Compositions, systems, and methods for allele-specific prime editing

Abstract

The present disclosure provides systems, methods, and compositions for modifying a target nucleic acid. Particularly, the present invention relates to modifying sequences in the 5' UTR near the Kozak sequence to silence a gene, for example, mutating a target rhodopsin allele start codon or deleting a region of the target rhodopsin allele comprising at least a portion of the start codon.

Description

COMPOSITIONS, SYSTEMS, AND METHODS FOR ALLELE-SPECIFIC PRIME EDITINGFIELD

[0001] The present invention relates to systems, methods, and compositions for modifying a target nucleic acid. Particularly, the present invention relates to modifying sequences in the 5’ UTR near the Kozak sequence to silence a gene, for example, silencing a disease-associated allele while leaving the wild-type allele unmodified.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 658,321, filed June 10, 2024, the content of which is herein incorporated by reference in its entirety.SEQUENCE LISTING STATEMENT

[0003] The content of the electronic sequence listing titled COLUM_43167_601_SequenceListing.xml (Size: 26,476 bytes; and Date of Creation: June 5, 2025) is herein incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under EY024698 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0005] Inherited retinal disorders (IRDs) represent a collection of genetically highly heterogeneous neurodegenerative disorders including retinitis pigmentosa that ultimately result in blindness. While there has been substantial preclinical progress in precision medicines for more common forms of retinal degenerations, therapeutic development for rare mutations unfortunately remains handicapped by burdensome developmental costs. Currently, there is only one FDA approved treatment for retinitis pigmentosa, which addresses a highly narrow subset of patients with mutations specifically in the RPE65 gene. For the remaining >100,000 patients in the United States alone, there exists no treatment. Thus, there remains a need for the development of methods and systems for use in broadly treating disease-associated, defective alleles.SUMMARY

[0006] Provided herein are systems for modifying a target rhodopsin allele. In some embodiments, the systems comprise: a Cas protein, or a nucleic acid encoding thereof; a reverse transcriptase, or a nucleic acid encoding thereof; one or more RNA polynucleotides comprising a spacer sequence and an extension sequence comprising a primer binding sequence (PBS) and a reverse transcriptase template (RTT) sequence; or one or more nucleic acids encoding thereof; and a nicking guide RNA (ngRNA), or a nucleic acid encoding thereof. In some embodiments, the RTT sequence encodes a sequence to mutate the target rhodopsin allele start codon or delete a region of the target rhodopsin allele comprising at least a portion of the start codon.

[0007] In some embodiments, the spacer sequence is configured to target rs7984(A / G) single nucleotide polymorphism in target rhodopsin allele. In some embodiments, the spacer sequence comprises SEQ ID NO: 1.

[0008] In some embodiments, the RTT sequence encodes a sequence to delete at least 31 basepairs of the target rhodopsin allele. In some embodiments, the RTT sequence comprises a sequence complementary to a sequence of the target rhodopsin allele downstream of the start codon. In some embodiments, the RTT sequence encodes a sequence to mutate the target rhodopsin allele start codon to a stop codon.

[0009] In some embodiments, the PBS comprises a sequence complementary to a sequence of the target rhodopsin allele upstream of the start codon. In some embodiments, the PBS comprises a sequence complementary to a sequence of the target rhodopsin allele upstream of site of cleavage by the Cas protein.

[0010] In some embodiments, the extension sequence comprises any of SEQ ID NOs: 2-14.

[0011] In some embodiments, the target rhodopsin allele comprises one or more disease- associated mutations.

[0012] In some embodiments, the spacer sequence and the extension sequence are contained within a single RNA polynucleotide.

[0013] In some embodiments, the Cas protein is Cas9 or a variant or fragment thereof. In some embodiments, the Cas protein is a Cas9 nickase and / or the Cas protein comprises a Cas protein variant configured to target an expanded range of PAM sequences. In some embodiments, the Cas protein and the reverse transcriptase are contained within a single fusion protein.

[0014] In some embodiments, the system further comprises at least one additional RNA polynucleotide comprising a spacer sequence and an extension sequence comprising a primer binding sequence (PBS) and a reverse transcriptase template (RTT) sequence; or one or more nucleic acids encoding thereof. In some embodiments the extension sequence comprises a sequence complementary to a sequence downstream of the start codon of the target rhodopsin allele. In some embodiments, the extension sequence targets a strand of the target rhodopsin allele opposite of that of the one or more RNA polynucleotides.

[0015] Also provided herein are methods for modifying a target rhodopsin allele comprising contacting a DNA encoding the rhodopsin allele with a system disclosed herein.

[0016] Further provided herein are methods for reducing levels of rhodopsin in a cell comprising introducing into the cell encoding a target rhodopsin allele a system disclosed herein.

[0017] In some embodiments, the target rhodopsin allele comprises one or more disease- associated mutations. In some embodiments, the target rhodopsin allele is an autosomal dominant disease-associated allele.

[0018] Additionally provided herein are methods of treating or preventing a disease or disorder in a subject in need thereof comprising administering of a system as disclosed herein to the subject. In some embodiments, the system is configured for delivery to retinal cells.

[0019] In some embodiments, the disease or disorder is caused or mitigated by a disease- associated allele of rhodopsin. In some embodiments, the disease-associated rhodopsin allele comprises one or more pathogenic mutations. In some embodiments, the system is configured to modify the disease-associated rhodopsin allele while not modifying a wild-type or non- pathogenic allele.

[0020] In some embodiments, the disease or disorder comprises retinal degeneration, retinitis pigmentosa, night blindness, macular dystrophy, vitelliform macular dystrophy, Leber congenital amaurosis, central areolar choroidal dystrophy, cone-rod dystrophy, or a combination thereof.

[0021] Also provided herein are systems and methods to modify a target gene. In some embodiments, the system comprise: a Cas protein, or a nucleic acid encoding thereof; a reverse transcriptase, or a nucleic acid encoding thereof; one or more RNA polynucleotides comprising a spacer sequence and an extension sequence comprising a primer binding sequence (PBS) and a reverse transcriptase template (RTT) sequence; or one or more nucleic acids encoding thereof; and a nicking guide RNA (ngRNA), or a nucleic acid encoding thereof. In some embodiments,the RTT sequence encodes a sequence to mutate a target gene start codon or delete a region of the target gene comprising at least a portion of the start codon.

[0022] In some embodiments, the target gene comprises a variation (e.g., a single nucleotide polymorphism) in the region surrounding the start codon. In select embodiments, the target gene comprises a variation (e g., a single nucleotide polymorphism) upstream of the start codon. In some embodiments, the target gene comprises contain one or more pathogenic or disease- associated mutations or alterations. In some embodiments, the one or more pathogenic or disease-associated mutations or alterations are linked to the variation (e.g., a single nucleotide polymorphism). In some embodiments, the target gene is sequenced to determine the one or more pathogenic or disease-associated mutations or alterations linked to the variation (e.g., a single nucleotide polymorphism). In some embodiments, the methods target and modify the disease-associated target gene allele while leaving the wild-type or non-pathogenic allele unmodified.

