WO2025085759A1 - Telomerase enhancement of gene editing - Google Patents

Abstract

Provided herein is a composition comprising a telomerase or a functional fragment thereof, and/or a telomerase activator, wherein the composition further comprises components for a gene editing system. Also provided are methods of increasing the efficiency of gene editing and methods of editing a gene in a subject.

Description

TELOMERASE ENHANCEMENT OF GENE EDITING

CROSS REFERENCE TO RELATED APPLICATIONS

[1] This application claims the benefit of priority to U.S. Provisional Application No. 63/591,535, filed October 19, 2023, which is incorporated by reference herein in its entirety.

BACKGROUND

[2] Gene editing technologies have unlocked a new class of treatments for diseases, both genetic and otherwise. With the advent of genomic sequencing over the last several decades, the genetic mutations or abnormalities at the root of genetic predispositions or diseases have come to light. It has further been discovered how damage to a genome via external agents or age can be the root cause of diseases such as cancer. However, traditional pharmacological approaches cannot address these root causes..

[3] Approaches to genetic engineering began to trickle in slowly with the development of zinc finger nucleases, a specially designed enzyme capable of cleaving DNA at a specific place. Gene editing began to build with the development of transcription activator-like effector nucleases, however the early iterations of these systems could only cut. The replacement or editing of a gene was largely controlled by endogenous DNA repair and could not be directed to make significant changes. Gene editing exploded with the discovery of CRISPR/Cas systems, a precise gene editing system derived from a naturally occurring bacterial defense mechanism. CRISPR/Cas began to allow for much more precise single-stranded or double-stranded breaks and, for the first time, the introduction of repair templates to correct a mutated gene. The utility of CRISPR/Cas systems continues to be expanded, with modified systems like prime editors and base editors being introduced for more specific types of gene edits. However, such treatments are still limited by their inaccuracies and inefficiencies of editing, and the time and resources required. Gene editing as a whole is difficult to direct, particularly when applied to a genome as lengthy as the human genome.

[4] The human genome - and most eukaryotic genomes - are protected by telomeres, which occur at the ends of linear chromosomes and prevent degradation of the primary genetic information of the chromosome. These telomeres are maintained by the ribonucleoprotein telomerase. Degradation of telomeres is understood to play a role in age-related disease and, as such, maintenance of telomerase may play a pivotal role in the prevention and treatment of diseases. However, telomerase is not commonly employed as a disease treatment.

[5] As such, there exists a need for improved gene editing and treatment of age-related and genetic diseases. These needs and others are at least partially satisfied by the present disclosure. SUMMARY

[6] In an aspect, provided is a composition comprising a telomerase or a functional fragment thereof, and/or a telomerase activator, wherein the composition further comprises components for a gene editing system.

[7] In another aspect, provided is a composition comprising a nucleic acid encoding a telomerase or functional fragment thereof and one or more nucleic acids encoding components for a gene editing system. In another aspect, provided is a vector encoding any of the disclosed nucleic acids. In some aspects, the vector comprises mRNA, modified mRNA, or circular RNA.

[8] In another aspect, provided is an adenovirus comprising any of the disclosed vectors. In another aspect, provided is a cell or cell component (e.g., exosome), or a vehicle (e.g., lipid nanoparticle) comprising any of the disclosed vectors.

[9] In another aspect, provided is a method of increasing efficiency of gene editing, the method comprising exposing a target nucleic acid to a gene editing system and a telomerase or functional fragment thereof, and/or a telomerase activator, wherein said telomerase or functional fragment thereof increases efficiency of gene editing with the target nucleic acid.

[10] In another aspect, provided is a method of editing a gene in a subject in need thereof, the method comprising the steps of: a) providing to the subject a gene editing system, a telomerase or functional fragment thereof, and/or a telomerase activator, under conditions for gene editing; b) allowing gene editing to occur within a cell of the subject, wherein said telomerase or functional fragment thereof increases efficiency of gene editing with the target nucleic acid.

[11] Other systems, methods, features and/or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and/or advantages be included within this description and be protected by the accompanying claims.

BRIEF DESCRIPTION OF DRAWINGS

[12] FIGURE 1 depicts telomerase (TERT) therapy reduced yH2A.X (DNA damage marker) in ECs of different organs in mice.

[13] FIGURE 2 depicts human telomerase (hTERT) normalized the growth rate of endothelial cells (ECs) derived from patients with Hutchison Gilford Progeria Syndrome (HGPS).

[14] FIGURE 3 depicts a heat map representative of transcriptome analysis between Control, progeria and hTERT treated progeria cells.

[15] FIGURE 4 depicts a Western blot analysis for lamin A, progerin and lamin C proteins. Cells were treated with different combinations of RNA and cell lysates were collected 2 weeks after treatment. From left to right: 1 -protein Ladder. 2-Cell lysate from Non-HGPS fibroblasts (Con = Control, non-HGPS fibroblasts). 3-Cell lysate from HGPS fibroblasts, treated with vehicle. 4-Cell lysate from HGPS fibroblasts previously treated with hTERT mRNA 5 -Cell lysate from HGPS fibroblasts previously treated with CRISPR-Cas9 mRNA 6-Cell lysate from HGPS fibroblasts previously treated with CRISPR-Cas9 and sgRNA and RNA templates of sense and antisense. 7-Cell lysate from HGPS fibroblasts previously treated with hTERT and CRISPR-Cas9 mRNA in addition to sgRNA and sense template. 8-Cell lysate from HGPS fibroblast previously treated with hTERT and CRISPR-Cas9 mRNA in addition to sgRNA, sense and antisense templates. 9-Cell lysate from HGPS fibroblast previously treated with hTERT and CRISPR-Cas9 mRNA in addition to sgRNA and antisense template. 10-protein Ladder. The cell lysate from the normal (Non-HGPS) control fibroblasts manifest both Lamin A and C, but no Progerin. All lysates from HGPS fibroblasts, in addition to Lamin A and C, show evidence of Progerin. The Progerin levels are reduced in HGPS fibroblasts previously treated with hTERT mRNA, or with CRISPR- Cas9 mRNA in addition to sgRNA, sense and/or antisense templates. Progerin levels are most reduced in cells treated with the combination of TERT mRNA, together with CRISPR-Cas9 mRNA with sgRNA, sense and antisense templates.

[16] FIGURE 5 depicts WT mice were administered empty LNPs intravenously at different dosages (0, 2, 4, 6, or 8 pg/g, n=5/group), and sacrificed 6 h post-administration via cardiac puncture. Blood serum analysis revealed a dose-dependent increase of liver markers, allowing identification of 2 pg/g as a safe dosage.

[17] FIGURE 6 depicts WT mice were administered DiD-labeled LNPs intravenously (retro-orbital) at a dosage of 2 pg/g, and sacrificed 6h post-administration, to assess LNPs biodistribution in organs (heart, lungs, liver, spleen, and kidneys were imaged with IVIS). Preferential accumulation of LNPs occurred in filtering organs (liver, spleen) and lungs.

[18] FIGURE 7 depicts that the properties of LNP-mTERT are maintained in comparison to the empty LNPs within 28 days. Zeta potential (the surface charge), concentration, encapsulation, size, and particle diameter distribution are maintained in LNP-mTERT.

[19] FIGURE 8 depicts a schematic abstract of treatment with LNP delivery of hTERT and CRISPR/Cas.

[20] FIGURE 9 depicts progeria protein in iPSCs derived ECs with & without hTERT.

[21] FIGURE 10 depicts PCR of total RNA from HEK 293T cells using primers for Lamin A or Progerin. Total RNA was collected 7 days after transfection with the gene editing complex. As a note, the PCR products for Lamin A and progerin had different sizes. [22] FIGURE 11 depicts PCR of total RNA from HEK 293 T cells collected 7 days after transfection with genome editing complex. In the absence of telomerase mRNA (the GFP group), the gene editing complex to induce the Lamin A mutation is ineffective.

[23] FIGURE 12 depicts normal HAEC total RNA collected 72h after transfection. As a note, the PCR products for Lamin A and progerin had different sizes.

[24] FIGURE 13 depicts total RNA from HAEC collected 6 days after transfection.

[25] FIGURE 14 depicts gene editing to correct the 1824C>T mutation in iPSC-HGPS ECs. These data are from DNA Sanger Sequencing and experiments were done in triplicate. All components of the treatment are in the form of RNA molecules. Cells were collected 48-72 hours after transfections. Cells were treated with total equal amount of RNA, either GFP, hTERT, Cas9 or in a combination as (Cas9+gRNA+S/AS), (hTERT+Cas9+gRNA-i-S), (hTERT+Cas9+gRNA+AS) and (hTERT+Cas9+gRNA+S/AS).

[26] FIGURE 15 depicts that base editing in the presence of TERT reduces progerin in HGPS-ECs.

DETAILED DESCRIPTION

[27] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. 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. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

Definitions

[28] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[29] Throughout the description and claims of this specification, the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and are not intended to exclude, for example, other additives, segments, integers, or steps. Furthermore, it is to be understood that the terms comprise, comprising, and comprises as they relate to various aspects, elements, and features of the disclosed invention also include the more limited aspects of “consisting essentially of’ and “consisting of.” [30] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a “polymer” includes aspects having two or more such polymers unless the context clearly indicates otherwise.

[31] Ranges can be expressed herein as from “about” one particular value and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It should be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[32] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[33] For the terms "for example" and "such as," and grammatical equivalences thereof, the phrase "and without limitation" is understood to follow unless explicitly stated otherwise.

[34] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to the order or sequence of nucleotides along a strand of nucleic acids. In some cases, the order of these nucleotides may determine the order of the amino acids along a corresponding polypeptide chain. The nucleic acid sequence thus codes for the amino acid sequence. The nucleic acid sequence may be single-stranded or double-stranded, as specified, or contain portions of both double-stranded and single-stranded sequences. The nucleic acid sequence may be composed of DNA, both genomic and cDNA, RNA, or a hybrid, where the sequence comprises any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil (U), adenine (A), thymine (T), cytosine (C), guanine (G), inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. It may include modified bases, including locked nucleic acids, peptide nucleic acids and others known to those skilled in the art.

