Methods and compositions for the treatment of androgenic alopecia

Abstract

Provided herein are methods and compositions for treating androgenic alopecia in a subject in need thereof. The method includes administering a therapeutically effective amount of a pharmaceutical composition to a hair follicle of the subject, wherein the pharmaceutical composition comprises a lipid nanoparticle encapsulating a gene-editing cassette and wherein the gene-editing cassette inactivates an androgen receptor in the hair follicle.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF ANDROGENIC ALOPECIACROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 638,114, filed April 24, 2024, the entire contents of which are incorporated herein by reference.SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The Sequence Listing XML file, created on April 24, 2025, is named FAN0007WO.xml and is 350,444 bytes in size.TECHNICAL FIELD

[0003] The present disclosure relates generally to the field of medical therapeutics and genome editing, specifically compositions and methods for editing and / or modifying expression of the androgen receptor for the treatment of androgenic alopecia.BACKGROUND

[0004] Androgenic alopecia, also known as pattern hair loss, is a common form of hair loss in both men and women. It is characterized by a receding hairline and / or balding on the scalp. The role of androgen receptors in androgenic alopecia is central to the condition's development and progression.

[0005] Individuals who do not suffer from androgenic alopecia have what are commonly referred to as androgen- independent hair follicles. In individuals with androgenic alopecia, hair follicles are sensitive to androgens, specifically DHT. This sensitivity is due to the presence of androgen receptors in the hair follicles. The androgen receptor is designed to bind to androgens, which are hormones such as testosterone and dihydrotestosterone (DHT). DHT is a more potentandrogen that plays a significant role in androgenic alopecia. When DHT binds to these receptors, it triggers a series of cellular events that lead to the miniaturization of the hair follicle. Overtime, miniaturization can lead to follicles that produce no hair.

[0006] Conventional treatments for androgenic alopecia aim to reduce the effects of DHT on hair follicles. For example, finasteride is a medication that inhibits the enzyme responsible for converting testosterone to DHT, thereby reducing DHT levels and its impact on hair follicles. However, finasteride causes a number of side effects, ranging from decreased libido to increased risk of cancer. Another approach is the use of topical treatments like minoxidil, which does not directly target DHT but promotes hair growth by widening blood vessels and opening potassium channels. However, minoxidil also comes with side effects, such as unwanted facial hair growth, hand and foot swelling, and unexplained weight gain. Other conventional treatments, such as hair transplants and anti-androgens, come with a host of side effects and complications.

[0007] Hair follicles are complex structures that go through extensive changes based on the growth phase the follicle is in. Topically applied substances are unable to enter a follicle that is not in an appropriate growth phase. Thus, as androgenic alopecia progresses, conventional topical treatments become less effective.

[0008] Accordingly, a need exists to administer treatments directly to the hair follicle to target hair follicles in various growth stages, and to limit treatments to the targeted site.SUMMARY

[0009] The instant disclosure is related to methods of treating androgenic alopecia in a subject in need thereof.

[0010] In an embodiment, the present disclosure is related to methods and compositions for treating androgenic alopecia in a subject in need thereof. The method includes administering a therapeutically effective amount of a pharmaceutical composition to a hair follicle of the subject, wherein the pharmaceutical composition comprises a lipid nanoparticle encapsulating a geneediting cassette and wherein the gene-editing cassette inactivates an androgen receptor in the hair follicle.DETAILED DESCRIPTION

[0011] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document.

[0012] Aspects of the present disclosure relate to the treatment of androgenic alopecia in a subject in need thereof by administering a therapeutically effective amount of a pharmaceutical composition to a hair follicle of the subject. Optionally, the pharmaceutical composition comprises a lipid nanoparticle encapsulating a gene-editing cassette. In some aspects, the gene-editing cassette inhibits an androgen receptor in the hair follicle. Optionally, the pharmaceutical composition is administered by intrafollicular injection.

[0013] While the following terms are believed to be well understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the presently-disclosed subject matter. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently- disclosed subject matter belongs. The following references provide a general definition of many of the terms used in this invention: Singleton et al. , Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991), the contents of each of which are incorporated by reference herein.

[0014] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0015] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of insome embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0016] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0017] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith or obtaining a desired pharmacologic and / or physiologic effect. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, diminishes, abrogates, abates, alleviates, decreases the intensity of, or cures a disease and / or adverse symptom attributable to the disease. In some embodiments, the effect is preventative, i.e., the effect protects or prevents an occurrence or reoccurrence of a disease or condition. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of a composition as described herein.

[0018] An “effective amount,” as used herein, refers to an amount of a substance (e.g., a therapeutic compound and / or composition) that elicits a desired biological response. In some embodiments, an effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay and / or alleviate one or more symptoms of the disease, disorder, and / or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of a formulation to treat a disease, disorder, and / or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of; reduces severity of and / or reduces incidence of one or more symptoms or features of the disease, disorder, and / or condition. Furthermore, an effectiveamount may be administered via a single dose or via multiple doses within a treatment regimen. In some embodiments, individual doses or compositions are considered to contain an effective amount when they contain an amount effective as a dose in the context of a treatment regimen. Those of ordinary skill in the art will appreciate that a dose or amount may be considered to be effective if it is or has been demonstrated to show statistically significant effectiveness when administered to a population of patients; a particular result need not be achieved in a particular individual patient in order for an amount to be considered to be effective as described herein.

[0019] The term “administering,” as used herein, refers to any route of administering an effective amount of a therapeutic agent, such as one of the compositions described herein. In embodiments, the administering includes, but is not limited to, intrafollicular administration, intravenous administration, topical administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, oral administration, intracerebral administration, intraspinal administration, intrathecal administration, subarachnoid administration, epidural administration, periocular administration, intraocular administration, and the like. In a specific embodiment, the pharmaceutical composition is administered intrafollicularly.

[0020] As used herein, the term “alteration” or “modification” refers to a change in the expression level, structure, or activity of an analyte, gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a change (e.g., increase or decrease) in expression levels. In embodiments, the increase or decrease in expression levels is by 10%, 25%, 40%, 50% or greater. In some embodiments, an alteration includes an insertion, deletion, or substitution of a nucleobase or amino acid (by, e.g., genetic engineering).

[0021] As used herein, “analog” and grammatical equivalents thereof refers to a molecule that is not identical but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog’s function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog’s protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

[0022] As used herein, a “gene-editing cassete” refers to an intermolecular complex for editing a nucleobase of a target nucleotide sequence. In some embodiments, a gene-editing cassette refers to an agent that binds a polynucleotide and has nucleobase modifying activity. In some embodiments, the gene-editing cassette includes a nucleotide binding domain (e.g., Cas9 or Cpfl) in conjunction with a guide polynucleotide (e.g., guide RNA (gRNA). In some embodiments, the gene-editing cassette includes a CRISPR-Cas9 system. The CRISPR-Cas9 system may include at least a guide RNA (gRNA) and a Cas9 nuclease.

[0023] In some embodiments, the gene-editing cassette is introduced to modify a nucleotide and designed to introduce genetic changes in a subject using gene-editing technologies, described in greater detail herein. Optionally, the gene-editing cassette employs a CRISPR-Cas9 system to modify the genome of a subject. In some embodiments, the gene-editing cassette is administered to a target site to modify the genome at the target site. Optionally, the target site is a hair follicle.Nucleotide Binding Domain

[0024] As used herein, a “nucleotide binding domain” refers to a protein that associates with a guide polynucleotide that guides the nucleotide binding domain to a target nucleic acid sequence. In some embodiments, the nucleotide binding domain is a Cas9 protein. In some embodiments, the nucleotide binding domain includes all or a portion (e.g., a functional portion) of Cas9 from Corynebacterium ulcerans (NCBI Refs: NC 015683.1, NC 017317.1); Corynebacterium diphtheria (NCBI Refs: NC 016782.1 , NC 016786.1 ); Spiroplasma syrphidicola (NCBI Ref: NC 021284.1 ); Prevotella intermedia (NCBI Ref: NC 017861.1); Spiroplasma taiwanense (NCBI Ref: NC 021846.1); Streptococcus iniae (NCBI Ref: NC 021314.1); Belliella baltica (NCBI Ref: NC 018010.1); Psychroflexus torquis (NCBI Ref: NC 018721.1); Streptococcus thermophilus (NCBI Ref: YP 820832.1); Listeria innocua (NCBI Ref: NP 472073.1); Campylobacter jejuni (NCBI Ref: YP 002344900.1); Neisseria meningitidis (NCBI Ref: YP 002342100.1), Streptococcus pyogenes, or Staphylococcus aureus.

