Enhanced nuclear delivery of DNA and compositions for use in practicing the same

Abstract

Methods and compositions for enhancing nuclear DNA delivery are provided. Aspects of the methods include contacting a cell with a DNA to be delivered to the nucleus of the cell and an agent that modulates the activity of protein Barrier-to-Autointegration Factor (BAF) in a cell. In addition, compositions, including reagents, devices and kits thereof, that find use in practicing embodiments of the subject methods are provided.

Description

[0001] ENHANCED NUCLEAR DELIVERY OF DNA AND COMPOSITIONS FOR

[0002] USE IN PRACTICING THE SAME

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of the United States Provisional Application Serial No. 63 / 563,878 filed on March 11, 2024; United States Provisional Application Serial No. 63 / 633,236 filed on April 12, 2024; and United States Provisional Application Serial No. 63 / 702,825 filed on October 3, 2024, the disclosures of which are herein incorporated by reference.

[0005] INTRODUCTION

[0006] Field of the Invention

[0007] This invention pertains to the delivery of nucleic acids to cells and tissues.

[0008] Background

[0009] There are many instances in which nuclear delivery of a nucleic acid is desired, where such instances include research, diagnostic and therapeutic applications. An example of such a therapeutic application is gene therapy. In the field of gene therapy, viral vectors, such as vectors based on the virus AAV, are commonly employed to deliver genes to the nucleus. However, AAV’s genome is limited in size, so any gene greater than 4.7kB will not be suitable for use with AAV vectors, which limits the utility of such vectors for many indications. In addition, vir al vectors, such as AAV, induce an adaptive immune response, such that they can only be delivered once, which is not suitable for some indications, such as indications in the liver where the cells are slowly dividing and will lose the transduced genome, thereby requiring redosing. Furthermore, viral vectors such as AAV are toxic at the doses that are required for a therapeutic benefit in some indications. Accordingly, what is needed is a new delivery vehicle for delivering nucleic acids, such as DNA, to cells, particularly in patients in need of gene therapy but also in vitro during research.

[0010] That next generation delivery vehicle is a nanoparticle. Of particular interest are lipid nanoparticles, given how LNPs have been de-risked by being used in Onpattro and the Co vid vaccines. The problem is that, in contrast to AAV’s ability to carry its DNA cargo all the way into the nucleus, nanoparticles only deliver their cargo to the cytoplasm. In the case of dividing cells, delivery to the cytosol may be sufficient as the nuclear envelope dissolves in the course of replication and new DNA gets captured as the nucleus re-forms in the daughter cells. However, it is a problem for nondividing or slowly dividing cells. What is needed to make LNPs into a successful delivery vehicle for DNA is a mechanism to enhance the transport of the DNA from the cytoplasm into the nucleus. The inventors have identified methods and compositions to satisfy the above need.

[0011] SUMMARY

[0012] Methods and compositions for enhancing nuclear DNA delivery are provided. Aspects of the methods include contacting a cell with a DNA to be delivered to the nucleus of the cell and an agent that modulates the activity of the protein Barrier-to-Autointegration Factor (BAF) in a cell. In addition, compositions, including reagents, devices and kits thereof, that find use in practicing embodiments of the subject methods are provided.

[0013] Aspects of the disclosure include compositions for the delivery of DNA to the nucleus of a cell are provided. In embodiments, the compositions include a cytosolic delivery vehicle, a DNA, and an agent that modulates the protein Barrier to Autointegration Factor (BAF).

[0014] In some embodiments, the agent is a protein having an amino acid sequence having a sequence identity of 90% or more to wild type BAF, or an mRNA encoding the protein having an amino acid sequence having a sequence identity of 90% or more to wild type BAF. In some instances, the protein includes a mutation such as a substitution at a residue relative to the wild type human BAF protein sequence selected from the group consisting of the threonine at residue 2, the threonine at residue 3, the serine at residue 4, the alanine at residue 12, the glutamic acid at residue 28, the glycine at residue 31, the lysine at residue 33, the glutamic acid at residue 36, the arginine at residue 37, the leucine at residue 58, the alanine at reside 71, or the glycine at residue 79of the wild type human BAF protein or a corresponding residue in a BAF ortholog. In some instances, the protein includes a mutation relative to wild type human BAF selected from the group consisting of T2D, T3D, S4D, A12T, E28D, G31S, K33R, E36D, R37K, L58R, A71K, and G79R. In some instances, the protein includes a heterologous UTR. In some embodiments, the agent is a BAF kinase or an mRNA encoding a BAF kinase. In some instances, the BAF kinase includes an amino acid sequence having a sequence identity of 90% or more to vaccinia virus Ankara strain B 1 protein, where in some instances the BAF kinase includes an amino acid sequence having a sequence identity of 90% or more to human VRK1, VRK1-X1, human VRK2A, human VRK2B, or human VRK3. In some embodiments, the agent is a siRNA that is specific for BAF. In some embodiments, the agent is a small molecule, e.g., a small molecule is selected from the group consisting of obtusilactone B, Mahubanolide, kotomolide B, epilitsenolide D2, and rabeprazole. In some embodiments, the agent includes a LEM domain or an mRNA encoding a LEM domain. In some instances, the LEM domain includes an animo acid sequence having a sequence identity of 90% or more to a sequence selected from the group consisting of: a. an emerin LEM domain b. a LAP2-beta LEM domain: and c. a MANI LEM domain:

[0015] In some embodiments, the agent does not include a LEM domain or a nucleic acid encoding a LEM domain.

[0016] In some embodiments, the cytosolic delivery vehicle includes a non-viral delivery vehicle. In some instances, the non-viral delivery vehicle is a nanoparticle, such as a lipid nanoparticle (LNP). In some instances, the non-viral delivery vehicle is a vesicle, such as a vesicle selected from the group consisting of an exosome, an extracellular vesicle, and a micelle. In some embodiments, the composition includes a single cytosolic delivery vehicle having both the DNA and the agent. In some instances, the composition includes a first cytosolic delivery vehicle that includes the DNA and a second cytosolic delivery vehicle that includes the agent.

[0017] In some embodiments, the composition further includes an mRNA encoding a nuclear' targeting factor (NTF), and the DNA includes a DNA nuclear targeting sequence (DTS). In some instances, a. the NTF is a Gal4 NTF and the DTS is a Gal4 binding sequence; or b. the NTF is a TetR NTF and the DTS is a TetR binding sequence.

