Prime editing compositions and methods of use

Abstract

The present disclosure relates to systems for prime editing and methods of use thereof. The present disclosure provides systems in which prime editing components are split between two expression cassettes in two vectors. The present disclosure further provides cardiac-specific promoters and expression cassettes in which a cardiac-specific promoter is operably linked to one or more prime editing component.

Description

Attorney Docket No.: TENA-070 / 03WO 334682-2515PRIME EDITING COMPOSITIONS AND METHODS OF USECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 706,496 filed October 11, 2024, U.S. Provisional Patent Application No. 63 / 802,335 filed May 8, 2025, and U.S. Provisional Patent Application No. 63 / 805,095 filed May 13, 2025, the entire disclosures of each of which are incorporated herein by reference in their entireties.STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is TENA_070_03WO_SeqList_ST26.xml. The XML file is 146,906 bytes, and was created on October 8, 2025, and is being submitted electronically via USPTO Patent Center.TECHNICAL FIELD

[0003] In some aspects, the present disclosure relates to systems for prime editing and methods of use thereof. In some aspects, the present disclosure provides systems in which prime editing components are split between two expression cassettes in two vectors. In some aspects, the expression cassettes comprise a cardiac-specific promoter operably linked to the prime editing components, and the system may be used for treatment of heart disease.BACKGROUND

[0004] Prime editing provides tools for precision editing of target genes without generation of double-stranded breaks. However, use of prime editing in vivo requires delivery of the prime editing machinery to target cells. There is a need for systems capable of delivering prime editing machinery to target cells. Design of such systems will require a vector, and promoters and expression cassettes will need to be designed to be compatible with the vector.Attorney Docket No.: TENA-070 / 03WO 334682-2515SUMMARY

[0005] In some aspects, the present disclosure provides modified cardiac troponin T (TNNT2) promoters. In some embodiments, the modified TNTT2 promoter comprises a polynucleotide comprising 350 or fewer nucleotides of SEQ ID NO: 5. In some embodiments, the polynucleotide comprises at least 120 nucleotides and not more than 350 nucleotides. In some embodiments, the polynucleotide comprises at least 300 nucleotides and not more than 350 nucleotides. In some embodiments, the polynucleotide comprises at least 120, at least 130, at least 140, or at least 150 nucleotides. In some embodiments, the polynucleotide consists of 304 bp. In some embodiments, the polynucleotide consists of SEQ ID NO: 8. In some embodiments, the polynucleotide consists of 350 bp. In some embodiments, the polynucleotide consists of SEQ ID NO: 7. In some embodiments, the polynucleotide consists of 150 bp. In some embodiments, the polynucleotide consists of SEQ ID NO: 9. In some embodiments, the polynucleotide consists of 131 bp. In some embodiments, the polynucleotide consists of SEQ ID NO: 10.

[0006] In some aspects, the present disclosure provides an expression cassette comprising a promoter described herein, operatively linked to a polynucleotide encoding a gene product. In some aspects, the present disclosure provides a vector comprising an expression cassette described herein. In some embodiments, the vector is an AAV vector. In some embodiments, the AAV vector is AAV9 or a variant thereof.

[0007] In some aspects, the present disclosure provides a system comprising:(i) a first vector comprising a first expression cassette comprising:(a) a first modified TNNT2 promoter operatively linked to a first polynucleotide encoding an N-terminal fragment of a fusion protein comprising an N-terminal fragment of an RNA-guided nickase and an N-terminal fragment of a split- intein;(ii) a second vector comprising a second expression cassette comprising:(a) a second modified TNNT2 promoter operatively linked to a second polynucleotide encoding a C-terminal fragment of the fusion protein comprising a C-terminal fragment of the RNA-guided nickase, a reverse transcriptase, and a C-terminal fragment of the split-intein; and(b) a third promoter operatively linked to a third polynucleotide encoding a prime editing guide RNA (pegRNA).Attorney Docket No.: TENA-070 / 03WO 334682-2515

[0008] In some embodiments, the first and second modified TNNT2 promoters are independently selected from any TNNT2 promoter descried herein and a TNNT2 promoter consisting of SEQ ID NO: 6. In some embodiments, the first expression cassette further comprises a fourth promoter operatively linked to a fourth polynucleotide encoding a nicking guide RNA (ngRNA). In some embodiments, the system expresses the fusion protein comprising the RNA-guided nickase and the reverse transcriptase.

[0009] In some embodiments, the C-terminal fragment of the fusion protein further comprises an RNA binding domain of small RNA binding exonuclease protection factor La. In some embodiments, the system expresses the fusion protein comprising the RNA-guided nickase, the reverse transcriptase, and the RNA binding domain of small RNA binding exonuclease protection factor La.

[0010] In some embodiments, the N-terminal fragment of the fusion protein comprises SEQ ID NO: 19 and the C-terminal fragment of the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-28 and 51-52. In some embodiments, the N-terminal fragment of the fusion protein comprises SEQ ID NO: 19 and the C-terminal fragment of the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 20-22, 24, 26, 28, 51, and 52.

[0011] In some embodiments, the first expression cassette comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 37 and the second expression cassette comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 38. In some embodiments, the first expression cassette comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 35 and the second expression cassette comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 36, 43, 44, 46, 48, 50, 53, and 54.

[0012] In some embodiments, the third promoter and / or fourth promoter are Pol III promoters. In some embodiments, the first and second vectors are adeno-associated virus (AAV) vectors.

[0013] In some embodiments, the first vector comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 37, a promoter operatively linked to an ngRNA, a left ITR, and a right ITR; and the second vector comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 38, a promoterAttorney Docket No.: TENA-070 / 03WO 334682-2515 operatively linked to a pegRNA, a left ITR, and a right ITR. In some embodiments, the first vector comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 35, a promoter operatively linked to an ngRNA, a left ITR, and a right ITR; and the second vector comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 36, 43, 44, 46, 48, 50, 53, and 54, a promoter operatively linked to a pegRNA, a left ITR, and a right ITR.

[0014] In some aspects, the present disclosure provides a method of editing a target gene in a cell comprising contacting the cell with a system described herein. In some embodiments, the cell is in vivo or ex vivo.BRIEF DESCRIPTION OF THE DRAWINGSE

[0015] FIGS. 1A-1B provide an alignment of modified TNNT2 promoter sequences (FIG. 1A) and a schematic of the design of a chimeric TNNT2+MYBPC3 promoter (FIG. IB).

[0016] FIGS. 2A-2B provide a schematic showing cloning of AAV transgene cassettes carrying each of the modified and chimeric promoters operationally linked to GFP (FIG. 2 A) and GFP expression levels of these cassettes in a pooled study in mouse hearts (FIG. 2B).

[0017] FIGs. 3A-3D show the design (FIGs. 3A-3C) and GFP expression levels in human iPSC-derived cardiomyocytes (FIG. 3D) of transgene cassette plasmids carrying different 3’ UTR and polyA signal sequences.

[0018] FIGs. 4A-4C show the design (FIGs. 4A-4B) and in vivo Dnmtl locus editing performance (FIG. 4C) of the VI 5 split-PE cassette design.

[0019] FIGs. 5A-5B show in vivo expression (FIG. 5A) of split-PE cassette design versions (V4, VI 3, VI 5, VI 6, and VI 8) and the design of the VI 3 cassette design (FIG. 5B).

[0020] FIGs. 6A-6B provide a schematic of an efficacy study (FIG. 6A) and ejection fractions (EF), left ventricle internal diameters at systolic stage (LVID;s), and left ventricle internal diameters at diastolic stage (LVID;d) at various time points (FIGs. 6B and 6C) after administration in a mouse Rbm20 cardiomyopathy model.

[0021] FIGs. 7A-7C show the design (FIG. 7A) and in vivo Dnmtl locus editing performance (FIG. 7B) of the VI 9 split-PE cassette compared to the VI 3 split-PE cassette andAttorney Docket No.: TENA-070 / 03WO 334682-2515 of the VI 9 split-PE cassette delivered by wild-type AAV9 and by an engineered AAV capsid.

[0022] FIGs. 8A-8B show the design (FIG. 8A) and in vitro gene editing efficiency (FIG. 8B) of split-PE designs VI 3, VI 9, V20, V21, V22, V23, V24, V25, and V26.

[0023] FIGs. 9A-9C provide a schematic of an efficacy study (FIG. 9A) and ejection fractions (EF), left ventricle internal diameters at systolic stage (LVID;s), and left ventricle internal diameters at diastolic stage (LVID;d) at various time points after administration (FIG. 9B) and at 20 weeks after administration (FIG. 9C) in a mouse Rbm20 cardiomyopathy model.

[0024] FIGs. 10A-10C provide a schematic of an efficacy study (FIG. 10A) and in vivo editing performance of the VI 9, V20, V22, and V24 split-PE designs, as measured in DNA reads (FIG. 10B) and RNA reads (FIG. 10C).

[0025] FIGs. 11A-11C show the design (FIG. 11 A) and in vitro gene editing efficiency of the VI 9, V20, V22, V24, V27, and V28 split-PE designs in cells using the peg-186 + ng-26 guide RNA pair (FIG. 11B) or the peg-189 + ng-26 guide RNA pair (FIG. 11C).DETAILED DESCRIPTION

[0026] The present technology relates to systems for prime editing and methods of use thereof. In some aspects, the present disclosure provides cardiac-specific promoters (e.g., a modified cardiac troponin 2 (TNNT2) promoter) and expression cassettes and vectors (e.g., an AAV vector) comprising such a promoter. In some aspects, the present disclosure provides two- vector systems wherein the first vector comprises an expression cassette that encodes an N- terminal fragment of an RNA-guided nickase and an N-terminal fragment of a split-intein, the second vector comprises an expression cassette that encodes a C-terminal fragment of the RNA- guided nickase, a reverse transcriptase, a C-terminal fragment of a split-intein, and a prime editing guide RNA (pegRNA), wherein the first and second vector each comprise one of the cardiac-specific promoters described herein. In some aspects, the present disclosure provides systems wherein the first expression cassette additionally encodes a nicking guide RNA (ngRNA). In some aspects, the present disclosure provides systems wherein the C-terminal fragment of the fusion protein further comprises the RNA binding domain of small RNA binding exonuclease protection factor La. In some aspects, the present disclosure provides methods of editing a target gene in a cell comprising contacting the cell with a system described herein.Attorney Docket No.: TENA-070 / 03WO 334682-2515Definitions

[0027] Unless the context indicates otherwise, the features of the invention can be used in any combination. Any feature or combination of features set forth can be excluded or omitted. Certain features of the invention, which are described in separate embodiments may also be provided in combination in a single embodiment. Features of the invention, which are described in a single embodiment may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are disclosed herein as if each and every combination were individually disclosed. All sub-combinations of the embodiments and elements are disclosed herein as if every such sub-combination were individually disclosed.

[0028] 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 this invention belongs. The detailed description is divided into sections only for the reader’s convenience and disclosure found in any section may be combined with that in another section. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the exemplary methods and materials are now described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. Reference to a publication is not an admission that the publication is prior art.

