cGAS-STING PATHWAY INHIBITORS AND USES THEREOF

Abstract

Disclosed herein are cGAS-STING pathway inhibitors, compositions, and uses including dsDNA delivery and genome-editing. The cGAS-STING pathway inhibitors reduce immune activation when introducing dsDNA to a cell. The disclosed cGAS-STING pathway inhibitors may be used in genome-editing applications.

Description

[0001] cGAS-STING PATHWAY INHIBITORS AND USES THEREOF

[0002] SEQUENCE LISTING

[0003] [1] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 20, 2025, is named 16290_0010-00304_SL.xml and is 58,000 bytes in size.

[0004] RELATED APPLICATIONS

[0005] [2] This Application claims the benefit and priority to US Provisional Application 63 / 622,849 filed June 21, 2024, entitled “cGAS-STING PATHWAY INHIBITORS AND USES THEREOF,” the contents of which are expressly incorporated therein by reference in its entirety.

[0006] FIELD OF THE DISCLOSURE

[0007] [3] This disclosure relates to cGAS-STING pathway inhibitors and methods of dsDNA delivery and genome-editing.

[0008] BACKGROUND

[0009] [4] Cells have evolved immune responses to the presence of cytoplasmic nucleic acids to defend against viral infection. The cyclic GMP-AMP synthase (cGAS)-STlNG pathway is one of the dominant pathways that senses cytosolic double-stranded DNA (dsDNA). cGAS initiates the early immune response to DNA viruses after binding dsDNA. This causes a conformational change in cGAS catalyzing the generation of 2’,3’-cGAMP, which act as a second messenger (Lee et al. Experimental & Molecular Medicine (2019) 51:153). The accumulation of 2’3’-cGAMP results in the dimerization of STING, which in turn is phosphorylated by TBK1 triggering the downstream activation of the transcription factors NF-KB, IFR3, and IFR7 (Lee et al. 2019). These transcription factors induce expression of type I interferons and pro-inflammatory cytokines, which activate immune cells, producing an inflammatory response.

[0010] [5] Nucleic acid-based medicines have become an increasingly important treatment modality for various medical indications. These approaches require the delivery of nucleic acids, which may include a dsDNA component. However, the administration of dsDNA may result in the activation of the cGAS-STING pathway, potentially leading to undesired immune response effects. In particular, genome-editing approaches have gained prominence in recent years as they have the potential to treat and even cure disease at the genetic level, as opposed to merely managing symptoms. Depending on the genome editing system and method of delivery, certain components of a genome-editing system can trigger a cell’s immune response due to the presence of an exogenous nucleic acid such as a dsDNA component. The inflammation caused by the immune response may limit the applicability or effectiveness of nucleic acid medicines and genome-editing systems. Accordingly, there is an unmet need for factors that prevent or reduce the immune response to dsDNA either as a nucleic acid medicine or a component of a genome-editing system to improve the ability to introduce these therapeutics to cells.

[0011] SUMMARY

[0012] [6] In some aspects, the present disclosure provides cGAS-STING pathway inhibitors and compositions for dsDNA delivery and genome-editing.

[0013] [7] In some aspects, the present disclosure provides compositions for dsDNA delivery. In some embodiments, the composition for dsDNA delivery comprises a cGAS-STING pathway inhibitor or nucleic acid encoding a cGAS-STING pathway inhibitor, dsDNA, and at least one acceptable carrier, wherein the cGAS-STING pathway inhibitor and dsDNA are encapsulated in lipid nanoparticles (LNPs). In some embodiments, the cGAS-STING pathway inhibitor reduces cGAS binding to DNA. In some embodiments, the cGAS-STING pathway inhibitor ligates ubiquitin to cGAS for degradation. In some embodiments, the cGAS-STING pathway inhibitor degrades 2’3’ cGAMP. In some embodiments, the cGAS-STING pathway inhibitor comprises a protein selected from Tables 1-3. In some embodiments, the cGAS-STING pathway inhibitor comprises one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor is encoded by a nucleic acid encoding an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor further comprises a nuclear localization signal (NLS). In some embodiments, the cGAS-STING pathway inhibitor comprises a poxin. In some embodiments, the cGAS-STING pathway inhibitor further comprises an intrinsically disordered region (IDR). In some embodiments, the cGAS-STING pathway inhibitor is encoded in nucleic acid. In some embodiments, the cGAS-STING pathway inhibitor is mRNA. In some embodiments, the cGAS-STING pathway inhibitor is circular' RNA.

[0014] [8] In some embodiments, the dsDNA comprises an AttD site.

[0015] [9] In some embodiments, the cGAS-STING pathway inhibitor and dsDNA are encapsulated in separate LNPs. In some embodiments, the cGAS-STING pathway inhibitor and dsDNA are co- encapsulated. In some embodiments, the LNPs contain RNA and DNA at a mass ratio of 1 : 1 , 2: 1 , 3:1, 10:1, 1:2, 1:3, or 1:10.

[0016]

[0010] In some aspects, the present disclosure provides compositions for genome-editing. In some embodiments, the composition for genome-editing comprises a cGAS-STING pathway inhibitor or nucleic acid encoding a cGAS-STING pathway inhibitor, a genome-editing enzyme or nucleic acid encoding a genome-editing enzyme, dsDNA, and at least one acceptable carrier. In some embodiments, the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are encapsulated in LNPs. In some embodiments, the cGAS-STING pathway inhibitor reduces cGAS binding to DNA. In some embodiments, the cGAS-STING pathway inhibitor ligates ubiquitin to cGAS for degradation. In some embodiments, the cGAS-STING pathway inhibitor degrades 2’3’ cGAMP. In some embodiments, the cGAS-STING pathway inhibitor comprises a protein selected from Table 1, Table 2, or Table 3. In some embodiments, the cGAS-STING pathway inhibitor comprises one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor is encoded by a nucleic acid encoding an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor further comprises a nuclear localization signal (NLS). In some embodiments, the cGAS-STING pathway inhibitor comprises a poxin. In some embodiments, the cGAS-STING pathway inhibitor further comprises an intrinsically disordered region (IDR). In some embodiments, the cGAS-STING pathway inhibitor is encoded in nucleic acid. In some embodiments, the cGAS-STING pathway inhibitor is mRNA. In some embodiments, the cGAS-STING pathway inhibitor is circular RNA.

[0017]

[0011] In some embodiments, the genome-editing enzyme comprises a large serine recombinase (LSR). In some embodiments, the genome-editing enzyme comprises a Cas polypeptide, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a tyrosine recombinase, or a transposon-encoded enzyme. In some embodiments, the transposon-encoded enzyme comprises TnpB, TnsB, a PiggyBac transposase, or a Sleeping Beauty transposase.

[0018]

[0012] In some embodiments, the cGAS-STING pathway inhibitor and genome-editing enzyme are encoded on a single nucleic acid. In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA comprises a P2A site between the cGAS-STING pathway inhibitor and the genome-editing enzyme. In some embodiments, the mRNA comprises a IRES between the cGAS-STING pathway inhibitor and the genome-editing enzyme. In some embodiments, the nucleic acid is circular RNA. In some embodiments, the circular RNA comprises an IRES.

[0019]

[0013] In some embodiments, the dsDNA comprises an AttD site.

[0020]

[0014] In some embodiments, the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are administered in lipid nanoparticles (LNPs). In some embodiments, the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are encapsulated in separate LNPs. In some embodiments, the cGAS-STING pathway inhibitor and genome-editing enzyme are coencapsulated in LNPs. In some embodiments, the genome-editing enzyme and dsDNA are coencapsulated in LNPs. In some embodiments, the cGAS-STING pathway inhibitor and dsDNA are co-encapsulated in LNPs. In some embodiments, the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are co-encapsulated in LNPs. In some embodiments, the LNPs contain RNA and DNA at a mass ratio of 1 :1, 2:1, 3:1, 10:1, 1:2, 1:3, or 1:10.

[0021]

[0015] In some aspects, the composition for genome-editing comprises means for reducing cGAS biding to DNA, ligating ubiquitin to cGAS for degradation, or degrading 2’3’ cGAMP; a genomeediting enzyme; dsDNA; and at least one acceptable carrier, wherein the composition is encapsulated in LNPs.

[0022]

[0016] In some aspects, the present disclosure provides a method of dsDNA delivery. The method comprises the administering a cGAS-STING pathway inhibitor and dsDNA to a cell. The compositions for dsDNA delivery disclosed herein are suitable for the method of dsDNA delivery. In some embodiments, the cGAS-STING pathway inhibitor and dsDNA are co-administered. In some embodiments, the cGAS-STING pathway inhibitor and dsDNA are administered sequentially. In some embodiments, the cGAS-STING pathway inhibitor is administered prior to the dsDNA. In some embodiments, the cGAS-STING pathway inhibitor is administered prior to and co-administered with the dsDNA. In some embodiments, the cGAS-STING pathway inhibitor is administered after the dsDNA.

[0023]

[0017] In some aspects, the present disclosure provides a method for genome-editing. The method comprises the administering a cGAS-STING pathway inhibitor and a genome-editing system comprising a genome-editing enzyme and dsDNA to a cell or subject. The compositions for genome-editing disclosed herein are suitable for the method of genome-editing. In some embodiments, the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are administered in LNPs. Tn some embodiments, the LNPs encapsulating the cGAS-STING pathway inhibitor arc administered to the cell separately from the LNPs encapsulating the dsDNA.

[0024]

[0018] In some embodiments, the LNPs encapsulating the cGAS-STING pathway inhibitor are administered to the cell separately from the LNPs encapsulating the dsDNA.

[0025]

[0019] In some embodiments, the cGAS-STING pathway inhibitor reduces IRF-3 expression, as compared to a control. In some embodiments, the cGAS-STING pathway inhibitor reduces cytokine response, as compared to a control. In some embodiments, the cytokine response comprises IP- 10 and / or IFN-y.

[0026]

[0020] In some embodiments, the cell to which the cGAS-STING pathway inhibitor is delivered is in a subject.

[0027] BRIEF DESCRIPTION OF THE FIGURES

[0028]

[0021] FIGS. 1A-1B show western blots of KSHV ORF52 and VACV B2R expression. THP1- dual cells were treated with KSHV ORF52 mRNA or VACV B2R mRNA alone or with DNA (mRNA was delivered by Messenger Max; DNA was delivered by LyoVec). 16 hours post treatment, the cellular expression of the reference protein actin (42 kDa), (Fig 1A) and KSHV ORF52 and VACV B2R proteins (Fig IB) was evaluated in western blots (lane 1: KSHV ORF52 (16 kDa); lane 2: VACV B2R (26 kDa); lane 3: no treatment; lane 4: KSHV ORF52 + DNA; lane 5: VACV B2R + DNA; lane 6: DNA only).