[0025] As used herein, the term “Cas9” or “Cas9 domain” refers to an RNA guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g, a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and / or the gRNA binding domain of Cas9). A Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (clusteredregularly interspaced short palindromic repeat) associated nuclease. In some embodiments, a Cas9 protein may associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif), described in greater detail herein.

[0026] It will be appreciated that Cas9 nuclease sequences and structures are known in the art (See, e.g., “Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et al., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by transencoded small RNA and host factor RNase III.” Deltcheva E., et al. , Nature 471 :602- 607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., et al., Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0027] Exemplary, non-limiting examples of nucleic acid programmable DNA binding proteins include Cas9 (e.g, dCas9 and nCas9), Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Cas 12 g, Casl2h, Casl2i, and Casl2j / CasD (Casl2j / Casphi). Nonlimiting examples of Cas enzymes include Casl, Cas IB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, CasSa, Cas8b, Cas8c, Cas9 (also known as Csnl or Csxl2), CaslO, CaslOd, Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, Casl2i, Casl2j / Cas<D, Cpfl, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csxl 1, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARE, DinG, homologues thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, although they may not be specifically listed in this disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct; 1 :325-336. doi:10.1089 / crispr.2018.0033; Van et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan 4;363(6422):88-91. doi: 10.1126 / science.aav7271, the entire contents of each are hereby incorporated by reference.

[0028] Generally, the Cas9 nuclease (SEQ ID NO: 395) has two functional endonuclease domains: RuvC and HNH. Cas9 undergoes a conformational change upon target binding that positions the nuclease domains to cleave opposite strands of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA. In some embodiments, the gene-editing cassette includes a Cas9 protein having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 395 Exemplary Cas-9 proteins suitable for use in the gene editing cassettes of the present disclosure may also be found US Patent Application Serial No. 18 / 699,800, the entire contents of which are incorporated herein by reference.

[0029] In some embodiments, the polynucleotide programmable nucleotide binding domain comprises a nickase domain. Herein the term “nickase” refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that is capable of cleaving only one strand of the two strands in a duplexed nucleic acid molecule (e.g., DNA). In some embodiments, a nickase can be derived from a fully catalytically active (e.g., natural) form of a polynucleotide programmable nucleotide binding domain by introducing one or more mutations into the active polynucleotide programmable nucleotide binding domain. For example, where a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can include a D10A mutation and a histidine at position 840. In such embodiments, the residue H840 retains catalytic activity and can thereby cleave a single strand of the nucleic acid duplex. In another example, a Cas9-derived nickase domain comprises an H840A mutation, while the amino acid residue at position 10 remains a D. In some embodiments, a nickase can be derived from a fully catalytically active (e.g, natural) form of a polynucleotide programmable nucleotide binding domain by removing all or a portion (e.g., a functional portion) of a nuclease domain that is not required for the nickase activity. For example, where a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can comprise a deletion of all or a portion (e.g., a functional portion) of the RuvC domain or the HNH domain.

[0030] In some embodiments, a Cas9 nuclease has an inactive (e.g, an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase, referred to as an “nCas9” protein (for “nickase” Cas9). The Cas9 nickase may be a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g, a duplexed DNA molecule). In some embodiments the Cas9 nickase cleaves the target strand of a duplexed nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is base paired to (complementary to) a gRNA (e.g, an sgRNA) that is bound to the Cas9.

[0031] As used herein, “Cas9 nickase” or “nCas9” refers to a variant of the standard Cas9 enzyme which is engineered to cut only a single strand of a nucleic acid duplex. Optionally, nickase Cas9 is used when off-target effects are a concern. Optionally, Cas9 nickase is derived from the wild-type Cas9 by mutating one of the two nuclease domains — either the RuvC-like nuclease domain or the HNH nuclease domain — rendering it inactive. This modification allows nCas9 to make a single-strand cut or "nick" in the DNA, rather than a double-stranded break. Optionally, the Cas9 nickase has a mutation in the D10A position of the RuvC domain.Guide Polynucleotides

[0032] Provided herein are compositions and methods for gene-editing in target cells. Further provided herein are compositions comprising a guide polynucleic acid sequence or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more guide polynucleotides as provided herein.

[0033] In some embodiments, a gene-editing cassette includes a guide polynucleotide, optionally gRNA. As used herein, “gRNA” or “guide RNA” refers to any nucleotide sequence that is used to guide the gene-editing cassette. In some embodiments, the gRNA is a short synthetic RNA sequence designed to be complimentary to a target region of DNA in the genome. By binding to a specific sequence within the genome, the gRNA ensures that the gene editing occurs at the intended location. In some embodiments, the gRNA specifically binds to a target polynucleotide sequence (i.e., via complementary base pairing between bases of the guide polynucleotide and bases of the target polynucleotide sequence). Thus, the gRNA serves as a guide for the Cas9 nuclease. The design of the gRNA aids the specificity and efficiency of the CRISPR-Cas9 editing process.

[0034] A guide polynucleotide may include natural or non-natural (or unnatural) nucleotides (e.g., peptide nucleic acid or nucleotide analogs). In some cases, the targeting region of a guide nucleic acid sequence can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. A targeting region of a guide nucleic acid can be between 10- 30 nucleotides in length, or between 15-25 nucleotides in length, or between 15-20 nucleotides in length.

[0035] In some embodiments, a guide polynucleotide is RNA and / or DNA. In some embodiments, the guide polynucleotide is gRNA. Exemplary, non-limiting guide RNAs that may be used with the gene-editing cassette include cripsrRNA (crRNA), trans-activating crRNA (tracrRNA), single guide RNA (sgRNA), dual RNA, pooled RNA, long single-guide RNA (IgRNA), and / or cyclic gRNA (cgRNA). In some embodiments, the gRNA is an sgRNA.

[0036] An RNA / Cas complex may assist in “guiding” a Cas protein to a target DNA. In nature, DNA-binding and cleavage typically requires protein and both trRNA and crRNA. The complex endonucleolytically cleaves linear or circular dsDNA at a target sequence complementary to the crRNA. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3'-5' exonucleolytically. However, single guide RNAs (“sgRNA”) may be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species.

[0037] In some embodiments, crRNA is derived from the CRISPR array of a bacterial cell.Optionally, the crRNA consists of multiple repeat sequences interspaced by unique sequences called spacers. Optionally, the spacers are derived from the DNA of viruses or plasmids that have previously infected the cell from which the CRISPR array is isolated.

[0038] In some embodiments, tracrRNA is derived from the CRISPR array of a bacterial cell. Optionally, the tracrRNA includes a base pairing region and a Cas9 binding site. In some embodiments, the base pairing region forms complementary base pairs with a region of the crRNA, optionally with the repeat sequence of the crRNA.

[0039] In some embodiments, sgRNA is engineered to direct the Cas9 nuclease to a specific location in the DNA for targeted gene modification. Optionally, the sgRNA combines crRNA and tracrRNA into a synthetic, streamlined molecule. Optionally, sgRNA includes repeat sequences. In some embodiments, the repeat sequences are fused to a Cas9 binding site.Optionally, the sgRNA has a length of from about 15 to about 25 nucleotides, including 16, 17, 18, 19, 20, 21, 22, 23, and 24 nucleotides, or any range having endpoints defined by any two of the aforementioned values.