[0018] In some embodiments, the composition includes a nuclease, an integrase or a transposase.IN some embodiments, the composition encodes a nuclease and the composition includes a guide RNA (gRNA).

[0019] In some embodiments, the DNA is 100 - 15,000 bp. In some embodiments, the DNA includes a coding sequence. In some embodiments, the DNA incldues an expression cassette comprising a promoter operably linked to the coding sequence. In some instances, the DNA includes sequences flanking the coding sequence or the expression cassette that are homologous to genomic sequences of the cell for which the DNA is configured to be used.

[0020] Aspects of the disclosure include methods, where the methods include contacting a cell with a DNA and an agent that modulates the activity of the protein Barrier to Autointegration Factor (BAF). In some embodiments, the agent is a protein that includes an amino acid sequence having a sequence identity of 90% or more to wild type BAF, or an mRNA encoding the protein that includes an amino acid sequence having a sequence identity of 90% or more to wild type BAF. In some instances, the protein includes a mutation such as a substitution at a residue relative to the wild type human BAF protein sequence selected from the group consisting of the threonine at residue 2, the threonine at residue 3, the serine at residue 4, the alanine at residue 12, the glutamic acid at residue 28, the glycine at residue 31, the lysine at residue 33, the glutamic acid at residue 36, the arginine at residue 37, the leucine at residue 58, the alanine at reside 71, or the glycine at residue 79of the wild type human BAF protein or a corresponding residue in a BAF ortholog. In some instances, the protein includes a mutation relative to wild type human BAF selected from the group consisting of T2D, T3D, S4D, A12T, E28D, G31S, K33R, E36D, R37K, L58R, A71K, and G79R. In some instances, the protein includes a heterologous UTR. In some embodiments, the agent is a BAF kinase or an mRNA encoding a BAF kinase. In some instances, the BAF kinase includes an amino acid sequence having a sequence identity of 90% or more to vaccinia virus Ankara strain B 1 protein, where in some instances the BAF kinase includes an amino acid sequence having a sequence identity of 90% or more to human VRK1, VRK1-X1, human VRK2A, human VRK2B, or human VRK3. In some embodiments, the agent is a siRNA that is specific for BAF. In some embodiments, the agent is a small molecule, e.g., a small molecule is selected from the group consisting of obtusilactone B, Mahubanolide, kotomolide B, epilitsenolide D2, and rabeprazole. In some embodiments, the agent includes a LEM domain or an mRNA encoding a LEM domain. In some instances, the LEM domain includes an animo acid sequence having a sequence identity of 90% or more to a sequence selected from the group consisting of: a. an emerin LEM domain b. a LAP2-beta LEM domain: and c. a MANI LEM domain:

[0021] In some embodiments, the agent does not include a LEM domain or a nucleic acid encoding a LEM domain.

[0022] In some embodiments of the methods, the cell is simultaneously or sequentially contacted with the DNA and the agent. In some instances, the DNA and the agent are present in a cytosolic delivery vehicle. In some instances, the DNA and the agent are present in the same cytosolic delivery vehicle. In some instances, the DNA and the agent are present in different cytosolic delivery vehicles. In some instances, the cytosolic delivery vehicle includes a nanoparticle, such as a lipid nanoparticle. In some instances, the cytosolic delivery vehicle includes a vesicle, such as a vesicle selected from the group consisting of an exosome, an extracellular vesicle, and a micelle.

[0023] In some embodiments, the methods include electroporating the cell. In some embodiments, the cell is in vivo. In some embodiments, the methods include orally, parenterally, subrctinally, intravitreally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenously, intra-arterially, intraperitoneally, intracranially, subcutaneously, intramuscularly, intranasally, intrathecally and / or intraarticularly delivering the DNA and the agent. In some embodiments, the cell is ex vivo. In some embodiments, the cell is selected from the group consisting of hepatocytes, stellate cells, T lymphocytes, B lymphocytes, NK cells, hematopoietic stem cells, skeletal muscle cells, cardiomyocytes, neurons, astrocytes, oligodendrocytes, dendritic cells and skin cells.

[0024] BRIEF DESCRIPTION OF THE FIGURES

[0025] Figures 1A, 1A’, IB, IB’, 1C, and 1C’ illustrate the formation of cytoplasmic puncta of DNA following transfection into the cell, and the subcellular localization of Barrier to Autointegration Factor (BAF) to these puncta. (A) and (A’): low and high magnification images of labeled DNA, following transfection in HepG2 cells. (B) and (B’): low and high magnification images of BAF protein in DNA- transfected HepG2 cells. (C) and (C’): low and high magnification images of BAF protein in untransfected HepG2 cells. Images were taken 16 hours after transfection.

[0026] Figures 2A-2F illustrate the subcellular localization of the nuclear envelope protein Emerin before and after transfection of cells with DNA or RNA. In these panels, Emerin protein is visualized with an Emerin-specific antibody (red) and the nuclei are stained with DAPI (blue). (A) Untreated HepG2 cells stained with an Emerin-specific antibody; (B) untreated HepG2 cells co-stained with DAPI; (C) mock transfected HepG2 cells (transfection reagents only) (D) HepG2 cells transfected with an mRNA encoding a nuclear translocating factor (NTF); (E) HepG2 cells transfected with DNA; (F) HepG2 cells transfected with DNA and the mRNA encoding an NTF.

[0027] Figures 3A-3C illustrate by confocal microscopy the envelope-type structure that forms from Emerin that is colocalized with transfected DNA. (A) Emerin protein, as visualized with an Emerin- specific antibody (red): (B) DNA, as visualized with an EdU (5-ethynyl-2'-deoxyuridine) label that was incorporated into the DNA prior to transfection (yellow); (C) merge of A and B. Nucleus is stained with DAPI (blue).

