an adjacent 20 bp protospacer sequence (which is complementary to the 5' end of the sgRNA).
  • Structural studies have suggested that the SpCas9-sgRNA-target DNA complex includes several SpCas9-mediated DNA contacts, including direct hydrogen bonds made by four SpCas9 residues (N497, R661, Q695, Q926) to the phosphate backbone of the target DNA strand. Alanine substitution of one, more, or all of those residues do not reduce on-target cleavage efficiency of SpCas9.
  • SpCas9-HFl with an additional Dl 135E substitution (dubbed SpCas9-HF2) retains 70% or more activity of wild-type SpCas9 in studies and may be included in the composition 101. Additionally, variants harboring additional L169A or Y450A substitutions (positions whose side chains are believed to mediate non-specific hydrophobic interactions with the target DNA on its PAM proximal end) may be included. See Kleinstiver, 2016, High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off targets, Nature 529(7587):490-495, incorporated by reference.
  • the Cas9 comprises at least one amino acid variation, relative to wild-type Cas9, comprising one or more of R780, K810, K848, K855, H982, K1003, or R1060.
  • the Cas9 includes one, two, or all three of K848A, K1003A, and R1060A.
  • the Cas9 is encoded by an mRNA sequence as set forth in SEQ ID NO.: 59, e.g., HF S.p. Cas9, SpCas9-HFl, and High-Fidelity variant #1, which are equivalent and are defined by SEQ ID NO.: 59.
  • the Cas9 is encoded by an mRNA sequence that has at least 85%, 90%, 95%, or 100% identity to SEQ ID NO. : 59.
  • Modified nucleases with enhanced specificity such as an enhanced specificity version of Cas9 ⁇ e.g., eSp(l . l) Cas9; SEQ ID NO. : 60) may be used in embodiments of the present specification.
  • the mRNA encoding eSp(l . l) preferably includes a 5' cap, a 5' UTR, a Kozak sequence, an ORF, a 3' UTR, and a poly- A tail.
  • Streptococcus pyogenes Cas9 crystal structures exhibit a positively-charged groove, positioned between the HNH, RuvC, and PAM-interacting domains in SpCas9, that is likely to be involved in stabilizing the non-target strand of the target DNA.
  • Neutralization of positively-charged residues in that non-target strand groove may weaken non-target strand binding and encourage re-hybridization between the target and non-target DNA strands, thereby requiring more stringent Watson-Crick base pairing between the RNA guide and the target DNA strand. Five substitutions within that groove may reduce activity at off-target sites compared to wild type SpCas9 while maintaining on-target cleavage efficiency.
  • Variants with both high efficiency (wild type levels of on-target indel formation) and specificity (no detectable indel formation at EMX(l) and VEGFA(l) off-targets) include:
  • SpCas9 K855A
  • SpCas9 K810A/K1003A/R1060A
  • SpCas9 K848A/K1003A/R1060A
  • Some embodiments include eSpCas9(1.0) or (1.1) encoded by an mRNA 113 in a composition 101.
  • the mRNA 113 may include one or a plurality of 5-methylcytidine (m5c), pseudouridine ( ⁇ ), or both.
  • Modified nucleosides may reduce innate immune activation and increase translation of mRNA. Unmodified mRNA may induce undesirable cytokine secretion.
  • Large quantities of RNA may be prepared by in vitro transcription from DNA templates using phage RNA polymerase or solid-phase chemical synthesis. Triphosphate-derivatives of pseudouridine ( ⁇ ) and 5-methylcytidine (m5C) (TriLink) may be used to generate RNA containing a modified nucleoside.
  • RNA-dependent protein kinase PDR
  • Modified nucleotides preferably include pseudouridine- ( ⁇ ), or 5-methylcytidine- (m5C), 5-methoxy-uridine, and ⁇ - (mSC/ ⁇ ) nucleoside modifications.
  • m5C ⁇ -nucleoside modified mRNA yields the least amount of RNA sensor activation and the highest level of translation.
  • HPLC purification removes dsRNA and other contaminants from in vitro-transcribed RNAs containing ⁇ or mSC/ ⁇ nucleosides, yielding RNA with high levels of translation. See Kariko, 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucleic Acids Res 39(21):el42, incorporated by reference.
  • the mRNAs may be transcribed to contain, e.g., 30, 51, or 120-nt long poly(A) tails.
  • Poly(A) tail may be added in a template-dependent fashion during transcription and/or may be added enzymatically post-transcription. For example, after transcription, additional poly(A) tail may be added with yeast poly(A) polymerase.
  • RNAs may be capped with, e.g., an m7G capping kit with or without 2'-0-methyltransferase (ScriptCap, CellScript) to obtain Capl or CapO. Capping may be done using TriLink' s CleanCap or AmpTec's enzymatic capping, for example, to result in Capl .
  • Immunogenicity of a therapeutic protein may reduce efficacy of the therapeutic by lowering half-life of the therapeutic through immune-based clearance following repeat exposure, via anti-drug antibodies (ADA).
  • Strategies to reduce ADA include: immunosuppression, patient tolerization (dose, route, and co-formulation), and modification of highly immunogenic T-cell epitopes within the protein.
  • a Cas9 or a modified Cas9 includes modifications to immunogenic T-cell epitopes of Cas9 or a modified Cas9.
  • mRNA-based therapeutics synthetic, directed post-translational chemical modification of the protein, e.g., PEGylation, is not possible. However, it is possible to alter critical amino acids within a predicted T-cell epitope to reduce immunogenicity and ADA.
  • Some embodiments of the present disclosure include nuclease sequences in which T-cell epitopes are modified relative to wild-type nuclease sequences. Modifications in nuclease epitopes T-cell epitopes may improve in vivo drug properties, reduce ADA, and facilitate repeat administration.
  • Another method to reduce immunogenicity is to reduce drug exposure by conjugating degradation peptides to Cas9 or sgRNA to reduce the half-life of CRISPR components.
  • the Cas endonuclease comprises a wild-type Streptococcus pyogenes Cas9 amino acid sequence as set forth in SEQ ID NO.: 63.
  • the Cas endonuclease comprises a wild-type Streptococcus pyogenes Cas9 amino acid sequence comprising an N-terminal and C-terminal nuclear localization sequences (NLS), e.g., SEQ ID NO.: 61.
  • NLS N-terminal and C-terminal nuclear localization sequences
  • additional Cas9 protein variants with different NLS and linker sequences may be included within the same mRNA molecule; e.g., an mRNA may include one of SEQ ID NOs. : 56-58, as well as any mRNA encoding the amino acid sequence as set forth in SEQ ID NO.: 61.
  • the mRNA molecule may encode more than one Cas9, e.g., more than one of SEQ ID NOs. : 54-60, 67-70, 77, and 78.
  • one or more Cas9 variants has enhanced nuclear localization relative to SEQ ID NO.: 61, resulting in higher endonuclease activity, higher potency, and allow administering lowering drug levels. Those benefits may contribute to reduced immunogenicity, as well as lower cost of drug manufacturing.
  • the Cas endonuclease is a variant Cas9 with reduced ADA and the optional terminal NLS sequences.
  • the Cas endonuclease comprises an amino acid sequence as set forth in SEQ ID NO. : 62.
  • the invention employs an mRNA that encodes a modified Cas endonuclease such as that of SEQ ID NO. : 62: .
  • SEQ ID NO. : 62 provides a Spy Cas9 protein sequence with N-terminal and C-terminal NLS engineered to have reduced ADA.
  • Predicted MHC II epitopes are underlined. Amino acids in bold text within each epitope indicate substitutions to reduce ADA risk.
  • some embodiments of the invention include the mutagenesis of potential MHC II-restricted CD4 T cell epitopes for HLA-DRB1.