[0023] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1A is a schematic of the prevalence of rs7984 SNP by geographical region. FIG. IB is a schematic of the potential genotype paradigms seen in general population. Heterozygous genotypes (1 & 2) are potentially treatable by SNV editing. FIG. 1C is a pie chart showing the estimated fraction of populations with genotypes amenable to treatment as determined by ClinVar Database.

[0025] FIG. 2A is sequencing results of plasmid engineering of CMV-hRHO constructs (ATG— >TAG and deletion) in addition to immunoblotting demonstrate that terminal editing products can effectively silence gene expression. CMB-hRHA - SEQ ID NOs: 19 and 20; ATG— >TAG modification is SEQ ID NO: 21. FIG. 2B is a graph of on target editing efficiency of start to stop codon conversion (ATG— >TAG) PE systems. FIG. 2C is a graph of on target editing efficiency of Start Codon deleting PE systems.

[0026] FIG. 3A is Sanger Sequencing results of prime edited cell lines homozygous for either SNV (rs7984_A / G). Reference Human RHO and WT HEK 293T (A / A) - SEQ ID NOs: 22 and 23; Engineered HEK293T (G / G) is SEQ ID NO: 24. FIG. 3B is a graph of OFF-Targetinganalysis of PE systems editing in the engineered HEK293T rs7984 G / G cell line. FIG. 3C is a graph of the ON:OFF ratio targeting as determined by NGS.

[0027] FIG. 4A is a schematic representation of PRIME-Del (Choi, J., Nat Biotechnol 40, 218-226 (2022)) strategy to excise region of the genome containing the Start Codon for Rhodopsin. FIG. 4B is a graph of ON-Targeting analysis of PRIME-Del PE systems editing in the HEK293T cells comparing recently published novel editors. FIGS. 4C and 4D are graphs of OFF-target analysis in G / G editing HEK293T cells (FIG. 4C) and the ratio between ON-target (A / A) and OFF-target editing efficiencies (FIG. 4D). FIG. 4E is a graph of the independent function of secondary pegRNA indicates high off-target effects independent of allele-specific pegRNA.

[0028] FIGS. 5 A and 5B are editing results, as determined by next generation sequencing, of on (FIG. 5 A) and off (FIG. 5B) target analysis for second round of optimized prime editing systems exploring various RTT and PBS lengths. FIG. 5C is a graph showing the optimized lead candidate system is able to achieve robust on target editing rates consistently (>40%) while minimizing off-target edits (-8-9%). FIG. 5D is a graph demonstrating feasibility of delivering these therapeutic components in dual vector systems compatible with AAV delivery.

[0029] FIG. 6A is a diagram detailing the CRISPR engineering design to humanize the Rhodopsin gene, including homologous arms and validating primers. FIG. 6B is PCR amplification demonstrating successful integration using primers specific to junctions and insertion fragment. FIG. 6C is GeneWiz sequencing results from Fl mice indicating successful humanization and successful installation of pathogenic P347L mutation. rRHO - SEQ ID NOs: 25 and 26; WT Fl - SEQ ID NO: 27; P347L Fl - SEQ ID NO: 28. FIG. 6D is qPCR analysis of mouse and human rhodopsin genes indicating successful and selective expression of transgene and silencing of endogenous mouse RHO.

[0030] FIG. 7A is a graph of rod responses of humanized and pathogenic P347L models compared to wildtype (wt) mice at 4, 8, and 12 weeks. FIG. 7B is a graph of cone responses of humanized and pathogenic P347L models compared to wildtype (wt) mice at 4, 8, and 12 weeks. FIG. 7C is graphs of max responses (a and b waves) of humanized and pathogenic P347L models compared to wildtype (wt) mice at 4, 8, and 12 weeks. FIG. 7D is a diagram of retinal cross sections used for histological evaluation of outer nuclear layer (ONL) thickness, with the appropriate row annotated by blue bars. FIG. 7E is a spidergrams detailing ONL thickness as afunction of distance from optic nerve head demonstrate histology in the humanized mice comparable to WT mice, with the pathogenic P347L mice showing a clear progressive thinning across the evaluated timepoints.

[0031] FIGS. 8 A and 8B show the rod responses (rod-driven b responses (FIG. 8 A) and maximum a responses (FIG. 8B)) in mice eyes treated with the lead candidate system, as in FIG. 5C but adapted to the split-intein design to be AAV compatible, as compared to untreated contralateral retinae at 8 weeks.

[0032] FIGS. 9A and 9B are histological retinal cross sections at 30X magnification used for evaluation of outer nuclear layer (ONL) thickness, as indicated, for a retina treated with the lead candidate system (FIG. 9A) and an untreated contralateral retina (FIG. 9B), as in FIG. 8.DETAILED DESCRIPTION

[0033] The work presented herein details a therapeutic prime editing strategy generalized to treat multiple mutations in the most implicated inherited retinal disease (IRD) gene, rhodopsin (RHO). While the collective autosomal dominant RHO mutation population ( / 7 / O-adRP patients) is quite sizable (>10,000 patients in the US), dozens of pathogenic variants have been identified, many with increasingly rare frequencies that make the clinical development of mutation-specific therapies financially prohibitive. Current CRISPR / Cas9 based approaches suffer shortcomings due to double-strand breaks, deletion sizes, and impacts on the wildtype allele.

[0034] Described herein is an allele-targeting, mutation-agnostic prime editing strategy with the potential to treat a substantial population of AHO-adRP patients independent of their underlying mutation. In the RHO gene, there are only two SNPs that occur with high frequency: rs2855558 (A / G) located deep within the 3’ UTR and rs7984 (A / G) located in the 5’ UTR near the translation start codon. Given the prevalence (-50% of the population is heterozygous) and positioning of rs7984, the disclosed methods can target this marker (FIG. 1). While not PAM generating, this SNP creates a mismatch in the most distal 3’ base of the protospacer sequence and can confer meaningful specificity.

[0035] By capitalizing on a highly heterozygous SNP, rs7984, found within the 5’ untranslated region (UTR) near the Kozak sequence, a target allele can be selectively silenced using prime editing to mutate the start codon and or confer deletions about the start codon, leaving the wildtype or non-target allele intact to support basic biological function. To assess themethods, a humanized mouse model of rhodopsin bearing the targeted SNP in addition to the clinically relevant, pervasive, and heterozygous P347L dominant negative mutation was developed. Mice treated with the disclosed systems show increased rod responses and thickening of the outer nuclear layer (ONL).

[0036] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.Definitions

[0037] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0038] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0039] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0040] As used herein, the terms “administering,” “providing,” and “introducing,” are used interchangeably herein and refer to the placement into a cell, organism, or subject by a method or route which results in at least partial localization to a desired site. Administration can be by any appropriate route which results in delivery to a desired location in the cell, organism, or subject.

[0041] The term “contacting” as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or of immediate or local proximity.