[0029]

[0022] FIGS. 2A-2D show reduction in IRF-3 activation (or luciferase induction) in THPl-dual cells treated with DNA + KSHV ORF52 mRNA or VACV B2R mRNA in LNPs compared to DNA-LNP alone. The reduction in IRF-3 induction is shown as fold over background (Fig 2A) and as raw value (Fig 2B). Cell viability is measured using a Cell Titer Gio assay, results were shown as percentage viability in respect to nontreated cells (Fig 2C) and as a raw value (Fig 2D).

[0030]

[0023] FIGS. 3A-3D show reduction in IRF-3 activation (or luciferase induction) following treatment with DNA + KSHV ORF52 mRNA or VACV B2R mRNA, either separately encapsulated or co-capsulated in LNPs at a mass ratio of 1:3 (mRNA:DNA) over a dose range. IRF-3 reduction is shown as fold over background (Fig 3A) and as a raw value (Fig 3B). Cell viability is shown in respect to nontreated cells using a Cell Titer Gio assay (Fig 3C) and as a raw value (Fig 3D).

[0031]

[0024] FIGS. 4A-4F show reduction in DNA-induced immunogenicity with VACV B2R mRNA treatment in human peripheral blood mononuclear cells (PBMCs). Relative mRNA levels of IP- 10 (Fig 4A) and IL-6 (Fig 4B) are shown for separate LNP treatments of DNA and VACV B2R at multiple ratios and for co-cncapsulation at a mass ratio of 1:3 (mRNA:DNA) by qPCR. Relative mRNA levels of IP- 10 (Fig 4C) and IFN-y (Fig 4D) are shown for separate LNP treatments of DNA and VACV B2R at multiple ratios and for co-encapsulation at a ratio of 1:3 (VACV B2R mRNA:DNA) by ddPCR. As a control, co-treatment of DNA with mCherry mRNA did not result in a similar reduction relative mRNA levels of IP- 10 (Fig 4E) and IFN-y (Fig 4F) by ddPCR.

[0032]

[0025] FIG 5 shows reduction in DNA immunogenicity with VACV B2R mRNA treatment in cynomolgus monkey PBMCs.

[0033]

[0026] FIG 6 shows a schematic of exemplary compositions comprising a cGAS-STING pathway inhibitor, dsDNA, and an LSR, including wherein the cGAS-STING pathway inhibitor and LSR are encoded as separate mRNAs (Fig 6A), wherein the cGAS-STING pathway inhibitor and LSR are encoded on a single mRNA with a P2A site (Fig 6B), and wherein the cGAS-STING pathway inhibitor and LSR are encoded on a single mRNA with an NLS (Fig 6C).

[0034] DETAILED DESCRIPTION

[0035]

[0027] Disclosed herein are cGAS-STING pathway inhibitors that may be used to reduce or prevent immune activation that may occur with administration of dsDNA to a cell. In some embodiments, the cGAS-STING pathway inhibitors are used in connection with genome editing systems that comprise a dsDNA component. The cGAS-STING pathway inhibitors disclosed herein may target various aspects of the cGAS-STING pathway. For example, the cGAS-STING pathway inhibitors may be capable of reducing cGAS binding to dsDNA, ligating ubiquitin to cGAS for degradation, or degrading 2’3’ cGAMP. The instant specification provides compositions that may be used in connection with dsDNA delivery. The compositions for dsDNA delivery may comprise a cGAS-STING pathway inhibitor, dsDNA, and an acceptable carrier. Also disclosed herein are methods and compositions for genome-editing in a cell or a subject. The methods and compositions comprise the use of a cGAS-STING pathway inhibitor and a genome-editing system comprising a genome-editing enzyme and dsDNA.

[0036] L DEFINITIONS

[0037]

[0028] Unless stated otherwise, the following terms and phrases have the meanings described below. The definitions are not meant to be limiting in nature and serve to provide a clearer understanding of certain aspects of the present disclosure.

[0029] Genome editing system: As used herein, the term “genome editing system” refers to one or more components that can modify the genomic DNA of a cell. Genome editing systems may comprise an enzyme that can modify one or both strands of a target DNA sequence. Examples of such enzymes include zinc fingers, recombinases (e.g., large serine recombinases), CRISPR- related enzymes (e.g., Cas9), and transposases.

[0038]

[0030] AttA site: As used herein, the term “AttA site” refers to the attachment site within an acceptor nucleic acid to which a large serine recombinase (LSR) binds. An AttA site may exist in the genome of a non-phage and non-bacterial organism, e.g., a human.

[0039]

[0031] AttD site: As used herein, the term “AttD site” refers to an attachment site in a nucleic acid to which a large serine recombinase binds. AttD sites may be engineered into a nucleic acid to facilitate recombination of the nucleic acid at a cognate AttA site.

[0040]

[0032] Identity: As used herein, the term “identity” in the context of sequence comparisons refers to the number of exact matches between two different sequences in a sequence alignment. Sequence alignment techniques and software include Basic Local Alignment Search Tool (BLAST, which includes e.g., BLASTP for protein sequences and BLASTN for nucleic acid sequences), ClustalOmega, MUSCLE, and MAFFT. Preferably, the sequence alignment is performed using BLAST.

[0041]

[0033] Inhibiting: As used herein, the term “inhibiting” or “inhibitor”, particularly in relation to cGAS-STING pathway inhibitors, includes reducing, preventing, or blocking the expression or activity of a cGAS-STING pathway component. Components of the cGAS-STING pathway include but are not limited to cGAS, STING, TBK1, IKKE, IRF3, and NF-KB.

[0042] II. cGAS-STING PATHWAY INHIBITORS

[0043]

[0034] The present disclosure provides cGAS-STING pathway inhibitors for use with delivery of dsDNA, including dsDNA that is a component of a genome editing system. The cGAS-STING pathway inhibitors of the present disclosure may comprise proteins, peptides, or small molecules, as well as nucleic acids encoding the proteins and peptides.

[0044]

[0035] The cGAS-STING pathway stalls with the activation of cGAS through binding to dsDNA. Reducing the binding of dsDNA by cGAS results in a reduction in activation of the signaling pathway. Certain dsDNA viruses express proteins that reduce the activation of the cGAS-STING pathway by preventing binding of dsDNA by cGAS. Such proteins, fragments, or variants thereof, or small molecule analogs, may be used in the present invention to reduce or prevent immune activation when applied in combination with a dsDNA or a genome editing system comprising a dsDNA component. In some embodiments, the cGAS-STING pathway inhibitor reduces cGAS binding to dsDNA. Exemplary cGAS-STING pathway inhibitors that reduce cGAS binding to dsDNA are provided in Table 1. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence with at least 90% (at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the amino acid sequence corresponding to one of the protein accession numbers for a cGAS-STING pathway inhibitor selected from Table 1.

[0045] Table 1: Exemplary cGAS-STING pathway inhibitors that reduce cGAS binding to dsDNA

[0046]

[0036] An example of such a protein is ORF52 (also referred to as KicGAS) from the Kaposi sarcoma-associated herpesvirus (KSHV), and other proteins that prevent interactions between dsDNA and cGAS are known in the art. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 99% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the cGAS-STING pathway inhibitor comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the cGAS-STING pathway inhibitor comprises an nucleic acid encoding a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the cGAS- STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 1 . In some embodiments, the cGAS- STING pathway inhibitor comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1. In some embodiments, the cGAS-STING pathway inhibitor comprises a nuclear localization signal (NLS). In some embodiments, the cGAS-STING pathway inhibitor comprises more than one NLS. In some embodiments, the cGAS-STING pathway inhibitor comprises two NLSs. In some embodiments, the cGAS-STING pathway inhibitor comprises three NLSs. In some embodiments, the cGAS-STING pathway inhibitor comprises four NLSs. In some embodiments, the cGAS-STING pathway inhibitor comprises five NLSs.

[0047]

[0037] In some embodiments, the NLS is from SV40. In some embodiments, the NLS is from myc. In some embodiments, the NLS is from p53. Exemplary sequences for NLSs are known in the art. See e.g., Lu et al., Cell Communication and Signaling 19, 60 (2021).

[0048]

[0038] In some embodiments, the variant of a cGAS-STING pathway inhibitor comprises the amino acid of one of SEQ ID NOs: 29-42. In some embodiments, the variants comprise the amino acid sequence of SEQ ID NO: 1, wherein the amino acid residue at position C101 is substituted. In some embodiments, the variants comprise the amino acid sequence of SEQ ID NO: 1, wherein the amino acid residue at position M14 is substituted.

[0049]

[0039] In some embodiments, the cGAS-STING pathway inhibitor comprises a chimeric fusion of a domain from two or more of the disclosed cGAS-STING pathway inhibitors in the instant application. In some embodiments, the cGAS-STING pathway inhibitor comprises a chimeric fusion of a domain from KSHV ORF52 and a domain from HSV-1 VP22.

[0050]

[0040] In some embodiments, the cGAS-STING pathway inhibitor comprises a chimeric fusion of a domain from KSHV OR52. In some embodiments, the domain from KSHV ORF52 is selected from any one of: the alpha 1 (al) helix, the al- a2 linker, the alpha 2 (a2) helix, and the alpha 3 (a3) helix. In some embodiments, the cGAS-STING pathway inhibitor comprises the alpha 1 (al) helix of KSHV ORF52. In some embodiments, the cGAS-STING pathway inhibitor comprises the alpha 2 (a2) helix of KSHV ORF52. In some embodiments, the cGAS-STING pathway inhibitor comprises the alpha 3 (a3) helix of KSHV ORF52. In some embodiments, the CGAS-STING pathway inhibitor comprises the alpha 1 (al) helix of KSHV ORF52 wherein amino acids L24, E27, N28, and L31 are conserved. In some embodiments, the CGAS-STING pathway inhibitor comprises the alpha 1 (al) helix of KSHV ORF52 wherein amino acids L17, 121, R32, and L35 are conserved. In some embodiments, the CGAS-STING pathway inhibitor comprises the alpha 1 (al) helix of KSHV ORF52 wherein amino acid L17 is conserved. Tn some embodiments, the CGAS-STING pathway inhibitor comprises the alpha 1 (al) helix and al- a2 linker of KSHV ORF52. In some embodiments, the CGAS-STING pathway inhibitor comprises the alpha 1 (al) helix and al - a2 linker of KSHV ORF52 wherein amino acids P41 and P45 are conserved. In some embodiments, the CGAS-STING pathway inhibitor comprises the alpha 1 (al) helix and al- a2 linker of MHV-68 ORF52 wherein amino acids 41 and 45 are each proline.