[35] As used herein, “amino acid” refers to a compound containing both amino ( — NH2) and carboxyl ( — COOH) groups generally separated by one carbon atom. The central carbon atom may contain a substituent which can be either charged, ionizable, hydrophilic or hydrophobic. Any of 22 basic building blocks of proteins having the formula NH2 — CHR — COOH, where R is different for each specific amino acid, and the stereochemistry is in the ‘L’ configuration. Additionally, the term “amino acid” can optionally include those with an unnatural ‘D’ stereochemistry and modified forms of the ‘D’ and ‘L’ amino acids.

[36] As used herein, “peptide” refers to a chain of amino acids in which each amino acid is connected to the next by a formation of an amide bond. Peptides are generally considered to consist of up to 30 amino acids, or alternatively up to 25 amino acids, or alternatively up to 20 amino acids, or alternatively up to 15 amino acids, or alternatively up to 10 amino acids, or alternatively up to 5 amino acids, or alternatively between about 5-10 amino acids, or alternatively between about 10-15 amino acids, while the term “protein” is applied to compounds containing longer amino acid chains. As used herein, the term “protein domain” refers to a unit of a protein that serves a single role (e.g., functional, structural, etc.). Proteins can include a single domain or multiple domains. As used herein, the term “enzyme” refers to a protein which can catalyze or facilitate a chemical reaction or biological process.

[37] As used herein, the term “cell” includes progeny. It is also understood that all progenies may not be precisely identical in D A content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological property, as screened for in the originally transformed cell, are included. The “cells” referred to in the present invention generally are prokaryotic or eukaryotic hosts.

[38] As used herein, the term “endogenous” refers to processes, moieties, or other phenomena that occur or are generated within a given cell, organism, or subject. In contrast, the term “exogenous” refers to processes, moieties, or other phenomena that occur or are generated outside of a given cell, organism, or subject.

[39] As used herein, the term “gene” refers to a segment of DNA arranged in a linear manner along a chromosome, which codes for a specific protein or segment of protein. A gene typically includes a promoter, a 5' untranslated region, one or more coding sequences (exons), optionally introns, and a 3’ untranslated region. The gene may further comprise a terminator, enhancers and/or silencers. In some aspects, the gene may be “mutated,” which refers to the replacement, absence, or presence of additional nucleic acids as compared to a control gene. In some aspects, the gene may be “abnormal,” which refers to an atypical presentation of a gene as compared to a control gene. In some aspects, the mutation or abnormality may have a negative effect on the expression of the gene.

[40] As used herein, the term “subject” includes animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some aspects, the subject is a human.

[41] As used herein, a “disease” or “disorder” or “condition” is an abnormal condition of an organism that impairs bodily functions, associated with specific symptoms and signs.

[42] As used herein, the term “telomerase” refers to a ribonucleoprotein (i.e., a protein that is conjugated to RNA) which can alter the length of a telomere. A telomerase can he any enzyme capable of affecting this result, including telomerases which are known in the art, functional fragments or derivatives of known telomerases, or synthetically derived, or artificially created, telomerases. The telomerase can be an enzyme with another function, such as a polymerase, that also has telomerase activity. Types of telomerases which are useful with the present invention are described in more detail below. The function of telomerase can refer to the entire process of altering the length of a telomere, or to individual steps or a series of steps involved in altering the length of a telomere including, but not limited to, identifying a nucleic acid sequence, nucleic acid sequence binding, addition or removal of bases to a nucleic acid sequence, etc.

[43] As used herein, the term “functional fragment” refers to any partial segment of a protein or nucleic acid sequence which at least partially retains the capability to perform a function or a part of a function of the full protein or full nucleic acid sequence. The functional fragment can be capable of performing multiple functions of the full protein or full nucleic acid sequence, a single function of the full protein or full nucleic acid sequence, or a part of one or more functions of the full protein or full nucleic acid sequence.

[44] A “telomere” is a region of repetitive nucleotide sequences associated with specialized proteins at the ends of linear chromosomes. Telomeres are a widespread genetic feature most commonly found in eukaryotes. In most, if not all species possessing them, they protect the terminal regions of chromosomal DNA from progressive degradation and ensure the integrity of linear chromosomes by preventing DNA repair systems from mistaking the very ends of the DNA strand for a double-strand break.

[45] “Gene editing,” “genome editing,” or “genome engineering,” is a type of genetic engineering in which nucleic acid is inserted, deleted, modified or replaced in a gene or a genome. The nucleic acid can be RNA, DNA, or a hybrid of both. It can comprise synthetic nucleic acids. Types of nucleic acids are described in more detail above. The gene or the genome can be found in a cell. Gene editing can be performed on a single cell, or multiple cells in vitro, it can be performed ex vivo, or it can be performed within cells of a tissue or an organism. Gene editing can be performed in vivo. Further details on gene editing are described below.

Compositions

[46] In an aspect, provided is a composition comprising a telomerase or a functional fragment thereof, and/or a telomerase activator, wherein the composition further comprises components for a gene editing system.

[47] As used herein, the term “components of a gene editing system” refers to one or more enzymes, proteins, compounds, or other elements needed to edit a gene. Specific gene editing systems are provided below, and any of the components listed below can be used as components of the gene editing system. [48] Gene editing systems

[49] In some aspects, the gene editing system is a CRISPR/Cas system. As used herein, the term “clustered regularly interspaced short palindromic repeats (CRISPR)ZCRISPR associated protein (Cas)” system or “CRISPR/Cas system” refers to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) proteins for editing a target nucleic acid sequence. CRISPR/Cas gene editing is known to those of skill in the art, and examples of its mechanism and utility can be found in (Zhang 2020) and (Liu 2022).

[50] As used herein, the term “target nucleic acid sequence” refers to a region of a nucleic acid sequence which is targeted by a gene editing system for gene editing. In some aspects, the CRISPR/Cas system comprises an endonuclease and a gRNA. As used herein, the term “endonuclease” refers to an enzyme which can cleave or split a nucleic acid sequence, typically by breaking at least one phosphodiester bond in the backbone of the nucleic acid sequence. These endonucleases can also include naturally occurring or engineered mutants, variants or derivatives thereof. In some aspects, the endonuclease comprises a Cas protein. In some aspects the Cas protein comprises Cas9, Casl l, Cas 12a Cas 13, or Cas 14. (Hillary 2022) provides examples of the different types of Cas systems.

[51] As used herein, the term “guide RNA” or “gRNA” or “single guide RNA” or “sgRNA” refers to an RNA strand which can bind to or near the target nucleic acid sequence. The gRNA can bring (i.e., “guide”) the endonuclease to the target nucleic acid sequence. In some aspects, the gRNA comprises a CRISPR RNA (crRNA) which binds to or near the target nucleic acid sequence and a trans-activating CRISPR RNA (tracrRNA) which binds to the endonuclease. Each of these is considered a component of a gene editing system.

[52] Specific components which may be included in a CRISPR/Cas gene editing system include, but are not limited to, an endonuclease, such as a Cas protein (e.g., Cas9, Casl 1, Cas 12a, Casl3, Cas 14), gRNA, and nucleic acid to be inserted (this can be referred to herein as a “payload.”) Such nucleic acid can be provided in a variety of ways, including, but not limited to, vectors. These are also considered components of the gene editing system. Examples of vectors can be found in Mengstie et al. (2022), and include adenoviral vectors (AdVs), adeno-associated viruses (AAVs), and lentivirus vectors (LVs). Also specifically contemplated herein is nanoparticle delivery of gene editing systems.

[53] In some aspects, the CRISPR/Cas system is a prime editing system. As used herein, the term “prime editing system” refers to a CRISPR-based system for the editing of target nucleic acid sequences without an exogenous repair template. In some aspects, the prime editing system further comprises pegRNA. As used herein, the term “prime editing guide RNA” or “pegRNA” refers to an RNA strand including a 5’ end that encodes a gRNA, which can bind to a first strand of a target nucleic acid sequence, and a 3’ end that encodes a primer binding sequence (PBS), which can bind to a reverse transcriptase and a second strand of the target nucleic acid sequence, and a template for the desired edit to the target nucleic acid sequence. Prime editing is known to those of skill in the art, and examples of its mechanism and utility can be found in (Lu 2022) and (Koeppel 2023).

[54] In some aspects, the prime editing system further comprises a reverse transcriptase. As used herein, the term “reverse transcriptase” (i.e., RNA-directed DNA polymerases) refers to an enzyme having reverse transcriptase activity (i.e., that catalyze synthesis of DNA from an RNA template). In general, such enzymes include, but are not limited to, retroviral reverse transcriptase, retrotransposon reverse transcriptase, non-LTR-retrotransposon reverse transcriptases, chromosomally encoded bacterial reverse transcriptases, and mobile group II intron reverse transcriptases, which are found in bacteria, archaea, and eukaryotic organelles, mitochondria and chloroplasts that evolved from endosymbiotic bacteria. These reverse transcriptases can also include naturally occurring or engineered mutants, variants or derivatives thereof.

[55] Specific components which may be included in a prime editing gene editing system include, but are not limited to, an endonuclease, such as a Cas protein (e.g., Cas9, Casl l, Casl2a, Casl3, Cas 14), pegRNA, and a reverse transcriptase or functional fragment thereof.

[56] In some aspects, the CRISPR/Cas system is a base editing system. As used herein, the term “base editing system” refers to a CRISPR-based system for the editing of target nucleic acid sequences by chemically modifying individual bases. In some aspects, the base editing system further comprises a base editor. As used herein, the term “base editor” refers to an enzyme which can convert a given base in a nucleic acid sequence to another. In some aspects, the base editor comprises a cytosine base editor (CBE). As used herein, the term “cytosine base editor” or “CBE” refers to a base editing system that can convert cytosine to thymine and/or guanine to adenine. In some aspects, the CBE comprises cytidine deaminase or a functional fragment thereof. In some aspects, the base editor is an adenine base editor (ABE). As used herein, the term “adenine base editor” or “ABE” refers to a base editing system that can convert adenine to guanine. In some aspects, the ABE comprises adenine deaminase or a functional fragment thereof. Base editing is known to those of skill in the art, and examples of its mechanism and utility can be found in (Rees 2018) and (Eid 2018).

