ATR-activating polypeptides and methods of use thereof

Abstract

Provided herein are non-naturally occurring Ataxia Telangiectasia and Rad3 (ATR)-activating polypeptides and nucleic acids encoding the same, and methods of using the same.

Description

[0001] ATR-ACTIVATING POLYPEPTIDES AND METHODS OF USE THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 549,733, filed February 5, 2024, the entire contents of which are herein incorporated by reference.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named 56747-0002W01_SL_ST26.xml. The XML file, created on February 5th, 2025, is 31,346 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] The present disclosure relates to the field of biotechnology, and more specifically, to ATR-activating polypeptides and methods of treating an ATR-related disease.

[0008] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0009] This invention was made with government support under the U.S. Department of Veterans Affairs. The Government has certain rights in the invention.

[0010] BACKGROUND

[0011] Cells are constantly exposed to endogenous and exogenous threats to their genomes, such as UV radiation, reactive oxygen species, DNA double-strand breaks, and DNA replication fork stalling. As such, cells have evolved DNA damage response (DDR) mechanisms to respond to such conditions and control cell cycle progression, apoptosis, transcription, DNA repair, and ultimately genome integrity. SUMMARY

[0012] Ataxia Telangiectasia and Rad3-related (ATR) is a DDR protein kinase which primarily responds to single-strand DNA breaks and its kinase cascade can result in checkpoint control of cell cycle progression. Hypomorphic mutations of ATR in humans are associated with Seckel Syndrome, characterized by severe microcephaly, growth retardation, and intellectual disability. Ablation of ATR signaling in adult mice is reported to result in stem and progenitor cell loss, ultimately leading to defects in tissue homeostasis and the onset of aging-associated phenotypes. Recent studies in mice have also demonstrated that ATR controls pre-synaptic firing of adult neurons. Moreover, research on ATR in muscle stem cells (MuSCs) has revealed a new role in the maintenance of stem cell quiescence, preventing stem cell exhaustion, and functional decline. Without wishing to be bound by any theory, the ability to hyperactivate or maintain ATR signaling may bring long-term therapeutic benefits for patients suffering from rare neuro-muscular diseases such as Seckel Syndrome, to progressive muscle wasting diseases, such as sarcopenia and muscular dystrophy. Increasing stem cell resilience may also have applications in stem cell therapeutics, such as CAR-T, by ensuring stem cell survival during arduous reprogramming and transplantation steps.

[0013] Thus, provided herein are nucleic acids including a sequence encoding a non- naturally occurring ATR-activating polypeptide, where the non-naturally occurring ATR-activating polypeptide includes: (i) a nuclear localization signal sequence; and (ii) an ataxia telangiectasia and a Rad3-related (ATR)-activating domain.

[0014] In some embodiments, the non-naturally occurring ATR-activating polypeptide includes a linker sequence disposed between the nuclear localization signal sequence and the ATR-activating domain. In some embodiments, the linker sequence includes (GS)X, (GGS)X, (GGGGS)X(SEQ ID NO: 6), or (GGSG)X(SEQ ID NO: 7), where x is an integer from 1 to 50.

[0015] In some embodiments, the non-naturally occurring ATR-activating polypeptide includes a replication protein A (RPA)-binding domain. In some embodiments, the RPA-binding domain in the non-naturally occurring ATR- activating polypeptide is positioned C-terminally relative to the ATR-activating domain. In some embodiments, the non-naturally occurring ATR-activating polypeptide includes a linker sequence disposed between the ATR-activating domain and the RPA-binding domain. In some embodiments, the linker sequence includes (GS)X, (GGS)X, (GGGGS)x (SEQ ID NO: 6), or (GGSG)X(SEQ ID NO: 7), where x is an integer from 1 to 50.

[0016] In some embodiments, the RPA-binding domain includes a sequence at least 80% identical to SEQ ID NO: 5. In some embodiments, the RPA-binding domain includes a sequence at least 90% identical to SEQ ID NO: 5. In some embodiments, the RPA-binding domain includes a sequence at least 95% identical to SEQ ID NO: 5. In some embodiments, the RPA-binding domain is SEQ ID NO: 5.

[0017] In some embodiments, the nuclear localization signal sequence includes SEQ ID NO: 8 (PKKKRRV) or SEQ ID NO: 9 (PKKKRKV).

[0018] In some embodiments, the ATR-activating domain includes a sequence that is at least 80% identical to SEQ ID NO: 11. In some embodiments, the ATR-activating domain includes a sequence that is at least 90% identical to SEQ ID NO: 11. In some embodiments, the ATR-activating domain includes a sequence that is at least 95% identical to SEQ ID NO: 11. In some embodiments, the ATR-activating domain is SEQ ID NO: 11.

[0019] In some embodiments, the non-naturally occurring ATR-activating polypeptide includes a cell-penetrating peptide at the N-terminus. In some embodiments, the cell-penetrating peptide is SEQ ID NO: 10.

[0020] In some embodiments, the non-naturally occurring ATR-activating polypeptide includes a sequence that is at least 80% identical to SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the non-naturally occurring ATR-activating polypeptide includes a sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the non-naturally occurring ATR-activating polypeptide includes a sequence that is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the non-naturally occurring ATR-activating polypeptide is SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the nucleic acid is DNA.

[0021] In some embodiments, the sequence encoding the non-naturally occurring ATR-activating polypeptide includes a sequence that is at least 80% identical to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the sequence encoding the non- naturally occurring ATR-activating polypeptide includes a sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the sequence encoding the non-naturally occurring ATR-activating polypeptide includes a sequence that is at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the sequence encoding the non-naturally occurring ATR-activating polypeptide is SEQ ID NO: 2 or SEQ ID NO: 4.

[0022] In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is mRNA.

[0023] Also provided herein are compositions including any of the nucleic acids described herein and a pharmaceutically acceptable excipient.

[0024] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition is formulated for local administration. In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for intravenous, subcutaneous, or intramuscular administration.

[0025] In some embodiments, the nucleic acid is formulated as a lipid nanoparticle. In some embodiments, the nucleic acid is formulated for unencapsulated delivery.

[0026] Also provided herein are non-naturally occurring ATR-activating polypeptides including: (i) a nuclear localization signal sequence; and (ii) an ataxia telangiectasia and a Rad3 -related (ATR)-activating domain.

[0027] In some embodiments, the non-naturally occurring ATR-activating polypeptide includes a linker sequence disposed between the nuclear localization signal sequence and the ATR-activating domain. In some embodiments, the linker sequence includes (GS)X, (GGS)X, (GGGGS)X(SEQ ID NO: 6), or (GGSG)X(SEQ ID NO: 7), where x is an integer from 1 to 50.

[0028] In some embodiments, the non-naturally occurring ATR-activating polypeptide includes a replication protein A (RPA)-binding domain. In some embodiments, the RPA-binding domain in the non-naturally occurring ATR- activating polypeptide is positioned C-terminally relative to the ATR-activating domain. In some embodiments, the non-naturally occurring ATR-activating polypeptide includes a linker sequence disposed between the ATR-activating domain and the RPA-binding domain. In some embodiments, the linker sequence includes (GS)X, (GGS)X, (GGGGS)x (SEQ ID NO: 6), or (GGSG)X(SEQ ID NO: 7), where x is an integer from 1 to 50.

[0029] In some embodiments, the RPA-binding domain includes a sequence at least 80% identical to SEQ ID NO: 5. In some embodiments, the RPA-binding domain includes a sequence at least 90% identical to SEQ ID NO: 5. In some embodiments, the RPA-binding domain includes a sequence at least 95% identical to SEQ ID NO: 5. In some embodiments, the RPA-binding domain is SEQ ID NO: 5.

[0030] In some embodiments, the nuclear localization signal sequence is SEQ ID NO: 8 (PKKKRRV) or SEQ ID NO: 9 (PKKKRKV).

[0031] In some embodiments, the ATR-activating domain includes a sequence that is at least 80% identical to SEQ ID NO: 11. In some embodiments, the ATR-activating domain includes a sequence that is at least 90% identical to SEQ ID NO: 11. In some embodiments, the ATR-activating domain includes a sequence that is at least 95% identical to SEQ ID NO: 11. In some embodiments, the ATR-activating domain is SEQ ID NO: 11.

[0032] In some embodiments, the non-naturally occurring ATR-activating polypeptide includes a cell-penetrating peptide at the N-terminus.

[0033] In some embodiments, the cell-penetrating peptide is SEQ ID NO: 10.

[0034] In some embodiments, the non-naturally occurring ATR-activating polypeptide includes a sequence that is at least 80% identical to SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the non-naturally occurring ATR-activating polypeptide includes a sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the non-naturally occurring ATR-activating polypeptide includes a sequence that is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the non-naturally occurring ATR-activating polypeptide is SEQ ID NO: 1 or SEQ ID NO: 3.

[0035] Also provided herein are compositions including any of the non-naturally occurring ATR-activating polypeptides described herein and a pharmaceutically acceptable excipient. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition is formulated for local administration. In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for intravenous, subcutaneous, or intramuscular administration.

[0036] In some embodiments, the nucleic acid is formulated as a lipid nanoparticle. In some embodiments, the nucleic acid is formulated for unencapsulated delivery.

[0037] Also provided herein are methods of treating an ATR-related disease in a subject, the method including administering to the subject an effective amount of any one of the nucleic acids described herein, any one of the non-naturally occurring ATR-activating polypeptides described herein, or any of the compositions described herein.

[0038] In some embodiments, the ATR-related disease is selected from the group consisting of: Seckel syndrome, sarcopenia, muscular dystrophy, and familial cutaneous telangiectasia and cancer syndrome. In some embodiments, the subject has been previously diagnosed or identified as having the ATR-related disease.

[0039] Also provided herein are methods of treating an ATR-related disease in a subject including administering to the subject a stem cell previously contacted with any of the nucleic acids described herein, any of the non-naturally occurring ATR- activating polypeptides described herein, or any of the compositions described herein.

[0040] In some embodiments, the method includes, prior to administering, a step of contacting a stem cell with any of the nucleic acids described herein, any of the non- naturally occurring ATR-activating polypeptides described herein, or any of the compositions described herein, to obtain the stem cell administered to the subject.

[0041] In some embodiments, the stem cell is a muscle stem cell. In some embodiments, the method includes, prior to the contacting step, a step of obtaining a stem cell that is used in the contacting step, from the subject. In some embodiments, the stem cell is an autologous stem cell. In some embodiments, the stem cell is an allogeneic stem cell.

[0042] In some embodiments, the ATR-related disease is selected from the group consisting of: Seckel syndrome, sarcopenia, muscular dystrophy, and familial cutaneous telangiectasia and cancer syndrome. In some embodiments, the subject has been previously diagnosed or identified as having the ATR-related disease.

[0043] Also provided herein are methods of transiently increasing at least one activity of ATR in a cell, the method including contacting the cell with an effective amount of any of the nucleic acids described herein, any one of the non-naturally occurring ATR activating polypeptides described herein, or any one of the compositions described herein.

[0044] In some embodiments, the contacting is performed in vitro.

[0045] In some embodiments, the method includes, after the contacting step, administering the cell to a subject. In some embodiments, the contacting is performed in a subject.

[0046] In some embodiments, the subject has an ATR-related disease. In some embodiments, the ATR-related disease is selected from the group consisting of Seckel syndrome, sarcopenia, muscular dystrophy, and familial cutaneous telangiectasia and cancer syndrome. In some embodiments, the subject has been previously identified or diagnosed as having an ATR-related disease.

[0047] In some embodiments, the cell is a stem cell. In some embodiments, the stem cell is a muscle stem cell.

[0048] Also provided herein are methods of increasing resilience of a stem cell, the method including contacting the stem cell with an effective amount of any one of the nucleic acids described herein, any one of the non-naturally occurring ATR-activating polypeptides described herein, or any one of the compositions described herein.

[0049] Also provided herein are methods of maintaining resilience of a stem cell, the method including contacting the stem cell with an effective amount of any one of the nucleic acids described herein, any one of the non-naturally occurring ATR-activating polypeptides described herein, or any one of the compositions described herein.

[0050] In some embodiments, the contacting is performed in vitro.

[0051] In some embodiments, the method includes, after the contacting step, administering the stem cell to a subject. In some embodiments, the contacting is performed in a subject.

[0052] In some embodiments, the subject has an ATR-related disease. In some embodiments, the ATR-related disease is selected from the group consisting of Seckel syndrome, sarcopenia, muscular dystrophy, and familial cutaneous telangiectasia and cancer syndrome. In some embodiments, the subject has been previously identified or diagnosed as having an ATR-related disease.

[0053] In some embodiments, the stem cell is a muscle stem cell.

[0054] Also provided herein are methods of preparing a stem cell for administration to a subject in need thereof, the method including contacting the stem cell with an effective amount of any of the nucleic acids described herein, any one of the non- naturally occurring ATR-activating polypeptides described herein, or any of the compositions described herein.