[0051]

[0041] In some embodiments, the cGAS-STING pathway inhibitor comprises a chimeric fusion of a domain from HSV-1 VP22. In some embodiments, the domain from HSV-1 VP22 is selected from the alpha 1 (al) helix, the alpha 2 (a2) helix, the alpha 3 (a3) helix, and the alpha 4 (a4) helix. In some embodiments, the cGAS-STING pathway inhibitor comprises the alpha 1 (al) helix of HSV-1 VP22. In some embodiments, the cGAS-STING pathway inhibitor comprises the alpha 2 (a2) helix of HSV-1-VP22. In some embodiments, the cGAS-STING pathway inhibitor comprises the alpha 3 (a3) helix of HSV-1 VP22. In some embodiments, the cGAS-STING pathway inhibitor comprises the alpha 4 (a4) helix of HSV-1 VP22. In some embodiments, the cGAS-STING pathway inhibitor comprises the alpha 2, 3, and 4 helices of HSV-1-VP22. In some embodiments, the domain from HSV-1 VP22 comprises amino acids 190-258. In some embodiments, the domain from HSV-1 VP22 comprises amino acids 174-281. In some embodiments, the domain from HSV-1 VP22 comprises amino acids 174-301.

[0052]

[0042] In some embodiments, the cGAS-STING pathway inhibitor comprises a chimeric fusion of the alpha 1 helix and al- a2 linker from KSHV OR52 and alpha 2, 3, and 4 helices of HSV-1 VP22.

[0053]

[0043] Another mechanism that dsDNA viruses may use to evade or reduce activation of the cGAS-STING pathway is to degrade cGAS. An example of such a mechanism is ubiquitination, where a protein will induce ligation or ligate ubiquitin to cGAS to direct it for degradation. Certain embodiments of the present invention include proteins, including fragments or variants thereof, or compounds that induce ligation of ligate ubiquitin to cGAS. In some embodiments, the cGAS- STING pathway inhibitor ligates ubiquitin to cGAS for degradation. Exemplary cGAS-STING pathway inhibitors that ligate ubiquitin to cGAS for degradation are provided in Table 2. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence with at least 90% (at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the amino acid sequence of one of the protein accession numbers for a cGAS-STTNG pathway inhibitor selected from Table 2.

[0054] Table 2: Exemplary cGAS-STING pathway inhibitors that ligate ubiquitin to cGAS for degradation

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0044] One non-limiting example of such a protein is the Vaccinia E5R. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 90% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 95% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 99% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the cGAS-STING pathway inhibitor comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO: 3. In some embodiments, the cGAS-STING pathway inhibitor further comprises an intrinsically disordered region (IDR).

[0062]

[0045] A further embodiment of cGAS-STING pathway inhibitors includes proteins, including fragments or variants thereof, or compounds that can degrade 2’3’ cGAMP, thus inhibiting activation of STING and the downstream factors. One such example of proteins is the poxin family of enzymes from the Poxvirade family of viruses that cleave the 3’-5’ bond in 2’3’ cGAMP converting the 2’3’ cGAMP into linear Gp[2’-5’]Ap[3’] (Eaglesham et al. Nature (2019) 566(7743):529-263). In some embodiments, the cGAS-STING pathway inhibitor degrades 2’3’ cGAMP. Exemplary cGAS-STING pathway inhibitors that degrade 2’3’ cGAMP are provided in Table 3. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence with at least 70% (at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the amino acid sequence of one of the protein accession numbers for a cGAS-STING pathway inhibitor selected from Table 3.

[0063] Table 3: Exemplary cGAS-STING pathway inhibitors that degrade 2’3’ cGAMP

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0046] In some embodiments, the cGAS-STING pathway inhibitor is a poxin (referred to as a poxvirus immune nuclease) (Eaglcsham ct al). In some embodiments, the cGAS-STING pathway inhibitor degrades 2’3’ cGAMP by hydrolysis of the cyclic ring.

[0075]

[0047] In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 70% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 75% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 80% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 85% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 90% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 95% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 99% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding the amino acid sequence of SEQ TD NO: 2. In some embodiments, the cGAS-STING pathway inhibitor further comprises an intrinsically disordered region (IDR).

[0076]

[0048] In some embodiments, the cGAS-STING pathway inhibitor is encoded by SEQ ID NO: 43 (B2R). In some embodiments, the cGAS-STING pathway inhibitor is encoded by SEQ ID NO: 44 (human codon-optimized B2R). In some embodiments, the cGAS-STING pathway inhibitor is encoded by SEQ ID NO: 45 (mouse codon-optimized B2R).

[0077]

[0049] In some embodiments, the cGAS-STING pathway inhibitor is encoded by SEQ ID NO: 47 (EVM153). In some embodiments, the cGAS-STING pathway inhibitor is encoded by SEQ ID NO: 48 (human codon-optimized EVM153). In some embodiments, the cGAS-STING pathway inhibitor is encoded by SEQ ID NO: 49 (mouse codon-optimized EVM153).

[0078]

[0050] In some embodiments, the cGAS-STING pathway inhibitor comprises a variant of a naturally-occurring protein comprising the amino acid sequence of one of SEQ ID NOs: 10-28. In some embodiments, the variants comprise the amino acid sequence of SEQ ID NO: 2, wherein the amino acid residue at position P155, W162, C71, or S90 is substituted.

[0079]

[0051] The cGAS-STING pathway inhibitor of the present disclosure can also inhibit the pathway downstream of cGAS and 2’3’ cGAMP. For example, TBK1 phosphorylates STING, which then interacts with IRF-3 and NF-KB to drive gene expression changes. In some embodiments, the cGAS-STING pathway inhibitor induces ligation of ubiquitin to TBK1 for degradation. In some embodiments, the cGAS-STING pathway inhibitor is NLRP14. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 90% identity to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the cGAS- STING pathway inhibitor comprises an amino acid sequence having 95% identity to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 99% identity to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the cGAS-STING pathway inhibitor comprises an mRNA encoding an amino acid sequence having 90% identity to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the cGAS-STING pathway inhibitor comprises an mRNA encoding an amino acid sequence having 95% identity to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the cGAS-STING pathway inhibitor comprises an mRNA encoding an amino acid sequence having 99% identity to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the cGAS-STING pathway inhibitor comprises an mRNA encoding the amino acid sequence of SEQ ID NO: 46.

[0080]

[0052] The efficacy of the cGAS-STING pathway inhibitors can be assessed using conventional methods. In some embodiments, the inhibition of cGAS-STING signaling activation is measured by IRF-3 expression. The IRF-3 expression level can be measured by luciferase expression in cell lines or conventional methods for measuring gene expression. In some embodiments, the cGAS-STING pathway inhibitor reduces IRF-3 expression, as compared to a control. In some embodiments, the cGAS-STING pathway inhibitor reduces cytokine response, as compared to a control. In some embodiments, the cytokine response comprises IL-6, IP- 10, IFN-a, IFN-0, MCP-1, RANTES, and / or IFN-y. The cytokine response can be measured using conventional methods, including e.g., ELISA, qPCR, ddPCR, or flow cytometry.

[0081] III. COMPOSITIONS FOR PRODUCTION OR ADMINISTRATION OF cGAS-STING PATHWAY INHIBITORS

[0082]

[0053] In one aspect, the cGAS-STING pathway inhibitors can be used in a composition for dsDNA delivery. In some embodiments, the composition for dsDNA delivery comprises a cGAS- STING pathway inhibitor, dsDNA, and at least one acceptable carrier. In additional embodiments, the composition for dsDNA delivery comprises a cGAS-STING pathway inhibitor and a genome editing system that comprises a dsDNA component. In some embodiments, the composition for dsDNA delivery comprises two or more cGAS-STING pathway inhibitors.

[0083]

[0054] The present disclosure provides cGAS-STING pathway inhibitors suitable for use in a composition for dsDNA delivery. In some embodiments, the cGAS-STING pathway inhibitor comprises an mRNA encoding an amino acid sequence with at least 90% (at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the amino acid sequence of one of the protein accession numbers for a cGAS-STING pathway inhibitor selected from Tables 1-3. In some embodiments, the cGAS-STING pathway inhibitor reduces cGAS binding to dsDNA. In some embodiments, the cGAS-STING pathway inhibitor ligates ubiquitin to cGAS for degradation. In some embodiments, the cGAS-STING pathway inhibitor degrades 2’3’ cGAMP. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 90% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 95% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS- STTNG pathway inhibitor comprises an amino acid sequence having 99% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90% with one of the amino acid sequences of SEQ ID NOs:

[0084] 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 95% with one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 99% with one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding one of the amino acid sequences of SEQ ID NOs: 1,

[0085] 2, 3, or 46. In some embodiments, the nucleic acid further comprises an NLS sequence. In some embodiments, the cGAS-STING pathway inhibitor comprises a poxin. In some embodiments, the cGAS-STING pathway inhibitor further comprises an IDR. In some embodiments, the cGAS- STING pathway inhibitor is encoded in nucleic acid. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nuclear acid is circular RNA. In some embodiments, the dsDNA comprises an AttD site.

[0086]

[0055] The compositions of the instant application can be delivered to a cell using lipid-based formulations. In some embodiments, the lipid-based formulation comprises a lipid nanoparticle (LNP). In some embodiments, the cGAS-STING pathway inhibitor, or a nucleic acid encoding a cGAS-STING pathway inhibitor, is encapsulated in LNPs. In some embodiments, the LNPs comprise an ionizable lipid, a helper lipid, cholesterol, and a PEG lipid. The preparation of LNPs is well-known in the art (see, e.g., PCT / EP2020 / 087254; PCT / US2014 / 029116; PCT / IB2012 / 003019; and PCT / US2019 / 025246). The concentration of nucleic acid within the LNPs can be determined using conventional techniques. In some embodiments, the concentration of nucleic acid within the LNPs is determined using RNA quantification methods (e.g., RiboGreen and PicoGreen). In some embodiments, the LNPs contain RNA and DNA at a mass ratio of 1:1, 2:1, 3:1, 10:1, 1:2, 1:3, or 1:10.

[0087]

[0056] In some embodiments, the cGAS-STING pathway inhibitor and dsDNA are encapsulated in LNPs. In some embodiments, the cGAS-STING pathway inhibitor and dsDNA are encapsulated in separate LNPs. In some embodiments, the LNPs encapsulating the cGAS-STING pathway inhibitors are administered separately from the LNPs encapsulating the dsDNA. In some embodiments, the cGAS-STING pathway inhibitor and dsDNA arc co-cncapsulatcd in LNPs.

[0088]

[0057] In some aspects, the instant application provides compositions for genome-editing comprising a cGAS-STING pathway inhibitor. In some embodiments, the composition for genome-editing comprises a cGAS-STING pathway inhibitor, a genome-editing system comprising a genome-editing enzyme and dsDNA, and at least one acceptable carrier.