  • a prediction tool may be used to predict MHC II T-cell epitopes in a protein sequence. Suitable prediction tools include, for example, SYFPETHI, IEDB (including the following variations: recommended, Consensus, NetMHCpan, NN_align, SMM_allign, Combinatorial library, Sturniolo), RANKPEP, ProPred, MULTIPRED2, MHCIIPred, MHC2SKpan, and NetMHCII.
  • SYFPETHI SYFPETHI
  • IEDB including the following variations: recommended, Consensus, NetMHCpan, NN_align, SMM_allign, Combinatorial library, Sturniolo
  • RANKPEP ProPred
  • MULTIPRED2 MHCIIPred
  • MHC2SKpan NetMHCII
  • SEQ ID NO.: 62 contains seven potential T cell epitopes as follows: epitope 1 includes amino acids 165-181 ; epitope 2 includes amino acids 251-265; epitope 3 includes amino acids 582-596; epitope 4 includes amino acids 737-751 ; epitope 5 includes amino acids 896-910; epitope 6 includes amino acids 968-982; and epitope 7 includes amino acids 1328-1342.
  • each epitope may have sub-sequences as follows (with reference to SEQ ID NO.
  • epitope 1 in epitope 1, of particular interest may be sub-region la (amino acids 168-176) or sub-region lb (amino acids 170-178); in epitope 2, sub-region 2a is amino acids 255-263; in epitope 3, sub-region 3a is amino acids 586-594; in epitope 4, sub-region 4a includes amino acids 741-749; in epitope 5, sub-region 5a includes amino acids 900-908 ; in epitope 6, sub-region 6a includes amino acids 972-980; in epitope 7, sub-region 7a includes amino acids 1332-1340.
  • the aforementioned epitope and sub-regions of Cas9 are of particular interest as they represent putative epitopes that may be highly-immunogenic.
  • many (e.g., at least half) or substantially all residues in epitopes 1-3 are mutated to alanine (and the mutations do not interfere with Cas9 structure / function).
  • certain specified residues within epitopes 5-7 are mutated, including one or more selected from (with reference to SEQ ID NO. : 63): W883, Q885, N888, V955, L1315, and N1317. Mutating charged residues to alanine may affect the local electrostatics and positioning of nearby residues that do directly contact the nucleic acid.
  • Exemplary Cas9 nucleic acid sequences of the present disclosure are shown in FIG. 16, while exemplary Cas9 protein sequences of the present disclosure are shown in FIG. 17.
  • the present disclosure provides compositions comprising a Cas endonuclease that targets a viral nucleic acid and one or more guide RNAs that target a viral nucleic acid, or a nucleic acid encoding such a Cas9 endonuclease and/or guide RNAs.
  • the present disclosure provides a composition that comprises (a) an mRNA encoding a Cas endonuclease and (b) one or more guide RNAs, for the treatment of a viral infection, such as an HBV or UPV infection.
  • the composition comprises a Cas9 endonuclease and one or more guide RNAs.
  • the composition comprises a ribonucleoprotein (RNP) complex, wherein the RNP comprises a Cas9 endonuclease complexed or associated with a guide R P.
  • RNP ribonucleoprotein
  • the mRNA and guide RNA may include particular sequences and modifications, and may be encapsulated by nanoparticles.
  • the guide RNA targets an HBV or HPV nucleic acid.
  • the composition includes mRNA encoding a modified
  • RNAs endonuclease as well as one or more guide RNAs that target the Cas endonuclease to the HPV or HBV genome.
  • Those RNAs are packaged in a lipid nanoparticle, solid nanoparticle, or liposome.
  • the RNA-encapsulated nanoparticles are optimally formulated for topical, mucosal, or local delivery to infected tissue, which avoids systemic delivery and circulation, thus minimizing drug exposure, off-target activity, and immunogenicity of the Cas endonuclease.
  • the nanoparticles are administered with a device such as, for example, a microneedle array to enhance RNA delivery to the basal epithelium.
  • the mRNA preferably encodes a programmable nuclease such as a Cas endonuclease or a modified Cas endonuclease.
  • the Cas endonuclease or modified endonuclease is a Cas9 endonuclease or modified Cas9 endonuclease.
  • the mRNA encodes a modified Cas endonuclease that is modified, relative to a wild-type version, for decreased immunogenicity.
  • the encoded, modified nuclease may include, relative to wild type, one or a plurality of substitutions within T-cell epitopes.
  • a therapeutic protein is delivered as an mRNA
  • a modified nucleotide sequence when the mRNA is translated into protein, the resulting protein can have, relative to wild-type, alternative or modified epitopes that do not trigger an anti-drug antibody (ADA) response.
  • the invention includes modified Cas endonuclease protein sequences in which one or more predicted T-cell epitopes have substitutions that avoid or decrease an immune response relative to wild-type Cas endonuclease.
  • the modified Cas endonuclease, with the modified T-cell epitopes may be delivered to cells encoded in a DNA vector, in protein form (e.g., as an active ribonucleoprotein (RNP) with the modified Cas endonuclease complexed with an antiviral guide RNA), or as an mRNA to be translated within the target cells.
  • the nuclease is delivered in mRNA form, e.g., along with one or more guide RNAs.
  • guide RNAs comprise targeting regions that are complementary to sites within a viral genome, e.g., the genomes of HPV16, HPV18, HBV, or EBV, which sites do not also appear in a human genome.
  • the guide RNAs may include features such as modified nucleotides that promote the delivery of the RNAs to, and retention within, infected cells.
  • modified nucleotides in RNAs may function to improve RNA stability, reduce immunogenicity, or improve specificity of the endonuclease activity.
  • the guide RNAs can include features such as one or more 2'-0-methyl groups on a ribose ring, one or more phosphorothioate bonds between nucleotides, or both, and particularly located proximal to the 5' and 3' termini of the guide RNAs, which may protect against exonuclease digestion.
  • the guide RNAs may optionally include one or more locked nucleic acid, bridged nucleic acid, or conformationally restricted nucleic acid— e.g., within the targeting region— which may stabilize binding to the target, reduce binding to non-viral targets (e.g., human DNA), or enhance specificity of endonuclease activity.
  • the Cas endonuclease can include mutations, relative to wild-type Cas9, that may enhance specificity and decrease off-target activity (e.g., by destabilizing
  • the RNAs may include modifications such as pseudouridine or 5-methyl- cytosine that may minimize an immune response by the patient.
  • the RNAs are packaged in lipid nanoparticles that include, for example, cationic lipids, which balance the charge of the phosphate backbone and promote penetration through tissue and into cells and release of RNA within the cell.
  • the lipid nanoparticles may further be provided in a topical formulation that contains a suitable gel or suspension, such as an aqueous suspension, which may include a tissue retention-enhancing or thickening agent such as, for example, hydroxyethyl cellulose or carboxymethyl cellulose.
  • the formulation may include excipients to enhance LNP stability such as, for example, sucrose or mannitol.
  • the formulation may include excipients to enhance tissue penetration such as, for example, sodium lauryl sulfate, ethanol, diethylene glycol monoethyl ether (Transcutol), propylene glycol, polyethylene glycol (PEG) esters, sucrose esters, or N-methyl pyrrolidone (NMP).
  • excipients to enhance tissue penetration such as, for example, sodium lauryl sulfate, ethanol, diethylene glycol monoethyl ether (Transcutol), propylene glycol, polyethylene glycol (PEG) esters, sucrose esters, or N-methyl pyrrolidone (NMP).
  • the formulated nanoparticles may be administered with a device to enhance RNA delivery to the basal epithelium, such as, for example, a microneedle array.
  • the composition is applied topically or locally to a site of infection such as a high-grade pre-cancerous lesion associated with an HPV infection.
  • the RNAs are released within the cells, and the mRNAs are translated by the cell's ribosomes to produce a Cas endonuclease.