[0028] Figure 4 shows the amount of human Factor IX protein that has accumulated in mouse plasma seven days after delivery of an LNP co-formulated with a DNA comprising an hFIX expression cassette and an mRNA encoding the BAF modulator BAF-L58R (groups 1-4), or an mRNA encoding BAF-L58R protein and an mRNA encoding a Gal4 nuclear translocating factor (group 5), or no additional mRNA agents (group 6). Groups 1-4: DNA (0.3mg / kg) co-formulated with decreasing amounts of BAF-L58R mRNA (0.6 mg / kg, 0.3 mg / kg, 0.1 mg / kg, 0.03 mg / kg). Group 5: DNA (0.3mg / kg) co-formulated with BAF-L58R mRNA (0.3 mg / kg) and Gal4 NTF (0.6 mg / kg). Group 6: DNA alone (0.3 mg / kg). Group 7: vehicle (PBS). Figure 5 shows the amount of human Factor IX protein that has accumulated in mouse plasma seven days after delivery of an LNP co-formulated with a DNA comprising an hFIX expression cassette and either an mRNA encoding the BAF-L58R protein based on mouse BAF ("mBAF-L58R") and an mRNA encoding a Gal4 nuclear' translocating factor (group 1), an mRNA encoding BAF-L58R protein based on human BAF (“hBAF-L58R”) and an mRNA encoding a Gal4 nuclear translocating factor (group 2), an mRNA encoding BAF-L58R protein based on mouse BAF but utilizing alternative UTRs to alter mRNA translation kinetics and an mRNA encoding a Gal4 nuclear translocating factor (group 3), or simply an mRNA encoding the BAF-L58R protein based on mouse BAF, i.e. without an mRNA encoding a Gal4 nuclear- translocating factor (group 4).

[0029] Figure 6 shows the amount of human Factor IX protein that has accumulated in mouse plasma seven days after delivery of an LNP co-formulated with a DNA comprising an hFIX expression cassette, an mRNA encoding a GAL4 nuclear translocating factor, and either an mRNA encoding mBAF-L58R (group 1) or an mRNA encoding mBAF-L58R with a C-terminal NLS (group 2).

[0030] Figure 7 shows the amount of human Factor IX protein that has accumulated in mouse plasma seven days after delivery of an LNP formulated with a DNA comprising an hFIX expression cassette, an mRNA encoding a GAL4 nuclear- translocating factor, and an mRNA encoding mouse wild type BAF (group 1).

[0031] Figure 8 shows the amount of human Factor IX protein that has accumulated in mouse plasma three and seven days after delivery of an LNP formulated with a DNA comprising an hFIX expression cassette, an mRNA encoding a Gal4 nuclear translocating factor and either: an mRNA encoding vaccinia virus Ankara strain Bl protein (group 1); an mRNA encoding a vaccinia virus Western Region strain Bl protein (group 2); or an mRNA encoding BAF-L58R protein based on mouse BAF. Group 4: vehicle (PBS).

[0032] Figures 9A-9D provide images of sections of mouse liver labeled with an antibody specific for human Factor IX at 15 or 27 days following i.v. injection of: (A) PBS; (B) an LNP formulated with a DNA comprising an hFIX expression cassette; (C) an LNP co-formulated with a DNA comprising an hFIX expression cassette and an mRNA encoding a GAL4 nuclear translocating factor; or (D) an LNP co- formulated with a DNA comprising an hFIX expression cassette, an mRNA encoding BAF-L58R protein based on mouse BAF, and an mRNA encoding a GAL4 nuclear translocating factor. Tissue sections in Figures 9A-C are from 27 days post-dose; tissue section in Figure 9D is from 15 days post-dose.

[0033] Figures 10A-10C illustrate the amount of human Factor IX protein that has accumulated in plasma between 3 and 14 days after delivery of an LNP formulated with a DNA comprising an hFIX expression cassette and an mRNA encoding a mutant human BAF protein fused to an NLS into (A) mice, (B) rats, or (C) NHPs. Figure 11 demonstrates the increase in GFP expression following delivery of LNPs formulated with a DNA comprising a GFP expression cassette and an mRNA encoding BAF variants of the present disclosure into primary human hepatocytes (PHHs).

[0034] INCORPORATION BY REFERENCE

[0035] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0036] DEFINITIONS

[0037] The terms "polypeptide," "polypeptide sequence," "peptide," "peptide sequence," "protein," "protein sequence" and "amino acid sequence" are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds, which series may include proteins, polypeptides, oligopeptides, peptides, and fragments thereof. The protein may be made up of naturally occurring amino acids and / or synthetic (e.g., modified or non-naturally occurring) amino acids. Thus "amino acid", or "peptide residue", as used herein means both naturally occurring and synthetic amino acids. The terms "polypeptide", "peptide", and "protein" includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, beta-galactosidase, luciferase, and the like. Furthermore, it should be noted that a dash at the beginning or end of an amino acid sequence indicates cither a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to a carboxyl or hydroxyl end group. However, the absence of a dash should not be taken to mean that such peptide bond or covalent bond to a carboxyl or hydroxyl end group is not present, as it is conventional in representation of amino acid sequences to omit such.

[0038] The term "polynucleotide," "polynucleotide sequence," "oligonucleotide," "oligonucleotide sequence," "oligomer," "oligo," "nucleic acid sequence" or "nucleotide sequence" used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer having purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0039] The terms "derivative" and "variant" refer without limitation to any compound such as nucleic acid or protein that has a structure or sequence derived from the compounds disclosed herein and whose structure or sequence is sufficiently similar to those disclosed herein such that it has the same or similar activities and utilities or, based upon such similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the referenced compounds, thereby also interchangeably referred to "functionally equivalent" or as "functional equivalents." Modifications to obtain "derivatives" or "variants" may include, for example, addition, deletion and / or substitution of one or more of the nucleic acids or amino acid residues.

[0040] The functional equivalent or fragment of the functional equivalent, in the context of a protein, may have one or more conservative amino acid substitutions. The term "conservative amino acid substitution" refers to substitution of an amino acid for another amino acid that has similar properties as the original amino acid. The groups of conservative amino acids are as follows:

[0041] Group Name of the amino acids

[0042] Aliphatic Gly, Ala, Vai, Leu, He

[0043] Hydroxyl or Sulfhydryl / Selenium-containing Ser, Cys, Thr, Met

[0044] Cyclic Pro

[0045] Aromatic Phc, Tyr, Trp

[0046] Basic His, Lys, Arg

[0047] Acidic and their Amide Asp, Glu, Asn, Gin

[0048] Conservative substitutions may be introduced in any position of a preferred predetermined peptide or fragment thereof. It may however also be desirable to introduce non-conservative substitutions, particularly, but not limited to, a non-conscrvativc substitution in any one or more positions. A non- conservative substitution leading to the formation of a functionally equivalent fragment of the peptide would for example differ substantially in polarity, in electric charge, and / or in steric bulk while maintaining the functionality of the derivative or variant fragment.

[0049] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may have additions or deletions (i.c., gaps) as compared to the reference sequence (which does not have additions or deletions) for optimal alignment of the two sequences. In some cases, the percentage can be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0050] The terms "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., the entire polypeptide sequences or individual domains of the polypeptides), when compared and aligned for maximum correspondence over a comparison window or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the complement of a test sequence.