[0055] In some embodiments, the stem cell is a muscle stem cell.

[0056] In some embodiments, the method includes, prior to the contacting step, a step of obtaining a stem cell that is used in the contacting step, from the subject. In some embodiments, the stem cell is an autologous stem cell. In some embodiments, the stem cell is an allogeneic stem cell.

[0057] In some embodiments, the method includes, either before or after the contacting step, introducing a therapeutic nucleic acid into the stem cell. In some embodiments, the method includes administering the stem cell to a subject in need thereof.

[0058] In some embodiments, the subject has an ATR-related disease. In some embodiments, the ATR-related disease is selected from the group consisting of: Seckel syndrome, sarcopenia, muscular dystrophy, and familial cutaneous telangiectasia and cancer syndrome. In some embodiments, the subject has been previously identified or diagnosed as having the ATR-related disease.

[0059] Also provided herein are methods of increasing efficiency of insertion of exogenous DNA into the genome of a mammalian cell, the method including: (a) contacting the mammalian cell with an effective amount of any one of the nucleic acids described herein, any one of the non-naturally occurring ATR-activating polypeptides described herein, or any one of the compositions described herein; and (b) contacting the mammalian cell with nucleic acid including the exogenous DNA.

[0060] In some embodiments, step (a) is performed before step (b). In some embodiments, step (b) is performed before step (a). In some embodiments, step (a) and step (b) are performed at substantially the same time. In some embodiments, steps (a) and (b) are performed in vitro.

[0061] In some embodiments, the method includes, after steps (a) and (b), a step of administering the mammalian cell to a subject. In some embodiments, steps (a) and (b) are performed in a subject.

[0062] In some embodiments, the mammalian cell is a stem cell. In some embodiments, the stem cell is a muscle stem cell.

[0063] In some embodiments, step (b) includes contacting the mammalian cell with a nucleic acid encoding a gene-editing enzyme. In some embodiments, the gene-editing enzyme is selected from the group consisting of a Cas9, a zinc-finger nuclease, and a transcription activator-like effector nuclease (TALEN).

[0064] In some embodiments, the nucleic acid, the non-naturally occurring ATR- activating polypeptide, or the composition is administered two or more times to the subject.

[0065] In some embodiments, the nucleic acid, the non-naturally occurring ATR- activating polypeptide, or the composition is administered locally. In some embodiments, the nucleic acid, the non-naturally occurring ATR-activating polypeptide, or the composition is administered systemically.

[0066] In some embodiments, the subject is a human subject.

[0067] Also provided herein are kits including: (a) any one of the compositions described herein; and (b) instructions for administering any one of the compositions to a subject in need thereof.

[0068] Also provided are cells comprising any of the nucleic acids described herein, any of the non-naturally occurring ATR-activating polypeptides described herein, and / or any of the compositions described herein. In some embodiments, the cell is a stem cell. In some embodiments, the stem cell is a muscle stem cell.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0070] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0071] BRIEF DESCRIPTION OF DRAWINGS

[0072] Figure 1 are schematic diagrams showing an activation mechanism of ATR kinase and ATR-activating DNA structures, ATR-activating domains of DNA topoisomerase II binding protein 1 (TOPBP1) and ETAA1 activator of ATR kinase (ETAA1), and a schematic representation of wild-type ETAA1.

[0073] Figure l is a schematic diagram showing wild-type ETAA1 and mRNA of the ATR activating domain.

[0074] Figure 3 is an exemplary schematic showing an AAD mRNA cell treatment workflow.

[0075] Figures 4A-4B show AAD mRNA treatment hyperactivates ATR signaling. Figure 4A shows a representative Western blot and Figure 4B shows quantification of the results showing increased levels of phosphorylated checkpoint kinase- 1 (p- Chkl) and Cyclin F after AAD mRNA treatment.

[0076] Figures 5A-5B show AAD mRNA treatment maintains quiescence and increases stem cell resilience. Figure 5A shows a decrease in percentage of Edu+ cells and TUNEL+ cells after mRNA treatment. Figure 5B shows results where the number of nuclei present in MHC+ cells / number of total nuclei per field was determined and expressed as percentage.

[0077] Figure 6 is a graph showing AAD mRNA treatment increases the ability of muscle stem cells (MuSCs) to reseed the stem cell niche.

[0078] Figure 7 shows a schematic representation of wild-type ETAA1 and the non- naturally occurring ATR-activating polypeptides described herein.

[0079] Figures 8A-8C shows flow cytometry analysis of phospho-Chkl and phospho-H2AX positive cells in K562 lymphoblast cells that were untreated, treated with endogenous AAD, SV40-NLS_AAD_RPA2, SV40-NLS_AAD_W107R_RPA2, SV40-NLS_AAD, and SV40-NLS_AAD_W107R. Figures 9A-9C shows flow cytometry analysis of phospho-Chkl and phospho-H2AX positive cells in human embryonic stem cells (Hl -ESC) that were untreated, treated with endogenous AAD, SV40-NLS_AAD_RPA2, SV40- NLS_AAD_W107R_RPA2, SV40-NLS_AAD, and SV40-NLS_AAD_W107R.

[0080] Figures 10A-C shows TALEN guided integration of CRISPi DNA sequences upon AAD mRNA treatment.

[0081] DETAILED DESCRIPTION

[0082] Provided herein are nucleic acids that include a sequence encoding a non- naturally occurring ATR-activating polypeptide, wherein the non-naturally occurring ATR-activating polypeptide comprises (i) a nuclear localization signal sequence; and (ii) an ataxia telangiectasia and a Rad3-related (ATR)-activating domain.

[0083] Also provided herein are non-naturally occurring ATR-activating polypeptides that include (i) a nuclear localization signal sequence; and (ii) an ataxia telangiectasia and a Rad3 -related (ATR)-activating domain.

[0084] Also provided herein are compositions that include any of the nucleic acids described herein, non-naturally occurring! ATR-activating polypeptides described herein, and a pharmaceutically acceptable excipient.

[0085] Also provided herein are methods of treating an ATR-related disease in a subject that include administering to the subject an effective amount of any of the nucleic acids described herein, the non-naturally occurring ATR-activating polypeptides described herein, or the compositions described herein. Also provided herein are methods of treating an ATR-related disease in a subject that include administering to the subject a stem cell previously contacted with any of the nucleic acids described herein, the non-naturally occurring ATR-activating polypeptides described herein, or the compositions described herein.

[0086] Also provided herein are methods of transiently increasing at least one activity of ATR in a cell that include contacting the cell with an effective amount of any of the nucleic acids described herein, the non-naturally occurring ATR-activating polypeptides described herein, or the compositions described herein. Also provided herein are methods of increasing resilience and / or maintaining resilience of a stem cell that include contacting the stem cell with an effective amount of any of the nucleic acids described herein, the non-naturally occurring ATR-activating polypeptides described herein, or the compositions described herein. Also provided herein are methods of preparing a stem cell for administration to a subject in need thereof that include contacting the stem cell with an effective amount of any of the nucleic acids described herein, the non-naturally occurring ATR-activating polypeptides described herein, or the compositions described herein.

[0087] Also provided herein are methods of increasing efficiency of insertion of exogenous DNA into the genome of a mammalian cell that include (a) contacting the mammalian cell with an effective amount of any of the nucleic acids described herein, the non-naturally occurring ATR-activating polypeptides described herein, or the compositions described herein; and (b) contacting the mammalian cell with nucleic acid comprising the exogenous DNA.

[0088] Various non-limiting aspects of these nucleic acids, compositions, and methods are described herein, and can be used in any combination without limitation. Additional aspects of various components of these nucleic acids, compositions, and methods are known in the art.

[0089] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0090] As used herein, the term “about”, when used in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0091] As used herein, the term “administration” typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. As can be understood by those skilled in the art, the route of administration will depend on the agent to be delivered. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion or infusion) for at least a selected period of time.

[0092] As used herein, a “cell” can refer to a eukaryotic cell, optionally obtained from a subject or a commercially available source.

[0093] As used herein, “delivering”, “gene delivery”, “gene transfer”, “transducing”, or “transfecting” can refer to the introduction of an exogenous polynucleotide into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (e.g., viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of “naked” polynucleotides (e.g., electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides). In some embodiments, an introduced polynucleotide may be transiently maintained.

[0094] As used herein, the term “expression” refers to the process by which polynucleotides are transcribed (and / or optionally processed, such as by one or more of splicing, capping, editing, etc) into mRNA and / or the process by which an mRNA is translated into peptides, polypeptides, or proteins. In some embodiments, expression level of a gene may be determined, for example, by measuring amount of mRNA, or protein encoded thereby, in a sample (e.g., a cell or tissue sample) in which the gene is expressed. As used herein, the term “encode” or “encoding” is used to describe the relationship between a nucleic acid and a polypeptide. For example, an mRNA whose sequence can be translated (e.g., by action of a ribosome) into a polypeptide is said to “encode” that polypeptide. Moreover, a nucleic acid (e.g., DNA or RNA) that, through one or more steps of replication (e.g., transcription, reverse transcription and / or other polymerization) and / or processing (e.g., splicing, capping, editing, etc) can be used to generate such an mRNA, is also said to “encode” the relevant polypeptide. A nucleic acid strand that encodes a polypeptide is referred to as a “coding” strand; its complement is an “antisense” strand.

[0095] As used herein, in its broadest sense, a “nucleic acid” refers to a compound and / or substance that is, or can be incorporated into, a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is, or can be incorporated into, a polynucleotide chain with a phosphodiester linkage. In some embodiments, “nucleic acid” can refer to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside), and in some embodiments, “nucleic acid” can refer to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, a “nucleic acid” is or comprises ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2’-amino-LNA having a 2’-amino functionalization, and 2’- amino-a-LNA having a 2’ -amino functionalization), or a combination thereof. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid residue analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in one or more residues, and in some embodiments, are linked together other than by a phosphodiester. For example, in some embodiments, a nucleic acid includes one or more phosphorothioate and / or phosphoroamidite (e.g., 5'-N-phosphoramidite) linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid includes one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 - propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2- thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), enzymatic synthesis in the absence of a complementary template, reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide.

[0096] As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, a pharmaceutical composition is suitable for administration to a human or animal subject, e.g., via a particular route of administration (e.g., parenteral). In some embodiments, an active agent is present in a pharmaceutical composition in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0097] As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, a subject does not display a particular, or in some embodiments any, symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to that has previously received a treatment for a disease, disorder, or condition. In some embodiments, the previously administered treatment was not deemed effective in the subject. In some embodiments, the subject has been previously identified or diagnosed as having the disease, disorder, or condition.

[0098] As used herein, the term “treatment” means to ameliorate or reduce the severity or frequency of at least one symptom of a disorder. Generally, the methods of treatment include administering a therapeutically effective amount of composition that reduces the severity or frequency of at least one symptom of a disorder in a subject who is in need of, or who has been determined to be in need of, such treatment.

[0099] Non-Naturally Occurring ATR-Activating Polypeptides

[0100] Serine / threonine-protein kinase ATR, also known as ataxia telangiectasia and Rad3-related protein (ATR) or FRAP-related protein 1 (FRP1), is an enzyme that is encoded by AXQ A TR gene. Ataxia Telangiectasia and Rad3-related protein (ATR) is a DNA damage response (DDR) protein kinase which primarily responds to singlestrand DNA breaks and its kinase cascade can result in checkpoint control of cell cycle progression. Hypomorphic mutations of ATR in humans have been associated with Seckel Syndrome, characterized by severe microcephaly, growth retardation, and intellectual disability. Furthermore, ATR ablation in adult mice is reported to result in stem and progenitor cell loss, ultimately leading to defects in tissue homeostasis and the onset of aging associated phenotypes.

[0101] Additionally, studies on ATR in muscle stem cells (MuSCs) have revealed a role of ATR in the maintenance of stem cell quiescence, preventing stem cell exhaustion and functional decline. In some embodiments, the ability to hyper-activate or maintain ATR signaling may bring long-term therapeutic benefits for patients suffering from rare neuro-muscular diseases such as Seckel Syndrome, to progressive muscle wasting diseases, such as sarcopenia and muscular dystrophy. In some embodiments, increasing stem cell resilience also has applications in stem cell therapeutics, such as CAR-T, by ensuring stem cell survival during arduous reprogramming and transplantation steps.

[0102] ATR can be activated by ETAA1 and TopBPl proteins, both of which contain a conserved ATR Activating Domain (AAD). As used herein, an “ATR-activating polypeptide” refers to a polypeptide that can activate or maintain ATR kinase activity. In some embodiments, an ATR-activating polypeptide can be transiently expressed in a cell, thereby activating ATR signaling to prevent diseases in which loss of ATR activity is a driving factor. In some embodiments, an ATR-activating polypeptide can be contacted with a stem cell prior to transplantation to increase stem cell resilience.