[0042] The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., a ribosomal or transfer RNA), a polypeptide, or a precursor of any of the foregoing. The RNA or polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained. Thus, a “gene” refers to a DNA or RNA, or portion thereof, that encodes a polypeptide or an RNA chain that has functional role to play in an organism. For the purpose of this disclosure, it may be considered that genes include regions that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0043] The terms “disease-associated” and “disease-causing” refer to any gene or mutations or polynucleotide whose products are expressed at an abnormal level or in an abnormal form in cells obtained from a disease-affected individual as compared with tissues or cells obtained from an individual not affected by the disease. For example, a disease-associated gene may be expressed at an abnormally high level or at an abnormally low level, where the altered expression correlates with the occurrence and / or progression of the disease. A disease-associated gene also refers to a gene, the mutation or genetic variation of which is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease.

[0044] A cell has been “genetically modified,” “transformed,” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0045] As used herein, a “nucleic acid” or a “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793- 800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA / RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and / or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and / or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or doublestranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0046] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies.

[0047] Nucleic acid or amino acid sequence “identity,” as described herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T- Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3x, FAS™, and S SEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad Sci. USA, 106( Q): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(1) 951- 960 (2005), Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).

[0048] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non- human) that may benefit from the administration of devices and systems contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods herein, the mammal is a human.

[0049] As used herein, “treat,” “treating,” and the like means a slowing, stopping, or reversing of progression of a disease or disorder. The term also includes a reversing of theprogression of such a disease or disorder to a point of eliminating or greatly reducing the disease. As such, “treating” means an application or administration where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.

[0050] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an “insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell.

[0051] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.Prime Editing Systems

[0052] Prime editing is a double-strand break (DSB)-independent clustered-regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system that can ameliorate both transition and transversion mutations in addition to small deletions and insertions. Generally, a prime editing guide RNA (pegRNA) is used in conjunction with a prime editor, e.g., a H840A Streptococcus pyogenes Cas9 (spCas9) nickase linked to an optimized Moloney murine leukemia virus (MMLV) reverse transcriptase (RT).

[0053] pegRNAs are similar to standard single-guide RNAs (sgRNAs) but differ due to a sequence comprising a primer binding site (PBS) and a reverse transcription template (RTT) sequence. The primer binding site hybridizes with the bases upstream of the prime editor generated nick, while the RTT encodes the information of the intended edits and directs reverse transcription. Together, the prime editor and the pegRNA form the prime editing 2 strategy (PE2). The Cas9 nickase is guided to the DNA target site by the pegRNA. After nicking by Cas9, the reverse transcriptase uses the pegRNA to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand. The edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand. Once the prime editor incorporates the edit into one strand, there is a mismatch between the original sequence on one strand and the edited sequence on the other strand. In some embodiments, an additional nicking guide RNA (ngRNA) is used to nick the non-edited strand,directing DNA repair enzymes to use the edited strand as a template to remake the mismatched strand. The prime editor, the pegRNA, and ngRNA form prime editing 3 (PE3) strategies.

[0054] Disclosed herein are methods and systems for modifying a gene. The methods and systems comprise a sequence-specific nuclease, or a nucleic acid encoding thereof; an RNA- dependent DNA polymerase, or a nucleic acid encoding thereof; one or more RNA polynucleotides comprising a spacer sequence and an extension sequence comprising a primer binding sequence (PBS) and a reverse transcriptase template (RTT) sequence, or one or more nucleic acids encoding thereof; and a nicking guide RNA (ngRNA), or a nucleic acid encoding thereof.

[0055] In some embodiments, the RTT sequence comprises a sequence to mutate the start codon. In some embodiments, the RTT sequence comprises a sequence to replace the start codon with a stop codon. In some embodiments, the RTT sequence comprises a sequence to delete a region of the gene comprising the start codon.

[0056] The methods may be applicable to modifying any gene. In some embodiments, the methods are used to modify genes which include alleles comprising a variation in the region upstream of the start codon. In some embodiments those alleles may contain one or more pathogenic or disease-associated mutations or alterations. Such, that the methods allow targeting and modification of the disease-associated alleles while leaving the wild-type or non-pathogenic alleles unmodified.

[0057] In some embodiments, the system and methods are configured to modify a rhodopsin gene. In some embodiments, the system is configured to target rs7984(A / G) single nucleotide polymorphism (SNP) in rhodopsin alleles. In some embodiments, the system is configured to target the rs7984(A / G) SNP in a disease-associated rhodopsin allele comprising one or more disease-associated or pathogenic mutations.1. Sequence-specific nuclease

[0058] Exemplary sequence-specific nucleases for use in the present invention include, but are not limited to, Cas proteins, Argonaute (Ago) proteins, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALEN). In some embodiments, the sequencespecific nuclease is a Cas protein.

[0059] Cas proteins are described in further detail in, e.g., Haft et al., PLoS Comput. Biol., 1(6): e60 (2005), incorporated herein by reference. The Cas protein may be any Casendonuclease, or fragment or naturally occurring or engineered variants thereof In some embodiments, the Cas endonuclease is a Class 2 Cas endonuclease. In some embodiments, the Cas endonuclease is a Type V Cas endonuclease. In some embodiments, the Cas protein is Cas9, Casl2a, otherwise referred to as Cpfl, or Casl4. In one embodiment, the Cas9 protein is a wildtype Cas9 protein. In some embodiments, the Cas9 protein is a Cas9 variant.

[0060] The Cas9 protein can be obtained or derived from any suitable microorganism, and a number of bacteria express Cas9 protein orthologs or variants. In some embodiments, the Cas9 is from Streptococcus pyogenes or Staphylococcus aureus. Cas9 proteins of other species are known in the art (see, e.g., U.S. Patent Application Publication 2017 / 0051312, incorporated herein by reference) and may be used in connection with the present disclosure. The amino acid sequences of Cas proteins from a variety of species are publicly available through the GenBank and UniProt databases.

[0061] In certain embodiments, a Cas nuclease can only cleave a target sequence if an appropriate PAM is present. See, for example Doudna et al., Science, 2014, 346(6213): 1258096, incorporated herein by reference. A PAM site is a nucleotide sequence in proximity to a target sequence. For example, PAM site may be a DNA sequence immediately following the DNA sequence targeted by the Cas protein. A PAM can be 5' or 3' of a target sequence. A PAM can be upstream or downstream of a target sequence. A PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. In certain embodiments, a PAM is between 2-6 nucleotides in length. Nonlimiting examples of the PAM sequences include: CC, CA, AG, GT, TA, AC, CA, GC, CG, GG, CT, TG, GA, AGG, TGG, T-rich PAMs (such as TTT, TTG, TTC, etc ), NGG, NGA, NAG, and NGGNG, where “N” is any nucleotide.

[0062] In some embodiments, the Cas protein comprises a Cas variant configured to target an expanded or altered range of PAM sequences which may facilitate essentially PAMless cleavage. In some embodiments, the Cas protein comprises a variant of the Streptococcus pyogenes Cas9 enzyme selected from xCas9, Cas9-VQR, SpG and SpRY. See, for example, Walton et al., Science. 2020 Apr 17;368(6488):290-296, Hu, et al., Nature 2018; 556 (57-63), Kleinstiver et al., Nature 2015; 523(7561):481-5, Hu et al., Mol Plant 2016; 9, 43-945, incorporated herein by reference in their entirety.