[57] Specific components which may be included in a base editing gene editing system include, but are not limited to, an endonuclease, such as a Cas protein (e.g., Cas9, Casl l, Cas 12a, Casl 3, Cas 14), gRNA, cytidine deaminase or a functional fragment thereof, adenine deaminase or a functional fragment thereof, and a base to be inserted. [58] In some aspects, the gene editing system is a zinc finger nuclease (ZNF) system. As used herein, the term “ZFN system” refers to a protein, or a domain within a larger protein, that binds nucleic acid sequences in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. In some aspects, the zinc finger nuclease system comprises a restriction endonuclease and a DNA binding domain. As used herein, the term “restriction endonuclease” refers to an enzyme which can cleave or split a nucleic acid sequence at a specific restriction site (e.g., short palindromic sequences), typically by breaking at least one phosphodiester bond in the backbone of the nucleic acid sequence. These restriction endonucleases can also include naturally occurring or engineered mutants, variants or derivatives thereof. In some aspects, the restriction endonuclease comprises Fokl. As used herein, the term “DNA binding domain” refers to a protein domain which can recognize and bind to either single stranded or double stranded DNA or other nucleic acids. In some aspects, the DNA binding domain comprises a zinc finger DNA binding domain. ZFN gene editing is known to those of skill in the art, and examples of its mechanism and utility can be found in (Kianianmomeni 2011) and (Carroll 2008).

[59] Specific components which may be included in a ZFN gene editing system include, but are not limited to, a restriction endonuclease, such as Fokl, a DNA binding domain, such as a zinc finger DNA binding domain, and a gene to be inserted.

[60] In some aspects, the gene editing system is a transcription activator-like effector nuclease (TALEN) system. As used herein, the term “TALEN system” refers to artificial restriction enzymes generated by fusing a TAL effector DNA binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) can be quickly engineered to bind practically any DNA sequence. The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that are engineered to work together to cleave DNA at the same site. In some aspects, the TALEN system comprises a restriction endonuclease and a DNA binding domain. In some aspects, the restriction endonuclease comprises Fokl. In some aspects, the DNA binding domain comprises a transcription activator-like (TAL) effector DNA binding domain. TALEN gene editing is known to those of skill in the art, and examples of its mechanism and utility can be found in (Joung 2012) and (Becker 2021).

[61] Specific components which may be included in a TALEN gene editing system include, but are not limited to, a restriction endonuclease, such as Fokl, a DNA binding domain, such as a transcription activator-like effector DNA binding domain, and a gene to be inserted. [62] Telomerase/telomerase activators

[63] Naturally occurring telomerase includes a telomerase reverse transcriptase (TERT) and telomerase RNA (TERC). The scientific community broadly considers TERT to specifically act at telomeres to extend them, thereby mitigating the “end replication problem” of telomere loss after each cell division. Telomerase across species can further include other species- specific proteins, for example, dyskerin in human telomerase. In some aspects, the telomerase or functional fragment thereof comprises TERT or a functional fragment thereof. In some aspects, the telomerase or functional fragment thereof comprises a TERT domain selected from essential N-terminal (TEN) domain, a TERT RNA-binding domain (TRBD), a reverse-transcriptase (RT) domain, a C- terminal extension (CTE), or any combinations thereof. In some aspects, the telomerase or functional fragment thereof comprises multiple TERT domains selected from the above-listed TERT domains. In some aspects, the multiple TERT domains are connected, conjugated, or complexed together in a naturally occurring telomerase. In some aspects, the multiple TERT domains are not connected, conjugated, or complexed together in a naturally occurring telomerase. In some aspects, the telomerase or functional fragment thereof comprises an entire TERT domain or multiple entire TERT domains selected from the above-listed TERT domains. In some aspects, the telomerase or functional fragment thereof comprises at least one partial TERT domain selected from the above-listed TERT domains.

[64] In some aspects, the telomerase or functional fragment thereof comprises TERC or a functional fragment thereof. In some aspects, the telomerase or functional fragment thereof comprises dyskerin or a functional fragment thereof. In some aspects, the telomerase or a functional fragment thereof comprises any combination of TERT or a functional fragment thereof, a full TERT domain or functional fragment thereof, a partial TERT domain or functional fragment thereof, TERC or a functional fragment thereof, and dyskerin or a functional fragment thereof.

[65] In some aspects, the telomerase or functional fragment thereof is naturally occurring. In some aspects, the telomerase or functional fragment thereof is endogenous to a cell comprising the composition. In some aspects, the telomerase or functional fragment thereof is exogenous to a cell comprising the composition. In some aspects, the telomerase or functional fragment thereof is not naturally occurring. In some aspects, the telomerase or functional fragment thereof further comprises one or more mutations to a naturally occurring telomerase or functional fragment of a naturally occurring telomerase.

[66] In some aspects, the telomerase or functional fragment thereof is derived from a mammal. In some aspects, the mammal is swine, human, bovine, or primate. In some aspects, the telomerase or functional fragment thereof is derived from a bird. In some aspects, the bird is poultry.

[67] As used herein, the term “telomerase activator” refers to a molecule, compound, complex, or combinations thereof which can increase the activity of any one or more functions of a telomerase or functional fragment thereof relative to a control telomerase or functional fragment thereof. In some aspects, the telomerase activator is naturally occurring. In some aspects, the telomerase activator is not naturally occurring. In some aspects, the telomerase activator comprises cycloastragenol, oleanolic acid, maslinic acid, or combinations thereof. In some aspects, the telomerase activator is a telomerase activator compound (TAC) as described in Shim et al., TERT activation targets DNA methylation and multiple aging hallmarks. Cell. 2024 Jul 25 ; 187(15):4030- 4O42.el3.

[68] In some aspects the telomerase activator increases activity of an endogenous telomerase or functional fragment thereof. In some aspects, the telomerase activator increases activity of an exogenous telomerase or functional fragment thereof. In some aspects, the telomerase activator is required so as to activate an exogenous telomerase or functional fragment thereof that is otherwise inactive in the absence of the telomerase activator.

[69] Combination of telomerase/telomerase activator and compositions

[70] In another aspect, provided is a composition comprising a nucleic acid encoding a telomerase or functional fragment thereof and one or more nucleic acids encoding components for a gene editing system.

[71] In some aspects, TERT replaces the reverse transcriptase in the gene editing system.

[72] TERT can also be used in the prime editing system as a replacement for the reverse transcriptase enzyme currently in use.

[73] In some aspects, the gene editing system is a CRISPR/Cas system. In some aspects, the CRISPR/Cas system comprises an endonuclease and a gRNA. In some aspects, the endonuclease comprises a Cas protein. In some aspects, the Cas protein comprises Cas9, Casl 1, Casl2a Casl3, or Cas 14. In some aspects, the Cas protein comprises a modified version of Cas9, known as “nCas9” (Cas9 nickase), which creates a single-strand break rather than the double-strand break generated by standard Cas9. In some aspects, the gRNA comprises a crispr RNA (crRNA) which binds to or near the target nucleic acid sequence and a trans-activating crispr RNA (tracrRNA) which binds to the endonuclease.

[74] In some aspects, the CRISPR/Cas system is a prime editing system. In some aspects, the prime editing system further comprises pegRNA. In some aspects, the prime editing system further comprises a reverse transcriptase. In some aspects, the reverse transcriptase is telomerase reverse transcriptase (TERT) or a functional fragment thereof.

[75] In some aspects, the gene editing system is a zinc finger nuclease (ZNF) system. In some aspects, the zinc finger nuclease system comprises a restriction endonuclease and a DNA binding domain. In some aspects, the restriction endonuclease comprises Fokl. In some aspects, the DNA binding domain comprises a zinc finger DNA binding domain.

[76] In some aspects, the gene editing system is a transcription activator-like effector nuclease (TALEN) system. In some aspects, the TALEN system comprises a restriction endonuclease and a DNA binding domain. In some aspects, the restriction endonuclease comprises Fokl. In some aspects, the DNA binding domain comprises a transcription activator-like (TAL) effector DNA binding domain.

[77] In some aspects, the telomerase or functional fragment thereof comprises TERT or a functional fragment thereof. In some aspects, the telomerase or functional fragment thereof comprises a TERT domain selected from essential N-terminal (TEN) domain, a TERT RNA- binding domain (TRBD), a reverse-transcriptase (RT) domain, a C-terminal extension (CTE), or any combinations thereof. In some aspects, the telomerase or functional fragment thereof comprises multiple TERT domains selected from the above-listed TERT domains. In some aspects, the multiple TERT domains are connected, conjugated, or complexed together in a naturally occurring telomerase. In some aspects, the multiple TERT domains are not connected, conjugated, or complexed together in a naturally occurring telomerase. In some aspects, the telomerase or functional fragment thereof comprises an entire TERT domain or multiple entire TERT domains selected from the above-listed TERT domains. In some aspects, the telomerase or functional fragment thereof comprises at least one partial TERT domain selected from the abovelisted TERT domains.

[78] In some aspects, the telomerase or functional fragment thereof comprises TERC or a functional fragment thereof. In some aspects, the telomerase or functional fragment thereof comprises dyskerin or a functional fragment thereof. In some aspects, the telomerase or a functional fragment thereof comprises any combination of TERT or a functional fragment thereof, a full TERT domain or functional fragment thereof, a partial TERT domain or functional fragment thereof, TERC or a functional fragment thereof, and dyskerin or a functional fragment thereof.

[79] In some aspects, the telomerase or functional fragment thereof is naturally occurring. In some aspects, the telomerase or functional fragment thereof is endogenous to a cell comprising the composition. In some aspects, the telomerase or functional fragment thereof is exogenous to a cell comprising the composition. In some aspects, the telomerase or functional fragment thereof is not naturally occurring. In some aspects, the telomerase or functional fragment thereof further comprises one or more mutations to a naturally occurring telomerase or functional fragment of a naturally occurring telomerase.

[80] In some aspects, the telomerase or functional fragment thereof is derived from a mammal. In some aspects, the mammal is swine, human, bovine, or primate. In some aspects, the telomerase or functional fragment thereof is derived from a bird. In some aspects, the bird is poultry.