[0089]

[0058] In some embodiments, the cGAS-STING pathway inhibitor has at least 90% (at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the amino acid sequence of one of the protein accession numbers of a cGAS-STING pathway inhibitor selected from Tables 1-3. In some embodiments, the cGAS-STING pathway inhibitor reduces cGAS binding to dsDNA. In some embodiments, the cGAS-STING pathway inhibitor ligates ubiquitin to cGAS for degradation. In some embodiments, the cGAS-STING pathway inhibitor degrades 2’3’ cGAMP. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 90% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 95% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 99% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS- STING pathway inhibitor comprises one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 95% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 99% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nuclear localization signaling (NLS). In some embodiments, the cGAS- STING pathway inhibitor comprises a poxin. In some embodiments, the cGAS-STING pathway inhibitor further comprises an IDR. In some embodiments, the cGAS-STING pathway inhibitor is encoded in nucleic acid. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is circular RNA.

[0090]

[0059] Many genome-editing systems are known in the ail. Genome-editing enzymes may recognize specific DNA sequences by binding affinity of the enzyme itself, by an associated component such as a DNA template that can hybridize to a target DNA sequence, or by a guide molecule such as an RNA. While genome-editing systems may have different mechanisms of creating a genome modification, multiple systems may incorporate dsDNA as a template sequence to introduce new genetic material into the genome. For example, large serine recombinases (LSRs) are a class of enzymes within the DNA integrase family that precisely catalyze the recombination of DNA in a controllable way. Generally found in phage and bacteria, these enzymes recognize pairs of distinct attachment sites and facilitate the integration of genetic elements into bacterial genomes via recombination at these cognate attachment sites. LSRs are capable of inserting dsDNA comprising an AttD site into a cognate AttA site in another nucleic acid molecule such as a genome or plasmid. Unlike other classes of integrases that permit bidirectional integration, LSRs typically drive unidirectional integration of nucleic acids between the cognate attachment sites. In some embodiments, the genome-editing enzyme comprises a large serine recombinase (LSR).

[0091]

[0060] Suitable LSRs for use in the disclosed genome editing systems may include LSRs known in the art, including e.g., PaOl, Bxbl, PhiC31, Pf80, Cp36, Dn29, BcelNTa, SscINTd, SacINTd, INT10, Dre, Vika, Bxbl, pC31, RDF, BT1, Rl, R2, R3, R4, R5, TP901-1, Al 18, FC1, <1>C1, MR11, TGI, 0370.1, Wp, BL3, SPBc, K38, or variants thereof. See e.g., Durrant et al., Nature Biotechnology 41, 488-499 (2023); Yarnall et al., Nature Biotechnology 41, 500-512 (2023); Xu et al., BMC Biotechnol, 13, 87 (2013).

[0092]

[0061] In some embodiments, the genome-editing enzyme comprises an LSR-fusion protein comprising an LSR and a fusion domain. In some embodiments, the fusion domain comprises a polypeptide, e.g., with which the LSR is not naturally linked. In some embodiments, the fusion domain comprises a protein domain, e.g., with which the LSR is not naturally linked. In some embodiments, the fusion domain comprises a protein, e.g., with which the LSR is not naturally linked. In some embodiments, the fusion domain comprises a cell-penetrating peptide. In some embodiments, the fusion domain comprises an arginine-rich peptide. In some embodiments, the fusion domain comprises an arginine-rich dipeptide repeat protein. In some embodiments, the fusion domain comprises a combination of a DNA-binding domain, a cell-penetrating peptide, an arginine-rich peptide, and an arginine-rich dipeptide repeat protein. Tn some embodiments, the LSR of the genome editing system is fused to a DNA-binding domain. In some embodiments, the DNA-binding domain comprises a catalytically inactive Cas polypeptide (dCas). In some embodiments, the dCas comprises dCas9 or dCasl2. In some embodiments, the DNA-binding domain comprises a Cas9 nickase. In some embodiments, the DNA-binding domain comprises a catalytically inactive zinc finger polypeptide (ZNF). In some embodiments, the DNA-binding domain comprises a catalytically inactive transcription activator- like effector nuclease (TALEN). In some embodiments, the LSR-fusion protein comprises a linker between the LSR and fusion domain. In some embodiments, the LSR-fusion protein does not comprise a linker between the LSR and fusion domain. In some embodiments, the LSR-fusion protein comprises a polymerase. In some embodiments, the LSR-fusion protein comprises a reverse transcriptase. In some embodiments, the LSR-fusion protein comprises one of the cGAS-STING pathway inhibitors disclosed herein. In some embodiments, the LSR-fusion protein comprises a domain from one of the cGAS-STING pathway inhibitors disclosed herein. In some embodiments, the LSR-fusion protein further comprises an NLS.

[0093]

[0062] Other genome-editing systems are also widely used. One non-limiting example is the CRISPR genome-editing system, which comprises a Cas polypeptide and a nucleic acid such as a guide RNA. The Cas polypeptide cuts or nicks a specific DNA sequence based on hybridization of the guide RNA to a corresponding target DNA. This allows the sequence at the cut site to be edited through homologous recombination, non-homologous end joining, or other DNA repair mechanisms. In some embodiments, the CRISPR genome-editing system comprises a polymerase. In some embodiments, the CRISPR genome-editing system comprises a reverse polymerase. Certain applications of CRISPR systems include administration of a dsDNA as a template for a desired new genomic sequence. In some embodiments, the genome-editing enzyme comprises a Cas polypeptide, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a tyrosine recombinase, or a transposon-encoded enzyme. In some embodiments, the transposon-encoded enzyme comprises TnpB, TnsB, a PiggyBac transposase, or a Sleeping Beauty transposase. Any of the aforementioned genome-editing systems may be used with an associated dsDNA component and a cGAS-STING pathway inhibitor as disclosed herein.

[0094]

[0063] In some embodiments, the cGAS-STING pathway inhibitor and genome-editing enzyme are encoded on a single nucleic acid molecule. In some embodiments, the nucleic acid is mRNA or circular RNA. In some embodiments, the mRNA comprises a P2A site between the cGAS- STING pathway inhibitor and genome-editing enzyme. In some embodiments, the mRNA comprises a T2A, E2A, or F2A site between the cGAS-STING pathway inhibitor and genomeediting enzyme. In some embodiments, the mRNA or circular RNA comprises an internal ribosome entry site (IRES) between the cGAS-STING pathway inhibitor and genome-editing enzyme. In some embodiments, the circular RNA comprises an IRES.

[0095]

[0064] In some embodiments, the dsDNA comprises an AttD site. In some embodiments, the dsDNA comprises more than one AttD site (i.e., an “array” of AttDs). In some embodiments, the dsDNA comprises two AttDs. In some embodiments, the dsDNA comprises three AttDs. In some embodiments, the dsDNA comprises four AttDs. In some embodiments, the dsDNA comprises five AttDs. In some embodiments, the dsDNA comprises six AttDs. In some embodiments, the dsDNA comprises seven AttDs. In some embodiments, the dsDNA comprises eight AttDs. In some embodiments, the dsDNA comprises nine AttDs. In some embodiments, the dsDNA comprises ten AttDs.

[0096]

[0065] In some embodiments, the dsDNA comprises an AttD site, and the genome-editing enzyme is an LSR.

[0097]

[0066] In some embodiments, the dsDNA comprises a sequence that encodes one or more gene products including but not limited to RNAs (e.g., such as tRNA, rRNA, microRNA, siRNA, and mRNA), proteins, or polypeptides. In some embodiments, the dsDNA comprises a non-coding sequence. In some embodiments, the dsDNA comprises a sequence that encodes a transcription or translational control element (e.g., promoter elements, activator sequences, repressor sequences). In some embodiments, the dsDNA comprises a sequence that encodes a therapeutic protein. In some embodiments, the dsDNA comprises a sequence that encodes a therapeutic RNA. In some embodiments, the dsDNA comprises one or more of a sequence that encodes for a transcriptional or translational control element, a sequence that encodes a therapeutic protein, and a sequence that encodes a therapeutic RNA. In some embodiments, the dsDNA comprises a sequence for a gene, a gene variant, or a portion thereof.

[0098]

[0067] In some embodiments, the dsDNA comprises a sequence that encodes a secreted protein. In some embodiments, the dsDNA comprises a sequence that encodes a membrane-bound protein. In some embodiments, the dsDNA comprises a sequence that encodes a clotting factor. In some embodiments, the dsDNA comprises a sequence that encodes a cytokine. In some embodiments, dsDNA comprises a sequence that encodes a hormone. In some embodiments, the dsDNA comprises a sequence that encodes an enzyme. In some embodiments, the dsDNA comprises a sequence that encodes an antibody, or a portion thereof. In some embodiments, the dsDNA comprises a sequence that encodes a chimeric antigen receptor (CAR). In some embodiments, the dsDNA comprises a sequence that encodes a T cell receptor (TCR). In some embodiments, the dsDNA comprises a sequence that encodes a B cell receptor (BCR). In some embodiments, the dsDNA comprises a sequence that encodes an immune cell activation or inhibitory receptor. In some embodiments, the dsDNA comprises a sequence that encodes a growth factor ligand. In some embodiments, the dsDNA comprises a sequence that encodes a transcription factor. In some embodiments, the dsDNA comprises a sequence that encodes a checkpoint inhibitor or agonist.

[0099]

[0068] In some embodiments, the dsDNA comprises a sequence that encodes a guide RNA. In some embodiments, the dsDNA comprises a sequence that encodes a guide RNA for DNA-based Cas systems (e.g., Cas9). In some embodiments, the dsDNA comprises a sequence that encodes a guide RNA for RNA-based Cas systems (e.g., Casl3). See e.g., Ding et al., Nature Communications 15: 1572 (2024). In some embodiments, the dsDNA comprises a sequence that encodes a micro RNA. In some embodiments, the dsDNA comprises a sequence that encodes a tRNA. In some embodiments, the dsDNA comprises a sequence that encodes a long non-coding RNA (Inc RNA). In some embodiments, the dsDNA comprises a sequence that encodes a circular RNA.

[0100]

[0069] In some embodiments, the dsDNA is non-integrating DNA. In some embodiments, the dsDNA is extrachromosomal DNA. In some embodiments, the dsDNA is a vector for episomal gene expression. In some embodiments, the dsDNA comprises cis elements that lead to episomal replication. In some embodiments, the dsRNA comprises a scaffold / matrix attachment region (S / MAR) motif for maintenance and replication (see e.g., Bozza et al., Sei Adv. 7(16): eabfl333 (2021).

[0101]

[0070] In some embodiments, the dsDNA may be partially double-stranded. In some embodiments, the dsDNA may comprise partially single- stranded DNA. In some embodiments, the dsDNA may comprise one or more nucleotide modifications.