[0040] In some embodiments, dual RNA uses separate crRNA and tracrRNA molecules. Optionally, dual RNA enables multiplexing, where multiple targets are edited simultaneously. Optionally, dual RNA allows for easier swapping of crRNAs targeting different sequences without the need to redesign the entire sgRNA. In some embodiments, a guide polynucleotide comprises two or more individual polynucleotides, which can interact with one another via for example complementary base pairing (e.g., a dual guide polynucleotide, dual gRNA).

[0041] In some embodiments, the gene-editing cassettes employ pooled gRNA to guide the gene-editing cassette. Pooled RNA involves mixtures of different gRNAs targeting multiple genes or sites within a genome. Optionally, pooled gRNA is used for large-scale genetic screens to identify the functions of various genes.

[0042] In some embodiments, IgRNA molecules are developed for more complex editing purposes. Optionally, IgRNAs are longer than typical sgRNAs. In some embodiments, IgRNAs include additional RNA elements that confer new functionalities, such as recruitment of transcriptional activators or repressors.

[0043] In some embodiments, the gene-editing cassette includes cgRNA. Optionally, the cgRNA provides enhanced stability to the gene-editing cassette. Optionally, cgRNA increases the duration in which the gene-editing cassette modifies the target sequence.

[0044] In some embodiments, a guide polynucleotide may include one or more modifications, including but not limited to a substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, or combination thereof. For example, and without being bound by theory, a guide polynucleotide may be modified by 5' adenylate, 5' guanosine-triphosphate cap, 5' N7-Methylguanosine-triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' thiophosphate, 5' phosphate, 5' thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9, 3 '-3' modifications, 2'-O-methyl thioPACE (MSP), 2'-O-methyl-PACE (MP), and constrained ethyl (S-cEt), 5 '-5' modifications, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dualbiotin, PC biotin, psoralen C2, psoralen C6, TINA, 3' DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY- 7, QSY-9, carboxyl linker, thiol linkers, 2'-deoxyribonucleoside analog purine, 2'- deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-O-methyl ribonucleoside analog, sugar modified analogs, wobble / universal bases, fluorescent dye label, 2'-fluoro RNA, 2'-O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5 '-triphosphate, 5'- methylcytidine-5 '-triphosphate, or combinations thereof.

[0045] In some embodiments, the gene-editing cassette includes a gRNA having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with one or more of SEQ ID NOs: 1-394. In some embodiments the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-394. In some embodiments, the gene-editing cassette includes a gRNA having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with one or more of SEQ ID NOs: 1-85. Optionally, the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-85. In some embodiments, the gene-editing cassette includes a gRNA having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with one or more of SEQ ID NOs: 1-29. In some embodiments, the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-29. In some embodiments, the gene-editing cassette includes a gRNA having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with one or more of SEQ ID NOs: 1-11. In some embodiments, the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-11. In some embodiments, the gene-editing cassette includes a gRNA having at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with one or more of SEQ ID NOs: 1-8. Optionally, the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-8. In some embodiments, the gene-editing cassette includes a gRNA having at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with one or more of SEQ ID NOs:l-5. In some embodiments, the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-5.Additional Elements

[0046] Optionally, the gene-editing cassette includes one or more additional elements. Exemplary additional elements include repair templates, selection markers, promoters and / or regulatory elements.

[0047] In some embodiments, the gene-editing cassette includes a repair template. For example, and without being bound by theory, for precise editing tasks, such as inserting a new gene or correcting a mutation, a piece of donor DNA is included in the cassette. This DNA may serve as a repair template for the cell's repair machinery (homology-directed repair) to use when fixing the double-stranded break made by Cas9. By designing the donor DNA with sequences that are homologous to the regions flanking the cut site, specific changes may be integrated into the genome.

[0048] In some embodiments, the gene-editing cassette includes one or more selection markers to facilitate the identification and selection of successfully edited cells. Optionally, the selection markers are genes that confer resistance to antibiotics or other selectable agents. Only cells that have incorporated the gene editing cassette (and therefore the selection marker) will survive in the presence of the selectable agent.

[0049] In some embodiments, the gene-editing cassette includes one or more promoters and / or regulatory elements to control the expression of the inserted genes or the Cas9 enzyme itself. These elements ensure that gene expression occurs at the desired levels, at specific times, and / or in specific tissues or cells.

[0050] In some embodiments, the gene-editing cassette comprises a modified CRISPR- Cas9 system. Optionally, the modified CRISPR-Cas9 system is a base-editing system or a primeediting system.Base-Editing System

[0051] In some embodiments, the gene-editing cassette comprises a base-editing system. As used herein, a “base-editing system” refers to a system that enables the direct conversion of one nucleotide base pair into another. Optionally, a base-editing system enables this conversion without breaking both strands of the nucleic acid. In some embodiments, a base-editing system can be used to correct a genetic mutation, restore normal gene function, and / or create a specific model of a disease.

[0052] In some embodiments, the base-editing system includes a polynucleotide programmable nucleotide binding domain as described herein, a nucleobase editing domain (e.g., a deaminase domain) for editing the nucleobase and a guide polynucleotide as described herein. It will be appreciated that components of the base editor system may be associated with each other via covalent bonds, noncovalent interactions, or any combination of associations and interactions thereof.

[0053] In some embodiments, the base-editing system is directed to the target sequence using gRNA. This gRNA is then complexed with a Cas9 protein that has been modified to not cleave the nucleotide strand (nuclease-dead Cas9) or to only nick one strand of the nucleic acid (Cas9 nickase). Upon binding to the target sequence, the Cas protein exposes the nucleotide bases within a small editable window. The proximity of the deaminase enzyme to the nucleotide strand allows the enzyme to act on the specific bases. The deaminase enzyme chemically alters the target base. Following this, cellular repair mechanisms process these changes, resulting in a permanent base substitution after replication.

[0054] In some embodiments, the base-editing system comprises a modified CRISPR- Cas9 system, wherein the CRISPR-Cas9 lacks the ability to create double-stranded breaks. In some embodiments, the base-editing system includes a base editor enzyme. Optionally, the base editor enzyme is attached to the Cas9 protein. In some embodiments, the base editor enzyme chemically converts one nucleotide base into another. Optionally, the base editing enzyme is derived by fusing a deaminase enzyme to a partially inactivated CRISPR-Cas system. Optionally, the partially inactivated CRISPR-Cas system is nuclease-dead Cas9 or a Cas9 nickase. See Porto, et al., Base editing: advances and therapeutic opportunities, Nature Reviews Drug Discovery 19:839-59 (2020); Rees, et al., Base editing: precision chemistry on the genome and transcriptome of living cells, Nat. Rev. Genet. 19(12): 770-88 (2018); and US 2023 / 0235309, published July 27, 2023 by Liu, et al., each of which is incorporated herein by reference in its entirety.

[0055] In some embodiments, the base-editing system is a cytidine base editor. Optionally, the cytidine base editor converts cytosine to uracil, which is treated as thymine during DNA replication or repair, thereby converting a cytosine residue to a thymine residue. This also results in converting guanine to adenine on the complementary strand. Optionally, the base editor enzyme is cytidine deaminase.

[0056] In some embodiments, a base editor disclosed herein comprises a cytidine deaminase capable of deaminating a target cytidine (C) base of a polynucleotide to produce uridine (U), which has the base pairing properties of thymine. In some embodiments, for example where the polynucleotide is double-stranded (e.g., DNA), the uridine base may be substituted with a thymidine base (e.g., by cellular repair machinery) to give rise to a C:G to a T:A transition. In some embodiments, deamination of a C to U in a nucleic acid by a base editor may not be accompanied by substitution of the U to a T.

[0057] The deamination of a target C in a polynucleotide to give rise to a U is a nonlimiting example of a type of base editing that can be executed by a base editor described herein. In another example, a base editor comprising a cytidine deaminase domain can mediate conversion of a cytosine (C) base to a guanine (G) base. For example, a U of a polynucleotide produced by deamination of a cytidine by a cytidine deaminase domain of a base editor may be excised from the polynucleotide by a base excision repair mechanism (e.g., by a uracil DNA glycosylase (UDG) domain), producing an abasic site. The nucleobase opposite the abasic site may then be substituted (e.g., by base repair machinery) with another base, such as a C, by for example a translesion polymerase. Although it is typical for a nucleobase opposite an abasic site to be replaced with a C, other substitutions (e.g., A, G or T) may also occur.