[81] In another aspect, provided is a vector encoding any of the disclosed nucleic acids. As used herein, the term “vector” refers to any moiety which can deliver a nucleic acid sequence into a cell or virus so that the nucleic acid sequence can be replicated and/or expressed by the cell or virus. In some aspects, the vector is a plasmid. In some aspects, the vector is a viral vector. In some aspects, the vector is a cosmid. In some aspects, the vector is an artificial chromosome. In some aspects, the vector comprises mRNA, modified mRNA, or circular mRNA. In another aspect, provided is an adenovirus comprising any of the disclosed vectors. In another aspect, provided is vehicle (e.g., lipid nanoparticle) comprising any of the disclosed vectors.

[82] In another aspect, provided is a cell comprising any of the disclosed vectors. In some aspects, the cell has been transfected with any of the disclosed vectors. In some aspects, the cell can deliver any of the disclosed vectors to another cell or virus. In some aspects, the cell is a bacterium, a yeast, an archaeon, or another prokaryotic or single-celled organism. In some aspects, the cell is a mesenchymal stem cell, an induced pluripotent stem cell, or an embryonic stem cell. In some aspects, the cell is naturally occurring within a subject. In some aspects, provided is a cell component (e.g., exosome) comprising any of the disclosed vectors.

Methods

[83] In an aspect, provided is a method of increasing efficiency of gene editing, the method comprising exposing a target nucleic acid to a gene editing system and a telomerase or functional fragment thereof, and/or a telomerase activator, wherein said telomerase or functional fragment thereof increases efficiency of gene editing with the target nucleic acid.

[84] As used herein, the term “increasing efficiency of gene editing” refers to improving the number of on-target edits by the gene editing system, reducing the number of off-target edits by the gene editing system, and/or reducing the time and/or resources required for the function of the gene editing system. In some aspects, increasing efficiency of gene editing by using a telomerase comprises an increase in the number of on-target edits of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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%, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 times (or more, less, or any amount in between) compared to gene editing without the use of telomerase. In a specific example, the efficiency is increased by about 10%.

[85] In some aspects, use of telomerase with the gene editing system decreases the number of off-target edits of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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%, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 times (or more, less, or any amount in between) compared to gene editing without the telomeric moiety. In a specific example, the reduction in off-target edits is decreased by about 10%.

[86] In another aspect, provided is a method of editing a gene in a subject in need thereof, the method comprising the steps of: a) providing to the subject a gene editing system, a telomerase or functional fragment thereof, and/or a telomerase activator, under conditions for gene editing; b) allowing gene editing to occur within a cell of the subject, wherein said telomerase or functional fragment thereof increases efficiency of gene editing with the target nucleic acid.

[87] As used herein, the term “conditions for gene editing” refers to one or more environmental factors which permit a gene editing system to have stability, reach, and edit a target nucleic acid sequence. Conditions for gene editing are known to those of skill in the art. Exemplary conditions for gene editing are given in (Liu 2022), (Carroll 2008), (Becker 2021), and (Lu 2022).

[88] As used herein, the term “allowing gene editing to occur” refers to providing sufficient time under conditions for gene editing for a gene editing system to reach and edit the target nucleic acid sequence. Allowing gene editing to occur is known to those of skill in the art. Examples of allowing gene editing to occur is given in (Liu 2022), (Carroll 2008), (Becker 2021), and (Lu 2022).

[89] In some aspects, the subject has a disease, disorder, or condition which can benefit from gene editing. In some aspects, a disease, disorder, or condition which can benefit from gene editing can refer to a disease, disorder, or condition caused by a mutation or abnormality in a gene or multiple genes of the subject. In some aspects, the disease is Hutchinson-Gilford Progeria Syndrome (progeria), autosomal dominant polycystic kidney disease, cystic fibrosis, sickle cell anemia, alpha- 1 antitrypsin deficiency, hemophilia, beta thalassemia, atherosclerosis, vascular dementia, or other genetic or age-related disorders. In some aspects, the gene to be edited is an LMNA gene.

[90] In some aspects, a disease, disorder, or condition which can benefit from gene editing can refer to a disease, disorder, or condition caused by damage to a gene or multiple genes of the subject. In some aspects, the damage to a gene or multiple genes of a subject is the result of radiation exposure. In some aspects, a disease, disorder, or condition which can benefit from gene editing can refer to a disease which can be targeted by genetic editing of one or more of a subject’s immune cells. In some aspects, the disease is cancer.

[91] In some aspects, the gene to be edited is replaced, deleted, modified, or inserted. In some aspects, when the gene is to be replaced or modified, the gene editing system further comprises a repair template.

[92] In some aspects, the gene editing system and telomerase or functional fragment thereof are encoded by a nucleic acid. In some aspects, the nucleic acid is in a vector. In some aspects, the vector is in an adenovirus. In some aspects, the vector is in a vehicle (e.g., lipid nanoparticle).

EXAMPLES

Example 1 - hTERT in Gene Editing

[93] ECs as well as VSMCs derived from HGPS are senescent. Using growth rate monitoring of cells, a study was conducted which showed that during the period (21 days) while cells are doubling, the accumulation of progerin, and associated DNA damage is more severe in ECs compared to VSMCs. Also, the number of senescent cells, indicated through b-gal staining, increases in ECs compared to VSMCs (Xu 2022). Furthermore, the senescent ECs release inflammatory cytokines that can adversely affect other cells. Therefore, progerin affects the patient’s cells either directly or through the systemic effects of inflammatory cytokines released by cells (such as ECs) manifesting a senescent associated secretory phenotype (SASP).

[94] hTERT is a human reverse transcriptase, that is widely believed to specifically have its primary action at the telomeres. It uses the long noncoding RNA TERC as a template to reverse transcribe the hexameric repeat TTAGGG into telomeric DNA, preserving telomere length. hTERT treatment has previously been shown to reduce nuclear blebbing (nuclear malformation) and to normalize cellular shape. Further, endothelial function (tube formation, nitric oxide production, and LDL uptake by HGPS-ECs) is restored after telomerase therapy. Therefore, the HGPS-ECs are reasonable targets for genome editing and could reduce the overall toxicity of progerin in the vasculature by reducing EC SASP, and the release of inflammatory cytokines into the systemic circulation.

[95] HGPS systemically affects all organs, causing cutaneous changes, loss of sub-dermal adipose tissue, joint stiffness, and skeletal and muscle abnormalities. Abnormal post-translational modification due to a lack of amino acid residues in the normal cleavage sites causes accumulation of progerin in the nuclear envelope and a disturbed nuclear shape, rigidity, and function leading to aberrant gene expression. mTERT delivery in progeria mice also significantly reduced DNA damage foci in isolated lung ECs, liver vasculature, and hepatocytes (FIG. 1). The improvement in the lifespan of the mice and their health, such as reduced aorta inflammatory signals, VCAM- 1, and pro-thrombotic factors, may partly be caused by reducing progerin expression through the delivery of telomerase therapy.

[96] Though the hTERT-mRNA transfection was transient in human cells, the effects were robust and significant (FIG. 2). RNA sequencing analysis showed that more than 250 genes were fully recovered (FIG. 3). Preliminary data from investigating the hTERT contribution of genome editing indicates that transient transfection of coding mRNA for hTERT, Cas9, sgRNA (sg), and donor template RNA (s: sense, AS: antisense) (Xu 2020) (Lee 2020) in progeria fibroblast significantly reduces progerin mRNA after a 48-h transfection. Furthermore, a western blot analysis of progerin protein two weeks later shows very low levels of progerin in cells transfected with hTERT+Cas9+S/AS (red star) compared to other combinations (FIG. 4). These data suggest three functions for hTERT : 1 ) hTERT has an indirect role in increasing homologous recombination activity beyond telomeric sites, 2) hTERT has a direct role in the repair process of DNA damage through promoting HDR, or 3) hTERT regulates splicing mechanisms partly by reducing cryptic splicing of lamin A, which leads to reduced progerin. Either way, it could help with the permanent correction of lamin A mutation in cells transfected with Cas9 and has therapeutic potential.

[97] Delivering nucleic acids to target cells requires a delivery system compatible with the properties of genetic cargo. LNPs exploiting a microfluidic approach that allows rapid and efficient mixing of an organic phase (containing lipids and phospholipids) with an aqueous phase at acidic pH were synthesized containing the nucleic acids of interest. During synthesis, the negatively charged mRNA complexes with the positively charged ionizable lipid of choice (DLin-MC3- DMA, protonated at the acidic pH of synthesis). The other helper lipids (DSPC, Cholesterol, and PEG1000-PE) nucleate around those complexes leading to self-assembly of nanoparticles with a size of -100 nm. Later, particles are dialyzed to bring the solution to a physiological pH and allow de-protonation of the ionizable lipid. [98] When administering LNPs intravenously to WT mice, increasing toxicity was observed with increasing levels of LNPs, as indicated by liver dysfunction marker enzymes (i.e., ALT, AST, BUN) in addition to serum LDL (FIG. 5). Nevertheless, at a dose of 2 mg/kg, these marker levels were comparable to those observed in mice given the vehicle PBS, and adding mRNA to the LNPs did not alter those levels. Lastly, biodistribution was assessed by delivering DiD-labeled LNPs to WT mice at a dosage of 2pg/g and measuring the fluorescence signal from LNPs in various organs (heart, lungs, liver, spleen, kidneys) at 6 h post-administration. As expected, IVIS imaging revealed that major off-target accumulation occurred mainly in filtering organs (liver and spleen) and lungs (FIG. 6). The study has also shown that the LNP-encapsulated luciferase mRNA maintained its integrity and can be translated. Further, others have shown that low-dose intravenous injections of LNPs encapsulating Cas9 can effectively target specific organs (Wei 2020).

Example 2 - Characterizing the role of hTERT in assisting Cas9 genome editing to correct the mutated lamin A gene and rejuvenate vascular cells

[99] A study can be conducted in which cells can be transfected with hTERT mRNA, Cas9 fused to GFP sequences, sgRNA, and template RNA (sense or antisense) to test whether 1) hTERT increases the efficiency of molecular modifications in the genome editing system Cas9, and 2) hTERT can permanently correct the lamin A mutation in HGPS cells. GFP expression in cells can facilitate the sorting of cells with active Cas9 and can indicate that the lamin A gene has been corrected. VSMCs and ECs derived from HGPS-iPSCs can be used in such experiments. Cells can be transfected once or multiple times, and the availability of hTERT and Cas9 protein (from the first transfection) can increase the efficiency of genome editing. The results can be compared to cells that have received hTERT -mRNA or Cas9 protein alone. Western blotting and PCR of lamin A from GFP-expressing cells can be analyzed to quantify the expression of progerin protein and the edited genome, respectively. Further analysis of nuclear morphology and functional assays for VSMCs and ECs can be performed to demonstrate the efficacy of genome editing and cellular health.

