Gene editing systems, compositions, and methods for treatment of vexas syndrome

Abstract

The present disclosure describes gene editing systems and therapeutics for use in treating VEXAS syndrome. In particular, the disclosure describes lipid nanoparticles that enhance the targeted delivery of gene editing systems and therapeutics to blood cell progenitor cells, enabling treatment of VEXAS syndrome, in vivo or ex vivo.

Description

GENE EDITING SYSTEMS, COMPOSITIONS, AND METHODS FOR TREATMENT OF VEXAS SYNDROMERELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Serial No. 63 / 580,221, filed September 1, 2023, U.S. Provisional Application Serial No. 63 / 592,888, filed October 24, 2023, U.S. Provisional Application Serial No. 63 / 623,233, filed January 20, 2024, and U.S. Provisional Application Serial No. 63 / 557,214, filed February 23, 2024, each of which are incorporated herein by reference in their entireties.TECHNICAL FIELD

[0002] The present disclosure generally relates to the field of treating VEXAS (vacuoles, El enzyme, X-linked, autoinflammatory. somatic) syndrome which is caused by mutations in the UBA1 gene (ubiquitin-like modifier-activating enzyme 1). The disclosure further relates to gene editing systems comprising gene editing compositions for correcting UBA1 mutations in cells which are causative of VEXAS syndrome and which may be administered under in vitro, ex vivo, or in vivo conditions. The disclosure further relates to gene editing compositions comprising delivery vehicles (e.g.. LNPs) formulated with RNA components, including various coding RNAs, including linear and / or circular mRNAs. and / or non-coding RNAs, including guide RNAs or other functional non-coding RNA components, which may be administered in an effective amount for the treatment of VEXAS syndrome.BACKGROUND

[0003] VEXAS syndrome is an adult-onset autoinflammatory disease caused by a somatic mutation in the UBA1 gene (ubiquitin-like modifier activating enzyme 1) which encodes UBA 1 in hematopoietic progenitor cells. The term “VEXAS’’ is derived from an acronym describing the main features of the disease: Vacuoles, El enzyme, X-linkcd, Autoinflammatory, Somatic. As an X-linkcd somatic disorder, it disproportionately impacts biological males, particularly those over the age of 50 years old. VEXAS patients often present with a wide array of inflammatory symptoms that affect connective tissues (e.g., cartilage), skin, joints, blood vessels and the lungs, and has been reported with clinical features that include skin lesions, fever, weight loss, arthritis, chondritis, venous thrombosis, and lymphadenopathy, along with other less common inflammatory conditions.Particular symptoms often include skin rashes, swelling and pain of joints and other cartilaginous structures (ears, nose), shortness of breath, coughing, inflammation of blood vessels, fever, and extreme fatigue. Patients often also present with anemia, low platelet counts and blood clots.

[0004] There are currently no known curative or standardized treatment models for VEXAS. Patients are typically treated by attempting to manage the associated inflammatory symptoms withcorticosteroids and other immunosuppressants, each of which is associated with significant toxicity. There remains a need in the art for methods of treating VEXAS syndrome, especially ones that have a minimal burden on the patient and which are curative. SUMMARY

[0005] Described herein are gene editing compositions, methods, processes, and kits for the treatment of VEXAS. In certain embodiments, the present disclosure contemplates the use of LNP- based gene editing systems and therapeutics comprising the same, for the treatment of VEXAS syndrome. In particular, described herein are compositions, methods, processes, and kits comprising nucleobase editing systems capable of executing one or more edits to the genome of a patient as part of an LNP formulation which may be delivered in vivo. Also contemplated herein are methods of treating VEXAS through ex vivo editing of a patient’s own cells to address the mutation underlying VEXAS syndrome, and then transplanting the modified cells back into the patient.

[0006] In other aspects, the present disclosure provides nucleic acid molecules encoding the gene editing systems and / or components thereof for treating VEXAS syndrome by repairing and / or correcting one more VEXAS-associated mutations in the UBA1 gene.

[0007] In still other aspects, the present disclosure provides gene editing systems for treating VEXAS syndrome by repairing and / or correcting one more VEXAS-associated mutations in the UBA1 gene wherein the gene editing system comprises a programmable nuclease (e.g., an RNA- guided nuclease, such as CRISPR-Cas Type II or Type V nuclease) and a guide RNA comprising a spacer sequence which is complementary to a portion of the UBA1 gene at a target site, and optionally one or more additional editing functionalities, such as, but not limited to a reverse transcriptase, a deaminase, a nuclease, a recombinase, or an invertase.

[0008] In yet other aspects, the present disclosure provides a prime editing system (or “reverse transcriptase based editing system”) for treating VEXAS syndrome by repairing and / or correcting one more VEXAS-associated mutations in the UBA1 gene wherein the gene editing system comprises a programmable nuclease (e.g., an RNA-guided nuclease, such as CRISPR-Cas Type II or Type V nuclease, and preferably a nickase which cuts only one of the two strands of DNA at any given target site) and a prime editing guide RNA (“pegRNA”) comprising a spacer sequence which is complementary to a portion of the UBA1 gene at a target site, a reverse transcriptase template (“RTT)” and a primer binding site (“PBS”), wherein the nuclease programmable nuclease complexes with the pegRNA and localizes to a target site in the UBA1 gene and nicks a strand to create an available 3’ end and then the reverse transcriptase synthesizes a new single strand DNA strand from the 3’end of the nick which is templated against the RTT and contains the repaired sequence. The newly synthesized strand of DNA or “DNA flap” comprises the regions of homology with the endogenous strand immediately downstream of the nick. Through the action of DNA repair processes in the cell, the DNA flap replaces the endogenous strand downstream of the nick, thereby installingthe repaired sequence on the nicked strand. Following further DNA repair and replication processes, the edited strand is incorporated into both strands thereby permanently installing the edit into the target site, and thereby correcting the UBA1 gene by repairing the targeted mutation.

[0009] In certain embodiments, the VEXAS-associated mutations correspond to the codon associated with Met-41 of the wildtype UBA1 protein of SEQ ID NO: 2. In other embodiments, the VEXAS- associated mutations are in the codon associated with His-55 in the wildtype UBA1 protein of SEQ ID NO: 2. In other embodiments, the VEXAS-associated mutations are in the codon associated with Ser-56 in the wildtype UBA1 protein of SEQ ID NO: 2. In other embodiments, the VEXAS- associated mutations are in the codon associated with Gly-477 in the wildtype UBA1 protein of SEQ ID NO: 2. In other embodiments, the VEXAS-associated mutations are in the codon associated with Ala-478 in the wildtype UBA1 protein of SEQ ID NO: 2. In other embodiments, the VEXAS- associated mutations are in the codon associated with Asp-506 in the wildtype UBA1 protein of SEQ ID NO: 2. In other embodiments, the VEXAS-associated mutations are in the codon associated with Ser-621 in the wildtype UBA1 protein of SEQ ID NO: 2. In other embodiments, the VEXAS- associated mutations is any mutation reported in the UBA1 gene that is described after the date of this filing and which is correctable by the gene editing systems disclosed herein.

[0010] In certain embodiments, the VEXAS-associated mutations correspond to the codon at nucleotide residues 121, 122, and / or 123 in SEQ ID NO: 1 which correspond to the codon of Met-41 of SEQ ID NO: 2. In one embodiment, the VEXAS-associated mutation is located a position 121 of SEQ ID NO: 1, or position 122 of SEQ ID NO: 1, or position 123 of SEQ ID NO: 1. In particular embodiments, the mutation is a T to C mutation at nucleotide residue 122 of codon 121-ATG-123 of SEQ ID NO: 1 corresponding to Met-41 of SEQ ID NO: 2, which converts the Met-41 to a Thr, i.e., a M41T mutation (converting the codon from ATG to TTG). In particular other embodiments, the mutation is a A to G mutation at nucleotide residue 121 of codon 121-ATG-123 of SEQ ID NO: 1 corresponding to Met-41 of SEQ ID NO: 2, which converts the Met-41 to a Val, i.e., a M41V mutation (converting the codon from ATG to GTG). In still other embodiments, the mutation is a A to C mutation at nucleotide residue 121 of codon 121-ATG-123 of SEQ ID NO: 1 corresponding to Met-41 of SEQ ID NO: 2, which converts the Met-41 to a Leu, i.e., a M41L mutation (converting the codon from ATG to CTG).

[0011] The particular UBA1 gene that is being targeted for editing may be a wildtype sequence, i.e., comprising no mutations. The particular UBA1 gene that is being targeted for editing may be a comprise at least one VEXAS-associated mutation. The particular UBA1 gene that is being targeted for editing may be a comprise at least two VEXAS-associated mutations. The particular UBA1 gene that is being targeted for editing may be a comprise at least three VEXAS-associated mutations. The particular UBA1 gene that is being targeted for editing may be a comprise at least four VEXAS- associated mutations. The particular UBA1 gene that is being targeted for editing may be a compriseat least five VEXAS-associated mutations. The particular UBA1 gene that is being targeted for editing may be a comprise at least six VEXAS-associated mutations. The particular UBA1 gene that is being targeted for editing may be a comprise at least seven VEXAS-associated mutations. The particular UBA1 gene that is being targeted for editing may be a comprise at least eight VEXAS- associated mutations. The particular UBA1 gene that is being targeted for editing may be a comprise more than eight VEXAS-associated mutations. These mutations may include result in the substitution of any one or more of wildtype M41, H55, S56, G477, A478, D506, D506, or S621 with another amino acid. The amino acid substitutions resulting from specific mutations in the underlying nucleotide sequence codons may be another amino acid with similar properties (e.g., a polar amino acid substituted for a polar amino acid) or with dissimilar properties (e.g., a nonpolar amino acid substituted for a polar amino acid). In some embodiments, the mutations correctable by the editing systems described herein may include mutations that result in M41T, M41V, M41L, S56F, G477A, A478S, D506G, D506N, or S621C substitutions.

[0012] The UBA1 sequences contemplated herein that may be edited by the methods and compositions described herein may be UBA1 comprising SEQ ID NO: 1, or any nucleotide sequence having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 99% sequence identity with SEQ ID NO: 1, and may contain one or more VEXAS- associated mutations, including include one or more of M41T, M41V, M41L, S56F, G477A, A478S, D506G, D506N, or S621C mutations. The UBA1 gene which may be edited may be in vivo, i.e., wherein the editing system is delivered to a patient and the editing occurs within the body of the patient. The UBA1 gene which may be edited may be in a cell ex vivo, i.e., wherein the editing system is delivered to a cell that is first isolated from a patient, edited, and then returned to the body of the patient.

[0013] The UBA1 sequences contemplated herein that may be edited by the methods and compositions described herein may be UBA1 comprising SEQ ID NO: 1, or any nucleotide sequence having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 99% sequence identity with SEQ ID NO: 1, and may contain one or more VEXAS- associated mutations, including include one or more of M41T, M41V, M41L, S56F, G477A, A478S, D506G, D506N, or S621C mutations.

[0014] In various embodiments, any previously reported mutation in the UBA1 gene or any VEXAS- associated mutations in UBA1 not yet reported may be corrected by the gene editing systems described herein. Previously reported mutations in UBA1 correspond with M41T, M41V, and M41L substitutions in the UBA1 protein and may be corrected by the gene editing systems described herein. In addition, previously reported mutations in UBA1 gene correspond with S56F, G477A, A478S, D506G, D506N, S621C substitutions in the UBA1 protein may be corrected by the gene editing systems described herein.

[0015] In other embodiments, any previously reported mutation in the UBA1 gene or any VEXAS- associated mutations in UBA1 not yet reported may be corrected by the prime editing systems described herein. Previously reported mutations in UBA1 correspond with M41T, M41V, and M41L substitutions in the UBA1 protein and may be corrected by the prime editing systems described herein. In addition, previously reported mutations in UBA1 gene correspond with S56F, G477A, A478S, D506G, D506N, S621C substitutions in the UBA1 protein may be corrected by the prime editing systems described herein.

[0016] In still other aspects, the disclosure provides guide RNA molecules for use in the gene editing systems described herein, wherein the guide RNA molecule is designed in accordance with the particular nucleic acid programmable nuclease that is being implemented in the gene editing system. For example, where the gene editing system includes a Type II CRISPR nuclease (e.g., Cas9), the gene editing system may comprise a guide RNA that is capable of complexing with said Type II nuclease and directing it to a target site of interest, e.g., a UBA1 mutant gene. In another example, where the gene editing system includes a Type V CRISPR nuclease (e.g., Cas12a), the gene editing system may comprise a guide RNA that is capable of complexing with said Type V nuclease and directing it to a target site of interest, e.g., a UBA1 mutant gene. In yet another example, where the gene editing system includes a TnpB nuclease, the gene editing system may comprise a guide RNA that is capable of complexing with said TnpB nuclease and directing it to a target site of interest, e.g., a UBA1 mutant gene. In still another example, where the gene editing system comprises a prime editor (which comprises a Cas9 nickase and reverse transcriptase in some embodiments), the gene editing system may comprise a prime editing guide RNA (“pegRNA”) that is capable of complexing with said prime editor (and specifically, with the Cas9 nickase component) and directing it to a target site of interest, e.g., a UBA1 mutant gene.

[0017] In prime editing embodiments, such as those described herein and exemplified in Example 9, the prime editing system may be capable of correcting a L41M mutation (i.e., reverting a Leu mutation back to a Met). In some embodiments, a prime editing system for restoring a L41M mutation may comprise as a pegRNA any one of the pegRNAs disclosed in Appendix A, which include SEQ ID NOs: 3-659. Thus, a prime editing system for correcting an L41M mutations in UBA1 may comprising a nucleic acid programmable nuclease nickase (e.g., Cas9 nickase), a reverse transcriptase, and a pegRNA selected from the group consisting of SEQ ID NOs: 3-659.

[0018] In prime editing embodiments, such as those described herein and exemplified in Example 9, the prime editing system may be capable of correcting a T41M mutation (i.e., reverting a Thr mutation back to a Met). In some embodiments, a prime editing system for restoring a T41M mutation may comprise as a pegRNA any one of the pegRNAs disclosed in Appendix B, which include SEQ ID NOs: 660-1319. Thus, a prime editing system for correcting a T41M mutations inUBA1 may comprising a nucleic acid programmable nuclease nickase (e.g., Cas9 nickase), a reverse transcriptase, and a pegRNA selected from the group consisting of SEQ ID NOs: 660-1319.

[0019] In other prime editing embodiments, such as those described herein and exemplified in Example 9, the prime editing system may be capable of correcting a V41M mutation (i.e., reverting a Val mutation back to a Met). In some embodiments, a prime editing system for restoring a V41M mutation may comprise as a pegRNA any one of the pegRNAs disclosed in Appendix C, which include SEQ ID NOs: 1320-1976. Thus, a prime editing system for correcting a V41M mutations in UBA1 may comprising a nucleic acid programmable nuclease nickase (e.g., Cas9 nickase), a reverse transcriptase, and a pegRNA selected from the group consisting of SEQ ID NOs: 1320-1976.

[0020] Other aspects relate to methods of correcting a L41M mutation in UBA1 using a gene editing system that comprises a nucleic acid programmable nuclease, an appropriate guide RNA having a space sequence the comprises any one of the spacer sequences (or a portion thereof) any one of the pegRNAs provided in Appendices A, B, or C, and optionally one or more addition functionalities, such as a reverse transcriptase, a deaminase, a recombinase, an invertase, or a nuclease. Such gene editing systems may include base editor systems or prime editor systems.

[0021] Still other aspects relate to methods of correcting a T41M mutation in UBA1 using a gene editing system that comprises a nucleic acid programmable nuclease, an appropriate guide RNA having a space sequence the comprises any one of the spacer sequences (or a portion thereof) any one of the pegRNAs provided in Appendices A, B, or C, and optionally one or more addition functionalities, such as a reverse transcriptase, a deaminase, a recombinase, an invertase, or a nuclease. Such gene editing systems may include base editor systems or prime editor systems.

[0022] Yet other aspects relate to methods of correcting a V41M mutation in UBA1 using a gene editing system that comprises a nucleic acid programmable nuclease, an appropriate guide RNA having a space sequence the comprises any one of the spacer sequences (or a portion thereof) any one of the pegRNAs provided in Appendices A, B, or C, and optionally one or more addition functionalities, such as a reverse transcriptase, a deaminase, a recombinase, an invertase, or a nuclease. Such gene editing systems may include base editor systems or prime editor systems.

[0023] Other aspects relate to methods of correcting a L41M mutation in UBA1 using a prime editing system that comprises a nucleic acid programmable nuclease (optionally a nickase), a reverse transcriptase, and any one of the pegRNA provided in Appendix A, and optionally one or more addition functionalities, such as a reverse transcriptase, a deaminase, a recombinase, an invertase, or a nuclease.

[0024] Still other aspects relate to methods of correcting a T41M mutation in UBA1 using a prime editing system that comprises a nucleic acid programmable nuclease (optionally a nickase), a reverse transcriptase, and any one of the pegRNA provided in Appendix B, and optionally one or moreaddition functionalities, such as a reverse transcriptase, a deaminase, a recombinase, an invertase, or a nuclease.

[0025] Yet other aspects relate to methods of correcting a V41M mutation in UBA1 using a prime editing system that comprises a nucleic acid programmable nuclease (optionally a nickase), a reverse transcriptase, and any one of the pegRNA provided in Appendix C, and optionally one or more addition functionalities, such as a reverse transcriptase, a deaminase, a recombinase, an invertase, or a nuclease.

[0026] The herein disclosed gene editing systems and methods may be conducted and / or administered such that they operate in vivo in certain embodiments. In other embodiments, the herein disclosed gene editing systems and methods may be ex vivo.

[0027] Other aspects relate to methods of correcting a L41M mutation in UBA1 ex vivo using a prime editing system that comprises a nucleic acid programmable nuclease (optionally a nickase), a reverse transcriptase, and any one of the pegRNA provided in Appendix A, and optionally one or more addition functionalities, such as a reverse transcriptase, a deaminase, a recombinase, an invertase, or a nuclease. As a first step, cells in which editing is desired can be isolated from a patient and then returned to the body after editing has been conducted.

[0028] Still other aspects relate to methods of correcting a T41M mutation in UBA1 ex vivo using a prime editing system that comprises a nucleic acid programmable nuclease (optionally a nickase), a reverse transcriptase, and any one of the pegRNA provided in Appendix B, and optionally one or more addition functionalities, such as a reverse transcriptase, a deaminase, a recombinase, an invertase, or a nuclease. As a first step, cells in which editing is desired can be isolated from a patient and then returned to the body after editing has been conducted.

[0029] Yet other aspects relate to methods of correcting a V41M mutation in UBA1 ex vivo using a prime editing system that comprises a nucleic acid programmable nuclease (optionally a nickase), a reverse transcriptase, and any one of the pegRNA provided in Appendix C, and optionally one or more addition functionalities, such as a reverse transcriptase, a deaminase, a recombinase, an invertase, or a nuclease. As a first step, cells in which editing is desired can be isolated from a patient and then returned to the body after editing has been conducted.

[0030] In a further aspect, the disclosure provides nucleic acid molecules encoding the described genome editing systems and said components thereof, as well as polypeptides making up the components of said genome editing systems. In yet another aspect, the disclosure provides vectors for transferring and / or expressing said genome editing systems, e.g., under in vitro, ex vivo, and in vivo conditions. In still another aspect, the disclosure provides cell-delivery compositions and methods, including compositions for passive and / or active transport to cells (e.g., plasmids), delivery by virus- based recombinant vectors (e.g., AAV and / or lentivirus vectors), delivery by non-virus-based systems (e.g., liposomes and LNPs), and delivery by virus-like particles.

[0031] Depending on the delivery system employed, the genome editing systems described herein may be delivered in the form of DNA (e.g., plasmids or DNA-based virus vectors), RNA (e.g., ncRNA and mRNA delivered by LNPs), a mixture of DNA and RNA, protein (e.g., virus-like particles), and ribonucleoprotein (RNP) complexes. Any suitable combinations of approaches for delivering the components of the herein disclosed genome editing systems may be employed. In one embodiment, each of the components of the genome editing systems disclosed herein is delivered by an all-RNA system, e.g., the delivery of one or more RNA molecules (e.g., mRNA and / or ncRNA) by one or more LNPs, wherein the one or more RNA molecules form the ncRNA and guide RNA (as needed) and / or are translated into the polypeptide components (e.g., the RT and a programmable nuclease). In yet another aspect, the disclosure provides methods for genome editing by introducing a genome editing system described herein into a cell (e.g., under in vitro, in vivo, or ex vivo conditions) comprising a target edit site (e.g., a mutant UBA1 gene), thereby resulting in an edit at the target site (i.e., and edited UBA1 gene). In other aspects, the disclosure provides formulations comprising any of the aforementioned components for delivery to cells and / or tissues, including in vitro, in vivo, and ex vivo delivery, recombinant cells and / or tissues modified by the recombinant retron-based genome modification systems and methods described herein, and methods of modifying cells by conducting genome editing using the herein disclosed genome modification systems. The disclosure also provides methods of making the recombinant genome modification systems, vectors, compositions and formulations described herein, as well as to pharmaceutical compositions and kits for modifying cells under in vitro, in vivo, and ex vivo conditions that comprise the herein disclosed genome editing and / or modification systems. Still further, the disclosure provides methods of treating VEXAS syndrome but conducting genome editing under ex vivo or in vivo conditions to correct one or more UBA1 mutations.

[0032] The following numbered paragraphs further are contemplated by the present disclosure: Paragraph 1. A gene editing system for editing a UBA1 gene, comprising: a) a nucleic acid programmable nuclease or a polynucleotide encoding the same; b) optionally an additional editing functionality; and c) at least one guide RNA comprising a spacer that targets the UBA1 gene, wherein the spacer is selected from a spacer from any one of the sequences from Appendix A, Appendix B, or Appendix C. Paragraph 2. The gene editing system of Paragraph 1, wherein the nucleic acid programmable nuclease is a CRISPR Type II nuclease, a CRISPR Type V, or a TnpB nuclease. Paragraph 3. The gene editing system of Paragraphs 1 or 2, wherein the nucleic acid programmable nuclease is a nickase.Paragraph 4. The gene editing system of any one of the preceding Paragraphs, wherein the additional editing functionality is reverse transcriptase, a recombinase, or a deaminase. Paragraph 5. The gene editing system of any one of the preceding Paragraphs, wherein the UBA1 gene comprises one or more mutations. Paragraph 6. The gene editing system of Paragraph 5, wherein the one or more mutations in the UBA1 gene results in a M41T, M41V, M41L, S56F, G477A, A478S, D506G, D506N, or S621C substitution. Paragraph 7. A prime editing system for editing a UBA1 gene, comprising: a) a nucleic acid programmable nuclease or a polynucleotide encoding the same; b) a reverse transcriptase; and c) at least one pegRNA that targets the UBA1 gene, wherein the pegRNA is selected from any one of the sequences from Appendix A, Appendix B, or Appendix C, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of the sequences from Appendix A, Appendix B, or Appendix C. Paragraph 8. The prime editing system of Paragraph 7, wherein the nucleic acid programmable nuclease is a CRISPR Type II nuclease, a CRISPR Type V, or a TnpB nuclease. Paragraph 9. The prime editing system of Paragraphs 7 or 8, wherein the nucleic acid programmable nuclease is a nickase. Paragraph 10. The prime editing system of any one of Paragraphs 7-9, wherein the reverse transcriptase (RT) is a retron RT or a viral RT. Paragraph 11. The prime editing system of any one of Paragraphs 7-9, wherein the viral RT is an MMLV RT. Paragraph 12. The prime editing system of any one of the preceding Paragraphs, wherein the UBA1 gene comprises one or more mutations.Paragraph 13. The prime editing system of Paragraph 12, wherein the one or more mutations in the UBA1 gene results in a M41T, M41V, M41L, S56F, G477A, A478S, D506G, D506N, or S621C substitution. Paragraph 14. A pharmaceutical composition comprising: a) at least one lipid nanoparticle (LNP) comprising at least one ionizable lipid selected from those listed in Tables (I), (II), (III), (IV) or (V); and b) the gene editing system of Paragraph 1. Paragraph 15. A pharmaceutical composition comprising: a) at least one lipid nanoparticle (LNP) comprising at least one ionizable lipid selected from those listed in Tables (I), (II), (III), (IV) or (V); and b) the prime editing system of Paragraph 7. Paragraph 16. The pharmaceutical composition of any one Paragraphs 14-15, wherein the at least one lipid nanoparticle further comprises: i) at least one structural lipid; ii) at least one phospholipid; and iii) at least one PEGylated lipid. Paragraph 17. The pharmaceutical composition of any one of Paragraphs 14-16, wherein the at least one structural lipid is selected from cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta-sitosterol-acetate and any combinations thereof. Paragraph 18. The pharmaceutical composition of any one of Paragraphs 14-17, wherein the at least one phospholipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC), 1,2-di-O- octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuc cinoyl- sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3-((((R)-2-(oleoyloxy)-3- (stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-α-phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Dielaidoyl-sn-phosphatidylethanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphate (18:1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate (18:1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3- phospho-(1’-myo-inositol) (DOPI; 18:1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2-dilinoleoyl-sn-glycero-3-phospho-L-serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- L-serine (16:0-18:1 PS; POPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18:1 PS), 1- stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycero-3- phospho-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin. Paragraph 19. The pharmaceutical composition of any one of Paragraphs 14-18, wherein the at least one PEGylated lipid is selected from (R)-2,3-bis(octadecyloxy)propyl-1- (methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG- PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG- DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-C11, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click C10, N(Carbonyl- methoxypolyethylenglycol-2000)-l,2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG- PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2-distearoyl-sn-glycero-3- phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE-PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE- PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol-polyethyleneglycol, Cl8PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R)-2,3- bis(octadecyloxy)propyl-1-(methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)-C-DOMG, PEG-C-DMA, and DSPE-PEG-X. Paragraph 20. The pharmaceutical composition of any one of Paragraphs 14-19, wherein the LNP further comprises at least one additional lipid component selected from 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3 PC), Acylcarnosine (AC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), N-oleoyl- sphingomyelin (SPM) (C18:l), N-lignoceryl SPM (C24:0), N-nervonoylshphingomyelin (C24:l), Cardiolipin (CL), l,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8-9PC), dicetyl phosphate (DCP), dihexadecyl phosphate (DCP1), 1,2-Dipalmitoylglycerol-3-hemisuccinate (DGSucc), short-chain bis-n-heptadecanoyl phosphatidylcholine (DHPC), dihexadecoyl- phosphoethanolamine (DHPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), l,2-dilauroyl- sn-glycero-3-PE (DLPE), dimyristoyl glycerol hemisuccinate (DMGS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleyloxybenzylalcohol (DOBA), 1,2-dioleoylglyceryl-3- hemisuccinate (DOGHEMS), N-[2-(2-{2-[2-(2,3-Bis-octadec-9-enyloxy-propoxy)-ethoxy]-ethoxy}- ethoxy)-ethyl]-3-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-ylsulfanyl)-propionamide (DOGP4αMan), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell- fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), histaminedistearoylglycerol (HDSG), 1,2-Dipalmitoylglycerol- hemisuccinate-Nα-Histidinyl-Hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10-12- pentacosadiynamide (h-Pegi-PCDA), 2-[l-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH), hydrogenatedsoybeanphosphatidylcholine (HSPC), 1,2-Dipalmitoylglycerol-O-α-histidinyl-Nα-hemisuccinate (IsohistsuccDG), mannosialized dipalmitoylphosphatidylethanolamine (ManDOG), l,2- Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (MCC-PE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1-myristoyl-2- hydroxy-sn-glycero-phosphocholine (MHPC), a thiol-reactive maleimide headgroup lipid e.g.1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)but-yramid (MPB-PE), Nervonic Acid (NA), sodium cholate (NaChol), l,2-dioleoyl-sn-glycero-3-[phosphoethanolamine-N- dodecanoyl (NC12-DOPE), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), phosphatidylethanolamine lipid (PE), PE lipid conjugated with polyethylene glycol(PEG) (e.g., polyethylene glycol-distearoylphosphatidylethanolamine lipid (PEG-PE)), phosphatidylglycerol (PG), partially hydrogenated soy phosphatidylchloline (PHSPC), phosphatidylinositol lipid (PI), phosphotidylinositol-4-phosphate (PIP), palmitoyloleoylphosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), phosphatidylserine (PS), lissamine rhodamineB- phosphatidylethanolamine lipid (Rh-PE), purified soy-derived mixture of phospholipids (SIOO), phosphatidylcholine (SM), 18-1-trans-PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), soybean phosphatidylcholine (SPC), sphingomyelins (SPM), alpha,alpha-trehalose-6,6'-dibehenate (TDB), l,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE), ((23S,5R)-3- (bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran- 2-yl)methylmethylphosphate, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, and dioleylphosphatidylethanolamine. Paragraph 21. The pharmaceutical composition of any one of Paragraphs 14-20, wherein the LNP further comprises one or more targeting moieties. Paragraph 22. A method of treating VEXAS syndrome in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition of any one of Paragraphs 14- 21. Paragraph 23. The pharmaceutical composition of any one of Paragraphs 14-21 for use as a medicament in the treatment of VEXAS syndrome.Paragraph 24. Use of a pharmaceutical composition of any one of Paragraphs 14-21 for the manufacture of a medicament for delivery of a gene editing system capable of treating VEXAS syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG.1 is a schematic depicting the UBA1 wildtype gene as represented by the GenBank Accession No. NP_003325.2 and having SEQ ID NO: 2. The schematic depicts the relative position of reported UBA1 mutations. For example, reference to “M41T” refers to a mutation in the corresponding UBA1 gene that converts the wildtype methionine to a threonine. This nomenclature applies to the other mutations shown as well. This schematic does not preclude that other VEXAS- associated mutations will be identified in the future and that the herein disclosed editing systems are capable of editing any other mutations discovered in the UBA1 gene to be associated with VEXAS- syndrome.

[0034] FIG.2 is a schematic depicting the concept that a UBA1 mutation may be corrected by a gene editing system disclosed herein to restore or repair the UBA1 gene to a wildtype sequence.

[0035] FIG.3 is a schematic depicting exemplary embodiments disclosed herein (and exemplified in Example 6) of using a prime editing system with an appropriate pegRNA (e.g., those disclosed in Appendices A, B, or C) to correct mutations in the UBA1 gene, such as those corresponding to a M41T mutation, M41V mutation, or a M41L mutation.