[0063] In some embodiments, the Cas protein is a Cas9 nickase (Cas9n). Wild-type Cas9 has two catalytic nuclease domains facilitating double- stranded DNA breaks. A Cas9 nickase proteinis typically engineered through inactivating point mutation(s) in one of the catalytic nuclease domains causing Cas9 to nick or enzymatically break only one of the two DNA strands using the remaining active nuclease domain. Cas9 nickases are known in the art (see, e.g., U.S. Patent Application Publication 2017 / 0051312, incorporated herein by reference) and include, for example, Streptococcus pyogenes with point mutations at DIO or H840.2. RNA-dependent DNA polymerase

[0064] RNA-dependent DNA polymerases (e.g., reverse transcriptases) synthesize complementary DNA using RNA as a template. Any RNA-dependent DNA polymerase, or variant, truncation, or single subunit thereof or enzyme having RNA-dependent DNA polymerase activity can be utilized in the systems and methods herein. Exemplary RNA- dependent DNA polymerases include retroviral reverse transcriptases, retrotransposon reverse transcriptases, bacterial reverse transcriptases, Tth DNA polymerase, Taq DNA polymerase, Tma DNA polymerase, and functional variants or fragments thereof. In some embodiments, the RNA-dependent DNA polymerases is a Moloney murine leukemia virus (MMLV) reverse transcriptase.

[0065] In some embodiments, the reverse transcriptase and the sequence-specific nucleases (e.g., Cas protein) comprise a fusion protein, also referred to herein as a prime editor. The reverse transcriptase can be fused to the sequence-specific nucleases (e.g., Cas protein) in any orientation and may be separated from the sequence-specific nucleases (e.g., Cas protein) with an amino acid linker.

[0066] In some embodiments, the reverse transcriptase and the sequence-specific nuclease (e.g., Cas protein) are provided in a split system. For example, the reverse transcriptase and the sequence-specific nuclease are provided as two or more different polypeptides (or nucleic acids encoding the two or more different polypeptides) such that two or more separate polypeptides together form a functional fusion protein or prime editor. In some embodiments, the sequences that encode the two or more separate polypeptides are present on the same vector. In some embodiments, they are present on two or more separate vectors. A split system can be used for any number of reasons such as overcoming a packing limit of vector or other delivery vehicle or regulating the active system by temporal or spatial introduction of the two or more vectors. Split systems include, but are not limited to, intein, MS2 or SunTag based systems. The split systemmay comprise more than one split system type (e.g., an intein based system and a SunTag based system) or more than one split system of a single type (e.g., one or more intein based systems).

[0067] In some embodiments, the split system is a split intein system. A “split intein” involves two or more complementary part inteins, for example two or more pairs of an N- intein and C-intein, that associate selectively and extremely tightly to form a full intein. As used herein, the word “intein” means a naturally occurring or artificially-constructed polypeptide sequence embedded within a precursor protein that can catalyze a splicing reaction during posttranslation processing of the protein. A list of known inteins is published at neb.com / inteins.html.

[0068] Inteins function as protein introns and are excised out of a protein while the remaining flaking regions (exteins) are joined by a peptide bond. Split inteins systems join two polypeptides without leaving a scar. In terms of split site selection particular attention is given to split sites which are surface exposed to lessen any steric hindrance during protein splicing. Thus, a functional reverse transcriptase and sequence-specific nuclease fusion protein can be reconstituted from two or more separate polypeptides by using a split-intein protein splicing strategy by respectively fusing dipartite domains that interact with each other on two ends of the two or more separate polypeptides desired to be joined.

[0069] In some embodiments, the prime editor is a variant prime editor containing a reverse transcriptase and / or Cas9 variant, including for example PE4, PE5, PE6a, PE6b, PE6c, PE6d, PE6e, PE6f, PE6g, and the like. See, for example, Peter J. Chen, et al., Cell, 2021 Oct 28;184(22):5635-5652.e29 and Jordan L Doman, et al., Cell, 2023 Aug 31 ; 186(18):3983- 4002. e26.3. pegRNA

[0070] The systems and methods disclosed herein include a spacer sequence and an extension sequence comprising a primer binding sequence (PBS) and a reverse transcriptase template (RTT) sequence, or one or more nucleic acids encoding thereof. In some embodiments, each of the spacer sequence, PBS, and RTT sequence are provided as a single prime editing guide RNA (pegRNA), or a nucleic acid encoding thereof. The spacer sequence directs the nuclease to bind to a DNA molecule having complementarity with the pegRNA, the PBS hybridizes with the bases upstream of the nuclease generated nick (e.g., upstream of the start codon), and the RTTencodes the information of the intended edits (e g., a mutation of the start codon or deletion of a region comprising at least a portion of the start codon) and directs reverse transcription.

[0071] The spacer sequence, the RTT sequence, and the PBS may be selected from any of the sequences disclosed herein or fragments or extensions thereof which lack or include one or more nucleotides from the 5’ and / or 3’ end. The extension sequence as a whole may also include one or more nucleotides from the 5’ and / or 3’ end.

[0072] In some embodiments, the spacer sequence is configured to target a sequence upstream of the start codon. In some embodiments, the spacer sequence is configured to target rs7984(A / G) single nucleotide polymorphisms in the rhodopsin gene. Thus, the spacer sequence can differentiate between two alleles having different SNPs in the rhodopsin gene. In some embodiments, the spacer sequence comprises SEQ ID NO: 1.

[0073] As described above the RTT sequence may encode a sequence to delete a region of a gene (e.g., the rhodopsin gene) comprising at least a portion of the start codon. In some embodiments, the RTT sequence encodes a sequence to delete at least 31 basepairs (bp) of the rhodopsin gene. The size of the region of the gene is dependent on the RTT sequence and the PBS. The deletion may be greater than 3 Ibp, for example, about 35bp, about 40bp, about 45bp, about 50bp, about 55bp, about 60bp, about 65pb, about 70bp, about 75bp, about 80bp, about 85bp, about 90bp, about lOObp, about 200bp, about 300bp, about 400bp, about 500bp, or more.

[0074] The RTT sequence comprises a sequence complementary to a sequence of the rhodopsin gene downstream of the start codon. Thus, when the prime editing ‘repairs’ the nick, the result is removal of the start codon and a product in which the region of the gene complementary to the RTT sequence (downstream of the start codon) is adjacent a sequence upstream of the start codon which is complementary to the PBS. Thus, the desired deletion can be tailored by the choice of lengths of PBS and RTT sequences, and the regions upstream and downstream of the start codon to which they are complementary.