[100] Design of mRNA N another apeutic: cGMP-grade modified mRNA (mmRNA) can be incorporated into clinical-grade LNPs that are FDA-approved and in clinical use. LNPmmRNA therapeutics can be synthesized using a GMP-compliant device such as the NanoassemblerTM (Precision NanoSystems, Inc.) in a GMP facility. The NanoassemblerTM enables rapid, reproducible, and scalable manufacturing of homogeneous next-generation LNPs, which can incorporate nucleic acids (Li 2019) using a microfluidic mixing cartridge, where lipid-containing solvent is pumped into one inlet and aqueous buffer into the other inlet. LNP formation takes place at the interface of the solvent and aqueous streams and is based on polarity change along with the chamber. The mixing is promoted by the design of the channel equipped with a staggered herringbone geometry, which provides controlled mixing of organic and aqueous phases. The physiochemical properties of LNPs can be controlled via flow rate alterations of the separate streams as well as the ratios of aqueous to organic phases during synthesis.

[101] Furthermore, the system can be scaled-up, such as by using parallel mixing cartridges, allowing its utilization as a high throughput method (Belli veau 2012). LNPs have been formulated with various characteristics (size ranges and zeta potentials). As an example, in FIG. 7, the properties of LNP-mTERT are maintained within 28 days, which indicates the proper encapsulation and maintenance of mRNA integrity for the delivery. As needed, the LNPs can be formulated to sizes of 50nm-200nm and zeta potential of -20m V to +20mV.

[102] The safety of LNP have been shown in Covid-19 vaccine, however the safety of intravenous application of the LNP can be investigated to assess the outcome when using different dosages compared to local intramuscular vaccine administration. Using the optimized protocol to assemble the LNP encapsulating mRNA, LNPs carrying different combinations of mRNA can be generated for injection; Including scramble LNP-mRNA, LNP-Cas9-sgRNA, LNP-mTERT-Cas9 and (sgRNA & template RNA), all in concentration of 2mg/kg, which have been shown to indicate minimal toxicity (FIG. 5) in mice.

[103] Research Design and Methods: The study can test whether hTERT increases the efficiency of molecular modifications using the genome editing system Cas9 and whether it can permanently correct the lamin A mutation in HGPS cells. Fibroblasts (obtained from the Progeria Foundation), ECs and VSMCs derived from HGPS iPSCs, and ECs and VSMCs derived from their parents can be used as control cells. Cells can be transfected with mRNA of hTERT (0-2 ug/ml) alone or in combination with Cas9 fused to GFP sequences, sgRNA, and template RNA (sense or antisense). GFP expression in cells can facilitate the sorting of cells with active Cas9 to facilitate evaluation of the lamin A gene editing. PCR or sequencing can confirm the correction. In addition, the viability of the cells can be determined to investigate the toxicity of combinatory mRNA. This therapy can be tested on all HGPS cells, including fibroblasts, to identify whether hTERT assistance to Cas9 correlates with telomere length or is independent since it has been demonstrated that some HGPS fibroblasts have normal telomere length (Li 2019). Cells can be transfected twice within 24 h to have hTERT and Cas9 protein (from the first transfection) already translated and available for genome editing as editing efficiency is increased when the Cas9 proteins are available. Cells can be collected 2 days and 1, 2, 3, and 4 weeks after transfection for further analysis. The results can be compared with cells that have received hTERT-mRNA or Cas9 alone. Western blotting and PCR of lamin A/progerin protein and mRNA from GFP-expressing cells can be analyzed to quantify the expression of progerin protein and the edited genome, respectively. Analysis of nuclear morphology, gene expression of proinflammatory factors and functional assays for VSMCs and ECs can be performed to demonstrate the efficiency of genome editing and cellular health. Although the mRNA components that can be used for this therapy that have a low chance of genomic integration, deep sequencing of the human genome can be performed to identify potential off-target effects in human cells, which can be compared with the mouse study.

[104] This approach has the advantage of direct targeting of progerin without suppressing the indispensable lamin A gene for human cells (Sanchez-Lopez 2021), distinguishing it from current studies. Further, an RNA template can be provided for editing the mutation, which can be degraded due to its short lifespan.

Example 3 - Identifying whether the lipid nanoparticle delivery of mRNA encoding hTERT, Cas9, and sgRNA can correct the mutation and improve lifespan in progeria mice

[105] It has previously been shown that lentiviral delivery of mTERT in a progeria mouse model significantly increases their lifespan. A study can be conducted which aims to optimize a safe L P formulation to be used as a delivery system for a mixture of mRN As to effectively correct the lamin A mutation in addition to rejuvenating the cells. A preliminary evaluation of in vivo dose-dependent toxicity of a single dose of empty LNPs in wild-type mice allowed the identification of a safe dosage, revealing no acute toxicity within 8 hours after injection while delivering intact mRNA that can be readily translated. Progeria mice can be administered LNPs as follows: 1) empty LNPs, 2) LNPs loaded with scramble mRNA, 3) LNPs loaded with Cas9 mRNA and sgRNA, or 4) LNPs loaded with mTERT, Cas9, sgRNA, mRNAs, and template RNA. Wildtype mice can be administered LNPs loaded with scramble mRNA. Liver enzyme studies can be performed to identify any toxicity. Serum and organs (liver, spleen, lung, and heart) can be collected for pathology and histology analyses while also sequencing for the lamin A gene. In particular, the aorta can be assessed for the correction of lamin A mutation as well as markers of vascular senescence: vascular adhesion molecule expression (VCAM1), inflammatory cytokines, reactive oxygen species, nitric oxide, cellular proliferation, and DNA damage. The remaining mice can be monitored for their weight and their lifespan.

[106] Research Design and Methods: It has previously been shown that lentivirus delivery of mTERT in a progeria mouse model (LMNA*G608G ) significantly increased lifespan (Varga 2006). The goal of this study is to optimize a safe LNP-mRNA delivery of a mixture of mRNAs to efficiently correct the lamin A mutation and rejuvenate the cells. The composition and dosage of the LNPs in mice has been optimized, which exhibit no acute toxicity within 8 hours after injection while delivering intact mRNA that can be readily translated. One-month-old progeria mice can be administered LNPs as follows: 1) empty LNPs, 2) LNPs loaded with scramble mRNA, 3) LNPs loaded with Cas9 mRNA and sgRNA, or 4) LNPs loaded with mTERT, Cas9, sgRNA mRNAs, and template RNA. Wildtype mice can be administered LNPs loaded with scramble mRNA. The expression of telomerase and Cas9 can be checked within 24-48 h of delivery. As HGPS is a progressive degenerative disorder, the editing effects on the pathophysiology of each organ can be investigated 2 and 6 months after injection.

[107] The sudden death observed in the previous studies using a AAV viral vector of CRISPR-Cas9 (Santiago-Fernandez 2019) (Beyret 2019) could be due to colonic complications.. To identify potential chronic toxicity with exposure an mRNA genome editing approach, several mice in each group can be sacrificed 2 and 6 months after injection of the different forms of LNP mRNA and can be analyzed for alterations in the expression of liver enzymes and gross pathology. Their serum can be collected and analyzed for markers of renal and hepatic function, CBC, and inflammatory cytokines. Organs (liver, spleen, lung, and heart) can be collected for gross pathological and histological analysis. In addition, TUNEL analysis can be performed to identify apoptotic cells in different organs. As HGPS patients suffer from heart failure and stroke, vascular tissue such as that of the aorta (aortic arch) can be assessed for the correction of lamin A mutation and maintenance of VSMCs, as well as reduction in markers of vascular senescence, such as VCAM1, inflammatory cytokines, reactive oxygen species, nitric oxide, cellular proliferation, and DNA damage. Sequencing of the target region in DNA isolated from the liver, heart, muscle, and lung can be performed to assess editing efficiency. A higher correction is expected in the liver as the biodistribution assay indicates that the highest LNP accumulation is in the liver. The most edited lamin A cells, using AAV9, are also observed in the liver of HGPS mice (Santiago- Fernandez 2019), potentially for the same reason.

[108] Additional mice in each group (as mentioned above) can be maintained for the survival and lifespan study. HGPS mice lose weight gradually after two months (Osorio 2011); their growth rate and body weight can be monitored and compared with controls to assess their improvement and whether the treatment attenuated weight loss. In addition, the heart rate in the HGPS mice can be assessed using electrocardiography for normal function maintenance. Detailed experiments for analyzing the musculoskeletal system, involving grip strength and running wheels for forelimb and hindlimb strength, can be performed since mobility is impaired in aging and in HGPS children. Example 4 - Generation of Disease Model and Correction of Progerin Mutation

[109] Markedly increased efficiency with human Telomerase rnRNA: A study was conducted which created a disease model using gene editing. Specifically, the study created a mutation in the Lamin A gene of human cells to induce Progeria. Hutchinson-Gilford Progeria Syndrome (HGPS) is an accelerated aging syndrome associated with premature vascular disease and death due to heart attack and stroke. In HGPS a mutation in lamin A (progerin), alters nuclear morphology and gene expression. As a result, they have accelerated cellular senescence and dysfunction. HGPS children typically appear normal at birth, but show growth retardation before the age of 2 years. Further manifestations include loss of hair, lipodystrophy, sclerodermatous skin, osteolysis, and progressive atherosclerosis leading to death at about 13 years due to myocardial infarction and/or stroke. Most HGPS patients carry the 1824C>T mutation (G608G), which activates a cryptic splice site resulting in the expression of the abnormal protein progerin, which is different from the normal protein Lamin A in that 50 amino acids are deleted near its C terminus (LA 50/progerin).

[110] FIG. 10 depicts the results of polymerase chain reaction (PCR) of total RNA from HEK 293T cells collected 7 days after transfection with the gene editing complex. The gene editing complex includes Cas9. The PCR was performed using primers for Lamin A or Progerin. The primers base pair with the normal or mutant sequences to generate PCR products of different molecular weights. Appearance of a mutant sequence in the PCR is an indication that a genomic mutation has been made by the gene editing complex. When mRNA encoding human Telomerase (TERT) is given together with the gene editing complex, the generation of mutant sequence is much greater.