  • the Cas9 endonuclease includes linker sequences and one or more nuclear localization sequences (NLS) at the N-terminus and/or C-terminus designed for optimal nuclear localization.
  • the Cas endonuclease complexes with the provided guide RNA or guide RNAs to form active RNP.
  • the RNP traffics to the nucleus and binds to the viral genome by virtue of sequence-specific interaction between the complementary portions of the guide RNA and the target within the viral genome.
  • the Cas endonuclease cleaves the viral genome.
  • Resultant viral DNA fragments may be degraded or repaired by cellular pathways, thereby clearing or disrupting the infection.
  • FIG. 1 shows an exemplary composition of the present disclosure 101 that includes an mRNA 113 encoding a Cas endonuclease and a guide RNA 121.
  • One or a plurality of nanoparticles 105 (which include a cationic lipid 107) encapsulate the mRNA 113 and the guide RNA 121.
  • the guide RNA 121 includes a targeting region 127, which is complementary to a target nucleic acid.
  • the guide RNA 121 comprises any one of SEQ ID NOs. : 1-38, 71-76, and 79-83 and the targeting region 127 is complementary to a target nucleic acid in a genome of a human papillomavirus (HPV).
  • HPV human papillomavirus
  • the guide RNA 121 comprises any one of SEQ ID NOs.: 1-30, 71-76, and 79-83, and the targeting region 127 is complementary to a target nucleic acid in a genome of a human papillomavirus (HPV).
  • the nanoparticles 105 are optionally carried by a carrier formulation 135, such as water, an aqueous solution, or a gel.
  • the carrier formulation 135 optionally includes one or more of an excipient 136 such as sodium lauryl sulfate, ethanol, diethylene glycol monoethyl ether (Transcutol), propylene glycol, polyethylene glycol (PEG) esters, sucrose esters, or N-methyl pyrrolidone.
  • the Cas9 mRNA comprises a nucleic acid as set forth in SEQ ID NOs.
  • the guide RNA comprises a nucleic acid as set forth in SEQ ID NOs.: 1, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 74, 75, and 76; and the Cas9 mRNA and guide RNA are encapsulated within a plurality of nanoparticles, wherein the plurality of nanoparticles are dispersed within a carrier liquid, oil, or gel provided by the carrier formulation.
  • the composition includes a Cas endonuclease, or a polynucleotide encoding the Cas endonuclease, with one or more substitutions within a portion of (relative to SEQ ID NO. : 63): amino acids 149-165 (aka epitope 1); amino acids 235-249 (aka epitope 2); amino acids 566-580 (aka epitope 3); amino acids 721-735 (aka epitope 4); amino acids 880-894 (aka epitope 5); amino acids 952-966 (aka epitope 6); or amino acids 1312-1326 (aka epitope 7).
  • the Cas endonuclease may include one more NLS (e.g., at either terminus) and in some embodiments, is represented by SEQ ID NO. : 62, and those same seven epitopes, with reference to SEQ ID NO. : 62 are as follows: epitope 1 includes amino acids 165-181; epitope 2 includes amino acids 251-265; epitope 3 includes amino acids 582-596; epitope 4 includes amino acids 737-751; epitope 5 includes amino acids 896-910; epitope 6 includes amino acids 968-982; and epitope 7 includes amino acids 1328-1342. With continuing reference to SEQ ID NO. : 62, those one or more substitutions may particularly be located within one or more of amino acids 168-176; amino acids 170-
  • the one or more substitutions may be, relative to SEQ ID NO.: 63, at R152, 1154, A157, F238, N240, A243, F569, K571, C574, W883, Q885, N888, V955, L1315, and N1317, and each may be a substitution to one of A, K, V, S, L, Q, R, E, T, N.
  • the substitutions include any of, and optionally, at least about five or eight of: R152K, I154V, A157S, F238L, N240Q, A243S, F569L, K571R, C574E, W883T, Q885N, N888Q, V955I, L1315I, and N1317Q.
  • compositions comprising multiple guide RNAs
  • the composition comprises more than one species of guide RNAs, and the species of guide RNAs have different polynucleotide sequences, modifications, or both.
  • different species of guide RNA molecules have different polynucleotide sequences.
  • the different species of guide RNAs bind to different targets in the same viral nucleic acid.
  • the different species of guide RNAs bind to targets in different viral nucleic acids.
  • the composition comprises more than one species of guide RNA and the first guide RNA includes: UGCAAUGUUU CAGGACCCAC GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU UU (SEQ ID NO.: 1), preferably with certain modifications.
  • the first three nucleotides at the 5' end each include a 2'-0-methyl group on the ribose ring.
  • the last three nucleotides at the 3' end each include a 2'-0-methyl group on the ribose ring.
  • the first three and the last three inter-nucleotide linkages include a phosphorothioate bond, (sequences are given using canonical RNA nomenclature using A, C, G, and U (in uppercase or lowercase) and any variations from canonical bases and/or backbone are described by the appropriate accompanying text.
  • the guide RNA 121 e.g., one of SEQ ID NOs.: 1-38, 71-76, and 79-83 further includes one or more
  • the phosphorothioate (PS) bond substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone of an oligo. This modification is preferably included to protect the inter-nucleotide linkage from nuclease degradation. Phosphorothioate bonds can be introduced between the last 3-5 nucleotides at the 5 '-end, the 3 '-end, or both of the oligo to inhibit exonuclease degradation. Including phosphorothioate bonds throughout the entire oligo will help reduce attack by endonucleases as well. Oligos with PS bonds can be ordered from, for example, Integrated DNA Technologies, Inc. (Coralville, IA).
  • Compositions of the invention may include a second guide RNA for use against HPV16.
  • the second guide RNA includes one of SEQ ID NO. : 1, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 4, 37, 74, 75, or 76, optionally along with any of those same modifications described for the first guide RNA.
  • the second guide RNA preferably also includes ten or more 2'-0-methyluridine to reduce innate immune response.
  • Compositions of the invention may include a third guide RNA.
  • the third guide RNA includes one of SEQ ID NO.: 1, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 4, or 37, optionally with any of those same modifications described for the first guide RNA and optionally for the second guide RNA.
  • the third guide RNA includes at least one bridged nucleic acid (BNA).
  • BNA bridged nucleic acid
  • the third guide RNA includes Locked Nucleic Acids or bridged nucleic acids at specific sites in DNA-binding region to increase specificity.
  • Compositions of the invention may include a fourth guide RNA for treating HPV18.
  • the fourth guide RNA includes one of SEQ ID NO. : 31, 33, and 35, and optionally includes any of the described modification, and preferably that the first three nucleotides at the 5' end each include a 2'-0-methyl group on the ribose ring. Similarly, the last three nucleotides at the 3' end each include a 2'-0-methyl group on the ribose ring. Additionally, the first three and the last three inter-nucleotide linkages include a phosphorothioate bond.
  • the fourth guide RNA may optionally include ten or more 2'-0-methyluridine.
  • the fourth guide RNA may optionally include one or more conformationally-restricted nucleotides (e.g., BNA or LNA) preferably within the targeting region.
  • the present disclosure provides a composition
  • a composition comprising: an mRNA encoding a Cas endonuclease; a guide RNA as described herein; a plurality of nanoparticles comprising a cationic lipid and encapsulating the mRNA and the guide RNA; and a carrier formulation.
  • the carrier formulation stabilizes the lipid nanoparticle and enhances topical or local delivery by promoting tissue retention and tissue penetration.
  • the guide RNA comprises any one of SEQ ID NOs.: 1-30, 71-76, and 79-83.
  • the composition comprises more than one species of guide RNAs, and the species of guide RNAs have different polynucleotide sequences, modifications, or both.