[0029] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to “a recombinant AAV virion” includes a plurality of such virions and reference to “the cardiac cell” includes one or more cardiac cells.

[0030] The conjunction “and / or” means both “and” and “or,” and lists joined by “and / or” encompasses all possible combinations of one or more of the listed items.

[0031] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to “a recombinant AAV virion” includes a plurality of such virions and reference to “the cardiac cell” includes one or more cardiac cells.

[0032] The conjunction “and / or” means both “and” and “or,” and lists joined by “and / or” encompasses all possible combinations of one or more of the listed items.

[0033] The use of numerical values in the various quantitative values specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the wordAttorney Docket No.: TENA-070 / 03WO 334682-2515"about." It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about.” It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios, such as about 2, about 3, and about 4, and sub-ranges, such as about 10 to about 50, about 20 to about 100, and so forth. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0034] The term “vector” refers to a macromolecule or complex of molecules comprising a polynucleotide or protein to be delivered to a cell. In some embodiments, a “vector” refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.

[0035] The term “variant” refers to a protein or nucleic acid having one or more genetic changes (e.g., insertions, deletions, substitutions, or the like) that returns all or substantially all of the functions of the reference protein or nucleic acid. For example, a variant of a therapeutic protein retains the same or substantially the same activity and / or provides the same or substantially the same therapeutic benefit to a subject in need thereof. A variant of a promoter sequence retains the ability to initiate transcription at the same, substantially the same, or an increased level as the reference promoter, and retains the same or substantially the same cell type specificity. In particular embodiments, polynucleotides variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence. In particular embodiments, proteinAttorney Docket No.: TENA-070 / 03WO 334682-2515 variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence.

[0036] The term “wild-type” or “WT” refers to the naturally-occurring polynucleotide sequence encoding a protein, or a portion thereof, or protein sequence, or portion thereof, respectively, as it normally exists in vivo in a normal or healthy subject.

[0037] ‘AAV” is an abbreviation for adeno-associated virus. The term covers all subtypes of AAV, except where a subtype is indicated, and to both naturally occurring and recombinant forms. The abbreviation “rAAV” refers to recombinant adeno-associated virus. “AAV” includes AAV or any subtype. “AAV5” refers to AAV subtype 5. “AAV9” refers to AAV subtype 9. The genomic sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), NC_006261 (AAV8), and AY530579 (AAV9). Publications describing AAV inclue Srivistava et al. (1983) J. Virol. 45:555; Chiorini et al. (1998) J. Virol. 71 :6823; Chiorini et al. (1999) J. Virol. 73:1309; Bantel-Schaal etal. (1999) J. Virol. 73:939; Xiao etal. (1999) J. Virol. 73:3994; Muramatsu et al. (1996) Virol. 221 :208; Shade et al. (1986) J. Virol. 58:921 ; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Mons et al. (2004) Virology 33:375-383; Int’l Pat. Publ Nos. WO2018 / 222503 Al, WO2012 / 145601A2, W02000 / 028061A2,WO 1999 / 61601A2, and WO1998 / 11244A2; U.S. Pat. Appl. Nos. 15 / 782,980 and 15 / 433,322; and U.S. Pat. Nos. 10,036,016, 9,790,472, 9,737,618, 9,434,928, 9,233,131, 8,906,675, 7,790,449, 7,906,111, 7,718,424, 7,259,151, 7,198,951, 7,105,345, 6,962,815, 6,984,517, and 6,156,303.

[0038] An “AAV vector” or “rAAV vector” as used in the art to refer either to the DNA packaged into in the rAAV virion or to the rAAV virion itself, depending on context. As used herein, unless otherwise apparent from context, rAAV vector refers to a nucleic acid (typically a plasmid) comprising a polynucleotide sequence capable of being packaged into an rAAV virion, but with the capsid or other proteins of the rAAV virion. Generally an rAAV vector comprises a heterologous polynucleotide sequence (i.e., a polynucleotide not of AAV origin) and one or two AAV inverted terminal repeat sequences (ITRs) flanking the heterologousAttorney Docket No.: TENA-070 / 03WO 334682-2515 polynucleotide sequence. Only one of the two ITRs may be packaged into the rAAV and yet infectivity of the resulting rAAV virion may be maintained. See Wu et al. (2010) Mol Ther. 18:80. An rAAV vector may be designed to generate either single-stranded (ssAAV) or self- complementary (scAAV). See McCarty D. (2008) Mo. Ther. 16:1648-1656; WO2001 / 11034; WO200 1 / 92551 ; WO2010 / 129021.

[0039] An “rAAV virion” refers to an extracellular viral particle including at least one viral capsid protein (e.g. VP1) and an encapsulated rAAV vector (or fragment thereof), including the capsid proteins.

[0040] The term “inverted terminal repeats” or “ITRs” as used herein refers to AAV viral cis-elements named so because of their symmetry. These elements are essential for efficient multiplication of an AAV genome.

[0041] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rdedition; Ausubel etal. eds. (2007) Current Protocols in Molecular Biology; Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson etal. (1991) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Lreshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; IRL Press (1986) Immobilized Cells and Enzymes; Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3rdedition (2002) Cold Spring Harbor Laboratory Press; Sohail (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press); and Sell (2013) Stem Cells Handbook.

[0042] The term “isolated” means separated from constituents, cellular and otherwise, inAttorney Docket No.: TENA-070 / 03WO 334682-2515 which the virion, cell, tissue, polynucleotide, peptide, polypeptide, or protein is normally associated in nature. For example, an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype.

[0043] As used herein, “sequence identity” or “identity” refers to the percentage of number of amino acids that are identical between a sequence of interest and a reference sequence. Generally identity is determined by aligning the sequence of interest to the reference sequence, determining the number of amino acids that are identical between the aligned sequences, dividing that number by the total number of amino acids in the reference sequence, and multiplying the result by 100 to yield a percentage. Sequences can be aligned using various computer programs, such BLAST, available at ncbi.nlm.nih.gov. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996); and Meth. Mol. Biol. 70: 173-187 (1997); J. Mol. Biol. 48: 44. Skill artisans are capable of choosing an appropriate alignment method depending on various factors including sequence length, divergence, and the presence of absence of insertions or deletions with respect to the reference sequence.

[0044] A “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. A “gene product” is a molecule resulting from expression of a particular gene. Gene products may include, without limitation, a polypeptide, a protein, an aptamer, an interfering RNA, or an mRNA. Gene-editing systems (e.g. a prime editing system) may be described as one gene product or as the several gene products required to make the system (e.g. a Cas protein, a reverse transcriptase, and a guide RNA).

[0045] A “control element” or “control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements include transcriptional regulatory sequences such as promoters and / or enhancers.

[0046] A “promoter” is a DNA sequence capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3 ’ direction) from the promoter.Attorney Docket No.: TENA-070 / 03WO 334682-2515

[0047] “Operatively linked” or “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.

[0048] The term “expression cassette” refers to a polynucleotide cassette comprising a coding sequence which encodes a gene product of interest used to effect the expression of the gene product in target cells. Unless otherwise specified, the expression cassette of an AAV vector includes only the polynucleotides between (and not including) the ITRs.

[0049] The terms “upstream” and “upstream end” refer to a portion of a polynucleotide that is, with reference to a transcription start site (TSS), 5' to the TSS on the sense strand (or coding strand) of the polynucleotide; and 3' to the TSS on the antisense strand of the polynucleotide. The terms “downstream” and “downstream end” refer to a portion of a polynucleotide that is, with reference to a TSS, 3' to TSS on the sense strand (or coding strand) of the polynucleotide; and 5' to the TSS on the antisense strand of the polynucleotide. Thus, a deletion from the upstream end of a promoter is a deletion of one or more base pairs in the nontranscribed region of the polynucleotide, 5' to the TSS on the sense strand (or equivalently, 3' to the TSS on the antisense strand). A deletion from the downstream end of a promoter is a deletion of one or more base pairs in the transcribed region of the polynucleotide, 3' to the TSS on the sense strand (or equivalently, 5' to the TSS on the antisense strand).

[0050] The term “transgene” refers to a nucleic acid sequence encoding a protein (e.g., a therapeutic protein), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal’s genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.

[0051] “Heterologous” means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced byAttorney Docket No.: TENA-070 / 03WO 334682-2515 genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. Thus, for example, an rAAV that includes a heterologous nucleic acid is an rAAV that includes a nucleic acid not normally included in a naturally -occurring AAV.

[0052] The terms “genetic alteration” and “genetic modification” (and grammatical variants thereof), are used interchangeably herein to refer to a process wherein a genetic element (e.g., a polynucleotide) is introduced into a cell other than by mitosis or meiosis. The element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell. Genetic alteration may be effected, for example, by transfecting a cell with a polynucleotide through any process known in the art, such as electroporation, calcium phosphate precipitation, or contacting with a polynucleotide-liposome complex. Genetic alteration may also be effected, for example, by transduction or infection with a vector.

[0053] A cell is said to be “stably” altered, transduced, genetically modified, or transformed with a polynucleotide sequence if the sequence is available to perform its function during extended culture of the cell in vitro. Generally, such a cell is “heritably” altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell.

[0054] The term “transfection” is as used herein refers to the uptake of an exogenous nucleic acid molecule by a cell. A cell has been “transfected” when exogenous nucleic acid has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acid molecules into suitable host cells.

[0055] The term “transduction” is as used herein refers to the transfer of an exogenous nucleic acid into a cell by a recombinant virion, in contrast to “infection” by a wild-type virion. When infection is used with respect to a recombinant virion, the terms “transduction” and “infectious” are synonymous, and therefore “infectivity” and “transduction efficiency” areAttorney Docket No.: TENA-070 / 03WO 334682-2515 equivalent and can be determined using similar methods.

[0056] Unless otherwise specified, all medical terminology is given the ordinary meaning of the term used by medical professional as, for example, in Harrison ’s Principles of Internal Medicine, 15ed., which is incorporated by reference in its entirety for all purposes, in particular the chapters on cardiac or cardiovascular diseases, disorders, conditions, and dysfunctions.

[0057] Treatment,” “treating,” and “treat” are defined as acting upon a disease, disorder, or condition with an agent to reduce or ameliorate harmful or any other undesired effects of the disease, disorder, or condition and / or its symptoms. In some embodiments, the term “treat,” “treating,” or “treatment” refers to alleviating one or more symptoms of the disease, disorder or condition. In some embodiments, the term “treat,” “treating,” or “treatment” refers to preventing, decreasing the likelihood of occurrence or recurrence of, the disease, disorder, or condition. In some embodiments, the term “treat,” “treating,” or “treatment” refers to slowing the progression or development of, or eliminating, reducing, or slowing the development of one or more symptoms associated with, the disease, disorder or condition. In some embodiments, the term “treat,” “treating,” or “treatment” means that one or more symptoms of the disease, disorder, or condition are alleviated in a subject receiving the agent as disclosed and described herein, compared to a subject who does not receive such treatment.