[111] The experimental groups in this example can include the endonuclease Cas9; plus the guide (g) RNA that directs Cas 9 to a specific site on the lamin A gene (LMNA) gRNA; plus an RNA construct (Sense) encoding the mutation(1824C>T mutation), which serves as the template for a reverse transcriptase to repair the cut DNA strand, so as to incorporate the mutation into the genomic DNA; and/or an Anti-sense construct, encoding the corresponding mutation of the complementary DNA strand (1824T>A mutation); in some cases including mRNA encoding human telomerase (TERT).

[112] FIG. 10 shows that in the GFP group (HEK cells transfected with Cas9+LMNA gRNA+ Sense) has induced negligible gene editing to generate the mutation, with very low levels of Progerin mRNA detected. Indeed, the levels of Progerin mRNA detected are no different from the No gRNA group (Cas 9 + Sense construct, with telomerase RNA, but in the absence of the guide RNA). In the absence of the guide RNA, Cas 9 cannot be directed to the lamin A gene for editing. By contrast, when TERT is included with the gene editing complex (with the Sense or Antisense construct) effective gene editing is observed, with substantially increased expression of Progerin, representing successful gene editing of the Lamin A gene.

[113] FIG. 11 is a histogram, showing the levels of Progerin expression in the GFP group (no guide RNA) are negligible, whereas there is a substantial increase in Progerin expression when TERT is included with the gene editing complex (with the Sense or Antisense construct).

[114] FIG. 12 is similar to FIG. 10, except in this case, the HEK cells are replaced by human aortic endothelial cells (HAECs). Again, there is very little gene editing when the guide RNA is not included (No gRNA), but substantial editing when TERT is added to the gene editing machinery, with a Sense and/or Antisense construct.

[115] FIG. 13 is a separate experiment, showing the results of transfection with the gene editing complex (Cas 9 + LMNA gRNA) + Sense construct + GFP mRNA substituting for TERT mRNA [GFP group]; Cas 9 without the gRNA + Sense construct + TERT mRNA [No gRNA group],

[116] Correction of Progerin Mutation in iPSC-ECs derived from Progeria patients: Another study was conducted which shows that the combination of the gene editing machinery with telomerase is effective in correcting the 1824C>T mutation in Lamin A, in iPSC-derived endothelial cells from patients with Progeria. These studies indicate that telomerase mRNA is essential to reverse the Progerin mutation.

[117] FIG. 14 shows the result of Sanger sequencing of genomic DNA from iPSC-ECs from Progeria patients. Cells were treated with total equal amount of RNA, either GFP, hTERT, Cas9 alone; or in combination, i.e., (Cas9+gRNA+S+AS), (hTERT+Cas9+gRNA+S), (hTERT+Cas9+gRNA+AS) and (hTERT+Cas9+gRNA+S+AS). HGPS-ECs were collected 72 hours post-transfection.

[118] The genomic DNA was harvested and sent for Sanger Sequencing of 24 samples. In the control groups, 2 of 12 samples showed a reversal of the C to T mutation, which may be artifactual. In the absence of TERT, the full complement of gene editing machinery showed editing in 0 of 3 samples. By contrast, 5 of 9 samples showed successful editing when TERT was included with the gene editing machinery (one of the 9 samples were undetermined; N).

[119] FIGS. 15 shows that, to confirm that effective base editing occurred with the addition of TERT mRNA, the study assessed Progerin mRNA expression in several conditions. When the study assessed Progerin expression, a reduction was observed when telomerase mRNA was combined with the gene editing machinery (Cas9+ LMNA gRNA+S+AS), as opposed to the addition of GFP mRNA alone, or to when TERT mRNA was combined with the gene editing machinery lacking the guide RNA (No gRNA group). All data is 48-72h post transfection. [120] These studies confirm that mRNA telomerase (TERT) markedly increases the efficiency of gene editing, so as to create a disease model ; or to correct a disease mutation. Studies are ongoing to understand the mechanism by which TERT mRNA has this non-canonical effect. Without being bound by theory, it is suspected that the reverse transcriptase activity of telomerase protein permits telomerase to use the mRNA Sense or Antisense constructs as templates to produce DNA that can replace the homologous segment that is excised by casRNA/gRNA complex.

Discussion

[121] HGPS is an accelerated premature aging syndrome due to a single mutation of lamin A, which generates a permanent famesylated protein called progerin (Ahmed 2018) (Gordon 2014). Since the cause of progeria syndrome has been identified, many efforts have been made to explore treatment options. Some treatments, such as famesyltransferase inhibitors (Fong 2006, Science) and resveratrol treatment to recover (SIRT1) (Liu 2012), have improved the premature aging phenotype and extended lifespan in murine progeria models. Drugs such as rapamycin (Cao 2011, Sci Transl Med) and metformin (Egesipe 2016) have reversed senescence in human HGPS cells. Currently lonafamib, a famesyltransferase inhibitor (FTI), is the only clinically approved drug. Whereas it reduces progerin toxicity, it only extends the lifespan of individuals with progeria by 1 to 2 years (Gordon 2018). Lonafarnib also has adverse side effects (Young 2013) and is not effective for all children (Gordon 2014) (Gordon 1993) (Blondel 2016) (Gordon 2016). Alternatively, a new study found that inhibition of progerin-lamin A binding (JH4), regardless of the famesylation status of mutated lamin A, effectively alleviates the pathological features of progeria and extends lifespan in mice (Kang 2021) (Lai 2020). This suggests that famesylation of progerin may not be the main pathology of HGPS.

[122] Recent advances in gene therapy have yielded more effective methods for removing progerin. The addition of morpholino oligos (Osorio 2011) or CRISPR-Cas9 (Cas9) gene editing (Santiago-Fernandez 2019) (Beyret 2019) to reduce lamin A and progerin has successfully reduced the aging phenotype and increased the lifespan of progeria mice by 26-40%, specifically because lamin A is dispensable for mice (Osorio 2011) (Fong 2006, Clin Invest). However, eliminating the lamin A gene for progeria patients may not be clinically applicable. Therefore, developing a treatment to address the root cause of HGPS and correct the mutation with no change in normal lamin A is necessary and can have more advantages than drugs alleviating the associated symptoms.

[123] The desirable efficiency of genome editing techniques, including Cas9 and other guided endonucleases techniques, is low, but these techniques have promising benefits for future drug therapy. Cas9 is more commonly used to delete mutant genes/proteins but can also be used for genome editing and to replace a targeted DNA sequence with the normal/native sequence while having minimal undesired genomic modifications or cellular stress (Duan 2021) (Komor 2017). Most programmable nuclease methods, including Cas9, function by creating a DNA double strands break (DSB) at the site of target loci in a cell. Subsequently, genome modification, such as insertion, deletion, or base editing through non-homologous end joining (NHEJ) or the homology- directed repair (HDR) pathway, can be achieved (Bao 2021). Providing a DNA template to apply designed changes by the Cas9 endonuclease approach has a potential risk of random DNA integration and off-target effects. Better strategies for this modification are needed to generate a higher rate of success for translational applications.

[124] In the Cas9 technique, a desired pathway for correcting a point mutation or precise deletion/integration is HDR, which uses donor DNA as a template (Chapman 2012). HDR is efficient, with reduced errors, and is active in dividing cells because it needs proteins that are expressed in the S and G2 phases of the cell cycle (Jasin 2013) (Lieber 2010) (Amoult 2017). Cas9 editing in HGPS mice suggests eliminating lamin A using this method may have an application in the clinic (Santiago-Fernandez 2019). However, HGPS vascular cells are already replicatively senescent and have all the hallmarks of aging, such as short telomeres, increased accumulated damage, and inflammation (Xu 2022). It has been shown (Xu 2022) (Mojiri 2021) that inflammatory cytokines that are released from senescent endothelial cells (ECs) induce proinflammatory signals in neighboring cells. While correcting the lamin A mutation could stop further cellular damage, it will not fully recover cellular health nor reverse telomere erosion, DNA damage, and some aberrant gene expression. It is crucial to restore proper gene expression and recover the cellular functions of progeria cells while also correcting for the mutation to gain impactful therapy for progeria children, which can be achieved by the combination therapy of hTERT-enhanced gene editing.

[125] Importantly, it has been shown that treating progeria cells with human telomerase (hTERT) mRNA rejuvenates the cells by ameliorating aging features. hTERT therapy normalizes proliferation of HGPS cells, restores telomere length, reduces global DNA damage (potentially by the promotion of HDR; Mao 2016), and reduces SASP. Collectively, the addition ofTERT-mRNA to HGPS cells results in genome editing and has a synergistic effect through rejuvenating cells, even those with no mutation. Advanced RNA technology has allowed the use of the RNA format for TERT, Cas9 complex, and guide RNA. The short lifespan of RNA and its potential clearance is a considerable advantage of the RNA system in reducing immune responses. It is proposed that nanoparticle delivery of a short donor RNA of lamin A, accompanied by telomerase mRNA and CRISPR-Cas9 mRNA, increases the correction of the mutation in progeria cells and rejuvenates senescent cells (FIG. 8). It further proposed that this increase in homologous recombination and the restoration of the cell’ s proliferation capacity by hTERT allows higher chance for the mutation to be corrected. Finally, lipid nanoparticle (LNP) delivery of RNA is safe as the LNPs are degraded after dissociation from their cargo, and all injected RNA are degraded due to their short lifespan (no genomic integration).

[126] Impact: Programmable genome editing is a promising tool to apply genetic changes at a specific site of any living cell while inducing minimal cellular agitation. Effective correction of the single mutation in the lamin A gene using genome editing tools relies on DNA damage-repair mechanisms, including homologous -direct recombination, if 1) the template is provided and 2) cells are in their proliferative state. hTERT can increase homologous recombination activity, rejuvenate senescent cells, and recover normal cell proliferation. Therefore, TERT-mRNA could contribute to genome correction and have a synergistic effect with Cas9, which together provide a promising therapeutic path for children with HGPS. While the large amount of required copy numbers of the virus to deliver the genome editing factors is alarming and associated with a high cancer risk, the delivery of coding mRNA using LNPs at the right dosage is a safe, transient, and translational genome editing strategy. Delivery of the combination therapy using optimized LNP doses and its components could achieve desirable restoration of normal lamin A. In vivo studies have shown significant improvements when a small portion of the targeted cells receive the gene therapy. Therefore, the portion of vascular cells that take up LNPs and correct lamin A RNA can significantly benefit the vasculature and lifespan of the progeria mice.