  • the composition comprises guide RNA molecules having different polynucleotide sequences, and the different species of guide RNAs bind to different targets in the same viral nucleic acid, or to targets in different viral nucleic acids.
  • the cationic lipid may comprise l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or N-[l-(2,3-dioleoyloxy)propyl]- ⁇ , ⁇ , ⁇ -trimethylammonium methyl sulfate (DOTAP).
  • DOPE dioleoyl-sn-glycero-3-phosphoethanolamine
  • DOTAP N-[l-(2,3-dioleoyloxy)propyl]- ⁇ , ⁇ , ⁇ -trimethylammonium methyl sulfate
  • the nanoparticles are PEG-ylated.
  • the plurality of nanoparticles are dispersed within the carrier formulation, and the carrier formulation comprises a carrier liquid, oil, or gel.
  • the Cas endonuclease may be a Cas9, and the Cas9 may optionally comprise between one and twenty-five amino acid substitutions relative to wild type Cas9.
  • Exemplary amino acid substitutions include R780, K810, K848, K855, H982, K1003, or R1060, as well as K848A, K1003A, and R1060A.
  • the coding sequence of the mRNA comprises a plurality of 5- methylcytidine, pseudouridine, or 5 methoxy-uridine.
  • the mRNA comprises SEQ ID NO.: 55, 56, 57, 58, 59,
  • the guide RNA comprises (a) any one of SEQ ID NOs. : 1-30, 71-76, and 79-83, or (b) a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs. : 1-30, 71-76, and 79-83; and the plurality of nanoparticles are dispersed within a carrier liquid, oil, or gel provided by the carrier formulation.
  • the mRNA comprises SEQ ID NO.: 55, 56, 57, 58, 59, 60, 67, 68, 69, 70, 77, or 78;
  • the guide RNA comprises any one of SEQ ID NOs. : 1-30, 71-76, and 79-83; and the plurality of nanoparticles are dispersed within a carrier liquid, oil, or gel provided by the carrier formulation.
  • the mRNA comprises SEQ ID NO. : 55, 56, 57,
  • the guide RNA comprises (a) any one of SEQ ID NOs. : 31-38, or (b) a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs.: 31-38; and the plurality of nanoparticle are dispersed within a carrier liquid, oil, or gel provided by the carrier formulation.
  • the mRNA comprises SEQ ID NO. : 55, 56, 57,
  • the guide RNA comprises any one of SEQ ID NOs. : 31- 38; and the plurality of nanoparticles are dispersed within a carrier liquid, oil, or gel provided by the carrier formulation.
  • the mRNA comprises SEQ ID NO. : 55, 56, 57, 58, 59, 60, 67, 68, 69, 70, 77, or 78;
  • the guide RNA comprises (a) any one of SEQ ID NOs. : 41-46 and 66, or (b) a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs.: 41-46 and 66; and the plurality of nanoparticles are dispersed within a carrier liquid, oil, or gel provided by the carrier formulation.
  • the mRNA comprises SEQ ID NO. : 55, 56, 57, 58, 59, 60, 67, 68, 69, 70, 77, or 78;
  • the guide RNA comprises any one of SEQ ID NOs. : 41- 46 and 66; and the plurality of nanoparticles are dispersed within a carrier liquid, oil, or gel provided by the carrier formulation.
  • the cationic lipid may comprise 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and N-[l-(2,3-dioleoyloxy)propyl]- ⁇ , ⁇ , ⁇ -trimethylammonium methyl sulfate (DOTAP).
  • DOPE 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine
  • DOTAP N-[l-(2,3-dioleoyloxy)propyl]- ⁇ , ⁇ , ⁇ -trimethylammonium methyl sulfate
  • the guide RNA may comprise SEQ ID NO.: 1, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 4, 37, 41, 43, 45, 74, 75, or 76, wherein a plurality of the nucleotides of the guide RNA are further substituted with nucleotides having a modified base, and each modified base is 2'-0-methylcytidine; 2'-0-methylguanosine; 2'-0- methyluridine; and 2'-0-methylpseudouridine; or 2'-0-methyladenosine.
  • the guide RNA may comprise SEQ ID NO.: 41, 43, 45, 74, 75, or 76 and wherein a plurality of the nucleotides of the guide RNA are further substituted with nucleotides having a modified base, and each modified base is 2'-0- methylcytidine; 2'-0-methylguanosine; 2'-0-methyluridine; and 2'-0-methylpseudouridine; or 2'-0-methyladenosine.
  • the guide RNA comprises (a) any one of SEQ ID NOs. : 1- 30, 71-76, and 79-83; (b) any one of SEQ ID NOs.: 31-38; or (c) any one of SEQ ID NOs.: 41-46 and 66.
  • the Cas is a Cas9, e.g., the Cas9 endonuclease comprises an amino acid sequence (a) as set forth in SEQ ID NO. : 61, 62, 63, or 84; or (b) encoded by a nucleic acid molecule as set forth in SEQ ID NO.
  • a pharmaceutical composition comprising a ribonucleoprotein and a pharmaceutically acceptable carrier is also contemplated by the present disclosure.
  • the guide RNAs and Cas mRNAs as disclosed herein are packaged in lipid nanoparticles that include, for example, cationic lipids, which balance the charge of the phosphate backbone and promote penetration through tissue and into cells and release of RNA within the cell.
  • the lipid nanoparticles may further be provided in a topical formulation that contains a suitable gel or suspension, such as an aqueous suspension, which may include a tissue retention-enhancing or thickening agent such as, for example, hydroxy ethyl cellulose or carboxymethyl cellulose.
  • the formulation may include excipients to enhance LNP stability such as, for example, sucrose or mannitol.
  • the formulation may include excipients to enhance tissue penetration such as, for example, sodium lauryl sulfate, ethanol, diethylene glycol monoethyl ether (Transcutol), propylene glycol, polyethylene glycol (PEG) esters, sucrose esters, or N-methyl pyrrolidone.
  • excipients to enhance tissue penetration such as, for example, sodium lauryl sulfate, ethanol, diethylene glycol monoethyl ether (Transcutol), propylene glycol, polyethylene glycol (PEG) esters, sucrose esters, or N-methyl pyrrolidone.
  • the formulated nanoparticles may be administered with a device to enhance RNA delivery to the basal epithelium, such as, for example, a microneedle array.
  • the compositions include a Cas endonuclease, or a polynucleotide encoding the Cas
  • Embodiments of the present disclosure also include an active RNP enveloped in a liposome, or a polynucleotide encapsulated in a lipid nanoparticle.
  • the liposomes or nanoparticles may further be provided in a suitable carrier such as a suspension or gel as described below.
  • Methods of the invention include delivering these compositions to a site of infection and preferably non-systemically.
  • compositions and methods employ a modified Cas endonuclease in which predicted T cell epitopes have been modified, relative to wild type, to attenuate an immune response that would otherwise be initiated by delivery or expression of the nuclease in the infected tissue.
  • the cationic lipid may include l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) or N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP).
  • DOPE dioleoyl-sn-glycero-3- phosphoethanolamine
  • DOTAP N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate
  • the plurality of nanoparticles are solid lipid nanoparticles dispersed within the carrier formulation, and the carrier formulation comprises a carrier liquid, oil, or gel.
  • the nanoparticles may be PEG-ylated.
  • the composition 101 includes a plurality of nanoparticles 105 comprising a cationic lipid 107.
  • the nanoparticles 105 encapsulate the mRNA 113 and the guide RNA 121. Any suitable nanoparticles may be included.
  • the nanoparticle 105 may be a solid lipid nanoparticle as shown in FIG. 1. Additionally or alternatively, liposomes may be used to deliver the mRNA 113 and the guide RNA 121 due to multiple cationic surface groups, which interact with anionic nucleic acids and form lipoplexes.