[0103] Also, described herein are nucleic acids comprising a sequence encoding a non-naturally occurring ATR-activating polypeptide. Also described herein are non- naturally occurring ATR-activating polypeptides. In some embodiments, a non- naturally occurring ATR-activating polypeptide includes (i) a nuclear localization signal sequence (e.g., any of the exemplary nuclear localization signal sequences described herein or known in the art), and (ii) an ATR-activating domain (e.g., any of the ATR-activating domains described herein or known in the art). In some embodiments, a non-naturally occurring ATR-activating polypeptide can further include a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) disposed between the nuclear localization signal sequence and the ATR-activating domain.

[0104] In some embodiments, a non-naturally occurring ATR-activating polypeptide can further include a replication protein A (RPA)-binding domain (e.g., any of the exemplary RPA-binding domains described herein or known in the art). In some embodiments, the RPA-binding domain is positioned C-terminally relative to the ATR-activating domain. In some embodiments, the non-naturally occurring ATR- activating polypeptide can further include a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) disposed between the ATR-activating domain and the RPA-binding domain. In some embodiments, a non-naturally occurring ATR-activating polypeptide can further include a cell -penetrating peptide (e.g., any of the cell-penetrating peptides described herein or known in the art) at the N-terminus.

[0105] In some embodiments, a non-naturally occurring ATR-activating polypeptide can lack one or more amino acids (e.g., at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 280, at least 290, at least 300, at least 320, at least 320, at least 340, at least 360, at least 380, at least 400, at least 420, at least 440, at least 460, at least 480, at least 500, at least 520, at least 540, at least 560, at least 580, at least 600, at least 620, at least 640, at least 640, at least 660, at least 680, or at least 700 amino acids) as compared to a wildtype ATR-activating polypeptide (e.g., a wildtype human ATR-activating polypeptide). In some embodiments, a non-naturally occurring ATR-activating polypeptide can include one or more contiguous amino acids (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids) not present in a wildtype ATR-activating polypeptide (e.g., a wildtype human ATR- activating polypeptide).

[0106] In some embodiments, a non-naturally occurring ATR-activating polypeptide comprises a total of about 170 amino acids to about 320 amino acids (e.g., about 170 to about 310, about 170 to about 300, about 170 to about 290, about 170 to about 280, about 170 to about 270, about 170 to about 260, about 170 to about 255, about 170 to about 250, about 170 to about 245, about 170 to about 240, about 170 to about 235, about 170 to about 230, about 170 to about 225, about 170 to about 220, about 170 to about 215, about 170 to about 210, about 170 to about 205, about 170 to about 200, about 170 to about 195, about 170 to about 190, about 170 to about 185, about 170 to about 180, about 175 to about 320, about 175 to about 310, about 175 to about 300, about 175 to about 290, about 175 to about 280, about 175 to about 270, about 175 to about 260, about 175 to about 255, about 175 to about 250, about 175 to about 245, about 175 to about 240, about 175 to about 235, about 175 to about 230, about 175 to about 225, about 175 to about 220, about 175 to about 215, about 175 to about 210, about 175 to about 205, about 175 to about 200, about 175 to about 195, about 175 to about 190, about 175 to about 185, about 180 to about 320, about 180 to about 310, about 180 to about 300, about 180 to about 290, about 180 to about 280, about 180 to about 270, about 180 to about 260, about 180 to about 255, about 180 to about 250, about 180 to about 245, about 180 to about 240, about 180 to about 235, about 180 to about 230, about 180 to about 225, about 180 to about 220, about 180 to about 215, about 180 to about 210, about 180 to about 205, about 180 to about 200, about 180 to about 195, about 180 to about 190, about 185 to about 320, about 185 to about 310, about 185 to about 300, about 185 to about 290, about 185 to about 280, about 185 to about 270, about 185 to about 260, about 185 to about 255, about 185 to about 250, about 185 to about 245, about 185 to about 240, about 185 to about 235, about 185 to about 230, about 185 to about 225, about 185 to about 220, about 185 to about 215, about 185 to about 210, about 185 to about 205, about 185 to about 200, about 185 to about 195, about 190 to about 320, about 190 to about 310, about 190 to about 300, about 190 to about 290, about 190 to about 280, about 190 to about 270, about 190 to about 260, about 190 to about 255, about 190 to about 250, about 190 to about 245, about 190 to about 240, about 190 to about 235, about 190 to about 230, about 190 to about 225, about 190 to about 220, about 190 to about 215, about 190 to about 210, about 190 to about 205, about 190 to about 200, about 195 to about 320, about 195 to about 310, about 195 to about 300, about 195 to about 290, about 195 to about 280, about 195 to about 270, about 195 to about 260, about 195 to about 255, about 195 to about 250, about 195 to about 245, about 195 to about 240, about 195 to about 235, about 195 to about 230, about 195 to about 225, about 195 to about 220, about 195 to about 215, about 195 to about 210, about 195 to about 205, about 200 to about 320, about 200 to about 310, about 200 to about 300, about 200 to about 290, about 200 to about 280, about 200 to about 270, about 200 to about 260, about 200 to about 255, about 200 to about 250, about 200 to about 245, about 200 to about 240, about 200 to about 235, about 200 to about 230, about 200 to about 225, about 200 to about 220, about 200 to about 215, about 200 to about 210, about 205 to about 320, about 205 to about 310, about 205 to about 300, about 205 to about 290, about 205 to about 280, about 205 to about 270, about 205 to about 260, about 205 to about 255, about 205 to about 250, about 205 to about 245, about 205 to about 240, about 205 to about 235, about 205 to about 230, about 205 to about 225, about 205 to about 220, about 205 to about 215, about 210 to about 320, about 210 to about 310, about 210 to about 300, about 210 to about 290, about 210 to about 280, about 210 to about 270, about 210 to about 260, about 210 to about 255, about 210 to about 250, about 210 to about 245, about 210 to about 240, about 210 to about 235, about 210 to about 230, about 210 to about 225, about 210 to about 220, about 215 to about 320, about 215 to about 310, about 215 to about 300, about 215 to about 290, about 215 to about 280, about 215 to about 270, about 215 to about 260, about 215 to about 255, about 215 to about 250, about 215 to about 245, about 215 to about 240, about 215 to about 235, about 215 to about 230, about 215 to about 225, about 220 to about 320, about 220 to about 310, about 220 to about 300, about 220 to about 290, about 220 to about 280, about 220 to about 270, about 220 to about 260, about 220 to about 255, about 220 to about 250, about 220 to about 245, about 220 to about 240, about 220 to about 235, about 220 to about 230, about 225 to about 320, about 225 to about 310, about 225 to about 300, about 225 to about 290, about 225 to about 280, about 225 to about 270, about 225 to about 260, about 225 to about 255, about 225 to about 250, about 225 to about 245, about 225 to about 240, about 225 to about 235, about 230 to about 320, about 230 to about 310, about 230 to about 300, about 230 to about 290, about 230 to about 280, about 230 to about 270, about 230 to about 260, about 230 to about 255, about 230 to about 250, about 230 to about 245, about 230 to about 240, about 235 to about 320, about 235 to about 310, about 235 to about 300, about 235 to about 290, about 235 to about 280, about 235 to about 270, about 235 to about 260, about 235 to about 255, about 235 to about 250, about 235 to about 245, about 240 to about 320, about 240 to about 310, about 240 to about 300, about 240 to about 290, about 240 to about 280, about 240 to about 270, about 240 to about 260, about 240 to about 255, about 240 to about 250, about 245 to about 320, about 245 to about 310, about 245 to about 300, about 245 to about 290, about 245 to about 280, about 245 to about 270, about 245 to about 260, about 245 to about 255, about 250 to about 320, about 250 to about 310, about 250 to about 300, about 250 to about 290, about 250 to about 280, about 250 to about 270, about 250 to about 260, about 260 to about 320, about 260 to about 310, about 260 to about 300, about 260 to about 290, about 260 to about 280, about 260 to about 270, about 270 to about 320, about 270 to about 310, about 270 to about 300, about 270 to about 290, about 270 to about 280, about 280 to about 320, about 280 to about 310, about 280 to about 300, about 280 to about 290, about 290 to about 320, about 290 to about 310, about 290 to about 300, about 300 to about 320, about 300 to about 310, or about 310 to about 320 amino acids)

[0107] Nuclear Localization Sequences

[0108] As used herein, a “nuclear localization sequence” refers to a short strech of amino acids that mediates the transport of a protein into a nucleus of a mammalian cell. In some embodiments, a nuclear localization sequence includes a high proportion of positively charged lysines or arginines exposed on the protein surface. In some embodiments, a nuclear localization sequence includes about 7 to 20 (e.g., about 8 to 20, about 9 to 20, about 10 to 20, about 12 to 20, about 14 to 20, about 16 to 20, about 18 to 20, about 7 to 18, about 8 to 18, about 9 to 18, about 10 to 18, about 12 to 18, about 14 to 18, about 16 to 18, about 7 to 16, about 8 to 16, about 9 to 16, about 10 to 16, about 12 to 16, about 14 to 16, about 7 to 14, about 8 to 14, about 9 to 14, about 10 to 14, about 12 to 14, about 7 to 12, about 8 to 12, about 9 to 12, about 10 to 12, about 7 to 10, about 8 to 10, about 9 to 10, about 7 to 9, about 8 to 9, or about 7 to 8) amino acids.

[0109] In some embodiments, a nuclear localization signal sequence comprises SEQ ID NO: 8 (PKKKRRV) or SEQ ID NO: 9 (PKKKRKV). In some embodiments, the nuclear localization sequence can comprise PKKRKV (SEQ ID NO: 18). Additional examples of nuclear localization sequences are known in the art.

[0110] A TR-Activating Domains

[0111] In mammalian cells, activation of ATR is mediated by two proteins, ETAA1 and TOPBP 1, both of which associate with the kinase through very similar ATR- activation domains (AADs). Both ETAA1 and TOPBP 1 contain experimentally- defined ATR activation domains (AADs) that are mostly unstructured and have minimal sequence similarity. A tryptophan residue in both AADs is required for ATR activation.

[0112] In some embodiments, an ATR-activating domain can include a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 11.

[0113] In some embodiments, an ATR-activating domain can include a total of about 8 to about 20 amino acids (e.g., about 8 to about 19, about 8 to about 18, about 8 to about 17, about 8 to about 16, about 8 to about 15, about 8 to about 14, about 8 to about 13, about 8 to about 12, about 8 to about 11, about 8 to about 10, about 9 to about 20, about 9 to about 19, about 9 to about 18, about 9 to about 17, about 9 to about 16, about 9 to about 15, about 9 to about 14, about 9 to about 13, about 9 to about 12, about 9 to about 11, about 10 to about 20, about 10 to about 19, about 10 to about 18, about 10 to about 17, about 10 to about 16, about 10 to about 15, about 10 to about 14, about 10 to about 13, about 10 to about 12, about 11 to about 20, about 11 to about 19, about 11 to about 18, about 11 to about 17, about 11 to about 16, about 11 to about 15, about 11 to about 14, about 11 to about 13, about 12 to about 20, about 12 to about 19, about 12 to about 18, about 12 to about 17, about 12 to about 16, about 12 to about 15, about 12 to about 14, about 13 to about 20, about 13 to about 19, about 13 to about 18, about 13 to about 17, about 13 to about 16, about 13 to about 15, about 14 to about 20, about 14 to about 19, about 14 to about 18, about 14 to about 17, about 14 to about 16, about 15 to about 20, about 15 to about 19, about 15 to about 18, about 15 to about 17, about 16 to about 20, about 16 to about 19, about 16 to about 18, about 17 to about 20, about 17 to about 19, or about 18 to about 20 amino acids).

[0114] Linker Sequences

[0115] In some embodiments, a non-naturally occurring ATR-activating polypeptide further includes a linker sequence. In some embodiments, a non-naturally occurring ATR-activating polypeptide further includes a linker sequence disposed between the nuclear localization signal sequence and the ATR-activating domain.