[127] When the RNA nanotherapeutic is administered to HGPS mice, vascular function and structure can be improved and thereby delay the progression of the major causes of morbidity and mortality in these children (i.e., coronary and carotid artery disease causing myocardial infarction and stroke). The therapy has the potential for repeated doses for higher efficacy if necessary. Notably, myocardial infarction and stroke are also the major causes of death worldwide. Vascular aging is associated with myocardial infarction and stroke in the general population and in HGPS. The methodologies may be widely applicable for other age-related diseases such as atherosclerosis and vascular dementia.

[128] Significance: Cas9 and other guided endonuclease techniques, such as base editing, have shown promising benefits of gene therapy in HGPS-derived cells or HGPS mouse models (Santiago-Fernandez 2019) (Beyret 2019) (Koblan 2021) (Vermeij 2021). However, the Cas9 guided endonuclease approach has a risk of random DNA integration and off- target effects (Bao 2021). Importantly, these nuclease-genome editing techniques depend on the HDR repair mechanism, which operates most proficiently in proliferative cells. However, the lack of replication in non-dividing or senescent cells can affect the efficiency of these techniques. HDR activity in HGPS cells/mice can be increased to enhance the efficiency and precision of Cas9 performance (Yeh 2019) (Song 2016) (Canny 2018) (Charpentier 2018) (Nambiar 2019).

Advanced modified CRISPR-endonucleases have improved the correction of diverse genetic defects with a lower risk of genome manipulation. In particular, Prime editors (PEs) are exemplified by a reverse transcriptase (RT) fused to an RNA-programmable nickase and a prime editing guide RNA (pegRNA) to copy genetic information directly from an extension on the pegRNA into the target genomic locus (Anzalone 2019).

[129] It has been suggested that telomeric DNA damage is irreparable and induces persistent DNA-damage-response activation (Fumagalli 2012). More recently, it has been shown that ECs derived from progeria iPSCs and transfected with hTERT-mRNA recover their functions and morphology. The beneficial effects are broader than just an extension of telomeres and telomeric damage repair (Mojiri 2021) (Cesare 2013). There may be a non-canonical role of TERT in repairing genomic DNA damage in cells with normal telomere length. Transcriptome analysis indicates that more than 250 genes fully normalize in HGPS-ECs treated with hTERT-mRNA (FIG. 3). Many of these genes, such as RECQL5 (Hu 2007), PALB2 (Buisson 2012), FANCC (Niedzwiedz 2004), DHX9 (Chakraborty 2021), and RBM6 (Machour 2021), belong to the DNA repair pathway and specifically to the HDR repair pathway. hTERT-mRNA also promotes normal proliferation in HGPS-ECs (FIG. 2), which means cells go through S and G2 phases more often than senescent cells, which results in higher chances for HDR. Therefore, hTERT-mRNA would enhance the Cas9 genome editing of lamin A, partly by promoting HDR.

[130] Age-related functional decline in the vascular system is associated with cardiovascular diseases. Progerin is a toxic protein when expressed in cells. Current HGPS mouse models generated by lamin A mutation or human progerin knock-in, which ubiquitously express progerin, suggest that the loss of VSMCs is a primary cause of vascular disease and progressive aging (McClintock 2006) (Zhang 2011). Indeed, VSMC-specific progerin knock-in mice have a normal lifespan but show vascular stiffness and calcification and progressive atherosclerosis when crossed with apolipoprotein E-deficient mice (Hamczyk 2018) (Kim 2018). However, in the EC-specific progerin mouse model, ECs are dysfunctional, express inflammatory proteins, and can cause ischemia due to reduced capillary density which leads to systemic aging. The shortened lifespan in mice with EC-specific progerin is similar to that in mice where progerin is ubiquitously expressed (Sun 2020) (Osmanagic-Myers 2019). Note however that the vascular disease manifested in HGPS mice (a loss of vascular smooth muscle in the media) is dissimilar to that in HGPS patients, where there is an occlusive disease due to myointimal hyperplasia and atherosclerosis resulting in myocardial infarction and stroke.

[131] Further, hTERT-mRNA therapy reduced SASP in HGPS-ECs and normal VSMCs when cocultured with progeria cells. TERT therapy can reduce progerin expression (FIG. 9), consistent with previous reports (Cao 2011, J Clin Invest), but the exact mechanism is unclear. Reduction of progerin levels suppresses the cycle of DNA damage/inflammation that gives rise to senescence. Therefore, hTERT-mRNA accompanied with genome editing would have synergistic effects to normalize vascular function and reduce the pathophysiological condition of HGPS children.

[132] Alternative Approaches: It is expected that LNPs deliver their components, including Cas9, with the biodistribution pattern shown (Kazemian 2022) (Rahimi 2022) (Liu 2020). The comparison of pathology and life extension amongst mice injected with empty LNPs, LNP with scramble TERT mRNA, or LNPs with the mRNA TERT and genome editing complex, can determine the potential toxicity from LNP alone, the mRNA itself, or the active complex . Modified mRNA has minimal toxicity due to immune activation (Kariko 2008) (Starostina 2021), and LNPs may be reformulated to reduce adverse effects.

[133] DNA sequencing and pathophysiological studies of the treatment groups injected with Cas9 mRNA complex alone or in combination with TERT can assess benefits (reduction in the number of cells with the lamin A mutation; reduced progerin levels; restored cellular functions) and toxicities (e.g. off-target edits of the DNA sequence), as well as limitations of the approach (e.g. lack of efficient tissue targeting). One can address any deficiencies or limitations using approaches familiar to those skilled in the art (e.g. different LNP formulation; modified concentration or dosing of the active principles).

[134] Recently, systemic delivery of Cas9-AAV9 for editing HGPS resulted in no reduction of progerin-positive cells in the lung, kidney, and aorta. However, mice showed overall significant life extension (Santiago-Fernandez 2019), indicating that a small number of cells receiving a permanent genome correction can be efficient and helpful. A modest level of genetic correction is expected in the major organs affected by progerin but with the synergistic effect of TERT, a significant increase in the health span is expected. This can support the idea that 100% of the cells do not have to have their genome corrected to improve life for HGPS patients. Additionally, these findings would open the door to treat similar genetic diseases.

[135] The concentrations of LNPs have been optimized and show no acute toxicity in mice 8 h after injection. However, mice can be monitored for chronic toxicity 2 and 6 months after injection. In addition, any abnormalities in the toxicity studies can trigger a more definitive histopathological analysis. These studies can permit a correlation of dosage with vascular (aortic) correction of lamin A and detection of any toxicity of the LNPs with or without TERTCas9- mRNA. Furthermore, these data can help assess biodistribution, and the route of injection can be optimized as both intravenous and intraperitoneal injections can be used for lamin A editing (Santiago-Fernandez 2019) (Beyret 2019).

[136] A preliminary biodistribution study revealed that while there is vascular (aortic) uptake, most of the LNPs administered systemically distribute to the liver and spleen. If comparable increases in aortic telomerase activity or lamin A editing are not observed as was observed in vitro with LNPs as a carrier for the mmRNA, an alternate approach would be to use “leukosomes”. Leukosomes are LNPs into which are integrated the membrane fraction of autologous leukocytes. Leukosomes have an affinity for sites of vascular inflammation (Martinez 2018) (Molinaro 2016) (Molinaro 2018). Preferential delivery of rapamycin to the inflamed endothelium overlying atheroma has been documented in apoE-/- hypercholesteremic mice (Boada 2019), which is mediated in part by endothelial VCAM expression. It was shown that endothelial VCAM expression is increased in the aortic endothelium of HGPS mice.

[137] The purpose of these studies is to edit the mutant lamin A gene; however, it will be of interest to investigate whether lamin A correction could protect against atherosclerosis. In this regard, one limitation of the LmnaG609G mouse is that it does not mimic the obstructive lesions seen in HGPS children. Therefore, a hypercholesterolemic Progeria model, e.g., the high-fat diet- fed Apoe-/-LmnaG609G/G609G mice, could be used, which develop a severe vascular pathology, including medial VSMC loss and lipid retention, adventitial fibrosis, and accelerated atherosclerosis, thus resembling cardiovascular disease in patients with HGPS (Hamczyk 2018). Hence, the synergistic effect of TERT-Cas9 as an atherogenic protective approach can be examined.

[138] The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein.

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Claims

What is claimed is:

1. A composition comprising a telomerase or a functional fragment thereof, and/or a telomerase activator, wherein the composition further comprises components for a gene editing system.

2. The composition of claim 1 , wherein the gene editing system is a CRISPR/Cas system.

3. The composition of claim 1, wherein the gene editing system is a zinc finger nuclease (ZNF) system.

4. The composition of claim 1 , wherein the gene editing system is a transcription activatorlike effector nuclease (TALEN) system.

5. The composition of claim 2, wherein the CRISPR/Cas system comprises an endonuclease and a gRNA.

6. The composition of claim 5, wherein the endonuclease comprises a Cas protein.

7. The composition of claim 6, wherein the Cas protein comprises Cas9, Casl 1 , Casl2a Casl3, or Casl4.

8. The composition of claim 2 or any one of claims 5-7, wherein the CRISPR/Cas system is a prime editing system.

9. The composition of claim 8, wherein the prime editing system further comprises pegRNA.

10. The composition of any one of claims 8-9, wherein the prime editing system further comprises a reverse transcriptase.

11. The composition of claim 10, wherein the reverse transcriptase is telomerase reverse transcriptase (TERT) or a functional fragment thereof.

12. The composition of claim 2 or any one of claims 5-7, wherein the CRISPR/Cas system is a base editing system.

13. The composition of claim 12, wherein the base editing system comprises a cytosine base editor (CBE), and wherein the CBE comprises cytidine deaminase or a functional fragment thereof.

14. The composition of claim 12, wherein the base editing system comprises an adenine base editor (ABE), and wherein the ABE comprises adenine deaminase or a functional fragment thereof.