[0102]

[0071] The composition for genome-editing can be delivered to a cell or subject using LNPs. In some embodiments, the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are encapsulated in LNPs. In some embodiments, the cGAS-STING pathway inhibitor, genome- editing enzyme, and dsDNA are encapsulated in separate LNPs. In some embodiments, the cGAS- STING pathway inhibitor and gcnomc-cditing enzyme arc co-cncapsulatcd in LNPs. In some embodiments, the cGAS-STING pathway inhibitor and dsDNA are co-encapsulated in LNPs. In some embodiments, the genome-editing enzyme and dsDNA are co-encapsulated in LNPs. In some embodiments, the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are co-encapsulated in LNPs. In some embodiments, the LNPs encapsulating the cGAS-STING pathway inhibitors are administered separately from the LNPs encapsulating the dsDNA.

[0103]

[0072] In some embodiments, the composition for genome-editing comprises a cGAS-STING pathway inhibitor comprising a nucleic acid sequence encoding an amino acid sequence having at least 90% identity to the amino acid of SEQ ID NO:1, a genome-editing enzyme comprising an LSR, dsDNA comprising an AttD site, and an acceptable carrier, wherein the cGAS-STING pathway inhibitor and LSR are encoded on a single mRNA molecule comprising a P2A site between the cGAS-STING pathway inhibitor and LSR. In some embodiments, the composition for genome-editing comprises a cGAS-STING pathway inhibitor comprising a nucleic acid sequence encoding an amino acid sequence having at least 90% identity to the amino acid of SEQ ID NO:2, a genome-editing enzyme comprising an LSR, dsDNA comprising an AttD site, and an acceptable carrier, wherein the cGAS-STING pathway inhibitor and LSR are encoded on a single mRNA molecule comprising a P2A site between the cGAS-STING pathway inhibitor and LSR. The composition for genome-editing can comprise any cGAS-STING pathway inhibitor and genome-editing combination from their respective columns in Table 5, wherein the genomeediting system comprises a dsDNA.

[0104] Table 5: cGAS-STING pathway inhibitor and genome-editing system combinations for compositions for genome-editing

[0105]

[0073] The instant application provides cGAS-STING pathway inhibitors as a means for reducing cGAS binding to dsDNA, ligating ubiquitin to cGAS for degradation, or degrading 2’3’ cGAMP. The support for such means for inhibiting the cGAS-STING pathway can be found throughout the instant specification as shown in Table 6. These means can be used in a composition for genomeediting. In some embodiments, a composition for genome-editing comprises a means for inhibiting cGAS binding to DNA, ligating ubiquitin to cGAS for degradation, or degrading 2’3’ cGAMP; a genome-editing enzyme; dsDNA; and at least one acceptable carrier.

[0106] Table 6: Exemplification of various Means for inhibiting cGAS-STING pathway components

[0107]

[0074] In some embodiments, the cGAS-STING pathway inhibitor is encoded in nucleic acid. In some embodiments, the cGAS-STING pathway inhibitor is encoded in mRNA or circular RNA. In some embodiments, the mRNA comprises a 5’ cap structure, or a modified 5’ cap structure. In some embodiments, the mRNA comprises a 3’ poly adenylation sequence. In some embodiments, the mRNA comprises a 5’ UTR. In some embodiments, the mRNA comprises a 3’ UTR. In some embodiments, the UTR comprises one or more viral internal ribosome entry sites (IRES) or eukaryotic IRES. In some embodiments, the mRNA or circular RNA comprises a miRNA binding site or a fragment thereof, a restriction site or a fragment thereof, an RNA editing motif or a fragment thereof, a zip code element or a fragment thereof, an RNA trafficking element or a fragment thereof, or a combination thereof. In some embodiments, the accessory element comprises a binding domain to an IRES transacting factor (ITAF). In some embodiments, the mRNA or circular RNA comprises a cis-regulatory element. In some embodiments, the cis- regulatory element acts as a safety switch for regulating expression of the integrated sequence. In some embodiments, the cis-rcgulatory clement is a miRNA binding site. In some embodiments, the cis-regulatory element is a conditional activator. In some embodiments, the cis-regulatory element is a repressor. In some embodiments the cis-regulatory element is an adenosine deaminase acting on RNA (ADAR) enzyme. In some embodiments, the mRNA comprises a polyA region, a polyC region, a poly AC region, a polyprimidine tract, or a combination or variant thereof. In some embodiments, the circular RNA comprises an IRES. In some embodiments, the cGAS-STING pathway inhibitor is a polypeptide.

[0108]

[0075] In some embodiments, the cGAS-STING pathway inhibitor disclosed herein may be prepared by in vitro synthesis. In some embodiments, the in vitro synthesis comprises introducing an expression vector encoding an epitope tagged cGAS-STING pathway inhibitor into a suitable cell line and purifying the cGAS-STING pathway inhibitor via chromatography. Non-limiting examples of epitope tags include His, Ha, FLAG, Glutathione S-Transferase (GST), and maltose binding protein.

[0109]

[0076] Suitable expression vectors include, but are not limited to, viral vectors. In certain embodiments, viral vectors may be used to express a nucleic acid or administer a nucleic acid to a cell or a patient. Numerous suitable expression vectors are known to those of skill in the art, and many are commercially available. Non-limiting examples of vectors include a plasmid, a minicircle, a nanoplasmid, an adenovirus, an AAV, a lentivirus, a HSV, a retrovirus, a doggybone DNA (dbDNA)™, and exosome, and a fusosome. Depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544). The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include epitope tags (e.g., His, Ha, FLAG, Glutathione S-Transferase (GST), maltose binding protein, etc.). In some embodiments, the cGAS-STING pathway inhibitor may be prepared as mRNA by in vitro transcription of DNA. In some embodiments, the mRNA comprising the cGAS-STING pathway inhibitor encodes the amino acid sequence of SEQ ID NO: 1-46. The mRNA comprising the cGAS-STING pathway inhibitor may be codon optimized for the desired cell and / or species in which it will be expressed. Codon optimization can be performed with known methods and may enhance protein expression. In some embodiments, the mRNA encoding a cGAS-STING pathway inhibitor with the amino acid sequence of SEQ ID NO: 1 -46 is codon optimized.

[0110]

[0077] Methods of introducing a nucleic acid into a host cell arc known in the art, and any known method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include, include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like.

[0111] IV. METHODS

[0112]

[0078] In some aspects, the present disclosure provides a method of dsDNA delivery to a cell. In some embodiments, the method of dsDNA delivery comprises administering a cGAS-STING pathway inhibitor and dsDNA to a cell.

[0113]

[0079] The instant application provides exemplary cGAS-STING pathway inhibitors for use in the method of dsDNA delivery. In some embodiments, the composition for genome-editing comprises a cGAS-STING pathway inhibitor with at least 90% (at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the amino acid sequence of the protein accession number of a cGAS-STING pathway inhibitor selected from Tables 1-3. In some embodiments, the cGAS- STING pathway inhibitor reduces cGAS binding to dsDNA. In some embodiments, the cGAS- STING pathway inhibitor ligates ubiquitin to cGAS for degradation. In some embodiments, the cGAS-STING pathway inhibitor degrades 2’3’ cGAMP. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 90% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 95% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 99% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 95% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 99% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises an NLS. In some embodiments, the cGAS-STING pathway inhibitor comprises a poxin. In some embodiments, the cGAS-STING pathway inhibitor further comprises an IDR. In some embodiments, the cGAS-STING pathway inhibitor is encoded in nucleic acid. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is circular RNA.

[0114]

[0080] In some embodiments, the dsDNA comprises an AttD site. In some embodiments, the dsDNA comprises an AttD site, and the genome-editing enzyme is an LSR.

[0115]

[0081] In some embodiments, the cGAS-STING pathway inhibitor and dsDNA are coencapsulated in LNPs. In some embodiments, the cGAS-STING pathway inhibitor and dsDNA are encapsulated in separate LNPs.

[0116]

[0082] In some embodiments, the cGAS-STING pathway inhibitor and dsDNA are coadministered. In some embodiments, the cGAS-STING pathway inhibitor and dsDNA are administered sequentially. In some embodiments, the cGAS-STING pathway inhibitor is administered prior to the dsDNA. In some embodiments, the cGAS-STING pathway inhibitor is administered prior to and co-administered with the dsDNA. In some embodiments, the cGAS- STING pathway inhibitor is administered after the dsDNA.

[0117]

[0083] In some aspects, the present disclosure provides a method of genome-editing. In some embodiments, the method of genome-editing comprises administering a cGAS-STING pathway inhibitor and a genome-editing system comprising a genome-editing enzyme and dsDNA to a cell or subject.

[0118]

[0084] The instant application provides exemplary cGAS-STING pathway inhibitors for use in the method of genome-editing. In some embodiments, the composition for genome-editing comprises a cGAS-STING pathway inhibitor with at least 90% (at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the amino acid sequence of a cGAS-STING pathway inhibitor selected from Tables 1-3. In some embodiments, the cGAS-STING pathway inhibitor reduces cGAS binding to dsDNA. In some embodiments, the cGAS-STING pathway inhibitor ligates ubiquitin to cGAS for degradation. In some embodiments, the cGAS-STING pathway inhibitor degrades 2’3’ cGAMP. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 90% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 95% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises an amino acid sequence having 99% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 95% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 99% identity to one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS-STING pathway inhibitor comprises a nucleic acid encoding one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, or 46. In some embodiments, the cGAS- STING pathway inhibitor comprises a nuclear localization signaling (NLS). In some embodiments, the cGAS-STING pathway inhibitor comprises a poxin. In some embodiments, the cGAS-STING pathway inhibitor further comprises an IDR. In some embodiments, the cGAS-STING pathway inhibitor is encoded in nucleic acid. In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA is circular RNA.

[0119]

[0085] In some embodiments, the genome-editing enzyme comprises a large serine recombinase. In some embodiments, the genome-editing enzyme comprises a Cas polypeptide, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZNF), a tyrosine recombinase, or a transposon-encoded enzyme. In some embodiments, the genome-editing enzyme comprises a polymerase. In some embodiments, the genome-editing enzyme comprises a reverse polymerase. In some embodiments, the transposon-encoded enzyme comprises TnpB, TnsB, a PiggyBac transposase, or a Sleeping Beauty transposase. In some embodiments, the dsDNA comprises an AttD site.

[0086] In some embodiments, the cGAS-STING pathway inhibitor and genome-editing enzyme arc encoded on a single nucleic acid molecule. In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA comprises a P2A site between the cGAS-STING pathway inhibitor and genome-editing enzyme. In some embodiments, the mRNA comprises an IRES site between the cGAS-STING pathway inhibitor and genome-editing enzyme. In some embodiments, the nucleic acid is circular RNA. In some embodiments, the circular RNA comprises an IRES.