[0116] In some embodiments, a linker sequence can be a flexible linker sequence. Non-limiting examples of linker sequences that can be used are described in Klein et al., Protein Engineering, Design & Selection, Vol. 27, No. 10, pp. 325-330, 2014; Priyanka et al., Protein Sci. 22(2): 153-167, 2013. In some examples, a linker sequence is a synthetic linker sequence. In some embodiments, a linker sequence can have a total length of 1 amino acid to about 100 amino acids, 1 amino acid to about 90 amino acids, 1 amino acid to about 80 amino acids, 1 amino acid to about 70 amino acids, 1 amino acid to about 60 amino acids, 1 amino acid to about 50 amino acids, 1 amino acid to about 45 amino acids, 1 amino acid to about 40 amino acids, 1 amino acid to about 35 amino acids, 1 amino acid to about 30 amino acids, 1 amino acid to about 25 amino acids, 1 amino acid to about 24 amino acids, 1 amino acid to about 22 amino acids, 1 amino acid to about 20 amino acids, 1 amino acid to about 18 amino acids, 1 amino acid to about 16 amino acids, 1 amino acid to about 14 amino acids, 1 amino acid to about 12 amino acids, 1 amino acid to about 10 amino acids, 1 amino acid to about 8 amino acids, 1 amino acid to about 6 amino acids, 1 amino acid to about 4 amino acids, about 2 amino acids to about 100 amino acids, about 2 amino acids to about 90 amino acids, about 2 amino acids to about 80 amino acids, about 2 amino acids to about 70 amino acids, about 2 amino acids to about 60 amino acids, about 2 amino acids to about 50 amino acids, about 2 amino acids to about 45 amino acids, about 2 amino acids to about 40 amino acids, about 2 amino acids to about 35 amino acids, about 2 amino acids to about 30 amino acids, about 2 amino acids to about 25 amino acids, about 2 amino acids to about 24 amino acids, about 2 amino acids to about 22 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 18 amino acids, about 2 amino acids to about 16 amino acids, about 2 amino acids to about 14 amino acids, about 2 amino acids to about 12 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 4 amino acids, about 4 amino acids to about 100 amino acids, about 4 amino acids to about 90 amino acids, about 4 amino acids to about 80 amino acids, about 4 amino acids to about 70 amino acids, about 4 amino acids to about 60 amino acids, about 4 amino acids to about 50 amino acids, about 4 amino acids to about 45 amino acids, about 4 amino acids to about 40 amino acids, about 4 amino acids to about 35 amino acids, about 4 amino acids to about 30 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 24 amino acids, about 4 amino acids to about 22 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 18 amino acids, about 4 amino acids to about 16 amino acids, about 4 amino acids to about 14 amino acids, about 4 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 6 amino acids, about 6 amino acids to about 100 amino acids, about 6 amino acids to about 90 amino acids, about 6 amino acids to about 80 amino acids, about 6 amino acids to about 70 amino acids, about 6 amino acids to about 60 amino acids, about 6 amino acids to about 50 amino acids, about 6 amino acids to about 45 amino acids, about 6 amino acids to about 40 amino acids, about 6 amino acids to about 35 amino acids, about 6 amino acids to about 30 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 24 amino acids, about 6 amino acids to about 22 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 18 amino acids, about 6 amino acids to about 16 amino acids, about 6 amino acids to about 14 amino acids, about 6 amino acids to about 12 amino acids, about 6 amino acids to about 10 amino acids, about 6 amino acids to about 8 amino acids, about 8 amino acids to about 100 amino acids, about 8 amino acids to about 90 amino acids, about 8 amino acids to about 80 amino acids, about 8 amino acids to about 70 amino acids, about 8 amino acids to about 60 amino acids, about 8 amino acids to about 50 amino acids, about 8 amino acids to about 45 amino acids, about 8 amino acids to about 40 amino acids, about 8 amino acids to about 35 amino acids, about 8 amino acids to about 30 amino acids, about 8 amino acids to about 25 amino acids, about 8 amino acids to about 24 amino acids, about 8 amino acids to about 22 amino acids, about 8 amino acids to about 20 amino acids, about 8 amino acids to about 18 amino acids, about 8 amino acids to about 16 amino acids, about 8 amino acids to about 14 amino acids, about 8 amino acids to about 12 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 24 amino acids, about 10 amino acids to about 22 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 18 amino acids, about 10 amino acids to about 16 amino acids, about 10 amino acids to about 14 amino acids, about 10 amino acids to about 12 amino acids, about 12 amino acids to about 100 amino acids, about 12 amino acids to about 90 amino acids, about 12 amino acids to about 80 amino acids, about 12 amino acids to about 70 amino acids, about 12 amino acids to about 60 amino acids, about 12 amino acids to about 50 amino acids, about 12 amino acids to about 45 amino acids, about 12 amino acids to about 40 amino acids, about 12 amino acids to about 35 amino acids, about 12 amino acids to about 30 amino acids, about 12 amino acids to about 25 amino acids, about 12 amino acids to about 24 amino acids, about 12 amino acids to about 22 amino acids, about 12 amino acids to about 20 amino acids, about 12 amino acids to about 18 amino acids, about 12 amino acids to about 16 amino acids, about 12 amino acids to about 14 amino acids, about 14 amino acids to about 100 amino acids, about 14 amino acids to about 90 amino acids, about 14 amino acids to about 80 amino acids, about 14 amino acids to about 70 amino acids, about 14 amino acids to about 60 amino acids, about 14 amino acids to about 50 amino acids, about 14 amino acids to about 45 amino acids, about 14 amino acids to about 40 amino acids, about 14 amino acids to about 35 amino acids, about 14 amino acids to about 30 amino acids, about 14 amino acids to about 25 amino acids, about 14 amino acids to about 24 amino acids, about 14 amino acids to about 22 amino acids, about 14 amino acids to about 20 amino acids, about 14 amino acids to about 18 amino acids, about 14 amino acids to about 16 amino acids, about 16 amino acids to about 100 amino acids, about 16 amino acids to about 90 amino acids, about 16 amino acids to about 80 amino acids, about 16 amino acids to about 70 amino acids, about 16 amino acids to about 60 amino acids, about 16 amino acids to about 50 amino acids, about 16 amino acids to about 45 amino acids, about 16 amino acids to about 40 amino acids, about 16 amino acids to about 35 amino acids, about 16 amino acids to about 30 amino acids, about 16 amino acids to about 25 amino acids, about 16 amino acids to about 24 amino acids, about 16 amino acids to about 22 amino acids, about 16 amino acids to about 20 amino acids, about 16 amino acids to about 18 amino acids, about 18 amino acids to about 100 amino acids, about 18 amino acids to about 90 amino acids, about 18 amino acids to about 80 amino acids, about 18 amino acids to about 70 amino acids, about 18 amino acids to about 60 amino acids, about 18 amino acids to about 50 amino acids, about 18 amino acids to about 45 amino acids, about 18 amino acids to about 40 amino acids, about 18 amino acids to about 35 amino acids, about 18 amino acids to about 30 amino acids, about 18 amino acids to about 25 amino acids, about 18 amino acids to about 24 amino acids, about 18 amino acids to about 22 amino acids, about 18 amino acids to about 20 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 20 amino acids to about 24 amino acids, about 20 amino acids to about 22 amino acids, about 22 amino acids to about 100 amino acids, about 22 amino acids to about 90 amino acids, about 22 amino acids to about 80 amino acids, about 22 amino acids to about 70 amino acids, about 22 amino acids to about 60 amino acids, about 22 amino acids to about 50 amino acids, about 22 amino acids to about 45 amino acids, about 22 amino acids to about 40 amino acids, about 22 amino acids to about 35 amino acids, about 22 amino acids to about 30 amino acids, about 22 amino acids to about 25 amino acids, about 22 amino acids to about 24 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 80 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 80 amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to about 60 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 90 amino acids, or about 90 amino acids to about 100 amino acids.

[0117] In some embodiments, a linker is rich in glycine (Gly or G) residues. In some embodiments, a linker is rich in serine (Ser or S) residues. In some embodiments, a linker is rich in glycine and serine residues. In some embodiments, a linker has one or more glycine-serine residue pairs (GS), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. In some embodiments, a linker has one or more Gly-Gly-Gly-Ser (GGGS) (SEQ ID NO: 17) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS (SEQ ID NO: 17) sequences. In some embodiments, a linker has one or more Gly-Gly-Gly- Gly-Ser (GGGGS) (SEQ ID NO: 6) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS (SEQ ID NO: 6) sequences. In some embodiments, a linker has one or more Gly-Gly-Ser-Gly (GGSG) (SEQ ID NO: 7) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG (SEQ ID NO: 7) sequences. In some embodiments, a linker sequence comprises (GS)X, (GGS)X, (GGGGS)X(SEQ ID NO: 6), or (GGSG)X(SEQ ID NO: 7), wherein x is an integer from 1 to 50. Replication Protein A (RP A) -Binding Domains

[0118] In some embodiments, a non-naturally occurring ATR-activating polypeptide can further include a replication protein A (RPA)-binding domain. Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair. In some embodiments, a non-naturally occurring ATR-activating polypeptide can include one or more (e.g.,one, two, three, or four) RPA-binding domains. In some embodiments, the one or more RPA-binding domains in the non-naturally occurring ATR-activating polypeptide is positioned C-terminally relative to the ATR-activating domain.

[0119] In some embodiments, a non-naturally occurring ATR-activating polypeptide can further include a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) disposed between the ATR-activating domain and the RPA-binding domain. In some embodiments where the non-naturally occurring ATR-activating polypeptide comprises two or more RPA-binding domains, the non-naturally occurring ATR-activating polypeptide can further include a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between any two RPA-binding domains. In some embodiments, a linker sequence comprises (GS)X, (GGS)X, (GGGGS)X(SEQ ID NO: 6), or (GGSG)X(SEQ ID NO: 7), wherein x is an integer from 1 to 50.

[0120] In some embodiments, an RPA-binding domain comprises a sequence at least 80% identical, at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 5.

[0121] In some embodiments, an RPA-binding domain comprises a total of about 12 to about 30 amino acids (e.g., about 12 to about 29, about 12 to about 28, about 12 to about 27, about 12 to about 26, about 12 to about 25, about 12 to about 24, about 12 to about 23, about 12 to about 22, about 12 to about 21, about 12 to about 20, about 12 to about 19, about 12 to about 18, about 12 to about 17, about 12 to about 16, about 12 to about 15, about 12 to about 14, about 13 to about 30, about 13 to about 29, about 13 to about 28, about 13 to about 27, about 13 to about 26, about 13 to about 25, about 13 to about 24, about 13 to about 23, about 13 to about 22, about 13 to about 21, about 13 to about 20, about 13 to about 19, about 13 to about 18, about 13 to about 17, about 13 to about 16, about 13 to about 15, about 14 to about 30, about 14 to about 29, about 14 to about 28, about 14 to about 27, about 14 to about 26, about 14 to about 25, about 14 to about 24, about 14 to about 23, about 14 to about 22, about 14 to about 21, about 14 to about 20, about 14 to about 19, about 14 to about 18, about 14 to about 17, about 14 to about 16, about 15 to about 30, about

[0122] 15 to about 29, about 15 to about 28, about 15 to about 27, about 15 to about 26, about 15 to about 25, about 15 to about 24, about 15 to about 23, about 15 to about

[0123] 22, about 15 to about 21, about 15 to about 20, about 15 to about 19, about 15 to about 18, about 15 to about 17, about 16 to about 30, about 16 to about 29, about 16 to about 28, about 16 to about 27, about 16 to about 26, about 16 to about 25, about

[0124] 16 to about 24, about 16 to about 23, about 16 to about 22, about 16 to about 21, about 16 to about 20, about 16 to about 19, about 16 to about 18, about 17 to about 30, about 17 to about 29, about 17 to about 28, about 17 to about 27, about 17 to about 26, about 17 to about 25, about 17 to about 24, about 17 to about 23, about 17 to about 22, about 17 to about 21, about 17 to about 20, about 17 to about 19, about

[0125] 18 to about 30, about 18 to about 29, about 18 to about 28, about 18 to about 27, about 18 to about 26, about 18 to about 25, about 18 to about 24, about 18 to about

[0126] 23, about 18 to about 22, about 18 to about 21, about 18 to about 20, about 19 to about 30, about 19 to about 29, about 19 to about 28, about 19 to about 27, about 19 to about 26, about 19 to about 25, about 19 to about 24, about 19 to about 23, about

[0127] 19 to about 22, about 19 to about 21, about 20 to about 30, about 20 to about 29, about 20 to about 28, about 20 to about 27, about 20 to about 26, about 20 to about 25, about 20 to about 24, about 20 to about 23, about 20 to about 22, about 21 to about 30, about 21 to about 29, about 21 to about 28, about 21 to about 27, about 21 to about 26, about 21 to about 25, about 21 to about 24, about 21 to about 23, about 22 to about 30, about 22 to about 29, about 22 to about 28, about 22 to about 27, about 22 to about 26, about 22 to about 25, about 22 to about 24, about 23 to about 30, about 23 to about 29, about 23 to about 28, about 23 to about 27, about 23 to about 26, about 23 to about 25, about 24 to about 30, about 24 to about 29, about 24 to about 28, about 24 to about 27, about 24 to about 26, about 25 to about 30, about 25 to about 29, about 25 to about 28, about 25 to about 27, about 26 to about 30, about 26 to about 29, about 26 to about 28, about 27 to about 30, about 27 to about 29, or about 28 to about 30 amino acids).