[0051] The term "complementary" or "substantially complementary," interchangeably used herein, means that a nucleic acid (e.g., DNA or RNA) has a sequence of nucleotides that enables it to non- covalently bind, i.e. form Watson-Crick base pairs and / or G / U base pairs to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid). As is known in the art, standard Watson-Crick base-parring includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C).

[0052] A DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA when placed under the control of appropriate regulatory sequences. A DNA polynucleotide may encode an RNA (mRNA) that is translated into protein, or a DNA polynucleotide may encode an RNA that is not translated into protein (e.g., tRNA, rRNA, or a guide RNA; also called "non-coding" RNA or "ncRNA"). A protein coding sequence or a sequence that encodes a particular protein or polypeptide, is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.

[0053] As used herein, "codon" refers to a sequence of three nucleotides that together form a unit of genetic code in a DNA or RNA molecule. As used herein the term "codon degeneracy" refers to the nature in the genetic code permitting var iation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Nucleotides are typically referred to as the following: A (adenine), G (guanine), C (cytosine), T (thymine), U (uracil), N (= A or C or G or T / U), B (= C or G or T / U), D (= A or G or T / U), H (= A or C or T / U), K (= G or T / U), M (= A or C), R (= A or G), S (= C or G), V(= A or C or G), W (= A or T / U), (= C or T / U). When describing the RNA herein, it will be appreciated by the ordinarily skilled artisan that in the course of transcription, a DNA sequence will be transcribed into an mRNA sequence having the same nucleotide sequence but for thymine, which will be encoded as uracil in the RNA. Accordingly, the ordinarily skilled artisan will readily be able to convert the DNA sequences disclosed herein or known in the art to mRNA sequences.

[0054] The term "codon-optimized" or "codon optimization" refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that ar e more frequently used in the genes of that organism. Codon usage tables are readily available, for example, at the "Codon Usage Database" available at www.kazusa.or.jp / codon / (visited Mar. 20, 2008). By utilizing the knowledge on codon usage or codon preference in each organism, one of ordinary skill in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide, but which uses codons optimal for a given species. Codon- optimized coding regions can be designed by various methods known to those skilled in the art.

[0055] The term "recombinant" or "engineered" when used with reference, for example, to a cell, a nucleic acid, a protein, or a vector, indicates that the cell, nucleic acid, protein or vector has been modified by or is the result of laboratory methods. Thus, for example, recombinant or engineered proteins include proteins produced by laboratory methods. Recombinant or engineered proteins can include amino acid residues not found within the native (non-recombinant or wild-type) form of the protein or can include amino acid residues that have been modified, e.g., labeled. The term can include any modifications to the peptide, protein, or nucleic acid sequence. Such modifications may include the following: any chemical modifications of the peptide, protein or nucleic acid sequence, including of one or more amino acids, deoxyribonucleotides, or ribonucleotides; addition, deletion, and / or substitution of one or more of amino acids in the peptide or protein; and addition, deletion, and / or substitution of one or more of nucleic acids in the nucleic acid sequence.

[0056] The term "genomic DNA" or "genomic sequence" refers to the DNA of a genome of an organism including, but not limited to, the DNA of the genome of a bacterium, fungus, archea, plant or animal.

[0057] As used herein, "transgene," "exogenous gene" or "exogenous sequence," in the context of nucleic acid, refers to a nucleic acid sequence or gene that was not present in the genome of a cell but artificially introduced into the genome, e.g., via genome-edition.

[0058] As used herein, "endogenous gene" or "endogenous sequence," in the context of nucleic acid, refers to a nucleic acid sequence or gene that is naturally present in the genome of a cell, without being introduced via any artificial means.

[0059] The term "expression cassette" refers to a DNA coding sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. The terms "recombinant expression vector," or "DNA construct" are used interchangeably herein to refer to a DNA molecule having a vector and at least one insert. Recombinant expression vectors are usually generated for the purpose of expressing and / or propagating the insert(s), or for the construction of other recombinant nucleotide sequences. The nucleic acid(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences. The term "operably linked" means that the nucleotide sequence of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence. The term "regulatory sequence" is intended to include, for example, promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the target cell, the level of expression desired, and the like.

[0060] A cell has been "genetically modified" or "transformed" or "transfected" by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. The genetically modified (or transformed or transfected) cells that have therapeutic activity, e.g., treating hemophilia A, can be used and referred to as therapeutic cells.

[0061] The term "concentration" used in the context of a molecule such as peptide fragment refers to an amount of molecule, e.g., the number of moles of the molecule, present in a given volume of solution.

[0062] The terms "individual," "subject" and "host" are used interchangeably herein and refer to any subject for whom diagnosis, treatment or therapy is desired. In some aspects, the subject is a mammal. In some aspects, the subject is a human being. In some aspects, the subject is a patient. In some aspects, the subject is a human patient. In some aspects, the subject can have or is suspected of having a disorder or health condition associated with a gene-of-interest (GOI). In some aspects, the subject is a human who is diagnosed with a risk of disorder or health condition associated with a GOI at the time of diagnosis or later. In some cases, the diagnosis with a risk of disorder or health condition associated with a GOI can be determined based on the presence of one or more mutations in the endogenous GOI or genomic sequence near the GOI in the genome that may affect the expression of GOI.

[0063] The term "treatment" referring to a disease or condition means that at least an attenuation (amelioration) of the symptoms associated with the condition afflicting an individual is achieved, where attenuation is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., a symptom, associated with the condition (e.g., hemophilia) being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or eliminated entirely such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. Thus, treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., reducing the rate of progression of disease; (iii) mitigating, that is, reducing the symptoms of, or completely inhibiting, an active disease.

[0064] The terms "effective amount," "pharmaceutically effective amount," or "therapeutically effective amount" as used herein mean a sufficient amount of the composition to provide the desired utility when administered to a subject having a particular condition. The term "therapeutically effective amount" therefore refers to an amount of therapeutic cells or a composition having therapeutic cells that is sufficient to promote a particular effect when administered to a subject in need of treatment. An effective amount would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using routine experimentation .

[0065] The term "pharmaceutically acceptable excipient" as used herein refers to any suitable substance that provides a pharmaceutically acceptable earlier, additive or diluent for administration of a compound(s) of interest to a subject. "Pharmaceutically acceptable excipient" can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.