  • FIG. 4 shows an exemplary liposome 401 that may be used to encapsulate the mRNA 113 and the guide RNA 121.
  • cationic lipids are classified into three major categories based on the head group structure: monovalent lipids such as N (l-(2,3-dioleyloxy) propyl)-N,N,N- trimethylammonium chloride (DOTMA) and 1,2-dioleyl -3 -trimethylammonium -propane (DOTAP); multivalent lipids such as dioctadecylamidoglycylspermine (DOGS); and cationic lipid derivatives such as 3P-(N-(N',N'-dimethylaminoethane)-carbamoyl) cholesterol (DC- Chol).
  • monovalent lipids such as N (l-(2,3-dioleyloxy) propyl)-N,N,N- trimethylammonium chloride (DOTMA) and 1,2-dioleyl -3 -trimethylammonium -propane (DOTAP)
  • DOTMA 1,2-dioleyl -3 -
  • the myristoyl (C14) chain is optimal for transfection compared to C16 and C18 chains.
  • Longer chains increase the phase transition temperature and reduce the fluidity of the lipid membrane, which may be unfavorable for lipid membrane fusion.
  • unsaturated alkyl chains with considerably higher lipid fluidity may lead to a higher transfection efficiency compared to saturated alkyl chain lipids.
  • Cationic lipids may be used as vectors to condense and deliver anionic nucleic acids through electrostatic interactions.
  • the excess cationic coating may aid binding of vectors with negatively charged cell surfaces and the endosomal membrane to help cytoplasmic delivery of nucleic acids.
  • Electrostatic interaction between the cationic lipid head group and the backbone of nucleic acids drives encapsulation of mRNA 121 and guide RNA 113 in cationic liposomes.
  • the nanoparticles 105 are PEG- ylated.
  • LNPs may typically range in size from 50-200 nm in diameter, and preferably range in size from 60-120 nm, and may optionally include a surface coating of a neutral polymer such as PEG to minimize protein binding and unwanted uptake.
  • the nanoparticles 105 are optionally carried by a carrier 135, such as water, an aqueous solution, suspension, or a gel.
  • LNPs may be included in a formulation or preparation for topical delivery such as a suspension or gel.
  • Such as a formulation may include chemical enhancers, such as fatty acids, surfactants, esters, alcohols, polyalcohols, pyrrolidones, amines, amides, sulfoxides, terpenes, alkanes and phospholipids (to enhance topical drug penetration by perturbing the highly ordered structure of the epithelium or stratum corneum).
  • chemical enhancers such as fatty acids, surfactants, esters, alcohols, polyalcohols, pyrrolidones, amines, amides, sulfoxides, terpenes, alkanes and phospholipids (to enhance topical drug penetration by perturbing the highly ordered structure of the epithelium or stratum corneum).
  • LNP Lipid-based nanoparticles
  • liposomes and solid-lipid nanoparticles may be used.
  • Topical drug delivery of nanoparticles may provide therapeutic action directly to the targeted site, potentially reducing unwanted systemic side effects.
  • nanoparticles can be formulated for topical use in the clinic, for example, as solution/liquid formulations, dry formulations, or viscous formulations ⁇ i.e., creams, lotions, gels, ointments).
  • the medium used to suspend the nanoparticles should be biocompatible and used to facilitate percutaneous absorption.
  • the suspending medium may also alter the release kinetics of the drug from the nanoparticles. See Goyal, 2016, Nanoparticles and nanofibers for topical drug delivery, J Control Release 240:77-92, incorporated by reference.
  • the medium may aid penetration of barriers such as stratum corneum.
  • stratum corneum includes corneocytes embedded in a double-layered matrix of free sterols, free fatty acids, triglycerides, and ceramides
  • skin penetration enhancers may increase the penetration of the RNA.
  • LNPs are suspended in a buffer.
  • the buffer may include a penetration enhancing agent such as sodium lauryl sulfate (SLS).
  • SLS is an anionic surfactant that enhances penetration into the skin by increasing the fluidity of epidermal lipids. The increase in lipid fluidity below the applied site may allow SLS to diffuse optimally. SLS could thus increase intra-epidermal drug delivery without increasing transdermal delivery.
  • Lipid nanoparticles optionally may be delivered via a gel, such as a polyoxyethylene-polyoxypropylene block copolymer gel (optionally with SLS).
  • Poloxamers are nonionic triblock copolymers composed of a central hydrophobic chain of
  • polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of
  • polyoxyethylene poly(ethylene oxide)
  • poly(ethylene oxide) poly(ethylene oxide)
  • P for poloxamer
  • the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core
  • Lipid nanoparticles may be freeze-dried ⁇ e.g., using dextrose (5% w/v) as a lyoprotectant).
  • L Ps may be held in an aqueous suspension or in an emulsification, e.g., with lecithin.
  • a method of treating a viral infection in a subject comprising administering to the subject a therapeutically effective amount of a guide RNA, DNA, vector, or Cas9-guide RNA composti on.
  • the present disclosure provides a method of treating a Human Papillomavirus (HPV) infection.
  • HPV Human Papillomavirus
  • Some HPV types can cause precancerous lesions, which are abnormal growths that can turn into cancer, or cancer.
  • Certain HPV types infect genital and other areas, including inside and outside the vagina, the penis, the anus or some areas of the head and neck.
  • "High-risk" HPVs are types of HPV that are more likely to cause cancer. Such an infection transforms normal cells into precancerous lesions or cancer.
  • Cancers associated with HPV include cervical cancer, oral cancer, anal cancer, vulvar cancer, vaginal cancer, and penile cancer.
  • Cervical cancer from HPV may manifest as a precancerous cervical lesion.
  • a precancerous cervical lesion which is also called an intraepithelial lesion, is an abnormality in the cells of the cervix that could eventually develop into cervical cancer.
  • cervical cells There are two main types of cervical cells, squamous and glandular, and abnormalities can occur in either type.
  • the most common types of precancerous cervical lesions include: atypical squamous cells (abnormalities in the squamous cells of the cervix); squamous intraepithelial lesion
  • the composition is applied topically or locally to a site of infection such as a high-grade pre-cancerous lesion associated with an HPV infection.
  • the RNAs are released within the cells, and the mRNAs are translated by the cell's ribosomes to produce a Cas endonuclease.
  • the Cas9 endonuclease includes linker sequences and one or more nuclear localization sequences ( LS) at the N-terminus and/or C-terminus designed for optimal nuclear localization.
  • the Cas endonuclease complexes with the provided guide RNA or guide RNAs to form active ribonucleoprotein (RNP).
  • the RNP traffics to the nucleus and binds to the viral genome by virtue of sequence-specific interaction between the complementary portions of the guide RNA and the target within the viral genome.
  • the Cas endonuclease cleaves the viral genome.
  • Resultant viral DNA fragments may be degraded or repaired by cellular pathways, thereby clearing or disrupting the infection.
  • FIG. 5 diagrams a method 501 of treating an HPV infection.
  • the method 501 includes providing 505 a composition 101 according to the embodiments described herein.
  • An effective amount of the composition 101 is administered 509 at a site of infection in a patient in need thereof an effective amount of a composition.
  • the composition 101 includes an mRNA 113 encoding a Cas endonuclease and a guide RNA 121 comprising one selected from the list consisting of SEQ ID NOs. : 1-38, 71-76, or 79-83.
  • a plurality of nanoparticles 105 encapsulate the mRNA and the guide RNA.
  • the composition 101 is administered 509 parenterally or topically, and systemic circulation is avoided.
  • the composition 101 may be applied directly to a surface of, or injected into, the site of infection, by such means, the method 501 may be used to treat a site of infection such as a squamous cell carcinoma lesion, e.g., a high-grade, pre-cancerous HPV lesion.