[0128] Cell-Penetrating Peptides

[0129] In some embodimetns, a non-naturally occurring ATR-activating polypeptide can further include a cell -penetrating peptide at the N-terminus. In some embodiments, the cell-penetrating peptide comprises SEQ ID NO: 10. In other embodiments, the cell-penetrating peptide can comprise RKKRRQRRR (SEQ ID NO: 19), RRRRRRRR (SEQ ID NO: 20), RKKRRRESRKKRRRES (SEQ ID NO: 21), GRPRESGI<I<RI<RI<RLI<P (SEQ ID NO: 22), RQII<IWFQNRRMI<WI<I< (SEQ ID NO: 23), GRRRRRRRRRPPQ (SEQ ID NO: 24), LLIILRRRIRKQAHAHSK (SEQ ID NO: 25), RVRVFVVHIPRLT (SEQ ID NO: 26), GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 27), KLALKLALKALKAALKLA (SEQ ID NO: 28), GWTLNSAGYLLGI<INLI<ALAALAI<I<IL (SEQ ID NO: 29), VSALK (SEQ ID NO: 30), CSIPPEVKFNPFVYLI (SEQ ID NO: 31), GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 32), or HGLASTLTRWAHYNALIRAF (SEQ ID NO: 33).

[0130] Additional examples of cell-penetrating peptides include those available in the CPPsite 2.0 database and described in Xie et al., Front. Pharmacol. 11 :697, 2020; Agrawal et al., Nucleic Acids Res. 44:D1O98-D1103, 2016; and Kauffman et al., Trends Biochem. Sci. 40:749-764, 2015.

[0131] In some embodiments, a cell-penetrating peptide can have a total of about 5 amino acids to about 30 amino acids (e.g., about 5 to about 28, about 5 to about 26, about 5 to about 24, about 5 to about 22, about 5 to about 20, about 5 to about 19, about 5 to about 18, about 5 to about 17, about 5 to about 16, about 5 to about 15, about 5 to about 14, about 5 to about 13, about 5 to about 12, about 5 to about 11, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 6 to about 30, about 6 to about 28, about 6 to about 26, about 6 to about 24, about 6 to about 22, about 6 to about 20, about 6 to about 19, about 6 to about 18, about 6 to about 17, about 6 to about 16, about 6 to about 15, about 6 to about 14, about 6 to about 13, about 6 to about 12, about 6 to about 11, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 7 to about 30, about 7 to about 28, about 7 to about 26, about 7 to about 24, about 7 to about 22, about 7 to about 20, about 7 to about 19, about 7 to about 18, about 7 to about 17, about 7 to about 16, about 7 to about 15, about 7 to about 14, about 7 to about 13, about 7 to about 12, about 7 to about 11, about 7 to about 10, about 7 to about 9, about 8 to about 30, about 8 to about 28, about

[0132] 8 to about 26, about 8 to about 24, about 8 to about 22, about 8 to about 20, about 8 to about 19, about 8 to about 18, about 8 to about 17, about 8 to about 16, about 8 to about 15, about 8 to about 14, about 8 to about 13, about 8 to about 12, about 8 to about 11, about 8 to about 10, about 9 to about 30, about 9 to about 28, about 9 to about 26, about 9 to about 24, about 9 to about 22, about 9 to about 20, about 9 to about 19, about 9 to about 18, about 9 to about 17, about 9 to about 16, about 9 to about 15, about 9 to about 14, about 9 to about 13, about 9 to about 12, about 9 to about 11, about 10 to about 30, about 10 to about 28, about 10 to about 26, about 10 to about 24, about 10 to about 22, about 10 to about 20, about 10 to about 19, about

[0133] 10 to about 18, about 10 to about 17, about 10 to about 16, about 10 to about 15, about 10 to about 14, about 10 to about 13, about 10 to about 12, about 11 to about 30, about 11 to about 28, about 11 to about 26, about 11 to about 24, about 11 to about 22, about 11 to about 20, about 11 to about 19, about 11 to about 18, about 11 to about 17, about 11 to about 16, about 11 to about 15, about 11 to about 14, about

[0134] 11 to about 13, about 12 to about 30, about 12 to about 28, about 12 to about 26, about 12 to about 24, about 12 to about 22, about 12 to about 20, about 12 to about 19, about 12 to about 18, about 12 to about 17, about 12 to about 16, about 12 to about 15, about 12 to about 14, about 13 to about 30, about 13 to about 28, about 13 to about 26, about 13 to about 24, about 13 to about 22, about 13 to about 20, about 13 to about 19, about 13 to about 18, about 13 to about 17, about 13 to about 16, about 13 to about 15, about 14 to about 30, about 14 to about 28, about 14 to about 26, about 14 to about 24, about 14 to about 22, about 14 to about 20, about 14 to about 19, about 14 to about 18, about 14 to about 17, about 14 to about 16, about 15 to about 30, about 15 to about 28, about 15 to about 26, about 15 to about 24, about 15 to about 22, about 15 to about 20, about 15 to about 19, about 15 to about 18, about 15 to about 17, about 16 to about 30, about 16 to about 28, about 16 to about 26, about 16 to about 24, about 16 to about 22, about 16 to about 20, about 16 to about 19, about 16 to about 18, about 17 to about 30, about 17 to about 28, about 17 to about 26, about 17 to about 24, about 17 to about 22, about 17 to about 20, about 17 to about 19, about 18 to about 30, about 18 to about 28, about 18 to about 26, about 18 to about 24, about 18 to about 22, about 18 to about 20, about 20 to about 30, about 20 to about 28, about 20 to about 26, about 20 to about 24, about 20 to about 22, about 22 to about 30, about 22 to about 28, about 22 to about 26, about 22 to about 24, about 24 to about 30, about 24 to about 28, about 24 to about 26, about 26 to about 30, about 26 to about 28, or about 28 to about 30 amino acids).

[0135] Exemplary Non-Naturally Occurring A TR-Activating Polypeptides

[0136] In some embodiments, a non-naturally occurring ATR-activating polypeptide comprises a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

[0137] In some embodiments, a nucleic acid sequence encoding the non-naturally occurring ATR-activating polypeptide comprises a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, a nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is mRNA. In some embodiments, the nucleic acid is DNA.

[0138] Compositions and Kits

[0139] Also provided herein are compositions (e.g., pharmaceutical compositions) that include at least one of any of the nucleic acids or any of the non-naturally occurring ATR-activating polypeptides described herein.

[0140] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition is suitable for administration to a human or animal subject, e.g., via a particular route of administration (e.g., parenteral). In some embodiments, an active agent is present in a pharmaceutical composition in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0141] In some embodiments, the pharmaceutical compositions are formulated for different routes of administration (e.g., intravenous, subcutaneous). In some embodiments, the pharmaceutical composition is formulated for local administration. In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for intravenous, subcutaneous, or intramuscular administration. In some embodiments, the pharmaceutical compositions can include a pharmaceutically acceptable excipient (e.g., phosphate buffered saline).

[0142] In some embodiments, the pharmaceutical compositions are formulated with a pharmaceutically acceptable excipient. The pharmaceutical compositions and formulations can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

[0143] In some embodiments, a pharmaceutical composition includes a nucleic acid, where the nucleic acid is formulated as a lipid nanoparticle. As used herein, a “lipid nanoparticle” refers to a nanoparticle composed of lipids, wherein a lipid nanoparticle comprises a solid lipid core matrix that can solubilize lipophilic molecules. Lipid nanoparticle (LNP) synthesis comprises (i) lipid component preparation, which involves the combination of different types of lipids to achieve the desired lipid composition; and (ii) aqueous phase preparation, which includes the substance to be encapsulated by the LNP. In some embodiments, a lipid nanoparticle can include a liposome, solid lipid nanoparticle, nanostructured lipid carrier, or cationic lipid- nucleic acid complex. In some embodiments, lipid nanoparticles can be used as a pharmaceutical drug delivery system. In some embodiments, LNPs can be used as a delivery vehicle for nucleic acids (e.g., mRNA). As used herein, the term “encapsulation” refers to the process of confining an individual mRNA molecule within a nanoparticle. In some embodiments, a lipid nanoparticle can encapsulate a nucleic acid comprising a sequence encoding a non-naturally occurring ATR- activating polypeptide. In some embodiments, the nucleic acid is formulated for unencapsulated delivery.

[0144] In some embodiments, single or multiple administrations of pharmaceutical compositions can be given to a subject in need thereof, depending on the dosage and frequency as required and tolerated by the subject. In some embodiments, the pharmaceutical formulation should provide a sufficient quantity of active agent to effectively treat or ameliorate conditions, diseases or symptoms.

[0145] Also provided herein are kits that include any of the nucleic acids or any of the non-naturally occurring ATR-activating polypeptides described herein. Also provided herein are kits that include any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, the kits can include instructions for performing any of the methods described herein. In some embodiments, the kits can include at least one dose of any of the pharmaceutical compositions described herein.

[0146] Cells

[0147] Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acids described herein that encode any of the non-naturally occurring ATR-activating polypeptides described herein.

[0148] In some embodiments of any of the methods described herein, the cell can be a eukaryotic cell. As used herein, the term “eukaryotic cell” refers to a cell having a distinct, membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human) cells. In some embodiments, a cell can be a stem cell. As used herein, a “stem cell” refers to an undifferentiated or partially differentiated cell that can differentiate into various types of cells. In some embodiments, a stem cell can be a muscle stem cell. In some embodiments, a stem cell can be an autologous stem cell. In some embodiments, a stem cell can be an allogeneic stem cell.

[0149] Methods of introducing nucleic acids and expression vectors into a cell (e.g., an eukaryotic cell) are known in the art. Non-limiting examples of methods that can be used to introduce a nucleic acid into a cell include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalefection, hydrodynamic delivery, magnetofection, viral transduction (e.g., adenoviral and lentiviral transduction), and nanoparticle transfection.

[0150] Methods of Treatment

[0151] Also provided herein are methods of treating a subject in need thereof (e.g., any of the exemplary subjects described herein or known in the art) that include administering to the subject a therapeutically effective amount of any of the nucleic acids, or any of the non-naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein.

[0152] In some embodiments of these methods, the subject has been identified or diagnosed as having an ATR-related disease. In some embodiments, the subject has been previously diagnosed or identified as having an ATR-related disease. Nonlimiting examples of ATR-related disease include: Seckel syndrome, sarcopenia, muscular dystrophy, and familial cutaneous telangiectasia and cancer syndrome. In some embodiments, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of the ATR-related disease in the subject (e.g., as compared to the number, severity, or frequency of the one or more symptoms of the ATR-related disease in the subject prior to treatment).

[0153] Also provided herein are methods of treating an ATR-related disease in a subject that include administering to the subject a stem cell previously contacted with any of the nucleic acids, or any of the non-naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, a method can further include, prior to administering, a step of contacting a stem cell with any of the nucleic acids, or any of the non-naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein, to obtain the stem cell administered to the subject. In some embodiments, the stem cell is a muscle stem cell.

[0154] In some embodiments, a method can further include, prior to the contacting step, a step of obtaining a stem cell that is used in the contacting step, from the subject. In some embodiments, the stem cell is an autologous stem cell. In some embodiments, the stem cell is an allogeneic stem cell.

[0155] Methods of Increasing ATR Activity

[0156] Also described herein are methods of transiently increasing at least one activity of ATR in a cell that include contacting a cell with an effective amount of any of the nucleic acids, or any of the non-naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein. As used herein, the term “transient increase” can refer to an increase in a protein activity (e.g., kinase activity) for a period of time that is less than the period of time protein activity is increased if the protein is constitutively expressed in the same cell type. In some embodiments, a method described herein can transiently increase at least one activity of ATR in a cell for about 1 hour to about 7 days (e.g., about 1 hour to about 6 days, about 1 hour to about 5 days, about 1 hour to about 4 days, about 1 hour to about 3 days, about 1 hour to about 60 hours, about 1 hour to about 48 hours, about 1 hour to 36 hours, about 1 hour to about 24 hours, about 1 hour to about 12 hours, about 1 hour to about 6 hours, about 6 hours to about 7 days, about 6 hours to about 6 days, about 6 hours to about 5 days, about 6 hours to about 4 days, about 6 hours to about 3 days, about 6 hours to about 60 hours, about 6 hours to about 48 hours, about 6 hours to 36 hours, about 6 hours to about 24 hours, about 6 hours to about 12 hours, about 12 hours to about 7 days, about 12 hours to about 6 days, about 12 hours to about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12 hours to about 60 hours, about 12 hours to about 48 hours, about 12 hours to 36 hours, about 12 hours to about 24 hours, about 24 hours to about 7 days, about 24 hours to about 6 days, about 24 hours to about 5 days, about 24 hours to about 4 days, about 24 hours to about 3 days, about 24 hours to about 60 hours, about 24 hours to about 48 hours, about 24 hours to 36 hours, about 36 hours to about 7 days, about 36 hours to about 6 days, about 36 hours to about 5 days, about 36 hours to about 4 days, about 36 hours to about 3 days, about 36 hours to about 60 hours, about 36 hours to about 48 hours, about 48 hours to about 7 days, about 48 hours to about 6 days, about 48 hours to about 5 days, about 48 hours to about 4 days, about 48 hours to about 3 days, about 48 hours to about 60 hours, about 60 hours to about 7 days, about 60 hours to about 6 days, about 60 hours to about 5 days, about 60 hours to about 4 days, about 60 hours to about 3 days, about 3 days to about 7 days, about 3 days to about 6 days, about 3 days to about 5 days, about 3 days to about 4 days, about 4 days to about 7 days, about 4 days to about 6 days, about 4 days to about 5 days, about 5 days to about 7 days, about 5 days to about 6 days, or about 6 days to about 7 days).