15. The composition of claim 3, wherein the zinc finger nuclease system comprises a restriction endonuclease and a DNA binding domain.

16. The composition of claim 15, wherein the restriction endonuclease comprises Fokl.

17. The composition of any one of claims 15-16, wherein the DNA binding domain comprises a zinc finger DNA binding domain.

18. The composition of claim 4, wherein the TALEN system comprises a restriction endonuclease and a DNA binding domain.

19. The composition of claim 18, wherein the restriction endonuclease comprises Fokl.

20. The composition of any one of claims 18-19, wherein the DNA binding domain comprises a transcription activator-like (TAL) effector DNA binding domain.

21. The composition of any one of claims 1-20, wherein the telomerase or functional fragment thereof comprises a TERT domain selected from essential N-terminal (TEN) domain, a TERT RNA-binding domain (TRBD), a reverse-transcriptase (RT) domain, a C-terminal extension (CTE), or any combinations thereof.

22. The composition of any one of claims 1-21 , wherein the telomerase or functional fragment thereof further comprises one or more mutations to a naturally occurring telomerase or functional fragment of a naturally occurring telomerase.

23. The composition of any one of claims 1-21 , wherein the telomerase or functional fragment of telomerase is derived from a mammal.

24. The composition of claim 23, wherein the mammal is swine.

25. The composition of claim 23, wherein the mammal is human.

26. The composition of any one of claims 1-25, wherein the telomerase activator comprises cycloastragenol, oleanolic acid, maslinic acid, or combinations thereof.

27. The composition of any one of claims 1-26, wherein the telomerase activator increases activity of an endogenous telomerase or functional fragment thereof.

28. The composition of any one of claims 1-26, wherein the telomerase activator increases activity of an exogenous telomerase or functional fragment thereof.

29. The composition of claim 28, wherein the telomerase activator is required so as to activate an exogenous telomerase or functional fragment thereof that is otherwise inactive in the absence of the telomerase activator

30. A composition comprising a nucleic acid encoding a telomerase or functional fragment thereof and one or more nucleic acids encoding components for a gene editing system.

31. The composition of claim 30, wherein the gene editing system is a CRISPR/Cas system.

32. The composition of claim 30, wherein the gene editing system is a zinc finger nuclease (ZNF) system.

33. The composition of claim 30, wherein the gene editing system is a transcription activatorlike effector nuclease (TALEN) system.

34. The composition of claim 31 , wherein the CRISPR/Cas system comprises an endonuclease and a gRNA.

35. The composition of claim 34, wherein the endonuclease comprises a Cas protein.

36. The composition of claim 35, wherein the Cas protein comprises Cas9, Casl 1, Cas 12a Casl3, or Cas 14.

37. The composition of claim 31 or any one of claims 34-36, wherein the CRISPR/Cas system is a prime editing system.

38. The composition of claim 37, wherein the prime editing system further comprises pegRNA.

39. The composition of any one of claims 37-38, wherein the prime editing system further comprises a reverse transcriptase.

40. The composition of claim 39, wherein the reverse transcriptase is telomerase reverse transcriptase (TERT) or a functional fragment thereof.

41. The composition of claim 31 or any one of claims 34-36, wherein the CRISPR/Cas system is a base editing system.

42. The composition of claim 41 , wherein the base editing system comprises a cytosine base editor (CBE), and wherein the CBE comprises cytidine deaminase or a functional fragment thereof.

43. The composition of claim 41 , wherein the base editing system comprises an adenine base editor (ABE), and wherein the ABE comprises adenine deaminase or a functional fragment thereof.

44. The composition of claim 32, wherein the zinc finger nuclease system comprises a restriction endonuclease and a DNA binding domain.

45. The composition of claim 44, wherein the restriction endonuclease comprises FokT.

46. The composition of any one of claims 44-45, wherein the DNA binding domain comprises a zinc finger DNA binding domain.

47. The composition of claim 33, wherein the TALEN system comprises a restriction endonuclease and a DNA binding domain.

48. The composition of claim 47, wherein the restriction endonuclease comprises Fokl.

49. The composition of any one of claims 47-48, wherein the DNA binding domain comprises a transcription activator-like (TAL) effector DNA binding domain.

50. The composition of any one of claims 30-49, wherein the telomerase or functional fragment thereof comprises a TERT domain selected from essential N-terminal (TEN) domain, a TERT RNA-binding domain (TRBD), a reverse- transcriptase (RT) domain, a C-terminal extension (CTE), or any combinations thereof.

51. The composition of any one of claims 30-50, wherein the nucleic acid encoding a telomerase or functional fragment thereof further comprises one or more mutations to nucleic acid encoding a naturally occurring telomerase or functional fragment of a naturally occurring telomerase.

52. The composition of any one of claims 30-51, wherein the telomerase or functional fragment of telomerase is derived from a mammal.

53. The composition of claim 52, wherein the mammal is swine.

54. The composition of claim 52, wherein the mammal is human.

55. A vector encoding the nucleic acids of any one of claims 30-54.

56. An adenovirus comprising the vector of claim 55.

57. A vehicle (e.g., lipid nanoparticle) comprising the vector of claim 55.

58. A cell or cell component (e.g., exosome) comprising the vector of claim 55.

59. A method of increasing efficiency of gene editing, the method comprising exposing a target nucleic acid to a gene editing system and a telomerase or functional fragment thereof, and/or a telomerase activator, wherein said telomerase or functional fragment thereof increases efficiency of gene editing with the target nucleic acid.

60. The method of claim 59, wherein increasing efficiency of gene editing with the target nucleic acid comprises an increase in the number of on-target edits of about 10% or more and/or a decrease in the number of off-target edits of about 10% or more compared to gene editing without the telomeric moiety.

61. The method of any one of claims 59-60, wherein the gene editing system is a CRISPR/Cas system.

62. The method of any one of claims 59-60, wherein the gene editing system is a zinc finger nuclease (ZNF) system.

63. The method of any one of claims 59-60, wherein the gene editing system is a transcription activator-like effector nuclease (TALEN) system.

64. The method of claim 61, wherein the CRISPR/Cas system comprises an endonuclease and a gRNA.

65. The method of claim 64, wherein the endonuclease comprises a Cas protein.

66. The method of claim 65, wherein the Cas protein comprises Cas9, Casl 1, Casl2a Casl3, or Cas 14.

67. The method of claim 61 or any one of claims 64-66, wherein the CRTSPR/Cas system is a prime method system.

68. The method of claim 67, wherein the prime editing system further comprises pegRNA.

69. The method of any one of claims 67-68, wherein the prime editing system further comprises a reverse transcriptase.

70. The method of claim 69, wherein the reverse transcriptase is telomerase reverse transcriptase (TERT) or a functional fragment thereof.

71. The composition of claim 61 or any one of claims 64-66, wherein the CRISPR/Cas system is a base editing system.

72. The composition of claim 71, wherein the base editing system comprises a cytosine base editor (CBE), and wherein the CBE comprises cytidine deaminase or a functional fragment thereof.

73. The composition of claim 71, wherein the base editing system comprises an adenine base editor (ABE), and wherein the ABE comprises adenine deaminase or a functional fragment thereof.

74. The method of claim 62, wherein the zinc finger nuclease system comprises a restriction endonuclease and a DNA binding domain.

75. The method of claim 74, wherein the restriction endonuclease comprises Fokl.

76. The method of any one of claims 74-75, wherein the DNA binding domain comprises a zinc finger DNA binding domain.

77. The method of claim 63, wherein the TALEN system comprises a restriction endonuclease and a DNA binding domain.

78. The method of claim 77, wherein the restriction endonuclease comprises Fokl.

79. The method of any one of claims 77-78, wherein the DNA binding domain comprises a transcription activator-like (TAL) effector DNA binding domain.

80. The method of any one of claims 59-79, wherein the telomerase or functional fragment thereof comprises a TERT domain selected from essential N-terminal (TEN) domain, a TERT RNA-binding domain (TRBD), a reverse-transcriptase (RT) domain, a C-terminal extension (CTE), or any combinations thereof.

81. The method of any one of claims 59-80, wherein the telomerase or functional fragment thereof further comprises one or more mutations to a naturally occurring telomerase or functional fragment of a naturally occurring telomerase.

82. The method of any one of claims 59-81, wherein the telomerase or functional fragment of telomerase is derived from a mammal.

83. The method of claim 82, wherein the mammal is swine.

84. The method of claim 82, wherein the mammal is human.

85. The method of any one of claims 59-84, wherein the telomerase activator comprises cycloastragenol, oleanolic acid, maslinic acid, or combinations thereof.

86. The method of any one of claims 59-85, wherein the telomerase activator increases activity of an endogenous telomerase or active fragment thereof.

87. The method of any one of claims 59-86, wherein the telomerase activator increases activity of an exogenous telomerase or functional fragment thereof.

88. The method of claim 87, wherein the telomerase activator is required so as to activate an exogenous telomerase or functional fragment thereof that is otherwise inactive in the absence of the telomerase activator

89. A method of editing a gene in a subject in need thereof, the method comprising the steps of: a) providing to the subject a gene editing system, a telomerase or functional fragment thereof, and/or a telomerase activator, under conditions for gene editing; b) allowing gene editing to occur within a cell of the subject, wherein said telomerase or functional fragment thereof increases efficiency of gene editing with the target nucleic acid.

90. The method of claim 89, wherein the subject has a disease, disorder, or condition which can benefit from gene editing.

91. The method of any one of claims 89-90, wherein the gene to be edited is replaced, deleted, modified, or inserted.

92. The method of claim 91 , wherein when the gene is to be replaced or modified, the gene editing system further comprises a repair template.

93. The method of any one of claims 89-92, wherein the gene editing system and telomerase or functional fragment thereof are encoded by a nucleic acid.

94. The method of claim 93, wherein the nucleic acid is in a vector.

95. The method of claim 94, wherein the vector is in an adenovirus.

96. The method of claim 94, wherein the vector is in a vehicle (e.g., lipid nanoparticle).

97. The method of any one of claims 90-96, wherein the disease is Hutchinson-Gilford

Progeria Syndrome (progeria), atherosclerosis, vascular dementia, or autosomal dominant polycystic kidney disease.

98. The method of claim 97, wherein the gene to be edited is an LMNA gene.