[0036] FIG.4 is a schematic depicting the generalized structure of a pegRNA in (A), which includes from the 5’ to 3’ direction a spacer (which is complementary to one of the strands of a target sequence), scaffold (which associates and / or complexes with a nucleic acid programmable nuclease; GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG TGGCACCGAGTCGGTGC; SEQ ID NO: 2076), RTT (the reverse transcriptase template which encodes the corrected sequence), PBS (the primer binding site which associates by duplex formation with the 3’ end of the nicked strand in the target sequence and provides a starting point for reverse transcriptase synthesis), and an optional linker for joining an optional stabilizing RNA motif, such as the one in each of the pegRNA sequences of Appendices A, B, and C (with motif having the sequence of CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA; SEQ ID NO: 2075). In the figure, part (B) and (C) describe the annotation of each of the sequences of Appendices A, B, and C as represented by exemplary SEQ ID NO: 3.

[0037] FIG.5 is a schematic depicting the experimentation used to screen and identify effective RTBE RNAs for using in prime editing of the UAB1 gene. For purposes herein, an “RTBE RNA” is equivalent to a pegRNA.

[0038] FIG.6A and 6B are bar graphs demonstrating that the UBA1 locus is accessible for prime editing in primary human HSPCs. Ten pegRNAs were tested for prime editing activity atthe UBA1 locus without nicking gRNAs. It is noted that 4-5 pegRNAs were capable of installing pathogenic UBA1 mutations associated with VEXAS in primary bone marrow derived human HSPCs with minimal indels.

[0039] FIG.7A and 7B are bar graphs demonstrating that addition of nicking gRNA increases editing efficiencies at UBA1 in human primary hematopoetic stem cells (HSPCs). HSPCs were co- electroporated with PEMax, pegRNA469 and one of four nicking gRNAs. Addition of nicking gRNAs increased exact intended edit frequencies between 7-30 fold. Indel frequencies were greater in samples treated with PE3 ngRNAs and at background levels for samples treated with PE3b ngRNAs (s-seed, ns-non seed).

[0040] FIG.8A and 8B are bar graphs demonstrating that addition of nicking gRNA increases editing efficiencies at UBA1 in human primary hematopoietic stem cells. HSPCs were co- electroporated with PEMax, epegRNA474 or 477 and one of four nicking gRNAs. Addition of nicking gRNAs increased exact intended edit frequencies for epegRNA477 up to eight-fold but did not increase editing efficiencies for epegRNA474. Indel frequencies were greater in samples treated with PE3 ngRNAs and at background levels for samples treated with PE3b ngRNAs (s-seed, ns-non seed).

[0041] FIG.9 is a set of schematics of UBA1 epegRNA spacers, RTTs, and PBSs.

[0042] FIG.10 is a set of schematics illustrating strategies for construction of a VEXAS model cell line. Strategy 1 or 2: THP1 or U937 cells will be nucleofected with (Strategy 1) prime editing mRNA, pegRNA and a ten-fold lower dose of GFP mRNA or (Strategy 2) CRISPR / Cas9 mRNA + ssODN and a ten-fold lower dose of GFP mRNA. Afterwards, a mixed population of modified and WT cells will be present. Most edited cells should contain EGFP, sorting on GFP fluorescence lead to isolation of mostly UBA1 mutant cells which can be single cell cloned and grown for further characterization.

[0043] FIG.11 is a set of schematics of the knock-in vector approach to Rosa26 “safe-harbor” locus. See Example 10.

[0044] FIG.12 is a set of schematics illustrating the knock-in vector and targeting approach at the UBA1 locus. Modified from Gou et al, Cell, 2017. See Example 10.

[0045] FIG.13 is a graph showing the % edits for installing UBA1 & HBB variants in macrophage cell lines, as outlined in Example 8.

[0046] FIG.14 is a set of graphs showing the % edits and indel% for installing UBA1 M41L and V in 293T cells, as outlined in Example 8.

[0047] FIGs.15A, 15B and 15C are images of isolated 293T cells after UBA1 editing illustrating various cellular morphologies of the edited cells. FIG.15A shows larger cell colonies, FIG.15B shows “droplets” in some clones, FIG.15C shows sparser colonies.

[0048] FIG.16 is a graph showing the percent of clones with the “droplet” phenotype, correlating with the percentage of UBA1 editing in bulk populations of 293T cells.

[0049] FIGs.17 and 18 are graphs showing 293T UBA1 M41L clones isolated from bulk edited populations measured by % edits (FIG.17) and % indels (FIG.18). Two of the clones isolated from the bulk edited cells contained detectable indels at UBA1 (FIG.18).

[0050] FIG.19 is a series of images of various harvested cell lines showing the “droplet” phenotype. Percentages above each image correspond to the amount of UBA1 M41L reads present in NGS data collected from each clone colony.

[0051] FIG.20 is a set of graphs showing installation of UBA1 M41 mutations in a mobilized CD34+ human HSPC. The data shows that the edited cells are robust with high product purity. The left-hand graph shows exact edit % and the right hand graph shows % indels.

[0052] FIG.21 is a set of graphs showing installation of UBA1 M41 mutations in cord blood derived CD34+ human HSPC. The data shows that the edited cells are robust with high product purity. The left hand graph shows exact edit % and the right hand graph shows % indels.

[0053] FIGs.22 and 23 are a set of images showing electroporation of PEMax mRNA alone (FIG. 22) or PEMax mRNA + epegRNA + nicking gRNA (FIG.23) led to a decrease in HSPC colony counts after culture, with corresponding graphs quantifying said decreases.

[0054] FIGs.24 and 25 illustrate a comparative analysis of the data in FIGs.22 and 23. FIG.24 compares the electroporation of PEMax mRNA alone or PEMax mRNA + epegRNA + nicking gRNA, showing that the HSPC colony counts do not change after culture in MethoCultTM. FIG.25 shows that different amounts of seeded cells does not change the colony counts after culture to a statistically relevant degree. Overall, this demonstrates that the installation of the UBA1 mutation did not impact the viability of the cells or impart toxicity.

[0055] FIG.26 shows the proportion of total HSPC clones edited for UBA1M41Ltwo weeks after editing. Demonstrates that the HSPC UBA1M41Lclones are long-lived.

[0056] FIG.27 demonstrates that UBA1M41LHSPC clones are mostly heterozygous for UBA1M41Lallele two weeks after editing. Demonstrates that the HSPC UBA1M41Lclones are long-lived.

[0057] FIG.28A identifies several pegRNA reagents correcting UBA1M41Lin 293T cells. The sequences for the indicated pegRNAs (1: nk10104 (SEQ ID NO: 2061); 2: nk10105 (SEQ ID NO: 2062); 3: nk10106 (SEQ ID NO: 2063)) can be found in Table 10A of Example 10.

[0058] FIG.28B is a bar graph showing that Indels are low frequency events using UBA1M41Lcorrective epegRNAs as reported in Example 10 of the present dislcosure. The sequences for the indicated pegRNAs (1: nk10104 (SEQ ID NO: 2061); 2: nk10105 (SEQ ID NO: 2062); 3: nk10106 (SEQ ID NO: 2063)) can be found in Table 10A of Example 10.

[0059] FIG.29A is a bar graph demonstrating that pathogenic UBA1 mutations robustly are installed in human HSPCs through the methods described in Example 10 of the present disclosure. Lane 1 –untransfected control. Lane 2 – no cargo control. Lane 3 – PEmax alone. Lane 4 – nk10044 + gRNA0172. Lane 4 – nk10056 + gRNA0088. Lane 5 – nk10056 + gRNA0088. Lane 6 – pegRNA469 + gRNA0172. Lane 7 – PEmax + syngRNA0172 + nk10081. Lane 8 – PEmax + syngRNA0172 + nk10087.

[0060] FIG.29B is a bar graph demonstrating that HSPC colony counts are not impacted by installation of UBA1M41Lmutations in human HSPCs, as determined by the methods reported in Example 10 of the present disclosure.DETAILED DESCRIPTION I. Introduction

[0061] First described in October 2020 by Beck, et al., VEXAS syndrome is an adult-onset autoinflammatory disease caused by a somatic mutation in the UBA1 gene (ubiquitin-like modifier activating enzyme 1) which encodes UBA1 in hematopoietic progenitor cells and which presents as a progressive systemic inflammatory disease. The term “VEXAS” is derived from an acronym describing the main features of the disease: Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic. As an X-linked somatic disorder, it disproportionately impacts biological males, and particularly those over the age of 50 years old. VEXAS patients often present with a wide array of inflammatory symptoms that affect connective tissues (e.g., cartilage), skin, joints, blood vessels and the lungs, and has been reported with clinical features that include skin lesions, fever, weight loss, arthritis, chondritis, venous thrombosis, and lymphadenopathy, along with other less common inflammatory conditions.

[0062] The wildtype human UBA1 cDNA coding sequence is as follows (SEQ ID NO: 1) (3177 nt, GenBank Accession No. NM_003334.4):

[0063] In the above sequence, the underlined and bolded “ATG” codon corresponds to wildtype Met41 in the UBA1 protein and is often reported as containing the VEXAS-associated mutations which cause substitution of wildtype Met with another amino acid, such Leu, Val, or Thr.

[0064] The present specification contemplates the editing of any UBA1 gene, mutant or otherwise, and which has a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 1. The target UBA1 gene may be in the patient’s cells in vivo or may be in a cell, e.g., a cell under ex vivo conditions wherein the cell was isolated from the body for editing and then later reintroduction to the patient’s body.

[0065] The wildtype human UBA1 protein sequence is as follows (SEQ ID NO: 2) (1058 aa, GenBank Accession No. NP_003325.2):

[0066] The present specification contemplates the editing of any UBA1 protein, mutant or otherwise, and which has a amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 2. The target UBA1 gene may be in the patient’s cells in vivo or may be in a cell, e.g., a cell under ex vivo conditions wherein the cell was isolated from the body for editing and then later reintroduction to the patient’s body.

[0067] The translation of SEQ ID NO: 1 visualized against SEQ ID NO: 2 is provided below for clarity as to the corresponding codons affected by the VEXAS-associated mutations reported to date. Shown in bold / underlined are codons in the nucleotide sequence corresponding M41, H55, S56, G477, A478, D506, and S621, which are codons and their corresponding amino acid residues which have been reported to date as being associated with VEXAS-associated mutations:

[0068] To date, nearly all pathogenic UBA1 mutations associated with VEXAS syndrome have been traced back to the nucleotides associated with Met41 residue of the protein (indicated as a bold underlined “ATG” in SEQ ID NO: 1 and a bold underlined “M” in SEQ ID NO: 2). As of this filing and relative to SEQ ID NOs: 1 and 2, about 50% of these disease-associated mutations relate to a T122C mutation in UBA1, corresponding to a M41T (Met41Thr) substitution. Another 21% of these disease-associated mutations correspond to an A121G mutation in UBA1, corresponding to a M41V (Met41Val) substitution, or a A121C mutation in UBA1, corresponding to a M41L (Met41Leu) substitution. The remaining 6% of pathogenic mutations are caused by a mutation in the splice acceptor site of exon 3, which houses M41 as the second amino acid residue. In other words, the most commonly reported VEXAS-associated mutations have been substitutions from ATG (methionine) to ACG (threonine), GTG (valine), or CTG (leucine). These mutations effectively block translation initiation at M41 thereby halting expression of the cytoplasmic form of UBA1, termed UBA1b. In the absence of UBA1b expression, an alternative shorter isoform, UBA1c, is expressed from translation initiation at Met67. However, the UBA1c isoform has diminished and insufficient catalytic activity. The lack of UBA1b and / or presence of UBA1c results in decreased ubiquitylation activity and hyperinflammation.

[0069] Other VEXAS-associated mutations observed have included a Ser56Phe (S56F) substitution in exon 3 as well as Gly477Ala (G477A), Ala478Ser (A478S), Asp506Gly (D506G), Asp506Asn (D506N), and Ser621Cys (S621C).

[0070] There is no known curative treatment or standardized treatment models for VEXAS. Patients are typically treated by attempting to manage the inflammatory symptoms with corticosteroids and other immunosuppressants, each of which are associated with significant toxicity. There remains a need in the art for methods of treating VEXAS syndrome, especially ones that have a minimal burden on the patient and which are curative.

[0071] The present disclosure describes systems, compositions, and methods of using gene therapy (e.g., gene editing and / or gene replacement) for treating VEXAS syndrome. These systems, compositions, and methods in general relate to the introduction of healthy copies of the UBA1 gene into cells and / or to the correction of VEXAS-causing mutations in the UBA1 gene in target cells (e.g., blood cell precursor cells, including but not limited to hematopoietic stem cells) under in vitro, ex vivo, or in vivo conditions. Such systems, composition, and methods disclosed herein may be used to correct for any VEXAS-causing mutation in the UBA1 gene, including, but not limited to: a M41 substitution (e.g., a M41T, M41V, or M41L substitution); a Ser56Phe (S56F) substitution; a Gly477Ala (G477A) substitution; an Ala478Ser (A478S) substitution; an Asp506Gly (D506G) substitution; an Asp506Asn (D506N) substitution; or a Ser621Cys (S621C) substitution, or any combination of two or more of these mutations / substitutions. In addition, the systems, compositions,and methods described herein may be used to correct any VEXAS-associated mutation that is identified after the date of this filing in UBA1.

[0072] The disclosure further relates to gene therapy compositions (e.g., gene replacement and / or gene editing compositions) comprising delivery vehicles (e.g., LNPs) formulated with protein and / or nucleic acid components (e.g., DNA or RNA), including various coding RNAs, including linear and / or circular mRNAs, and / or non-coding RNAs, including guide RNAs or other functional non- coding RNA components, which may be administered in an effective amount for the treatment of VEXAS syndrome either by resulting in the replacement of a defective UBA1 gene, the installation of a healthy copy (or multiple copies) of the UBA1 gene into the genome (e.g., at a safe harbor site), or the correction of the defective UBA1 gene sequence.

[0073] Described herein are gene editing systems for use in treating disease (e.g., VEXAS syndrome) and / or otherwise modifying the sequence and / or expression of target nucleotide sequences. Further described herein are pharmaceutical compositions comprising said gene therapy systems (e.g., gene editor or gene replacement systems) formulated in a delivery vehicle, such as, but not limited to a lipid nanoparticle (LNP).

[0074] In various aspects, the disclosure provides LNPs capable of delivering a gene therapy system (e.g., a gene editor or gene replacement system) to blood cell precursor cells, including but not limited to hematopoietic stem cells. The gene therapy systems (e.g., gene editing systems or gene replacement systems) of the present disclosure are preferably delivered to a patient under in vivo conditions (e.g., administered to a subject in an effective amount), but can also be delivered to target cells (e.g., hematopoietic stem cells) under ex vivo conditions. The disclosure also provides in various aspects therapeutic or pharmaceutical compositions comprising LNPs comprising gene therapy systems (e.g., gene editing systems or gene replacement systems) or one or more components thereof for use in treating disease (e.g., VEXAS syndrome) and / or otherwise modifying the sequence and / or expression of target nucleotide sequences, including VEXAS-causing mutations in the UBA1 gene, including, but not limited to: a M41 substitution (e.g., a M41T, M41V, or M41L substitution); a Ser56Phe (S56F) substitution; a Gly477Ala (G477A) substitution; an Ala478Ser (A478S) substitution; an Asp506Gly (D506G) substitution; an Asp506Asn (D506N) substitution; or a Ser621Cys (S621C) substitution, or any combination of two or more of these mutations / substitutions.

[0075] In certain embodiments, the disclosure also provides methods of using the gene therapy systems (e.g., gene editing systems or gene replacement systems) to treat a disease (e.g., VEXAS syndrome), ex vivo. In some embodiments, the present disclosure provides methods comprising extracting and culturing a population of a patient’s own cells (e.g., a hematopoietic stem cell population), contacting said cells with a gene editing system of the present disclosure, ex vivo, to modify the cells, and then transplanting the modified cells back into the patient. In someembodiments, the method further comprises treating the patient in such a way that increases the likelihood of acceptance of the transplanted modified cells.

[0076] The gene therapy systems (e.g., gene editing systems or gene replacement systems) may comprise DNA components, RNA components, protein components, nucleoprotein components, or combinations thereof. In other aspects, the disclosure provides nucleic acid molecules that encode various componentry of the deliverable gene therapy systems (e.g., gene editing systems or gene replacement systems) contemplated herein. In addition, other aspects of the disclosure provide nucleic acid molecules as components of the herein contemplated gene therapy systems (e.g., gene editing systems or gene replacement systems), such as, but not limited to plasmids or vectors encoding one or more components of a gene editing system, RNAs encoding one or more components of a gene editing system (e.g., mRNAs coding for a nuclease domain of a gene editing system), and non-coding RNAs (e.g., guide RNAs capable of complexing with and targeting a nucleic acid- programmable DNA binding domain to a specific target nucleotide sequence or a retron ncRNA, depending on the gene editing system being deployed). The disclosure, in other aspects, provides for the various protein components of the various gene editing systems contemplated herein, including, but not limited to, user-programmable DNA binding proteins and various effector proteins, such as nucleases, polymerases, reverse transcriptases, recombinases, integrases, endonucleases, exonucleases, transposases, and deaminases. The disclosure also describes nucleoprotein components of the gene therapy systems (e.g., gene editing systems or gene replacement systems) contemplated herein, such as, but not limited to a nuclease-guide RNA complexes. The disclosure also provides methods of modifying the sequence and / or expression level of a target nucleic acid molecule through the delivery and / or administration of an pharmaceutical composition described herein that may comprise in various embodiments a delivery vehicle (e.g., an LNP) formulated with a gene therapy system (e.g., gene editing system or gene replacement system) or components thereof. Still further, the disclosure provides methods of treating a disease by administering a therapeutically effective amount of a gene therapy system (e.g., gene editing systems or gene replacement systems) described herein that results in the modification in the sequence and / or expression level of a target nucleic acid molecule (e.g., a disease-associated gene).

[0077] The compositions (e.g., LNP-formulated gene editing systems) described herein may include a variety of coding RNA molecules that code for the various components of gene editing systems or gene replacement systems. In various aspects, the coding RNA may be linear mRNA. In other embodiments, the coding RNA may be circular mRNA. In various aspects that involve LNP- formulated compositions, the LNPs include improved LNPs that protect linear and / or circular mRNA cargos from degradation and clearance while achieving targeted systemic or local delivery for use as enhanced gene editing / replacement platforms and / or therapeutic agents.

[0078] In various other aspects, the compositions (e.g., LNP-formulated gene editing systems) described herein may also include a repair template, e.g., an HDR donor single or double stranded DNA.

[0079] Accordingly, the instant specification describes compositions (e.g., LNP-formulated compositions), methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and / or use of gene editing and / or gene replacement systems as therapeutic compositions for the treatment of VEXAS syndrome. Further described herein are compositions (e.g., LNP-formulated compositions), methods, processes, kits and devices for the selection, design, preparation, manufacture, formulation, and / or use of gene editing and / or gene replacement systems for the prophylactic and / or therapeutic treatment of one or more diseases or a symptom thereof (e.g., VEXAS syndrome).

[0080] In various embodiments, the compositions comprising a delivery vehicle, such as, but not limited to an LNP. The components capable of being encapsulated by or otherwise incorporated by the delivery vehicles (e.g., LNPs) described herein may be referred to as “payloads” (e.g., LNP payloads) and may include all of the biological materials described above, including DNA molecules, RNA molecules (coding and / or non-coding), proteins, and nucleoproteins (e.g., Cas / guide RNA complexes).

[0081] In certain embodiments, the LNP compositions selectively and effectively deliver the gene editing payloads to specific cell types that allow for the VEXAS syndrome to be treated. In certain embodiments, the LNPs of the present disclosure deliver to red blood cell progenitor cells. In certain embodiments, the LNPs of the present disclosure deliver to hematopoietic stem cells. II. Gene editor systems

[0082] The present disclosure describes systems, compositions, and methods of using gene editing for treating VEXAS syndrome. These systems, compositions, and methods in general relate to the correction of VEXAS-causing mutations in the UBA1 gene in target cells (e.g., blood cell precursor cells, including but not limited to hematopoietic stem cells) under in vitro, ex vivo, or in vivo conditions. Such systems, composition, and methods disclosed herein may be used to correct for any VEXAS-causing mutation in the UBA1 gene, including, but not limited to: a M41 substitution (e.g., a M41T, M41V, or M41L substitution); a Ser56Phe (S56F) substitution; a Gly477Ala (G477A) substitution; an Ala478Ser (A478S) substitution; an Asp506Gly (D506G) substitution; an Asp506Asn (D506N) substitution; or a Ser621Cys (S621C) substitution, or any combination of two or more of these mutations / substitutions. In addition, the systems, compositions, and methods described herein may be used to correct any VEXAS-associated mutation that is identified after the date of this filing in UBA1.

[0083] In various aspects, such systems, composition, and methods described herein are capable of executing one or more edits or modifications that enable treatment of VEXAS syndrome. In certainembodiments, the gene editing systems edit or modify a somatic mutation in the Ubiquitin Activating Enzyme UBA1 at position Met41 of the protein. In certain embodiments, the gene editing systems address the production of the mutant inactive isoform UBA1c, correcting the decreased ubiquitylation activity and hyperinflammation caused by UBA1c.

[0084] Such systems, composition, and methods disclosed herein may be used to correct for any VEXAS-causing mutation in the UBA1 gene, including, but not limited to: a M41 substitution (e.g., a M41T, M41V, or M41L substitution); a Ser56Phe (S56F) substitution; a Gly477Ala (G477A) substitution; an Ala478Ser (A478S) substitution; an Asp506Gly (D506G) substitution; an Asp506Asn (D506N) substitution; or a Ser621Cys (S621C) substitution, or any combination of two or more of these mutations / substitutions.

[0085] Such systems, composition, and methods disclosed herein may be used to correct for any VEXAS-causing mutation in the UBA1 gene, including, but not limited to any UBA1 mutation that is identified as associated with VEXAS syndrome after the date of this filing.

[0086] Genome editing and / or replacement tools encompass a diverse set of technologies that can make many types of genomic alterations in various contexts. These technologies have evolved over the last couple of decades to provide a range of user-programmable editing tools that include ZFN (zinc finger) nuclease editing systems, meganuclease editing systems, and TALENS (transcription activator-like effector nucleases). The past decade has seen an explosive growth in a new generation of genome editing systems based on components from bacterial immune pathways, including CRISPR (clustered regularly interspaced short palindromic repeats) and the associated CRISPR-associated proteins (e.g., CRISPR-Cas9) (Jinek et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, Vol.337 (6096), pp.816-821), meganuclease editors (Boissel et al., “megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering,” Nucleic Acids Research 42: pp.2591-2601) and bacterial retron systems (Schubert et al., “High-throughput functional variant screens via in vivo production of single-stranded DNA,” PNAS, April 27, 2021, Vol.118(18), pp.1-10). In particular, CRISPR-Cas9 has been derivatized in numerous ways to expand upon its guide RNA-based programmable double-strand cutting activity to form systems ranging from finding alternative CRISPR Cas nuclease enzymes having different PAM requirements and cutting properties (e.g., Cas12a, Cas12f, Cas13a, and Cas13b) to base editing (Komor et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature, May 19, 2016, 533 (7603); pp.420-424 [cytosine base editors or CBEs] and Gaudelli et al., “Programmable base editing of A-T to G-C in genomic DNA without DNA cleavage,” Nature, Vol.551, pp.464-471 [adenine base editors or ABEs]) to prime editing (Anzalone et al., “Search-and-replace genome editing without double-strand breaks or donor DNA,” Nature, Dec 2019, 576 (7789): pp.149-157) to twin prime editing (Anzalone et al., “Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing,” NatureBiotechnology, Dec 9, 2021, vol.40, pp.731-740) to epigenetic editing (Kungulovski and Jeltsch, “Epigenome Editing: State of the Art, Concepts, and Perspective,” Trends in Genetics, Vol.32, 206, pp.101-113) to CRISPR-directed integrase editing (Yarnell et al., “Drag-and-drop genome insertion of large sequences without double-stranded DNA cleavage using CRISPR-directed integrases,” Nature Biotechnology, Nov 24, 2022, (“PASTE”)), among others.  In various embodiments, such editing systems are implemented for VEXAS syndrome by replacement and / or correction of a defective UBA1 gene, including, but not limited to a UBA1 gene comprising one or more mutations which result in the UBA1 protein: a M41 substitution (e.g., a M41T, M41V, or M41L substitution); a Ser56Phe (S56F) substitution; a Gly477Ala (G477A) substitution; an Ala478Ser (A478S) substitution; an Asp506Gly (D506G) substitution; an Asp506Asn (D506N) substitution; or a Ser621Cys (S621C) substitution, or any combination of two or more of these mutations / substitutions.

[0087] In various aspects, the compositions (e.g., LNP-formulated compositions) described herein may be used to deliver a payload of interest to a biological target, e.g., to a cell or a bodily tissue. The term “payload” refers to an active substance, such as a small molecule, polypeptide, peptide, carbohydrate, or nucleic acid molecule, and includes, without limitation, mRNA molecules (including linear and circular mRNA) and non-coding RNAs (e.g., guide RNAs) which are encapsulated within a delivery vehicle (e.g., LNP) described herein. In various embodiments, the payload is an RNA molecule, which may be linear or circular and may comprise one or more functional nucleotide sequences of interest, which may include, but are not limited to coding and non-coding nucleotide sequences. In various embodiments, the non-coding nucleotide sequences may comprise regulatory elements that influence RNA post-transcriptional processing, nuclear translation control sequences, and sequences which encode one or more biological products of interest, e.g., a therapeutic protein or nucleobase editing system, among other sequence elements that may impact the functioning of the RNA or its encoded products. As used herein, the term “coding region of interest” or “product coding region” or the like may be used to refer to the encoded one or more biological products of interest. Equivalently, a product coding region may be referred to as a “product expression sequence.”

[0088] In various embodiments, the gene editing and / or replacement systems described herein may be formulated in LNPs. There remain numerous challenges associated with the delivery of gene editing tools—including, but not limited to, CRISPR-Cas9 and alternative Cas nuclease editors, retron editors, base editors, prime editors, twin prime editors, epigenetic editors, and integrase editors—to achieve safe and effective therapeutic application of such tools in cells and patients for treating disease and / or otherwise modifying the nucleotide sequence of a target nucleic acid molecule (e.g., a gene or genome). That said, the use of lipid nanoparticles (LNPs) has emerged as a leading delivery option for the safe, effective, and targeted delivery of gene editing tools to target tissues and cells. However, there remains a need for improved LNPs, including better performing ionizable lipids, that will enhance the targeted delivery of LNP-based gene editing tools. Preferably, such improved LNPswould protect payloads from degradation and clearance while achieving targeted delivery, be suitable for systemic or local delivery, and provide delivery of a wide variety of gene editing tools, such as those mentioned above. In addition, such improved LNP-based therapeutics should exhibit low toxicity and provide an adequate therapeutic index, such that patient treatment at an effective dose of the LNP minimizes risk to the patient while maximizing therapeutic benefit. The present disclosure provides these and related advantages.

[0089] In one embodiment relating to a gene replacement approach, the present disclosure provides a gene replacement system for transient expression of healthy UBA1 comprising one or more coding RNAs (e.g., a linear or circular mRNA), or one or more vectors encoding said coding RNAs, which once delivered to an affected target cell, allow for the expression of a healthy copy of UBA1. In this way, defective UBA1 protein in the cell—due to the presence of one or more VEXAS-associated mutations in the UBA1 gene resulting in a variant UBA1 having a M41 substitution (e.g., a M41T, M41V, or M41L substitution); a Ser56Phe (S56F) substitution; a Gly477Ala (G477A) substitution; an Ala478Ser (A478S) substitution; an Asp506Gly (D506G) substitution; an Asp506Asn (D506N) substitution; or a Ser621Cys (S621C) substitution, or any combination of two or more of these mutations / substitutions—may be supplemented by a functional healthy source of UBA1 protein expressed from the delivered coding RNAs and / or vectors.

[0090] In another embodiment relating to a gene replacement approach, the present disclosure provides a gene replacement system for permanent expression of healthy UBA1 comprising a vector comprising a first sequence encoding a healthy copy of UBA1 and one or more second sequences flanking the first sequence which comprise one or more regions of homology with a target safe harbor site in the genome of the target cell which allow for the integration of the healthy copy of the UBA1 gene into the safe harbor site. The healthy copy of the UBA1 gene may also comprising one or more regulatory sequences (e.g., a promoter, enhancer, and / or transcription factor binding sites) operably linked to the UBA1 gene such that the expression (transcription and / or translation) of the UBA1 gene is controlled. In this way, defective UBA1 protein in the cell—due to the presence of one or more VEXAS-associated mutations in the UBA1 gene resulting in a variant UBA1 having a M41 substitution (e.g., a M41T, M41V, or M41L substitution); a Ser56Phe (S56F) substitution; a Gly477Ala (G477A) substitution; an Ala478Ser (A478S) substitution; an Asp506Gly (D506G) substitution; an Asp506Asn (D506N) substitution; or a Ser621Cys (S621C) substitution, or any combination of two or more of these mutations / substitutions—may be supplemented by a functional healthy source of UBA1 protein expressed from the genome from the integrated UBA1 gene.

[0091] In other embodiments, the disclosure provides gene editing approaches for correcting a defective UBA1 gene in a target cell genome. The gene editing system is not particularly limited and can including any suitable gene editing system that results in the repair of the patient’s VEXAS- associated mutation, including any of those resulting in a variant UBA1 having a M41 substitution(e.g., a M41T, M41V, or M41L substitution); a Ser56Phe (S56F) substitution; a Gly477Ala (G477A) substitution; an Ala478Ser (A478S) substitution; an Asp506Gly (D506G) substitution; an Asp506Asn (D506N) substitution; or a Ser621Cys (S621C) substitution, or any combination of two or more of these mutations / substitutions.

[0092] The gene editing systems contemplated herein may comprise a programmable nuclease which introduces a double-stranded or single-stranded break at a specific target site in a defective UBA1 gene.

[0093] In various embodiments, the programmable nuclease may be an amino acid programmable nuclease, such as a nuclease comprising a zinc finger binding domain. In some embodiments, the nuclease cuts both strands at the target site. In other embodiments, the nuclease cuts only a single strand at the target site. In still other embodiments, the nuclease lacks a nuclease activity and does not cut the target site at all.