[0157] In some embodiments, a cell is contacted with an effective amount of any of the nucleic acids, or any of the non-naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein, wherein the contacting is performed in vitro. In some embodiments, the method can further include, after the contacting step, administering the cell to a subject.

[0158] In some embodiments, a cell is contacted with an effective amount of any of the nucleic acids, or any of the non-naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein, wherein the contacting is performed in a subject (e.g., in vivo). In some embodiments, the subject has an ATR-related disease. In some embodiments, the cell is a stem cell. In some embodiments, the stem cell is a muscle stem cell.

[0159] Methods of Increasing / Maintaining Resilience of a Stem Cell

[0160] Also provided herein are methods of increasing and / or maintaining resilience of a stem cell that include contacting the stem cell with an effective amount of any of the nucleic acids, or any of the non-naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein.

[0161] In some embodiments, a stem cell is contacted with an effective amount of any of the nucleic acids, or any of the non-naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein, wherein the contacting is performed in vitro. In some embodiments, the method can further include, after the contacting step, administering the stem cell to a subject. In some embodiments, prior to the contacting step, the stem cell has been obtained from a subject (e.g., the same subject to which the contacted stem cell is later administered).

[0162] In some embodiments, a stem cell is contacted with an effective amount of any of the nucleic acids, or any of the non-naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein, wherein the contacting is performed in a subject (e.g., in vivo). In some embodiments, the subject has an ATR-related disease (e.g., any of the exemplary ATR-related diseases described herein). In some embodiments, the subject has been previously identified or diagnosed as having an ATR-related disease.

[0163] In some embodiments, the stem cell is a muscle stem cell.

[0164] Also provided herein are methods of preparing a stem cell for administration to a subject in need thereof that include contacting the stem cell with an effective amount of any of the nucleic acids, or any of the non-naturally occurring ATR- activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, the stem cell is a muscle stem cell.

[0165] In some embodiments, the method can further include, prior to the contacting step, a step of obtaining a stem cell that is used in the contacting step, from the subject. In some embodiments, the stem cell is an autologous stem cell.

[0166] In some embodiments, the stem cell is an allogeneic stem cell.

[0167] In some embodiments, the method can further include, either before or after the contacting step, introducing a therapeutic nucleic acid into the stem cell. In some embodiments, the method can further include administering the stem cell to a subject in need thereof, wherein the subject has an ATR-related disease (e.g., any of the exemplary ATR-related diseases described herein).

[0168] In some embodiments, any of the nucleic acids, or any of the non-naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein, is administered two or more times to the subject. In some embodiments, any of the nucleic acids, or any of the non- naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein, is administered locally. In some embodiments, any of the nucleic acids, or any of the non-naturally occurring ATR- activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein, is administered systemically. In some embodiments, the subject is a human subject.

[0169] Methods of Increasing Efficiency of Insertion of Exogenous DNA

[0170] Also provided herein are methods of increasing efficiency of insertion of exogenous DNA into the genome of a mammalian cell that include (a) contacting the mammalian cell with an effective amount of any of the nucleic acids, or any of the non-naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein; and (b) contacting the mammalian cell with a nucleic acid comprising the exogenous DNA.

[0171] In some embodiments, the mammalian cell is contaced with a nucleic acid comprising the exogenous DNA before being contacted with an effective amount of any of the nucleic acids, or any of the non-naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, the mammalian cell is contacted with an effective amount of any of the nucleic acids, or any of the non-naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein before being contaced with a nucleic acid comprising the exogenous DNA. In some embodiments, the mammalian cell is contacted with an effective amount of any of the nucleic acids, or any of the non-naturally occurring ATR-activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein and a nucleic acid comprising the exogenous DNA, at substatially the same time.

[0172] In some embodiments, the mammalian cell is contacted with an effective amount of any of the nucleic acids, or any of the non-naturally occurring ATR- activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein and a nucleic acid comprising the exogenous DNA, wherein the contacting is performed in vitro. In some embodiments, the method can further include administering the mammalian cell to a subject, after the contacting step.

[0173] In some embodiments, the mammalian cell is contacted with an effective amount of any of the nucleic acids, or any of the non-naturally occurring ATR- activating polypeptides, or any of the compositions (e.g., pharmaceutical compositions) described herein and a nucleic acid comprising the exogenous DNA, wherein the contacting is performed in a subject (e.g., in vivo).

[0174] In some embodiments, the mammalian cell is a stem cell. In some embodiments, the stem cell is a muscle stem cell.

[0175] In some embodiments, the method comprises contacting the mammalian cell with nucleic acid comprising the exogenous DNA, wherein the contacting can further include contacting the mammalian cell with a nucleic acid encoding a gene-editing enzyme. As used herein, a “gene-editing enzyme” can refer to an agent that can target and bind to a specific sequence in DNA. In some embodiments, a gene-editing enzyme comprises CRISPR / Cas9 components. As used herein, the term “CRISPR” refers to a technique of sequence-specific genetic manipulation relying on the clustered regularly interspaced short palindromic repeats pathway, which unlike RNA interference regulates gene expression at a transcriptional level. In some embodiments, a gene-editing enzyme can include a CRISPR-associated enzyme or protein that uses CRISPR sequences as a guide to recognize and cleave specific nucleic acid strands that are complementary to the CRISPR sequence. For example, a gene-editing Cas effector can associate with a CRISPR RNA sequence to bind to and alter DNA or RNA target sequences. In some embodiments, a gene-editing enzyme comprises a Cas9 protein, a Casl3b protein, or a Casl3d protein. In some embodiments, a gene-editing enzyme can be a Cas9 endonuclease that makes a double-stranded break in a target DNA sequence. In some embodiments, a geneediting enzyme can be a Casl2a nuclease that also makes a double-stranded break in a target DNA sequence. In some embodiments, a gene-editing enzyme can be a Cas 13 nuclease which targets RNA. In some embodiments, a gene-editing enzyme comprises a Cas9 protein, a Casl3b protein, or a Casl3d protein. In some embodiments, the gene-editing enzyme comprises a nuclease dead Cas9 (dCas9) protein. In some embodiments, the gene-editing enzyme comprises a Cas 13b protein. In some embodiments, the gene-editing enzyme comprises a Cast 3d protein. In some embodiments, the gene-editing enzyme is selected from the group consisting of a Cas9, CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), RNA- targeting CRISPR (Casl3, djCasl3, or hifiCasl3), a zinc-finger nuclease, and a transcription activator-like effector nuclease (TALEN).

[0176] EXAMPLES

[0177] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0178] Example 1: AAD mRNA Preparation and Transfection

[0179] Briefly, DNA constructs were purchased as gBlocks from Integrated DNA Technologies (IDT). Lyophilized DNA was resuspended in TE buffer at 10 ng / uL and amplified from resuspended using a DNA polyermerase with primers (SEQ ID NOs: 12-16) to generate the constructs shown in Figure 7.

[0180] Next, RNA was reverse transcribed from the generated DNA templates using HiScribe™ T7 High Yield RNA Synthesis KiT (NEB #E2040S) and precipitated with LiCl to a final concentration of 2.5 M. Poly(A) tails were added to RNA constructs using E. coli Poly(A) Polymerase (NEB: M0276L).

[0181] The RNA was transfected one hour prior to transfection cell growth media was replaced with Opti-Mem (Gibco 31985062) containing 0.2mg / mL Recombinant B 18R Protein (Stem Cell Technologies 78075). Briefly, Lipofectamine MessengerMax™ was diluted in Opti-Mem media and incubated for lOmins at room temperature. RNA was diluted in Opti-Mem media to 0.02 pg / uL. Lipofectamine mixture and RNA mixture were combined and incubated for 5 minutes. Varying volumes of RNA-lipid mixture were added to cells and incubated for at least 24 hours.

[0182] Example 2: AAD mRNA Hyperactives ATR Signaling and Maintains Quiescence and Increases Stem Cell Resilience

[0183] Figures 4A-4B show that transfection of AAD mRNA, prepared according to Example 1, hyperactivates ATR signaling. Figure 4A shows a representative Western blot and Figure 4B shows quantification of the results showing increased levels of phosphorylated checkpoint kinase-1 (p-Chkl) and Cyclin F after AAD mRNA treatment. p-Chkl and Cyclin F are known targets of ATR signaling. Further, Figure 5 A shows a decrease in the percentage of Edu+ cells and TUNEL+ cells after transfection of AAD mRNA. Assays for Edu+ and TUNEL+ cells are commonly used to determine whether cells proliferation is occurring. The data demonstrate that transfection with AAD mRNA reduces the number of Edu+ and TUNEL+ cells as compared to transfection with GFP mRNA i.e., transfection with AAD mRNA maintains cell quiescence. Figure 5B shows results where the number of nuclei present in MHC+ cells / number of total nuclei per field was determined and expressed as percentage. Transfection with AAD mRNA shows a reduced percentage as compared to transfection with GFP mRNA.

[0184] Figure 6 is a graph showing transfection with either AAD mRNA or GPF mRNA increases the ability of muscle stem cells (MuSCs) to reseed the stem cell niche. Collectively the data demonstrate that transfection of AAD mRNA hyperactives ATR signaling, maintains cell cycle quiescence, and increases stem cell resilience.

[0185] Additionally, K562 lymphoblast cells or human embryonic stem cells (Hl- ESC) were transfected with AAD mRNA, prepared according to Example 1, and prepared for flow cytometry analysis as described below. Flow Cytometry

[0186] Briefly, cells were trypsonized using Accutase (Stem Cell Technologies 07920) for 15 mins at 37°C followed by inactivation. Cells were washed and fixed in 4% formaldehyde for 5 mins at room temperature and washed with PBS. Primary antibodies (Phospho-Chkl (Ser345) (133D3) Rabbit mAb, Cell Signaling Technology 2348S; Phospho-Histone H2A.X (Serl39) (D7T2V) Mouse mAb, Cell Signaling Technology 80312S) were diluted in buffer and incubated overnight at 4 °C. The next day the cells were centrifuged and washed in buffer. Secondary antibodies were diluted in buffer and incubated for 1 hour at room temperature and washed. After the final wash, the cells were resuspended in PBS. Flow cytometry analysis of phospho- Chkl and phospho-H2AX positive cells was conducted on a Agilent Novocyte Quanteon flow cytometry machine and analyzed using Flow Jo.

[0187] Figures 8A-8C shows flow cytometry analysis of phospho-Chkl and phospho-H2AX positive cells in K562 lymphoblast cells that were untreated or treated with endogenous AAD, SV40-NLS_AAD_RPA2, SV40- NLS AAD W107R RPA2, SV40-NLS_AAD, and SV40-NLS_AAD_W107R. Endogenous AAD, SV40-NLS-W107R_RPA2, and SV40-NLS_AAD_W107R are control constructs, as well as, untreated cells. Cells transfected with either SV40- NLS_AAD_RPA2 or SV40-NLS_AAD showed an increase in pCHkl positive and yH2AX positive cells as compared to cells transfected with control constructs.

[0188] Figures 9A-9C shows flow cytometry analysis of phospho-Chkl and phospho-H2AX positive cells in human embryonic stem cells (Hl -ESC) that were untreated, treated with endogenous AAD, SV40-NLS_AAD_RPA2, SV40- NLS_AAD_W107R_RPA2, SV40-NLS_AAD, and SV40-NLS_AAD_W107R. Endogenous AAD, SV40-NLS-W107R_RPA2, and SV40-NLS_AAD_W107R are control constructs, as well as, untreated cells. Cells transfected with either SV40- NLS_ETAA1_AAD_RPA2 or SV4O-NLS_ETAA1_AAD demonstrated an increased dose response as compared to cells transfected with control constructs.