[0058] Accordingly, in some embodiments a base editor described herein comprises a deamination domain (e.g., cytidine deaminase domain) capable of deaminating a target C to a U in a polynucleotide. Further, as described below, the base editor may comprise additional domains which facilitate conversion of the U resulting from deamination to, in some embodiments, a T or a G. For example, a base editor comprising a cytidine deaminase domain may further comprise auracil glycosylase inhibitor (UGI) domain to mediate substitution of a U by a T, completing a C- to-T base editing event. In embodiments, the base editor may comprise a uracil stabilizing protein as described herein. In another example, a base editor may incorporate a translesion polymerase to improve the efficiency of C-to-G base editing, since a translesion polymerase may facilitate incorporation of a C opposite an abasic site (i.e., resulting in incorporation of a G at the abasic site, completing the C-to-G base editing event).

[0059] A base editor comprising a cytidine deaminase as a domain may deaminate a target C in any polynucleotide, including DNA, RNA and DNA-RNA hybrids. Typically, a cytidine deaminase catalyzes a C nucleobase that is positioned in the context of a single-stranded portion of a polynucleotide. In some embodiments, the entire polynucleotide comprising a target C is single-stranded. For example, and without being bound by theory, in some embodiments, a cytidine deaminase incorporated into a base editor deaminates a target C in a single-stranded RNA polynucleotide. In some embodiments, a base editor comprising a cytidine deaminase domain may act on a double-stranded polynucleotide, but the target C may be positioned in a portion of the polynucleotide which at the time of the deamination reaction is in a single-stranded state. For example, in embodiments where the NAGPB domain comprises a Cas9 domain, several nucleotides may be left unpaired during formation of the Cas9-gRNA-target DNA complex, resulting in formation of a Cas9 “R-loop complex.” These unpaired nucleotides may form a bubble of single-stranded DNA that serves as a substrate for a single-strand specific nucleotide deaminase enzyme (e.g., cytidine deaminase).

[0060] In some embodiments, a cytidine deaminase of a base editor comprises all or a portion (e.g., a functional portion) of an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. APOBEC is a family of evolutionarily conserved cytidine deaminases. Members of this family are C-to-U editing enzymes. The N- terminal domain of APOBEC like proteins is the catalytic domain, while the C-terminal domain is a pseudocatalytic domain. More specifically, the catalytic domain is a zinc dependent cytidine deaminase domain and is important for cytidine deamination. APOBEC family members include APOBEC 1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (“APOBEC3E” now refers to this), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and Activation-induced (cytidine) deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of an APOBEC 1 deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) ofAP0BEC2 deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of is an APOBEC3 deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of an APOBEC3A deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of APOBEC3B deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of APOBEC3C deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of APOBEC3D deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of APOBEC3E deaminase. In some embodiments, a deaminase incorporated into abase editor comprises all or a portion (e.g., a functional portion) of APOBEC3F deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of APOBEC3G deaminase. In some embodiments, a deaminase incorporated into abase editor comprises all or a portion (e.g., a functional portion) of APOBEC3H deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of APOBEC4 deaminase. In some embodiments, a deaminase incorporated into abase editor comprises all or a portion (e.g., a functional portion) of activation-induced deaminase (AID). In some embodiments a deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of cytidine deaminase 1 (CDA1). It should be appreciated that a base editor can comprise a deaminase from any suitable organism (e.g., a human or a rat). In some embodiments, a deaminase domain of a base editor is from a human, chimpanzee, gorilla, monkey, orangutan, alligator, pig, cow, dog, rat, or mouse. In some embodiments, the deaminase domain of the base editor is derived from rat (e.g., rat APOBEC1). In some embodiments, the deaminase domain of the base editor is derived from an orangutan polypeptide (e.g., a Pongo pygmaeus (Orangutan) APOBEC). In some embodiments, the deaminase domain of the base editor is derived from a golden snub-nosed monkey polypeptide (e.g., a Rhinopithecus roxellana (golden snub-nosed monkey) APOBEC3F (A3F)). In some embodiments, the deaminase domain of the base editor is derived from an American Alligator polypeptide (e.g., an Alligator mississippiensis (American alligator) APOBEC1). In some embodiments, the deaminase domain of the base editor is derived from a pig polypeptide (e.g., a Sus scrofa (pig) APOBEC3B). In some embodiments, the deaminase domain of the base editor is human APOBEC 1. In some embodiments, the deaminase domain of the base editor is pmCDAL

[0061] In some embodiments, a base editor described herein comprises an adenosine deaminase domain. Such an adenosine deaminase domain of a base editor may facilitate the editing of an adenine (A) nucleobase to a guanine (G) nucleobase by deaminating the A to form inosine (I), which exhibits base pairing properties of G. Adenosine deaminase is capable of deaminating (i.e., removing an amine group) adenine of a deoxyadenosine residue in deoxyribonucleic acid (DNA). In some embodiments, an A-to-G base editor further comprises an inhibitor of inosine base excision repair, for example, a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine specific nuclease. Without wishing to be bound by any particular theory, the UGI domain or catalytically inactive inosine specific nuclease can inhibit or prevent base excision repair of a deaminated adenosine residue (e.g, inosine), which can improve the activity or efficiency of the base editor. A base editor comprising an adenosine deaminase may act on any polynucleotide, including DNA, RNA and DNA-RNA hybrids. In certain embodiments, a base editor comprising an adenosine deaminase may deaminate a target A of a polynucleotide comprising RNA. For example, the base editor can comprise an adenosine deaminase domain capable of deaminating a target A of an RNA polynucleotide and / or a DNA- RNA hybrid polynucleotide. In an embodiment, an adenosine deaminase incorporated into a base editor comprises all or a portion (e.g., a functional portion) of adenosine deaminase acting on RNA (ADAR, e.g., AD ARI or ADAR2) or tRNA (AD AT). A base editor comprising an adenosine deaminase domain may also be capable of deaminating an A nucleobase of a DNA polynucleotide. In some embodiments, an adenosine deaminase domain of a base editor comprises all or a portion (e.g., a functional portion) of an AD AT comprising one or more mutations which permit the AD AT to deaminate a target A in DNA. For example, the base editor can comprise all or a portion (e.g., a functional portion) of an AD AT from Escherichia coli (EcTadA) comprising one or more of the following mutations: D108N, A106V, D147Y, E155V, L84F, H123Y, I156F, or a corresponding mutation in another adenosine deaminase. In some embodiments, the adenosine deaminase is a TadA.

[0062] The adenosine deaminase may be derived from any suitable organism (e.g, E. coli). In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, theadenosine deaminase is from E. coli. Exemplary, nonlimiting adenosine deaminase sequences and modifications suitable for use with embodiments of the present disclosure may be found in US Patent Application Serial No. 18 / 699,800, which is incorporated herein by reference in its entirety.Prime-Editing System

[0063] In some embodiments, the gene-editing cassette comprises a prime-editing system. As used herein, a “prime-editing system” refers to a system that enables the insertion, deletion, and replacement of nucleotide sequences. Optionally, a prime-editing system enables this conversion without breaking both strands of the nucleic acid. In some embodiments, a primeediting system may be used to correct a genetic mutation, restore normal gene function, and / or create a specific model of a disease. Optionally, the prime-editing system may be used for more complex alterations. In some embodiments, the prime-editing system includes a prime-editing guide RNA (pegRNA), a Cas9 nickase, and a reverse transcriptase.

[0064] In some embodiments, a prime-editing system directly writes new genetic information into a target site using a catalytically impaired Cas9, optionally fused to an engineered reverse transcriptase. In some embodiments, the catalytically impaired Cas9 protein has an inactivated UNH domain. Optionally, the catalytically impaired Cas9 is a Cas9 H840A nickase. In some embodiments, the reverse transcriptase is a murine leukemia virus reverse transcriptase, optionally a Moloney murine leukemia virus (M-MLV) reverse transcriptase. In some embodiments, the reverse transcriptase is a modified M-MLV reverse transcriptase, optionally M- MLV RT(D200N / L603 W / T330P / T306K / W313F).