[0066] DETAILED DESCRIPTION

[0067] Methods and compositions for enhancing nuclear DNA delivery are provided. Aspects of the methods include contacting a cell with a DNA to be delivered to the nucleus of the cell and an agent that modulates the activity of the protein Barrier-to-Autointegration Factor (BAF) in a cell. In addition, compositions, including reagents, devices and kits thereof, that find use in practicing embodiments of the subject methods are provided.

[0068] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0069] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0070] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near' or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0071] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0072] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0073] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0074] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.

[0075] METHODS

[0076] As summarized above, methods for enhancing nuclear DNA delivery are provided. Aspects of the methods include contacting a cell with a DNA to be delivered and an agent that modulates the activity of the protein Barrier-to- Autointegration Factor (BAF) in a cell. By "enhancing" nuclear DNA delivery is meant that embodiments of the invention provide for increased DNA delivery to the nucleus, e.g., compared to a suitable control. The magnitude of such increase may vary, where in some instances the magnitude of enhancement is 2-fold or greater, such as 5-fold or greater, including 10-fold or greater, 25- fold or greater, 50-fold or greater, 100-fold greater, or 1000-fold greater. Embodiments of the methods provide for transportation of a DNA, which may be a cargo nucleic acid, from the cytosol of a cell into the nucleus of cell. As such, embodiments of methods of invention may be characterized as methods of facilitating or promoting transport of a DNA into the nucleus of a cell. Where the DNA includes a coding sequence, practicing embodiments of the methods of the invention may result in enhancement of expression of the coding sequence of the DNA. By "enhancing" expression of the coding sequence is meant expression of the coding sequence of the DNA is increased, e.g., compared to a suitable control. The magnitude of such increase may vary, where in some instances the magnitude of enhancement is 2- fold or greater, such as 5-fold or greater, including 10-fold or greater, 25-fold or greater, 50-fold or greater, or 100-fold or greater. Embodiments of methods of the invention may provide for an improvement, or an enhancement, in delivery and / or expression of DNA to the nucleus over DNA delivery methods that depend on the delivery of DNA that is not provided in conjunction with an agent that modulates BAF. In some instances, the improvement is a 2-fold increase or more in the amount of DNA in the nucleus relative to the amount of DNA that would be found in the nucleus if that DNA was not provided in conjunction with an agent that modulates BAF, for example, a 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, or 50-fold increase in the amount of DNA in the nucleus. This may be detected as an increase in the expression of a coding sequence of the DNA, for example, as a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 15-fold, 20-fold, 30- fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold or 1000-fold increase or more in RNA or protein encoded by the DNA over that expressed in a cell that has not been contacted with DNA or a cell that has been contacted with a DNA not in conjunction with the agent that modulates BAF .

[0077] Various aspects of the methods are now reviewed in greater detail.

[0078] Modulation of the protein Barrier to Autointegration Factor (BAF)

[0079] As reviewed above, aspects of embodiments of the invention include contacting the cell with a DNA to be delivered and an agent that modulates the activity of the protein Barrier to Autointegration Factor (BAF). Barrier- to- Autointegration Factor (BAF, also referred to as BAF nuclear assembly factor, or BANF, encoded by the BANF1 gene, the human ortholog having the protein sequence and the mouse ortholog having the protein sequence is known in the art to be a conserved nuclear envelope (NE) component that binds chromatin in a 3d-structure dependent, sequence-independent fashion and helps it to anchor to the nuclear envelope. During mitosis, phosphorylation by vaccinia-related kinase 1 (VRK1 ) or 2 (VRK2) causes BAF to release chromosomal DNA, allowing the nuclear envelope to dissemble. BAF dephosphorylation by protein phosphatase 2 A (PP2A) at the end of mitosis allows BAF to recapture the DNA and, working in concert with LEM-domain containing proteins, mediate its packaging within a new nuclear envelope. BAF has also been observed in the art to recognize and bind exogenous DNA entering the cell and nucleate the formation of a nuclear envelope-like structure called an exclusome around the DNA. For the sake of clarity, “BAF” does not refer to the “BRG1 - or BRM-associated factor” chromatin remodeling complex, also known in the art as the mammalian SWI / SNF complex.

[0080] By an agent that modulates BAF (referred to herein as a “BAF modulator”), it is meant an agent that suppresses, reduces, attenuates, inhibits, promotes, agonizes, supplements or otherwise modifies one or more activities of BAF in a manner that provides for a desired result, e.g., an enhancement of nuclear delivery and / or expression of a DNA, such as described above. In some embodiments, BAF modulator agents are agents that reduce the affinity of BAF for DNA. In some embodiments, BAF modulator agents are agents that reduce the ability of BAF to dimerize. In some embodiments, BAF modulator agents are agents that reduce the affinity of BAF for LEM domain-containing proteins such as LAP2, Emcrin, and MANI. In some embodiments, BAF modulator agents are agents that compete with endogenous BAF to inhibit functions of endogenous BAF. In some embodiments, BAF modulator agents are agents that interact with a BAF-interacting protein to modulate the interaction between BAF and that BAF- interacting protein, thereby altering the activity of BAF on DNA, e.g. as it relates to BAF’s ability to condense DNA, localize DNA to a region of the cell, or otherwise modify the ability of DNA to be re- localized to the nucleus of a cell. In some embodiments, BAF modulator agents are agents that interact directly with a DNA, so as to modify the ability of DNA to be re-localized to the nucleus of a cell, e.g. by altering the condensation of DNA, the association of DNA with other proteins, the subcellular localization of the DNA to a region of the cell, and the like.

[0081] In some instances, the BAF modulator is a protein or a nucleic acid, e.g. a DNA or an mRNA, encoding a protein. In some such instances, the BAF modulator is a wild type BAF protein or a nucleic acid, e.g., a DNA or an mRNA, encoding a wild type BAF protein, e.g. a human BAF protein having the protein sequence or the mouse ortholog having the protein sequence Thus, for example, the BAF modulator may be a DNA encoding a polypeptide comprising the human BAF protein, e.g. a deoxyribopolynucleotide having a sequence identity of 65% or more to the sequence or to the sequence: or an mRNA encoding a polypeptide comprising human BAF protein sequence, e.g. a ribopolynucleotide having a sequence identity of 65% or more to the sequence:

[0082] As another example, the BAF modulator may be a DNA encoding a polypeptide comprising the mouse BAF protein, e.g. a deoxyribopolynucleotide having a sequence identity of 65% or more to the sequence; or to the sequence: polypeptide comprising mouse BAF protein sequence, e.g. a ribopolynucleotide having a sequence identity of 65% or more to the sequence:

[0083] In other instances, the BAF modulator is a BAF mutant protein or a nucleic acid, e.g., a DNA or an mRNA, encoding a BAF mutant protein. BAF mutant proteins finding use in embodiments of the invention may vary, where examples of BAF mutant proteins include proteins comprising an amino acid sequence having a sequence identity of 75% or more, such as 80% or more, including 90% or more, e.g., 95% or more, to a wild type BAF. In some instances, the BAF mutant comprises a mutation, e.g. a substitution or deletion, relative to wild type BAF that mimics hyperphosphorylation of BAF, that reduces the ability of BAF to be dephosphorylated, that reduces the ability of BAF to bind DNA and / or that reduces the ability of BAF to interact with LEM-domain containing proteins. In some embodiments, the mutation includes a substitution at a residue relative to the wild type human BAF protein sequence selected from the group consisting of the threonine at residue 2, the threonine at residue 3, the serine at residue 4, the alanine at residue 12, the glutamic acid at residue 28, the glycine at residue 31, the lysine at residue 33, the glutamic acid at residue 36, the arginine at residue 37, the leucine at residue 58, the alanine at reside 71, or the glycine at residue 79of the wild type human BAF protein or a corresponding residue in a BAF ortholog. For example, the mutation may include a heterologous amino acid (relative to wild type human BAF) selected from the group consisting of an aspartic acid at residue 2, an aspartic acid at residue 3, an aspartic acid at residue 4, a threonine at residue 12, an aspartic acid at residue 28, a serine at reside 31, an arginine at residue 33, an aspartic acid at residue 36, a lysine at residue 37, an arginine at residue 58, a lysine at reside 71, and an arginine at residue 79; or in a corresponding residue in a BAF ortholog, for example, T2D, T3D, S4D, A12T, E28D, G31S, K33R, E36D, R37K, L58R, A71K, G79R, and the like. In other embodiments, the BAF modulator is a barrier to autointegration factor-like (BAFL) protein, also referred to as BAF2 or BANF2, or a nucleic acid, e.g., a DNA or an mRNA, encoding a BAFL protein, including proteins comprising an amino acid sequence having a sequence identity of 75% or more, such as 80% or more, including 90% or more, e.g., 95% or more, to wild type BAFL In yet other embodiments, the BAF modulator is a kinase that targets BAF, i.e. a kinase that phosphorylates BAF or nucleic acid, e.g., a DNA or an mRNA, encoding a kinase that phosphorylates BAF, referred to herein as a “BAF kinase” or a “B AF-targeting kinase”. Examples of BAF kinases finding use in embodiments of the invention include any polypeptide that phosphorylates BAF, including, but not limited to, Bl proteins of Poxviridae, and mutants thereof having sequence identity of 75% or more, such as 80% or more, including 90% or more, e.g., 95% or more, 96% or more, 97% or more to a B 1 ortholog that retain kinase activity on BAF, and polypeptides comprising an amino acid sequence having a sequence identity of 75% or more, such as 80% or more, including 90% or more, e.g., 95% or more, 96% or more, or 97% or more to a Bl ortholog. Bl proteins of Poxviridae are serine / threonine kinases that phosphorylate BAF at the N terminus, thereby inhibiting BAF binding to DNA. Bl orthologs are highly conserved within the members of the Poxviridae family that infect mammals, and include but are not limited to the Bl protein from vaccinia virus Ankara strain (GenBank Accession No. 057252): as encoded by, for example, an mRNA having the sequence:

[0084] The Bl protein from vaccinia virus Western Reserve strain (GenBank Accession No. YP_233065): as encoded by, for example, an mRNA having the sequence:

[0085] The horsepox virus OPG187 protein (GenBank Accession No. YP_010509385):

[0086] The cowpox CPXV196 protein (GenBank Accession No. ADZ29301): the variola virus protein (GenBank Accession No. ABF22933): and the Monkeypox OPG187 protein (GenBank Accession No. UZV11619.1):

[0087] Examples of B AF kinases finding use in embodiments of the invention also include, but are not limited to, mammalian VRK proteins, as well as mutants thereof that retain kinase activity on BAF. VRK proteins are a group of eukaryotic serine / threonine kinases having homology (~40% identity) to the Poxviridae Bl-like proteins, including, e.g. the vaccinia-related kinase 1 (VRK1) protein (GenBank Accession no. NM_003384.3) having a sequence: the vaccinia-related kinase 1-X1 (VRK1-X1) protein (GenBank Accession no. XM_047431752), having a sequence: the vaccinia-related kinase 2A (VRK2A) protein (GenBank Accession no. NM_006296.7) having a sequence: the vaccinia-related kinase 2B (VRK2B) protein, having a sequence:

[0088] In some embodiments, the VRK protein is a mutant that does not comprise a functional NLS, e.g. the sequence of VRK1 , for example a deletion of the NLS or a substitution in the NLS that renders it inactive. In some embodiments, the VRK protein is a mutant comprising a modification that reduces the ability of an modulator to inhibit the kinase, e.g. the binding of an modulator of the kinase, for example, the binding of RAN-GDP. Kinase-active mutants of B 1 proteins and their orthofogs, including the VRKL VRK2, and VRK3 proteins, can be readily determined by one of ordinary skill in the art by using well developed methods for detecting the presence of a phosphate group on a target protein to assess the ability of the subject kinase to phosphorylate BAF protein.

[0089] In some embodiments, the BAF modulator comprises a LEM domain or a nucleic acid, e.g., a DNA or an mRNA, encoding a LEM domain. As is known in the art, a LEM domain is a ~40-residue helix-loop-helix fold structure that directly binds to BAF. While the LEM domain may vary, LEM domains of interest include, but are not limited to: an Emerin LEM domain ; a LAP2-beta LEM domain: ; a MANI LEM domain and the like. In other embodiments, the BAF modulator does not comprise a LEM domain or a nucleic acid, e.g., a DNA or an mRNA, encoding a LEM domain. In some such embodiments, the BAF modulator does not comprise a LEM-domain containing protein 2 (LEMD2) polypeptide. In some such embodiments, the BAF modulator docs not comprise a LEM-domain containing protein 3 (LEMD3) polypeptide. In some embodiments, the protein agent that is the BAF modulator, e.g. as described herein, comprises a domain that reduces the half-life of the protein, e.g. a degradation domain. Examples of degradation domains include, but are not limited to