[0075] The PBS may be at least 5bp, at least lObp, at least 15bp, at least 20bp, at least 25 or more in length. In some embodiments, the PBS is about 5bp to about 20bp in length. In some embodiments, the PBS is 5bp, 6bp, 7bp, 8bp, 9bp, lObp, 1 Ibp, 12bp, 13bp, 14bp, 15bp, 16bp, 17bp, 18bp, 19bp, or 20bp in length.

[0076] The RTT sequence may be at least 20bp, at least 25bp, at least 30bp, at least 35bp, at least 40bp, at least 45bp, at least 50bp, at least 55bp, at least 60bp, or more in length. In someembodiments, the RTT sequence is about 25bp to about 45bp in length. Tn some embodiments, the RTT sequence is 25bp, 26bp, 27bp, 28bp, 29bp, 3Obp, 31bp, 32bp, 33bp, 34bp, 35bp, 36bp, 37bp, 38bp, 39bp, 40bp, 41bp, 42bp, 43bp, 44bp, or 45 in length.

[0077] In some embodiments, the extension sequence comprises any of SEQ ID NOs: 2-14, or the sequences for the RTT or PBS as described herein.

[0078] “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule, which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization.

[0079] In some embodiments, the systems disclosed herein further comprise at least one additional pegRNA, or additional RNA polynucleotide(s) comprising a spacer sequence and an extension sequence (e.g., a PBS, and an RTT), or a nucleic acid encoding thereof, for targeting the opposite strand of the one or more RNA polynucleotides described above. For example, the at least one additional pegRNA can be used to delete any size portion or the entirety of the targeted rhodopsin gene (approximately 7 kb) using PRIME-Del and twinPE. Accordingly, combining a first pegRNA, or components thereof, targeting the rs7984(A / G) single nucleotide polymorphisms in the rhodopsin gene with a second pegRNA, or components thereof, targeting a opposite strand at the 3’ end of the target allele, in the 3’ UTR, or downstream of the 3’ UTR facilitates allele specific deletion of the entire gene or substantially the entire gene.

[0080] The pegRNAs may comprise additional structural elements or sequences including a gRNA scaffold responsible for Cas9 binding, a transcription termination sequence that the 3 ’ end of the molecule, and mutations or structural motifs that increase editing efficiency or enhance RNA stability or prevent RNA degradation. For example, the pegRNA may further comprise: a triple helix forming sequence (e.g., triple helix terminators from a long non-coding RNAs (IncRNAs), e.g., metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)); a tRNA- like sequence; a pseudoknot (e.g., a modified prequeosinei-1 riboswitch aptamer, (evopreQi) or the frameshifting pseudoknot from Moloney murine leukemia virus (MMLV)); and silent mutations near the intended edit (e.g., less than 10 bp away). See, for example, Nelson, et al. Nat Biotechnol. 2022 Mar;40(3):402-410, Chen, et al., Cell. 2021 Oct 28;184(22):5635-5652.e29,International Patent Publication No. W02022067130, each of which is incorporated herein by reference in its entirety.

[0081] The additional structural elements or sequences may be present at any location in the pegRNA which does not interfere with the function of the spacer sequence, primer binding sequence (PBS), and a reverse transcriptase template (RTT) sequence. In some embodiments, the additional structural elements or sequences are at the 3’ end of the pegRNA.4. Nicking guide RNA (ngRNA)

[0082] In some embodiments, the systems and methods comprise a nicking guide RNA (ngRNA) that complexes with the sequence-specific nuclease and introduces a nick in the nonedited DNA stand. In certain embodiments, the nick induced by using the ngRNA is on the opposite strand as the initial nick. In certain embodiments, the nick induced by using the ngRNA is on the same strand as the initial nick. Thus, the ngRNA sequence may target the same or different strand as the spacer sequence.

[0083] The ngRNA may be selected from any of the sequences disclosed herein or fragments or extensions thereof which lack or add one or more nucleotides from the 5’ and / or 3’ end. In some embodiments, the ngRNA comprises one, two, three, four, five, six, seven, eight, nine, or ten additional nucleotides on the 5’ and / or 3’ end as compared to the sequences disclosed herein.

[0084] The systems and methods may further include an engineered DNA mismatch repair (MMR)-inhibitor (e.g., protein or silencing RNA), or a nucleic acid encoding thereof. See Chen, et al., Cell. 2021 Oct 28;184(22):5635-5652.e29, incorporated herein by reference.Nucleic Acids

[0085] The present disclosure also provides for one or more nucleic acids encoding the systems disclosed herein, or components thereof, vectors containing these nucleic acids and cells containing the vectors. The vectors may be used to propagate the segment in an appropriate cell and / or to allow expression from the segment (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence. In some embodiments, the one or more nucleic acids comprise one or more messenger RNAs, one or more vectors, or any combination thereof.

[0086] Nucleic acids of the present disclosure (e.g., nucleic acids encoding the sequence specific nuclease, the RNA-dependent DNA polymerases, pegRNA, ngRNA, nucleic acids encoding pegRNA and ngRNA) can comprise any of a number of promoters, including, but notlimited to, constitutive, regulatable or inducible, cell type specific, tissue-specific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter / regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EFla (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta-actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit betaglobin splice acceptor), TRE (Tetracycline response element promoter), Hl (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the present system, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1- alpha (EFl -a) promoter with or without the EFl -a intron. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell.

[0087] Moreover, inducible expression can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible promoter / regulatory sequence. Promoters that are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter / regulatory sequence that is capable of driving expression of the desired protein operably linked thereto.

[0088] The present disclosure also provides for vectors containing the nucleic acids and cells containing the nucleic acids or vectors, thereof. The vectors may be used to propagate the nucleic acid in an appropriate cell and / or to allow expression from the nucleic acid (e.g., an expressionvector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.

[0089] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6: 187, incorporated herein by reference). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference.

[0090] The vectors of the present disclosure may direct the expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining.

[0091] Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene for selection of stable or transient transfectants in host cells; transcription termination and RNA processing signals; 5’-and 3 ’-untranslated regions; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and reporter gene for assessing expression of the chimeric receptor. Suitable vectors and methods for producing vectorscontaining transgenes are well known and available in the art. Selectable markers include chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, neomycin, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. cerevisiae.

[0092] When introduced into a cell, the vectors may be maintained as an autonomously replicating sequence or extrachromosomal element or may be integrated into host DNA.

[0093] Thus, the disclosure further provides for cells comprising a system for modifying a target nucleic acid, or one or more nucleic acids or vectors encoding thereof, as disclosed herein.

[0094] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids into cells, tissues, or a subject. Such methods can be used to administer the nucleic acids to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), a nucleic acid, and a nucleic acid complexed with a delivery vehicle.

[0095] Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. A variety of viral constructs may be used to deliver the present nucleic acids to the cells, tissues, and / or a subject. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated, baculoviral, and herpes simplex viral vectors. Nonlimiting examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant baculoviruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7(1 ):33-40; and Walther W. and Stein U., 2000 Drugs, 60(2): 249-71, incorporated herein by reference.

[0096] Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of cells. Transfection refers to the taking up of a vector by a cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-precipitation, electroporation, DEAE-dextran treatment, microinjection, viralinfection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and / or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.