[0065] In some embodiments, the pegRNA identifies the target nucleotide sequence to be edited and encodes new genetic information to replace the target sequence. In some embodiments, the pegRNA includes an extended single guide RNA (sgRNA) containing a primer binding site and a reverse transcriptase template. In some embodiments, the primer binding site is a short sequence that is complementary to the target nucleotide sequence. After the nucleotide strand is nicked, the primer-binding site hybridizes with the exposed strand, serving as a primer for the next stage of editing. During genome editing, the primer binding site allows the 3’ end of the nicked DNA strand to hybridize to the pegRNA, while the RT template serves as a template for the synthesis of edited genetic information. In some embodiments, the reverse transcriptase template contains the desired edits and is used as template to synthesize new nucleotides at the target site.The length and sequence of the reverse transcriptase template are designed based on the specific insertions, deletions, or substitutions intended in the genomic DNA. Optionally, the reverse transcriptase template is similar to the gRNA in the previously-described CRISPR-Cas9 system.

[0066] Once the target nucleotide is nicked, the primer binding site of the pegRNA hybridizes with the complementary strand of the nicked nucleotide, binding the pegRNA to the target sequence. The bound pegRNA then serves as a template for the reverse transcriptase that is tethered to the Cas9 protein. This enzyme extends the nicked nucleotide end using the reverse transcriptase template on the pegRNA, incorporating the edited sequence into the target genome. The edited strand is eventually integrated into the existing nucleic acid strand through the cell's natural repair mechanisms, replacing the old sequence with the new, edited sequence. See Tu, et al., Prime Editing: An All-Rounder for Genome Editing, Int. J. Mol. Sci. 23(17): 9862 (2022); Anzalone, et al., Search-and-replace genome editing without double-strand breaks or donor DNA, Nature 576: 146-57 (2019); and US 2023 / 0357766, published Nov. 9, 2023 by Tiu, et al., each of which is incorporated herein by reference in its entirety.Delivery Systems

[0067] A composition including the gene-editing cassette may comprise a nucleotide binding domain polypeptide and a combination of one or more of any guide RNAs provided herein. Such a composition may be used to effect base editing in a cell through different delivery approaches, for example, electroporation, nucleofection, viral transduction or transfection.

[0068] The gene-editing cassette may be delivered to cell of interest via a variety of delivery techniques known in the art, such as a nanoparticle, a viral vector, or electroporation. Exemplary delivery systems include lipid-based delivery systems, such as liposomes, lipid micro and nanoparticles, and cationic lipids; polymer-based delivery systems, such as dendrimers and polymer particles; peptide-based delivery systems, such as cell-penetrating peptides; inorganic particles, such as gold and silica particles; and viral vectors.

[0069] In some embodiments, the gene-editing cassette is delivered via a nanoparticle, such as an inorganic nanoparticle or a lipid-based nanoparticle. In some embodiments, the compositions of the present disclosure include a lipid nanoparticle, optionally encapsulating the gene-editing cassette.

[0070] Optionally, the entire cassette is introduced into cells through a delivery method, such as lipid nanoparticles, viral vectors, plasmids, or physical methods like electroporation. In some embodiments, lipid nanoparticles are employed as a delivery system for the gene-editing cassettes described herein. Optionally, the lipid nanoparticles aid in transporting the gene-editing cassette into the cells. For example, and without being bound by theory, when the lipid nanoparticle is administered to a subject, the lipid nanoparticles are taken into a target cell by endocytosis, where the lipid nanoparticle is enclosed in an endosome.

[0071] Various lipids are suitable for use in the lipid nanoparticles. In embodiments, the lipid nanoparticle includes a lipid selected from one or more of lecithin, phosphatidylcholines, phosphatidic acid, phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, cardiolipins, lipid- polyethyleneglycol conjugates, and combinations thereof. In embodiments, the lipids of the lipid monolayer may be PEGylated, at least in part, in order to facilitate the avoidance of immune clearance of the LNP. In embodiments, the lipid monolayer may further comprise cholesterol as a stabilizer.

[0072] Lipid nanoparticles offer a non-viral, efficient, and relatively safe method for delivering the gene-editing cassette into target cells. In some embodiments, the lipid nanoparticles protect the gene-editing cassette components from degradation in the biological environment, facilitate their uptake by cells, and enable their release into the cytoplasm. Several types of lipid nanoparticles may be tailored for delivery of the gene-editing cassette to optimize the delivery of nucleic acids such as guide RNA (gRNA) and the Cas9 mRNA or protein. In some embodiments, the lipid nanoparticle is a solid lipid nanoparticle. Generally, a solid lipid nanoparticle includes one or more lipids that is a solid at body temperature.

[0073] Optionally, the lipid nanoparticles aid in transporting the gene-editing cassette into the cells. For example, and without being bound by theory, when the lipid nanoparticle is administered to a subject, the lipid nanoparticles are taken into a target cell by endocytosis, where the lipid nanoparticle is enclosed in an endosome. Once the gene-editing cassette is released from the endosome, the gRNA binds to the Cas9 protein, forming a complex that binds to a target sequence in the genome, as described in greater detail herein.

[0074] Optionally, the lipid nanoparticle includes at least one lipid. Optionally the lipid is an ionizable lipid. In some embodiments, the lipid is a cationic lipid. Optionally, the lipidnanoparticle also includes a structural lipid. In some embodiments, the lipid nanoparticle includes one or more PEGylated lipids. In some embodiments, the lipid nanoparticle include cholesterol. In some embodiments, the lipid nanoparticle includes one or more lipid-polymer conjugates.

[0075] In some embodiments, the lipid nanoparticle is an ionizable lipid nanoparticle. Optionally, these lipid nanoparticles contain ionizable lipids that are neutral at physiological pH but become positively charged in the acidic environment of the endosome. In some embodiments, the change in the acidic environment facilitates endosomal release of the gen-editing cassette into the cytoplasm. In some embodiments, the lipid nanoparticle is a cationic lipid nanoparticle. Generally, cationic lipid nanoparticles carry a positive charge, which enhances their interaction with negatively charged nucleic acids and promotes cellular uptake via electrostatic interactions with the negatively charged cell membranes.