[0094] In various other embodiments, the programmable nuclease may be an amino acid programmable nuclease, such as a nuclease comprising a TALE domain (i.e., a TALEN). In some embodiments, the nuclease cuts both strands at the target site. In other embodiments, the nuclease cuts only a single strand at the target site. In still other embodiments, the nuclease lacks a nuclease activity and does not cut the target site at all.

[0095] In still other embodiments, the programmable nuclease may be a nucleic acid programmable nuclease, such as a CRISPR nuclease which is programmed to bind and cut a specific nucleotide sequence (e.g., the defective UBA1 gene) when complexed with a guide RNA that comprises a sequence that is complementary to the target site (or a strand thereof). In some embodiments, the nuclease cuts both strands at the target site. In other embodiments, the nuclease cuts only a single strand at the target site. In still other embodiments, the nuclease lacks a nuclease activity and does not cut the target site at all.

[0096] In some embodiments, the single-strand or double-strand nuclease cut introduced into a target defective UBA1 gene results in a A. Nucleic acid payloads

[0097] In various embodiments, the LNP compositions described herein can be used to deliver a nucleic acid or polynucleotide payload, e.g., a linear or circular mRNA.

[0098] In some embodiments, a LNP is capable of delivering a polynucleotide to a target cell, tissue, or organ. A polynucleotide, in its broadest sense of the term, includes any compound and / or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybridsthereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc. RNAs useful in the compositions and methods described herein can be selected from the group consisting of but are not limited to, shortimers, antagomirs, antisense, ribozymes, short interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, a polynucleotide is mRNA. In some embodiments, a polynucleotide is circular RNA. In some embodiments, a polynucleotide encodes a protein, e.g., a nucleobase editing enzyme. A polynucleotide may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A polypeptide may be of any size and may have any secondary structure or activity. In some embodiments, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.

[0099] In other embodiments, a polynucleotide is an siRNA. An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest. For example, an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a nanoparticle composition including the siRNA. An siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0100] In some embodiments, a polynucleotide is an shRNA or a vector or plasmid encoding the same. An shRNA may be produced inside a target cell upon delivery of an appropriate construct to the nucleus. Constructs and mechanisms relating to shRNA are well known in the relevant arts.

[0101] A polynucleotide may include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5'-terminus of the first region (e.g., a 5'-UTR), a second flanking region located at the 3'-terminus of the first region (e.g., a 3'- UTR), at least one 5'-cap region, and a 3'-stabilizing region. In some embodiments, a polynucleotide further includes a poly-A region or a Kozak sequence (e.g., in the 5'-UTR). In some cases, polynucleotides may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide. In some embodiments, a polynucleotide (e.g., an mRNA) may include a 5'cap structure, a chain terminating nucleotide, a stem loop, a polyA sequence, and / or a polyadenylation signal. Any one of the regions of a nucleic acid may include one or more alternative components (e.g., an alternative nucleoside). For example, the 3'-stabilizing region may contain an alternative nucleoside such as an L-nucleoside, an inverted thymidine, or a 2'-O-methyl nucleoside and / or the coding region, 5'-UTR, 3'-UTR, or cap region may include an alternative nucleoside such as a 5- substituted uridine (e.g., 5-methoxyu ridine), a 1-substituted pseudouridine (e.g., 1-methyl pseudouridine or 1-ethyl-pseudouridine), and / or a 5-substituted cytidine (e.g., 5-methyl-cytidine). In some embodiments, a polynucleotide contains only naturally occurring nucleosides.

[0102] In some cases, a polynucleotide is greater than 30 nucleotides in length. In another embodiment, the poly nucleotide molecule is greater than 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 50 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides, or greater than 5000 nucleotides.

[0103] In some embodiments, a polynucleotide molecule, formula, composition or method associated therewith comprises one or more polynucleotides comprising features as described in WO2002 / 098443, WO2003 / 051401, WO2008 / 052770, WO2009 / 127230, WO2006 / 122828, WO2008 / 083949, WO2010 / 088927, WO2010 / 037539, WO2004 / 004743, WO2005 / 016376, WO2006 / 024518, WO2007 / 095976, WO2008 / 014979, WO2008 / 077592, WO2009 / 030481, WO2009 / 095226, WO2011 / 069586, WO2011 / 026641, WO2011 / 144358, WO2012 / 019780, WO2012 / 013326, WO2012 / 089338, WO2012 / 113513, WO2012 / 116811, WO2012 / 116810, WO2013 / 113502, WO2013 / 113501, WO2013 / 113736, WO2013 / 143698, WO2013 / 143699, WO2013 / 143700, WO2013 / 120626, WO2013 / 120627, WO2013 / 120628, WO2013 / 120629, WO2013 / 174409, WO2014 / 127917, WO2015 / 024669, WO2015 / 024668, WO2015 / 024667, WO2015 / 024665, WO2015 / 024666, WO2015 / 024664, WO2015 / 101415, WO2015 / 101414,WO2015 / 024667, WO2015 / 062738, WO2015 / 101416, all of which are incorporated by reference herein.

[0104] In some embodiments, a polynucleotide comprises one or more microRNA binding sites. In some embodiments, a microRNA binding site is recognized by a microRNA in a non-target organ. In some embodiments, a microRNA binding site is recognized by a microRNA in the liver. In some embodiments, a microRNA binding site is recognized by a microRNA in hepatic cells.

[0105] In certain embodiments, an RNA of the present disclosure comprises one or more phosphonate modifications selected from a phosphorothioate linkage (PS), phosphorodithioate linkage (PS2), methylphosphonate linkage (MP), methoxypropylphosphonate linkage (MOP), 5’-(E)- vinylphosphonate linkage (5’-(E)-VP), 5’-Methyl Phosphonate linkage (5’-MP), (S)-5’-C-methyl with phosphate linkage, 5’-phosphorothioate linkage (5’-PS), and a peptide nucleic acid linkage (PNA). In certain embodiments, an RNA of the present disclosure comprises one or more ribose modifications selected from a 2’-O-methyl (2’-OMe), 2’-O-methoxyethyl (2’-O-MOE), 2’-deoxy-2’-fluoro (2’-F), 2’-arabino-fluoro (2’-Ara-F), 2’-O-benzyl, 2’-O-methyl-4-pyridine (2’-O-CH2Py(4)), Locked nucleic acid (LNA), (S)-cET-BNA, tricyclo-DNA (tcDNA), PMO, Unlocked Nucleic Acid (UNA) and glycol nucleic acid (GNA). In certain embodiments, the RNA comprises a Locked Nucleic Acid (LNA) comprising a methyl bridge, an ethyl bridge, a propyl bridge, a butyl bridge or an optionally substituted variant of any of the aforementioned. In certain embodiments, an RNA of the present disclosure comprises one or more modified bases selected from a pseudouridine (ψ), 2’thiouridine (s2U), N6’-methyladenosine (m6A), 5’methylcytidine (m5C), 5’fluoro2’-deoxyuridine, N- ethylpiperidine 7’-EAA triazole modified adenine, N-ethylpiperidine 6’triazole modified adenine, 6’pheynlpyrrolo-cytosine (PhpC), 2’,4’-difluorotoluyl ribonucleoside (rF), and 5’-nitroindole. B. Linear mRNA payloads

[0106] In various embodiments, the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein can be used to deliver an RNA payload that is a linear mRNA molecule.

[0107] Ribonucleic acid (RNA) is a molecule that is made up of nucleotides, which are ribose sugars attached to nitrogenous bases and phosphate groups. The nitrogenous bases include adenine (A), guanine (G), uracil (U), and cytosine (C). Generally, RNA mostly exists in the single-stranded form but can also exists double-stranded in certain circumstances. The length, form and structure of RNA is diverse depending on the purpose of the RNA. For example, the length of an RNA can vary from a short sequence (e.g., siRNA) to a long sequences (e.g., lncRNA), can be linear (e.g., mRNA) or circular (e.g., oRNA), and can either be a coding (e.g., mRNA) or a non-coding (e.g., lncRNA) sequence.

[0108] In various embodiments, the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein can be used to deliver a mRNA payload that is a linear mRNA molecule. In embodiments, the mRNA payload may comprise one or more nucleotide sequences that encode a product of interest, such as, but not limited to a component of a gene editing system (e.g. an endonuclease, a prime editor, etc.) and / or a therapeutic protein.

[0109] In some embodiments, the RNA payload may be a linear mRNA. As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide which encodes a protein of interest and which is capable of being translated to produce the encoded protein of interest in vitro, in vivo, in situ or ex vivo.

[0110] Generally, a mRNA molecule comprises at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail. In some aspects, one or more structural and / or chemical modifications or alterations may be included in the RNA which can reduce the innate immune response of a cell in which the mRNA is introduced. As used herein, a "structural" feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in a nucleic acid without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to affect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G".

[0111] Generally, a coding region of interest in an mRNA used herein may encode a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In another embodiment, the mRNA may encode a peptide of 2-30 amino acids, e.g. 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The mRNA may encode a peptide of at least 10, 11, 12, 13, 14, 15, 17, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, or a peptide that is no longer than 10, 11, 12, 13, 14, 15, 17, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

[0112] Generally, the length of the region of the mRNA encoding a product of interest is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides).

[0113] In some embodiments, the mRNA has a total length that spans from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1 ,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1 ,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000 nucleotides).

[0114] In some embodiments, the region or regions flanking the region encoding the product of interest may range independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).

[0115] In some embodiments, the mRNA comprises a tailing sequence which can range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Where the tailing region is a polyA tail, the length may be determined in units of or as a function of polyA Binding Protein binding. In this embodiment, the polyA tail is long enough to bind at least 4 monomers of PolyA Binding Protein. PolyA Binding Protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are functional.

[0116] In some embodiments, the mRNA comprises a capping sequence which comprises a single cap or a series of nucleotides forming the cap. The capping sequence may be from 1 to 10, e.g.2-9, 3- 8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, the caping sequence is absent.

[0117] In some embodiments, the mRNA comprises a region comprising a start codon. The region comprising the start codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.

[0118] In some embodiments, the mRNA comprises a region comprising a stop codon. The region comprising the stop codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.

[0119] In some embodiments, the mRNA comprises a region comprising a restriction sequence. The region comprising the restriction sequence may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.Untranslated Regions (UTRs)

[0120] In various embodiments, the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise at least one untranslated region (UTR) which flanks the region encoding the product of interest and / or is incorporated within the mRNA molecule. UTRs are transcribed by not translated. The mRNA payloads can include 5’ UTR sequences and 3’ UTR sequences, as well as internal UTRs.

[0121] The RNA payloads of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region. Where nucleic acids are designed to encode at least one polypeptide of interest, the nucleic acid may comprise one or more of these untranslated regions (UTRs). Wild-type untranslated regions of a nucleic acid are transcribed but not translated. In mRNA, the 5′ UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3′ UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into the RNA payload molecules (e.g., linear and circular mRNA molecules) of the present disclosure to, among other things, enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites. A variety of 5′UTR and 3′UTR sequences are known and available in the art.

[0122] In various embodiments, the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise at least one UTR that may be selected from any UTR sequence listed in Tables 19 or 20 of U.S. Patent No. 10,709,779, which is incorporated herein by reference. 5' UTR regions

[0123] In various embodiments, the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise at least one 5′ UTR.

[0124] A 5′ UTR is region of an mRNA that is directly upstream (5′) from the start codon (the first codon of an mRNA transcript translated by a ribosome). A 5′ UTR does not encode a protein (is non- coding). Natural 5′UTRs have features that play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG (SEQ ID NO: 1977), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ‘G’.5′UTR also have been known to form secondary structures which are involved in elongation factor binding.5’ UTR sequences are also known to be important for ribosome recruitment to the mRNA and have been reported to play arole in translation (Hinnebusch A, et al., (2016) Science, 352:6292: 1413-6). In addition, 5’ UTR sequences may confer increased half-life, increased expression and / or increased activity of a polypeptide encoded by the RNA payload described herein.

[0125] In various embodiments, the RNA payload constructs contemplated herein may include 5’UTRs that are found in nature and those that are not. For example, the 5’UTRs can be synthetic and / or can be altered in sequence with respect to a naturally occurring 5’UTR. Such altered 5’UTRs can include one or more modifications relative to a naturally occurring 5’UTR, such as, for example, an insertion, deletion, or an altered sequence, or the substitution of one or more nucleotide analogs in place of a naturally occurring nucleotide.

[0126] The 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3 'UTR starts immediately following the stop codon and continues until the transcriptional termination signal. While not wishing to be bound by theory, the UTRs may have a regulatory role in terms of translation and stability of the nucleic acid.

[0127] Natural 5' UTRs usually include features which have a role in translation initiation as they tend to include Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'.5'UTR also have been known to form secondary structures which are involved in elongation factor binding.

[0128] In an embodiment, the 5’ UTR comprises a sequence provided in Table X or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a 5’ UTR sequence provided in Table X, or a variant or a fragment thereof (e.g., a fragment that lacks the first one, two, three, four, five, or six nucleotides of the 5’ UTR sequence provided in Table X). In an embodiment, the 5’ UTR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1978, SEQ ID NO: 1979, SEQ ID NO: 1980, SEQ ID NO: 1981, SEQ ID NO: 1982, SEQ ID NO: 1983, SEQ ID NO: 1984, SEQ ID NO: 1985, SEQ ID NO: 1986, SEQ ID NO: 1987, SEQ ID NO: 1988, SEQ ID NO: 1989, SEQ ID NO: 1990, SEQ ID NO: 1991, SEQ ID NO: 1992, SEQ ID NO: 1993, SEQ ID NO: 1994, SEQ ID NO: 1995, SEQ ID NO: 1996, SEQ ID NO: 1997, SEQ ID NO: 1998, SEQ ID NO: 1999, SEQ ID NO: 2000, SEQ ID NO: 2001, SEQ ID NO: 2002, SEQ ID NO: 2003, SEQ ID NO: 2004, SEQ ID NO: 2005, or SEQ ID NO: 2006.

[0129] Table X – Exemplary nucleotide sequences of 5’ UTRs

[0130] In some embodiments of the disclosure, a 5′ UTR is a heterologous UTR, i.e., is a UTR found in nature associated with a different mRNA. In another embodiment, a 5′ UTR is a synthetic UTR, i.e., does not occur in nature. Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic. Exemplary 5′ UTRs include Xenopus or human derived alpha-globin or beta-globin (e.g., US8,278,063 and US9,012,219), human cytochrome b-245 polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus. CMV immediate-early 1 (IE1) gene (see US20140206753 and WO2013 / 185069), the sequence GGGAUCCUACC (SEQ ID NO: 2007) (WO2014144196) may also be used. In another embodiment, 5′ UTR of a TOP gene is a 5′ UTR of a TOP gene lacking the 5′ TOP motif (the oligopyrimidine tract) (e.g., WO / 2015101414, WO2015101415, WO / 2015 / 062738, WO2015024667, WO2015024667; 5′ UTR element derived from ribosomal protein Large 32 (L32) gene (WO / 2015101414, WO2015101415, WO / 2015 / 062738)), 5′ UTR element derived from the 5′UTR of an hydroxysteroid (17-β) dehydrogenase 4 gene (HSD17B4) (WO2015024667), or a 5′ UTR element derived from the 5′ UTR of ATP5A1 (WO2015024667) can be used. In one embodiment, an internal ribosome entry site (IRES) is used as a substitute for a 5′ UTR.

[0131]

[0132] In some embodiments, a 5′ UTR of the present disclosure comprises a sequence selected from SEQ ID NO: 2008 (GGGAAAUAAG AGAGAAAAGA AGAGUAAGAA GAAAUAUAAG AGCCACC), SEQ ID NO:2009 (GGGAAATAAG AGAGAAAAGA AGAGTAAGAA GAAATATAAG AGCCACC), SEQ ID NO:2010 (GGGAAAUAAG AGAGAAAAGA AGAGUAAGAA GAAAUAUAAG AGCCACC) and SEQ ID NO:2011 (GGGAAATAAG AGAGAAAAGA AGAGTAAGAA GAAATATAAG AGCCACC). 3' UTR regions

[0133] In various embodiments, the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise at least one 3′ UTR.3′ UTRs may be heterologous or synthetic.

[0134] A 3′ UTR is region of an mRNA that is directly downstream (3′) from the stop codon (the codon of an mRNA transcript that signals a termination of translation). A 3′ UTR does not encode a protein (is non-coding). Natural or wild type 3′ UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) (SEQ ID NO: 35) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-α. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well- studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3′ UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.

[0135] 3' UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al., 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of AREs include GM- CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family,most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.

[0136] Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of the mRNA payloads described herein. For example, one or more copies of an ARE can be introduced to make mRNA less stable and thereby curtail translation and decrease production of the resultant protein. Alternatively, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.

[0137] In some embodiments, the introduction of features often expressed in genes of target organs the stability and protein production of the mRNA can be enhanced in a specific organ and / or tissue. As a non-limiting example, the feature can be a UTR. As another example, the feature can be introns or portions of introns sequences.

[0138] Those of ordinary skill in the art will understand that 5′ UTRs that are heterologous or synthetic may be used with any desired 3′ UTR sequence. For example, a heterologous 5′ UTR may be used with a synthetic 3′ UTR with a heterologous 3′ UTR.

[0139] Non-UTR sequences may also be used as regions or subregions within an RNA payload construct. For example, introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. Incorporation of intronic sequences may increase protein production as well as nucleic acid levels.

[0140] Combinations of features may be included in flanking regions and may be contained within other features. For example, the polypeptide coding region of interest in an mRNA payload may be flanked by a 5′ UTR which may contain a strong Kozak translational initiation signal and / or a 3′ UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail.5′ UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes such as the 5′ UTRs described in US Patent Application Publication No.20100293625 and PCT / US2014 / 069155, herein incorporated by reference in its entirety

[0141] It should be understood that any UTR from any gene may be incorporated into the regions of an RNA payload molecule (e.g., a linear mRNA). Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5′ or 3′ UTR may be inverted, shortened, lengthened, made with one or more other 5′ UTRs or 3′ UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3′ UTR or 5′ UTR may be altered relative to a wild-type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion ofnucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3′ or 5′) comprise a variant UTR.

[0142] In some embodiments, a double, triple or quadruple UTR such as a 5′ UTR or 3′ UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. For example, a double beta-globin 3′ UTR may be used as described in US Patent publication 20100129877, the contents of which are incorporated herein by reference in its entirety.

[0143] It is also within the scope of the present disclosure to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level.

[0144] In some embodiments, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.

[0145] The untranslated region may also include translation enhancer elements (TEE). As a non- limiting example, the TEE may include those described in US Application No.20090226470, herein incorporated by reference in its entirety, and those known in the art. 5' Capping

[0146] In various embodiments, the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise a 5’ cap structure.

[0147] The 5' cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns removal during mRNA splicing.

[0148] Endogenous mRNA molecules may be 5'-end capped generating a 5'-ppp-5'-triphosphate linkage between a terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap may then be methylated to generate an N7-methyl- guanylate residue. The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-0-methylated.5'-decapping through hydrolysis andcleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[0149] Modifications to mRNA may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp-5' phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a- thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap.

[0150] Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.

[0151] Additional modifications include, but are not limited to, 2'-0-methylation of the ribose sugars of 5 '-terminal and / or 5'-anteterminal nucleotides of the mRNA (as mentioned above) on the 2'- hydroxyl group of the sugar ring. Multiple distinct 5 '-cap structures can be used to generate the 5 '- cap of a nucleic acid molecule, such as an mRNA molecule.

[0152] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wild-type or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e. non-enzymatically) or enzymatically synthesized and / or linked to a nucleic acid molecule.

[0153] For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5 '-5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-0-methyl group (i.e., N7,3'-0-dimethyl-guanosine-5'-triphosphate-5 '-guanosine (m7G-3'mppp-G; which may equivalently be designated 3' O-Me-m7G(5')ppp(5')G). The 3'-0 atom of the other, unmodified, guanine becomes linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g. an mRNA). The N7- and 3'-0-methlyated guanine provides the terminal moiety of the capped nucleic acid molecule (e.g. mRNA).

[0154] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-0-methyl group on guanosine (i.e., N7,2'-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0155] While cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5 '-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.

[0156] mRNA may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5'-cap structures. As used herein, the phrase "more authentic" refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a"more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and / or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 5 'cap structures are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and / or reduced 5'decapping, as compared to synthetic 5 'cap structures known in the art (or to a wild-type, natural or physiological 5 'cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0-methyltransferase enzyme can create a canonical 5 '-5 '-triphosphate linkage between the 5 '-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5 '-terminal nucleotide of the mRNA contains a 2'-0-methyl. Such a structure is termed the Capl structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro- inflammatory cytokines, as compared, e.g., to other 5 'cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5*)ppp(5*)N,pN2p (cap 0), 7mG(5*)ppp(5*)NlmpNp (cap 1), and 7mG(5*)-ppp(5')NlmpN2mp (cap 2).

[0157] In some embodiments, the 5' terminal caps may include endogenous caps or cap analogs.

[0158] In some embodiments, a 5' terminal cap may comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2'fluoro-guanosine, 7-deaza- guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. IRES Sequences

[0159] In various embodiments, the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise one or more IRES sequences.

[0160] In some embodiments, the mRNA may contain an internal ribosome entry site (IRES). First identified as a feature Picorna virus RNA, IRES plays an important role in initiating protein synthesis in absence of the 5' cap structure. An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. An mRNA that contains more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes. Non-limiting examples of IRES sequences that can be used include without limitation, those from picornaviruses (e.g. FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).

[0161] In some embodiments, the IRES is from Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus,Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kip1, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV- Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E / D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Salivirus A BNS, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVBS, EVA71, CVA3, CVA12, EV24 or an aptamer to eIF4G. Poly-A tails and 3’ stabilizing region

[0162] In various embodiments, the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise a poly-A tail.

[0163] During RNA processing, a long chain of adenine nucleotides (poly-A tail) may be added to a polynucleotide such as an mRNA molecules in order to increase stability. Immediately after transcription, the 3' end of the transcript may be cleaved to free a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the free 3' hydroxyl end. The process, called polyadenylation, adds a poly-A tail of a certain length.

[0164] In some embodiments, the length of a poly-A tail is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450,500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides) and no more than about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 3000 nucleotides in length. In some embodiments, the mRNA includes a poly-A tail from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1 ,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

[0165] In some embodiments, the poly-A tail is designed relative to the length of the overall mRNA. This design may be based on the length of the region coding for a target of interest, the length of a particular feature or region (such as a flanking region), or based on the length of the ultimate product expressed from the mRNA.

[0166] In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the mRNA or feature thereof. The poly-A tail may also be designed as a fraction of mRNA to which it belongs. In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A tail. Further, engineered binding sites and conjugation of mRNA for poly-A binding protein may enhance expression.

[0167] Additionally, multiple distinct mRNA may be linked together to the PABP (Poly-A binding protein) through the 3'-end using modified nucleotides at the 3 '-terminus of the poly-A tail. Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72 hr and day 7 post-transfection.

[0168] In some embodiments, the mRNA are designed to include a polyA-G Quartet. The G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail. Stop Codons

[0169] In various embodiments, the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise one or more translation stop codons. Translational stop codons, UAA, UAG, and UGA, are an important component of the genetic code and signal the termination of translation of an mRNA. During protein synthesis, stop codons interact with protein release factors and this interaction can modulateribosomal activity thus having an impact translation (Tate WP, et al., (2018) Biochem Soc Trans, 46(6):1615-162).

[0170] A stop element as used herein, refers to a nucleic acid sequence comprising a stop codon. The stop codon can be selected from TGA, TAA and TAG in the case of DNA, or from UGA, UAA and UAG in the case of RNA. In an embodiment, a stop element comprises two consecutive stop codons. In an embodiment, a stop element comprises three consecutive stop codons. In an embodiment, a stop element comprises four consecutive stop codons. In an embodiment, a stop element comprises five consecutive stop codons.

[0171] In some embodiments, the mRNA may include one stop codon. In some embodiments, the mRNA may include two stop codons. In some embodiments, the mRNA may include three stop codons. In some embodiments, the mRNA may include at least one stop codon. In some embodiments, the mRNA may include at least two stop codons. In some embodiments, the mRNA may include at least three stop codons. As non-limiting examples, the stop codon may be selected from TGA, TAA and TAG.

[0172] In other embodiments, the stop codon may be selected from one or more of the following stop elements of Table Y: Table Y: Additional stop elements

[0173] In some embodiments, the mRNA includes the stop codon TGA and one additional stop codon. In a further embodiment the addition stop codon may be TAA.MicroRNA binding sites and other regulatory elements

[0174] In various embodiments, the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein, may comprise one or more regulatory elements, including, but not limited to microRNA (miRNA) binding sites, structured mRNA sequences and / or motifs, artificial binding sites to bind to endogenous nucleic acid binding molecules, and combinations thereof. Chemically unmodified nucleotides

[0175] In some embodiments, the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein are not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g. A, G, C, or U). In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC, or dT). Chemically modified nucleotides

[0176] In some embodiments, the mRNA payloads of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein comprise, in some embodiments, comprises at least one chemical modification.

[0177] The terms “chemical modification” and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population. Generally, these terms do not refer to the ribonucleotide modifications in naturally occurring 5′-terminal mRNA cap moieties. With respect to a polypeptide, the term “modification” refers to a modification relative to the canonical set 20 amino acids. Polypeptides, as provided herein, are also considered “modified” of they contain amino acid substitutions, insertions or a combination of substitutions and insertions.

[0178] Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise various (more than one) different modifications. In some embodiments, a particular region of a polynucleotide contains one, two or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified polynucleotide. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response).

[0179] Modifications of polynucleotides include, without limitation, those described herein. Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) may comprisemodifications that are naturally-occurring, non-naturally-occurring or the polynucleotide may comprise a combination of naturally-occurring and non-naturally-occurring modifications. Polynucleotides may include any useful modification, for example, of a sugar, a nucleobase, or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage or to the phosphodiester backbone).

[0180] Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post- synthesis of the polynucleotides to achieve desired functions or properties. The modifications may be present on an internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.

[0181] The present disclosure provides for modified nucleosides and nucleotides of a polynucleotide (e.g., RNA polynucleotides, such as mRNA polynucleotides). A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.

[0182] Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non- standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base / sugar or linker may be incorporated into polynucleotides of the present disclosure.

[0183] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0184] In some embodiments, modified nucleobases in polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are selected from the group consisting of pseudouridine (ψ), N1- methylpseudouridine (m1ψ), N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine. In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0185] In some embodiments, modified nucleobases in polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are selected from the group consisting of 1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine and α-thio-adenosine. In some embodiments, polynucleotides includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0186] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise pseudouridine (ψ) and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 1-methyl- pseudouridine (m1ψ). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 2-thiouridine (s2U). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise methoxy-uridine (mo5U). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 2′-O-methyl uridine. In some embodiments polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise 2′-O-methyl uridine and 5-methyl-cytidine (m5C). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise N6-methyl-adenosine (m6A). In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise N6-methyl-adenosine (m6A) and 5-methyl-cytidine (mC).

[0187] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a polynucleotide can be uniformly modified with 5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5C). Similarly, a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

[0188] Exemplary nucleobases and nucleosides having a modified cytosine include N4-acetyl- cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl- cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.

[0189] In some embodiments, a modified nucleobase is a modified uridine. Exemplary nucleobases and In some embodiments, a modified nucleobase is a modified cytosine. nucleosides having a modified uridine include 5-cyano uridine, and 4′-thio uridine.

[0190]

[0191] The polynucleotides of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a polynucleotide of the disclosure, or in a given predetermined sequence region thereof (e.g., in the mRNA including or excluding the polyA tail). In some embodiments, all nucleotides X in a polynucleotide of the present disclosure (or in a given sequence region thereof) are modified nucleotides, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+CorA+G+C.

[0192] The polynucleotide may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

[0193]

[0194] The polynucleotides may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the polynucleotides may contain a modified pyrimidine such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the polynucleotide is replaced with a modified uracil (e.g., a 5-substituted uracil). The modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality ofcompounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the polynucleotide is replaced with a modified cytosine (e.g., a 5-substituted cytosine). The modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). C. Circular mRNA payloads

[0195] In various embodiments, the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions thereof described herein can be used to deliver an RNA payload that is a circular mRNA molecule or “oRNA.” The circular mRNA molecule may encode a CROI, such as a nucleobase editing system, or therapeutic protein as described in this specification.

[0196] In some embodiments, the RNA payload is a circular RNA (oRNA). As used herein, the terms “oRNA” or “circular RNA” are used interchangeably and can refer to a RNA that forms a circular structure through covalent or non-covalent bonds.

[0197] Circular RNA described herein are polyribonucleotides that form a continuous structure through covalent or non-covalent bonds. Due to the circular structure, oRNAs have improved stability, increased half-life, reduced immunogenicity, and / or improved functionality (e.g., of a function described herein) compared to a corresponding linear RNA.

[0198] In some embodiments, an oRNA binds a target. In some embodiments, an oRNA binds a substrate. In some embodiments, an oRNA binds a target and binds a substrate of the target. In some embodiments, an oRNA binds a target and mediates modulation of a substrate of the target. In some embodiments, an oRNA brings together a target and its substrate to mediate modification of the substrate, e.g., post-translational modification. In some embodiments, an oRNA brings together a target and its substrate to mediate a cellular process (e.g., alters protein degradation or signal transduction) involving the substrate. In some embodiments, a target is a target protein and a substrate is a substrate protein.

[0199] In some embodiments, an oRNA comprises a conjugation moiety for binding to a chemical compound. The conjugation moiety can be a modified polyribonucleotide. The chemical compound can be conjugated to the oRNA by the conjugation moiety. In some embodiments, the chemical compound binds to a target and mediates modulation of a substrate of the target. In some embodiments, an oRNA binds a substrate of a target and a chemical compound conjugated to the oRNA by the conjugation moiety binds the target to bring together the target and its substrate to mediate modification of the substrate, e.g., post-translational modification. In some embodiments, an oRNA binds a substrate of a target and a chemical compound conjugated to the oRNA by the conjugation moiety binds the target to bring together the target and its substrate to mediate modification of the substrate to mediate a cellular process (e.g., alters protein degradation or signaltransduction) involving the substrate. In some embodiments, a target is a target protein and a substrate is a substrate protein.

[0200] In some embodiments, the oRNA may be non-immunogenic in a mammal (e.g., a human, non-human primate, rabbit, rat, and mouse).