[0097] Methods of delivering vectors to cells may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110(6): 2082-2087, incorporated herein by reference); or viral transduction. In some embodiments, the vectors are delivered to host cells by viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the construct containing the one or more transgenes can be delivered by any method appropriate for introducing nucleic acids into a cell.

[0098] Additionally, delivery vehicles such as nanoparticle- and lipid-based delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1 : 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1 ;459(1 -2):70-83), incorporated herein by reference.

[0099] As such, the disclosure provides an isolated cell comprising the vector(s) or nucleic acid(s) disclosed herein. Preferred cells are those that can be easily and reliably grown, have reasonably fast growth rates, have well characterized expression systems, and can be transformed or transfected easily and efficiently. Examples of suitable prokaryotic cells include, but are not limited to, cells from the genera Bacillus (such as Bacillus subtilis and Bacillus brevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces, Salmonella, and Envinia. Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. Examples of suitable yeast cells include those from the genera Kluyveromyces, Pichia, Rhino-sporidium, Saccharomyces, and Schizosaccharomyces. Exemplary insect cells include Sf-9 and HIS (Invitrogen, Carlsbad, Calif.) and are described in, for example, Kitts et al., Bioiechniques, 14'. 810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4 564-572 (1993); andLucklow et al., J. Virol., 67'. 4566-4579 (1993), incorporated herein by reference. A number of suitable mammalian and human host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, Va.). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR-cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), as well as the CV-1 cell line (ATCC No. CCL70).Further exemplary mammalian host cells include primate, rodent, and human cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, HEK, A549, HepG2, mouse L- 929 cells, and BHK or HaK hamster cell lines. Methods for selecting suitable mammalian cells and methods for transformation, culture, amplification, screening, and purification of cells are known in the art.

[0100] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo and delivery to the cell comprises administration to a subject.Methods

[0101] The disclosure also provides methods of modifying a rhodopsin (Rho) allele. The phrase “modifying a nucleic acid sequence,” as used herein, refers to modifying at least one physical feature of a nucleic acid sequence of interest. Nucleic acid modifications include, for example, single or double strand breaks, deletion, or insertion of one or more nucleotides, and other modifications that affect the structural integrity or nucleotide sequence of the nucleic acid sequence.

[0102] The disclosure also provides methods of decreasing rhodopsin levels in a cell or subject comprising introducing into the cell encoding a target rhodopsin allele with the disclosed system. The reduction may be about a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 97.5%, 99% or more reduction in rhodopsin, or a disease-associated rhodopsin variant. For example, the reduction may include a complete elimination, for example of the presence of a pathological form of rhodopsin.

[0103] In some embodiments, the methods comprise contacting DNA encoding a rhodopsin allele with the disclosed system. In some embodiments, the methods comprise contacting DNA encoding the rhodopsin allele with a Cas9 protein; a reverse transcriptase; one or more RNA polynucleotides comprising a spacer sequence and an extension sequence comprising a primer binding sequence (PBS) and a reverse transcriptase template (RTT) sequence; and a nicking guide RNA (ngRNA). In some embodiments, the RTT sequence comprises a sequence to mutate the start codon (e.g., to a stop codon) or a sequence to delete a region of the gene comprising the start codon.

[0104] In some embodiments, the DNA encoding the rhodopsin allele is in a cell. In some embodiments, the rhodopsin allele is endogenous to a target cell. In some embodiments, the rhodopsin allele is genomic. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is ex vivo. In some embodiments the cell is in vivo.

[0105] In some embodiments, contacting DNA encoding the rhodopsin allele comprises introducing into the cell: a sequence-specific nuclease (e.g. Cas protein), or a nucleic acid encoding thereof; a reverse transcriptase or a nucleic acid encoding thereof; and one or more RNA polynucleotides comprising a spacer sequence and an extension sequence comprising a primer binding sequence (PBS) and a reverse transcriptase template (RTT) sequence, or a nucleic acid encoding thereof; and a nicking guide RNA (ngRNA), or a nucleic acid encoding thereof. In some embodiments, the RTT sequence comprises a sequence to mutate the start codon (e.g., to a stop codon) or a sequence to delete a region of the gene comprising the start codon. In some embodiments, introducing into the cell comprises administering to a subject.

[0106] Also disclosed herein are methods for treating or preventing a disease or disorder in a subject by modifying a target rhodopsin gene with a system as disclosed herein. The methods comprise administering to a subject: a sequence-specific nuclease (e.g. Cas protein), or a nucleic acid encoding thereof; an RNA-dependent DNA polymerase, or a nucleic acid encoding thereof; one or more RNA polynucleotides comprising a spacer sequence and an extension sequence comprising a primer binding sequence (PBS) and a reverse transcriptase template (RTT) sequence, or one or more nucleic acids encoding thereof; and a nicking guide RNA (ngRNA), ora nucleic acid encoding thereof, wherein the RTT sequence encodes a sequence to mutate the rhodopsin gene start codon (e.g., to a stop codon) or delete a region of the rhodopsin gene comprising at least a portion of the start codon.

[0107] In some embodiments, the disease or disorder is an ocular disease or disorder. In some embodiments, the disease or disorder is a retinal disease or disorder. In some embodiments, the disease or disorder is an inherited retinal disorder (IRD). In some embodiments, the disease or disorder comprises disease or disorder comprises retinal degeneration, retinitis pigmentosa, night blindness, macular dystrophy, vitelliform macular dystrophy, Leber congenital amaurosis, central areolar choroidal dystrophy, cone-rod dystrophy, or a combination thereof.

[0108] In some embodiments, the disease or disorder is caused or mitigated by a disease- associated allele of rhodopsin (e.g., autosomal dominant disease-associated allele of rhodopsin). As described above, the disclosed system is agnostic to the individual mutations as it relies on modifying or removing the start codon to render the gene non-functional. Thus, the disease- associated rhodopsin allele may comprise one or more pathogenic mutations or variations.

[0109] In some embodiments, the system is configured to modify the disease-associated rhodopsin allele while not modifying a wild-type or non-pathogenic allele. Thus, the system may target the disease-associated rhodopsin allele based on the appropriate rs7984(A / G) SNP. In some embodiments, the methods further comprise determining the rs7984(A / G) SNP associated with the disease-associated rhodopsin allele and the wild-type or non-pathogenic allele. In instances where the SNP is different between the disease-associated rhodopsin allele and the wild-type or non-pathogenic allele, the pegRNA can be designed to target the disease-associated rhodopsin allele. In some embodiments, the methods further comprise determining the location and nature of the pathogenic mutations or variations in the rhodopsin gene.

[0110] In some embodiments, the disease or disorder is caused or mitigated by an imbalance in the amount of rhodopsin. Accordingly, the methods disclosed herein may be used to treat or prevent a disease or disorder by reducing the level of rhodopsin in the subject.[OHl] In some embodiments, the disease or disorder caused or mitigated by mutations in the rhodopsin gene or a decrease in the level of the rhodopsin gene. In some embodiments, the subject has a disease-associated variant of the rhodopsin gene.