[0076] In some embodiments, the lipid nanoparticle includes a cationic lipid suitable for complexing with the nucleic acid in the core. As used herein, the term “cationic lipid” encompasses any of a number of lipid species that carry a net positive charge at physiological pH, which can be determined using any method known to one of skill in the art. Such lipids include, but are not limited to, the cationic lipids of formula (I) disclosed in International Application No. PCT / US2009 / 042476, entitled “Methods and Compositions Comprising Novel Cationic Lipids,” filed on May 1, 2009, and is herein incorporated by reference in its entirety. These include, but are not limited to, N-methyl-N-(2-(arginoylamino) ethyl)- N, N- Di octadecyl aminium chloride or di stearoyl arginyl ammonium chloride] (DS AA), N,N-di-myristoyl-N-methyl-N-2[N’ -(N6- guanidino-L-lysinyl)] aminoethyl ammonium chloride (DMGLA), N,N-dimyristoyl-N-methyl-N- 2[N2-guanidino-L- lysinyl] aminoethyl ammonium chloride, N,N-dimyristoyl-N-methyl-N-2[N’- (N2, N6- di-guanidino- L-lysinyl)] aminoethyl ammonium chloride, and N, N-di-stearoyl-N- methyl-N-2[N’-(N6- guanidino-L-lysinyl)] aminoethyl ammonium chloride (DSGLA). Other non-limiting examples of cationic lipids that can be present in the liposome or lipid bilayer of the presently disclosed lipid nanoparticles include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3- dioleoyloxy) propyl)-N,N,N -trimethylammonium chloride (DOTAP); N- (2,3- dioleyloxy) propyl) -N,N,N-trimethylammonium chloride (DOTMA) or other N- (N,N-1- dialkoxy)-alkyl-N,N,N-trisubstituted ammonium surfactants; N,N-distearyl- N,N- dimethylammonium bromide (DDAB); 3-(N-(N',N'-dimethylaminoethane)- carbamoyl) cholesterol (DC-Choi) and N-(l,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N -hydroxy ethyl ammonium bromide (DMRIE); 1,3 -dioleoyl- 3- trimethylammonium-propane, N-(l-(2,3-dioleyloxy)propyl)-N-(2- (sperminecarboxamido)ethyl)-N,N-dimethy- 1 ammonium trifluoroacetate (DOSPA); GAP-DLRIE; DMDHP; 3-p[4N-(H8N-diguanidino spermidine)- carbamoyl] cholesterol (BGSC); 3-P[N,N-diguanidinoethyl-aminoethane)-carbamoyl] cholesterol (BGTC); N,N\N2,N3 Tetra-methyltetrapalmitylspermine (cellfectin); N-t-butyl-N'- tetradecyl-3-tetradecyl-aminopropion-amidine (CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB); l,3-dioleoyloxy-2-(6-carboxyspermyl)-propyl amide (DOSPER); 4-(2,3-bis- palmitoyloxy-propyl)- 1 -methyl- lEI-imidazole (DPIM) N,N,N',N'- tetramethyl-N,N'-bis(2- hydroxyethyl)-2,3 dioleoyloxy- 1 ,4- butanediammonium iodide) (Tfx- 50); 1,2 dioleoyl-3-(4'- trimethylammonio) butanol-sn- glycerol (DOBT) or cholesteryl (4'trimethylammonia) butanoate (ChOTB) where the trimethylammonium group is connected via a butanol spacer arm to either the double chain (for DOTB) or cholesteryl group (for ChOTB); DE-l,2-dioleoyl-3- dimethylaminopropyl-P-hydroxy ethylammonium (DORI) or DL- l,2-0-dioleoyl-3- dimethylaminopropyl-P-hydroxyethylammonium (DORIE); l,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such as dioctadecylamidoglycylspermine (DOGS) and dipalmitoyl phosphatidylethanolamylspermine (DPPES); cholesteryl-3P- carboxyl-amido- ethylenetrimethylammonium iodide; 1- dimethylamino-3- trimethylammonio-DL-2-propyl- cholesteryl carboxylate iodide; cholesteryl-3- p- carboxyamidoethyleneamine; cholesteryl-3-P- oxysuccinamido- ethylenetrimethylammonium iodide; l-dimethylamino-3 -trimethylammonio- DL-2- propyl-cholesteryl-3-P-oxysuccinate iodide; 2-(2 -trimethylammonio)- ethylmethylamino ethyl-cholesteryl-3-P-oxysuccinate iodide; 3- p-N-(polyethyleneimine)- carbamoylcholesterol, DC-cholesterol; and N4-cholesteryl-spermine HC1 salt (GL67).

[0077] In some embodiments, the lipid nanoparticle is a PEGylated lipid nanoparticle. Optionally, a PEGylated lipid nanoparticle reduces immunogenicity in a subject. These lipid nanoparticles generally incorporate a polyethylene glycol (PEG)-lipid conjugate which forms a protective and stabilizing corona around the lipid nanoparticle. The corona increases circulation time by reducing opsonization and clearance by the mononuclear phagocyte system. In some embodiments, PEGylation is used in combination with ionizable and / or cationic lipids to improve pharmacokinetics and biodistribution of the lipid nanoparticles. In some embodiments, the lipid core matrix further comprises cholesterol as a stabilizer.Pharmaceutical Compositions

[0078] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the gene-editing cassette described herein. Optionally, the gene-editing cassette may be used to prevent, inhibit, or reverse hair loss and / or promote hair growth. In some embodiments, the pharmaceutical composition comprises a pharmaceutically-acceptable excipient, such as a diluent, carrier, stabilizer, solvent, buffer, emulsifier, preservative, osmotic agents, viscosity enhancer, absorption enhancer, etc. In some embodiments, the pharmaceutical composition further comprises one or more nutrients, vitamins, peptides, growth factors, combinations thereof, and the like.

[0079] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject. In some embodiments, the pharmaceutical composition is formulated for parenteral and / or topical delivery. In some embodiments, the pharmaceutical composition is formulated for injection. In some embodiments, the pharmaceutical composition is formulated for intrafollicular injection.

[0080] The gene-editing cassette may be present in the pharmaceutical composition at a therapeutically effective concentration. Optionally, the therapeutically effective concentration is determined by the amount of guide polynucleotide and / or the amount of the nucleotide binding domain. In some embodiments, a therapeutically effective concentration of the guide polynucleotide is from about 10 pM to about 1000 pM, including about 25 pM, about 50 pM, about 75 pM, about 100 pM, about 125 pM, about 150 pM, about 175 pM, about 200 pM, about 225, about 250 pM, about 275 pM, about 300 pM, about 325 pM, about 350 pM, about 375 pM, about 400 pM, about 425 pM, about 450 pM, about 475 pM, about 500 pM, about 525 pM, about 550 pM, about 575 pM, about 600 pM about 625 pM, about 650 pM, about 675 pM, about 700 pM, about 725 pM, about 750 pM, about 775 pM, about 800 pM, about 825 pM, about 850 pM, about 875 pM, about 900 pM, about 925 pM, about 950 pM, and about 975 pM, including any subrange having endpoints defined by any two of the aforementioned values. In some embodiments, a therapeutically effective concentration of the nucleotide binding domain is from about 1 ng / pL to about 500 ng / pL, including about 5 ng / pL, about 10 ng / pL, about 20 ng / pL, about 25 ng / pL, about 30 ng / pL, about 40 ng / pL, about 50 ng / pL, about 75 ng / pL, about 100 ng / pL, about 125 ng / pL, about 150 ng / pL, about 175 ng / pL, about 200 ng / pL, about 225 ng / pL,about 250 ng / pE, about 275 ng / pL, about 300 ng / pL, about 325 ng / pL, about 350 ng / pL, about 375 ng / pL, about 400 ng / pL, about 425 ng / pL, about 450 ng / pL, and about 475 ng / pL including any subrange having endpoints defined by any two of the aforementioned values.

[0081] In some embodiments, the pharmaceutical composition is administered to a subject suffering from hair loss, optionally androgenic alopecia. The pharmaceutical composition may be injected into the scalp at a plurality of injections sites in the area affected by alopecia. In some embodiments, the injection locations may be separated by about 0.5 cm to about 2.0 cm. In some embodiments, the pharmaceutical composition is injected into each follicle. In some embodiments, the pharmaceutical composition is administered via an injection alternative, such as a mesotherapy device or a jet injector.

[0082] In some embodiments, the pharmaceutical composition is administered to the hair follicle of a subject. Optionally, the pharmaceutical composition is administered in an amount ranging from about 0.1 pL to about 100 pL, including 0.2 pL, 0.3 pL, 0.4 pL, 0.5 pL, 0.6 pL, 0.7 pL, 0.8 pL, 0.9 pL, 1 pL, 1.5 pL, 2 pL, 2.5 pL, 3 pL, 3.5 pL, 4 pL, 4.5 pL, 5 pL, 5.5 pL, 6 pL, 6.5 pL, 7 pL, 7.5 pL, 8 pL, 8.5 pL, 9 pL, 9.5 pL, 10 pL, 11 pL, 12 pL, 13 pL, 14 pL, 15 pL, 16 pL, 17 pL, 18 pL, 19 pL, 20 pL, 25 pL, 30 pL, 35 pL, 40 pL, 45 pL, 50 pL, 55 pL, 60 pL, 65 pL, 70 pL, 75 pL, 80 pL, 85 pL, 90 pL, and 95 pL, including any range having endpoints defined by any two of the aforementioned values.Editing of Target Sequence

[0083] The compositions provided herein may be used to edit the genome of a subject. Genome editing involves the molecular manipulation of genetic material by deleting, replacing, or inserting a nucleotide sequence of a target gene. In some embodiments, to produce the gene edits described herein, cells, such as keratinocytes, dermal papilla cells, melanocytes, stem cells (bulge stem cells), etc., are contacted in vivo or in vitro with a gene-editing cassette as described herein. In some embodiments, the gene-editing cassette specifically binds to a target sequence within the cell.