[0201] In some embodiments, the oRNA may be capable of replicating or replicates in a cell from an aquaculture animal (e.g., fish, crabs, shrimp, oysters etc.), a mammalian cell, a cell from a pet or zoo animal (e.g., cats, dogs, lizards, birds, lions, tigers and bears etc.), a cell from a farm or working animal (e.g., horses, cows, pigs, chickens etc.), a human cell, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (e.g., normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non- mitotic cells, or any combination thereof.

[0202] In one aspect, provided herein is a pharmaceutical composition comprising: a circular RNA comprising, in the following order, a 3’ group I intron fragment, an Internal Ribosome Entry Site (IRES), an expression sequence encoding a polypeptide (e.g., a nucleobase editing system, therapeutic protein, such as a chimeric antigen receptor (CAR) or T cell receptor (TCR) complex protein), and a 5’ group I intron fragment, and a transfer vehicle comprising at least one of (i) an ionizable lipid, (ii) a structural lipid, and (iii) a PEG-modified lipid, wherein the transfer vehicle is capable of delivering the circular RNA polynucleotide to a cell (e.g., a human cell, such as an immune cell present in a human subject), such that the polypeptide is translated in the cell.

[0203] In some embodiments, the pharmaceutical composition is formulated for intravenous administration to the human subject in need thereof. In some embodiments, the 3’ group I intron fragment and 5’ group I intron fragment are Anabaena group I intron fragments.

[0204] In certain embodiments, the 3’ intron fragment and 5’ intron fragment are defined by the L9a-5 permutation site in the intact intron. In certain embodiments, the 3’ intron fragment and 5’ intron fragment are defined by the L8-2 permutation site in the intact intron.

[0205] In some embodiments, the IRES is from Taura syndrome virus, Tiiatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus , Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, HumanBCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIFl alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E / D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SHI, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA 16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV- PK15C, SF573 Dicistravirus, Hubei Picoma-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24 or an aptamer to eIF4G.

[0206] In some embodiments, the IRES comprises a CVB3 IRES or a fragment or variant thereof. In some embodiments, the pharmaceutical composition comprises a first internal spacer between the 3’ group I intron fragment and the IRES, and a second internal spacer between the expression sequence and the 5’ group I intron fragment. In certain embodiments, the first and second internal spacers each have a length of about 10 to about 60 nucleotides.

[0207] In some embodiments, the circular mRNA comprises a nucleotide sequence encoding a polypeptide of interest, such as a nucleobase editing system or therapeutic protein (e.g., a CAR or TCR complex protein).

[0208] In embodiments where the therapeutic protein encoded by the herein RNA payload (e.g., circular or linear mRNA) is a CAR or TCR complex protein, the CAR or TCR complex protein comprises an antigen binding domain specific for an antigen selected from the group: CD 19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule- 1, CD33, epidermal growth factor receptor variant III (EGFRvIII), disialoganglioside GD2, disaloganglioside GD3, TNF receptor family member, B cell maturation antigen (BCMA), Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)), prostate- specific membrane antigen (PSMA), Receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms- Like Tyrosine Kinase 3 (FLT3), Tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, Carcinoembryonic antigen (CEA), Epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD 117), Interleukin- 13 receptor subunit alpha-2, mesothelin, Interleukin 11 receptor alpha (IL-l lRa), prostate stem cell antigen (PSCA), Protease Serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis(Y) antigen, CD24, Platelet-derived growth factor receptor beta(PDGFR-beta), Stage-specific embryonic antigen-4 (SSEA-4), CD20, Folate receptor alpha, HER2, HER3, Mucin 1, cell surface associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), Prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), Ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2), glycoprotein 100 (gplOO), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type- A receptor 2 (EphA2), Fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight-melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (0AcGD2), Folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7 -related (TEM7R), claudin 6 (CLDN6), claudin 18.2 (CLDN18.2), thyroid stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, and CD179a.

[0209] In further embodiments where the therapeutic protein encoded by the herein RNA payload (e.g., circular or linear mRNA) is a CAR or TCR complex protein, the CAR or TCR complex protein comprises a CAR comprising an antigen binding domain specific for CD19. In some embodiments, the CAR or TCR complex protein comprises a CAR comprising a costimulatory domain selected from the group CD28, 4-1BB, 0X40, CD27, CD30, ICOS, GITR, CD40, CD2, SLAM, and combinations thereof. In some embodiments, the CAR or TCR complex protein comprises a CAR comprising a CD3zeta signaling domain. In some embodiments, the CAR or TCR complex protein comprises a CAR comprising a CH2CH3, CD28, and / or CD8 spacer domain. In some embodiments, the CAR or TCR complex protein comprises a CAR comprising a CD28 or CD8 transmembrane domain.

[0210] In some embodiments, the CAR or TCR complex protein comprises a CAR comprising: an antigen binding domain, a spacer domain, a transmembrane domain, a costimulatory domain, and an intracellular T cell signaling domain.

[0211] In some embodiments, the CAR or TCR complex protein comprises a multispecific CAR comprising antigen binding domains for at least two different antigens. In some embodiments, the CAR or TCR complex protein comprises a TCR complex protein selected from the group TCRalpha, TCRbeta, TCRgamma, and TCRdelta.

[0212] In some embodiments, the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein further comprise a targeting moiety. In certain embodiments, the targeting moiety mediates receptor-mediated endocytosis or direct fusion of the delivery vehicle (LNPs) into selected cells of a selected cell population or tissue in the absence of cell isolation or purification. In certain embodiments, the targeting moiety is capable of binding to a protein selected from the group CD3, CD4, CD8, CDS, CD7, PD-1, 4-1BB, CD28, Clq, and CD2. Incertain embodiments, the targeting moiety comprises an antibody specific for a macrophage, dendritic cell, NK cell, NKT, or T cell antigen. In certain embodiments, the targeting moiety comprises a scFv, nanobody, peptide, minibody, polynucleotide aptamer, heavy chain variable region, light chain variable region or fragment thereof.

[0213] In some embodiments, the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein are administered in an amount effective to treat a disease in the human subject (e.g., wherein the disease can be cancer, muscle disorder, or CNS disorder, etc.). In some embodiments, the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions have an enhanced safety profile when compared to a pharmaceutical composition comprising T cells or vectors comprising exogenous DNA encoding the same polypeptide, e.g., a CAR complex protein.

[0214] In some embodiments, the LNP-based nucleobase editing systems and pharmaceutical compositions thereof are administered in an amount effective to mount an immunogenic response in a human subject for the vaccination against an infectious agent and / or cancer. In some embodiments, the LNP-based nucleobase editing systems and pharmaceutical compositions have an enhanced safety profile when compared to state of the art gene editing delivery compositions.

[0215] In another aspect, the present disclosure provides a circular RNA comprising, in the following order, a 3’ group I intron fragment, an Internal Ribosome Entry Site (IRES), an expression sequence encoding a polypeptide (e.g., a nucleobase editing system, therapeutic protein, such as a chimeric antigen receptor (CAR) or T cell receptor (TCR) complex protein), and a 5’ group I intron fragment.

[0216] In some embodiments, the 3’ group I intron fragment and 5’ group I intron fragment are Anabaena group I intron fragments. In certain embodiments, the 3’ intron fragment and 5’ intron fragment are defined by the L9a-5 permutation site in the intact intron. In certain embodiments, the 3’ intron fragment and 5’ intron fragment are defined by the L8-2 permutation site in the intact intron. In certain embodiments, the IRES comprises a CVB3 IRES or a fragment or variant thereof.

[0217] In some embodiments, the circular RNA comprises a first internal spacer between the 3’ group I intron fragment and the IRES, and a second internal spacer between the expression sequence and the 5’ group I intron fragment.

[0218] In certain embodiments, the first and second internal spacers each have a length of about 10 to about 60 nucleotides.

[0219] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein consists of natural nucleotides. In some embodiments, the circular RNA further comprises a second expression sequence encoding a therapeutic protein. In some embodiments, the therapeutic protein comprises a checkpoint inhibitor. In certain embodiments, the therapeutic protein comprises a cytokine.

[0220] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein consists of natural nucleotides.

[0221] In some embodiments, the circular RNA payload LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein comprises a nucleotide sequence that is codon optimized, either partially or fully. In some embodiments, the circular RNA is optimized to lack at least one microRNA binding site present in an equivalent pre-optimized polynucleotide. In some embodiments, the circular RNA is optimized to lack at least one endonuclease susceptible site present in an equivalent pre-optimized polynucleotide. In some embodiments, the circular RNA is optimized to lack at least one RNA-editing susceptible site present in an equivalent pre-optimized polynucleotide.

[0222] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein has an in vivo functional half- life in humans greater than that of an equivalent linear RNA having the same expression sequence. In some embodiments, the circular RNA has a length of about 100 nucleotides to about 10 kilobases. In some embodiments, the circular RNA has a functional half-life of at least about 20 hours. In some embodiments, the circular RNA has a duration of therapeutic effect in a human cell of at least about 20 hours. In some embodiments, the circular RNA has a duration of therapeutic effect in a human cell greater than or equal to that of an equivalent linear RNA comprising the same expression sequence. In some embodiments, the circular RNA has a functional half-life in a human cell greater than or equal to that of an equivalent linear RNA comprising the same expression sequence.

[0223] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein has a half-life of at least that of a linear counterpart. In some embodiments, the oRNA has a half-life that is increased over that of a linear counterpart. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or greater. In some embodiments, the oRNA has a half- life or persistence in a cell for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer or any time therebetween. In some embodiments, the oRNA has a half-life or persistence in a cell for no more than about 10 mins to about 7 days, or no more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours (1 day), 36hours (1.5 days), 48 hours (2 days),60 hours (2.5 days), 72 hours (3 days), 4 days, 5 days, 6 days, or 7 days.

[0224] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein has a half-life or persistence in a cell while the cell is dividing. In some embodiments, the oRNA has a half-life or persistence in a cell post division.

[0225] In certain embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein has a half-life or persistence in a dividing cell for greater than about 10 minutes to about 30 days, or at least about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer or any time therebetween.

[0226] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein modulates a cellular function, e.g., transiently or long term. In certain embodiments, the cellular function is stably altered, such as a modulation that persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer. In certain embodiments, the cellular function is transiently altered, e.g., such as a modulation that persists for no more than about 30 mins to about 7 days, or no more than about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours(3 days), 4 days, 5 days, 6 days, or 7 days.

[0227] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides. In some embodiments, the oRNA may be of a sufficient size to accommodate a binding site for a ribosome.

[0228] In some embodiments, the maximum size of the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein may be limited by the ability of packaging and delivering the RNA to a target. In some embodiments, the size of the oRNA is a length sufficient to encode polypeptides, and thus, lengths of at least 20,000 nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, or at least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, at least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, at least 100 nucleotides may be useful.

[0229] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein comprises one or more elements described elsewhere herein. In some embodiments, the elements may be separated from one another by a spacer sequence or linker. In some embodiments, the elements may be separated from one another by 1 nucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 80 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, up to about 1 kb, at least about 1000 nucleotides.

[0230] In some embodiments, one or more elements are contiguous with one another, e.g., lacking a spacer element.

[0231] In some embodiments, one or more elements is conformationally flexible. In some embodiments, the conformational flexibility is due to the sequence being substantially free of a secondary structure.

[0232] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein comprises a secondary or tertiary structure that accommodates a binding site for a ribosome, translation, or rolling circle translation.

[0233] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein comprises particular sequence characteristics. For example, the oRNA may comprise a particular nucleotide composition. In some such embodiments, the oRNA may include one or more purine rich regions (adenine or guanosine). In some such embodiments, the oRNA may include one or more purine rich regions(adenine or guanosine).In some embodiments, the oRNA may include one or more AU rich regions or elements (AREs). In some embodiments, the oRNA may include one or more adenine rich regions.

[0234] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein comprises one or more modifications described elsewhere herein.

[0235] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein comprises one or more expression sequences and is configured for persistent expression in a cell of a subject in vivo. In some embodiments, the oRNA is configured such that expression of the one or more expression sequences in the cell at a later time point is equal to or higher than an earlier time point. In such embodiments, the expression of the one or more expression sequences can be either maintained at a relatively stable level or can increase over time. The expression of the expression sequences can be relatively stable for an extended period of time. For instance, in some cases, the expression of the one or more expression sequences in the cell over a time period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days does not decrease by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In some cases, in some cases, the expression of the one or more expression sequences in the cell is maintained at a level that does not vary by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% for at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days. Regulatory Elements

[0236] In some embodiments, the circular RNA payload of the LNP-based nucleobase editing systems, RNA therapeutics and pharmaceutical compositions described herein comprises one or more regulatory elements. As used herein, a "regulatory element" is a sequence that modifies expression of an expression sequence, e.g., a nucleotide sequence encoding a nucleobase editing system or a therapeutic protein, i.e., a coding region of interest (CROI). The regulatory element may include a sequence that is located adjacent to a coding region of interest encoded on the circular RNA payload. The regulatory element may be operatively linked to a nucleotide sequence of the circular RNA that encodes a coding region of interest (e.g., a nucleobase editing system or therapeutic polypeptide).

[0237] In some embodiments, a regulatory element may increase an amount of expression of a coding region of interest encoded on the circular RNA payload as compared to an amount expressed when no regulatory element exists.

[0238] In some embodiments, a regulatory element may comprise a sequence to selectively initiates or activates translation of a coding sequence of interest encoded on the circular RNA payload.

[0239] In some embodiments, a regulatory element may comprise a sequence to initiate degradation of the oRNA or the payload or cargo. Non-limiting examples of the sequence to initiate degradation includes, but is not limited to, riboswitch aptazyme and miRNA binding sites.

[0240] In some embodiments, a regulatory element can modulate translation of a coding region of interest encoded on the oRNA. The modulation can create an increase (enhancer) or decrease (suppressor) in the expression of the coding region of interest. The regulatory element may be located adjacent to the CROI (e.g., on one side or both sides of the CROI). Translation Initiation Sequence

[0241] In some embodiments, a translation initiation sequence functions as a regulatory element. In some embodiments, the translation initiation sequence comprises an AUG / ATG codon. In some embodiments, a translation initiation sequence comprises any eukaryotic start codon such as, but not limited to, AUG / ATG, CUG / CTG, GUG / GTG, UUG / TTG, ACG, AUC / ATC, AUU, AAG, AUA / ATA, or AGG. In some embodiments, a translation initiation sequence comprises a Kozak sequence. In some embodiments, translation begins at an alternative translation initiation sequence, e.g., translation initiation sequence other than AUG / ATG codon, under selective conditions, e.g., stress induced conditions. As a non-limiting example, the translation of the circular polyribonucleotide may begin at alternative translation initiation sequence, such as ACG. As another non-limiting example, the circular polyribonucleotide translation may begin at alternative translationinitiation sequence, CUG / CTG. As another non-limiting example, the translation may begin at alternative translation initiation sequence, GUG / GTG. As yet another non-limiting example, the translation may begin at a repeat-associated non-AUG (RAN) sequence,such as an alternative translation initiation sequence that includes short stretches of repetitive RNA e.g. CGG, GGGGCC, CAG, CTG.

[0242] In some embodiments, the oRNA encodes a polypeptide or peptide and may comprise a translation initiation sequence. The translation initiation sequence may comprise, but is not limited to a start codon, a non-coding start codon, a Kozak sequence or a Shine-Dalgarno sequence. The translation initiation sequence may be located adjacent to the payload or cargo (e.g., on one side or both sides of the coding region of interest).

[0243] In some embodiments, the translation initiation sequence provides conformational flexibility to the oRNA. In some embodiments, the translation initiation sequence is within a substantially single stranded region of the oRNA.

[0244] The oRNA may include more than 1 start codon such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or more than 15 start codons. Translation may initiate on the first start codon or may initiate downstream of the first start codon.

[0245] In some embodiments, the oRNA may initiate at a codon which is not the first start codon, e.g., AUG. Translation of the circular polyribonucleotide may initiate at an alternative translation initiation sequence, such as, but not limited to, ACG, AGG, AAG, CUG / CTG, GUG / GTG, AUA / ATA, AUU / ATT, UUG / TTG. In some embodiments, translation begins at an alternativetranslation initiation sequence under selective conditions, e.g., stress induced conditions. As a non- limiting example, the translation of the oRNA may begin at alternative translation initiation sequence, such as ACG. As another non-limiting example, the oRNA translation may begin at alternative translation initiation sequence, CUG / CTG. As yet another non-limiting example, the oRNA translation may begin at alternative translation initiation sequence, GTG / GUG. As yet another non- limiting example, the oRNA may begin translation at a repeat-associated non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA e.g. CGG, GGGGCC, CAG, CTG. IRES Sequences

[0246] In some embodiments, the oRNA described herein comprises an internal ribosome entry site (IRES) element capable of engaging an eukaryotic ribosome. In some embodiments, the IRES element is at least about 5 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 350 nucleotides, or at least about 500 nucleotides. In one embodiment, the IRES element is derived from the DNA of an organism including, but not limited to, a virus, a mammal, and a Drosophila. Such viral DNA may be derived from, but is not limited to, picornavirus complementary DNA (cDNA), with encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA. In one embodiment, Drosophila DNA from which an IRES element is derived includes, but is not limited to, an Antennapedia gene from Drosophila melanogaster.

[0247] In some embodiments, the IRES element is at least partially derived from a virus, for instance, it can be derived from a viral IRES element, such as ABPV_IGRpred, AEV, ALPV_IGRpred, BQCV_IGRpred, BVDV1_1-385, BVDV1_29-391, CrPV_5NCR, CrPV_IGR, crTMV_IREScp, crTMV_IRESmp75, crTMV_IRESmp228, crTMV_IREScp, crTMV_IREScp, CSFV, CVB3, DCV_IGR, EMCV-R, EoPV_5NTR, ERAV 245-961, ERBV 162-920, EV71_1-748, FeLV-Notch2, FMDV_type_C, GBV-A, GBV-B, GBV-C, gypsy_env, gypsyD5, gypsyD2, HAV_HM175, HCV_type_1a, HiPV_IGRpred, HIV-1, HoCV1_IGRpred, HRV-2, IAPV_IGRpred, idefix, KBV_IGRpred, LINE-1_ORF1_-101_to_-1, LINE-1_ORF1-302_to_-202, LINE-1_ORF2- 138_to_-86, LINE-1_ORF1_-44to_-1, PSIV_IGR, PV_type1_Mahoney,PV_type3_Leon, REV-A, RhPV_5NCR, RhPV_IGR, SINV1_IGRpred, SV40_661-830, TMEV, TMV_UI_IRESmp228, TRV_5NTR, TrV_IGR, or TSV_IGR. In some embodiments, the IRES element is at least partially derived from a cellular IRES, such as AML1 / RUNX1, Antp-D, Antp-DE, Antp-CDE, Apaf-1, Apaf-1, AQP4, AT1R_var1, AT1R_var2, AT1R_var3, AT1R_var4, BAG1_p36delta236 nt, BAG1_p36, BCL2, BiP_-222_-3, c-IAP1_285-1399, c-IAP1_1313-1462, c-jun, c-myc, Cat-1224, CCND1, DAPS, eIF4G, eIF4GI-ext, eIF4GII, eIF4GII-long, ELG1, ELH, FGF1A,FMR1, Gtx-133-141, Gtx-1-166,Gtx-1-120, Gtx-1-196, hairless, HAP4, HIF1a, hSNM1, Hsp101, hsp70, hsp70, Hsp90, IGF2_leader2, Kv1.4_1.2, L-myc, LamB1_-335_-1, LEF1, MNT_75-267, MNT_36-160, MTG8a, MYB, MYT2_997-1152, n-MYC, NDST1, NDST2, NDST3, NDST4L, NDST4S, NRF_-653_-17, NtHSF1, ODC1, p27kip1, 03_128-269, PDGF2 / c-sis, Pim-1, PITSLRE_p58, Rbm3, reaper, Scamper, TFIID, TIF4631, Ubx_1-966, Ubx_373-961, UNR, Ure2, UtrA, VEGF-A-133-1, XIAP_5-464, XIAP_305- 466, or YAP1.

[0248] In another embodiment, the IRES is an IRES sequence from Coxsackievirus B3 (CVB3), the protein coding region encodes Guassia luciferase (Gluc) and the spacer sequences are polyA-C.

[0249] In some embodiments, the IRES, if present, is at least about 50 nucleotides in length. In one embodiment, the vector comprises an IRES that comprises a natural sequence. In one embodiment, the vector comprises an IRES that comprises a synthetic sequence.

[0250] An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. A polynucleotide containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes (e.g., multicistronic mRNA). When polynucleotides are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the present disclosure include without limitation, those from picornaviruses (e.g., FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical Swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV). Termination Element

[0251] In some embodiments, the oRNA includes one or more coding regions of interest (i.e., also referred to as product expression sequences) which encode polypeptides of interest, including but not limited to nucleobase editing system and therapeutic proteins. In various embodiments, the product expression sequences may or may not have a termination element.

[0252] In some embodiments, the oRNA includes one or more product expression sequences that lack a termination element, such that the oRNA is continuously translated.

[0253] Exclusion of a termination element may result in rolling circle translation or continuous expression of the encoded peptides or polypeptides as the ribosome will not stall or fall-off. In such an embodiment, rolling circle translation expresses continuously through the product expression sequence.

[0254] In some embodiments, one or more product expression sequences in the oRNA comprise a termination element.

[0255] In some embodiments, not all of the product expression sequences in the oRNA comprise a termination element. In such instances, the product expression sequence may fall off the ribosome when the ribosome encounters the termination element and terminates translation.Rolling Circle Translation

[0256] In some embodiments, once translation of the oRNA is initiated, the ribosome bound to the oRNA does not disengage from the oRNA before finishing at least one round of translation of the oRNA. In some embodiments, the oRNA as described herein is competent for rolling circle translation. In some embodiments, during rolling circle translation, once translation of the oRNA is initiated, the ribosome bound to the oRNA does not disengage from the oRNA before finishing at least 2 rounds, at least 3 rounds, at least 4 rounds, at least 5 rounds, at least 6 rounds, at least 7 rounds, at least 8 rounds,at least 9 rounds, at least 10 rounds, at least 11 rounds, at least 12 rounds, at least 13 rounds, at least 14 rounds, at least 15 rounds, at least 20 rounds, at least 30 rounds, at least 40 rounds, at least 50 rounds, at least 60 rounds, at least 70 rounds, at least 80 rounds, at least 90 rounds, at least 100 rounds, at least 150 rounds, at least 200 rounds, at least 250 rounds, at least 500 rounds, at least 1000 rounds, at least 1500 rounds, at least 2000 rounds, at least 5000 rounds, at least 10000 rounds, at least 10.sup.5 rounds, or at least 10.sup.6 rounds of translation of the oRNA.

[0257] In some embodiments, the rolling circle translation of the oRNA leads to generation of polypeptide that is translated from more than one round of translation of the oRNA. In some embodiments, the oRNA comprises a stagger element, and rolling circle translation of the oRNA leads to generation of polypeptide product that is generated from a single round of translation or less than a single round of translation of the oRNA. Circularization

[0258] In one embodiment, a linear RNA may be cyclized, or concatemerized. In some embodiments, the linear RNA may be cyclized in vitro prior to formulation and / or delivery. In some embodiments, the linear RNA may be cyclized within a cell.

[0259] In some embodiments, the mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed. The newly formed 5'- / 3'-linkage may be intramolecular or intermolecular.

[0260] In the first route, the 5'-end and the 3 '-end of the nucleic acid contain chemically reactive groups that, when close together, form a new covalent linkage between the 5 '-end and the 3 '-end of the molecule. The 5 '-end may contain an NHS-ester reactive group and the 3 '-end may contain a 3'- amino-terminated nucleotide such that in an organic solvent the 3'-amino-terminated nucleotide on the 3 '-end of a synthetic mRNA molecule will undergo a nucleophilic attack on the 5 '-NHS-ester moiety forming a new 5 '- / 3 '-amide bond.

[0261] In the second route, T4 RNA ligase may be used to enzymatically link a 5'-phosphorylated nucleic acid molecule to the 3'-hydroxyl group of a nucleic acid forming a new phosphorodiester linkage. In an example reaction, ^g of a nucleic acid molecule is incubated at 37°C for 1 hour with 1- 10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) according to the manufacturer'sprotocol. The ligation reaction may occur in the presence of a split oligonucleotide capable of base- pairing with both the 5'-and 3'-region in juxtaposition to assist the enzymatic ligation reaction.

[0262] In the third route, either the 5 '-or 3 '-end of the cDNA template encodes a ligase ribozyme sequence such that during in vitro transcription, the resultant nucleic acid molecule can contain an active ribozyme sequence capable of ligating the 5 '-end of a nucleic acid molecule to the 3 '-end of a nucleic acid molecule. The ligase ribozyme may be derived from the Group I Intron, Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment). The ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37°C.

[0263] In some embodiments, the oRNA is made via circularization of a linear RNA.

[0264] In some embodiments, the following elements are operably connected to each other and, in some embodiments, arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a protein coding or noncoding region, d.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and e.) a 3′ homology arm. In certain embodiments said vector allows production of a circular RNA that is translatable and / or biologically active inside eukaryotic cells. In some embodiments, the biologically active RNA is, for example, an miRNA sponge, or long noncoding RNA.

[0265] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) optionally, a 5′ spacer sequence, d.) optionally, an internal ribosome entry site (IRES), e.) a protein coding or noncoding region, f.) optionally, a 3′ spacer sequence, g.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and h.) a 3′ homology arm. In certain embodiments, said vector allows production of a circular RNA that is translatable and / or biologically active inside eukaryotic cells.

[0266] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a 5′ spacer sequence, d.) an internal ribosome entry site (IRES), e.) a protein coding or noncoding region, f.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm. In some embodiments, said vector allows production of a circular RNA that is translatable and / or biologically active inside eukaryotic cells.

[0267] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a 5′ spacer sequence, d.) a protein coding or noncoding region, e.) a 3′ spacer sequence, f.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm. In some embodiments, said vector allows production of a circular RNA that is translatable and / or biologically active inside eukaryotic cells.

[0268] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) an internal ribosome entry site (IRES), d.) a protein coding or noncoding region, e.) a 3′ spacer sequence, f) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm. In some embodiments, said vector allows production of a circular RNA that is translatable and / or biologically active inside eukaryotic cells.

[0269] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a protein coding or noncoding region, d.) a 3′ spacer sequence, e.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and f.) a 3′ homology arm. In some embodiments, said vector allows production of a circular RNA that is translatable and / or biologically active inside eukaryotic cells.

[0270] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a 5′ spacer sequence, d.) a protein coding or noncoding region, e.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and f.) a 3′ homology arm. In some embodiments, said vector allows production of a circular RNA that is translatable and / or biologically active inside eukaryotic cells.

[0271] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) an internal ribosome entry site (IRES), d.) a protein coding or noncoding region, e.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and f) a 3′ homology arm. In some embodiments, said vector allows production of a circular RNA that is translatable and / or biologically active inside eukaryotic cells.

[0272] In some embodiments, the following elements are operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) a 5′ spacer sequence, d.) an internal ribosome entry site (IRES), e.) a protein coding or noncoding region, f) a 3′ spacer sequence, g.) a 5′ group I intron fragment containing a 5′ splice site dinucleotide, and h.) a 3′ homology arm. In some embodiments, said vector allowing production of a circular RNA that is translatable and / or biologically active inside eukaryotic cells.

[0273] In one embodiment, the 3′ group I intron fragment and / or the 5′ group I intron fragment is from a Cyanobacterium Anabaena sp. pre-tRNA-Leu gene or T4 phage Td gene.

[0274] In one embodiment, the 3′ group I intron fragment and / or the 5′ group I intron fragment is from a Cyanobacterium Anabaena sp. pre-tRNA-Leu gene.

[0275] In one embodiment, the protein coding region encodes a protein of eukaryotic or prokaryotic origin. In another embodiment, the protein coding region encodes human protein or non-human protein. In some embodiments, the protein coding region encodes one or more antibodies. For example, in some embodiments, the protein coding region encodes human antibodies. In one embodiment, the protein coding region encodes a protein selected from hFIX, SP-B, VEGF-A, human methylmalonyl-CoA mutase (hMUT), CFTR, cancer self-antigens, and additional gene editing enzymes like Cpf1, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). In another embodiment, the protein coding region encodes a protein for therapeutic use. In one embodiment, the human antibody encoded by the protein coding region is an anti-HIV antibody. In one embodiment, the antibody encoded by the protein coding region is a bispecific antibody. In one embodiment, the bispecific antibody is specific for CD19 and CD22. In another embodiment, the bispecific antibody is specific for CD3 and CLDN6. In one embodiment, the protein coding region encodes a protein for diagnostic use. In one embodiment, the protein coding region encodes Gaussia luciferase (Gluc), Firefly luciferase (Fluc), enhanced green fluorescent protein (eGFP), human erythropoietin (hEPO), or Cas9 endonuclease.

[0276] In one embodiment, the 5′ homology arm is about 5-50 nucleotides in length. In another embodiment, the 5′ homology arm is about 9-19 nucleotides in length. In some embodiments, the 5′ homology arm is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the 5′ homology arm is no more than 50, 45, 40, 35, 30, 25 or 20 nucleotides in length. In some embodiments, the 5′ homology arm is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length.

[0277] In one embodiment, the 3′ homology arm is about 5-50 nucleotides in length. In another embodiment, the 3′ homology arm is about 9-19 nucleotides in length. In some embodiments, the 3′ homology arm is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the 3′ homology arm is no more than 50, 45, 40, 35, 30, 25 or 20 nucleotides in length. In some embodiments, the 3′ homology arm is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length.

[0278] In one embodiment, the 5′ spacer sequence is at least 10 nucleotides in length. In another embodiment, the 5′ spacer sequence is at least 15 nucleotides in length. In a further embodiment, the 5′ spacer sequence is at least 30 nucleotides in length. In some embodiments, the 5′ spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the 5′ spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 5′ spacer sequence is between 20 and 50 nucleotides in length. In certain embodiments, the 5′ spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49 or 50 nucleotides in length. In one embodiment, the 5′ spacer sequence is a polyA sequence. In another embodiment, the 5′ spacer sequence is a polyA-C sequence.