[0090] In some instances, it is advantageous to engineer into the BAF modulator the functionalities of a nuclear targeting factor (NTF) as known in the art and as described herein. Thus, while in some embodiments, the protein agent that is the BAF modulator, e.g. as described herein, does not comprise a heterologous nuclear localization sequence (NLS) domain, in other instances, the protein agent that is the BAF modulator is engineered to comprise an NLS domain. In some instances, the NLS is engineered to be within the center of the protein, i.e. the NLS is flanked on both N and C termini by 20 or more amino acids endogenous to the BAF modulator. In some instances, the NLS is engineered to be at the N terminus. In some instances,, the NLS is engineered to be at the C terminus. In some instances, the NLS is engineered to be at both the N terminus and the C terminus. When engineered to reside at a terminus, the NLS may be engineered to be within 0-20 amino acids of the terminus, in some instances, within 0-10 amino acids of the terminus, in certain instances within 0-5 amino acids of the terminus, in some such cases, at the terminus. In some instances, the NLS may be flanked by one or more amino acids, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some instances, the BAF modulator will comprise one NLS, in other instances multiple NLSs. In some instances, in which multiple NLSs are employed, the same NLS is employed multiple times. In other instances, in which multiple NLSs are employed, different NLSs are used. In embodiments, when multiple NLSs are employed, they will be separated by a linker, e.g., a 6xK linker. Exemplary NLSs and their origins are provided in Table 5. Surprisingly, it has been observed that unlike other nuclear translocating DNA systems as known in the art and described herein, a BAF protein variant that is engineered to comprise an NLS does not require a specific nucleotide sequence, i.e. a DTS, on the DNA that it is translocating in order to be able to exert its additional function as an NTF. Thus, in such instance, the DNA does not comprise a BAF-modulator-specific DTS.

[0091] In some embodiments, the BAF modulator is a nucleic acid. BAF modulator nucleic acids may include DNA or RNA molecules, for example a DNA or RNA molecule encoding a protein that modulates BAF as described herein, some certain embodiments of which are described in Table 10 herein. In certain embodiments, the nucleic acids modulate, e.g., inhibit or reduce, the activity of a gene or protein, e.., by reducing or downregulating the expression of the gene. The nucleic acid may be single stranded or double-stranded and may include modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof. In some cases, the BAF modulator includes intracellular' gene silencing molecules by way of RNA splicing and molecules that provide an antisense oligonucleotide effect or an RNA interference (RNAi) effect useful for inhibiting gene function. In some cases, gene silencing molecules, such as, e.g., antisense RNA, short temporary RNA (stRNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), tiny non- coding RNA (tncRNA), snRNA, snoRNA, and other RNAi-like small RNA constructs, may be used to target a protein-coding as well as non-protein-coding genes. In some cases, the nucleic acids include aptamers (e.g., spiegelmers). In some cases, the nucleic acids include antisense compounds. In some cases, the nucleic acids include molecules which may be utilized in RNA interference (RNAi) such as double stranded RNA including small interfering RNA (siRNA), locked nucleic acid (LNA) modulators, peptide nucleic acid (PNA) modulators, etc. In some cases, the nucleic acids include aptamers.

[0092] In some instances, the BAF modulator is a small molecule agent that exhibits the desired activity, e.g., inhibiting BAF expression and / or activity. Naturally occurring or synthetic small molecule compounds of interest include numerous chemical classes, such as organic molecules, e.g., small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate agents comprise functional groups for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents may include cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Such molecules may be identified, among other ways, by employing screening protocols. Small molecule BAF modulators include, but are not limited to, obtusilactone B, Mahubanolide, kotomolide B, epilitsenolide D2, rabeprazole, and the like.

[0093] Deoxyribonucleic Acids (DNAs)

[0094] As reviewed above, embodiments of the methods include contacting a cell with a DNA. DNAs are double stranded deoxyribonucleic acids that may vary in length, ranging in some instances from 15nt to 15,000 nt, such as 100 to 10,000 nt and including 100 to 5000nt. Where the DNA is double-stranded, the DNA may vary in length, ranging in some instances from 15 to 15,000 bp, such as 100 to 15,000 bp, e.g., 100 to 10,000 bp, including 100 to 5000 bp. The DNA may vary as desired. In some instances, the DNA is 500 nt or more, for example 1 kb, 2 kb, 3 kb, 4 kb, or 5 kb or more, e.g., 6 kb, 7 kb, 8 kb, 9 kb, 10 kb or more, in some cases, 15 kb or more. The DNA may have any desired sequence. In some instances, the DNA may include one or more of coding sequences, promoters, sequences homologous to the genomic DNA of the targeted nucleus (e.g., to provide for genomic integration of the cargo nucleic acid), untranslated sequences (5' UTR, 3’ UTR), poly adenylation sequences, and the like.

[0095] A given DNA to be delivered to the nucleus may be configured to be maintained episomally or integrated into the genome, as desired. As such, in some instances a DNA is configured to be maintained episomally in the nucleus of a target cell, such that it is not genomically integrated. In other instances, the DNA may be configured to be integrated into the genome of a target cell. In such instances, integration may be accomplished using any convenient protocol, such as by using a gene editing system, e.g., as described in greater detail below.

[0096] In some instances, the DNA includes a coding sequence. By a coding sequence it is meant a nucleic acid sequence that encodes for any gene product, e.g., micro RNA (miRNA), small hairpin RNA (shRNA), circular RNA (circRNA), long noncoding RNA (IncRNA), mRNA, peptide, polypeptide, protein. Where desired, a given coding sequence can encode multiple gene products, e.g., separated by IRES sequence, 2A sequence, or the like. For example, a coding sequence to be delivered to the nucleus by a DNA can be configured so that it can be integrated into the genome in operable linkage with its native promoter, for example to replace a mutant coding sequence or to be in operable linkage with an active promoter at a safe harbor (in which instances, the cargo nucleic acid need not include, and in some instances does not include, a promoter).

[0097] In some instances, a DNA may include a promoter. As used herein, the term "promoter" refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving transcription of the nucleic acid sequence, which can be a heterologous target gene encoding a protein or an RNA. Promoters can be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter can also contain genetic elements at which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Various promoters, including inducible promoters, may be used to drive the expression of transgenes. A promoter sequence may be bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Promoters useful in DNAs of embodiments of the invention include, for example constitutively active promoters, such as the CMV promoter, CAG promoter (which combines the CMV enhancer with the chicken 0-actin (CBA) promoter), 0-actin promoter, SV-40 promoter, 4xGRM6-SV40, hTTR, hAAT, 3x- Serpina, ubiquitin B / C, EFl -Alpha, EFS, and HBV promoter, etc. Promoters useful in DNAs of embodiments of the invention also include promoters having more cell-type specific expression patterns, for example for hepatocytes, may include, without limitation the TTR, hAAT (and derivatives), 3xSerpina-TTR, HBV, UbiC, and P3-hybrid promoter. A cargo nucleic acid may include a promoter sequence to be delivered to the nucleus so that it can be integrated into the genome, for example to replace a mutant promoter.