[0058] ‘Administration,” “administering” and the like, when used in connection with a composition of the invention refer both to direct administration (administration to a subject by a medical professional or by self-administration by the subject) and / or to indirect administration (prescribing a composition to a patient). Typically, an effective amount is administered, which amount can be determined by one of skill in the art. Any method of administration may be used. Administration to a subject can be achieved by, for example, intravenous, intra-arterial, intramuscular, intravascular, or intramyocardial delivery.

[0059] As used herein the term “effective amount” and the like in reference to an amount of a composition refers to an amount that is sufficient to induce a desired physiologic outcome (e.g., reprogramming of a cell or treatment of a disease). An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period which the individual dosage unit is to be used, the bioavailability of the composition, the route of administration, etc. It is understood, however, that specific amounts of the compositions for any particular subjectAttorney Docket No.: TENA-070 / 03WO 334682-2515 depends upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the composition combination, severity of the particular disease being treated and form of administration.

[0060] The terms “individual,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates (e.g., simians); mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (e.g., dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0061] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0062] The term “purified” as used herein refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e. impurities, including native materials from which the material is obtained.

[0063] The term “therapeutic gene” as used herein refers to a gene that, when expressed, confers a beneficial effect on the cell or tissue in which it is present, or on a mammal in which the gene is expressed. Examples of beneficial effects include amelioration of a sign or symptom of a condition or disease, prevention or inhibition of a condition or disease, or conferral of a desired characteristic. Therapeutic genes include genes that partially or wholly correct a genetic deficiency in a cell or mammal.

[0064] As used herein the term “cardiac cell” refers to any cell present in the heart that provides a cardiac function, such as heart contraction or blood supply, or otherwise serves to maintain the structure of the heart. Cardiac cells as used herein encompass cells that exist in the epicardium, myocardium or endocardium of the heart. Cardiac cells also include, for example, cardiac muscle cells or cardiomyocytes, and cells of the cardiac vasculatures, such as cells of a coronary artery or vein. Other non-limiting examples of cardiac cells include epithelial cells, endothelial cells, fibroblasts, cardiac stem or progenitor cells, cardiac conducting cells and cardiac pacemaking cells that constitute the cardiac muscle, blood vessels and cardiac cell supporting structure. Cardiac cells may be derived from stem cells, including, for example,Attorney Docket No.: TENA-070 / 03WO 334682-2515 embryonic stem cells or induced pluripotent stem cells.

[0065] The term “cardiomyocyte” or “cardiomyocytes” as used herein refers to sarcomere-containing striated muscle cells, naturally found in the mammalian heart, as opposed to skeletal muscle cells. Cardiomyocytes are characterized by the expression of specialized molecules, e.g., proteins like myosin heavy chain, myosin light chain, cardiac a-actinin. The term “cardiomyocyte” as used herein is an umbrella term comprising any cardiomyocyte subpopulation or cardiomyocyte subtype, e.g., atrial, ventricular and pacemaker cardiomyocytes.

[0066] The term “cardiomyocyte-like cells” is intended to mean cells sharing features with cardiomyocytes, but which may not share all features. For example, a cardiomyocyte-like cell may differ from a cardiomyocyte in expression of certain cardiac genes.

[0067] As used herein, the term “equivalents thereof’ in reference to a polypeptide or nucleic acid sequence refers to a polypeptide or nucleic acid that differs from a reference polypeptide or nucleic acid sequence, but retains essential properties (e.g., biological activity). A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, deletions, additions, fusions and truncations in the polypeptide encoded by the reference sequence. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.

[0068] The term “genetic modification” refers to a permanent or transient genetic change induced in a cell following introduction of new nucleic acid (i.e., nucleic acid exogenous to the cell). Genetic change can be accomplished by incorporation of the new nucleic acid into the genome of the cardiac cell, or by transient or stable maintenance of the new nucleic acid as an extrachromosomal element. Where the cell is a eukaryotic cell, a permanent genetic change can be achieved by introduction of the nucleic acid into the genome of the cell. Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like.

[0069] Unless stated otherwise, the abbreviations used throughout the specification haveAttorney Docket No.: TENA-070 / 03WO 334682-2515 the following meanings: AAV, adeno-associated virus, rAAV, recombinant adeno-associated virus; FACS, fluorescence activated cell sorting; GFP, green fluorescence protein; kg, killigram; pg, microgram; pl, microliter; mg, milligram; ml, milliliter; msec, millisecond; min, minute; PBS, phosphate buffered saline; qPCR, quantitative polymerase chain reaction; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; RNA, ribonucleic acid; RNA-seq, RNA sequencing; RT-PCR, reverse transcriptase polymerase chain reaction; sec, second.Modified cardiac troponin promoters

[0070] In some embodiments, the present disclosure provides modified cardiac troponin T promoters. Cardiac troponin T can be abbreviated as cTnT or TNNT2. In some embodiments, the modified TNNT2 promoter is a truncated TNNT2 promoter comprising 350 or fewer nucleotides. In some embodiments, the modified TNNT2 promoter is a chimeric promoter comprising a portion of nucleotides from the TNNT2 promoter and a portion of nucleotides from the MYBPC3 promoter.

[0071] Illustrative polynucleotide sequences of the cardiac TNNT2 promoter are shown in Table 1 below.Table 1. Exemplary TNNT2 promotersAttorney Docket No.: TENA-070 / 03WO 334682-2515

[0072] In some embodiments, the modified TNNT2 promoter comprises 350 or fewer nucleotides in length. In some embodiments, the modified TNNT2 promoter comprises a polynucleotide comprising at least 120 nucleotides and not more than 350 nucleotides. In some embodiments, the modified TNNT2 promoter comprises a polynucleotide comprising at least 300 nucleotides and not more than 350 nucleotides. In some embodiments, the modified TNNT2Attorney Docket No.: TENA-070 / 03WO 334682-2515 promoter comprises a polynucleotide comprising at least 120, at least 130, at least 140, or at least 150 nucleotides. In some embodiments, the modified TNNT2 promoter comprises a polynucleotide consisting of 304 bp. In some embodiments, the modified TNNT2 promoter comprises a polynucleotide consisting of SEQ ID NO: 8. In some embodiments, the modified TNNT2 promoter comprises a polynucleotide consisting of 350 bp. In some embodiments, the modified TNNT2 promoter comprises a polynucleotide consisting of SEQ ID NO: 7. In some embodiments, the modified TNNT2 promoter comprises a polynucleotide consisting of 150 bp. In some embodiments, the modified TNNT2 promoter comprises a polynucleotide consisting of SEQ ID NO: 9. In some embodiments, the modified TNNT2 promoter comprises a polynucleotide consisting of 131 bp. In some embodiments, the modified TNNT2 promoter comprises a polynucleotide consisting of SEQ ID NO: 10.

[0073] In some embodiments, the modified TNNT2 promoter is modified by the deletion of polynucleotides with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 5) having about 600 base pairs. A modification may include one, two, three or more deletions. Each deletion may be a deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 40 base pairs, 42 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 254 base pairs, 275 base pairs, 300 base pairs, 325 base pairs, 350 base pairs, 375 base pairs, 400 base pairs, 425 base pairs, 450 base pairs, or 475 base pairs with respect to a reference TNNT2 promoter (SEQ ID NO: 5) having about 600 base pairs.

[0074] In some embodiments, the modified TNNT2 promoter comprises a polynucleotide comprising a sequence that shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% identity to any one of SEQ ID NOs: 6-10. In some embodiments, the polynucleotide comprises a sequence that shares at least 80% identity to any one of SEQ ID NOs: 6-10. In some embodiments, the polynucleotide comprises a sequence that shares at least 90% identity to any one of SEQ ID NOs: 6-10. In some embodiments, the polynucleotide comprises a sequence that shares 100% identity to any one of SEQ ID NOs: 6-10. In some embodiments, the polynucleotide comprises a sequence that shares at least 80% identity to SEQ ID NO: 6. In some embodiments, the polynucleotide comprises a sequence that shares at least 90% identity to SEQ ID NO: 6. In some embodiments, the polynucleotide comprises a sequence that shares 100% identity to SEQ ID NO: 6. In some embodiments, the polynucleotide comprises a sequence thatAttorney Docket No.: TENA-070 / 03WO 334682-2515 shares at least 80% identity to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a sequence that shares at least 90% identity to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a sequence that shares 100% identity to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a sequence that shares at least 80% identity to SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a sequence that shares at least 90% identity to SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a sequence that shares 100% identity to SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a sequence that shares at least 80% identity to SEQ ID NO: 9. In some embodiments, the polynucleotide comprises a sequence that shares at least 90% identity to SEQ ID NO: 9. In some embodiments, the polynucleotide comprises a sequence that shares 100% identity to SEQ ID NO: 9.

[0075] In some embodiments, the modified TNNT2 promoter comprises a polynucleotide sequence from a TNNT2 promoter and a polynucleotide sequence from a second cardiac-specific promoter. In some embodiments, the second cardiac-specific promoter is a cardiac myosin-binding protein C (MYBPC3) promoter. In some embodiments, the modified TNNT2 promoter comprises a first polynucleotide sequence comprising, in order, 50 nucleotides of the TNNT2 promoter, 50 nucleotides of the MYBPC3 promoter, and 31 nucleotides of the TNNT2 promoter. In some embodiments, the polynucleotide comprises a sequence that shares at least 80% identity to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a sequence that shares at least 90% identity to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a sequence that shares 100% identity to SEQ ID NO: 10.

[0076] In some embodiments, the modified TNNT2 promoters are incorporated into an expression cassette described herein. The modified TNNT2 promoters described herein provide advantages over other cardiac-specific promoters known in the art. The promoters described herein are small (350 or fewer nucleotides) and are able to mediate transgene expression at levels similar to or increased compared to other promoters known in the art. These truncated promoters therefore leave more space in a vector for additional components. This is particularly relevant for vector systems comprising multiple components, such as the prime editing systems described herein (comprising nucleic acid programmable DNA binding proteins, multiple guide RNAs, a reverse transcriptase, and additional proteins).Attorney Docket No.: TENA-070 / 03WO 334682-2515Prime editing systems

[0077] As used herein, prime editing refers to a genome editing method that directly writes new genetic information into a specified DNA site using an RNA-guided nickase (e.g., a Cas9 nickase) working in association with a polymerase (e.g., a reverse transcriptase). In some embodiments, the present disclosure provides a system comprising a (i) a first vector comprising a first expression cassette comprising: (a) a first modified TNNT2 promoter operatively linked to a first polynucleotide encoding an N-terminal fragment of a fusion protein comprising an N- terminal fragment of an RNA-guided nickase and an N-terminal fragment of a split-intein; (ii) a second vector comprising a second expression cassette comprising: (a) a second modified TNNT2 promoter operatively linked to a second polynucleotide encoding a C-terminal fragment of the fusion protein comprising a C-terminal fragment of the RNA-guided nickase, a reverse transcriptase, and a C-terminal fragment of the split-intein; and (b) a third promoter operatively linked to a third polynucleotide encoding a prime editing guide RNA (pegRNA). The system results in the expression of the multi-component prime editing system comprising (i) a fusion protein comprising an RNA-guided nickase and a reverse transcripase; and (ii) one or more guide RNAs that direct the fusion protein to the target DNA sequence.