[0279] In one embodiment, the 3′ spacer sequence is at least 10 nucleotides in length. In another embodiment, the 3′ spacer sequence is at least 15 nucleotides in length. In a further embodiment, the 3′ spacer sequence is at least 30 nucleotides in length. In some embodiments, the 3′ spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the 3′ spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some embodiments the 3′ spacer sequence is between 20 and 50 nucleotides in length. In certain embodiments, the 3′ spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In one embodiment, the 3′ spacer sequence is a polyA sequence. In another embodiment, the 5′ spacer sequence is a polyA-C sequence. Extracellular Circularization

[0280] In some embodiments, the linear RNA is cyclized, or concatemerized using a chemical method to form an oRNA. In some chemical methods, the 5'-end and the 3'-end of the nucleic acid (e.g., a linear RNA) includes chemically reactive groups that, when close together, may form a new covalent linkage between the 5'-end and the 3'-end of the molecule. The 5'-end may contain an NHS- ester reactive group and the 3'-end may contain a 3'-amino-terminated nucleotide such that in an organic solvent the 3'-amino-terminated nucleotide on the 3'-end of a linear RNA will undergo a nucleophilic attack on the 5'-NHS-ester moiety forming a new 5'- / 3'-amide bond.

[0281] In one embodiment, a DNA or RNA ligase may be used to enzymatically link a 5'- phosphorylated nucleic acid molecule (e.g., a linear RNA) to the 3'-hydroxyl group of a nucleic acid (e.g., a linear nucleic acid) forming a new phosphorodiester linkage. In an example reaction, a linear RNA is incubated at 37C for 1 hour with 1-10 units of T4 RNA ligase according to the manufacturer's protocol. The ligation reaction may occur in the presence of a linear nucleic acid capable of base- pairing with both the 5'-and 3'-region in juxtaposition to assist the enzymatic ligation reaction. In one embodiment, the ligation is splint ligation where a single stranded polynucleotide (splint), like a single stranded RNA, can be designed to hybridize with both termini of a linear RNA, so that the two termini can be juxtaposed upon hybridization with the single-stranded splint. Splint ligase can thus catalyze the ligation of the juxtaposed two termini of the linear RNA, generating an oRNA.

[0282] In one embodiment, a DNA or RNA ligase may be used in the synthesis of the oRNA. As a non-limiting example, the ligase may be a circ ligase or circular ligase.

[0283] In one embodiment, either the 5'-or 3'-end of the linear RNA can encode a ligase ribozyme sequence such that during in vitro transcription, the resultant linear RNA includes an active ribozyme sequence capable of ligating the 5'-end of the linear RNA to the 3'-end of the linear RNA. The ligaseribozyme may be derived from the Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment).

[0284] In one embodiment, a linear RNA may be cyclized or concatemerized by using at least one non-nucleic acid moiety. In one aspect, the at least one non-nucleic acid moiety may react with regions or features near the 5' terminus and / or near the 3' terminus of the linear RNA in order to cyclize or concatermerize the linear RNA. In another aspect, the at least one non-nucleic acid moiety may be located in or linked to or near the 5' terminus and / or the 3' terminus of the linear RNA. The non-nucleic acid moieties contemplated may be homologous or heterologous. As a non-limiting example, the non-nucleic acid moiety may be a linkage such as a hydrophobic linkage, ionic linkage, a biodegradable linkage and / or a cleavable linkage. As another non-limiting example, the non-nucleic acid moiety is a ligation moiety. As yet another non-limiting example, the non-nucleic acid moiety may be an oligonucleotide or a peptide moiety, such as an aptamer or a non-nucleic acid linker as described herein.

[0285] In one embodiment, a linear RNA may be cyclized or concatemerized due to a non-nucleic acid moiety that causes an attraction between atoms, molecular surfaces at, near or linked to the 5' and 3' ends of the linear RNA. As a non-limiting example, one or more linear RNA may be cyclized or concatemerized by intermolecular forces or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole- induced dipole forces, Van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonant bonds, agnostic bonds, dipolar bonds, conjugation, hyperconjugation and antibonding.

[0286] In one embodiment, the linear RNA may comprise a ribozyme RNA sequence near the 5' terminus and near the 3' terminus. The ribozyme RNA sequence may covalently link to a peptide when the sequence is exposed to the remainder of the ribozyme. In one aspect, the peptides covalently linked to the ribozyme RNA sequence near the 5' terminus and the 3' terminus may associate with each other causing a linear RNA to cyclize or concatemerize. In another aspect, the peptides covalently linked to the ribozyme RNA near the 5' terminus and the 3' terminus may cause the linear RNA to cyclize or concatemerize after being subjected to ligated using various methods known in the art such as, but not limited to, protein ligation.

[0287] In some embodiments, the linear RNA may include a 5' triphosphate of the nucleic acid converted into a 5' monophosphate, e.g., by contacting the 5' triphosphate with RNA 5' pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase (apyrase). Alternately, converting the 5' triphosphate of the linear RNA into a 5' monophosphate may occur by a two-step reaction comprising: (a) contacting the 5' nucleotide of the linear RNA with a phosphatase (e.g., Antarctic Phosphatase, Shrimp Alkaline Phosphatase, or Calf Intestinal Phosphatase) to remove all threephosphates; and (b) contacting the 5' nucleotide after step (a) witha kinase (e.g., Polynucleotide Kinase) that adds a single phosphate.

[0288] In some embodiments, RNA may be circularized using the methods described in WO2017222911 and WO2016197121, the contents of each of which are herein incorporated by reference in their entirety.

[0289] In some embodiments, RNA may be circularized, for example, by back splicing of a non- mammalian exogenous intron or splint ligation of the 5' and 3 ' ends of a linear RNA. In one embodiment, the circular RNA is produced from a recombinant nucleic acid encoding the target RNA to be made circular. As a non-limiting example, the method comprises: a) producing a recombinant nucleic acid encoding the target RNA to be made circular, wherein the recombinant nucleic acid comprises in 5' to 3 ' order: i) a 3 ' portion of an exogenous intron comprising a 3' splice site, ii) a nucleic acid sequence encoding the target RNA, and iii) a 5 ' portion of an exogenous intron comprising a 5 ' splice site; b) performing transcription, whereby RNA is produced from the recombinant nucleic acid; and c) performing splicing of the RNA, whereby the RNA circularizes to produce a oRNA.

[0290] While not wishing to be bound by theory, circular RNAs generated with exogenous introns are recognized by the immune system as "non-self" and trigger an innate immune response. On the other hand, circular RNAs generated with endogenous introns are recognized by the immune system as "self" and generally do not provoke an innate immune response, even if carrying an exon comprising foreign RNA.

[0291] Accordingly, circular RNAs can be generated with either an endogenous or exogenous intron to control immunological self / non-self discrimination as desired. Numerous intron sequences from a wide variety of organisms and viruses are known and include sequences derived from genes encoding proteins, ribosomal RNA (rRNA), or transfer RNA (tRNA).

[0292] Circular RNAs can be produced from linear RNAs in a number of ways. In some embodiments, circular RNAs are produced from a linear RNA by backsplicing of a downstream 5' splice site (splice donor) to an upstream 3' splice site (splice acceptor). Circular RNAs can be generated in this manner by any nonmammalian splicing method. For example, linear RNAs containing various types of introns, including self-splicing group I introns, self-splicing group II introns, spliceosomal introns, and tRNA introns can be circularized. In particular, group I and group II introns have the advantage that they can be readily used for production of circular RNAs in vitro as well as in vivo because of their ability to undergo self-splicing due to their autocatalytic ribozyme activity.

[0293] In some embodiments, circular RNAs can be produced in vitro from a linear RNA by chemical or enzymatic ligation of the 5' and 3' ends of the RNA. Chemical ligation can be performed, for example, using cyanogen bromide (BrCN) or ethyl-3-(3'-dimethylaminopropyl) carbodiimide(EDC) for activation of a nucleotide phosphomonoester group to allow phosphodiester bond formation. See e.g., Sokolova (1988) FEBS Lett 232: 153-155; Dolinnaya et al. (1991) Nucleic Acids Res., 19:3067-3072; Fedorova (1996) Nucleosides Nucleotides Nucleic Acids 15: 1137-1147; herein incorporated by reference. Alternatively, enzymatic ligation can be used to circularize RNA. Exemplary ligases that can be used include T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl 1), and T4 RNA ligase 2 (T4 Rnl 2).

[0294] In some embodiments, splint ligation using an oligonucleotide splint that hybridizes with the two ends of a linear RNA can be used to bring the ends of the linear RNA together for ligation. Hybridization of the splint, which can be either a DNA or a RNA, orientates the 5 '-phosphate and 3' - OH of the RNA ends for ligation. Subsequent ligation can be performed using either chemical or enzymatic techniques, as described above. Enzymatic ligation can be performed, for example, with T4 DNA ligase (DNA splint required), T4 RNA ligase 1 (RNA splint required) or T4 RNA ligase 2 (DNA or RNA splint). Chemical ligation, such as with BrCN or EDC, in some cases is more efficient than enzymatic ligation if the structure of the hybridized splint-RNA complex interferes with enzymatic activity.

[0295] In some embodiments, the oRNA may further comprise an internal ribosome entry site (IRES) operably linked to an RNA sequence encoding a polypeptide. Inclusion of an IRES permits the translation of one or more open reading frames from a circular RNA. The IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20: 102-110; Kobayashi et al., BioTechniques (1996) 21 :399-402; and Mosser et al., BioTechniques 199722150-161).

[0296] In some embodiments, the circularization efficiency of the circularization methods provided herein is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100%. In some embodiments, the circularization efficiency of the circularization methods provided herein is at least about 40%. Splicing Element

[0297] In some embodiments, the oRNA includes at least one splicing element. The splicing element can be a complete splicing element that can mediate splicing of the oRNA or the spicing element can be a residual splicing element from a completed splicing event. For instance, in some cases, a splicing element of a linear RNA can mediate a splicing event that results in circularization of the linear RNA, thereby the resultant oRNA comprises a residual splicing element from such splicing-mediated circularization event. In some cases, the residual splicing element is not able to mediate any splicing. In other cases, the residual splicing element can still mediate splicing under certain circumstances. Insome embodiments, the splicing element is adjacent to at least one expression sequence. In some embodiments, the oRNA includes a splicing element adjacent each expression sequence. In some embodiments, the splicing element is on one or both sides of each expression sequence, leading to separation of the expression products, e.g., peptide(s) and or polypeptide(s).

[0298] In some embodiments, theoRNA includes an internal splicing element that when replicated the spliced ends are joined together. Some examples may include miniature introns (<100 nt) with splice site sequences and short inverted repeats (30-40 nt) such as AluSq2, AluJr, and AluSz, inverted sequences in flanking introns, Alu elements in flanking introns, and motifs found in (suptable4 enriched motifs) cis-sequence elements proximal to backsplice events such as sequences in the 200 bp preceding (upstream of) or following (downstream from) a backsplice site with flanking exons. In some embodiments, the oRNA includes at least one repetitive nucleotide sequence described elsewhere herein as an internal splicing element. In such embodiments, the repetitive nucleotide sequence may include repeated sequences from the Alu family of introns. See, e.g., US Patent No. 11,058,706.

[0299] In some embodiments, the oRNA may include canonical splice sites that flank head-to-tail junctions of the oRNA.

[0300] In some embodiments, the oRNA may include a bulge-helix-bulge motif, comprising a 4-base pair stem flanked by two 3-nucleotide bulges. Cleavage occurs at a site in the bulge region, generating characteristic fragments with terminal 5'-hydroxyl group and 2', 3'-cyclic phosphate. Circularization proceeds by nucleophilic attack of the 5'-OH group onto the 2', 3'-cyclic phosphate of the same molecule forming a 3', 5'-phosphodiester bridge.

[0301] In some embodiments, the oRNA may include a sequence that mediates self-ligation. Non- limiting examples of sequences that can mediate self-ligation include a self-circularizing intron, e.g., a 5' and 3' slice junction, or a self-circularizing catalytic intron such as a Group I, Group II or Group III Introns. Non-limiting examples of group I intron self-splicing sequences may includeself-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td, and the intervening sequence (IVS) rRNA of Tetrahymena. Other Circularization Methods

[0302] In some embodiments, linear RNA may include complementary sequences, including either repetitive or nonrepetitive nucleic acid sequences within individual introns or across flanking introns. In some embodiments, the oRNA includes a repetitive nucleic acid sequence. In some embodiments, the repetitive nucleotide sequence includes poly CA or poly UG sequences. In some embodiments, the oRNA includes at least one repetitive nucleic acid sequence that hybridizes to a complementary repetitive nucleic acid sequence in another segment of the oRNA, with the hybridized segment forming an internal double strand. In some embodiments, repetitive nucleic acid sequences and complementary repetitive nucleic acid sequences from two separate oRNA that hybridize to generatea single oRNA, with the hybridized segments forming internal double strands. In some embodiments, the complementary sequences are found at the 5' and 3' ends of the linear RNA. In some embodiments, the complementary sequences include about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more paired nucleotides.

[0303] In some embodiments, chemical methods of circularization may be used to generate the oRNA. Such methods may include, but are not limited to click chemistry (e.g., alkyne- and azide- based methods, or clickable bases), olefin metathesis, phosphoramidate ligation, hemiaminal-imine crosslinking, base modification, and any combination thereof. In some embodiments, enzymatic methods of circularization may be used to generate the oRNA. In some embodiments, a ligation enzyme, e.g., DNA or RNA ligase, may be used to generate a template of the oRNA or complement, a complementary strand of the oRNA, or the oRNA. Any of the circular polynucleotides as taught in for example U.S. Patent No.10,709,779, which is incorporated by reference herein in its entirety, may be used herein. In addition, any of the circular RNAs, methods for making circular RNAs, circular RNA compositions that are described in the following publications are contemplated herein and are incorporated by reference in their entireties are part of the instant specification: US Patents US 11,352,640, US 11,352,641, US 11,203,767, US 10,683,498, US 5,773,244, and US 5,766,903; US Application Publications US 2022 / 0177540, US 2021 / 0371494, US 2022 / 0090137, US 2019 / 0345503, and US 2015 / 0299702; and PCT Application Publications WO 2021 / 226597, WO 2019 / 236673, WO 2017 / 222911, WO2016 / 187583, WO2014 / 082644 and WO 1997 / 007825. D. Gene editing systems

[0304] As described herein, the LNPs of the present disclosure comprise a gene editing system. As used herein, the term “gene editing system” (used interchangeably herein with the term “nucleobase editing system”) generally refers to a composition having one or more gene editing system components which are capable of independently or co-dependently editing, modifying, or altering a target polynucleotide sequence or a target transcript comprising a nucleic acid sequence and / or modifying the epigenome to effect a change in gene regulation. In certain embodiments, gene editing systems of the present disclosure include any editing systems known in the art.

[0305] For example, the LNP compositions herein may be used to deliver any gene editing system including CRISPR (clustered regularly interspaced short palindromic repeats) and the associated CRISPR-associated proteins (e.g., CRISPR-Cas9) (Jinek et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, Vol.337 (6096), pp.816-821), meganuclease editors (Boissel et al., “megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering,” Nucleic Acids Research 42: pp.2591-2601) and bacterial retron systems (Schubert et al., “High-throughput functional variant screens via in vivo production of single-stranded DNA,” PNAS, April 27, 2021, Vol.118(18), pp.1-10). In particular, CRISPR-Cas9 has beenderivatized in numerous ways to expand upon its guide RNA-based programmable double-strand cutting activity to form systems ranging from finding alternative CRISPR Cas nuclease enzymes having different PAM requirements and cutting properties (e.g., Cas12a, Cas12f, Cas13a, and Cas13b) to base editing (Komor et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature, May 19, 2016, 533 (7603); pp.420-424 [cytosine base editors or CBEs] and Gaudelli et al., “Programmable base editing of A-T to G-C in genomic DNA without DNA cleavage,” Nature, Vol.551, pp.464-471 [adenine base editors or ABEs]) to prime editing (Anzalone et al., “Search-and-replace genome editing without double-strand breaks or donor DNA,” Nature, Dec 2019, 576 (7789): pp.149-157) to twin prime editing (Anzalone et al., “Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing,” Nature Biotechnology, Dec 9, 2021, vol.40, pp.731-740) to epigenetic editing (Kungulovski and Jeltsch, “Epigenome Editing: State of the Art, Concepts, and Perspective,” Trends in Genetics, Vol.32, 206, pp.101-113) to CRISPR-directed integrase editing (Yarnell et al., “Drag- and-drop genome insertion of large sequences without double-stranded DNA cleavage using CRISPR- directed integrases,” Nature Biotechnology, Nov 24, 2022, (“PASTE”). Each of these gene editing systems may be packaged up in the LNP compositions described herein and delivered to target organs, tissues, and cells to bring about the modification of a target sequence or the expression of a target gene.

[0306] The gene editing systems deliverable by the herein disclosed LNPs can be any gene editing system. In certain embodiments, the LNPs of the present disclosure are used to delivery gene editing systems capable of editing, modifying or altering a target polynucleotide sequence that results in treatment of VEXAS syndrome. In still other embodiments, the gene editing systems are preferably, but not limited to, those disclosed herein. The gene editing systems contemplated herein can include (A) nucleobase gene editing systems which result in one or more the modifications to the sequence of target nucleic acid molecule, (B) an epigenetic editing system which results in one or more modifications to the epigenome to bring about an effect on gene expression without altering the sequence of a nucleic acid molecule, and (C) gene editing systems that combine the features of nucleobase editing systems and epigenetic editing systems.

[0307] Nucleobase editing systems include a wide array of configurations with various combinations of protein functionalities and / or nucleic acid molecule components, all of which are contemplated herein. In general, nucleobase editing systems comprise at least a (i) DNA binding domain that is user-programmable to target a specific sequence in a nucleic acid molecule and optionally (ii) one or more effector domains that facilitate the modification of the sequence of the nucleic acid molecule. User-programmability may comprise amino acid sequence-programmable DNA binding domains (e.g., TALENS and zinc finger-binding domains) or nucleic acid sequence-programmable DNA binding domains (e.g., CRISPR Cas9, CRISPR Cas12a, CRISPR Cas12f, CRISPR Cas13a, CRISPRCas13b, IscB, IsrB, or TnpB). Similarly, epigenetic editing systems comprise at least a (i) DNA binding domain that targets a specific sequence in a nucleic acid molecule and (ii) one or more effector domains that facilitates the modification of one or more epigenomic features of the nucleic acid molecule.

[0308] Gene editing systems may comprise one or more effector domains that provide various functionalities that facilitate changes in nucleotide sequence and / or gene expression, such as, but not limited to, single-strand DNA binding proteins, nucleases, endonucleases, exonucleases, deaminases (e.g., cytidine deaminases or adenosine deaminases), polymerases (e.g., reverse transcriptases), integrases, recombinases, etc., and fusion proteins comprising one or more functional domains linked together). In certain embodiments, the nucleobase editing systems include, but are not limited to, systems comprising a clustered regularly interspaced short palindromic repeats (“CRISPR”)- associated (“Cas”) protein, a zinc finger nuclease (“ZFN”), a transcription activator-like effector nuclease (“TALEN”), an adenosine deaminase acting on RNA (“ADAR”) enzyme, an adenosine deaminase acting on transfer RNA (“ADAT”) enzyme, an activation induced cytidine deaminase (“AID”) / apolipoprotein B editing complex (“APOBEC”) enzyme, a meganuclease, IscB, IsrB, TnpB, or a restriction enzyme.

[0309] In some embodiments, the nucleobase editing system edits, modifies, or alters the target polynucleotide sequence ex vivo. In some embodiments, the nucleobase editing system edits, modifies, or alters the target polynucleotide sequence in vivo. In some embodiments, the nucleobase editing system edits, modifies, or alters the target polynucleotide sequence in a cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0310] In some embodiments, the target polynucleotide sequence is a gene. In some embodiments, the target transcript comprising a nucleic acid sequence is a product of gene transcription. In some embodiments, the target transcript comprising a nucleic acid sequence is an RNA transcript such as a messenger RNA transcript, microRNA transcript or transfer RNA transcript.

[0311] The originator constructs and benchmark constructs of the present disclosure may comprise, encode or be conjugated to a cargo which is a nucleobase editing tool. As used herein, the term “nucleobase editing tool” is used interchangeably with “nucleobase editing system component” and generally refers to a compound or substance which is capable of independently or co-dependently editing, modifying, or altering a target polynucleotide sequence or a target transcript comprising a nucleic acid sequence. Nucleobase editing tools for the present disclosure include all nucleobase editing tools known in the art. In certain embodiments, the nucleobase editing tools include, but not limited to, effector proteins which modify DNA or RNA, guide elements which guide effector proteins to specific DNA or RNA sequence, repair elements which encode a nucleic acid sequence template, and supportive elements which activate or modulate the activity of another nucleobase editing tool, or activates or modulates host DNA repair enzymes.

[0312] In some embodiments, the cargo may comprise a nucleobase editing tool or a polynucleotide encoding a nucleobase editing tool. In some embodiments, the cargo may comprise one or more polynucleotides encoding a nucleobase editing tool. In some embodiments, the cargo may comprise a polynucleotide encoding one or more nucleobase editing tools. In some embodiments, the cargo may comprise a polynucleotide that is a component of the nucleobase editing tool. In some embodiments, the cargo may comprise a polynucleotide encoding one or more protein or peptide components in the nucleobase editing tool.

[0313] In some embodiments, the cargo may comprise an effector protein capable of modifying a target DNA or RNA sequence. In some embodiments, the cargo may comprise a polynucleotide encoding an effector protein. In certain embodiments, the effector proteins include polymerases, nucleases, and mutator enzymes. As used herein, the term “polymerases,” includes enzymes which catalyze the synthesis of DNA or RNA polymers. As used herein, the term “nucleases,” includes enzymes which cleave nucleobases. In certain embodiments, nucleases include enzymes which create single-stranded breaks (“SSB”) or double-stranded breaks (“DSB”) in nucleic acid sequences. As used herein, the term “mutator enzymes,” in its broadest sense, includes enzymes which mutate nucleic acid sequences. In certain embodiments, the cargo may comprise nucleases such as effector proteins include clustered regularly interspaced short palindromic repeats (“CRISPR”)-associated (“Cas”) proteins, zinc finger nucleases (“ZFNs”), transcription activator-like effector nucleases (“TALENs”), adenosine deaminase acting on RNA (“ADAR”) enzymes, adenosine deaminase acting on transfer RNA (“ADAT”) enzymes, activation induced cytidine deaminase (“AID”) / apolipoprotein B editing complex (“APOBEC”) enzymes, meganucleases, IscB, IsrB, TnpB, or restriction enzymes.

[0314] In some embodiments, the cargo may comprise a guide element which guide effector proteins to target a DNA or RNA sequence. In some embodiments, the cargo may comprise a polynucleotide encoding a guide element. In certain embodiments, guide elements include guide RNAs (“gRNAs”), CRISPR RNAs (“crRNAs”), prime editing guide RNAs (“pegRNAs”), transcription activator-like effectors (TALEs), or antisense oligomers.

[0315] In some embodiments, the cargo may further comprise a repair element which encodes a sequence repair template. In some embodiments, the cargo may further comprise a polynucleotide encoding a repair element or sequence repair template.

[0316] In some embodiments, the cargo may further comprise a supportive element which activates or modulates the editing system. In some embodiments, the cargo may further comprise a supportive element which activates or modulates the effector protein. In some embodiments, the cargo may further comprise a polynucleotide encoding a supportive element. Non-limiting categories of supportive elements include trans-activating RNA (“tracrRNA”). CRISPR-Cas editors

[0317] In some embodiments, the LNPs may be used to deliver a CRISPR-Cas gene editing system.

[0318] In general, nucleobase editing systems comprise at least a (i) DNA binding domain that is user-programmable to target a specific sequence in a nucleic acid molecule and optionally (ii) one or more effector domains that facilitate the modification of the sequence of the nucleic acid molecule. User-programmability may comprise amino acid sequence-programmable DNA binding domains (e.g., TALENS and zinc finger-binding domains) or nucleic acid sequence-programmable DNA binding domains (e.g., CRISPR Cas9, CRISPR Cas12a, CRISPR Cas12f, CRISPR Cas13a, CRISPR Cas13b, or TnpB), and including a guide RNA which targets the programmable DNA binding protein to target sequence.

[0319] In some embodiments, the CRISPR-Cas system comprises a Cas or Cas-derived protein.

[0320] In other embodiments, the amino acid sequence-programmable DNA binding domains (e.g., RNA-guided nuclease) used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease. Any RNA-guided Cas nuclease capable of catalyzing site- directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system Class 1, Type I, II, or III Cas nucleases; Class 2, Type II nuclease (such as Cas9); a Class 2, Type V nuclease (such as Cpfl), or a Class 2, Type VI nuclease (such as C2c2). Examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof.

[0321] In some embodiments, a Class 1, type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to perform genome modification as described herein. The Cas9 need not be physically derived from an organism but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP 002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP 002344900), Campylobacter coli (WP 060786116); Campylobacter fetus (WP 059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP 033919308); Spiroplasma syrphidicola (NC 021284); Prevotella intermedia (NC 017861); Spiroplasma taiwanense (NC 021846); Streptococcus iniae (NC 021314); Belliella baltica (NC 018010); Psychroflexus torquisl (NC O 18721); Streptococcus thermophilus (YP 820832), Streptococcus mutans (WP 061046374, WP 024786433); Listeria innocua (NP 472073); Listeria monocytogenes (WP 061665472); Legionellapneumophila (WP 062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP 033919308); Lactobacillus rhamnosus (WP 048482595, WP_032965177); and Neisseria meningitidis (WP_061704949, YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference in their entireties. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res.42(4):2577-90; Kapitonov et al. (2015) J. Bacterid.198(5): 797-807, Shmakov et al. (2015) Mol. Cell.60(3):385- 397, and Chylinski et al. (2014) Nucleic Acids Res.42(10):6091-6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[0322] In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpfl, or Cas12a) is used. Cpfl is another class II CRISPR / Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cpfl does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cpfl for targeting than Cas9. Cpfl is capable of cleaving either DNA or RNA. The PAM sites recognized by Cpfl have the sequences 5ʹ-YTN-3ʹ (where “Y” is a pyrimidine and “N” is any nucleobase) or 5ʹ-TTN-3ʹ, in contrast to the G-rich PAM site recognized by Cas9. Cpfl cleavage of DNA produces double-stranded breaks with a sticky-ends having a 4 or 5 nucleotide overhang. For a discussion of Cpfl, see, e.g., Ledford et al. (2015) Nature.526 (7571):17-17, Zetsche et al. (2015) Cell.163 (3):759-771, Murovec et al. (2017) Plant Biotechnol. J.15(8):917-926, Zhang et al. (2017) Front. Plant Sci.8:177, Fernandes et al. (2016) Postepy Biochem.62(3):315-326; herein incorporated by reference.

[0323] C2c1 (Cas12b) is another class II CRISPR / Cas system RNA-guided nuclease that may be used. C2cl, similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites. See, e.g., Shmakov et al. (2015) Mol Cell.60(3):385-397, Zhang et al. (2017) Front Plant Sci.8:177; herein incorporated by reference.

[0324] In one aspect, a nucleic acid sequence-programmable DNA binding domain can be associated with or complexed with at least one guide nucleic acid (e.g., guide RNA or a pegRNA), which localizes the DNA binding domain to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid, or a portion thereof (e.g., the spacer of a guide RNA which anneals to the protospacer of the DNA target). In other words, the guide nucleic-acid “programs” the DNA binding domain (e.g., Cas9 or equivalent) to localize and bind to complementary sequence of the protospacer in the DNA.

[0325] Any suitable nucleic acid sequence-programmable DNA binding domain may be used in the prime editors described herein. In various embodiments, the nucleic acid sequence-programmable DNA binding domain may be any Class 2 CRISPR-Cas system, including any type II, type V, or type VI CRISPR-Cas enzyme. Given the rapid development of CRISPR-Cas as a tool for genome editing, there have been constant developments in the nomenclature used to describe and / or identify CRISPR- Cas enzymes, such as Cas9 and Cas9 orthologs. CRISPR-Cas nomenclature is extensively discussed in Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol.1. No.5, 2018, the entire contents of which are incorporated herein by reference.

[0326] Without being bound by theory, the mechanism of action of certain CRISPR Cas enzymes contemplated herein includes the step of forming an R-loop whereby the Cas protein induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the Cas protein. The guide RNA spacer then hybridizes to the “target strand” at a region that is complementary to the protospacer sequence of the DNA. In some embodiments, the Cas protein may include one or more nuclease activities, which then cut the DNA leaving various types of lesions. For example, the Cas protein may comprises a nuclease activity that cuts the non-target strand at a first location, and / or cuts the target strand at a second location. Depending on the nuclease activity, the target DNA can be cut to form a “double-stranded break” whereby both strands are cut. In other embodiments, the target DNA can be cut at only a single site, i.e., the DNA is “nicked” on one strand. Exemplary Cas proteins with different nuclease activities include “Cas9 nickase” (“nCas9”) and a deactivated Cas9 having no nuclease activities (“dead Cas9” or “dCas9”).

[0327] The below description of various Cas proteins which can be used in connection with the presently disclosed LNP-delivered gene editing systems is not meant to be limiting in any way. The gene editing systems may comprise the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein—including any naturally occurring variant, mutant, or otherwise engineered version of Cas9—that is known or which can be made or evolved through a directed evolutionary or otherwise mutagenic process. In various embodiments, the Cas9 or Cas9 variants have a nickase activity, i.e., only cleave one strand of the target DNA sequence. In other embodiments, the Cas9 or Cas9 variants have inactive nucleases, i.e., are “dead” Cas9 proteins. Other variant Cas9 proteins that may be used are those having a smaller molecular weight than the canonical SpCas9 (e.g., for easier delivery) or having modified or rearranged primary amino acid structure.

[0328] The gene editing systems described herein may also comprise Cas9 equivalents, including Cas12a (Cpf1) and Cas12b1 proteins. The Cas proteins usable herein (e.g., SpCas9, Cas9 variant, or Cas9 equivalents) may also contain various modifications that alter / enhance their PAM specificities. The present disclosure contemplates any Cas9, Cas9 variant, or Cas9 equivalent which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity to a reference Cas9 sequence, such as a reference SpCas9 canonical sequence of Streptococcus pyogenes M1 (Accession No. Q99ZW2).