[0120]

[0087] The components for the method for genome-editing can be delivered to a cell or subject using LNPs. In some embodiments, the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are encapsulated in LNPs. In some embodiments, the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are encapsulated in separate LNPs. In some embodiments, the cGAS-STING pathway inhibitor and genome-editing enzyme are coencapsulated in LNPs. In some embodiments, the cGAS-STING pathway inhibitor and dsDNA are co-encapsulated in LNPs. In some embodiments, the genome-editing enzyme and dsDNA are co-encapsulated in LNPs. In some embodiments, the cGAS-STING pathway inhibitor, genomeediting enzyme, and dsDNA are co-encapsulated in LNPs. In some embodiments, the LNPs encapsulating the cGAS-STING pathway inhibitors are administered separately from the LNPs encapsulating the dsDNA.

[0121]

[0088] In some embodiments, the method of genome-editing comprises administering a cGAS- STING pathway inhibitor and a genome-editing system comprising a genome-editing enzyme and dsDNA to a cell or subject, wherein the genome-editing system is a CRISPR genome-editing system. In some embodiments, the CRISPR genome-editing system comprises Cas9. In some embodiments, the CRISPR genome-editing system comprises a PASTE system (see e.g., Yarnall et al., Name Biotechnology 41: 500-512 (2023)). In some embodiments, the CRISPR genomeediting system comprises a prime editing system (see e.g., Anzalone et al., Nature 576: 149-157 (2019)). In some embodiments, the CRISPR genome-editing system comprises a retron (see e.g., Zhao et al., CRISPR J 5(1): 31-29 (2022)).

[0122]

[0089] In some embodiments, the method of genome-editing comprises administering a cGAS- STING pathway inhibitor comprising a nucleic acid encoding an amino acid sequence with at least 90% to the identity of the amino acid sequence of SEQ ID NO: 1 and a genome-editing system comprising an LSR and dsDNA comprising an AttD site, wherein the cGAS-STING pathway inhibitor and LSR are encoded on a single mRNA molecule comprising a P2A site between the cGAS-STING pathway inhibitor and LSR. In some embodiments, the method of genome-editing comprises administering a cGAS-STING pathway inhibitor comprising a nucleic acid encoding an amino acid sequence with at least 90% to the identity of the amino acid sequence of SEQ ID NO: 2 and a genome-editing system comprising an LSR and dsDNA comprising an AttD site, wherein the cGAS-STING pathway inhibitor and LSR are encoded on a single mRNA molecule comprising a P2A site between the cGAS-STING pathway inhibitor and LSR.

[0123]

[0090] In some embodiments, the methods provided herein are administered to a non-dividing cell. In some embodiments, the methods provided herein are administered to a dividing cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a hepatocyte.

[0124]

[0091] In some aspects, the present disclosure provides methods for treating a disease in a subject by administering a composition disclosed herein to a patient, thereby generating an engineered cell within the subject. In some aspects, the present disclosure provides methods for treating a disease in a subject by generating an engineered cell using a composition disclosed herein and administering the engineered cell to the subject, thereby treating the disease. In some embodiments, a method of treating a disease in a subject, comprises (i) contacting isolated cells with an effective amount of a composition disclosed herein to yield engineered cells and (ii) administering the engineered cells to the subject, thereby treating the disease. In some embodiments, the method comprises contacting the isolated cells by electroporation. In some embodiments, the method comprises contacting the isolated cells by microinjection. In some embodiments, the method comprises contacting the isolated cells with an LNP. In some embodiments, the method occurs in vitro. In some embodiments, the method occurs ex vivo. In some embodiments, the subject is a human. In some embodiments, the disease is caused by a genetic mutation.

[0125] V. PHARMACEUTICAL COMPOSITIONS

[0126]

[0092] The cGAS-STING pathway inhibitor of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers, diluents, or vehicles. Pharmaceutically acceptable vehicles may be vehicles approved by a regulatory agency of the Lederal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans. Such pharmaceutical vehicles can be lipids or LNPs, including suitable solvents, buffers, diluents, or stabilizers. In some embodiments, the cGAS-STING pathway inhibitors of the present disclosure can be delivered by viral vectors. As such, administration of a cGAS-STING pathway inhibitor disclosed herein can be achieved in various ways, including intravenous, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, or intraocular administration. The active agent may be systemic after administration or may be localized by the use of regional administration or targeted delivery mechanisms.

[0127] General Considerations

[0128]

[0093] At various places in the present disclosure, substituents, or properties of compounds of the present disclosure are disclosed in groups or in ranges. It is intended that the present disclosure comprise each and every individual or sub-combination of the members of such groups and ranges, and that such groups or ranges include the endpoints. By way of nonlimiting example, if a group or range is from about 1 to about 10, then the group or range includes both the value of about 1 and the value of about 10.

[0129]

[0094] Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that comprise "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure can include embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure can include embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

[0130]

[0095] Any embodiment of the present disclosure that falls within the prior ail may be explicitly excluded from any one or more of the claims. Any embodiment of the agents, methods, and / or compositions of the present disclosure can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0131]

[0096] The present specification will control in instances where publications, patent applications, patents, and other references mentioned herein are incorporated by reference and are in conflict with the present specification.

[0132]

[0097] Section headings, materials, methods, and examples are illustrative only and not intended to be limiting. EXAMPLES

[0133] Example 1: Expression of cGAS-STING pathway inhibitors

[0134]

[0098] Expression of KSHV ORF52 mRNA and VACV B2R mRNA was evaluated in THP1- dual cells with and without treatment with DNA. The KSHV ORF52 mRNA construct included: a 5’UTR, V5 tag for western blot detection, open reading frame (ORF), 3’UTR, and a poly(A) tail. The KSHV ORF52 mRNA construct included: a 5’UTR, V5 tag for western blot detection, open reading frame (ORF), 3’UTR, and a poly(A) tail. The amino acid sequence encoded in the KSHV ORF52 mRNA was SEQ ID NO: 1. The VACV B2R mRNA construct included: a 5’UTR, V5 tag for western blot detection, open reading frame (ORF), 3’UTR, and poly(A) tail. The amino acid sequence encoded in the VACV B2R mRNA was SEQ ID NO: 2. mRNA encoding KSHV ORF52 and VACV B2R were prepared with in-vitro transcription using a DNA template. The DNA construct included: Bxbl attP, mCherry, bGHpA, AmpR, and pUC. The DNA construct sequence was SEQ ID NO: 5.

[0135]

[0099] Expression of VACV E5R ligase is evaluated in THP-1 dual cells with and without treatment with DNA in the same manner as above. The amino acid sequence encoded in the VACV E5R mRNA is SEQ ID NO: 3.

[0136]

[0100] Expression of Ectromelia virus EVM153 poxin is evaluated in THP-1 dual cells with and without treatment with DNA in the same manner as above. The amino acid sequence encoded in the EVM153 mRNA is SEQ ID NO: 4.

[0137]

[0101] LNP formulations: Lipid formulations were prepared as follows. Four lipids (an ionizable lipid, a helper lipid (DSPC (BP-25623)), cholesterol (BP-26125), and a PEG lipid (DMG-PEG 2000 (BP-25496))), all purchased from Broadpharm, were dissolved in ethanol at a ratio of 50:38:10:2. The lipid mixture was combined with acid aqueous buffer containing mRNA and / or DNA at a volumetric (aqueous:ethanol) ratio of 3:1 using a microfluidic mixer (Ignite, Precision Nanosystems). Formulations were dialyzed against 50 mM Tris (pH 7.5), 75 mM NaCl, and 10% sucrose in dialysis cassettes for at least 18 hr. The concentration of the nucleic acid within the LNPs was determined with RiboGreen (R 11491, ThermoFisher). Formulations were stored at -80°C until further use.

[0138]

[0102] THPl-dual cells (thpd-nfis, InvivoGen) were seeded at 500,000 cells / well in a 24- well plate in 1 mL of RPMI 1640 media (11875135, ThermoFisher) consisting of 9% FBS (A5209501, ThermoFisher), 50 mM HEPES buffer (15630080, ThermoFisher), and 1% pen-strep (15070063, ThermoFisher). ApoE (4144-AE, biotechne) was added to each well to a final concentration of 3 pg / mL. DNA-LNPs were added to each well at 2.5 g / well, and RNA-LNP was added at 0.83 pg / well. Controls included mRNA only conditions, a DNA only condition, and a non-treated condition.

[0139]

[0103] Western Blot: After 16 hours incubation, cells were harvested, washed, and frozen in liquid nitrogen. After thawing, the protein was extracted with a Ripa buffer (89900, ThermoFisher) containing protease inhibitor (78441, ThermoFisher). The protein concentration was quantified with a BCA assay (23225, ThermoFisher). LDS sample buffer (NP0008, Invitrogen) was added to the proteins 1:4 and the samples were vortexed, boiled for 3 minutes, vortexed, and centrifuged for 10 minutes. Proteins were separated through gel electrophoresis (WXP41220BOX, ThermoFisher) and compared against the Chameleon Dual molecular ladder (92860000, Licor). The proteins were then transferred to a PVDF membrane (Trans-Blot Turbo Transfer System, BioRad). Primary (451098, ThermoFisher) and secondary antibodies (926-32210, LI-COR) were added, and the gel was imaged (Odyssey DLx, LI-COR) to detect the V5-tagged proteins and cellular actin reference.

[0140]

[0104] Results in FIG 1A show expression of actin at the expected molecular weight (42 kDa). Results in FIG IB show expression of KSHV ORF52 and VACV B2R at the expected molecular weights. Lane 1: KSHV ORF52 (16 kda); Lane 2: VACV B2R (26 kda); Lane 3: KSHV ORF52 + DNA; Lane 4: VACV B2R + DNA; Lane 5: DNA only; Lane 6: no treatment. Results are images of the same gel.

[0141] Example 2: cGAS / STING pathway inhibitors reduce DNA-induced immunogenicity in THPl-dual cells

[0142]

[0105] The ability of KSHV ORF52 mRNA or VACV B2R mRNA to reduce DNA-induced immunogenicity was evaluated by measuring reduction in IRF-3 induction in THPl-dual cells through cotreatment with each mRNA and DNA delivered in separate LNP formulations. The mRNA, DNA, and LNP formulations were prepared as in Example 1.

[0143]

[0106] The THPl-dual cells were seeded at 50,000 cells / well in a 96- well plate. THPl-dual cells were treated with (250 ng / well) of DNA-LNP and a range of mRNA-LNP concentrations (10, 20, 40, 80, 160 ng / well). Controls included mcherry mRNA-LNP and non-treated cells.

[0144]

[0107] Results for reduction of IRF-3 are shown in FIG 2A (fold over background) and FIG 2B (raw values). Fold over background was calculated by dividing the luminescence signal of a sample well by the luminescence signal of non-treated cells. Viability results are shown in FIG 2C (% viability) and FIG 2D (raw values) and indicate that cotrcatmcnt with the mRNAs had minimum impact on cell viability. The % viability was calculated by dividing the luminescence signal of a sample well by the luminescence of non-treated cells.