[0084] As described herein, a gene-editing cassette may comprise a protein domain that is capable of binding a nucleotide sequence, optionally a target sequence, that contains a canonical or non-canonical protospacer adjacent motif (PAM) sequence. A PAM sequence is a nucleotide sequence in proximity to a target polynucleotide sequence. As used herein, the term “protospaceradjacent motif (PAM)” refers to a 2-6 base pair nucleotide sequence immediately following the target polynucleotide sequence targeted by the Cas9 nuclease of the gene-editing cassette. In some embodiments, the PAM can be a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (i.e., located downstream of the 5' end of the protospacer). The PAM sequence may be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGTT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNNGATT, NNAGAAW, or NAAAAC, wherein Y is a pyrimidine; N is any nucleotide base; W is A or T.

[0085] In some embodiments, the nucleotide binding domain directs cleavage of one or both strands at a target sequence, such as within a target sequence and / or within a complement of a target sequence. For example, a CRISPR enzyme can direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, the gene-editing cassette induces a break in the target sequence. Repair of the break by non-homologous end joining (NHEJ) or homology directed repair (HDR) introduces insertions, deletions, or point mutations at the site of the break.

[0086] In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is an RNA sequence. In some embodiments, the target sequence is a sequence in the genome of a bacteria, yeast, fungi, insect, plant, or animal. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3' end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is immediately adjacent to a non-canonical PAM sequence. In some embodiments, the guide nucleic acid (e.g., guide RNA) is complementary to a sequence in a gene of interest (e.g., a gene associated with a disease or disorder).

[0087] In some embodiments, the target sequence is within the androgen receptor. In some embodiments, modification of the target sequences results in reduced expression of the gene encoded by the target sequence. For example, in some embodiments, administering a gene-editing cassette according to the present disclosure to a subject experiencing androgenic alopecia resultsin down regulation or decreased expression of the androgen receptor. Exemplary gRNAs for use in the gene-editing cassettes for targeting the androgen receptor are represented in SEQ ID NOs: 1-394.

[0088] In some embodiments, modification of the target sequence results in at least 10% reduction of the gene targeted expression. In some embodiments, the gene editing efficiency may result in at least 10% reduction of the gene targeted expression. In some embodiments, the gene editing efficiency may result in at least 20% reduction of the gene targeted expression. In some embodiments, the gene editing efficiency may result in at least 25% reduction of the gene targeted expression. In some embodiments, the gene editing efficiency may result in at least 30% reduction of the gene targeted expression. In some embodiments, the base editing efficiency may result in at least 40% reduction of the gene targeted expression. In some embodiments, the base editing efficiency may result in at least 50% reduction of the gene targeted expression.EXAMPLES

[0089] Example 1

[0090] A composition is prepared comprising a lipid nanoparticle and a gene editing cassette. The lipid nanoparticle is comprised of an ionizable lipid component. The lipid nanoparticle encapsulates a gene editing cassette comprising a Cas protein and a guide RNA comprising a nucleotide sequence selected from one of SEQ ID NOs: 1-394. The composition further comprises one or more pharmaceutically acceptable excipients and is formulated to be administered via intrafollicular injection.

[0091] The subject to be treated is a human patient suffering from androgenic alopecia. The area to be injected is prepared according to methods known in the art. Follicles are selected for injection and the composition is injected into each selected follicle at a concentration of from 10-1000 pM of the guide RNA.

[0092] The subject cares for the injection site for a recovery period according to standard practices. The patient is seen for a follow-up appointment after 3 months and hair regrowth at the injection sites is observed.

[0093] Example 2

[0094] Eleven compositions are prepared comprising a lipid nanoparticle and a gene editing cassette. The lipid nanoparticle is comprised of an ionizable lipid component and / or a pegylated lipid. The lipid nanoparticle encapsulates the gene editing cassette. Each composition includes a gene-editing cassette comprising a guide RNA selected from SEQ ID NOs: 1-11. The composition further comprises one or more pharmaceutically acceptable excipients and is formulated to be administered via intrafollicular injection.

[0095] The compositions are injected into a murine model of androgenic alopecia. Exemplary models may be found, for example, in Crabtree, et al., (2010) Endocrinology, 151, (No. 5), 2373-2380 or Fu, et al., (2021). Biomed. Pharma. 137, 111247, the contents of each of which are incorporated herein by reference in their entireties. Follicles are selected for injection and the composition is injected into each selected follicle at a concentration of from about 10- 1000 pM of the guide RNA.

[0096] Digital photographs are taken on days 0, 7, 14, and 21 using a DSLR camera. Animals are sacrificed by cervical dislocation before hair and tissue samples are collected.

[0097] On day 21, at least 70 hairs per mouse are collected from at least three mice in each group, as described in, for example in Valerie, A., et al. (2001). Eur. J. Dermatol. 11 (No. 4), 315— 320, the content of which is incorporated herein by reference in its entirety.

[0098] To evaluate skin morphology and hair regrowth, skin samples are collected on day 21 (after depilation). Samples are fixed in 4% paraformaldehyde and then embedded in paraffin blocks. Eongitudinal and horizontal sections are prepared and stained with hematoxylin and eosin (H&E). Photographs of H&E-stained sections are taken using an inverted microscope. The longitudinal sections are stained using Masson’s trichrome staining to evaluate the area covered by melanin in the hair bulb. The sections are evaluated for follicular length, follicular diameters, and follicular density. The horizontal sections are evaluated for anagen follicle (A) and telogen follicle (T), and the A / T ratio is calculated using an inverted microscope.

[0099] Four parameters: 1) number of hair follicles; 2) hair bulb diameter; 3) follicular density (number of hair follicles per mm2); and 4) number of anagen (A) and telogen (T) follicles per millimeter area, are measured to evaluate hair growth using imaging software.

[0100] Example 3

[0101] Eight compositions are prepared comprising a lipid nanoparticle and a gene editing cassette. The lipid nanoparticle is comprised of an ionizable lipid component and / or a pegylated lipid. The lipid nanoparticle encapsulates the gene editing cassette. Each composition includes a gene-editing cassette comprising a guide RNA selected from SEQ ID NOs: 1-8. The composition further comprises one or more pharmaceutically acceptable excipients and is formulated to be administered via intrafollicular injection.

[0102] The compositions are injected into a murine model of androgenic alopecia. Exemplary models may be found, for example, in Crabtree, et al., (2010) Endocrinology, 151, 2373-2380 or Fu, et al., (2021). Biomed. Pharma. 137, 111247, the contents of each of which are incorporated herein by reference in their entireties. Follicles are selected for injection and the composition is injected into each selected follicle at a concentration of from about 10-1000 pM of the guide RNA.

[0103] Digital photographs are taken on days 0, 7, 14, and 21 using a DSLR camera. Animals are sacrificed by cervical dislocation before hair and tissue samples are collected.

[0104] On day 21, at least 70 hairs per mouse are collected from at least three mice in each group, as described in, for example in Valerie, A., et al. (2001). Eur. J. Dermatol. 11 (No. 4), 315— 320, the content of which is incorporated herein by reference in its entirety.

[0105] To evaluate skin morphology and hair regrowth, skin samples are collected on day 21 (after depilation). Samples are fixed in 4% paraformaldehyde and then embedded in paraffin blocks. Eongitudinal and horizontal sections are prepared and stained with hematoxylin and eosin (H&E). Photographs of H&E-stained sections are taken using an inverted microscope. The longitudinal sections are stained using Masson’s trichrome staining to evaluate the area covered by melanin in the hair bulb. The sections are evaluated for follicular length, follicular diameters, and follicular density. The horizontal sections are evaluated for anagen follicle (A) and telogen follicle (T), and the A / T ratio is calculated using an inverted microscope.