[0112] In some embodiments, the systems or components thereof are configured for delivery to retinal cells. In some embodiments, the system is configured for delivery to rod and conephotoreceptor cells. For example, in some embodiments, the nucleic acids encoding the components may comprise a retinal cell (e.g., rod and / or cone photoreceptor cell) promoter which directs expression of the components in the retinal cells. Suitable retinal, rod, and / or cone photoreceptor cell promoters include, but are not limited to: 770En_454P(hG7?A / 6), a human GRM6 gene-derived, short promoter; promoters based on the 2.1 -kb human L-opsin promoter (pR2. 1); promoter derived from the rhodopsin kinase (RK) gene; promoter derived from the rhodopsin gene; a promoter derived from the Nrl gene; murine rhodopsin promoter (mOP); G-protein-coupled receptor protein kinase 1 (GRK1) promoter; retinol -binding protein 3, interstitial (RBP3) promoter; RPE65 promoter; human inter-photoreceptor retinoid binding protein / retinol-binding protein 3 (IRBP) promoter; and retinaldehyde binding protein 1 (RLBP1) promoter. Additionally, or alternatively, the systems or components are configured for administration to the eye and / or retina, rather than systemic administration.

[0113] Administration may be through any suitable mode of administration, including but not limited to: intravenous, intra-arterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, topical, and inhalation. In some embodiments, the systems or components are delivered to the tissue(s) of interest. Such delivery may be either via a single dose, or multiple doses.

[0114] In some embodiments, an effective amount of the components of the systems, methods or compositions as described can be administered. As used herein the term “effective amount” may be used interchangeably with the term “therapeutically effective amount” and refers to that quantity that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “effective amount” refers to that quantity of the components of the system such that successful modification of the rhodopsin gene or successful modulation of rhodopsin expression (e.g., a disease-causing rhodopsin mutant) is achieved.

[0115] When utilized as a method of treatment, the effective amount may depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effectiveamount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human.Examples

[0116] The following are examples of the present invention and are not to be construed as limiting.Example 1 Allele Specific Silencing

[0117] Multiple editing paradigms that could achieve the desired allele-specific silencing, including precise mutations of the start codon and larger deletions of the start codon, were explored. Plasmid engineering was performed to validate that the proposed editing outcomes would indeed ablate expression. Starting with a plasmid containing full-length RHO and GFP reporter proteins driven by a CMV promoter, site directed mutagenesis was employed to synthesize plasmids containing either the ATG MAG conversion or larger deletion surrounding the start codon (sequencing results shown in FIG. 2A). Plasmids were transfected into HEK293T cells, incubated for 72 hours, lysed and then immunoblotted for rhodopsin expression to demonstrate that both deletion and precise modification of start codon result in silencing relative to wildtype, unedited plasmid (FIG. 2A).

[0118] HEK293T cells were engineered via transduction with plasmids encoding for prime editing systems capable of installing either form of the targeted rs7984(A / G) SNV. From there, triple plasmid systems delivering separate nicking gRNA, pegRNA, and editing machinery were transfected into the appropriate cell line (AA for “ON-target” and GG for “OFF-target” analysis) to determine editing efficiencies and specificities. Initially, two potential editing routes were explored — precise modifications of the start codon (“ATG”“TAG” conversions) as well as deletions of the start codon and surrounding genome. Post transfection, cells were incubated for 72 hours, DNA was harvested and collected, NGS-compatible primers were used to amplify the region of interest, and amplicon-EZ services were performed by Genewiz. Results were then analyzed via CRISPResso2 to determine the percentage of modified reads.

[0119] A large screening of various combinations of nicking gRNA (ngRNA) and pegRNAs was performed via plasmid transfections, modulating the 3’ extension for PBS and RTT lengths. The 3’ extensions encoded for the desire genetic manipulation (either ATG — TAG or a largerdeletion including the start codon) within the RTT. Using barcoded PCR primers with partial Illumina adapters, samples were pooled for NGS analysis. An NGS demultiplexing pipeline was designed that segregated reads by barcode, screened for read quality, removed primer-dimer, and passed the following datasets into CRISPResso2 for NGS analysis / visualization of editing outcomes.

[0120] To achieve precise deletions when using prime editing, pegRNAs are programmed to have 3’ extensions encoding for complementarity to regions of DNA further downstream of the start codon. Upon reverse transcription into DNA, this region can then hybridize with the downstream loci, allowing for the cell’s endogenous repair mechanisms to prune out the internal flanked sequence, generating a precise genomic deletion. Multiple nicks by various pegRNAs, were used to test for differences in nick positioning and size of deletion. Various deletion sizes, ranging from the absolute minimum deletion size possible (31 base pairs, up-to and including only the start codon) to larger deletions (up to ~70 bp in size to allow for the amplicons to remain within NGS limits) were explored. The combinations of nicking gRNA (ngRNA) and pegRNAs (RTT and PBS) tested are described below.

[0121] One deletion and nicking site combination that performed well deleted 58bp from the genome. Keeping the size of the deletion and the nicking site fixed, subsequent rounds of modification on the pegRNA were performed to identify the effect of PBS and RTT length on editing efficiency. A PBS of 9 base pairs in length and a RTT of 36 base pairs achieved nearly 50% editing efficiency. In summary, a handful of ATG^TAG candidates achieved appreciable on-target editing by NGS (FIG. 2B) and a deletion strategy resulted in high editing efficiencies, up to 50% (FIG. 2C).

[0122] Protospacer sequence included in pegRNA: uucuugggugggagcagcc(AZG) (SEQ ID NO: 1), wherein (A / G) is the location of SNP and will change depending on the desired target allele.

[0123] The 3’ extension will change with the size of the deletion. Assuming a fixed size, the corresponding PBS and RTT lengths can then be varied.

[0124] Assuming a standard RTT length of 35 and a PBS length of 13, deletion sizes were explored using the following sequences protospacer features.

[0125] Using the 58 bp deletion size and Nick 1, the RTT and PBS sizes were modified with the following combinations.

[0126] Combination capable of achieving >40% efficiency.

[0127] A major component of an editing system is not only editing efficiency, but also specificity. To determine the allele-specificity, HEK293T cells were engineered via prime editing to be homozygous for either form of the SNV (rs7984 A or rs7984 G). Sanger sequencing results shown in FIG. 3A demonstrate pure colonies following multiple rounds of clonal isolation. Using the same candidates from the above screening, G / G homozygous HEK293T were transfected and analyzed for editing efficiency. While Nick 2 data sets performed best in terms of ON-target efficiency, OFF-targeting was substantial. As a result, ON / OFF-tar eting was used as a threshold and the best performing candidates were performed in triplicate, which facilitated identification of a combination of pegRNA 4 and Nick 3, capable of achieving -25-30% editing efficiency with a near 5-to-l ON:OFF ratio.