[0329] The Cas proteins contemplated herein embrace CRISPR Cas 9 proteins, as well as Cas9 equivalents, variants (e.g., Cas9 nickase (nCas9) or nuclease inactive Cas9 (dCas9)) homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and may include a Cas9 equivalent from any Class 2 CRISPR system (e.g., type II, V, VI), including Cas12a (Cpf1), Cas12e (CasX), Cas12b1 (C2c1), Cas12b2, Cas12c (C2c3), C2c4, C2c8, C2c5, C2c10, C2c9 Cas13a (C2c2), Cas13d, Cas13c (C2c7), Cas13b (C2c6), and Cas13b. Further Cas-equivalents are described in Makarova et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299) and Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?,” The CRISPR Journal, Vol.1. No.5, 2018, the contents of which are incorporated herein by reference.

[0330] The terms “Cas9” or “Cas9 nuclease” or “Cas9 moiety” or “Cas9 domain” embrace any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a Cas9, naturally-occurring or engineered. The term Cas9 is not meant to be particularly limiting and may be referred to as a “Cas9 or equivalent.” Exemplary Cas9 proteins are further described in the art and are incorporated herein by reference. As noted herein, Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A.98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602- 607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E.Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference).

[0331] In certain embodiments, a polynucleotide programmable nucleotide binding domain of a nucleobase editor itself comprises one or more domains. In one embodiment, a polynucleotide programmable nucleotide binding domain comprises one or more nuclease domains. In some embodiments, the nuclease domain of a polynucleotide programmable nucleotide binding domain comprises an endonuclease or an exonuclease. In some embodiments, the endonuclease cleaves a single strand of a double-stranded nucleobase. In some embodiments, the endonuclease cleaves both strands of a double-stranded nucleobase molecule. In some embodiments, the polynucleotideprogrammable nucleotide binding domain is a deoxyribonuclease. In some embodiments, the polynucleotide programmable nucleotide binding domain is a ribonuclease.

[0332] In some embodiments, the nuclease domain of a polynucleotide programmable nucleotide binding domain can cut zero, one, or two strands of a target polynucleotide. In some embodiments, the polynucleotide programmable nucleotide binding domain comprises a nickase domain. Herein the term “nickase” refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that is capable of cleaving only one strand of the two strands in a duplexed nucleobase molecule (e.g., DNA). In some embodiments, the nickase is derived from a fully catalytically active (e.g., natural) form of a polynucleotide programmable nucleotide binding domain by introducing one or more mutations into the active polynucleotide programmable nucleotide binding domain. In certain embodiments, where a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9.

[0333] In some embodiments, the Cas9-derived nickase has one or more mutations in the RuvC-1 domain. In one embodiment, the Cas9-derived nickase has a D10A mutation in the RuvC-1 domain. In some embodiments, the Cas9-derived nickase has one or more mutations in the REC Lobe domain. In one embodiment, the Cas9-derived nickase has a N497A, R661A, and / or Q695A mutation in the REC Lobe domain. In some embodiment, the Cas9-derived nickase has one or more mutations in the HNH domain. In one embodiment, the Cas9-derived nickase has H840A, N863A, and / or D839A in the HNH domain.

[0334] In certain embodiments, in the SpCas9-derived nickase, the residue H840 retains catalytic activity and can thereby cleave a single strand of the nucleobase duplex. In certain embodiments, a Cas9-derived nickase domain can comprise an H840A mutation, while the amino acid residue at position 10 remains a D. In certain embodiments, a Cas9-derived nickase domain can comprise an N863A mutation, while the amino acid residue at position 10 remains a D. In some embodiments, the nickase is derived from a fully catalytically active (e.g., natural) form of a polynucleotide programmable nucleotide binding domain by removing all or a portion of a nuclease domain that is not required for the nickase activity. In certain embodiments, where a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain comprises a deletion of all or a portion of the RuvC domain or the HNH domain.

[0335] In certain embodiments, the nucleobase editing system is or comprises a CRISPR-Cas editor or Cas9 disclosed and described in one or more of US Application Publications US2015 / 0045546A1, US2019 / 0264232A1, US2018 / 0258417A1, and PCT Publications WO2013141680A1 and WO2021173359A1, each of which is incorporated by reference herein in their entirety.

[0336] Any of the above CRISPR-Cas editor embodiments or any variants, modifications, or derivatives thereof are contemplated herein to be delivered by the LNP systems disclosed in this specification for gene editing in cells, tissues, and / or organs under in vitro, ex vivo, or in vivoconditions. The various components described herein may be configured and delivered in any suitable manner. Any of the descriptions presented in this section are not intended to be strictly limiting. Base editors

[0337] In other embodiments, the LNPs may be used to deliver a base editing system. Base editors are generally composed of an engineered deaminase and a catalytically impaired CRISPR–Cas9 variant and enzymatically convert one base to another base at a specific target site with the assistance of endogenous DNA repair systems in the cell.

[0338] Base editing was first described in Komor et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage,” Nature, May 19, 2016, 533 (7603); pp.420- 424 in the form of cytosine base editors or CBEs followed by the disclosure of Gaudelli et al., “Programmable base editing of A-T to G-C in genomic DNA without DNA cleavage,” Nature, Vol. 551, pp.464-471 describing adenine base editors or ABEs. Subsequently, base editing has been described in numerous scientific publications, including, but not limited to (i) Kim JS. Precision genome engineering through adenine and cytosine base editing. Nat Plants.2018 Mar;4(3):148-151. doi: 10.1038 / s41477-018-0115-z. Epub 2018 Feb 26. PMID: 29483683.; (ii) Wei Y, Zhang XH, Li DL. The "new favorite" of gene editing technology-single base editors. Yi Chuan.2017 Dec 20;39(12):1115-1121. doi: 10.16288 / j.yczz.17-389. PMID: 29258982; (iii) Tang J, Lee T, Sun T. Single-nucleotide editing: From principle, optimization to application. Hum Mutat.2019 Dec;40(12):2171-2183. doi: 10.1002 / humu.23819. Epub 2019 Sep 15. PMID: 31131955; PMCID: PMC6874907; (iv) Grünewald J, Zhou R, Lareau CA, Garcia SP, Iyer S, Miller BR, Langner LM, Hsu JY, Aryee MJ, Joung JK. A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing. Nat Biotechnol.2020 Jul;38(7):861-864. doi: 10.1038 / s41587-020-0535-y. Epub 2020 Jun 1. PMID: 32483364; PMCID: PMC7723518; (v) Sakata RC, Ishiguro S, Mori H, Tanaka M, Tatsuno K, Ueda H, Yamamoto S, Seki M, Masuyama N, Nishida K, Nishimasu H, Arakawa K, Kondo A, Nureki O, Tomita M, Aburatani H, Yachie N. Base editors for simultaneous introduction of C-to-T and A-to-G mutations. Nat Biotechnol.2020 Jul;38(7):865-869. doi: 10.1038 / s41587-020- 0509-0. Epub 2020 Jun 2. Erratum in: Nat Biotechnol.2020 Jun 5;: PMID: 32483365; (vi) Fan J, Ding Y, Ren C, Song Z, Yuan J, Chen Q, Du C, Li C, Wang X, Shu W. Cytosine and adenine deaminase base-editors induce broad and nonspecific changes in gene expression and splicing. Commun Biol.2021 Jul 16;4(1):882. doi: 10.1038 / s42003-021-02406-5. PMID: 34272468; PMCID: PMC8285404; (vii) Zhang S, Yuan B, Cao J, Song L, Chen J, Qiu J, Qiu Z, Zhao XM, Chen J, Cheng TL. TadA orthologs enable both cytosine and adenine editing of base editors. Nat Commun.2023 Jan 26;14(1):414. doi: 10.1038 / s41467-023-36003-3. PMID: 36702837; PMCID: PMC988000; and (viii) Zhang S, Song L, Yuan B, Zhang C, Cao J, Chen J, Qiu J, Tai Y, Chen J, Qiu Z, Zhao XM, Cheng TL. TadA reprogramming to generate potent miniature base editors with high precision. NatCommun.2023 Jan 26;14(1):413. doi: 10.1038 / s41467-023-36004-2. PMID: 36702845; PMCID: PMC987999, each of which are incorporated herein by reference in their entireties.

[0339] Amino acid and nucleotide sequences of base editors, including adenosine base editors, cytidine base editors, and others are readily available in the art. For example, exemplary base editors that may be delivered using the LNP compositions described herein can be found in the following published patent applications, each of their contents (including any and all biological sequences) are incorporated herein by reference: US 2023 / 0021641 A1 CAS9 VARIANTS HAVING NON-CANONICAL PAM SPECIFICITIES AND USES THEREOF US 11542496 B2 Cytosine to guanine base editor US 11542509 B2 Incorporation of unnatural amino acids into proteins using base editing US 2022 / 0315906 A1 BASE EDITORS WITH DIVERSIFIED TARGETING SCOPE US 2022 / 0282275 A1 G-TO-T BASE EDITORS AND USES THEREOF US 2022 / 0249697 A1 AAV DELIVERY OF NUCLEOBASE EDITORS

[0340] In some embodiments, the LNP cargo comprises a base editing system or a polynucleotide encoding a CRISPR-Cas base editing system. In some embodiments, the cargo comprises a component of a base editing system or a polynucleotide encoding a component of a base editing system.

[0341] Base editing does not require double-stranded DNA breaks or a DNA donor template. In some embodiments, base editing comprises creating an SSB in a target double-stranded DNA sequence and then converting a nucleobase. In some embodiments, the nucleobase conversion is an adenosine to a guanine. In some embodiments, the nucleobase conversion is a thymine to a cytosine. In some embodiments, the nucleobase conversion is a cytosine to a thymine. In some embodiments, the nucleobase conversion is a guanine to an adenosine. In some embodiments, the nucleobase conversion is an adenosine to inosine. In some embodiments, the nucleobase conversion is a cytosine to uracil.

[0342] A base editing system comprises a base editor which can convert a nucleobase. The base editor (“BE”) comprises a partially inactive Cas protein which is connected to a deaminase that precisely and permanently edits a target nucleobase in a polynucleotide sequence. A base editor comprises a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g., adenosine deaminase or cytosine deaminase). In some embodiments, the partially inactive Cas protein is a Cas nickase. In some embodiments, the partially inactive Cas protein is a Cas9 nickase (also referred to as “nCas9”).

[0343] A polynucleotide programmable nucleotide binding domain, when in conjunction with a bound guide polynucleotide (e.g., gRNA), can specifically bind to a target polynucleotide sequence(i.e., via complementary base pairing between bases of the bound guide nucleobase and bases of the target polynucleotide sequence) and thereby localize the nucleobase editor to the target polynucleotide sequence desired to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid.

[0344] In certain embodiments, polynucleotide programmable nucleotide binding domains also include nucleobase programmable proteins that bind RNA. In certain embodiments, the polynucleotide programmable nucleotide binding domain can be associated with a nucleobase that guides the polynucleotide programmable nucleotide binding domain to an RNA.

[0345] In some embodiments, the LNP-deliverable base editors may comprise a deaminase domain that is a cytidine deaminase domain. A cytidine deaminase domain may also be referred to interchangeably as a cytosine deaminase domain. In some embodiments, the cytidine deaminase catalyzes the hydrolytic deamination of cytidine (C) or deoxycytidine (dC) to uridine (U) or deoxyuridine (dU), respectively. In some embodiments, the cytidine deaminase domain catalyzes the hydrolytic deamination of cytosine (C) to uracil (U). In some embodiments, the cytidine deaminase catalyzes the hydrolytic deamination of cytidine or cytosine in deoxyribonucleic acid (DNA). Without wishing to be bound by any particular theory, fusion proteins comprising a cytidine deaminase are useful inter alia for targeted editing, referred to herein as “base editing,” of nucleic acid sequences in vitro and in vivo.

[0346] One exemplary suitable type of cytidine deaminase is a cytidine deaminase, for example, of the APOBEC family. The apolipoprotein B mRNA-editing complex (APOBEC) family of cytidine deaminase enzymes encompasses eleven proteins that serve to initiate mutagenesis in a controlled and beneficial manner (see, e.g., Conticello S G. The AID / APOBEC family of nucleic acid mutators. Genome Biol.2008; 9(6):229). One family member, activation-induced cytidine deaminase (AID), is responsible for the maturation of antibodies by converting cytosines in ssDNA to uracils in a transcription-dependent, strand-biased fashion (see, e.g., Reynaud C A, et al. What role for AID: mutator, or assembler of the immunoglobulin mutasome, Nat Immunol.2003; 4(7):631-638). The apolipoprotein B editing complex 3 (APOBEC3) enzyme provides protection to human cells against a certain HIV-1 strain via the deamination of cytosines in reverse-transcribed viral ssDNA (see, e.g., Bhagwat A S. DNA-cytosine deaminases: from antibody maturation to antiviral defense. DNA Repair (Amst).2004; 3(1):85-89).

[0347] Some aspects of this disclosure relate to the recognition that the activity of cytidine deaminase enzymes such as APOBEC enzymes can be directed to a specific site in genomic DNA. Without wishing to be bound by any particular theory, advantages of using a nucleic acid programmable binding protein (e.g., a Cas9 domain) as a recognition agent include (1) the sequencespecificity of nucleic acid programmable binding protein (e.g., a Cas9 domain) can be easily altered by simply changing the sgRNA sequence; and (2) the nucleic acid programmable binding protein (e.g., a Cas9 domain) may bind to its target sequence by denaturing the dsDNA, resulting in a stretch of DNA that is single-stranded and therefore a viable substrate for the deaminase. It should be understood that other catalytic domains of napDNAbps, or catalytic domains from other nucleic acid editing proteins, can also be used to generate fusion proteins with Cas9, and that the disclosure is not limited in this regard.

[0348] In some embodiments, the cytidine deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the cytidine deaminase is an APOBEC1 deaminase. In some embodiments, the cytidine deaminase is an APOBEC2 deaminase. In some embodiments, the cytidine deaminase is an APOBEC3 deaminase. In some embodiments, the cytidine deaminase is an APOBEC3A deaminase. In some embodiments, the cytidine deaminase is an APOBEC3B deaminase. In some embodiments, the cytidine deaminase is an APOBEC3C deaminase. In some embodiments, the cytidine deaminase is an APOBEC3D deaminase. In some embodiments, the cytidine deaminase is an APOBEC3E deaminase. In some embodiments, the cytidine deaminase is an APOBEC3F deaminase. In some embodiments, the cytidine deaminase is an APOBEC3G deaminase. In some embodiments, the cytidine deaminase is an APOBEC3H deaminase. In some embodiments, the cytidine deaminase is an APOBEC4 deaminase. In some embodiments, the cytidine deaminase is an activation-induced deaminase (AID). In some embodiments, the cytidine deaminase is a vertebrate cytidine deaminase. In some embodiments, the cytidine deaminase is an invertebrate cytidine deaminase. In some embodiments, the cytidine deaminase is a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse deaminase. In some embodiments, the cytidine deaminase is a human cytidine deaminase. In some embodiments, the cytidine deaminase is a rat cytidine deaminase, e.g., rAPOBEC1.

[0349] In some embodiments, the nucleic acid editing domain is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the cytidine deaminase domain examples above.

[0350] In other embodiments, the LNP-deliverable base editors may comprise a deaminase domain that is an adenosine deaminase domain.

[0352] The disclosure provides fusion proteins that comprise one or more adenosine deaminases. In some aspects, such fusion proteins are capable of deaminating adenosine in a nucleic acid sequence (e.g., DNA or RNA). As one example, any of the fusion proteins provided herein may be base editors, (e.g., adenine base editors). Without wishing to be bound by any particular theory, dimerization of adenosine deaminases (e.g., in cis or in trans) may improve the ability (e.g., efficiency) of the fusion protein to modify a nucleic acid base, for example to deaminate adenine. In some embodiments, any of the fusion proteins may comprise 2, 3, 4 or 5 adenosine deaminases. In some embodiments, any ofthe fusion proteins provided herein comprise two adenosine deaminases. Exemplary, non-limiting, embodiments of adenosine deaminases are provided herein. It should be appreciated that the mutations provided herein (e.g., mutations in ecTadA) may be applied to adenosine deaminases in other adenosine base editors, for example those provided in U.S. Patent Publication No. 2018 / 0073012, published Mar.15, 2018, which issued as U.S. Pat. No.10,113,163, on Oct.30, 2018; U.S. Patent Publication No.2017 / 0121693, published May 4, 2017, which issued as U.S. Pat. No. 10,167,457 on Jan.1, 2019; International Publication No. WO 2017 / 070633, published Apr.27, 2017; U.S. Patent Publication No.2015 / 0166980, published Jun.18, 2015; U.S. Pat. No.9,840,699, issued Dec.12, 2017; and U.S. Pat. No.10,077,453, issued Sep.18, 2018, all of which are incorporated herein by reference in their entireties.

[0353] In some embodiments, any of the adenosine deaminases provided herein is capable of deaminating adenine. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine in a deoxyadenosine residue of DNA. The adenosine deaminase may be derived from any suitable organism (e.g., E. coli). In some embodiments, the adenosine deaminase is a naturally-occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). One of skill in the art will be able to identify the corresponding residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. Accordingly, one of skill in the art would be able to generate mutations in any naturally- occurring adenosine deaminase (e.g., having homology to ecTadA) that corresponds to any of the mutations described herein, e.g., any of the mutations identified in ecTadA. In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from E. coli.

[0354] Any two or more of the adenosine deaminases described herein may be connected to one another (e.g. by a linker) within an adenosine deaminase domain of the fusion proteins provided herein. For instance, the fusion proteins provided herein may contain only two adenosine deaminases. In some embodiments, the adenosine deaminases are the same. In some embodiments, the adenosine deaminases are any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminases are different. In some embodiments, the first adenosine deaminase is any of the adenosine deaminases provided herein, and the second adenosine is any of the adenosine deaminases provided herein, but is not identical to the first adenosine deaminase. In some embodiments, the fusion protein comprises two adenosine deaminases (e.g., a first adenosine deaminase and a second adenosine deaminase). In some embodiments, the fusion protein comprises a first adenosine deaminase and a second adenosine deaminase. In some embodiments, the first adenosine deaminase isN-terminal to the second adenosine deaminase in the fusion protein. In some embodiments, the first adenosine deaminase is C-terminal to the second adenosine deaminase in the fusion protein. In some embodiments, the first adenosine deaminase and the second deaminase are fused directly or via a linker.

[0355] In some embodiments, the base editor comprises a deaminase enzyme. In some embodiments, the base editor comprises a cytidine deaminase. In some embodiments, the base editor comprises a Cas9 protein fused to a cytidine deaminase enzyme. In some embodiments, the base editor comprises an adenosine deaminase. In some embodiments, the base editor comprises a Cas9 protein fused to an adenosine deaminase enzyme.

[0356] In some embodiments, the base editing system comprises an uracil glycosylase inhibitor. In some embodiments, the base editing system comprises a Cas9 protein fused to an uracil glycosylase inhibitor. In some embodiments, the cargo comprises an uracil glycosylase inhibitor or a polynucleotide encoding an uracil glycosylase inhibitor. In some embodiments, the cargo comprises a Cas9 protein fused to an uracil glycosylase inhibitor or a polynucleotide encoding a Cas9 protein fused to an uracil glycosylase inhibitor.

[0357] A variety of nucleobase modifying enzymes are suitable for use in the nucleobase systems disclosed herein. In some embodiments, the nucleobase modifying enzyme is a RNA base editor. In some embodiments, the RNA base editor can be a cytidine deaminase, which converts cytidine into uridine. Non-limiting examples of cytidine deaminases include cytidine deaminase 1 (CDA1), cytidine deaminase 2 (CDA2), activation-induced cytidine deaminase (AICDA), apolipoprotein B mRNA-editing complex (APOBEC) family cytidine deaminase (e.g., APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4), APOBEC1 complementation factor / APOBEC1 stimulating factor (ACF1 / ASF) cytidine deaminase, cytosine deaminase acting on RNA (CDAR), bacterial long isoform cytidine deaminase (CDDL), and cytosine deaminase acting on tRNA (CDAT). In other embodiments, the RNA base editor can be an adenosine deaminase, which converts adenosine into inosine, which is read by polymerase enzymes as guanosine. In certain embodiments, adenosine deaminases include tRNA adenine deaminase, adenosine deaminase, adenosine deaminase acting on RNA (ADAR), and adenosine deaminase acting on tRNA (ADAT).

[0358] In some embodiments, in the nucleobase editing systems disclosed herein, the Cas effector may associate with one or more functional domains (e.g., via fusion protein or suitable linkers). In some embodiments, the effector domain comprises one or more cytidine or nucleotide deaminases that mediate editing of via hydrolytic deamination. In certain embodiments, the effector domain comprises the adenosine deaminase acting on RNA (ADAR) family of enzymes. In certain embodiments, the adenosine deaminase protein or catalytic domain thereof capable of deaminating adenosine or cytidine in RNA or is an RNA specific adenosine deaminase and / or is a bacterial,human, cephalopod, or Drosophila adenosine deaminase protein or catalytic domain thereof, preferably TadA, more preferably ADAR, optionally huADAR, optionally (hu)ADAR1 or (hu)ADAR2, preferably huADAR2 or catalytic domain thereof.

[0359] In some embodiments, the cytidine deaminase is a human, rat or lamprey cytidine deaminase. In some embodiments, the cytidine deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase, an activation-induced deaminase (AID), or a cytidine deaminase 1 (CDA1).

[0360] In certain embodiments, the adenosine deaminase is adenosine deaminase acting on RNA (ADAR). In certain embodiments, the ADAR is ADAR (ADAR1), ADARB1 (ADAR2) or ADARB2 (ADAR3) (see, e.g., Savva et al. Genon. Biol.2012, 13(12):252).

[0361] In some embodiments, the gene editing system comprises AID / APOBEC (apolipoprotein B editing complex) family of enzymes deaminates cytidine to uridine, leading to mutations in RNA and DNA.

[0362] In some embodiments, the nucleobase editing system comprises ADAR and an antisense oligonucleotide. In certain embodiments, the antisense oligonucleotide is chemically optimized antisense oligonucleotide. In certain embodiments, the antisense oligonucleotide is administered for the nucleobase editing, wherein the antisense oligonucleotide activates human endogenous ADAR for nucleobase editing. Such ADAR and antisense oligonucleotide editing system provides a safer site- directed RNA editing with low off-target effect. See, e.g., Merkle et al., Nature Biotechnology, 2019, 37, 133-138.

[0363] Any of the above base editor embodiments or variants, modifications, or derivatives thereof are contemplated herein to be delivered by the LNP systems disclosed in this specification for gene editing in cells, tissues, and / or organs under in vitro, ex vivo, or in vivo conditions. The various components described herein may be configured and delivered in any suitable manner. Any of the descriptions presented in this section are not intended to be strictly limiting. Prime editors

[0364] In various embodiments, the herein disclosed LNPs may contain a prime editing system or components thereof and which may be used to conduct prime editing of target nucleic acid sequences in cells, tissues, and organs in an ex vivo or in vivo manner.

[0365] Prime editing technology is a gene editing technology that can make targeted insertions, deletions, and all transversion and transition point mutations in a target genome. Without wishing to be bound by any particular theory, the prime editing process may search and replace endogenous sequences in a target polynucleotide. The spacer sequence of a prime editing guide RNA (“PEgRNA” or “pegRNA”) recognizes and anneals with a search target sequence in a target strand of a double stranded target polynucleotide, e.g., a double stranded target DNA. A prime editing complex may generate a nick in the target DNA on the edit strand which is the complementary strand of the targetstrand. The prime editing complex may then use a free 3’ end formed at the nick site of the edit strand to initiate DNA synthesis, where a “primer binding site sequence” (PBS) of the PEgRNA complexes with the free 3’ end, and a single stranded DNA is synthesized (by reverse transcriptase) using an editing template of the PEgRNA as a template. As used herein, a “primer binding site” is a single- stranded portion of the PEgRNA that comprises a region of complementarity to the PAM strand (i.e., the non-target strand or the edit strand). The PBS is complementary or substantially complementary to a sequence on the PAM strand of the double stranded target DNA that is immediately upstream of the nick site.

[0366] The term “prime editor (PE)” refers to the polypeptide or polypeptide components involved in prime editing, or any polynucleotide(s) encoding the polypeptide or polypeptide components. In various embodiments, a prime editor includes a polypeptide domain having DNA binding activity and a polypeptide domain having DNA polymerase activity. In some embodiments, the prime editor further comprises a polypeptide domain having nuclease activity. In some embodiments, the polypeptide domain having DNA binding activity comprises a nuclease domain or nuclease activity. In some embodiments, the polypeptide domain having nuclease activity comprises a nickase, or a fully active nuclease. As used herein, the term “nickase” refers to a nuclease capable of cleaving only one strand of a double-stranded DNA target. In some embodiments, the prime editor comprises a polypeptide domain that is an inactive nuclease. In some embodiments, the polypeptide domain having programmable DNA binding activity comprises a nucleic acid guided DNA binding domain, for example, a CRISPR-Cas protein, for example, a Cas9 nickase, a Cpf1 nickase, or another CRISPR-Cas nuclease. In some embodiments, the polypeptide domain having DNA polymerase activity comprises a template-dependent DNA polymerase, for example, a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase. In some embodiments, the DNA polymerase is a reverse transcriptase. In some embodiments, the prime editor comprises additional polypeptides involved in prime editing, for example, a polypeptide domain having 5’ endonuclease activity, e.g., a 5' endogenous DNA flap endonucleases (e.g., FEN1), for helping to drive the prime editing process towards the edited product formation. In some embodiments, the prime editor further comprises an RNA-protein recruitment polypeptide, for example, a MS2 coat protein.

[0367] A prime editor may be engineered. In some embodiments, the polypeptide components of a prime editor do not naturally occur in the same organism or cellular environment. In some embodiments, the polypeptide components of a prime editor may be of different origins or from different organisms. In some embodiments, a prime editor comprises a DNA binding domain and a DNA polymerase domain that are derived from different species. In some embodiments, a prime editor comprises a Cas polypeptide (DNA binding domain) and a reverse transcriptase polypeptide (DNA polymerase) that are derived from different species. For example, a prime editor may comprisea S. pyogenes Cas9 polypeptide and a Moloney murine leukemia virus (M-MLV) reverse transcriptase polypeptide.

[0368] In some embodiments, polypeptide domains of a prime editor may be fused or linked by a peptide linker to form a fusion protein. In other embodiments, a prime editor comprises one or more polypeptide domains provided in trans as separate proteins, which are capable of being associated to each other through non-peptide linkages or through aptamers or recruitment sequences. For example, a prime editor may comprise a DNA binding domain and a reverse transcriptase domain associated with each other by an RNA-protein recruitment aptamer, e.g., a MS2 aptamer, which may be linked to a PEgRNA. Prime editor polypeptide components may be encoded by one or more polynucleotides in whole or in part. In some embodiments, a single polynucleotide, construct, or vector encodes the prime editor fusion protein. In some embodiments, multiple polynucleotides, constructs, or vectors each encode a polypeptide domain or portion of a domain of a prime editor, or a portion of a prime editor fusion protein. For example, a prime editor fusion protein may comprise an N-terminal portion fused to an intein-N and a C-terminal portion fused to an intein-C, each of which is individually encoded by an AAV vector.

[0369] The editing template may comprise one or more intended nucleotide edits compared to the endogenous double stranded target DNA sequence. Accordingly, the newly synthesized single stranded DNA also comprises the nucleotide edit(s) encoded by the editing template. Through removal of the editing target sequence on the edit strand of the double stranded target DNA and DNA repair mechanism, the newly synthesized single stranded DNA replaces the editing target sequence, and the desired nucleotide edit(s) are incorporated into the double stranded target DNA.

[0370] Prime editing was first described in Anzalone et al., “Search-and-replace genome editing without double-strand breaks or donor DNA,” Nature, Dec 2019, 576 (7789): pp.149-157, which is incorporated herein in its entirety. Prime editing has subsequently been described and detailed in numerous follow-on publications, including, for example, (i) Liu et al., “Prime editing: a search and replace tool with versatile base changes,” Yi Chuan, Nov.20, 2022, 44(11): 993-1008; (ii) Lu C et al., “Prime Editing: An All-Rounder for Genome Editing. Int J Mol Sci.2022 Aug 30;23(17):9862; (iii) Velimirovic M, Zanetti LC, Shen MW, Fife JD, Lin L, Cha M, Akinci E, Barnum D, Yu T, Sherwood RI. Peptide fusion improves prime editing efficiency. Nat Commun.2022 Jun 18;13(1):3512. doi: 10.1038 / s41467-022-31270-y. PMID: 35717416; PMCID: PMC9206660; (iv) Velimirovic M, Zanetti LC, Shen MW, Fife JD, Lin L, Cha M, Akinci E, Barnum D, Yu T, Sherwood RI. Peptide fusion improves prime editing efficiency. Nat Commun.2022 Jun 18;13(1):3512. doi: 10.1038 / s41467-022- 31270-y. PMID: 35717416; PMCID: PMC9206660; (v) Habib O, Habib G, Hwang GH, Bae S. Comprehensive analysis of prime editing outcomes in human embryonic stem cells. Nucleic Acids Res.2022 Jan 25;50(2):1187-1197. doi: 10.1093 / nar / gkab1295. PMID: 35018468; PMCID: PMC8789035; (vi) Marzec M, Brąszewska-Zalewska A, Hensel G. Prime Editing: A New Way forGenome Editing. Trends Cell Biol.2020 Apr;30(4):257-259. doi: 10.1016 / j.tcb.2020.01.004. Epub 2020 Jan 27. PMID: 32001098; (vii) Tao R, Wang Y, Jiao Y, Hu Y, Li L, Jiang L, Zhou L, Qu J, Chen Q, Yao S. Bi-PE: bi-directional priming improves CRISPR / Cas9 prime editing in mammalian cells. Nucleic Acids Res.2022 Jun 24;50(11):6423-6434. doi: 10.1093 / nar / gkac506. PMID: 35687127; PMCID: PMC9226529; (viii) Nelson JW, Randolph PB, Shen SP, Everette KA, Chen PJ, Anzalone AV, An M, Newby GA, Chen JC, Hsu A, Liu DR. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol.2022 Mar;40(3):402-410. doi: 10.1038 / s41587-021-01039-7. Epub 2021 Oct 4. Erratum in: Nat Biotechnol.2021 Dec 8; PMID: 34608327; PMCID: PMC8930418; (ix) Doman JL, Sousa AA, Randolph PB, Chen PJ, Liu DR. Designing and executing prime editing experiments in mammalian cells. Nat Protoc.2022 Nov;17(11):2431-2468. doi: 10.1038 / s41596-022- 00724-4. Epub 2022 Aug 8. PMID: 35941224; PMCID: PMC9799714; (x) Jiao Y, Zhou L, Tao R, Wang Y, Hu Y, Jiang L, Li L, Yao S. Random-PE: an efficient integration of random sequences into mammalian genome by prime editing. Mol Biomed.2021 Nov 18;2(1):36. doi: 10.1186 / s43556-021- 00057-w. PMID: 35006470; PMCID: PMC8607425; and (xi) Awan MJA, Ali Z, Amin I, Mansoor S. Twin prime editor: seamless repair without damage. Trends Biotechnol.2022 Apr;40(4):374-376. doi: 10.1016 / j.tibtech.2022.01.013. Epub 2022 Feb 10. PMID: 35153078, all of which are incorporated herein by reference.