[0106] Four parameters: 1) number of hair follicles; 2) hair bulb diameter; 3) follicular density (number of hair follicles per mm2); and 4) number of anagen (A) and telogen (T) follicles per millimeter area, are measured to evaluate hair growth using imaging software.

[0107] Example 4

[0108] Five compositions are prepared comprising a lipid nanoparticle and a gene editing cassette. The lipid nanoparticle is comprised of an ionizable lipid component and / or a pegylated lipid. The lipid nanoparticle encapsulates the gene editing cassette. Each composition includes a gene-editing cassette comprising a guide RNA selected from SEQ ID NOs: 1-5. The composition further comprises one or more pharmaceutically acceptable excipients and is formulated to be administered via intrafollicular injection.

[0109] The compositions are injected into a murine model of androgenic alopecia. Exemplary models may be found, for example, in Crabtree, et al., (2010) Endocrinology, 151, 2373-2380 or Fu, et al., (2021). Biomed. Pharma. 137, 111247, the contents of each of which are incorporated herein by reference in their entireties. Follicles are selected for injection and the composition is injected into each selected follicle at a concentration of from about 10-1000 pM of the guide RNA.

[0110] Digital photographs are taken on days 0, 7, 14, and 21 using a DSLR camera. Animals are sacrificed by cervical dislocation before hair and tissue samples are collected.

[0111] On day 21, at least 70 hairs per mouse are collected from at least three mice in each group, as described in, for example in Valerie, A., et al. (2001). Eur. J. Dermatol. 11 (No. 4), 315— 320, the content of which is incorporated herein by reference in its entirety.

[0112] To evaluate skin morphology and hair regrowth, skin samples are collected on day 21 (after depilation). Samples are fixed in 4% paraformaldehyde and then embedded in paraffin blocks. Eongitudinal and horizontal sections are prepared and stained with hematoxylin and eosin (H&E). Photographs of H&E-stained sections are taken using an inverted microscope. The longitudinal sections are stained using Masson’s trichrome staining to evaluate the area covered by melanin in the hair bulb. The sections are evaluated for follicular length, follicular diameters, and follicular density. The horizontal sections are evaluated for anagen follicle (A) and telogen follicle (T), and the A / T ratio is calculated using an inverted microscope.

[0113] Four parameters: 1) number of hair follicles; 2) hair bulb diameter; 3) follicular density (number of hair follicles per mm2); and 4) number of anagen (A) and telogen (T) follicles per millimeter area, are measured to evaluate hair growth using imaging software.

[0114] It is noted that the terms “substantially” and “about” may be utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. These terms are also utilized herein to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue. The term “substantially” is used herein also to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue. Thus, it is used to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation, referring to an arrangement of elements or features that, while in theory would be expected to exhibit exact correspondence or behavior, may in practice embody something less than exact.

[0115] It is an aspect of the present disclosure, alone or in combination with any other aspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition to a hair follicle of the subject, wherein the pharmaceutical composition comprises a lipid nanoparticle encapsulating a gene-editing cassette and wherein the gene-editing cassette inactivates an androgen receptor in the hair follicle.

[0116] It is an aspect of the present disclosure, alone or in combination with any other aspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia wherein the gene-editing cassette comprises a nucleotide binding domain and a guide polynucleotide.

[0117] It is an aspect of the present disclosure, alone or in combination with any other aspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia wherein the nucleotide binding domain is a Cas protein.

[0118] It is an aspect of the present disclosure, alone or in combination with any other aspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia wherein the pharmaceutical composition comprises from about 10 pM to about 1000 pM of the guide polynucleotide.

[0119] It is an aspect of the present disclosure, alone or in combination with any otheraspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia wherein the pharmaceutical composition comprises from about 1 ng / pL to about 500 ng / pL of the nucleotide binding domain.

[0120] It is an aspect of the present disclosure, alone or in combination with any other aspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia, wherein the lipid nanoparticle is selected from the group consisting of ionizable lipid nanoparticles, cationic lipid nanoparticles, PEGylated lipid nanoparticles, and solid lipid nanoparticles.

[0121] It is an aspect of the present disclosure, alone or in combination with any other aspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia wherein the gene-editing cassette comprises a CRISPR-Cas9 system comprising a gRNA.

[0122] It is an aspect of the present disclosure, alone or in combination with any other aspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia wherein the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-394.

[0123] It is an aspect of the present disclosure, alone or in combination with any other aspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia wherein the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-85.

[0124] It is an aspect of the present disclosure, alone or in combination with any other aspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia wherein the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-29.

[0125] It is an aspect of the present disclosure, alone or in combination with any other aspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia, wherein the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-11.

[0126] It is an aspect of the present disclosure, alone or in combination with any other aspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia wherein the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-8.

[0127] It is an aspect of the present disclosure, alone or in combination with any other aspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia wherein the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-5.

[0128] It is an aspect of the present disclosure, alone or in combination with any other aspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia wherein the gene-editing cassette comprises a base-editing system.

[0129] It is an aspect of the present disclosure, alone or in combination with any other aspects provided herein, wherein the present disclosure relates to a method of treating androgenic alopecia wherein the gene-editing cassette comprises a prime-editing system.

[0130] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting. As used in the specification and appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0131] It is noted that one or more of the following claims utilize the term “wherein” as a transitional phrase. For the purposes of defining the present technology, it is noted that this term is introduced in the claims as an open-ended transitional phrase that is used to introduce a recitation of a series of characteristics of the structure and should be interpreted in like manner as the more commonly used open-ended preamble term “comprising.”

[0132] It should be understood that where a first component is described as “comprising” or “including” a second component, it is contemplated that, in some embodiments, the first component “consists” or “consists essentially of’ the second component. Additionally, the term “consisting essentially of’ is used in this disclosure to refer to quantitative values that do not materially affect the basic and novel characteristic (s) of the disclosure.

[0133] It should be understood that any two quantitative values assigned to a property or measurement may constitute a range of that property or measurement, and all combinations of ranges formed from all stated quantitative values of a given property or measurement are contemplated in this disclosure.

[0134] While particular embodiments have been illustrated and described herein, it should be understood that various other changes and modifications may be made without departing from scope of the claimed subject matter. Moreover, although various aspects of the claimed subject matter have been described herein, such aspects need not be utilized in combination. It is therefore intended that the appended claims cover all such changes and modifications that are within the scope of the claimed subject matter.

Claims

CLAIMS1. A method of treating androgenic alopecia in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition to a hair follicle of the subject, wherein the pharmaceutical composition comprises a lipid nanoparticle encapsulating a gene-editing cassette and wherein the gene-editing cassette inactivates an androgen receptor in the hair follicle.

2. The method of claim 1, wherein the gene-editing cassette comprises a nucleotide binding domain and a guide polynucleotide.

3. The method of claim 2, wherein the nucleotide binding domain is a Cas protein.

4. The method of claim 2, wherein the pharmaceutical composition comprises from about 10 pM to about 1000 pM of the guide polynucleotide.

5. The method of claim 2, wherein the pharmaceutical composition comprises from about 1 ng / pL to about 500 ng / pL of the nucleotide binding domain.

6. The method of claim 1, wherein the lipid nanoparticle is selected from the group consisting of ionizable lipid nanoparticles, cationic lipid nanoparticles, PEGylated lipid nanoparticles, and solid lipid nanoparticles.

7. The method of claim 1, wherein the gene-editing cassette comprises a CRISPR-Cas9 system comprising a gRNA.

8. The method of claim 7, wherein the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-394.

9. The method of claim 7, wherein the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-85.

10. The method of claim 7, wherein the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-29.

11. The method of claim 7, wherein the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-11.

12. The method of claim 7, wherein the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-8.

13. The method of claim 7, wherein the gRNA has a nucleotide sequence selected from SEQ ID NOs: 1-5.

14. The method of claim 1, wherein the gene-editing cassette comprises a base-editing system.

15. The method of claim 1, wherein the gene-editing cassette comprises a prime-editing system.