[0078] In some embodiments, the systems described herein comprise a prime editing guide RNA (“pegRNA”). PegRNAs comprise a spacer sequence that binds to the target DNA sequence, a scaffold sequence capable of binding the RNA-guided nickase, a template sequence comprising the desired edit, and a primer binding site. Through DNA repair and / or replication machinery, the endogenous strand of the target site is replaced by the newly synthesized replacement strand containing the desired edit.

[0079] Any suitable RNA-guided nickase may be used in the systems described herein. In various embodiments, the RNA-guided nickase may be any Class 2 CRISPR-Cas system having a nickase activity (e.g., only cleaves of strand of the target DNA sequence), including any type II, type V, or type VI CRISPR-Cas enzyme. In some embodiments, the RNA-guided nickase is an SaCas9 nickase. In some embodiments, the RNA-guided nickase is an SpCas9 nickase.

[0080] The RNA-guided nickase may also comprise nickase variants of Cas9 equivalents, including Casl2a (Cpfl), Casl2e (CasX), Casl2bl (C2cl), Casl2b2, Casl2c (C2c3), C2c4, C2c8, C2c5, C2cl0, C2c9 Casl3a (C2c2), Casl3d, Casl3c (C2c7), Casl3b (C2c6), and Casl3b.Attorney Docket No.: TENA-070 / 03WO 334682-2515Further Cas-equivalents are described in Makarova et al., Science 2016; 353(6299) and Makarova et al., The CRISPR Journal, Vol.l. No.5, 2018, the contents of which are incorporated herein by reference. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A in reference to the canonical SpCas9 sequence, or to equivalent amino acid positions in other Cas9 variants or Cas9 equivalents.

[0081] In some embodiments, the system comprises a fusion protein comprising an RNA- guided nickase and a reverse transcriptase.

[0082] Reverse transcriptases are multi-functional enzymes typically with three enzymatic activities including RNA- and DNA-dependent DNA polymerization activity, and an RNaseH activity that catalyzes the cleavage of RNA in RNA-DNA hybrids. Some mutants of reverse transcriptases have disabled the RNaseH moiety. These enzymes synthesize complementary DNA (cDNA) using RNA as a template. More recently, mutants and fusion proteins have been created in the quest for improved properties such as thermostability, fidelity and activity. Any of the wild type, variant, and / or mutant forms of reverse transcriptase which are known in the art or which can be made using methods known in the art are contemplated herein.

[0083] Non-limiting examples of reverse transcriptases include Moloney Murine Leukemia Virus (M-MLV); Human Immunodeficiency Virus (HIV) reverse transcriptase and avian Sarcoma-Leukosis Virus (ASLV) reverse transcriptase, which includes but is not limited to Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV reverse transcriptase, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A reverse transcriptase, Avian Sarcoma Virus UR2 Helper Virus UR2AV reverse transcriptase, Avian Sarcoma Virus Y73 Helper Virus YAV reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, and Myeloblastosis Associated Virus (MAV) reverse transcriptase.

[0084] In some embodiments, the reverse transcriptase comprises one or more mutations or deletions in the RNase H domain. As mentioned above, one of the intrinsic properties ofAttorney Docket No.: TENA-070 / 03WO 334682-2515 reverse transcriptases is the RNase H activity, which cleaves the RNA template of the RNA:cDNA hybrid concurrently with polymerization. The RNase H activity can be unnecessary for certain applications. The RNase H activity may also lower reverse transcription efficiency, presumably due to its competition with the polymerase activity of the enzyme. Thus, the present disclosure contemplates any reverse transcriptase variants that comprise a modified RNaseH activity or lack the RNase H domain.

[0085] In some embodiments, the reverse transcriptase is an M-MLV reverse transcriptase lacking the RNase H domain. In some embodiments the reverse transcriptase is an evolved Tfl reverse transcriptase. In some embodiments the reverse transcriptase is an evolved and engineered Tfl reverse transcriptase. In some embodiments the reverse transcriptase is an evolved and engineered M-MLV reverse transcriptase lacking the RNase H domain.

[0086] In some embodiments, the present disclosure provides a fusion protein comprising an RNA-guided nickase and a reverse transcriptase described herein. In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, an RNA-guided nickase and a reverse transcriptase. In some embodiments, the fusion protein further comprises a linker.

[0087] In some embodiments, the fusion protein described herein may be divided into two or more fragments which become assembled inside the cell into the mature fusion protein. In some embodiments, the fusion protein is divided into an N-terminal fragment and a C-terminal fragment. In some embodiments, the fusion protein N-terminal fragment comprises an N- terminal fragment of the RNA-guided nickase disclosed herein and a split intein domain. In some embodiments, the fusion protein C-terminal fragment comprises a C-terminal fragment of the RNA-guided nickase, a reverse transcriptase, and a split intein domain.

[0088] In some embodiments, the N-terminal fragment of the fusion protein comprises SEQ ID NO: 19 and the C-terminal fragment of the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-28 and 51-52. In some embodiments, the N-terminal fragment of the fusion protein comprises SEQ ID NO: 19 and the C-terminal fragment of the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-22, 24, 26, 28, 51, and 52.

[0089] Once delivered or expressed within a cell, the split intein domains of the different fragments associate and bind to one another, and then undergo trans-splicing, which results in the excision of the split-intein domains from each of the fragments, and a concomitant formationAttorney Docket No.: TENA-070 / 03WO 334682-2515 of a peptide bond between the fragments, thereby resulting in the mature version of the fusion protein comprising the RNA-guided nickase and the reverse transcriptase (the mature fusion protein is also referred to herein as a “prime editor”). Non-limiting examples of split inteins are described in Stevens et al., PNAS, 2017, Vol.114: 8538-8543; Iwai et al., , FEBS Lett, 580: 1853-1858, each of which are incorporated herein by reference. Additional split intein sequences can be found, for example, in WO 2013 / 045632, WO 2014 / 055782, WO 2016 / 069774, and EP2877490, the contents each of which are incorporated herein by reference.

[0090] In some embodiments, the present disclosure provides a first expression cassette comprising: (a) a first promoter described herein operatively linked to a first polynucleotide encoding an N-terminal fragment of a fusion protein comprising an N-terminal fragment of an RNA-guided nickase described herein and an N-terminal fragment of a split-intein. In some embodiments, the first expression cassette comprises a first truncated TNNT2 promoter operatively linked to a first polynucleotide encoding an N-terminal fragment of a fusion protein comprising an N-terminal fragment of an RNA-guided nickase and an N-terminal fragment of a split-intein.

[0091] In some embodiments, the present disclosure further provides a second expression cassette comprising a second promoter described herein operatively linked to a second polynucleotide encoding a C-terminal fragment of a fusion protein comprising a C-terminal fragment of an RNA-guided nickase described herein, a polymerase described herein, and a C- terminal fragment of a split-intein. In some embodiments, the second expression cassette further comprises a third promoter described herein operatively linked to a third polynucleotide encoding a prime editing guide RNA (pegRNA). In some embodiments, the second expression cassette comprises (a) a second truncated TNNT2 promoter operatively linked to a second polynucleotide encoding a C-terminal fragment of the fusion protein comprising a C-terminal fragment of the RNA-guided nickase, a reverse transcriptase, and a C-terminal fragment of the split-intein; and (b) a third promoter operatively linked to a third polynucleotide encoding a prime editing guide RNA (pegRNA).

[0092] In some embodiments, the first expression cassette further comprises a fourth promoter operatively linked to a fourth polynucleotide encoding a nicking guide RNA (ngRNA).

[0093] In some embodiments, the C-terminal fragment of the fusion protein further comprises an RNA binding domain of small RNA binding exonuclease protection factor LaAttorney Docket No.: TENA-070 / 03WO 334682-2515(e.g., as described in Yan et al. Nature. 2024; 628(8008):639-647.) In some embodiments, the small RNA binding exonuclease protection factor La is according to Gene ID: 6741 and / or UmProt ID P05455.

[0094] In some embodiments, the first expression cassette comprises a sequence with at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 33, 35, 37, 39, and 41. In some embodiments, the first expression cassette comprises any one of SEQ ID NOs: 33, 35, 37, 39, and 41. In some embodiments, the first expression cassette further comprises a promoter described herein controlling expression of a ngRNA. In some embodiments, the first expression cassette is flanked by a left ITR on the 5 ’ end of the expression cassette and a right ITR on the 3 ’ end of the expression cassette. In some embodiments, the left ITR comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 31, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 31. In some embodiments, the right ITR comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 32, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 32.

[0095] In some embodiments, the second expression cassette comprises a sequence with at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 34, 36, 38, 40, 42-50, 53, and 54. In some embodiments, the second expression cassette comprises any one of SEQ ID NOs: SEQ ID NOs: 34, 36, 38, 40, 42-50, 53, and 54. In some embodiments, the second expression cassette further comprises a promoter described herein controlling expression of a pegRNA. In some embodiments, the first expression cassette is flanked by a left ITR on the 5’ end of the expression cassette and a right ITR on the 3’ end of the expression cassette. In some embodiments, the left ITR comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 31, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 31. In some embodiments, the right ITR comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 32, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, atAttorney Docket No.: TENA-070 / 03WO 334682-2515 least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 32.

[0096] In some embodiments, the first expression cassette comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 37 and the second expression cassette comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 38. In some embodiments, the first expression cassette comprises SEQ ID NO: 37 and the second expression cassette comprises SEQ ID NO: 38.

[0097] In some embodiments, the first expression cassette comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 35 and the second expression cassette comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 36, 43, 44, 46, 48, 50, 53, and 54.

[0098] In some embodiments, the first and second expression cassettes are selected from those shown in Table 2. In the table, “Orientation” refers whether the polynucleotide encoding the N-terminal or C-terminal fragment of the fusion protein face the same direction (tail-to-head) or opposite direction (tail-to-tail) as the polynucleotide encoding the gRNA.Table 2. Exemplary Expression CassettesAttorney Docket No.: TENA-070 / 03WO 334682-2515

[0099] The polynucleotides, expression cassettes, and / or vectors contemplated herein may be combined with other sequences, such as promoters, enhancers, untranslated regions (UTRs), introns, signal sequences, Kozak sequences, polyadenylation (poly(A)) signals, post- transcriptional regulatory elements, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, polynucleotides encoding selfcleaving polypeptides, epitope tags, and / or any other regulatory elements as disclosed elsewhere herein or as known in the art. In some embodiments, the polynucleotides, expression cassettes, and / or vectors described herein may also contain a ribosome binding site for translation initiation, a transcription terminator, and / or polynucleotide sequences for amplifying expression. The expression cassette may be flanked by one or more inverted terminal repeatsAttorney Docket No.: TENA-070 / 03WO 334682-2515(ITRs). The ITRs in an expression cassette serve as markers used for viral packaging of the expression cassette. The expression cassette can be integrated into the host cell genome, thereby expressing the transgene within a host cell.