  • the method 501 may be used to prevent the onset of a cancer, such as cervical, anal, oral, penile, or vaginal cancer.
  • the method is used to prevent cervical cancer.
  • the guide RNA 121 comprising one selected from the list consisting of SEQ ID NOs. : 1-38, 71-76, or 79-83 preferably includes a targeting region 127 substantially complementary to a region in an HPV genome.
  • the E6 and/or E7 genes of HPV are targeted.
  • a method of treating an HPV infection in a subject comprising administering a therapeutically effective amount of a guide RNA comprising SEQ ID NO.80 and an mRNA encoding a Cas9 endonuclease.
  • FIG. 6 is a map of HPV E6 and E7 genes on the HPV gene.
  • E7 genes may be used as targets using programmable nucleases in antiviral treatments. Since E6 and E7 proteins may be oncogenic it may be valuable to target their respective genes for destructions by the nuclease.
  • each gene is scanned for the protospacer adjacent motif (PAM) of the nuclease (e.g., 5'-NGG-3' for Cas9).
  • PAM protospacer adjacent motif
  • the match criteria may be the requirement of no perfect match.
  • the targeting sequence is 20-nt + PAM (e.g., 23-nt for Cas9) for which there is no 23 nt string within a human genome that matches > 70%.
  • the targeting sequence is 20-nt + PAM for which there is no 20 nt string within the human genome that is followed by the PAM and wherein the 20 nt of human genome matches the 20 nt of targeting sequence by > 70% (e.g., if Cas9 is the nuclease, a 20 nt string of human genome with 14 or more matching bases followed by the PAM would rule out use of a given targeting sequence).
  • aspects of the invention provide a method of making a medicament for the treatment of a viral infection.
  • FIG. 10 diagrams methods 1001 of making a medicament (e.g., the composition 101) for treatment of an HPV infection.
  • the method 1001 includes preparing 1005 an mRNA encoding a Cas endonuclease; preparing 1009 one or more guide RNA comprising one selected from the list consisting of SEQ ID NOs. : 1-38, 71-76, or 79-83; and encapsulating 1013 the mRNA and the guide RNA in a plurality of nanoparticles comprising a cationic lipid.
  • the nanoparticles may be introduced 1019 into a pharmaceutically acceptable carrier, e.g., a gel or suspension such as an aqueous suspension.
  • the guide RNA comprises one selected from one of SEQ ID NOs. : 1, 5, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 4, and 37, optionally with one or a plurality of modifications.
  • the guide RNA is preferably synthesized by solid-phase synthesis. Solid-phase synthesis is carried out on a solid support that may be held between filters, in columns that enable all reagents and solvents to pass through freely. With solid- phase synthesis, a large excesses of solution-phase reagents can be used to drive reactions quickly to completion. Impurities and excess reagents are washed away and no purification is required. The process may be automated and is amenable to automation on computer- controlled solid-phase synthesizers.
  • Solid supports are the insoluble particles, typically 50-200 ⁇ in diameter, to which the oligonucleotide is bound. Suitable supports include controlled pore glass and polystyrene. Solid supports are typically manufactured with a loading of 20-30 ⁇ of nucleoside per gram of resin. Any suitable method may be used including, for example, the H-phosphonate and phosphotriester methods, and Khorana's phosphodiester approach. In some embodiments, the phosphoramidite method using solid-phase technology and automation is used.
  • Phosphoramidite oligo synthesis proceeds in the 3'- to 5 '-direction with one nucleotide is added per synthesis cycle.
  • Building blocks used for synthesis are commonly referred to as "monomers", which are activated RNA nucleosides (phosphoramidites).
  • the dimethoxytrityl (DMT) group is used to protect the 5 '-end of the nucleoside, a ⁇ -cyanoethyl group protects the 3 '-phosphite moiety, and may also include additional groups that serve to protect reactive primary amines in the heterocyclic nucleo bases.
  • the protecting groups are selected to prevent branching or other undesirable side reactions from occurring during synthesis.
  • Oligonucleotides are synthesized on solid supports.
  • the support is a small column filled with control pore glass (CPG), polystyrene or a membrane.
  • CPG control pore glass
  • the oligonucleotide is usually synthesized from the 3 'to the 5'. The synthesis begins with the addition of a reaction column loaded with the initial support-bound protected nucleotide into the column holder of the synthesizer.
  • the first nucleotide building block or monomer is usually anchored to a long chain alkylamine-controlled pore glass (LCAA-CPG).
  • LCAA-CPG long chain alkylamine-controlled pore glass
  • the phosphoramidite approach to oligonucleotide synthesis proceeds in four steps on solid support, usually controlled pore glass (CPG) or polystyrene.
  • Synthesis is initiated with cleavage of the 5 '-trityl group by brief treatment with dichloroacetic acid (DC A) dissolved in dichloromethane (DCM).
  • DC A dichloroacetic acid
  • DCM dichloromethane
  • the monomer activated with tetrazole is coupled to the available 5'-hydroxyl resulting in a phosphite linkage.
  • phosphite oxidation by treatment with iodine using a THF/pyridine /H20 solution yields a phosphate backbone.
  • the capping step with acetic anhydride which terminates undesired failure sequences, completes the cycle of oligonucleotide synthesis. See McBride, 1983, An investigation of several deoxynucleoside phosphoramidites useful for synthesizing deoxyoligonucleotides.
  • the mRNA encoding a Cas endonuclease is prepared by synthesizing the mRNA. Any suitable synthesis method may be used.
  • the mRNA is made by in vitro transcription. In vitro transcription uses a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium ions, and an appropriate phage RNA polymerase.
  • the DNA template preferably includes a double-stranded promoter for binding of the phage polymerase.
  • the template may include plasmid constructs engineered by cloning, cDNA templates generated by first- and second-strand synthesis from an RNA precursor, or linear templates generated by PCR or by annealing chemically synthesized oligonucleotides.
  • the template may be an (e.g., linearized) plasmid.
  • Many plasmids include phage polymerase promoters. Any suitable promoter may be used, e.g., the promoter for any of three common polymerases, SP6, T7 or T3, may be used.
  • TriLink a linearized plasmid template is for "Run-off Transcription," which transcription stops when RNA polymerase falls off the DNA.
  • the plasmid encodes approximate Poly(A)80 tail.
  • the process co-transcriptionally adds methylated 5' cap (“Capl”).
  • This process is offered under the proprietary name CleanCap, by TriLink.
  • the process can use normal or modified NTPs (5mC, 5-methoxy -Uridine (5moU)) in any ratio.
  • mRNA is phosphatase treated to remove any 5' triphosphates from uncapped mRNAs. HPLC purifies final mRNA product.
  • PCR primers are used to amplify double stranded DNA template from only the mRNA-encoding region of a plasmid.
  • One PCR primer contains the Poly(A)120 tail, to prevent issues of Poly(A) tail loss in plasmid.
  • This process can use normal or modified NTPs (5mC, ⁇ ) in any ratio, and can do capping co- transcriptionally, or post-transcriptionally and enzymatically.
  • mRNA is phosphatase treated to remove any 5' tri-phosphates from uncapped mRNAs.
  • the mRNA is purified via spin columns to remove dsRNAs. This process is offered by Amp-Tec.
  • plasmid vectors for transcription templates may be linearized by restriction enzyme digestion. Because transcription proceeds to the end of the DNA template, linearization ensures that RNA transcripts of a defined length and sequence are generated. PCR products can also function as templates for transcription. A promoter can be added to the PCR product by including the promoter sequence at the 5' end of either the forward or reverse PCR primer.