[0189] Example 4: Exogenous DNA Integration Improves Post-AAD mRNA Treatment

[0190] Cells were transfected with TALEN® and exogenous DNA using FuGene® using manufacturer’s instructions with modifications. Briefly, a 3: 1 ratio of FuGene® to DNA ratio was used and 4: 1 : 1 ratio of DNA to Talen arms for 2 pg total DNA (all constructs combined). The mixture was incubated for 15 minutes. RNA-lipid-Opti- Mem media was replaced with Opti-Mem media. FuGene®-TALEN®-DNA mixture was added to cells. For stem cells and for DNA constructs containing selection, after 72 hours, media was replaced with media containing mTESR media (Stem Cell Technologies 85850) and Geneticin (Gibco 10131027 50ug / uL final concentration). Flow cytometry analysis of GFP positive cells was conducted Agilent Novocyte Quanteon flow cytometry machine 24 hours after selection and analyzed using FlowJo. Figures 10A-10B show the constucts containing AAD domains (i.e., SV40- NLS_ETAA1_AAD_RPA2 and SV-4ONLS_ETAA1_AAD) had improved exogenous DNA integration as compared to control constructs including endogenous ATR AAD, SV40-NLS_ETAAl_W107R_RPA2 (negative control), and untreated cells. The data desmonstrate that cells transfected with either SV4O-NLS_ETAA1_AAD_RPA2 or SV4O-NLS_ETAA1_AAD had improved exongenous DNA integration than cells transfected with control constructs. Figure 10C is a control experiment. SEQUENCE APPENDIX

[0191] SEQ ID NO: 1 - SV40-NLS ETAA1 AAD

[0192] MPKKKRRVGGGGSGGGGSGGGGS YETPKRALKMDSLS S SF S SPNDPDGQNDI FWDQNSPLTKQLGKGRKKQIYTTDSDEISHIVNRIAPQDEKPTTNSMLDMWIG ETAIPCTPSVAKGKSRAKISCTKLKTQSQEEELMKLAKQFDKNMEELDVIQEQ NKRNYDFTQMISETEILSNYKDNIQMW

[0193] SEQ ID NO: 2 - SV40-NLS ETAA1 AAD atgccgaaaaaaaaacgccgcgtgtaaggaggcggcggaagtgggggtggcggatccggagggggtggaagctatg aaaccccgaaacgcgcgctgaaaatggatagcctgagcagcagctttagcagcccgaacgatccggatggccagaacg atattttttgggatcagaacagcccgctgaccaaacagctgggcaaaggccgcaaaaaacagatttataccaccgatagcg atgaaattagccatattgtgaaccgcattgcgccgcaggatgaaaaaccgaccaccaacagcatgctggatatgtggattg gcgaaaccgcgattccgtgcaccccgagcgtggcgaaaggcaaaagccgcgcgaaaattagctgcaccaaactgaaaa cccagagccaggaagaagaactgatgaaactggcgaaacagtttgataaaaacatggaagaactggatgtgattcaggaa cagaacaaacgcaactatgattttacccagatgattagcgaaaccgaaattctgagcaactataaagataacattcagatgtg gtaa

[0194] SEQ ID NO: 3 - SV4O-NLS_ETAA1_AAD_RPA2

[0195] MPKKKRRVGGGGSGGGGSGGGGS YETPKRALKMDSLS S SF S SPNDPDGQNDI FWDQNSPLTKQLGKGRKKQIYTTDSDEISHIVNRIAPQDEKPTTNSMLDMWIG ETAIPCTPSVAKGKSRAKISCTKLKTQSQEEELMKLAKQFDKNMEELDVIQEQ NKRNYDFTQMISETEILSNYKDNIQMWSGSETPGTSESATPESIQRKRQEALV RRMAK

[0196] SEQ ID NO: 4 - SV4O-NLS_ETAA1_AAD_RPA2

[0197] Atgccgaaaaaaaaacgccgcgtgtaaggaggcggcggaagtgggggtggcggatccggagggggtggaagctatg aaaccccgaaacgcgcgctgaaaatggatagcctgagcagcagctttagcagcccgaacgatccggatggccagaacg atattttttgggatcagaacagcccgctgaccaaacagctgggcaaaggccgcaaaaaacagatttataccaccgatagcg atgaaattagccatattgtgaaccgcattgcgccgcaggatgaaaaaccgaccaccaacagcatgctggatatgtggattg gcgaaaccgcgattccgtgcaccccgagcgtggcgaaaggcaaaagccgcgcgaaaattagctgcaccaaactgaaaa cccagagccaggaagaagaactgatgaaactggcgaaacagtttgataaaaacatggaagaactggatgtgattcaggaa cagaacaaacgcaactatgattttacccagatgattagcgaaaccgaaattctgagcaactataaagataacattcagatgtg gtccggcagcgaaacccctggaacaagcgagagcgccaccccagagtctattcagcgcaaacgccaggaagcgctggt gcgccgcatggcgaaataa

[0198] SEQ ID NO: 5

[0199] IQRKRQEALVRRMAK

[0200] SEQ ID NO: 6 Linker Sequence

[0201] (GGGGS)x

[0202] SEQ ID NO: 7 Linker Sequence

[0203] (GGSG)X

[0204] SEQ ID NO: 8 Nuclear Localization Signal Sequence

[0205] PKKKRRV

[0206] SEQ ID NO: 9 Nuclear Localization Signal Sequence

[0207] PKKKRKV

[0208] SEQ ID NO: 10 TAT

[0209] YGRKKRRQRRR

[0210] SEQ ID NO: 11 ATR Activating Domain

[0211] GQNDIFWDQNS

[0212] SEQ ID NO: 12 SV40-NLS ETAA 1 _AAD -ALL

[0213] 5'-ATGCCGAAAAAAAAACGCCGCGTGTAA-3’

[0214] SEQ ID NO: 13 - SV40-NLS ETAA 1 AAD RPA REV

[0215] 5'-TTATTTCGCCATGCGGCGCAC-3'

[0216] SEQ ID NO: 14 - SV40-NLS ETAA 1 AAD REV

[0217] 5'- TTACCACATCTGAATGTTATCTTTATAGTTGCTCAGAATTTCGG -3' SEQ ID NO: 15 - Endogenous AAD FWD

[0218] 5'- ATGAGGTATGAAACACCAAA -3'

[0219] SEQ ID NO: 16 - Endogenous AAD REV

[0220] 5'- TTACTGGGTAAAATCATAATTCCT C -3'

[0221] SEQ ID NO: 17 - Linker Sequence

[0222] GGGS

[0223] SEQ ID NO: 18- SV40 NLS

[0224] PKKRKV

[0225] SEQ ID NO: 19 - TAT cell-penetrating peptide

[0226] RKKRRQRRR

[0227] SEQ ID NO: 20 - R8 cell-penetrating peptide

[0228] RRRRRRRR

[0229] SEQ ID NO: 21- DPV3 cell-penetrating peptide

[0230] RI<I<RRRESRI<I<RRRES

[0231] SEQ ID NO: 22- DPV6 cell-penetrating peptide

[0232] GRPRESGI<I<RI<RI<RLI<P

[0233] SEQ ID NO: 23- Penetratin cell-penetrating peptide

[0234] RQH<IWFQNRRMI<WI<I<

[0235] SEQ ID NO: 24- R9-TAT cell-penetrating peptide GRRRRRRRRRPPQ

[0236] SEQ ID NO: 25- pVEC cell-penetrating peptide

[0237] LLIILRRRIRKQAHAHSK SEQ ID NO: 26- ARF (19-31) cell-penetrating peptide

[0238] RVRVFVVHIPRLT

[0239] SEQ ID NO: 27- MPG cell-penetrating peptide GALFLGFLGAAGSTMGAWSQPKKKRKV

[0240] SEQ ID NO: 28- MAP cell-penetrating peptide KLALKLALKALKAALKLA

[0241] SEQ ID NO: 29- Transportan cell-penetrating peptide

[0242] GWTLNSAGYLLGI<INLI<ALAALAI<I<IL

[0243] SEQ ID NO: 30- Bip4 cell-penetrating peptide

[0244] VSALK

[0245] SEQ ID NO: 31- C105Y cell-penetrating peptide

[0246] CSIPPEVKFNPFVYLI

[0247] SEQ ID NO: 32- Melittin cell-penetrating peptide GIGAVLKVLTTGLPALISWIKRKRQQ

[0248] SEQ ID NO: 33- gH625 cell-penetrating peptide

[0249] HGLASTLTRWAHYNALIRAF

[0250] OTHER EMBODIMENTS

[0251] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A nucleic acid comprising a sequence encoding a non-naturally occurring ATR-activating polypeptide, wherein the non-naturally occurring ATR-activating polypeptide comprises:(i) a nuclear localization signal sequence; and(ii) an ataxia telangiectasia and a Rad3-related (ATR)-activating domain.

2. The nucleic acid of claim 1, wherein the non-naturally occurring ATR- activating polypeptide further comprises a linker sequence disposed between the nuclear localization signal sequence and the ATR-activating domain.

3. The nucleic acid of claim 2, wherein the linker sequence comprises (GS)X, (GGS)X, (GGGGS)x (SEQ ID NO: 6), or (GGSG)X(SEQ ID NO: 7), wherein x is an integer from 1 to 50.

4. The nucleic acid of any one of claims 1-3, wherein the non-naturally occurring ATR-activating polypeptide further comprises a replication protein A (RPA)-binding domain.

5. The nucleic acid of claim 4, wherein the RPA-binding domain in the non- naturally occurring ATR-activating polypeptide is positioned C-terminally relative to the ATR-activating domain.

6. The nucleic acid of claim 5, wherein the non-naturally occurring ATR- activating polypeptide further comprises a linker sequence disposed between the ATR-activating domain and the RPA-binding domain.

7. The nucleic acid of claim 6, wherein the linker sequence comprises (GS)X, (GGS)X, (GGGGS)X(SEQ ID NO: 6), or (GGSG)X(SEQ ID NO: 7), wherein x is an integer from 1 to 50.

8. The nucleic acid of any one of claims 4-7, wherein the RPA-binding domain comprises a sequence at least 80% identical to SEQ ID NO: 5.

9. The nucleic acid of claim 8, wherein the RPA-binding domain comprises a sequence at least 90% identical to SEQ ID NO: 5.

10. The nucleic acid of claim 9, wherein the RPA-binding domain comprises a sequence at least 95% identical to SEQ ID NO: 5.

11. The nucleic acid of claim 10, wherein the RPA-binding domain comprises SEQ ID NO: 5.

12. The nucleic acid of any one of claims 1-11, wherein the nuclear localization signal sequence comprises SEQ ID NO: 8 (PKKKRRV) or SEQ ID NO: 9 (PKKKRKV).

13. The nucleic acid of any one of claims 1-12, wherein the ATR-activating domain comprises a sequence that is at least 80% identical to SEQ ID NO: 11.

14. The nucleic acid of claim 13, wherein the ATR-activating domain comprises a sequence that is at least 90% identical to SEQ ID NO: 11.

15. The nucleic acid of claim 14, wherein the ATR-activating domain comprises a sequence that is at least 95% identical to SEQ ID NO: 11.

16. The nucleic acid of claim 15, wherein the ATR-activating domain comprises SEQ ID NO: 11.

17. The nucleic acid of any one of claims 1-16, wherein the non-naturally occurring ATR-activating polypeptide further comprises a cell-penetrating peptide at the N-terminus.

18. The nucleic acid of claim 17, wherein the cell-penetrating peptide comprises SEQ ID NO: 10.

19. The nucleic acid of any one of claims 1-16, wherein the non-naturally occurring ATR-activating polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

20. The nucleic acid of claim 19, wherein the non-naturally occurring ATR- activating polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

21. The nucleic acid of claim 20, wherein the non-naturally occurring ATR- activating polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

22. The nucleic acid of claim 21, wherein the non-naturally occurring ATR- activating polypeptide comprises SEQ ID NO: 1 or SEQ ID NO: 3.

23. The nucleic acid of any one of claims 1-22, wherein the nucleic acid is DNA.

24. The nucleic acid of claim 23, wherein the sequence encoding the non- naturally occurring ATR-activating polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 2 or SEQ ID NO: 4.

25. The nucleic acid of claim 24, wherein the sequence encoding the non- naturally occurring ATR-activating polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 4.

26. The nucleic acid of claim 25, wherein the sequence encoding the non- naturally occurring ATR-activating polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 2 or SEQ ID NO: 4.

27. The nucleic acid of claim 26, wherein the sequence encoding the non- naturally occurring ATR-activating polypeptide comprises SEQ ID NO: 2 or SEQ ID NO: 4.

28. The nucleic acid of any one of claims 1-22, wherein the nucleic acid is RNA.

29. The nucleic acid of claim 28, wherein the RNA is mRNA.

30. A composition comprising the nucleic acid of any one of claims 1-29 and a pharmaceutically acceptable excipient.

31. The composition of claim 30, wherein the composition is a pharmaceutical composition.

32. The composition of claim 31, wherein the pharmaceutical composition is formulated for local administration.

33. The composition of claim 31, wherein the pharmaceutical composition is formulated for systemic administration.

34. The composition of claim 31, wherein the pharmaceutical composition is formulated for intravenous, subcutaneous, or intramuscular administration.