[0100] As used herein, the term “regulatory element” refers to those non-translated regions of the vector (e.g., origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Kozak sequence), introns, poly(A) sequences, 5' and 3' untranslated regions) which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. The transcriptional regulatory element may be functional in either a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., bacterial or archaeal cell). In some embodiments, a polynucleotide sequence described herein is operably linked to multiple control elements that allow expression of the polynucleotide in both prokaryotic and eukaryotic cells.Poly (A) sequences

[0101] In some embodiments, the vector and / or expression cassettes described herein further comprises one or more poly(A) sequences. The term “poly(A) sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by addition of a poly(A) tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency. Cleavage and polyadenylation are directed by a poly(A) sequence in the RNA. The core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage-polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5’ cleavage product. In some embodiments, the core poly(A) sequence is an ideal poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). Non-limiting examples of poly(A) sequences include SV40 poly(A) sequence, bovine growth hormone (BGH) poly(A) sequence, rabbit 0-globin poly(A) sequence (r0gpA), variants thereof, and other suitable heterologous or endogenous poly(A) sequences known in the art. Exemplary poly (A) sequences are provided in Table 3 below.Table 3. Exemplary poly(A) sequencesAtorney Docket No.: TENA-070 / 03WO 334682-2515Attorney Docket No.: TENA-070 / 03WO 334682-2515

[0102] In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 11, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 11.

[0103] In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 12, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 12.

[0104] In some embodiments, the poly(A) sequence comprises a SV40 poly(A) sequence. In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 13, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13.

[0105] In some embodiments, the poly(A) sequence comprises a SV40 poly(A) sequence. In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 14, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14.

[0106] In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 15, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 15.

[0107] In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 16, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at leastAttorney Docket No.: TENA-070 / 03WO 334682-251598%, at least 99%, or 100% identity to SEQ ID NO: 16.

[0108] In some embodiments, the poly(A) sequence comprises a SV40 poly(A) sequence. In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 17, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17

[0109] In some embodiments, the poly(A) sequence comprises a SV40 poly(A) sequence. In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 18, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 18.ITRs

[0110] In some embodiments, the expression cassette is flanked by AAV inverted terminal repeats (ITRs) at the 5’ and 3’ ends. ITRs function as recognition sites for replication and markers used for viral packaging of the expression cassette. ITRs form T-shaped secondary structures by two adjacent inverted repeats separated by an unpaired nucleotide. ITRs are required for packaging the expression cassette into an rAAV virion, which provide the function of expressing the transgene after a host cell is targeted by the rAAV virion. The ITRs contain tetranucleotide repeat motifs called Rep-binding elements (RBE) that act as contact points for the Rep68 / 78 proteins encoded by the rep gene. The ITRs also contain a packaging signal for genome encapsidation, which directs 3’ genomic transport into preassembled capsids by Rep proteins. Any naturally occurring or synthetically derived ITRs described herein or known in the art can be used.

[0111] In some embodiments, the ITRs flanking the expression cassette are ITRs of the same AAV serotype as the Rep protein used in making the virions described herein. For example, where a Rep protein from AAV9 is used, the transgene expression cassette used in the expression system comprises ITRs from AAV9 as well. In another example, where a Rep protein from AAV2 is used, the transgene expression cassette used in the expression system comprises ITRs from AAV2 as well. In another example, where a Rep protein from AAV5 is used, the transgene expression cassette used in the expression system comprises ITRs from AAV5 as well. The ITRs may be of the same or different serotype as the capsid protein used inAttorney Docket No.: TENA-070 / 03WO 334682-2515 packaging the virion described herein.

[0112] In some embodiments, the first and / or second expression cassettes may each be flanked by one or more inverted terminal repeats (ITRs). In some embodiments, the first and second expression cassettes are each flanked by a left ITR on the 5’ end of the expression cassette and a right ITR on the 3’ end of the expression cassette. In some embodiments, the left ITR comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 31, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 31. In some embodiments, the right ITR comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 32, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 32. In some embodiments, the firstand second expression cassette are each flanked on the 5’ end of the expression cassette by a nucleotide sequence comprising SEQ ID NO: 31 and flanked on the 3’ end of the expression cassette by a nucleotide sequence comprising SEQ ID NO: 32. In some embodiments, the ITR sequences comprise, consist of, or consist essentially of a nucleotide sequence set forth in Table 4 below.

[0113] In some embodiments, the expression cassettes further comprise a junction sequence. In some embodiments, the expression cassette is flanked by one or both of a left ITR and junction sequence on the 5’ end of the expression cassette and a right ITR and junction sequence on the 3’ end of the expression cassette. In some embodiments, the left ITR and junction sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 29, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 29. In some embodiments, the right ITR and junction sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 30, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 30. In some embodiments, the first and second expression cassette are each flanked on the 5’ end of the expression cassette by a nucleotide sequence comprising SEQ ID NO: 29 and flanked on the 3 ’ end of the expression cassette by a nucleotide sequence comprising SEQ ID NO: 30. In some embodiments, the ITR plus junction sequences comprise, consist of, or consist essentially of a nucleotide sequence set forth in Table 4 below.Attorney Docket No.: TENA-070 / 03WO 334682-2515Table 4. Exemplary ITR sequencesSplit PE Systems

[0114] In some embodiments, the present disclosure provides a system comprising a first vector comprising a first expression cassette described herein and a second vector comprising a second expression cassette described herein.

[0115] The vector can be any viral vector or non-viral vector known in the art or described herein. In some embodiments, the vector is a viral vector. In some embodiments the viral vector is an adeno-associated virus vector (AAV), an adenoviral vector, a lentiviral vector, a retroviral vector, a herpes simplex virus vector (HSV), or a poxvirus vector.

[0116] As used herein, the term “retrovirus” or “retroviral” refers an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Retrovirus vectors are a common tool for gene delivery. Once the virus is integrated into the host genome, it is referred to as a “provirus.” The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules encoded by the virus. In some embodiments, a retroviral vector is altered so that it does not integrate into the host cell genome. Illustrative retroviruses include, but are not limited to, (1) genus gammaretrovirus, such as, Moloney murine leukemia virus (M-MuLV or M-MLV), Moloney murine sarcoma virus (MoMSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), and feline leukemia virus (FLV); (2) genus spumavirus,Attorney Docket No.: TENA-070 / 03WO 334682-2515 such as, simian foamy virus; and (3) genus lentivirus, such as, human immunodeficiency virus- 1 and simian immunodeficiency virus.

[0117] As used herein, the term “lentiviral” or “lentivirus” refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include but are not limited to, human immunodeficiency virus (HIV), including HIV type 1, and HIV type 2; visna-maedi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0118] In some embodiments, the viral vector is an adenoviral vector. The genetic organization of adenovirus includes an approximate 36 kb, linear, double-stranded DNA virus, which allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb.

[0119] In some embodiments, the viral vector is an AVV vector, such as an AAV vector selected from the group consisting of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rh.10, rh.20, rh.74, and a variant or chimeric AAV derived thereof. In some embodiments, the AAV expression vector is pseudotyped to enhance targeting. A pseudotyping strategy can promote gene transfer and sustain expression in a target cell type. For example, the AAV2 genome can be packaged into the capsid of another AAV serotype such as AAV5, AAV7, or AAV8, producing pseudotyped vectors such as AAV2 / 5, AAV2 / 7, and AAV2 / 8 respectively, as described in Balaji et al., J. Surg. Res. Sep. (2013) 184(l):691-698. In some embodiments, an AAV9 may be used to target expression in myofibroblast-like lineages, as described in Piras et al., Gene Therapy (2016) 23:469-478. In some embodiments, AAV1, AAV6, or AAV9 is used, and in some embodiments, the AAV is engineered, as described in Asokari et al., Hum. Gene Ther. Nov. (2013) 24(1 l):906-913; Pozsgai etal., Mol. Ther. (2017) 25(4): 855-869; Kotterman, M.A. and D.V. Schaffer, Nature Reviews Genetics (2014) 15:445-451; and US20160340393A1 to Schaffer et al. In some embodiments, the viral vector is AAV engineered to increase target cell infectivity as described in US20180066285A1. In some embodiments, the vector is an AAV9 vector.

[0120] In some embodiments, the vector is a non-viral vector. In some embodiments, the non- viral vector is a naked DNA (e.g., a DNA plasmid). In some embodiments, the non-viral vector is a plasmid. In some embodiments, the non-viral vector is a liposome or lipid vector comprising plasmid DNA and a lipid solution.Attorney Docket No.: TENA-070 / 03WO 334682-2515

[0121] In some embodiments, the vector is a recombinant vector. In some embodiments, the viral vectors described herein are replication incompetent, in that it cannot independently further replicate and package its genome. For example, when a cardiac cell is targeted with a virion, the transgene is expressed in the targeted cardiac cell, however, since the targeted cardiac cell lacks packaging and accessory function genes, the virion is not able to replicate. In some embodiments, the viral vectors described herein are replication competent.

[0122] In some embodiments, the vectors described herein are capable of being delivered to both dividing and non-dividing cells. In some embodiments, the vectors described herein are capable of being delivered to non-dividing cells. In some embodiments, the vectors described herein are capable of being delivered to dividing cells.

[0123] In some of these embodiments, the vector is an AAV vector or a variant thereof. In some of these embodiments, the vector is an AAV9 vector or a variant thereof. In some of these embodiments, the vector is an AAV5 vector or a variant thereof. In some of these embodiments, the vector is an AAV2 vector or a variant thereof.

[0124] The capsid proteins of AAV largely determine the immunogenicity and tropism of AAV vectors. In some embodiments, the AAV is an AAV subtype 9 (AAV9). In some embodiments, AAV9 is a preferred AAV vector due to its ability to transduce the heart following systemic delivery. While AAV9 can achieve moderate transduction of the heart, the majority of vector traffics to the liver. Moreover, in order to achieve therapeutic levels of transduction in the heart, relatively high systemic doses are required, potentially leading to systemic inflammation and in turn, toxicity.

[0125] Methods of introducing polynucleotides into a host cell are known in the art, and any known method can be used to introduce the polynucleotides described herein into a cell. Suitable methods include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery, microfluidics delivery methods, and the like.

[0126] In some embodiments, the first vector comprising a first expression cassette comprising a first promoter described herein operatively linked to a polynucleotide encoding anAttorney Docket No.: TENA-070 / 03WO 334682-2515N-terminal fragment of a fusion protein described herein. In some embodiments, the N-terminal fragment of a fusion protein comprises an N-terminal fragment of an RNA-guided nickase. In some embodiments, the N-terminal fragment of a fusion protein additionally comprises an N- terminal fragment of a split-intein. In some embodiments, the first expression cassette further comprises a fourth promoter operatively linked to a fourth polynucleotide encoding a nicking guide RNA (ngRNA).