[0371] In addition, prime editing has been described and disclosed in numerous published patent applications, each of which their entire contents, amino acid sequences, nucleotide sequences, and all disclosures therein are incorporated herein by reference in their entireties:

[0372] In some embodiments, the cargo comprises a prime editing system or a polynucleotide encoding a prime editing system. In some embodiments, the cargo comprises a component of a prime editing system or a polynucleotide encoding a component of a prime editing system.

[0373] Prime editing is a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas fused to an engineered reverse transcriptase, also referred to as a prime editor, which is programmable using a prime editing guide RNA (“pegRNA”) that both specifies the target site and encodes the desired edit (see, e.g., Anzalone et al., Nature 2019). Prime editing bypasses the need for DNA donor templates by using a prime editor having nickase or catalytically impaired enzymatic activity.

[0374] A prime editing system comprises a prime editor. The prime editor (“PE”) comprises a catalytically impaired Cas protein fused to an engineered reverse transcriptase which can precisely and permanently edit one or more target nucleobases in a target polynucleotide.

[0375] In some embodiments, the prime editor comprises an engineered Moloney murine leukemia virus (“M-MLV”) reverse transcriptase (“RT”) fused to a Cas-H840A nickase (called “PE2”). In some embodiments, the prime editor comprises an engineered M-MLV RT fused to a Cas9-H840A nickase. In some embodiments, the prime editor comprises an engineered M-MLV RT fused to a Streptococcus pyogenes Cas9 (spCas9)-H840A nickase. PE modifications include increased PAM flexibility to increase the utility of PE2 editing, expanding the coverage of targetable pathogenic variants in the ClinVar database that can now be prime edited to 94.4%.

[0376] In some embodiments, the prime editing system further comprises a prime editing guide RNA (“pegRNA”). In some embodiments, the cargo comprises a pegRNA or a polynucleotide encoding a pegRNA.

[0377] In some embodiments, the prime editing system further comprises a second guide RNA targeting the complementary strand, allowing the Cas9 nickase to also nick the non-edited strand (called “PE3”), which biases mismatch DNA repair in favor of the edited sequence. In some embodiments, the second guide RNA is designed to recognize the complementary strand of DNA only after the PE3 edit has occurred (called “PE3b”), which reduces indel formation.

[0378] In some embodiments, the prime editing system comprises an uracil glycosylase inhibitor. In some embodiments, the prime editing system comprises a Cas9 protein fused to an uracil glycosylase inhibitor. In some embodiments, the cargo comprises an uracil glycosylase inhibitor or a polynucleotide encoding an uracil glycosylase inhibitor. In some embodiments, the cargo comprises a Cas9 protein fused to an uracil glycosylase inhibitor or a polynucleotide encoding a Cas9 protein fused to an uracil glycosylase inhibitor.

[0379] Any of the above prime editor embodiments or variants, modifications, or derivatives thereof are contemplated herein to be delivered by the LNP systems disclosed in this specification for gene editing in cells, tissues, and / or organs under in vitro, ex vivo, or in vivo conditions. The various components described herein may be configured and delivered in any suitable manner. Any of the descriptions presented in this section are not intended to be strictly limiting. Retron editors

[0380] In still other embodiments, the herein disclosed LNPs may be used to encapsulate and deliver a retron editing system. A retron editing system in various embodiments may comprise (a) a retron reverse transcriptase, or a nucleic acid molecule encoding a retron reverse transcriptase, (b) a retron ncRNA (or a nucleic acid molecule encoding same) comprising a modified msd region to include a sequence that is reverse transcribed to form a single strand template DNA sequence (RT-DNA), (c) a nucleic acid programmable nuclease (e.g., a CRISPR Cas9 or Cas12a), and (d) a guide RNA to target the nuclease to a desired target site.

[0381] Retrons are defined by their unique ability to produce an unusual satellite DNA known as msDNA (multicopy single-stranded DNA). DNA encoding retrons includes a reverse trancriptase(RT)-coding gene (ret) and a nucleic acid sequence encoding the non-coding RNA (ncRNA), which contains two contiguous and inverted non-coding sequences referred to as the msr and msd. The ret gene and the non-coding RNA (including the msr and msd) are transcribed as a single RNA transcript, which becomes folded into a specific secondary structure following post-transcriptional processing. Once translated, the RT binds the RNA template downstream from the msd locus, initiating reverse transcription of the RNA towards its 5ʹ end, assisted by the 2’OH group present in a conserved branching guanosine residue that acts as a primer. Reverse transcription halts before reaching the msr locus, and the resulting DNA, the msDNA, remains covalently attached to the RNA template via a 2’- 5ʹ phosphodiester bond and base-pairing between the 3ʹ ends of the msDNA and the RNA template. The external regions, at the 5ʹ and 3ʹ ends of the msd / msr transcript (a1 and a2, respectively) are complementary and can hybridize, leaving the structures located in the msr and msd regions in internal positions. The msr locus, which is not reverse transcribed, forms one to three short stem-loops of variable size, ranging from 3 to 10 base pairs, whereas the msd locus folds into a single / double long hairpin with a highly variable long stem of 10-50 bp in length that is also present in the final msDNA form.

[0382] It has recently been reported that retrons may be utilized as a means to provide donor DNA template for HDR-dependent genome editing (e.g., see Lopez et al., “Precise genome editing across kingdoms of life using retron-derived DNA,” Nature Chemical Biology, December 12, 2021, 18, pages199–206 (2022)), however, producing sufficient levels of donor DNA template intracellularly to sufficiently support efficient HDR-dependent editing remains a significant challenge.

[0383] Retrons have previously been described in the scientific literature, including in the context of retron editing. For example, retrons have been described in the following references, each of which are incorporated herein by reference:

[0384] In addition, retrons have previously been described in the patent literature, including in the context of retron editing. For example, retrons have been described in the following references, each of which are incorporated herein by reference:

[0385] In some embodiments, the LNP-based retron editing system can be used for genome editing a desired site. A retron is engineered with a heterologous nucleic acid sequence encoding a donor polynucleotide (“template or donor nucleotide sequence” or “template DNA”) suitable for use with nuclease genome editing system. The nuclease is designed to specifically target a location proximal to the desired edit (the nuclease should be designed such that it will not cut the target once the edit is properly installed). The nuclease (e.g., CAS or non-CAS) is linked to the retron, either by direct fusion to the RT or by fusion of the msDNA to the gRNA (only applicable for RNA-guided nucleases). A heterologous nucleic acid sequence is inserted into the retron msd.

[0386] In some embodiments, the heterologous nucleic acid sequence has 10-100 or more bp of homologous nucleic acid sequence to the genome on both sides of the desired edit. The desired edit (insertion, deletion, or mutation) is in between the homologous sequence.

[0387] In some embodiments, donor polynucleotides comprise a sequence comprising an intended genome edit flanked by a pair of homology arms responsible for targeting the donor polynucleotide to the target locus to be edited in a cell. The donor polynucleotide typically comprises a 5ʹ homology arm that hybridizes to a 5ʹ genomic target sequence and a 3ʹ homology arm that hybridizes to a 3ʹ genomic target sequence. The homology arms are referred to herein as 5ʹ and 3ʹ (i.e., upstream and downstream) homology arms, which relate to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5ʹ and 3ʹ homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the “5ʹ target sequence” and “3ʹ target sequence,” respectively.

[0388] The homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus. For example, a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence comprising the intended edit can be integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., having sufficient complementary for hybridization) by the 5ʹ and 3ʹ homology arms.

[0389] In some embodiments, the corresponding homologous nucleotide sequences in the genomic target sequence (i.e., the “5ʹ target sequence” and “3ʹ target sequence”) flank a specific site for cleavage and / or a specific site for introducing the intended edit. The distance between the specific cleavage site and the homologous nucleotide sequences (e.g., each homology arm) can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher gene targeting rate. In some embodiments, the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.

[0390] A homology arm can be of any length, e.g.10 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc. In some instances, the 5ʹ and 3ʹ homology arms are substantially equal in length to one another. However, in some instances the 5ʹ and 3ʹ homology arms are not necessarily equal in length to one another. For example, one homology arm may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology arm, or only a few nucleotides less than the other homology arm. In other instances, the 5ʹ and 3ʹ homology arms are substantially different in length from one another, e.g. one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.

[0391] The donor polynucleotide may be used in combination with an RNA-guided nuclease, which is targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by a guide RNA. A target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site. For example, the gRNA can be designed with a sequence complementary to the sequence of a minor allele to target the nuclease-gRNA complex to the site of a mutation. The mutation may comprise an insertion, a deletion, or a substitution. For example, the mutation may include a single nucleotide variation, gene fusion, translocation, inversion, duplication, frameshift, missense, nonsense, or other mutation associated with a phenotype or disease of interest. The targeted minor allele may be a common genetic variant or a rare genetic variant. In some embodiments, the gRNA is designed to selectively bind to a minor allele with single base-pair discrimination, for example, to allow binding of the nuclease-gRNA complex to a single nucleotidepolymorphism (SNP). In particular, the gRNA may be designed to target disease-relevant mutations of interest for the purpose of genome editing to remove the mutation from a gene. Alternatively, the gRNA can be designed with a sequence complementary to the sequence of a major or wild-type allele to target the nuclease-gRNA complex to the allele for the purpose of genome editing to introduces a mutation into a gene in the genomic DNA of the cell, such as an insertion, deletion, or substitution. Such genetically modified cells can be used, for example, to alter phenotype, confer new properties, or produce disease models for drug screening.

[0392] In some embodiments, the RNA-guided nuclease used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease. Any RNA-guided Cas nuclease capable of catalyzing site- directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system Class 1, Type I, II, or III Cas nucleases; Class 2, Type II nuclease (such as Cas9); a Class 2, Type V nuclease (such as Cpfl), or a Class 2, Type VI nuclease (such as C2c2). Examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof.

[0393] In some embodiments, a Class 1, type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to perform genome modification as described herein. The Cas9 need not be physically derived from an organism but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP 002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP 002344900), Campylobacter coli (WP 060786116); Campylobacter fetus (WP 059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP 033919308); Spiroplasma syrphidicola (NC 021284); Prevotella intermedia (NC 017861); Spiroplasma taiwanense (NC 021846); Streptococcus iniae (NC 021314); Belliella baltica (NC 018010); Psychroflexus torquisl (NC O 18721); Streptococcus thermophilus (YP 820832), Streptococcus mutans (WP 061046374, WP 024786433); Listeria innocua (NP 472073); Listeria monocytogenes (WP 061665472); Legionella pneumophila (WP 062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP 033919308); Lactobacillus rhamnosus (WP 048482595, WP_032965177); and Neisseria meningitidis (WP_061704949,YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference in their entireties. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res.42(4):2577-90; Kapitonov et al. (2015) J. Bacterid.198(5): 797-807, Shmakov et al. (2015) Mol. Cell.60(3):385- 397, and Chylinski et al. (2014) Nucleic Acids Res.42(10):6091-6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[0394] The genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA and may further comprise a protospacer adjacent motif (PAM). In some embodiments, the target site comprises 20-30 base pairs in addition to a 3 or more base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two or more other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide. In some embodiments, the allele targeted by a gRNA comprises a mutation that creates a PAM within the allele, wherein the PAM promotes binding of the Cas9-gRNA complex to the allele.

[0395] In some embodiments, the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15- 25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. The guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.

[0396] In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpfl, or Cas12a) is used. Cpfl is another class II CRISPR / Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cpfl does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cpfl for targeting than Cas9. Cpfl is capable of cleaving either DNA or RNA. The PAM sites recognized by Cpfl have the sequences 5ʹ-YTN-3ʹ (where “Y” is a pyrimidine and “N” is any nucleobase) or 5ʹ-TTN-3ʹ, in contrast to the G-rich PAM site recognized by Cas9. Cpfl cleavage of DNA produces double-stranded breaks with a sticky-ends having a 4 or 5 nucleotide overhang. For a discussion of Cpfl, see, e.g., Ledford et al. (2015) Nature.526 (7571):17-17, Zetsche et al. (2015) Cell.163 (3):759-771, Murovec et al. (2017) Plant Biotechnol. J.15(8):917-926, Zhang et al. (2017) Front. Plant Sci.8:177, Fernandes et al. (2016) Postepy Biochem.62(3):315-326; herein incorporated by reference.

[0397] C2c1 (Cas12b) is another class II CRISPR / Cas system RNA-guided nuclease that may be used. C2cl, similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites. See, e.g., Shmakov et al. (2015) Mol Cell.60(3):385-397, Zhang et al. (2017) Front Plant Sci.8:177; herein incorporated by reference.

[0398] In yet another embodiment, an engineered RNA-guided Fokl nuclease may be used. RNA- guided Fokl nucleases comprise fusions of inactive Cas9 (dCas9) and the Fokl endonuclease (FokI- dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on Fokl. For a description of engineered RNA-guided Fold nucleases, see, e.g., Havlicek et al. (2017) Mol. Ther.25(2):342-355, Pan et al. (2016) Sci Rep.6:35794, Tsai et al. (2014) Nat Biotechnol.32(6):569-576; herein incorporated by reference.

[0399] In other embodiments, any other Cas enzymes and variants described in other sections of the application (all incorporated herein) can be used similarly.

[0400] In some embodiments, the RNA-guided nuclease is provided in the form of a protein, optionally where the nuclease is complexed with a gRNA to form a ribonucleoprotein (RNP) complex. In some embodiments, the RNA-guided nuclease is provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector). In some embodiments, the RNA-guided nuclease and the gRNA are both provided by vectors, such as the vectors and the vector system described in other parts of the application (all incorporated herein by reference). Both can be expressed by a single vector or separately on different vectors. The vectors encoding the RNA-guided nuclease and gRNA may be included in the vector system comprising the engineered retron msr gene, msd gene and ret gene sequences. In some embodiments, the RNA- guided nuclease is fused to the RT and / or the msDNA.

[0401] The RNP complex may be administered to a subject or delivered into a cell by methods known in the art, such as those described in U.S. Pat. No.11,390,884, which is incorporated by reference herein in its entirety. In some embodiments, the endonuclease / gRNA ribonucleoprotein (RNP) complexes are delivered to cells by electroporation. Direct delivery of the RNP complex to a subject or cell eliminates the need for expression from nucleic acids (e.g., transfection of plasmids encoding Cas9 and gRNA). It also eliminates unwanted integration of DNA segments derived from nucleic acid delivery (e.g., transfection of plasmids encoding Cas9 and gRNA). An endonuclease / gRNA ribonucleoprotein (RNP) complex usually is formed prior to administration.

[0402] Codon usage may be optimized to further improve production of an RNA-guided nuclease and / or reverse transcriptase (RT) in a particular cell or organism. For example, a nucleic acid encoding an RNA-guided nuclease or reverse transcriptase can be modified to substitute codons having a higher frequency of usage in a yeast cell, a bacterial cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the RNA-guidednuclease or reverse transcriptase is introduced into cells, the protein can be transiently, conditionally, or constitutively expressed in the cell.

[0403] In some embodiments, the engineered retron used for genome editing with nuclease genome editing systems can further include accessory or enhancer proteins for recombination. Examples of recombination enhancers can include nonhomologous end joining (NHEJ) inhibitors (e.g., inhibitor of DNA ligase IV, a KU inhibitor (e.g., KU70 or KU80), a DNA-PKc inhibitor, or an artemis inhibitor) and homologous directed repair (HDR) promoters, or both, that can enhance or improve more precise genome editing and / or the efficiency of homologous recombination. In some embodiments, the recombination accessory or enhancers can comprise C-terminal binding protein interacting protein (CtIP), cyclinB2, Rad family members (e.g. Rad50, Rad51, Rad52, etc).

[0404] CtIP is a transcription factor containing C2H2 zinc fingers that are involved in early steps of homologous recombination. Mammalian CtIP and its orthologs in other eukaryotes promote the resection of DNA double-strand breaks and are essential for meiotic recombination. HDR may be enhanced by using Cas9 nuclease associated (e.g. fused) to an N-terminal domain of CtIP, an approach that forces CtIP to the cleavage site and increases transgene integration by HDR. In some embodiments, an N-terminal fragment of CtIP, called HE for HDR enhancer, may be sufficient for HDR stimulation and requires the CtIP multimerization domain and CDK phosphorylation sites to be active. HDR stimulation by the Cas9-HE fusion depends on the guide RNA used, and therefore the guide RNA will be designed accordingly.

[0405] Using the gene editing system described herein, any target gene or sequence in a host cell can be edited or modified for a desired trait, including but not limited to: Myostatin (e.g., GDF8) to increase muscle growth; Pc POLLED to induce hairlessness; KISS1R to induce bore taint; Dead end protein (dnd) to induce sterility; Nano2 and DDX to induce sterility; CD163 to induce PRRSV resistance; RELA to induce ASFV resilience; CD18 to induce Mannheimia (Pasteurella) haemolytica resilience; NRAMPl to induce tuberculosis resilience; Negative regulators of muscle mass (e.g., Myostatin) to increase muscle mass.

[0406] Any of the above retron editor embodiments or variants, modifications, or derivatives thereof are contemplated herein to be delivered by the LNP systems disclosed in this specification for gene editing in cells, tissues, and / or organs under in vitro, ex vivo, or in vivo conditions. The various components described herein may be configured and delivered in any suitable manner. Any of the descriptions presented in this section are not intended to be strictly limiting. TnpB editors

[0407] In other embodiments, the herein disclosed LNPs may be used to encapsulate and deliver a TnpB editing system and / or components thereof. A TnpB editing system in various embodiments may comprise (a) a TnpB protein, or a nucleic acid molecule encoding a TnpB protein, (b) a TnpB guide RNA known as an “reRNA” or “right end RNA”, and optionally one or more additionalcomponents, including (c) an effector domain or otherwise accessory protein, and (d) a DNA template (e.g., a DNA donor for HDR-dependent repair at the TnpB-cut target site.

[0408] In various embodiments, the TnpB protein can be naturally occurring or the TnpB can be an engineered variant thereof and can be used in various applications, including precision gene editing in cells, tissues, organs, or organisms. The TnpB-based gene editing systems comprise a TnpB polypeptide and a nucleic acid component capable of forming a complex with the TnpB polypeptide which directs the complex to a target nucleotide sequence (e.g., a genomic target sequence such as a disease-associated gene). The TnpB gene editing systems contemplated herein may also be modified with one or more additional effector or accessory functions, such as a nuclease, recombinase, ligase, reverse transcriptase, polymerase, deaminase, etc. to provide additional genome editing functionality. In addition, the TnpB gene editing systems contemplated herein can utilize a nuclease-limited or nuclease-deficienty TnpB variant. Normal TnpB nuclease activity cuts both strands of a target DNA, however, TnpB nickases (having only the ability to cut one of the two strands but not both strands) and nuclease-inactive or “dead” TnpB (which does not cut either strand) may also be used into the TnpB systems described herein, particularly when combined with at least another genome editing functionality, such as a deaminase (for base editing functionality) or a reverse transcriptase (for prime editing functionality). Thus, disclosed herein are TnpB systems that may function as nuclease, nickases, or catalytically inactive polynucleotide binding proteins that can be coupled with other functional domains, such as deaminases, recombinase, ligases, polymerases (e.g., reverse transcriptase), nucleases, or reverse transcriptases.

[0409] In one embodiment, the TnpB systems and related compositions may specifically target single-strand or double-strand DNA. In one embodiment, the TnpB system may bind and cleave double-strand DNA. In one embodiment, the TnpB system may bind to double-stranded DNA without introducing a break to either of the strands. In one embodiment, the TnpB polypeptides or nuclease / nucleic acid component complexes may open, disrupting the continuity of one of the two DNA strands, thereby introducing a nick of the double stranded DNA. In an embodiment, and without being bound by theory, the size and configuration of the TnpB systems allows exposure to the non- targeting strand, which may be in single-stranded form, to allow for for the ability to modify, edit, delete or insert polynucleotides on the non-target strand. In an embodiment, this accessibility further allows for enhanced editing outcomes on the target and / or non-target strand, e.g., increased specificity, enhanced editing efficiency.

[0410] In one aspect, embodiments disclosed herein are directed to compositions comprising a TnpB and a reRNA capable of forming a complex with the TnpB and directing site-specific binding of the TnpB to a target sequence on a target polynucleotide.TnpB polypeptides

[0411] Any TnpB polypeptide may be utilized with the compositions described herein. The below description of various TnpBs which can be used in connection with the presently disclose TnpB editing systems is not meant to be limiting in any way. The TnpB editing systems disclosed herein may comprise a canonical or naturally-occuring TnpB, or any ortholog TnpB protein, or any variant TnpB protein—including any naturally occurring variant, mutant, or otherwise engineered version of TnpB—that is known or which can be made or evolved through a directed evolutionary or otherwise mutagenic process. In various embodiments, the TnpB or TnpB variants can have a nickase activity, i.e., only cleave of strand of the target DNA sequence. In other embodiments, the TnpB or TnpB variants have inactive nucleases, i.e., are “dead” TnpB proteins. Other variant TnpB proteins that may be used are those having a smaller molecular weight than the canonical TnpB (e.g., for easier delivery) or having modified amino acid sequences or substitutions.

[0412] Examples of TnpB proteins are provided as follows; however, these specific examples are not meant to be limiting. The TnpB editing systems of the present disclosure may use any suitable TnpB protein.

[0413] In various other embodiments, the TnpB editing systems of the present disclosure may include one or more TnpB polypeptides and reRNAs disclosed in any of the following published applications, or a polypeptide (or reRNA as the case may be) having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with one or more of the TnpB polypeptides or reRNAs disclosed therein: US2023 / 0056577; US2023 / 0051396 A1; US11578313 B2; US2023 / 0040216 A1; WO2023 / 015259 A2; US2023 / 0032369 A1; US2023 / 0033866 A1; WO2023 / 004430 A1; US11560555 B2; WO2023 / 275601 A1; WO2022 / 253903 A1; WO2022 / 248607 A2; US2022 / 0372525 A1; US2022 / 0348929 A1; US2022 / 0348925 A1; US11453866 B2; WO2022 / 173830 A1; WO2022 / 174144 A1; WO2022 / 159892 A1; WO2022 / 150651 A1; US11384344 B2; WO2022 / 140572 A1; US2022 / 0195503 A1; WO2022 / 098923 A1; WO2022 / 087494 A1; WO2022 / 086846 A2; WO2022 / 076425 A1; WO2022 / 076890 A1; WO2021 / 257997 A2; WO2021 / 247924 A1; US2021 / 0380956 A1; US11180751 B2; WO2021 / 188729 A1; WO2021 / 188286 A2; WO2021 / 183807 A1; WO2021 / 159020 A2; US2021 / 0214697 A1; US2021 / 0166783 A1; WO2021 / 050601 A1; EP3009511 B2; US2020 / 0291395 A1; US2020 / 0239896 A1; WO2019 / 178428 A1; US2012 / 0178668 A1; US7608450 B2; US2004 / 0091856 A1; US2004 / 0009477 A1; US2003 / 0134302 A1; US6562958 B1; and WO1999 / 051766 A1, each of which are incorporated in their entireties by reference.

[0414] In certain example embodiments, the TnpB polypeptides are between 175 and 800 amino acids in size, between 200 and 790 amino acids in size, between 200 and 780 amino acids in size, between 200 and 770 amino acids in size, between 200 and 760 amino acids in size, between 200 and 750 amino acids in size, between 200 and 740 amino acids in size, between 200 and 730 amino acidsin size, between 200 and 720 amino acids in size, between 200 and 720 amino acids in size, between 200 and 710 amino acids in size, between 200 and 700 amino acids in size, between 200 and 690 amino acids in size, between 200 and 680 amino acids in size, between 200 and 670 amino acids in size, between 200 and 660 amino acids in size, between 200 and 650 amino acids in size, between 200 and 640 amino acids in size, between 200 and 630 amino acids in size, between 200 and 620 amino acids in size, between 200 and 610 amino acids in size, between 200 and 600 amino acids in size, between 200 and 590 amino acids in size, between 200 and 580 amino acids in size, between 200 and 570 amino acids in size, between 200 and 560 amino acid, between 200 between 550 amino acids, between 200 and 540 amino acids, between 200 and 530 amino acids, between 200 and 520 amino acids, between 200 and 510 amino acids, between 200 and 500 amino acids, between 200 and 490 amino acids, between 200 and 480 amino acids, between 200 and 470 amino acids, between 200 and 460 amino acids, between 200 and 450 amino acids, between 200 and 440 amino acids, between 200 and 430 amino acids, between 200 and 420 amino acids, between 200 and 410 amino acids, between 210 and 500 amino acids, between 220 and 500 amino acids. Between 230 and 500 amino acids, between 240 and 500 amino acids, between 250 and 500 amino acids, between 260 and 500 amino acids, between 270 and 500 amino acids, between 280 and 500 amino acids, between 290 and 500 amino acids, between 300 and 500 amino acids, between 250 and 490 amino acids, between 250 and 480 amino acids, between 250 and 490 amino acids, or between 250 and 600 amino acids. In one embodiment, the TnpB polypeptide is between 300 and 500 amino acids, or between 350 and 450 amino acids.

[0415] In one embodiment, the TnpB polypeptides may comprise a modified naturally occurring protein, functional fragment or truncated version thereof, or a non-naturally occurring protein. In one embodiment, the TnpB polypeptide comprises one or more domains originating from other TnpB polypeptides, more particularly originating from different organisms. In one embodiment, the TnpB polypeptides may be designed by in silico approaches. Examples of in silico protein design have been described in the art and are therefore known to a skilled person.

[0416] The TnpB polypeptides also encompass homologs or orthologs of TnpB polypeptides whose sequences are specifically described herein (such as the sequences of Table A). The terms “ortholog” and “homolog” are well known in the art. By means of further guidance, a “homolog” of a protein as used herein is a protein of the same species which performs the same or a similar function as the protein it is a homolog of. Homologous proteins may but need not be structurally related, or are only partially structurally related. An “ortholog” of a protein as used herein is a protein of a different species which performs the same or a similar function as the protein it is an orthologue of. Orthologous proteins may be, but may not always be, structurally related or are only partially structurally related. In particular embodiments, the homolog or ortholog of a TnpB polypeptide such as referred to herein has a sequence homology or identity of at least 80%, at least 81%, at least 82%,at least 83%, at least 84% at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with a TnpB polypeptide, more specifically with a TnpB sequence identified in Table A. In particular embodiments, a homolog or ortholog is identified according to its domain structure and / or function. Sequence alignments conducted as described herein, as well as folding studies and domain predictions can aid in the identification of a homolog or ortholog with the structural and functional characteristics identifying TnpB polypeptides, particularly those with conserved residues, including catalytic residues, and domains of TnpB polypeptides.

[0417] In one embodiment, the TnpB polypeptide comprises at least at least one RuvC-like nuclease domain. The RuvC domain may comprise conserved catalytic amino acids indicative of the RuvC catalytic residue. In an example embodiment, the RuvC catalytic residue may be referenced relative to D191, E278, and D361 of the TnpB of D. radiodurans or a corresponding amino acid in an aligned sequence. In an aspect, the RuvC domain may comprise multiple subdomains, e.g., RuvC-I, RuvC-II and RuvC-III. The subdomains may be separated by intervening amino acid sequence of the protein.

[0418] In one embodiment, examples of the RuvC domain include any polypeptides a structural similarity and / or sequence similarity to a RuvC domain described in the art. In some examples, the RuvC domain may have an amino acid sequence that share at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with RuvC domains known in the art. One of ordinary skill in the art can modify, substitute, or otherwise alter the activity of the RuvC domain to alter the nuclease activity, such as whether and / or where the nuclease cuts the DNA.

[0419] In embodiments, the TnpB polypeptide has a nuclease activity. In one embodiment, the TnpB and the targeting RNA (e.g., the reRNA) can direct sequence-specific nuclease activity. The cleavage may result in a 5’ overhang. The cleavage may occur distal to a target-adjacent motif (TAM), and may occur at the site of the spacer (i.e., the spacer of the reRNA which is complementary to the target sequences) annealing site or 3’ of the target sequence. In an aspect, the TnpB cleaves at multiple positions within and beyond the nucleic acid component annealing site. In an aspect, DNA cleavage occurs 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more base pairs distal to the TAM and results in a 5’ overhang. In various embodiments, the TnpB has a nuclease activity against single-stranded DNA. In other embodiments, the TnpB has a nuclease activity against double-stranded DNA. TnpB modifications

[0420] In various aspects, the present disclosure provides one or more modifications of TnpB comprising TnpB fusions, TnpB mutations to increase sufficiency and / or efficiency and modification of TnpB reRNA. In some embodiments, one or more domains of the TnpB are modified, e.g., wedge domain, corresponding to the β-barrel, REC – helical bundle, RuvC – RuvC domain with the inserted helical hairpin (HH) and the zinc-finger domain (ZnF).

[0421] Without intending to be limited to any particular theory, TnpB operates as a homodimer with one DNA molecule and for some orthologs, its ability to form this conformation may be efficacy limiting. Takeda, Satoru N et al. “Structure of the miniature type V-F CRISPR-Cas effector enzyme.” Molecular cell vol.81,3 (2021): 558-570.e3.

[0422] Karvelis et al. demonstrated Deinococcus radiodurans ISDra2 TnpB to be an RNA-directed nuclease guided by RE-derived RNA (reRNA) to cleave DNA next to the 5' TTGAT transposon associated motif (TAM). Karvelis, T., Druteika, G., Bigelyte, G. et al. Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease. Nature 599, 692–696 (2021).