[0145]

[0108] VACV B2R mRNA was further tested at a mass ratio of 1:3 (mRNA:DNA) for evaluation of co-encapsulation of mRNA and DNA in an LNP formulation as compared to separately encapsulated mRNA and DNA. THPl-dual cells were treated with LNPs as previously described. DNA-LNP only and non-treated cells were included as control. After 16 hours, IRF-3 induction was measured by luminescence and viability was tested using a Cell Titer Gio assay.

[0146]

[0109] VACV B2R mRNA showed complete IRF-3 reduction at all three DNA dose levels tested in FIG 3A (fold over background) and FIG 3B (raw values). Results shown in FIG 3C (% viability) and FIG 3D (raw values) indicate viral mRNA-LNPs had minimum impact on cell viability. No substantial differences were observed between separate and co-encapsulated LNP formulations.

[0147] Example 3: VACV B2R mRNA reduces DNA-induced immunogenicity in PBMCs

[0148] [HO] The ability of VACV B2R mRNA to reduce DNA-induced immunogenicity was evaluated by measuring reduction in cytokines in human peripheral blood mononuclear cells (PBMCs) when cotreated with mRNA and DNA delivered in separate or co-encapsulated LNP formulations. The mRNA, DNA, and LNP formulations were prepared as in Example 1.

[0149] [Ill] The PMBCs were obtained from Stemcell Technologies. PBMCs were thawed and rested overnight in TheraPEAK™X-VIVO™ 15 (Lonza, BP04-744Q) supplemented with 2% human AB serum (Fisher, NC9310328) and lOOIU / mL human recombinant IL-2 (Stemcell Technologies, 78145.2). The cells were seeded at 100,000 cells / well in a 96-well plate. On Day 1, PMBCs were treated with LNPs as shown in Table 7 co-delivered with 4 pg / mL ApoE (4144-AE, Biotechne). Controls included media only (PBMC only), DNA only (1 pg / mL), B2R only (3 pg / mL), and mCherry only (3 pg / mL).

[0150] Table 7: VACV B2R mRNAzDNA samples in PBMCs

[0151]

[0112] Samples were collected 4 hours after treatment and RNA was extracted with RNeasy kit (Qiagen, 74181) following manufacturer’s protocol. Isolated RNA samples were further evaluated for relative mRNA levels of cytokines by RT-qPCR and RT-ddPCR.

[0152]

[0113] RT-qPCR was performed with CFX Opus 96 Real-Time PCR system using iTaq™ Universal SYBR® Green One-Step Kit (Bio-Rad, 1725151) following manufacturer’s protocol. The gene expression of IP- 10 and IL-6 were calculated with double delta Ct analysis. The relative mRNA levels are reported relative to actin levels and normalized with PBMC only control group. Primers were ordered as RxnReady Primer Pools from IDT.

[0153]

[0114] RT-ddPCR was performed with QX600 AutoDG Droplet Digital PCR System (BioRad) using One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad, 1864022) following the manufacturer’s protocol. Data analysis was performed with the QX Manager. The relative mRNA levels were calculated as the concentration of cytokine divided by that of a reference gene and reported relative to the PBMC only control. Primers and probes were designed using PrimerQuest (IDT) by default setting and ordered from IDT.

[0154]

[0115] Results in human PBMCs show a reduction in relative mRNA levels of cytokines with cotreatment of VACV B2R for separate and co-encapsulated formulations as shown in FIG 4A (IP- 10) and FIG 4B (IL-6) by qPCR and in FIG 4C (IP- 10) and FIG 4D (IFN-y) by ddPCR. Similar results were observed by qPCR and ddPCR as illustrated by evaluated relative mRNA levels of IP- 10. The mCherry mRNA cotreatment did not reduce the immune response as shown in FIG 4E (IP- 10) and FIG 4F (IFN-y) by ddPCR.

[0155]

[0116] The ability of VACV B2R mRNA to reduce DNA-induced immunogenicity was also evaluated by measuring reduction in cytokines in cynomolgus monkey PBMCs when cotreated with mRNA and DNA delivered in separate LNP formulations. The mRNA, DNA, and LNP formulations were prepared as in Example 1.

[0156]

[0117] The cynomolgus monkey PMBCs were obtained from IQBiosciences (IQB-MnPB102, Lot# P23L2006). Cyno PBMCs were thawed in RPMI medium 1640 (Gibco, 11875-093) with 10% FBS (Gibco, A31605-02) and seeded in 96-well plates. Concurrently, PMBCs were treated with B2R LNP (3 pg / mL), and DNA LNP (1 pg / mL) simultaneously at 3:1 ratio and co-dclivcrcd with 4 pg / mL ApoE (4144-AE, Biotechne). Controls included media only (PBMC only), DNA only (1 pg / mL), B2R only (3 pg / mL), DNA (1 pg / mL) and mCherry (3 pg / mL) delivered simultaneously, and mCherry only (3 pg / mL).

[0157]

[0118] Samples were collected 4 hours after treatment and RNA was extracted with RNeasy kit (Qiagen, 74181) following manufacturer’s protocol. Isolated RNA samples were further evaluated for relative mRNA levels of cytokines by RT-qPCR.

[0158]

[0119] RT-qPCR was performed with CFX Opus 96 Real-Time PCR system using iTaq™ Universal SYBR® Green One-Step Kit (Bio-Rad, 1725151) following manufacturer’s protocol. The gene expression of IP- 10 and IL-6 were calculated with double delta Ct analysis. The relative mRNA levels are reported relative to actin levels and normalized with PBMC only control group. Primers were ordered from IDT.

[0159]

[0120] Results in cyno PBMCs show a reduction in relative mRNA levels of cytokines with cotreatment of VACV B2R for separate formulations as shown in FIG 5A (IP- 10) and FIG 5B (IL-6) by qPCR.

[0160] Example 4; Variants of VACV B2R and KSHV ORF52

[0161]

[0121] Variants of VACV B2R and KSHV ORF52 were identified by positive zero-shot ESM wild-type marginal score of mutation effect in any of seven models (esmlb_t33_650M_UR50S, esmlv_t33_650M_UR90S_l , esmlv_t33_650M_UR90S_2, esml v_t33_650M_UR90S_3, esmlv_t33_650M_UR90S_4, esmlv_t33_650M_UR90S_5 and esm2_tl2_35M_UR50D). The wildtype VACV B2R sequence is SEQ ID NO: 2. The wildtype KSHV ORF52 sequence is SEQ ID NO: 1.

[0162]

[0122] For VACV B2R, the following variants were identified: P155D (SEQ ID NO: 10), W162K (SEQ ID NO: 11), P155E (SEQ ID NO: 12), C7 IK (SEQ ID NO: 13), C71E, C71D (SEQ ID NO: 14), C71N (SEQ ID NO: 15), C71A (SEQ ID NO: 16), C71V (SEQ ID NO: 17), C71P (SEQ ID NO: 18), C71L (SEQ ID NO: 19), C71R (SEQ ID NO: 20), C71I (SEQ ID NO: 21), C71S (SEQ ID NO: 22), C71G (SEQ ID NO: 23), C71T (SEQ ID NO: 24), W162E (SEQ ID NO: 25), C71Q (SEQ ID NO: 26), W162D (SEQ ID NO: 27), and S90G (SEQ ID NO: 28).

[0163]

[0123] For KSHV ORF52, the following valiants were identified: C101A (SEQ ID NO: 29), C101L (SEQ ID NO: 30), C101E (SEQ ID NO: 31), C101P (SEQ ID NO: 32), C101D (SEQ ID NO: 33), C101T (SEQ ID NO: 34), C101V (SEQ ID NO: 35), C1O1S (SEQ ID NO: 36), C101G (SEQ ID NO: 37), C101R (SEQ ID NO: 38), C101K (SEQ ID NO: 39), M14V (SEQ ID NO: 40), C101Q (SEQ ID NO: 41), and C101I (SEQ ID NO: 42).

[0164] SEQUENCE LISTING

Claims

CLAIMS1. A method of genome-editing, the method comprising administering a cGAS-STING pathway inhibitor and a genome-editing system to a cell, wherein the genome-editing system comprises a genome-editing enzyme and double-stranded DNA (dsDNA).

2. The method of claim 1, wherein the cGAS-STING pathway inhibitor reduces cGAS binding to dsDNA.

3. The method of claim 2, wherein the cGAS-STING pathway inhibitor comprises a protein selected from Table 1.

4. The method of claim 2, wherein the cGAS-STING pathway inhibitor comprises the amino acid sequence of SEQ ID NO: 1.

5. The method of claim 2, wherein the cGAS-STING pathway inhibitor comprises nucleic acid encoding an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 1.

6. The method of any one of claims 2-5, wherein the cGAS-STING pathway inhibitor further comprises a nuclear localization signal (NLS).

7. The method of claim 1, wherein the cGAS-STING pathway inhibitor ligates ubiquitin to cGAS for degradation.

8. The method of claim 7, wherein the cGAS-STING pathway inhibitor comprises a protein selected from Table 2.

9. The method of claim 7, wherein the cGAS-STING pathway inhibitor comprises the amino acid sequence of SEQ ID NO: 3.

10. The method of claim 7, wherein the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 3.

11. The method of claim 1, wherein the cGAS-STING pathway inhibitor degrades 2’3’ cGAMP.

12. The method of claim 11, wherein the cGAS-STING pathway inhibitor comprises a protein selected from Table 3.

13. The method of claim 11, wherein the cGAS-STING pathway inhibitor comprises a poxin.

14. The method of claim 11, wherein the cGAS-STING pathway inhibitor comprises the amino acid sequence of SEQ ID NO: 2.

15. The method of claim 11, wherein the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 2.

16. The method of any one of claims 7-15, wherein the cGAS-STING pathway inhibitor further comprises an intrinsically disordered region (IDR).

17. The method of any one of claims 1-16, wherein the cGAS-STING pathway inhibitor is encoded in nucleic acid.

18. The method of claim 17, wherein the nucleic acid is mRNA.

19. The method of claim 17, wherein the nucleic acid is circular RNA.

20. The method of any one of claims 1-19, wherein the genome-editing enzyme comprises a large serine recombinase (LSR).

21. The method of any one of claims 1-19, wherein the genome-editing enzyme comprises a Cas polypeptide, a transcription activator-like effector nuclease (TALEN), a zine-finger nuclease (ZFN), a tyrosine recombinase, or a transposon-encoded enzyme.

22. The method of claim 21, wherein the transposon-encoded enzyme comprises TnpB, TnsB, a PiggyBac transposase, or a Sleeping Beauty transposase.

23. The method of any one of claims 1-22, wherein the cGAS-STING pathway inhibitor and genome-editing enzyme are encoded on a single nucleic acid.

24. The method of claim 23, wherein the nucleic acid is mRNA.

25. The method of claim 24, wherein the mRNA comprises a P2A site between the cGAS- STING pathway inhibitor and the genome-editing enzyme.