[0127] In some embodiments, the second vector comprises a second expression cassette comprising a second promoter described herein operatively linked to a polynucleotide encoding a C-terminal fragment of a fusion protein described herein. In some embodiments, the C- terminal fragment of a fusion protein comprises a C-terminal fragment of an RNA-guided nickase. In some embodiments, the C-terminal fragment of a fusion protein additionally comprises a reverse transcriptase. In some embodiments, the C-terminal fragment of a fusion protein additionally comprises a C-terminal fragment of a split-intein. In some embodiments, the second expression cassette further comprises a third promoter described herein operatively linked to a polynucleotide encoding a prime editing guide RNA (gRNA). In some embodiments, the C-terminal fragment of the fusion protein further comprises an RNA binding domain of small RNA binding exonuclease protection factor La.

[0128] In some embodiments, the present disclosure provides a system comprising(i) a first vector comprising a first expression cassette comprising:(a) a first modified TNNT2 promoter operatively linked to a first polynucleotide encoding an N-terminal fragment of a fusion protein comprising an N-terminal fragment of an RNA-guided nickase and an N-terminal fragment of a split-intein;(ii) a second vector comprising a second expression cassette comprising:(a) a second modified TNNT2 promoter operatively linked to a second polynucleotide encoding a C-terminal fragment of the fusion protein comprising a C- terminal fragment of the RNA-guided nickase, a reverse transcriptase, and a C-terminal fragment of the split-intein; and(b) a third promoter operatively linked to a third polynucleotide encoding a prime editing guide RNA (pegRNA).

[0129] In some embodiments, the first and second modified TNNT2 promoters areAttorney Docket No.: TENA-070 / 03WO 334682-2515 independently selected from (a) all TNNT2 promoters described herein, including those in Table 1, and (b) a TNNT2 promoter consisting of SEQ ID NO: 6.

[0130] In some embodiments, the first expression cassette further comprises a fourth promoter operatively linked to a fourth polynucleotide encoding a nicking guide RNA (ngRNA). In some embodiments, the ngRNA causes the nickase to nick the non-edited strand and increase the efficiency of prime editing. Without wishing to be bound by theory, the ngRNA-mediated nicking of the non-edited strand is thought to bias DNA repair mechanisms towards replacing the non-edited strand.

[0131] In some embodiments, the third promoter and / or fourth promoter are Pol III promoters. Without wishing to be bound by theory, the vector embodiments described herein enable expressing the pegRNA from one vector and the ngRNA from a second vector. This enables the use of the same promoter to each gRNA. Expressing the two gRNAs from the same vector requires using different promoters for these two transcription units (e.g., a human U6 promoter and a murine U6 promoter), as use of two identical promoters in one cassette may cause cassette instability as a result of the homology regions between the two identical promoters. Splitting the gRNAs into two different vectors allows the use of the same human promoter to express both.

[0132] In some embodiments, the first and second vectors are adeno-associated virus (AAV) vectors.

[0133] In some embodiments, the first vector comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 37, a promoter described herein operatively linked to an ngRNA, a left ITR disclosed herein, and a right ITR disclosed herein. In some embodiments, the second vector comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 38, a promoter described herein operatively linked to an pegRNA, a left ITR disclosed herein, and a right ITR disclosed herein.

[0134] In some embodiments, the first vector comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 35, a promoter described herein operatively linked to an ngRNA, a left ITR disclosed herein, and a right ITR disclosed herein. In some embodiments, the second vector comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 36, 43, 44, 46, 48, 50, 53, and 54, a promoter described herein operatively linked to a pegRNA, a left ITRAttorney Docket No.: TENA-070 / 03WO 334682-2515 disclosed herein, and a right ITR disclosed herein.

[0135] In some embodiments, the system expresses the fusion protein comprising the RNA-guided nickase and the reverse transcriptase. In some embodiments, the system expresses the fusion protein comprising the RNA-guided nickase, the reverse transcriptase, and the RNA binding domain of small RNA binding exonuclease protection factor La.Methods

[0136] In some embodiments, the disclosure herein provides methods of editing a target gene in a cell comprising contacting the cell with a system described herein. In some embodiments, the cell is in vivo or ex vivo. The target gene may be any suitable as determined by a person skilled in the art. Non-limiting examples of target genes include myosin binding protein C (MYBPC3), DWORF, junctophilin (e.g., JPH2), BAG3, phospholamban (PLN), alpha-crystallin B chain (CRY AB), LMNA (such as Lamin A and Lamin C isoforms), troponin I type 3 (TNNI3), lysosomal-associated membrane protein 2 (LAMP2, such as LAMP2a, LAMP2b and LAMP2c isoforms), desmoplakin (DSP, such as DPI and DPII isoforms), desmoglein 2 (DSG2), junction plakoglobin (JUP), plakophilin-2 (PKP2), matrix metallopeptidase 11 (MMP11), synaptopodin 2 like (SYNPO2L) (e.g., SYNPO2LA or SYNPO2LA), or an RNA binding motif protein 20 (RBM20).

[0137] In some embodiments, the disclosure herein provides methods of treating a cardiac pathology in a subject in need thereof, comprising administering a therapeutically effective amount of a system or a recombinant AAV (rAAV) virus or virion described herein.

[0138] The terms “cardiac pathology” or “cardiac dysfunction” are used interchangeably and refer to any impairment in the heart’s pumping function. This includes, for example, impairments in contractility, impairments in ability to relax (sometimes referred to as diastolic dysfunction), abnormal or improper functioning of the heart’s valves, diseases of the heart muscle (sometimes referred to as cardiomyopathies), diseases such as angina pectoris, myocardial ischemia and / or infarction characterized by inadequate blood supply to the heart muscle, infiltrative diseases such as amyloidosis and hemochromatosis, global or regional hypertrophy (such as may occur in some kinds of cardiomyopathy or systemic hypertension), and abnormal communications between chambers of the heart.

[0139] As used herein, the term “cardiomyopathy” refers to any disease or dysfunction that affects myocardium directly. The etiology of the disease or disorder may be, for example,Attorney Docket No.: TENA-070 / 03WO 334682-2515 inflammatory, metabolic, toxic, infiltrative, fibroplastic, hematological, genetic, or unknown in origin. Two fundamental forms are recognized (1) a primary type, consisting of heart muscle disease of unknown cause; and (2) a secondary type, consisting of myocardial disease of known cause or associated with a disease involving other organ systems. “Specific cardiomyopathy” refers to heart diseases associated with certain systemic or cardiac disorders; examples include hypertensive and metabolic cardiomyopathy. The cardiomyopathies include dilated cardiomyopathy (DCM), a disorder in which left and / or right ventricular systolic pump function is impaired, leading to progressive cardiac enlargement; hypertrophic cardiomyopathy, characterized by left ventricular hypertrophy without obvious causes such as hypertension or aortic stenosis; and restrictive cardiomyopathy, characterized by abnormal diastolic function and excessively rigid ventricular walls that impede ventricular filling. Cardiomyopathies also include left ventricular non-compaction, arrhythmogenic right ventricular cardiomyopathy, and arrhythmogenic right ventricular dysplasia.Recombinant AA V virions

[0140] In some embodiments, polynucleotides encoding a prime editor and one or more gRNAs for use in the present technology are in the form of a recombinant AAV (rAAV) virus or virion, for example, to deliver the expression cassettes described herein to cardiac cells.

[0141] In some embodiments, the AAV is any AAV known in the art or described herein. In some embodiments, the AAV is an AAV selected from the group consisting of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rh.10, rh.20, rh.74, or a chimeric or variant AAV derived therefrom. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.20, AAVrh.74, or a variant thereof.

[0142] In some embodiments, the rAAV virus or virion comprises an AAV capsid protein and an expression cassette as described herein. Capsid proteins are structural proteins that make up the assembled icosahedral packaging of the rAAV virion that contains the expression cassette. Capsid proteins are classified by the serotype. Wild-type capsid serotypes in rAAV virions can be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.20, or AAVrh.74. Engineered capsid types include chimeric capsids and mosaic capsids. Capsids are selected for rAAV virions basedAttorney Docket No.: TENA-070 / 03WO 334682-2515 on their ability to transduce specific tissue or cell types.

[0143] Any capsid protein that can facilitate rAAV virion transduction into cardiac cells for delivery of a transgene, as described herein, can be used. Capsid proteins used in rAAV virions for transgene delivery to cardiac cells that result in high expression can be, for example, AAV4, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the AAV capsid protein described herein is a wild-type AAV capsid protein from AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rh.10, rh.20, rh.74, or a variant thereof. In some embodiments, the AAV is AAV9 or a variant thereof. In some embodiments, the AAV is AAV5 or a variant thereof. In some embodiments, the AAV is AAV2 or a variant thereof.

[0144] Artificial capsids, such as chimeric capsids generated through combinatorial libraries, can also be used for transgene delivery to cardiac cells that results in high expression. Other capsid proteins with various features can also be used in the rAAV virions of the disclosure. AAV vectors and capsids are provided in U.S. Pat. Pub. Nos. US10011640B2; US7892809B2, US8632764B2, US8889641B2, US9475845B2, US10889833B2,US10480011B2, and US10894949B2, the entire contents of each of which are incorporated by reference herein; and Int’l Pat. Pub. Nos. WO2020198737A1, WO2019028306 A2, WO2016054554A1, WO2018152333A1, WO2017106236A1, WO2008124724A1,W02017212019A1, W02020117898A1, WO2017192750A1, W02020191300A1, and W02017100671 Al, the entire contents of each of which are incorporated by reference herein.

[0145] In some embodiments, the AAV capsid protein is an engineered capsid protein, comprising two or more non-naturally occurring amino acid motifs (also called “variant amino acid sequences”) relative to a corresponding wild-type or parental capsid protein. The wild-type or parental capsid protein can be any wild-type, chimeric, or mosaic capsid protein as described herein or as known in the art, or a variant thereof. In some embodiments, the wild -type or parental capsid protein is an AAV9, AAV5, AAVrh.10, or AAVrh.74 capsid. Non-limiting examples of engineered capsid proteins are provided in IntT Pat. Pub. Nos. WO2021216456A2 and W02023201207A1 , the entire contents of each of which are incorporated by reference herein.EXAMPLESAttorney Docket No.: TENA-070 / 03WO 334682-2515Example 1. Developing short promoters for cardiac expression.