[0423] Without being bound by theory, it is contemplated that TnpB likely operates as a homodimer. Recent studies show that Cas9-Cas9 fusions displayed higher levels of genome modification and a higher proportion of th...

Claims

CLAIMS 1. A gene editing system for editing a UBA1 gene, comprising: a) a nucleic acid programmable nuclease or a polynucleotide encoding the same; b) optionally an additional editing functionality; and c) at least one guide RNA comprising a spacer that targets the UBA1 gene, wherein the spacer is selected from: i) a sequence selected from SEQ ID NOs: 2077-2090, or a sequence having at least 95% sequence identity with any of said sequences; or ii) a spacer from a sequence selected from those disclosed in Appendix A (SEQ ID NO: 3-659), Appendix B (SEQ ID NO:660-1319), or Appendix C (SEQ ID NO: 1320-1976), or a sequence having at least 95% sequence identity with any of said sequences.

2. The gene editing system of claim 1, wherein the nucleic acid programmable nuclease is a CRISPR Type II nuclease, a CRISPR Type V, or a TnpB nuclease.

3. The gene editing system of claim 1, wherein the nucleic acid programmable nuclease is a nickase.

4. The gene editing system of any one of claims 1-3, wherein the additional editing functionality is reverse transcriptase, a recombinase, or a deaminase.

5. The gene editing system of any one of claims 1-4, wherein the UBA1 gene comprises one or more mutations.

6. The gene editing system of claim 5, wherein the one or more mutations in the UBA1 gene results in at least one mutation independently selected from the group consisting of a M41T, M41V, M41L, S56F, G477A, A478S, D506G, D506N, and S621C substitution.

7. A prime editing system for editing a UBA1 gene, comprising: a) a nucleic acid programmable nuclease or a polynucleotide encoding the same; b) a reverse transcriptase (RT); and c) at least one pegRNA that targets the UBA1 gene, wherein the pegRNA is selected from any one of the sequences from Appendix A (SEQ ID NO: 3-659), Appendix B (SEQ ID NO:660-1319), or Appendix C (SEQ ID NO: 1320-1976), or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of the sequences from Appendix A, Appendix B, or Appendix C.

8. The prime editing system of claim 7, wherein the nucleic acid programmable nuclease is a CRISPR Type II nuclease, a CRISPR Type V, or a TnpB nuclease.

9. The prime editing system of claim 7, wherein the nucleic acid programmable nuclease is a nickase.

10. The prime editing system of any one of claims 7-9, wherein the reverse transcriptase (RT) is a retron RT or a viral RT.

11. The prime editing system of any one of claims 7-9, wherein the RT is an MMLV RT.

12. The prime editing system of any one of claims 7-11, wherein the UBA1 gene comprises one or more mutations.

13. The prime editing system of claim 12, wherein the one or more mutations in the UBA1 gene results in at least one mutation independently selected from the group consisting of a M41T, M41V, M41L, S56F, G477A, A478S, D506G, D506N, and S621C substitution.

14. A pharmaceutical composition comprising: a) at least one lipid nanoparticle (LNP) comprising: i. one or more ionizable lipids; ii. one or more phospholipids, in an amount of about 20 mol% to about 60 mol% of the total lipid content of the lipid nanoparticle; iii. one or more structural lipids; and iv. one or more PEGylated lipids; and b) the gene editing system of any one of claims 1-6 or the prime editing system of any one of claims 7-13.

15. A pharmaceutical composition comprising: a) at least one lipid nanoparticle (LNP) comprising at least one ionizable lipid selected from those listed in Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X); and b) the gene editing system of any one of claims 1-6 or the prime editing system of any one of claims 7-13.

16. The pharmaceutical composition of claim 15, wherein the at least one lipid nanoparticle further comprises: i) at least one structural lipid; ii) at least one phospholipid; and iii) at least one PEGylated lipid.

17. The pharmaceutical composition of claims 14 or 16, wherein the at least one structural lipid is selected from cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta- sitosterol-acetate and any combinations thereof.

18. The pharmaceutical composition of any one of claims 14, 16, or 17, wherein the at least one phospholipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC), 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuc cinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn- glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3- ((((R)-2-(oleoyloxy)-3-(stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-α- phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl- phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell- fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Dielaidoyl-sn- phosphatidylethanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl- phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphate (18:1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate (18:1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DOPI; 18:1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2-dilinoleoyl-sn-glycero-3-phospho-L- serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0-18:1 PS; POPS), 1- stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycero-3- phospho-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin.

19. The pharmaceutical composition of any one of claims 14 or 16-18, wherein the at least one PEGylated lipid is selected from (R)-2,3-bis(octadecyloxy)propyl-1- (methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG- PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG- DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-C11, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click C10, N(Carbonyl- methoxypolyethylenglycol-2000)-l,2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG- PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2-distearoyl-sn-glycero-3- phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE-PEGMA 500, DSPE- PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE- PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol-polyethyleneglycol, Cl8PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R)-2,3- bis(octadecyloxy)propyl-1-(methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)-C-DOMG, PEG-C-DMA, and DSPE-PEG-X.

20. The pharmaceutical composition of any one of claims 14-19, wherein the LNP further comprises at least one additional lipid component selected from 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3 PC), Acylcarnosine (AC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), N-oleoyl-sphingomyelin (SPM) (C18:l), N-lignoceryl SPM (C24:0), N-nervonoylshphingomyelin (C24:l), Cardiolipin (CL), l,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8-9PC), dicetyl phosphate (DCP), dihexadecyl phosphate (DCP1), 1,2-Dipalmitoylglycerol-3-hemisuccinate (DGSucc), short-chain bis-n-heptadecanoyl phosphatidylcholine (DHPC), dihexadecoyl- phosphoethanolamine (DHPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), l,2-dilauroyl- sn-glycero-3-PE (DLPE), dimyristoyl glycerol hemisuccinate (DMGS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleyloxybenzylalcohol (DOBA), 1,2-dioleoylglyceryl-3- hemisuccinate (DOGHEMS), N-[2-(2-{2-[2-(2,3-Bis-octadec-9-enyloxy-propoxy)-ethoxy]-ethoxy}- ethoxy)-ethyl]-3-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-ylsulfanyl)-propionamide (DOGP4αMan), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell- fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), histaminedistearoylglycerol (HDSG), 1,2-Dipalmitoylglycerol- hemisuccinate-Nα-Histidinyl-Hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10-12- pentacosadiynamide (h-Pegi-PCDA), 2-[l-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH), hydrogenatedsoybeanphosphatidylcholine (HSPC), 1,2-Dipalmitoylglycerol-O-α-histidinyl-Nα- hemisuccinate (IsohistsuccDG), mannosialized dipalmitoylphosphatidylethanolamine (ManDOG), l,2- Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (MCC-PE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1-myristoyl-2- hydroxy-sn-glycero-phosphocholine (MHPC), a thiol-reactive maleimide headgroup lipid e.g.1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)but-yramid (MPB-PE), Nervonic Acid (NA), sodium cholate (NaChol), l,2-dioleoyl-sn-glycero-3-[phosphoethanolamine-N- dodecanoyl (NC12-DOPE), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), phosphatidylethanolamine lipid (PE), PE lipid conjugated with polyethylene glycol(PEG) (e.g., polyethylene glycol-distearoylphosphatidylethanolamine lipid (PEG-PE)), phosphatidylglycerol (PG), partially hydrogenated soy phosphatidylchloline (PHSPC), phosphatidylinositol lipid (PI), phosphotidylinositol-4-phosphate (PIP), palmitoyloleoylphosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), phosphatidylserine (PS), lissamine rhodamineB- phosphatidylethanolamine lipid (Rh-PE), purified soy-derived mixture of phospholipids (SIOO), phosphatidylcholine (SM), 18-1-trans-PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE),soybean phosphatidylcholine (SPC), sphingomyelins (SPM), alpha,alpha-trehalose-6,6'-dibehenate (TDB), l,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE), ((23S,5R)-3- (bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran- 2-yl)methylmethylphosphate, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, and dioleylphosphatidylethanolamine.

21. The pharmaceutical composition of any one of claims 14-20, wherein the LNP further comprises one or more targeting moieties.

22. A method of treating VEXAS syndrome in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition of any one of claims 14-21.

23. The pharmaceutical composition of any one of claims 14-21 for use as a medicament in the treatment of VEXAS syndrome.

24. Use of a pharmaceutical composition of any one of claims 14-21 for the manufacture of a medicament for delivery of a gene editing system capable of treating VEXAS syndrome.

25. A method of correcting one or more VEXAS-associated mutations in a mutant UBA1 gene comprising: administering to a subject in need thereof an effective amount of a composition comprising (i) a prime editor or a polynucleotide encoding a prime editor, (ii) a pegRNA comprising a spacer that targets the UBA1 gene and a DNA synthesis template encoding an edit to correct the one or more VEXAS-associated mutations, and (iii) optionally, a second stranding nicking guide, wherein the spacer is selected from a spacer from any one the sequences from: (a) a sequence selected from SEQ ID NOs: 2077-2090, or a sequence having at least 95% sequence identity with any of said sequences; or (b) Appendix A, Appendix B, or Appendix C, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequenceidentity with any one of the sequences from Appendix A, Appendix B, or Appendix C.

26. The method of claim 25, wherein the prime editor comprises a nucleic acid programmable nuclease and a reverse transcriptase.

27. The method of claim 26, wherein the nuclease is a CRISPR type II nickase or a CRISPR type V nickase.

28. The method of claim 25, wherein the one or more VEXAS-associated UBA1 mutations is selected from the group consisting of: M41T, M41V, M41L, S56F, G477A, A478S, D506G, D506N, and S621C.

29. The method of claim 26, wherein the reverse transcriptase (RT) is a retron RT or a viral RT.

30. The method of claim 29, wherein the viral RT is an MMLV RT.

31. The method of any one of claims 25-30, wherein the composition is an LNP composition comprising at least one ionizable lipid selected from those listed in Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X).

32. The method of claim 31, wherein the LNP composition further comprises: i) at least one structural lipid; ii) at least one phospholipid; and iii) at least one PEGylated lipid.

33. The method of claim 32, wherein the at least one structural lipid is selected from the group consisting of cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta- sitosterol-acetate and any combinations thereof.

34. The method of claim 32, wherein the at least one phospholipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC), 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuc cinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn- glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3- ((((R)-2-(oleoyloxy)-3-(stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-α- phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl- phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell- fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Dielaidoyl-sn- phosphatidylethanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl- phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn- glycero-3-phosphate (18:1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate (18:1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DOPI; 18:1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2-dilinoleoyl-sn-glycero-3-phospho-L- serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0-18:1 PS; POPS), 1- stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycero-3- phospho-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin.

35. The method of claim 32, wherein the at least one PEGylated lipid is selected from the group consisting of (R)-2,3-bis(octadecyloxy)propyl-1- (methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG- PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG- DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-C11, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEGDMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click C10, N(Carbonyl- methoxypolyethylenglycol-2000)-l,2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG- PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2-distearoyl-sn-glycero-3- phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE-PEGMA 500, DSPE- PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE- PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol-polyethyleneglycol, Cl8PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R)-2,3- bis(octadecyloxy)propyl-1-(methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)-C-DOMG, PEG-C-DMA, and DSPE-PEG-X.

36. The method of claim 32, wherein the LNP further comprises at least one additional lipid component selected from the group consisting of 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3 PC), Acylcarnosine (AC), 1- hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), N-oleoyl-sphingomyelin (SPM) (C18:l), N- lignoceryl SPM (C24:0), N-nervonoylshphingomyelin (C24:l), Cardiolipin (CL), l,2-bis(tricosa- 10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8-9PC), dicetyl phosphate (DCP), dihexadecyl phosphate (DCP1), 1,2-Dipalmitoylglycerol-3-hemisuccinate (DGSucc), short-chain bis-n- heptadecanoyl phosphatidylcholine (DHPC), dihexadecoyl-phosphoethanolamine (DHPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), l,2-dilauroyl-sn-glycero-3-PE (DLPE), dimyristoyl glycerol hemisuccinate (DMGS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleyloxybenzylalcohol (DOBA), 1,2-dioleoylglyceryl-3-hemisuccinate (DOGHEMS), N-[2-(2-{2-[2-(2,3-Bis-octadec-9- enyloxy-propoxy)-ethoxy]-ethoxy}-ethoxy)-ethyl]-3-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro- pyran-2-ylsulfanyl)-propionamide (DOGP4αMan), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl- phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), histaminedistearoylglycerol (HDSG), 1,2-Dipalmitoylglycerol-hemisuccinate-Nα-Histidinyl- Hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10-12-pentacosadiynamide (h-Pegi- PCDA), 2-[l-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH), hydrogenatedsoybeanphosphatidylcholine (HSPC), 1,2-Dipalmitoylglycerol-O-α-histidinyl-Nα- hemisuccinate (IsohistsuccDG), mannosialized dipalmitoylphosphatidylethanolamine (ManDOG), l,2- Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (MCC-PE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1-myristoyl-2- hydroxy-sn-glycero-phosphocholine (MHPC), a thiol-reactive maleimide headgroup lipid e.g.1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)but-yramid (MPB-PE), Nervonic Acid (NA), sodium cholate (NaChol), l,2-dioleoyl-sn-glycero-3-[phosphoethanolamine-N- dodecanoyl (NC12-DOPE), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), phosphatidylethanolamine lipid (PE), PE lipid conjugated with polyethylene glycol(PEG) (e.g., polyethylene glycol-distearoylphosphatidylethanolamine lipid (PEG-PE)), phosphatidylglycerol (PG), partially hydrogenated soy phosphatidylchloline (PHSPC), phosphatidylinositol lipid (PI), phosphotidylinositol-4-phosphate (PIP), palmitoyloleoylphosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), phosphatidylserine (PS), lissamine rhodamineB- phosphatidylethanolamine lipid (Rh-PE), purified soy-derived mixture of phospholipids (SIOO), phosphatidylcholine (SM), 18-1-trans-PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), soybean phosphatidylcholine (SPC), sphingomyelins (SPM), alpha,alpha-trehalose-6,6'-dibehenate (TDB), l,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE), ((23S,5R)-3- (bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran- 2-yl)methylmethylphosphate, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, and dioleylphosphatidylethanolamine.

37. The method of any one of claims 25-36, wherein the administering is in vivo.

38. The method of any one of claims 25-36, wherein the administering is in vitro.

39. The method of any one of claims 25-36, wherein the administering is ex vivo.

40. The method of any one of claims 25-36, wherein the administering is to a cell in vivo, wherein a transformed cell comprises an edited or installed VEXAS-associated mutation in the UBA1 gene.

41. The method of claim 40, further comprising harvesting the cell and culturing the cell to form a cell line comprising a UBA1 gene with an edited VEXAS-associated mutation.

42. A lipid nanoparticle comprising: i. a cargo comprising one or more nucleic acid molecules encoding and / or constituting a gene editing system for editing a UBA1 gene; ii. one or more ionizable lipids; iii. one or more phospholipids, in an amount of about 20 mol% to about 60 mol% of the total lipid content of the lipid nanoparticle; iv. one or more structural lipids; and v. one or more PEG lipids.

43. The lipid nanoparticle of claim 42, wherein the one or more nucleic acid molecules are RNA molecules.

44. The lipid nanoparticle of any one of claims 42-43, wherein the lipid nanoparticle has an N:P ratio of between about 5:1 to about 8:

1.

45. The lipid nanoparticle of any one of claims 42-44, wherein the lipid nanoparticle comprises about 25 mol% to about 45 mol% of the one or more ionizable lipids, as a proportion of the total lipid content of the lipid nanoparticle.

46. The lipid nanoparticle of any one of claims 42-45, wherein the lipid nanoparticle comprises about 15 mol% to about 35 mol% of the one or more structural lipids, as a proportion of the total lipid content of the lipid nanoparticle.

47. The lipid nanoparticle of any one of claims 42-46, wherein the lipid nanoparticle comprises about 1 mol% to about 3 mol% of the one or more PEG lipids, as a proportion of the total lipid content of the lipid nanoparticle.

48. The lipid nanoparticle of any one of claims 42-47, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of the one or more PEG lipids; (b) about 15 mol% to about 35 mol% of the one or more structural lipids; (c) about 30 mol% to about 60 mol% of the one or more phospholipids; and (d) about 25 mol% to about 45 mol% of the one or more ionizable lipids.

49. The lipid nanoparticle of any one of claims 42-48, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of the one or more PEG lipids; (b) about 20 mol% to about 30 mol% of the one or more structural lipids; (c) about 35 mol% to about 45 mol% of the one or more phospholipids; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

50. The lipid nanoparticle of any one of claims 42-49, wherein the ionizable lipid is selected from any one of the compounds described in Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X).

51. The lipid nanoparticle of any one of claims 42-50, wherein the phospholipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC), 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuc cinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn- glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3- ((((R)-2-(oleoyloxy)-3-(stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-α- phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl- phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell- fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Dielaidoyl-sn- phosphatidylethanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn- glycero-3-phosphate (18:1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate (18:1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DOPI; 18:1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2-dilinoleoyl-sn-glycero-3-phospho-L- serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0-18:1 PS; POPS), 1- stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycero-3- phospho-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin, or combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the above phospholipids.

52. The lipid nanoparticle of any one of claims 42-51, wherein the structural lipid is selected from cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha- tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta-sitosterol- acetate and any combinations thereof.

53. The lipid nanoparticle of any one of claims 42-52, wherein the PEG lipid is selected from (R)- 2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG- DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG- DMG, PEG-DMA, PEG-Ceramide C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-C11, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click C10, N(Carbonyl-methoxypolyethylenglycol-2000)-l,2-distearoyl-sn-glycero3- phosphoethanolamine, Myrj52, mPEG-PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE-PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE-PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG- PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DOPE- PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol-polyethyleneglycol, Cl8PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000,C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-PEG2000, (R)-2,3-bis(octadecyloxy)propyl-1- (methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)-C-DOMG, PEG-C-DMA, and DSPE- PEG-X, and any combinations thereof.

54. The lipid nanoparticle of any one of claims 42-53, wherein the one or more phospholipids comprises one or more selected from phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and sphingoid lipids or a combination thereof.

55. The lipid nanoparticle of any one of claims 42-54, wherein the one or more phospholipids comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), sphingomyelin or a combination thereof.

56. The lipid nanoparticle of any one of claims 42-55, wherein the one or more phospholipids comprises two or more phospholipids, such that no single phospholipid makes up more than 30 mol% of the total lipid content of the nanoparticle.

57. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises about 40 mol% 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

58. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises about 40 mol% sphingomyelin.

59. The lipid nanoparticle of any one of claims 42-55, wherein the phospholipid is sphingomyelin, the structural lipid is cholesterol, and the PEG lipid is DMG-PEG2k.

60. The lipid nanoparticle of any one of claims 42-55, wherein the ionizable lipid is any compound from Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X), the phospholipid is sphingomyelin, the structural lipid is cholesterol, and the PEG lipid is DMG-PEG2k.

61. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% sphingomyelin; and (d) about 25 mol% to about 45 mol% of the one or more ionizable lipids.

62. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% sphingomyelin; and (d) about 25 mol% to about 45 mol% of an ionizable lipid selected from those in Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X), or any combinations thereof.

63. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG-PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% sphingomyelin; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

64. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG-PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% sphingomyelin; and (d) about 28 mol% to about 40 mol% of an ionizable lipid selected from those in Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X), or any combinations thereof.

65. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol; (c) about 40 mol% sphingomyelin; and (d) about 33 mol% of an ionizable lipid selected from those in Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X), or any combinations thereof.

66. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol; (c) about 40 mol% sphingomyelin; and (d) about 33 mol% of the one or more ionizable lipids.

67. The lipid nanoparticle of any one of claims 1-38, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% DSPC; and (d) about 25 mol% to about 45 mol% of the one or more ionizable lipids.

68. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% DSPC; and (d) about 25 mol% to about 45 mol% of an ionizable lipid selected from those in Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X), or any combinations thereof.

69. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG-PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% DSPC; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

70. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG-PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% DSPC; and (d) about 28 mol% to about 40 mol% of an ionizable lipid selected from those in Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X), or any combinations thereof.

71. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol; (c) about 40 mol% DSPC; and (d) about 33 mol% of an ionizable lipid selected from those in Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X), or any combinations thereof.

72. The lipid nanoparticle of any one of claims 42-55, wherein the one or more phospholipids comprises a mixture of sphingomyelin and DSPC, the structural lipid is cholesterol, and the PEG lipid is DMG -PEG2k.

73. The lipid nanoparticle of any one of claims 42-55, wherein the ionizable lipid is any compound from Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X), the one or more phospholipids comprise a mixture of sphingomyelin and DSPC, the structural lipid is cholesterol, and the PEG lipid is DMG-PEG2k.

74. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% of a combination of sphingomyelin and DSPC; and (d) about 25 mol% to about 45 mol% of the one or more ionizable lipids.

75. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 1 mol% to about 3 mol% of DMG-PEG2k; (b) about 15 mol% to about 35 mol% cholesterol; (c) about 30 mol% to about 60 mol% of a combination of sphingomyelin and DSPC; and (d) about 25 mol% to about 45 mol% of an ionizable lipid selected from those in Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X), or any combinations thereof.

76. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG-PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% of a combination of sphingomyelin and DSPC; and (d) about 28 mol% to about 40 mol% of the one or more ionizable lipids.

77. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 1.5 mol% to about 2.5 mol% of DMG-PEG2k; (b) about 20 mol% to about 30 mol% cholesterol; (c) about 35 mol% to about 45 mol% of a combination of sphingomyelin and DSPC; and (d) about 28 mol% to about 40 mol% of an ionizable lipid selected from those in Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X), or any combinations thereof.

78. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol; (c) about 40 mol% of a combination of sphingomyelin and DSPC; and (d) about 33 mol% of an ionizable lipid selected from those in Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X), or any combinations thereof.

79. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises (a) about 2 mol% of DMG-PEG2k; (b) about 25 mol% cholesterol; (c) about 40 mol% of a combination of sphingomyelin and DSPC; and (d) about 33 mol% of the one or more ionizable lipids.

80. The lipid nanoparticle of any one of claims 42-55, wherein the lipid nanoparticle comprises about 20 mol% 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and about 20 mol% sphingomyelin.

81. The lipid nanoparticle of any one of claims 42-80, wherein the one or more nucleic acid molecules encoding and / or constituting a gene editing system for editing a UBA1 gene comprises the gene editing system of any one of claims 1-6 or the prime editing system of any one of claims 7-13.

82. The lipid nanoparticle of any one of claims 42-80, wherein the LNP further comprises one or more targeting moieties.

83. The lipid nanoparticle of claim 82, wherein the targeting moiety has affinity for an HSC or surface protein thereof.

84. The lipid nanoparticle of claim 83, wherein the HSC surface protein is selected from the group consisting of: CD2; 2B4 / CD244 / SLAMF4; ABCG2; Aldehyde Dehydrogenase 1- A1 / ALDH1A1; BMI-1; C1qR1 / CD93; CD34; CD38; CD44; CD45; CD48 / SLAMF2; CD90 / Thy1; CD117 / c-kit; CD133; CDCP1; CXCR4; Endoglin / CD105; EPCR; Erythropoietin R; ESAM; EVI- 1;Flt-3 / Flk-2; GATA-2; GFI-1; Hematopoietic Lineage Marker; Integrin alpha 6 / CD49f; Mcl-1; MYB; PLZF; Podocalyxin; Prominin 2; PTEN; PU.1 / Spi-1; Sca-1 / Ly6; SLAM / CD150; Spi-B; STAT5a / b; STAT5a; STAT5b; VCAM-1 / CD106; and VEGFR2 / KDR / Flk-1.

85. A method of treating VEXAS syndrome in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising the lipid nanoparticle of any one of claims 42-84.

86. A pharmaceutical composition comprising the lipid nanoparticle of any one of claims 42- 84for use as a medicament in the treatment of VEXAS syndrome.

87. Use of a pharmaceutical composition comprising the lipid nanoparticle of any one of claims 42-84 for the manufacture of a medicament for delivery of a gene editing system capable of treating VEXAS syndrome.

88. A method of installing one or more VEXAS-associated mutations in a mutant UBA1 gene comprising: contacting with a cell an effective amount of a composition comprising (i) a prime editor or a polynucleotide encoding a prime editor, (ii) a pegRNA comprising a spacer that targets the UBA1 gene and a DNA synthesis template encoding an edit to correct the one or more VEXAS-associated mutations, and (iii) optionally, a second stranding nicking guide, wherein the spacer is selected from a spacer from any one the sequences from: (a) a sequence selected from SEQ ID NOs: 2077-2090, or a sequence having at least 95% sequence identity with any of said sequences; or (b) Appendix A, Appendix B, or Appendix C, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of the sequences from Appendix A, Appendix B, or Appendix C.

89. The method of claim 88, wherein the prime editor comprises a nucleic acid programmable nuclease and a reverse transcriptase.

90. The method of claim 89, wherein the nuclease is a CRISPR type II nickase or a CRISPR type V nickase.

91. The method of claim 88, wherein the one or more VEXAS-associated UBA1 mutations is selected from the group consisting of: M41T, M41V, M41L, S56F, G477A, A478S, D506G, D506N, and S621C.

92. The method of claim 89, wherein the reverse transcriptase (RT) is a retron RT or a viral RT.

93. The method of claim 92, wherein the viral RT is an MMLV RT.

94. The method of any one of claims 88-93, wherein the composition is an LNP composition comprising at least one ionizable lipid selected from those listed in Tables (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), and (X).

95. The method of claim 94, wherein the LNP composition further comprises: i) at least one structural lipid; ii) at least one phospholipid; and iii) at least one PEGylated lipid.

96. The method of claim 95, wherein the at least one structural lipid is selected from the group consisting of cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta- sitosterol-acetate and any combinations thereof.

97. The method of claim 95, wherein the at least one phospholipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC), 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuc cinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn- glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3- ((((R)-2-(oleoyloxy)-3-(stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-α- phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl- phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell- fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Dielaidoyl-sn- phosphatidylethanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl- phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn- glycero-3-phosphate (18:1 PA; DOPA), ammonium bis((S)-2-hydroxy-3-(oleoyloxy)propyl) phosphate (18:1 DMP; LBPA), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol) (DOPI; 18:1 PI), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2-dilinoleoyl-sn-glycero-3-phospho-L- serine (18:2 PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (16:0-18:1 PS; POPS), 1- stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18:1 PS), 1-stearoyl-2-linoleoyl-sn-glycero-3- phospho-L-serine (18:0-18:2 PS), 1-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:1 Lyso PS), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin.

98. The method of claim 95, wherein the at least one PEGylated lipid is selected from the group consisting of (R)-2,3-bis(octadecyloxy)propyl-1- (methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG- PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG- DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-C11, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click C10, N(Carbonyl- methoxypolyethylenglycol-2000)-l,2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG- PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE,mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2-distearoyl-sn-glycero-3- phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE-PEGMA 500, DSPE- PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE- PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol-polyethyleneglycol, Cl8PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R)-2,3- bis(octadecyloxy)propyl-1-(methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)-C-DOMG, PEG-C-DMA, and DSPE-PEG-X.

99. The method of claim 95, wherein the LNP further comprises at least one additional lipid component selected from the group consisting of 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3 PC), Acylcarnosine (AC), 1- hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), N-oleoyl-sphingomyelin (SPM) (C18:l), N- lignoceryl SPM (C24:0), N-nervonoylshphingomyelin (C24:l), Cardiolipin (CL), l,2-bis(tricosa- 10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8-9PC), dicetyl phosphate (DCP), dihexadecyl phosphate (DCP1), 1,2-Dipalmitoylglycerol-3-hemisuccinate (DGSucc), short-chain bis-n- heptadecanoyl phosphatidylcholine (DHPC), dihexadecoyl-phosphoethanolamine (DHPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), l,2-dilauroyl-sn-glycero-3-PE (DLPE), dimyristoyl glycerol hemisuccinate (DMGS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleyloxybenzylalcohol (DOBA), 1,2-dioleoylglyceryl-3-hemisuccinate (DOGHEMS), N-[2-(2-{2-[2-(2,3-Bis-octadec-9- enyloxy-propoxy)-ethoxy]-ethoxy}-ethoxy)-ethyl]-3-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro- pyran-2-ylsulfanyl)-propionamide (DOGP4αMan), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl- phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), histaminedistearoylglycerol (HDSG), 1,2-Dipalmitoylglycerol-hemisuccinate-Nα-Histidinyl- Hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10-12-pentacosadiynamide (h-Pegi-PCDA), 2-[l-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH), hydrogenatedsoybeanphosphatidylcholine (HSPC), 1,2-Dipalmitoylglycerol-O-α-histidinyl-Nα- hemisuccinate (IsohistsuccDG), mannosialized dipalmitoylphosphatidylethanolamine (ManDOG), l,2- Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (MCC-PE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1-myristoyl-2- hydroxy-sn-glycero-phosphocholine (MHPC), a thiol-reactive maleimide headgroup lipid e.g.1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)but-yramid (MPB-PE), Nervonic Acid (NA), sodium cholate (NaChol), l,2-dioleoyl-sn-glycero-3-[phosphoethanolamine-N- dodecanoyl (NC12-DOPE), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), phosphatidylethanolamine lipid (PE), PE lipid conjugated with polyethylene glycol(PEG) (e.g., polyethylene glycol-distearoylphosphatidylethanolamine lipid (PEG-PE)), phosphatidylglycerol (PG), partially hydrogenated soy phosphatidylchloline (PHSPC), phosphatidylinositol lipid (PI), phosphotidylinositol-4-phosphate (PIP), palmitoyloleoylphosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), phosphatidylserine (PS), lissamine rhodamineB- phosphatidylethanolamine lipid (Rh-PE), purified soy-derived mixture of phospholipids (SIOO), phosphatidylcholine (SM), 18-1-trans-PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), soybean phosphatidylcholine (SPC), sphingomyelins (SPM), alpha,alpha-trehalose-6,6'-dibehenate (TDB), l,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE), ((23S,5R)-3- (bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran- 2-yl)methylmethylphosphate, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, and dioleylphosphatidylethanolamine.

100. The method of any of claims 88-99, wherein the cell is from a cell line described in Example 8.

101. A cell line produced in accordance with the method of claim 100.

102. A mouse model produced in accordance with the method of Example 10.

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