26. The method of claim 24, wherein the mRNA comprises a IRES site between the cGAS- STING pathway inhibitor and genome-editing enzyme.

27. The method of claim 23, wherein the nucleic acid is circular RNA.

28. The method of claim 27, wherein the circular RNA comprises an IRES.

29. The method of any one of claims 1-20 and claims 23-28, wherein the dsDNA comprises an AttD site.

30. The method of any one of claims 1-29, wherein the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are administered in lipid nanoparticles (LNPs).

31. The method of claim 30, wherein the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are encapsulated in separate LNPs.

32. The method of claim 30, wherein the cGAS-STING pathway inhibitor and genomeediting enzyme are co-encapsulated in LNPs.

33. The method of claim 30, wherein the genome-editing enzyme and dsDNA are coencapsulated in LNPs.

34. The method of claim 30, wherein the cGAS-STING pathway inhibitor and dsDNA are co-encapsulated in LNPs.

35. The method of claim 30, wherein the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are co-encapsulated in LNPs.

36. The method of any one of claims 30-33, wherein the LNPs encapsulating the cGAS- STING pathway inhibitor are administered to the cell separately from the LNPs encapsulating the dsDNA.

37. The method of any one of claims 30-36, wherein the LNPs contain RNA and DNA at a mass ratio of 1:1, 2:1, 3:1, 10:1, 1:2, 1:3, or 1:10.

38. The method of any one of claims 1-37, wherein the cGAS-STING pathway inhibitor reduces IRF-3 expression, as compared to a control.

39. The method of any one of claims 1-37, wherein the cGAS-STING pathway inhibitor reduces cytokine response, as compared to a control.

40. The method of claim 39, wherein the cytokine response comprises IP- 10 and / or IFN-y.

41. The method of claims 1-40, wherein the cell is in a subject.

42. A composition for genome-editing comprising:a) a cGAS-STING pathway inhibitor or nucleic acid encoding a cGAS-STING pathway inhibitor, b) a genome-editing enzyme or nucleic acid encoding a genome-editing enzyme, c) dsDNA, and d) at least one acceptable carrier, wherein the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are encapsulated in LNPs.

43. The composition of claim 42, wherein the cGAS-STING pathway inhibitor reduces cGAS from binding to DNA.

44. The composition of claim 43, wherein the cGAS-STING pathway inhibitor comprises a protein selected from Table 1.

45. The composition of claim 43, wherein the cGAS-STING pathway inhibitor comprises the amino acid sequence of SEQ ID NO: 1.

46. The composition of claim 43, wherein the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 1.

47. The composition of any one of claims 43-46, wherein the cGAS-STING pathway inhibitor further comprises an NLS.

48. The composition of claim 42, wherein the cGAS-STING pathway inhibitor ligates ubiquitin to cGAS .

49. The composition of claim 48, wherein the cGAS-STING pathway inhibitor comprises a protein selected from Table 2.1650. The composition of claim 48, wherein the cGAS-STING pathway inhibitor comprises an amino acid sequence of SEQ ID NO: 3.

51. The composition of claim 48, wherein the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 3.

52. The composition of claim 42, wherein the cGAS-STING pathway inhibitor degrades 2’3’ cGAMP.

53. The composition of claim 52, wherein the cGAS-STING pathway inhibitor comprises a protein selected from Table 3.

54. The composition of claim 52, wherein the cGAS-STING pathway inhibitor comprises the amino acid sequence of SEQ ID NO: 2.

55. The composition of claim 52, wherein the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 2.

56. The composition of any one of claims 48-55, wherein the cGAS-STING pathway inhibitor further comprises an IDR.

57. The composition of any one of claims 42-56, wherein the cGAS-STING pathway inhibitor is encoded in nucleic acid.

58. The composition of claim 57, wherein the nucleic acid is mRNA.

59. The composition of claim 57, wherein the nucleic acid is circular RNA.

60. The composition of any one of claims 42-59, wherein the genome-editing enzyme comprises an LSR.

61. The composition of any one of claims 42-59, wherein the genome-editing enzyme comprises a Cas polypeptide, a TALEN, a ZFN, a tyrosine recombinase, or a transposon- encoded enzyme.

62. The composition of claim 61, wherein the transposon-encoded enzyme comprises TnpB, TnsB, a PiggyBac transposase, or a Sleeping Beauty transposase.

63. The composition of any one of claims 42-62, wherein the cGAS-STING pathway inhibitor and genome-editing enzyme are encoded on a single nucleic acid molecule.

64. The composition of claim 63, wherein the nucleic acid is mRNA.

65. The composition of claim 64, wherein the mRNA comprises a P2A site between the cGAS-STING pathway inhibitor and genome-editing enzyme.

66. The composition of claim 65, wherein the mRNA comprises an IRES between the cGAS- STING pathway inhibitor and genome-editing enzyme.

67. The composition of claim 63, wherein the nucleic acid is circular RNA.

68. The composition of claim 67, wherein the circular RNA comprises an IRES.

69. The composition of any one of claims 42-60 and claims 63-68, wherein the dsDNA comprises an AttD site.

70. The composition of any one of claims 42-69, wherein the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are encapsulated in separate LNPs.71 . The composition of any one of claims 70, wherein the cGAS-STING pathway inhibitor and gcnomc-cditing enzyme arc co-cncapsulatcd.

72. The composition of any one of claims 70, wherein the genome-editing enzyme and dsDNA are co-encapsulated.

73. The composition of any one of claims 70, wherein the cGAS-STING pathway inhibitor and dsDNA are co-encapsulated.

74. The composition of any one of claims 70, wherein the cGAS-STING pathway inhibitor, genome-editing enzyme, and dsDNA are encapsulated together in LNPs.

75. The composition of any one of claims 70-74, wherein the LNPs contain RNA and DNA at a mass ratio of 1:1, 2:1, 3:1, 10:1, 1:2, 1:3, or 1:10.

76. A composition for genome-editing comprising: a) means for reducing cGAS binding to DNA, ligating ubiquitin to cGAS for degradation, or degrading 2’3’ cGAMP. b) a genome-editing enzyme, c) dsDNA, and d) at least one acceptable carrier, wherein the composition is encapsulated in LNPs.

77. A method of dsDNA delivery to a cell, the method comprising administering a cGAS- STING pathway inhibitor and dsDNA.

78. The method of claim 77, wherein the cGAS-STING pathway inhibitor reduces cGAS binding to dsDNA.

79. The method of claim 78, wherein the cGAS-STING pathway inhibitor comprises a protein selected from Table 1.

80. The method of claim 78, wherein the eGAS-STING pathway inhibitor comprises the amino acid sequence of SEQ ID NO: 1.

81. The method of claim 78, wherein the eGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 1.

82. The method of any one of claims 78-81, wherein the eGAS-STING pathway inhibitor further comprises an NLS.

83. The method of claim 77, wherein the eGAS-STING pathway inhibitor ligates ubiquitin to cGAS for degradation.

84. The method of claim 83, wherein the eGAS-STING pathway inhibitor comprises a protein selected from Table 2.

85. The method of claim 84, wherein the eGAS-STING pathway inhibitor comprises the amino acid sequence of SEQ ID NO: 3.

86. The method of claim 84, wherein the eGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 3.

87. The method of claim 77, wherein the eGAS-STING pathway inhibitor degrades 2’3’ cGAMP.

88. The method of claim 87, wherein the eGAS-STING pathway inhibitor comprises a protein selected from Table 3.

89. The method of claim 87, wherein the eGAS-STING pathway inhibitor comprises a poxin.

90. The method of claim 87, wherein the eGAS-STING pathway inhibitor comprises the amino acid sequence of SEQ ID NO: 2.

91. The method of claim 87, wherein the eGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 2.

92. The method of any one of claims 83-91, wherein the eGAS-STING pathway inhibitor further comprises an IDR.

93. The method of any one of claims 77-92, wherein the eGAS-STING pathway inhibitor is encoded in nucleic acid.

94. The method of claim 93, wherein the nucleic acid is mRNA.

95. The method of claim 93, wherein the nucleic acid is circular RNA.

96. The method of any one of claims 77-95, wherein the eGAS-STING pathway inhibitor and dsDNA are co-administered.

97. The method of any one of claims 77-95, wherein the eGAS-STING pathway inhibitor and dsDNA are administered sequentially.

98. A composition for dsDNA delivery to a cell comprising: a) a eGAS-STING pathway inhibitor or nucleic acid encoding a eGAS-STING pathway inhibitor, b) dsDNA, and c) at least one acceptable carrier, wherein the eGAS-STING pathway inhibitor and dsDNA are encapsulated in LNPs.

99. The composition of claim 98, wherein the cGAS-STING pathway inhibitor reduces cGAS from binding to DNA.

100. The composition of claim 99, wherein the cGAS-STING pathway inhibitor comprises a protein selected from Table 1.

101. The composition of claim 99, wherein the cGAS-STING pathway inhibitor comprises the amino acid sequence of SEQ ID NO: 1.

102. The composition of claim 99, wherein the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 1.

103. The composition of any one of claims 99-102, wherein the cGAS-STING pathway inhibitor further comprises an NLS.

104. The composition of claim 98, wherein the cGAS-STING pathway inhibitor ligates ubiquitin to cGAS .

105. The composition of claim 104, wherein the cGAS-STING pathway inhibitor comprises a protein selected from Table 2.

106. The composition of claim 104, wherein the cGAS-STING pathway inhibitor comprises an amino acid sequence of SEQ ID NO: 3.

107. The composition of claim 104, wherein the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 3.

108. The composition of claim 98, wherein the cGAS-STING pathway inhibitor degrades 2’3’ cGAMP.

109. The composition of claim 108, wherein the eGAS-STING pathway inhibitor comprises a protein selected from Table 3.

110. The composition of claim 108, wherein the cGAS-STING pathway inhibitor comprises the amino acid sequence of SEQ ID NO: 2.

111. The composition of claim 108, wherein the cGAS-STING pathway inhibitor comprises a nucleic acid encoding an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 2.

112. The composition of any one of claims 104-111, wherein the cGAS-STING pathway inhibitor further comprises an IDR.

113. The composition of any one of claims 98-112, wherein the cGAS-STING pathway inhibitor is encoded in nucleic acid.

114. The composition of claim 113, wherein the nucleic acid is mRNA.

115. The composition of claim 113, wherein the nucleic acid is circular RNA.

116. The composition of any one of claims 98-115, wherein the dsDNA comprises an AttD site.

117. The composition of any one of claims 98-116, wherein the cGAS-STING pathway inhibitor and dsDNA are encapsulated in separate LNPs.

118. The composition of any one of claims 98-116, wherein the cGAS-STING pathway inhibitor and dsDNA are co-encapsulated.

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