[0146] In vivo prime editing requires fitting prime editor coding sequences and regulatory elements, and guide RNA transcription unit(s) in AAV vector(s), which lead to size constrain as AAV packaging efficiency, genome integrity, and / or potency drop dramatically when the recombinant genome size exceeds a certain limit. In this example, short promoter sequences were developed that mediate efficient gene expression in the heart. Alignment of the TNNT2 derived promoters is shown in FIG. 1A. Starting from the 400bp TNNT2 promoter (pTNNT2 (400bp), SEQ ID NO: 6), the most upstream 50bp region was trimmed to generate the 350bp TNNT2 promoter (pTNNT2 (350bp), SEQ ID NO: 7. Evolutionarily less conserved regions were further trimmed off, resulting in the 304bp TNNT2 promoter (pTNNT2 (304bp), SEQ ID NO: 8). Finally, the 150bp TNNT2 promoter (pTNNT2 (150bp) SEQ ID NO: 9) was generated by removing base pairs 1 -50 and 101-300 from the 400bp version. A chimeric 13 Ibp TNNT2 +MYBPC3 promoter SEQ ID NO: 10, which consists of two TNNT2 derived fragments and one MYBPC3 derived fragment, was developed and is shown in FIG. IB.Example 2. In vivo pooled comparison of short promoters.

[0147] The five short promoters described in Example 1 were cloned into AAV transgene cassette plasmids upstream of a GFP coding sequence and associated with 1 -3 unique barcode sequences in the 3’ UTR region (FIG. 2A). The resulting AAV cassettes were packaged in AAV9 capsids and administered to mice in a pooled manner through retro -orbital (RO) injection. At 3-week post- injection, heart samples were collected. The relative expression levels of barcodes were measured by next-generation sequencing (NGS) analysis and normalized to their abundance in the initial virus pool (FIG. 2B). The 350bp and 304bp promoters demonstrated expression levels in mouse heart that were at least comparable to the expression level of the 400bp promoter, making them valuable options for strong expression and saving 50bp to 96bp of space. Despite their substantially shorter length, the 150bp TNNT2 promoter and 13 Ibp TNNT2+MYBPC3 chimeric promoter yielded expression levels at about 50% of those observed for the 400bp TNNT2 promoter. These data demonstrate that these shorter promoters are suitable for cardiac expression cassettes where the size is severely constrained, precluding use of promoters between 304bp and 400bp in length.Attorney Docket No.: TENA-070 / 03WO 334682-2515Example 3. Comparing expression levels in cardiomyocytes mediated by various 3 ’ UTR and polyA signal sequences.

[0148] This example describes studies identifying 3 ’ UTR and polyA signal sequences that mediate high transgene expression levels and potentially facilitate in vivo prime editing applications. Five AAV transgene expression cassettes were constructed using different 3 ’ UTR and polyA signal sequences: pZC659 (SEQ ID NO: 11), pZC688 (SEQ ID NO: 12), pZC689(SEQ ID NO: 13), pZC695 (SEQ ID NO: 18), pZC690 (SEQ ID NO: 14), ZC691 (SEQ ID NO: 15), pZC692 (SEQ ID NO: 16), and pZC693 (SEQ ID NO: 17). Each of these cassettes used the same promoter (pTNNT2 (400bp) - SEQ ID NO: 6) and GFP coding sequence.

[0149] The 3’ UTR and polyA sequences were derived from either the bovine GH1 gene alone or bovine GH1 and human ACTC1. FIG. 3A shows an alignment of 3’ UTR and polyA signal sequences that were derived solely from the bovine GH1 gene. Transgene cassettes plasmids carrying chimeric 3’ UTR and polyA sequences derived from ACTC1 and bovine GH1 and are shown in FIG. 3B (pZC692) and FIG. 3C (pZC693), respectively. The relative GFP expression levels from these AAV transgene cassette plasmids (normalized to expression with the pZC695 vector) in human iPSC-derived cardiomyocytes (iPSC-CMs) are shown in FIG. 3D. pZC659, pZC688, pZC689, pZC695, and pZC690 all showed comparable expression. pZC691, pZC692, and pZC693 show higher expression than others, suggesting these 3’ UTR and polyA sequences are promising options for in vivo prime editing applications.Example 4. A dual AA V-based prime editing strategy edits the Dnmtl locus in vivo in mouse heart.

[0150] This example describes a study generating and validating a split prime editing (split-PE) cassette design that is compatible with dual-AAV delivery and can install desired edits in vivo in the heart. FIG. 4A shows the VI 5 split-PE cassette design used in this study. The prime editing (PE) machinery is divided into two parts expressed by two AAV vectors, each using the pTNNT2 (304bp) promoter (identified in FIG. 1 and FIG. 2) and the 3’ UTR + polyA signal from pZC691 (shown in FIG. 3).

[0151] The N-terminal PE vector (FIG. 4A, top, SEQ ID NO: 37) encodes approximately 75% of a SpCas9 nickase fused to the N-terminal split intein. The C-terminal PE vector (FIG. 4A, bottom SEQ ID NO: 38) encodes the C-terminal split intein, the remaining approximately 25% of SpCas9 nickase, and the M-MLV reverse transcriptase. The split inteins can mediateAttorney Docket No.: TENA-070 / 03WO 334682-2515 post-translational protein splicing, leading to the formation of full-length PE machinery (e.g., the SpCas9 nickase fused to the M-MLV reverse transcriptase). Additionally, the N-terminal PE vector and C-terminal PE vector express guide RNAs, ngRNA and pegRNA, respectively, which are responsible for directing the PE machinery to the target locus and determining the post-edit sequence and efficiency.

[0152] As shown in FIG. 4B, two PE cassettes were packaged into two separate AAVs with cardiac tropism (MyoAAV-4E capsid, described in WO 2021 / 077000A1, incorporated herein by reference) and co -administered to C57BL / 6 mice through RO-injection at 3.5E13 vg / kg each (7E13 vg / kg total). At 3-weeks post-injection, heart samples were collected, and the target region was amplified from genomic DNA and sequenced by NGS. FIG. 4C shows percentages of reads carrying the desired post-edit sequence with each dot representing one animal. Animals injected with negative control PE vectors (not targeting Dnmtl locus) showed no detectable editing at the Dnmtl locus. Animals injected with Dnmtl PE vectors showed -20% DNA editing efficiency at the Dnmtl locus in mouse heart. Expression and editing resulting from these vectors is expected to be localized to cardiomyocytes in the heart due to the 7,W7'2-derived promoter. However, the NGS data shown in FIG. 4C includes all cell types in the heart. For this reason, the actual editing efficiency in cardiomyocytes is likely to be significantly higher than suggested in FIG. 4C.Example 5. Optimization of split-PE cassettes.

[0153] This example describes a study that compares in vivo expression levels of 5 split- PE cassette design versions (V4, V13, V15, V16, and VI 8). These cassette design versions vary by promoter, relative orientation of PE machinery and guide RNA transcription units, and 3 ’ UTR + polyA signal whether the split-PE and gRNA transcription units face the same direction (tail-to-head) or opposite directions (tail-to-tail).Table 5. Split-PE Cassette DesignsAtorney Docket No.: TENA-070 / 03WO 334682-2515Atorney Docket No.: TENA-070 / 03WO 334682-2515Attorney Docket No.: TENA-070 / 03WO 334682-2515

[0154] Selected N- and C-terminal cassettes were packaged in AAVs comprising the MyoAAV-4E capsid and co-administered to wildtype C57BL / 6 mice through RO-injection at 3.5E13 vg / kg each (7E13 vg / kg total). Two to six mice were injected per cassette design version. Mouse hearts were harvested 3 weeks post-injection and homogenized. Cas9 was detected in heart lysates by western blot (FIG. 5A), with samples labeled as “cassette design version” - “animal ID” (e.g., “VI 3 -2”), “X” marking irrelevant lanes, and SpCas9-GFP protein spiked into blank lysate (cells not expressing Cas9) as a positive control (Cas9-GFP). VI 3 cassette design (FIG. 5B) tends to generate higher full length PE machinery expression level in mouse heart compared to other design versions.Example 6. Dual- AAV based prime editing rescues cardiac function in a Rbm20 cardiomyopathy mouse model

[0155] This example describes a study to test efficacy of prime editing treatment in the mouse Rbm20R636Q model, for which a schematic is shown in FIG. 6A. VI 3 split-PE cassettes carrying an exemplary pegRNA and ngRNA targeting mouse Rbm20 (“mRbm20-PE”) were packaged in the AAV ZC734 capsid (an engineered capsid that has a VR-VIII site variant sequence). A second pair of VI 3 dual-AAV split-PE vectors were generated as a negative control targeting mouse Dnmtl locus (“mDnmtl -PE”). N-terminal and C-terminal PE vectors were co-administered to 3.5-week-old Rbm20R636~ mice through RO injection at 4.64E13 vg / kg each (9.28E13 vg / kg total). Heart function of treated and control animals was measured by echocardiogram (Echo) once every 3 weeks, beginning at 4 weeks post-injection. FIGs 6B and 6C plot ejection fractions (EF), left ventricle internal diameters at systolic stage (LVID;s), and left ventricle internal diameters at diastolic stage (LVID;d) measured at pre-treatment baseline and post-injection time points. While heart function of Rbm20l<636~ mice slightlyAttorney Docket No.: TENA-070 / 03WO 334682-2515 declined after injection and remained roughly stable afterwards, mRbm20-PE treatment improved heart function relative to pre-treatment baseline, suggesting that the in vivo dual-AAV based prime editing method described herein is an efficacious strategy to rescue RBM20 cardiomyopathy.

[0156] A study was conducted to test efficacy of prime editing treatment in the mouse Rbm20R636^ model, for which a schematic is shown in FIG. 9A. VI 9 split-PE cassettes carrying pegRNA (m-peg-29) and ngRNA (m-ng-4.1) targeting mouse Rbm20 were packaged in the AAV ZC755 capsid (an engineered capsid that has a VR-VIII site variant sequence and a 716- 720 region variant sequence). The drug is referred to herein as “PE-mRbm20-5.3”. N-terminal and C-terminal PE vectors were co-injected to 3.5-week-old Rbm20R636Q / + mice through RO injection at 4.64E13 vg / kg per vector (9.28E13 vg / kg total). Heart function of treated, untreated, and wildtype animals was measured by echocardiogram (Echo) once every 4 weeks, beginning at 4 weeks post-injection. FIG. 9B plots ejection fractions (EF), left ventricle internal diameters at systolic stage (LVID;s), and left ventricle internal diameters at diastolic stage (LVID;d) measured at pre-treatment baseline and post-injection time points. FIG. 9C plots ejection fractions (EF), left ventricle internal diameters at systolic stage (LVID;s), and left ventricle internal diameters at diastolic stage (LVID;d) measured at the 20-week post-injection time point. PE-mRbm20-5.3 treatment improved heart function relative to the untreated (“Vehicle”) group, suggesting our in vivo dual-AAV based prime editing is an efficacious strategy to rescue RBM20 cardiomyopathy.Example 7. Incorporating the RNA-binding domain of small RNA binding exonuclease protection factor La improves dual-AA V based in vivo cardiac prime editing efficiency

[0157] This example describes a study to improve efficiency of our split-PE. The RNA- binding, N-terminal fragment of La was fused to the C-terminus of the VI 3 split-PE construct, generating a VI 9 split-PE design (FIG. 7A). An in vivo study was performed to compare PE efficiencies of dual-AAV based VI 9 split-PE cassette design against VI 3, both with cardiac specific TNNT2 promoter. The ZC734 AAV capsid (an engineered capsid that has a VR-VIII site variant sequence) was used to package VI 3 and VI 9 cassettes carrying pegRNA and ngRNA targeting mouse Dnmtl locus. PE-N and PE-C AAVs were co -administered to C57BL / 6 mice through RO-injection at 1E13 vg / kg each (2E13 vg / kg total). At 3-week post-injection, heart samples were collected, and the target region was amplified from genomic DNA and sequencedAttorney Docket No.: TENA-070 / 03WO 334682-2515 by NGS. FIG. 7B shows percentages of reads carrying the desired post-edit sequence with each dot representing one animal. VI 9 split -PE cassette design shows higher in vivo PE efficiency than VI 3.