  • the template DNA is then transcribed by a T7, T3 or SP6 RNA phage polymerase in the presence of ribonucleoside triphosphates (rNTPs). The polymerase traverses the template strand and uses base pairing with the DNA to synthesize a
  • RNA polymerase travels from the 3 ' ⁇ 5' end of the DNA template strand, to produce an RNA molecule in the 5' ⁇ 3 ' direction. See Jani, 2012, In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection, J Vis Exp 61 :3702, incorporated by reference.
  • Encapsulating the mRNA and the guide RNA in a plurality of nanoparticles comprising a cationic lipid may proceed by any suitable method.
  • Methods for preparation may include direct mixing between cationic liposomes and mRNA in solution, or rehydration of a thin-layer lipid membrane with mRNA in solution.
  • the dispersion of cationic lipid/mRNA complexes in the aqueous solution often results in heterogeneous complexes, sometimes still referred to as cationic liposomes, but more accurately called lipoplexes.
  • Lipoplexes can encapsulate nucleic acid cargos up to 90% of the input dose. See Wang, 2015, Delivery of oligonucleotides with lipid nanoparticles, Adv Drug Deliv Rev 87:68-80, incorporated by reference.
  • modified mRNA e.g., prepared with a T7 polymerase- based IVT kit with a yield of - 60 ⁇ g/reaction
  • DOTAP l,2-dioleoyl-3-trimethylammonium-propane
  • cholesterol (1 : 1 molar ratio
  • Methods for preparation may include direct mixing between cationic liposomes and RNA in solution, or rehydration of a thin-layer lipid membrane with RNA in solution.
  • the dispersion of cationic lipid/RNA complexes in the aqueous solution may result in heterogeneous complexes, sometimes still referred to as cationic liposomes, aka lipoplexes.
  • Lipoplexes can encapsulate nucleic acid cargos up to 90% of the input dose. See Wang, 2015, Delivery of oligonucleotides with lipid nanoparticles, Adv Drug Deliv Rev 87:68-80, incorporated by reference.
  • modified mRNA e.g., prepared with a T7 polymerase- based IVT kit with a yield of - 60 ⁇ g/reaction
  • DOTAP l,2-dioleoyl-3-trimethylammonium-propane
  • cholesterol (1 : 1 molar ratio
  • the electrostatic interaction promotes the self-assembly by inducing lipid bilayers to collapse on the core structure, resulting in spherical, solid, liposomal nanoparticles with a core/membrane structure.
  • the nanoparticle further comprises N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP).
  • DOTAP N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate
  • the nanoparticles may include l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).
  • HPLC-purified 1-methylpseudouridine-containing mRNA may be encapsulated in LNPs using a self-assembly process.
  • LNPs are prepared using ionizable lipid L319, distearoylphosphatidylcholine (DSPC), cholesterol and PEG- DMG at a molar ratio of 55 : 10:32.5 :2.5 (L319:DSPC:cholesterol:PEG-DMG).
  • the mRNA is introduced at a lipid nitrogen to siRNA phosphate ratio of 3, corresponding to a total lipid to mRNA weight ratio of -10: 1.
  • a spontaneous vesicle formation process is used to prepare the LNPs.
  • mRNA is diluted to -1 mg/ml in 10 mmol/1 citrate buffer, pH 4.
  • the lipids are solubilized and mixed in the appropriate ratios in ethanol.
  • Syringe pumps are used to deliver the mRNA solution and lipid solution at 15 and 5 ml/min, respectively.
  • the syringes containing mRNA solution and lipid solution are connected to a union connector (0.05 in thru hole, #P-728; IDEX Health & Science, Oak Harbor, WA) with PEEK high-performance liquid chromatography tubing (0.02 in ID for siRNA solution and 0.01 in ID for lipid solution).
  • a length of PEEK high-performance liquid chromatography tubing (0.04 in ID) is connected to the outlet of the union connector and led to a collection tube.
  • LNPs are filtered through a 0.2 ⁇ sterile filter.
  • L Ps preferably contain an ionizable cationic lipid/phosphatidylcholine/cholesterol/PEG-lipid (50: 10:38.5: 1.5 mol/mol), encapsulated RNA-to-total lipid ratio of -0.05 (wt/wt) and a diameter of -80 nm.
  • mRNA-L P formulations may be stored at -80° C at a concentration of mRNA of— 1 ⁇ g/ ⁇ l.
  • mRNA-L P formulations may be stored at -80° C at a concentration of mRNA of— 1 ⁇ g/ ⁇ l.
  • FIG. 11 shows a strategy for verifying specificity of the RNP.
  • the CRISPR components are electroporated into target cells along with double-stranded oligo- deoxynucleoside (dsODN).
  • dsODN double-stranded oligo- deoxynucleoside
  • the cells are incubated with the RNP and breaks introduced by Cas9 capture the dsODN.
  • genomic DNA gDNA
  • sample prep includes adding adapters to create a sequencing library. Sequencing is used to identify the captured dsODN.
  • Off-target activity may be assessed by the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GU DE-seq) method.
  • GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR- Cas nucleases. Nat Biotechnol 33 : 187-197, incorporated by reference.
  • Those GUIDE-seq methods of Tsai, 2015, were used to determine off target effects of the composition 101. But such means, the compositions 101 may be shown to have acceptable low off-target effects and high on-target specificity.
  • the disclosed compositions and particularly mRNAs encapsulated in lipid nanoparticles exhibit good penetration into and release within target tissues and cells, where release of the mRNA allows expression through, e.g., translation into active protein.
  • Epitope analysis was conducted for a wild-type Streptococcus pyogenes Cas9 amino acid sequence, SEQ ID NO.: 63.
  • Multiple in silico prediction tools were used to identify potential MHC II-restricted CD4 T cell epitopes for HLA-DRB1 for mutagenesis, including: SYFPETHI, IEDB (analysis completed with default settings, Consensus,
  • NetMHCpan NetMHCpan, NN_align, SMM_allign, Combinatorial library, and Sturniolo
  • RA KPEP ProPred
  • MULTIPRED2 MHCIIPred
  • MHC2SKpan NetMHCII
  • epitope 1 (includes amino acids 149-165); epitope 2 (includes amino acids 235-249); epitope 3 (includes amino acids 566-580); epitope 4 (includes amino acids 721-735); epitope 5 (includes 880-894; epitope 6 includes amino acids 952-966); and epitope 7 includes amino acids (1312-1326).
  • epitope 1 includes amino acids 165-181; epitope 2 includes amino acids 251-265; epitope 3 includes amino acids 582-596; epitope 4 includes amino acids 737-751; epitope 5 includes amino acids 896-910; epitope 6 includes amino acids 968-982; and epitope 7 includes amino acids 1328-1342.
  • each epitope may have sub- sequences as follows (with reference to SEQ ID NO. : 62): in epitope 1, of particular interest may be sub-region la (amino acids 168-176) or sub-region lb (amino acids 170-178); in epitope 2, sub-region 2a is amino acids 255-263; in epitope 3, sub-region 3a is amino acids 586-594; in epitope 4, sub-region 4a includes amino acids 741-749; in epitope 5, sub-region 5a includes amino acids 900-908 ; in epitope 6, sub-region 6a includes amino acids 972-980; in epitope 7, sub-region 7a includes amino acids 1332-1340.
  • the aforementioned epitope and sub-regions of Cas9 are of particular interest because they were identified as potential epitopes that may be highly-immunogenic.
  • each epitope is examined with respect to apo- Cas9 3D crystal structure data, the sgRNA-bound Cas9 structure, and the sgRNA-Cas9- dsDNA structure to determine the importance of the bold critical-residues in each epitope. For example, it may be found that most residues can be mutated to alanine to reduce the charge issues on the epitopes without likely impacting structure or function. It may also be found that residues that are alanine can be conservatively mutated to serine. EXAMPLE 2
  • a genetically encoded mRNA DNA was provided for transcription by a T7 phage polymerase, while guide RNA was synthesized by phosphoramidite synthesis.