35. The composition of any one of claims 30-34, wherein the nucleic acid is formulated as a lipid nanoparticle.

36. The composition of any one of claims 30-34, wherein the nucleic acid is formulated for unencapsulated delivery.

37. A non-naturally occurring ATR-activating polypeptide comprising:(i) a nuclear localization signal sequence; and(ii) an ataxia telangiectasia and a Rad3-related (ATR)-activating domain.

38. The non-naturally occurring ATR-activating polypeptide of claim 37, wherein the non-naturally occurring ATR-activating polypeptide further comprises a linker sequence disposed between the nuclear localization signal sequence and the ATR-activating domain.

39. The non-naturally occurring ATR-activating polypeptide of claim 38, wherein the linker sequence comprises (GS)X, (GGS)X, (GGGGS)X(SEQ ID NO: 6), or (GGSG)X(SEQ ID NO: 7), wherein x is an integer from 1 to 50.

40. The non-naturally occurring ATR-activating polypeptide of any one of claims 37-39, wherein the non-naturally occurring ATR-activating polypeptide further comprises a replication protein A (RPA)-binding domain.

41. The non-naturally occurring ATR-activating polypeptide of claim 40, wherein the RPA-binding domain in the non-naturally occurring ATR-activating polypeptide is positioned C-terminally relative to the ATR-activating domain.

42. The non-naturally occurring ATR-activating polypeptide of claim 41, wherein the non-naturally occurring ATR-activating polypeptide further comprises a linker sequence disposed between the ATR-activating domain and the RPA-binding domain.

43. The non-naturally occurring ATR-activating polypeptide of claim 42, wherein the linker sequence comprises (GS)X, (GGS)X, (GGGGS)X(SEQ ID NO: 6), or (GGSG)X(SEQ ID NO: 7), wherein x is an integer from 1 to 50.

44. The non-naturally occurring ATR-activating polypeptide of any one of claims 40-43, wherein the RPA-binding domain comprises a sequence at least 80% identical to SEQ ID NO: 5.

45. The non-naturally occurring ATR-activating polypeptide of claim 44, wherein the RPA-binding domain comprises a sequence at least 90% identical to SEQ ID NO: 5.

46. The non-naturally occurring ATR-activating polypeptide of claim 45, wherein the RPA-binding domain comprises a sequence at least 95% identical to SEQ ID NO: 5.

47. The non-naturally occurring ATR-activating polypeptide of claim 46, wherein the RPA-binding domain comprises SEQ ID NO: 5.

48. The non-naturally occurring ATR-activating polypeptide of any one of claims 37-47, wherein the nuclear localization signal sequence comprises SEQ ID NO: 8 (PKKKRRV) or SEQ ID NO: 9 (PKKKRKV).

49. The non-naturally occurring ATR-activating polypeptide of any one of claims 37-48, wherein the ATR-activating domain comprises a sequence that is at least 80% identical to SEQ ID NO: 11.

50. The non-naturally occurring ATR-activating polypeptide of claim 49, wherein the ATR-activating domain comprises a sequence that is at least 90% identical to SEQ ID NO: 11.

51. The non-naturally occurring ATR-activating polypeptide of claim 50, wherein the ATR-activating domain comprises a sequence that is at least 95% identical to SEQ ID NO: 11.

52. The non-naturally occurring ATR-activating polypeptide of claim 51, wherein the ATR-activating domain comprises SEQ ID NO: 11.

53. The non-naturally occurring ATR-activating polypeptide of any one of claims 37-52, wherein the non-naturally occurring ATR-activating polypeptide further comprises a cell-penetrating peptide at the N-terminus.

54. The non-naturally occurring ATR-activating polypeptide of claim 53, wherein the cell-penetrating peptide comprises SEQ ID NO: 10.

55. The non-naturally occurring ATR-activating polypeptide of any one of claims 37-52, wherein the non-naturally occurring ATR-activating polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

56. The non-naturally occurring ATR-activating polypeptide of claim 55, wherein the non-naturally occurring ATR-activating polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

57. The non-naturally occurring ATR-activating polypeptide of claim 56, wherein the non-naturally occurring ATR-activating polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

58. The non-naturally occurring ATR-activating polypeptide of claim 57, wherein the non-naturally occurring ATR-activating polypeptide comprises SEQ ID NO: 1 or SEQ ID NO: 3.

59. A composition comprising the non-naturally occurring ATR-activating polypeptide of any one of claims 37-58 and a pharmaceutically acceptable excipient.

60. The composition of claim 59, wherein the composition is a pharmaceutical composition.

61. The composition of claim 60, wherein the pharmaceutical composition is formulated for local administration.

62. The composition of claim 60, wherein the pharmaceutical composition is formulated for systemic administration.

63. The composition of claim 60, wherein the pharmaceutical composition is formulated for intravenous, subcutaneous, or intramuscular administration.

64. The composition of any one of claims 59-63, wherein the nucleic acid is formulated as a lipid nanoparticle.

65. The composition of any one of claims 59-63, wherein the nucleic acid is formulated for unencapsulated delivery.

66. A method of treating an ATR-related disease in a subject, the method comprising administering to the subject an effective amount of the nucleic acid of any one of claims 1-29, the non-naturally occurring ATR-activating polypeptide of any one of claims 37-58, or the composition of any one of claims 30-36 or 59-65.

67. The method of claim 66, wherein the ATR-related disease is selected from the group consisting of: Seckel syndrome, sarcopenia, muscular dystrophy, and familial cutaneous telangiectasia and cancer syndrome.

68. The method of claim 66 or 67, wherein the subject has been previously diagnosed or identified as having the ATR-related disease.

69. A method of treating an ATR-related disease in a subject comprising administering to the subject a stem cell previously contacted with the nucleic acid of any one of claims 1-29, the non-naturally occurring ATR-activating polypeptide of any one of claims 37-58, or the composition of any one of claims 30-36 or 59-65.

70. The method of claim 69, wherein the method further comprises, prior to administering, a step of contacting a stem cell with the nucleic acid of any one of claims 1-29, the non-naturally occurring ATR-activating polypeptide of any one ofclaims 37-58, or the composition of any one of claims 30-36 or 59-65, to obtain the stem cell administered to the subject.

71. The method of claim 69 or 70, wherein the stem cell is a muscle stem cell.

72. The method of any one of claims 69-71, wherein the method further comprises, prior to the contacting step, a step of obtaining a stem cell that is used in the contacting step, from the subject.

73. The method of claim 72, wherein the stem cell is an autologous stem cell.

74. The method of any one of claims 69-71, wherein the stem cell is an allogeneic stem cell.

75. The method of any one of claims 69-74, wherein the ATR-related disease is selected from the group consisting of: Seckel syndrome, sarcopenia, muscular dystrophy, and familial cutaneous telangiectasia and cancer syndrome.

76. The method of any one of claims 69-75, wherein the subject has been previously diagnosed or identified as having the ATR-related disease.

77. A method of transiently increasing at least one activity of ATR in a cell, the method comprising contacting the cell with an effective amount of the nucleic acid of any one of claims 1-29, the non-naturally occurring ATR activating polypeptide of any one of claims 37-58, or the composition of any one of claims 30- 36 or 59-65.

78. The method of claim 77, wherein the contacting is performed in vitro.

79. The method of claim 78, wherein the method further comprises, after the contacting step, administering the cell to a subject.

80. The method of claim 77, wherein the contacting is performed in a subject.

81. The method of claim 79 or 80, wherein the subject has an ATR-related disease.

82. The method of claim 81, wherein the ATR-related disease is selected from the group consisting of: Seckel syndrome, sarcopenia, muscular dystrophy, and familial cutaneous telangiectasia and cancer syndrome.

83. The method of claim 81 or 82, wherein the subject has been previously identified or diagnosed as having an ATR-related disease.

84. The method of any one of claims 77-83, wherein the cell is a stem cell.

85. The method of claim 84, wherein the stem cell is a muscle stem cell.

86. A method of increasing resilience of a stem cell, the method comprising contacting the stem cell with an effective amount of the nucleic acid of any one of claims 1-29, the non-naturally occurring ATR-activating polypeptide of any one of claims 37-58, or the composition of any one of claims 30-36 or 59-65.

87. A method of maintaining resilience of a stem cell, the method comprising contacting the stem cell with an effective amount of the nucleic acid of any one of claims 1-29, the non-naturally occurring ATR-activating polypeptide of any one of claims 37-58, or the composition of any one of claims 30-36 or 59-65.

88. The method of claim 86 or 87, wherein the contacting is performed in vitro.

89. The method of claim 88, wherein the method further comprises, after the contacting step, administering the stem cell to a subject.

90. The method of claim 86 or 87, wherein the contacting is performed in a subject.

91. The method of claim 89 or 90, wherein the subject has an ATR-related disease.

92. The method of claim 91, wherein the ATR-related disease is selected from the group consisting of: Seckel syndrome, sarcopenia, muscular dystrophy, and familial cutaneous telangiectasia and cancer syndrome.

93. The method of claim 91 or 92, wherein the subject has been previously identified or diagnosed as having an ATR-related disease.

94. The method of any one of claims 86-93, wherein the stem cell is a muscle stem cell.

95. A method of preparing a stem cell for administration to a subject in need thereof, the method comprising contacting the stem cell with an effective amount of the nucleic acid of any one of claims 1-29, the non-naturally occurring ATR- activating polypeptide of any one of claims 37-58, or the composition of any one of claims 30-36 or 59-65.

96. The method of claim 95, wherein the stem cell is a muscle stem cell.

97. The method of claim 95 or 96, wherein the method further comprises, prior to the contacting step, a step of obtaining a stem cell that is used in the contacting step, from the subject.

98. The method of claim 97, wherein the stem cell is an autologous stem cell.

99. The method of claim 95 or 96, wherein the stem cell is an allogeneic stem cell.

100. The method of any one of claims 95-99, wherein the method further comprises, either before or after the contacting step, introducing a therapeutic nucleic acid into the stem cell.

101. The method of any one of claims 95-100, wherein the method further comprises administering the stem cell to a subject in need thereof.

102. The method of claim 101, wherein the subject has an ATR-related disease.

103. The method of claim 102, wherein the ATR-related disease is selected from the group consisting of: Seckel syndrome, sarcopenia, muscular dystrophy, and familial cutaneous telangiectasia and cancer syndrome.

104. The method of claim 102 or 103, wherein the subject has been previously identified or diagnosed as having the ATR-related disease.

105. A method of increasing efficiency of insertion of exogenous DNA into the genome of a mammalian cell, the method comprising:(a) contacting the mammalian cell with an effective amount of the nucleic acid of any one of claims 1-29, the non-naturally occurring ATR-activating polypeptide of any one of claims 37-58, or the composition of any one of claims 30-36 or 59-65; and(b) contacting the mammalian cell with nucleic acid comprising the exogenous DNA.

106. The method of claim 105, wherein step (a) is performed before step (b).

107. The method of claim 105, wherein step (b) is performed before step (a).

108. The method of claim 105, wherein step (a) and step (b) are performed at substantially the same time.

109. The method of any one of claims 105-108, wherein steps (a) and (b) are performed in vitro.

110. The method of claim 109, wherein the method further comprises, after steps (a) and (b), a step of administering the mammalian cell to a subject.

111. The method of any one of claims 105-108, wherein steps (a) and (b) are performed in a subject.

112. The method of any one of claims 105-111, wherein the mammalian cell is a stem cell.

113. The method of claim 112, wherein the stem cell is a muscle stem cell.

114. The method of any one of claims 105-113, wherein step (b) further comprises contacting the mammalian cell with a nucleic acid encoding a gene-editing enzyme.

115. The method of claim 114, wherein the gene-editing enzyme is selected from the group consisting of: a Cas9, a zinc-finger nuclease, and a transcription activator-like effector nuclease (TALEN).

116. The method of any one of claims 66-68, 80, 90, and 111, wherein the nucleic acid, the non-naturally occurring ATR-activating polypeptide, or the composition is administered two or more times to the subject.

117. The method of any one of claims 66-68, 80, 90, 111, and 116, wherein the nucleic acid, the non-naturally occurring ATR-activating polypeptide, or the composition is administered locally.

118. The method of any one of claims 66-68, 80, 90, 111, and 116, wherein the nucleic acid, the non-naturally occurring ATR-activating polypeptide, or the composition is administered systemically.

119. The method of any one of claims 66-76, 79-83, 89-93, 95-104, 110, 111, and 116-118, wherein the subject is a human subject.

120. A kit comprising:(a) a composition of any one of claim 30-36 and 59-65; and(b) instructions for administering the composition to a subject in need thereof.

121. A cell comprising the nucleic acid of any one of claims 1-29, the non- naturally occurring ATR-activating polypeptide of any one of claims 37-58, and / or the composition of any one of claims 30-36 or 59-65.

122. The cell of claim 121, wherein the cell is a stem cell.

123. The cell of claim 122, wherein the stem cell is a muscle stem cell.

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