[0158] In an additional study, a wild-type AAV9 and an engineered capsid that has a VR- VIII site variant sequence and a 716-720 region variant sequence were used to package a VI 9 split-PE gene editing system carrying pegRNA and ngRNA targeting the mouse Dnmtl locus. PE-N and PE-C AAVs were co -administered to C57BL / 6 mice systemically at 1E13 vg / kg each (2E13 vg / kg total). At 3-week post-injection, heart samples were collected, and the target region was amplified from genomic DNA and sequenced by NGS. FIG. 7C shows percentages of reads carrying the desired post-edit sequence with each dot representing one animal.Example 8. Comparing editing efficiencies of various split-PE machinery designs

[0159] This example describes a study in which new split-PE designs were designed to provide comparable or higher efficiency than VI 3 and / or VI 9 split-PE. FIG. 8A presents split- PE machinery diagrams of VI 3, VI 9, and V20 to V26 designs. To compare the efficiencies of these split-PEs, an in vitro study was performed in HEK293T cells using slightly modified V13, VI 9, and V20 to V26 designs in which the cardiac specific TNNT2 promoter was replaced by the ubiquitous CAG promoter. All split-PE versions were tested in combination with an exemplary pegRNA and an exemplary ngRNA. HEK293T cells were transfected using lipofectamine 3000 with lOOng of DNA consisting of PE-N plasmid, PE-C plasmid, ngRNA encoding DNA fragment, and pegRNA encoding DNA fragment in a 1 : 1 :5: 5 ratio. Cells were harvested 72 hours post-transfection, and the editing target region was amplified from genomic DNA and sequenced by NGS. FIG. 8B plots % reads with designed edits. VI 9, V20, V22, V24, and V26 split-PE machineries show high efficiencies.

[0160] A study was conducted to compare in vivo prime editing efficiencies of split-PE designs V19, V20, V22, and V24, and a schematic is shown in FIG.10A. The AAV ZC755 capsid was used to package VI 9, V20, V22, and V24 cassettes (for which diagrams are provided in FIG. 8A) carrying pegRNA (m-peg-29) and ngRNA (m-ng-4.1) targeting mouse Rbm20 locus. PE-N and PE-C AAV vectors were co-injected to C57BL / 6 mice through RO-injection at 2.8E13 vg / kg per vector (5.6E13 vg / kg total). At 8 weeks post-injection, heart samples were collected, and the target region was amplified from cardiac DNA and RNA samples and sequenced by NGS. FIG. 10B shows percentages of reads carrying desired edit(s) from DNAAttorney Docket No.: TENA-070 / 03WO 334682-2515 sequencing. FIG. IOC shows percentages of reads carrying desired edit(s) from RNA sequencing. In these vectors, PE machinery is expressed from a TNNT2 derived promoter that is expected to preferably express in cardiomyocytes (with absent, or lower, expression occurring in other cell types). Consequently, prime editing is expected to happen in cardiomyocytes at a higher frequency than in other cell types in the heart. Because the Rbm20 gene is primarily transcribed in cardiomyocytes, measurement of Rbm20 editing efficiency in RNA samples is expected to provide a more accurate assessment of actual prime editing efficiency in the target cell type (cardiomyocytes) compared to measurement of Rbm20 editing efficiency in DNA samples. Measurement of Rbm20 editing efficiency in DNA samples is expected to underestimate the level of actual prime editing, because tissue processing, DNA extraction, and NGS analysis capture genome copies from all cell types in the heart. V20, V22, and V24 split- PE designs provided the highest prime editing efficiencies.

[0161] Additional split-PE designs were generated, as shown in FIG. 11A. To compare the prime editing efficiencies of these designs, an in vitro study was performed in HEK293T cells using slightly modified VI 3, VI 9, and V20 to V26 designs in which the cardiac-specific TNN2 promoter was replaced by the ubiquitous CAG promoter. All split-PE versions were tested in combination with two guide RNA pairs targeting the human RBM20 locus (peg-186 + ng-26 and peg-189 + ng-26). Cells were seeded in 96-well plates, then transfected 24 hours later using lipofectamine 3000 with lOOng of DNA consisting of PE-N plasmid, PE-C plasmid, ngRNA encoding DNA fragment, and pegRNA encoding DNA fragment in a 1 : 1 :5:5 ratio. Cells were harvested 72 hours post-transfection, and wells of the same condition were combined. The editing target region was amplified from genomic DNA and sequenced by NGS. The percentage of reads with designed edits are shown for cells treated with the peg- 186 + ng-26 guide RNA pair (FIG. 11B) or the peg-189 + ng-26 guide RNA pair (FIG. 11C). V19, V20, V22, V24, V27, and V28 split-PE machineries all showed efficient prime editing in vitro, supporting their potential use in vivo.

Claims

Attorney Docket No.: TENA-070 / 03WO 334682-2515CLAIMS1. A modified cardiac troponin T (TNNT2) promoter, comprising a polynucleotide comprising 350 or fewer nucleotides of SEQ ID NO: 5.

2. The TNNT2 promoter of claim 1, wherein the polynucleotide comprises at least 120 nucleotides and not more than 350 nucleotides3. The TNNT2 promoter of claim 1 or claim 2, wherein the polynucleotide comprises at least 300 nucleotides and not more than 350 nucleotides.

4. The TNNT2 promoter of any one of claims 1 -3, wherein the polynucleotide comprises at least 120, at least 130, at least 140, or at least 150 nucleotides.

5. The TNNT2 promoter of any one of claims 1 -4, wherein the polynucleotide consists of 304 bp.

6. The TNNT2 promoter of claim 5, wherein the polynucleotide consists of SEQ ID NO: 8.

7. The TNNT2 promoter of any one of claims 1 -4, wherein the polynucleotide consists of350 bp.

8. The TNNT2 promoter of claim 7, wherein the polynucleotide consists of SEQ ID NO: 7.

9. The TNNT2 promoter of any one of claims 1 -4, wherein the polynucleotide consists of150 bp.

10. The TNNT2 promoter of claim 9, wherein the polynucleotide consists of SEQ ID NO: 9.

11. The TNNT2 promoter of any one of claims 1 -4, wherein the polynucleotide consists of131 bp.

12. The TNNT2 promoter of claim 11, wherein the polynucleotide consists of SEQ ID NO: 10.

13. An expression cassette comprising the promoter of any one of claims 1 -12, operatively linked to a polynucleotide encoding a gene product.

14. A vector comprising the expression cassette of claim 13.

15. The vector of claim 14, wherein the vector is an AAV vector.Attorney Docket No.: TENA-070 / 03WO 334682-251516. The vector of claim 15, wherein the AAV vector is AAV9 or a variant thereof.

17. A system comprising:(i) a first vector comprising a first expression cassette comprising:(a) a first modified TNNT2 promoter operatively linked to a first polynucleotide encoding an N-terminal fragment of a fusion protein comprising an N-terminal fragment of an RNA-guided nickase and an N-terminal fragment of a split- intein;(ii) a second vector comprising a second expression cassette comprising:(a) a second modified TNNT2 promoter operatively linked to a second polynucleotide encoding a C-terminal fragment of the fusion protein comprising a C-terminal fragment of the RNA-guided nickase, a reverse transcriptase, and a C-terminal fragment of the split-intein; and(b) a third promoter operatively linked to a third polynucleotide encoding a prime editing guide RNA (pegRNA).

18. The system of claim 17, wherein the first and second modified TNNT2 promoters are independently selected from:(a) the TNNT2 promoter of any one of claims 1 -12; and(b) a TNNT2 promoter consisting of SEQ ID NO: 6.

19. The system of claim 17 or 18, wherein the first expression cassette further comprises a fourth promoter operatively linked to a fourth polynucleotide encoding a nicking guide RNA (ngRNA).

20. The system of any one of 17-19, wherein the system expresses the fusion protein comprising the RNA-guided nickase and the reverse transcriptase.

21. The system of any one of 17-20, wherein the C-terminal fragment of the fusion protein further comprises an RNA binding domain of small RNA binding exonuclease protection factor La.

22. The system of claim 21, wherein the system expresses the fusion protein comprising the RNA-guided nickase, the reverse transcriptase, and the RNA binding domain of small RNA binding exonuclease protection factor La.Attorney Docket No.: TENA-070 / 03WO 334682-251523. The system of any one of claims 17-22, wherein the N-terminal fragment of the fusion protein comprises SEQ ID NO: 19 and the C-terminal fragment of the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-28 and 51-52.

24. The system of any one of claims 17-22, wherein the N-terminal fragment of the fusion protein comprises SEQ ID NO: 19 and the C-terminal fragment of the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 20-22, 24, 26, 28, 51, and 52.

25. The system of any one of claims 17-24, wherein the first expression cassette comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 37 and the second expression cassette comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 38.

26. The system of any one of claims 17-24, wherein the first expression cassette comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 35 and the second expression cassette comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 36, 43, 44, 46, 48, 50, 53, and 54.

27. The system of any one of claims 17-26, wherein the third promoter and / or fourth promoter are Pol III promoters.

28. The system of any one of claims 17-27, wherein the first and second vectors are adeno- associated virus (AAV) vectors.

29. The system of claim 28, wherein the first vector comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 37, a promoter operatively linked to an ngRNA, a left ITR, and a right ITR; and the second vector comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 38, a promoter operatively linked to a pegRNA, a left ITR, and a right ITR.

30. The system of claim 28, wherein the first vector comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 35, a promoter operatively linked to an ngRNA, a left ITR, and a right ITR; and the second vector comprises a DNA sequence that is at least 70%, 80%, 90%, 95%, 99%, or 100% identical to a sequence selected from SEQ IDAttorney Docket No.: TENA-070 / 03WO 334682-2515NOs: 36, 43, 44, 46, 48, 50, 53, and 54, a promoter operatively linked to a pegRNA, a left ITR, and a right ITR.

31. A method of editing a target gene in a cell comprising contacting the cell with the system of any one of claims 17-30.

32. The method of claim 31, wherein the cell is in vivo or ex vivo.

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