[0128] Dual pegRNA strategies (TwinPE) have been shown to substantially improve the efficiency of larger deletions, otherwise known as PRIME-Del. Multiple rounds of TwinPE screening were performed with various editing systems including recently released prime editors, PE6c and PE6d. While PRIME-Del substantially improved the ON-target efficiency of the editing, the second pegRNA functioned too well independently, exhibiting a high degree of OFF- targeting that is not desirable (FIG. 4). However, it was observed that PE6c and PE6d have the potential to improve editing efficiency and PE6d was used for in vivo testing as it had been previously validated in the mouse brain.

[0129] Leveraging the PE6D editor, a final round of pegRNA modifications was performed. The nicking position and edit size were kept consistent with the previous round’s best performing candidate (FIGS. 2 and 3). From these results, a system capable of performing robust target edits (>40% by NGS) while minimizing off-targeting in the complementary cell line (more than 4x decrease in efficiency) was identified, as summarized in FIGS. 5A-5C.

[0130] Currently, the most explored route for in vivo delivery of prime editing to the retina remains a dual AAV system. Thus, the capability to translate prime editing systems into multiplasmid and split intein systems was demonstrated herein (FIG. 5D).Example 2 Mouse Model

[0131] As there is limited homology between mouse and human RHO genes, a humanized model carrying the human genomic sequence is essential for clinical development. The mouse model contains human cDNA inserted in the endogenous mouse RHO loci with the clinicallyrelevant P347L mutation installed in heterozygosity that is responsible for model pathology in addition to the non-pathogenic rs_7984(A / G) SNV located in the 5’ UTR. Mouse embryos were CRISPR engineered via microinjections to insert the desired genomic payload (either the healthy humanized allele or the pathogenic humanized P347L allele) as shown in FIG. 6A. Mosaic founders were then further bred to generate reserves of homozygous humanized wildtype RHO and homozygous P347L mice. These mice are then crossed to generate the final model containing fully humanized alleles, the targeted SNV in heterozygosity, as well as the pathogenic variant also in heterozygosity.

[0132] The model was characterized by several metrics including functional and histological analysis. The humanized P347L mouse model accurately phenocopies the clinical presentation of RHO-adRP while allowing for the assessment of human-targeting gRNAs. Demonstrating a similar degeneration when compared to previously published humanized models of RHO-adRP such as the Cl 10R mouse, the model exhibited a progressive functional deficit as measured by electroretinogram (ERG) that positively correlated with histopathology, supporting a rod-cone dystrophy. Meanwhile, humanization (mice homozygous for wildtype human RHO genes) has been shown to have no impact on function or histology, indicating pathology is driven by the mutant protein’s expression and not a lack of gene expression. Results measuring outer nuclear layer (ONL) thickness, a key metric of photoreceptor counts, demonstrated a clear degeneration with progressive thinning as a function of time. Results are summarized in FIG. 7.Example 3 In vivo therapeutic

[0133] The lead candidate system comprising the preferred nicking, RTT, and PBS sequences, as determined from in vitro analysis described above, was adapted to a split-intein design and packaged into a dual AAV therapeutic. An additional third AAV was also manufactured and co-injected to allow for detection of GFP signal and confirm positive transduction in vivo via quantitative fundus autofluorescence (qAF). Humanized mice, such those as described in Example 2, were subretinally injected with the therapeutic mixture at 3 weeks of age and functionally characterized using electroretinograms (ERGs). Treated mouse eyes experienced an increase in rod-driven b responses (FIG. 8A) as well as maximum a responses (FIG. 8B) when compared by paired t-test to untreated contralateral retinae at 8 weeks. Representative histological analysis was carried out on both treated and untreatedcontralateral retinae, as shown in FIGS. 9A and 9B. The ONL in the treated retina is about twice as thick as the untreated retina (64.45 pm versus 30.13 pm, respectively).

[0134] The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention.

[0135] Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

CLAIMSWhat is claimed is:

1. A system for modifying a target rhodopsin allele comprising: a Cas protein, or a nucleic acid encoding thereof; a reverse transcriptase, or a nucleic acid encoding thereof; one or more RNA polynucleotides comprising a spacer sequence and an extension sequence comprising a primer binding sequence (PBS) and a reverse transcriptase template (RTT) sequence; or one or more nucleic acids encoding thereof; and a nicking guide RNA (ngRNA), or a nucleic acid encoding thereof, wherein the RTT sequence encodes a sequence to mutate the target rhodopsin allele start codon or delete a region of the target rhodopsin allele comprising at least a portion of the start codon.

2. The system of claim 1, wherein the spacer sequence is configured to target rs7984(A / G) single nucleotide polymorphism in the target rhodopsin allele.

3. The system of claim 1 or 2, wherein the spacer sequence comprises SEQ ID NO: 1.

4. The system of any of claims 1-3, wherein the RTT sequence: encodes a sequence to delete at least 31 basepairs of the target rhodopsin allele or a sequence to mutate the target rhodopsin allele start codon to a stop codon; and / or comprises a sequence complementary to a sequence of the target rhodopsin allele downstream of the start codon.

5. The system of any of claims 1-4, wherein the PBS comprises a sequence complementary to a sequence of the target rhodopsin allele upstream of the start codon and / or a sequence complementary to a sequence of the target rhodopsin allele upstream of site of cleavage by the Cas protein.

6. The system of any of claims 1-5, wherein the extension sequence comprises any of SEQ ID NOs: 2-14.

7. The system of any of claims 1-6, wherein the target rhodopsin allele comprises one or more disease-associated mutations.

8. The system of any of claims 1-7, wherein the spacer sequence and the extension sequence are contained within a single RNA polynucleotide and / or the Cas protein and the reverse transcriptase are contained within a single fusion protein.

9. The system of any of claims 1-8, wherein the Cas protein is a Cas9 nickase and / or the Cas protein comprises a Cas protein variant configured to target an expanded range of PAM sequences.

10. A method for modifying a target rhodopsin allele comprises contacting a DNA encoding the rhodopsin allele with a system of any of claims 1-9.

11. A method for reducing levels of rhodopsin in a cell comprising introducing into the cell encoding a target rhodopsin allele a system of any of claims 1-9.

12. The method of claim 10 or 11, wherein the target rhodopsin allele comprises one or more disease-associated mutations and / or is an autosomal dominant disease-associated allele.

13. A method of treating or preventing a disease or disorder in a subject in need thereof comprising administering of a system of any of claims 1-9 to the subject, wherein the disease or disorder is an inherited retinal disease or disorder, optionally selected from the group consisting of retinal degeneration, retinitis pigmentosa, night blindness, macular dystrophy, vitelliform macular dystrophy, Leber congenital amaurosis, central areolar choroidal dystrophy, cone-rod dystrophy, and combinations thereof, and wherein the system is optionally configured for delivery to retinal cells.

14. The method of claim 13, wherein the disease or disorder is caused or mitigated by a disease- associated allele of rhodopsin, comprising one or more pathogenic mutations.

15. The method of claim 14, wherein the system is configured to modify the disease-associated rhodopsin allele while not modifying a wild-type or non-pathogenic allele.

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