  • FIG. 8 shows results of an in vitro assay showing that delivery (by electroporation) of Cas9 (SEQ ID NO. : 54) and guide RNA (SEQ ID NO.: 2) reduces copy number of HPV genome in SiHa cells.
  • A Viral DNA levels were lower in cells provided an HPV16-targeting guide RNA compared to cells provided with a non-specific guide RNA.
  • B Cell death was greater in HPV16+ SiHa cells provided an HPV16-targeting guide RNA compared to HPV16+ cells provided with non-specific guide RNA (left). Cell death was much lower in HPV16 negative 293 cells, regardless of whether an HPV16-targeting guide RNA and or a non-specific guide RNA was provided (right).
  • FIG. 9 demonstrates that CRISPR/Cas9 may be used to kill HPV16+ SiHa cancer cells in vitro. Cytoxicity was greater for cells electroporated with Cas9 mRNA (SEQ ID NO. : 54) and HPV16-targeting guide RNAs (SEQ ID NO. : 2), compared to cells receiving non-specific guide RNA. Cell killing tended to increase in days following treatment (left) and with increasing amounts of Cas9 mRNA (right).
  • FIG. 12 shows the detected specificity of guide RNAs in targeting portions of the HPV16 genome (SEQ ID NOs.: 64 and 65), at different doses by GUIDE- Seq as described in Example 5.
  • SEQ ID NO.: 64 or SEQ ID NO.: 65 is targeted, the specificity is 99.9% to 100%.
  • FIG. 13 show the results of measurement of on-target (left) and off-target (right) DNA cleavage efficiency of a Cas endonuclease for HPV16-targeting guide RNAs 1.1.1 (SEQ ID NO.: 2) (circles); 1.1.3 (SEQ ID NO. : 4) (squares), and E6-1 BNA/LNA (SEQ ID NO.: 83) (diamonds) using a cell-free DNA cleavage assay.
  • Cas9 protein and gRNAs are pre-incubated to form a ribonucleoprotein complex.
  • Double stranded DNA containing the gRNA target sequence is then added, and the CRISPR complex was allowed to cleave the DNA for a short period of time before the reaction was quenched.
  • the cleaved and uncleaved DNA products are separated by capillary gel electrophoresis and are quantified.
  • BNA/LNA modified nucleotides in the protospacer reduce activity toward off-target DNA while maintaining activity toward on-target DNA.
  • FIG. 14 shows the in-cell DNA cleavage specificity by GUIDE-Seq for UPV- targeting guide RNAs 1.1.1 (SEQ ID NO.: 2) (A), 1.1.3 (SEQ ID NO.: 4) (B), and E6-1 BNA/LNA (SEQ ID NO. : 83) (C).
  • the results show that including at least one
  • conformationally -restricted nucleotide such as a locked nucleic acid or a bridged nucleic acid residue can significantly improve in vivo cleavage specificity.
  • FIG. 15 shows successful tissue penetration of lipid nanoparticles containing mRNA encoding green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • GFP indicates active, expressed protein translated from LNP-delivered mRNA.
  • the arrows indicate regions where greater fluorescence is evident for LNP 1 A and LNP 1 B than for PBS or Turbofect. The results show that LNP 1 A and LNP 1 B result in extensive distribution of the expressed protein in cervical epithelium.
  • FIG. 7 demonstrates successful incorporation of the dsODN.
  • the upper panel is a schematic of dsODN (open bars) incorporation into genomic DNA (hatched bars).
  • the lower panel shows two distinct PCR reactions and their corresponding gel electrophoresis images demonstrating incorporation of the 35bp dsODN into SiHa genomic DNA.
  • genomic DNA was isolated and sheared by ultrasonification, and adapters were then ligated to sheared DNA.
  • dsODN-specific amplification and sequencing was performed on a Mi Seq next generation sequencing machine. Processing of sequences was performed as described at https://github.com/aryeelab/guideseq. Specificity was calculated as the percentage of on- target "reads" (i.e., by dividing the number of on-target reads by the total number of reads, xlOO).
  • target sequence cleavage was highly specific, showing no off-target effects, and on-target cleavage at a rate of more than 99% when the Cas9 mRNA corresponding to SEQ ID NO. : 68 was used with gRNA (SEQ ID NO. : 6) (upper table) in HPV16+ SiHa cells.
  • gRNA SEQ ID NO. : 6
  • No off-target reads were detected in 2 HPV16 negative cell lines (C33a, 293 T) using the same RNAs.
  • the on-target cleavage rate was even higher when the high-fidelity Cas9 mRNA sequences were delivered (lower table) with the same gRNA (SEQ ID NO.: 6) (100% were on target).
  • Conformationally restricted nucleotides (CRN)-modified nucleotides have a reduced tolerance for making non-Watson Crick base pairs compared to unmodified nucleotides. CRN modifications were therefore added to the targeting region of gRNAs and tested to confirm if they retained high on-target activity compared to unmodified nucleotides as well as increased specificity at predicted off-target DNA sites.
  • the effectiveness of a CRN-modified guide RNA in targeting HPV16 was assessed using a cell-free cleavage assay.
  • a 2kb double stranded DNA containing either the E7 gene target or a known potential off-target site for HPV16 guide 1.2 was incubated with a pre-formed ribonucleoprotein consisting of Cas9 protein (SEQ ID NO.: 61) and a guide RNA that was either unmodified (SEQ ID NO. : 6) or contained either BNA or LNA modifications in the protospacer at specifically selected positions (SEQ ID NO.: 79).
  • the guide RNA used as a control was a guide RNA that targets the EBV genome (SEQ ID NO. : 47).
  • the amount of target DNA cleaved was determined by separating the cleaved and uncleaved DNA production via capillary gel electrophoresis. As shown in FIG. 24A, when RNP is formed with BNA or LNA modified guide RNAs, activity toward Off-target DNA is reduced relative to RNP formed with unmodified guide RNA.
  • RNAs were delivered to HPV16+ SiHa cells via electroporation (nucleofection). Cells were incubated for 24 hours at 37 °C with 5% C0 2 . Total genomic DNA was then harvested and specific probes were used to quantitatively amplify the E7 gene target and the housekeeping gene, PPIA. Each sample was normalized to the amount of PPIA. As shown in FIG. 24B, CRN-modified guide RNAs function nearly as efficiently as unmodified guide RNAs against HPV16 E7 in cells, demonstrating that reduced off-target activity is not merely due to reduced overall activity.
  • RNAs were delivered to HPV16+ SiHa cells via electroporation (nucleofection). Cells were incubated for 8 days at 37 °C with 5% C0 2 after which cells were lifted and counted by flow cytometry. The relative fraction of viable cells on day 8 is shown in FIG. 24C, normalized to cells nucleofected with buffer only. CRN- modified guide RNAs function nearly as efficiently as unmodified guide RNAs in a SiHa killing assay.

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Abstract

La présente invention concerne des ARN guides destinés à être utilisés dans le clivage d'acides nucléiques viraux cibles. La présente invention concerne en outre des compositions qui comprennent un ARNm codant pour une endonucléase Cas et un ARN guide ayant des séquences et des modifications particulières, les deux ARN étant encapsulés par des nanoparticules, pour le traitement d'une infection par le VPH. Dans certains modes de réalisation, les ARN guides et le ARNm codant pour une endonucléase Cas sont conditionnés dans une nanoparticule lipidique, qui peut être mise en suspension dans une formulation de vecteurs pour une administration topique ou locale à un tissu infecté. Dans certains modes de réalisation, l'ARN guide et/ou le ARNm codant pour l'endonucléase Cas comprennent également des éléments tels que des nucléotides modifiés qui favorisent l'administration des ARN, et la rétention en leur sein, à des cellules infectées.
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