[0098] In some instances, the DNA includes an expression cassette. By an expression cassette it is meant a nucleic acid sequence comprising a promoter, e.g., as described above, operably linked to a coding sequence, e.g., as described above (also referred to herein as a transgene). In some instances, the expression cassette may also comprise one or more nucleic acid sequences including a 5’ untranslated region (5’ UTR), 3’ untranslated region (3’ UTR), polyA tail, miRNA regulatory element. In embodiments, the expression cassette may include a transgene and one or more regulatory sequences that allows and / or controls the expression of the transgene, e.g., where the expression cassette can include one or more of, e.g., in this order: an enhancer / promoter, an ORF reporter (transgene), a post-transcription regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyA). The expression cassette can also comprise an internal ribosome entry site (IRES) and / or a 2A element. The cis-regulatory elements include, but are not limited to, a promoter, a riboswitch, an insulator, a mir- regulatable element, a post-transcriptional regulatory element, a tissue- and cell type-specific promoter and an enhancer. As desired, the expression cassette can comprise 4000 or more nucleotides, 5000 or more nucleotides, 10,000 or more nucleotides or 20,000 or more nucleotides, or 30,000 or more nucleotides, or 40,000 or more nucleotides or 50,000 or more nucleotides, and in some instances may range between 4000-10,000 nucleotides or 10,000-50,000 nucleotides, or more than 50,000 nucleotides. The expression cassette to be delivered to the nucleus may be configured so that it can be maintained episomally. Alternatively, an expression cassette to be delivered to the nucleus may be configured so that it can be integrated into the genome.

[0099] The coding sequence e.g., transgene, of the expression cassette may vary. In some embodiments, the expression cassette can include a transgene in the range of 500 to 50,000 nucleotides in length. In some embodiments, the expression cassette can include a transgene in the range of 500 to 75,000 nucleotides in length. In some embodiments, the expression cassette can include a transgene which is in the range of 500 to 10,000 nucleotides in length. In some embodiments, the expression cassette can include a transgcnc which is in the range of 1000 to 10,000 nucleotides in length. In some embodiments, the expression cassette can include a transgene which is in the range of 500 to 5,000 nucleotides in length. The DNA constructs of embodiments of the invention do not have the size limitations of encapsidated AAV vectors, and thus enable nuclear delivery of large-size expression cassettes to provide efficient transgene.

[0100] A given expression cassette can include, for example, an expressible exogenous sequence (e.g., open reading frame) or transgene that encodes a protein that is either absent, inactive, or insufficient activity in the recipient subject or a gene that encodes a protein having a desired biological or a therapeutic effect. The transgene can encode a gene product that can function to correct the expression of a defective gene or transcript. In principle, the expression cassette can include any gene that encodes a protein, polypeptide or RNA that is either reduced or absent due to a mutation or which conveys a therapeutic benefit when overexpressed is considered to be within the scope of the disclosure. The expression cassette can include any transgene useful for treating a disease or disorder in a subject.

[0101] DNAs, e.g., as described herein, can be used to deliver and express any gene of interest in a subject, where such genes include, but are not not limited to, nucleic acids encoding polypeptides, or non- coding nucleic acids (e.g., RNAi, miRs etc.), as well as exogenous genes and nucleotide sequences, including virus sequences in a subjects' genome, e.g., HIV virus sequences and the like. In some instances, a DNA (e.g., as disclosed herein) is used for therapeutic purposes (e.g., for medical, diagnostic, or veterinary uses) or immunogenic polypeptides. In certain embodiments, a DNA is useful to express any gene of interest in the subject, which includes one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, or RNAs (coding or non-coding; e.g., siRNAs, shRNAs, micro-RNAs, and their antisense counterparts (e.g., antagoMiR)), antibodies, antigen binding fragments, or any combination thereof. As such, expression cassettes can encode polypeptides, sense or antisense oligonucleotides, or RNAs (coding or non-coding; e.g., siRNAs, shRNAs, micro-RNAs, and their antisense counterparts (e.g., antagoMiR)). Expression cassettes can include an exogenous sequence that encodes a reporter protein to be used for experimental or diagnostic purposes, such as P-lactamase, P-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, EPO, and others well known in the art.

[0102] Sequences provided in a given expression cassette, expression construct of a DNA, e.g., as described herein, can be codon optimized for the target host cell. As used herein, the term "codon optimized" or "codon optimization" refers to the process of modifying a nucleic acid sequence for enhanced expression in the cells of the vertebrate of interest, e.g., mouse or human, by replacing at least one, more than one, or a significant number of codons of the native sequence (e.g., a prokaryotic sequence) with codons that arc more frequently or most frequently used in the genes of that vertebrate. Various species exhibit par ticular bias for certain codons of a particular amino acid. Typically, codon optimization does not alter the amino acid sequence of the original translated protein. In some embodiments, a transgene expressed by the DNA is a therapeutic gene. In some embodiments, a therapeutic gene is an antibody, or antibody fragment, or antigen-binding fragment thereof, e.g., a neutralizing antibody or antibody fragment and the like. In some instances, a therapeutic gene is one or more therapeutic agent(s), including, but not limited to, for example, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen binding fragments, as well as variants, and / or active fragments thereof, for use in the treatment, prophylaxis, and / or amelioration of one or more symptoms of a disease, dysfunction, injury, and / or disorder. A coding sequence to be delivered to the nucleus so that it can be integrated into the genome in operable linkage with its native promoter, for example to replace a mutant coding sequence or to be in operable linkage with an active promoter at a safe harbor (in which instances, the polynucleotide does not comprise a promoter); or the coding sequence of an expression cassette to be delivered to the nucleus to be either maintained episomally or integrated into the genome. By a coding sequence it is meant a nucleic acid sequence that encodes for any gene product, e.g., micro RNA (miRNA), small hairpin RNA (shRNA), circular’ RNA (circRNA), long noncoding RNA (IncRNA), mRNA, peptide, polypeptide, protein. Can encode multiple gene products, e.g., separated by IRES sequence, 2A sequence, or the like.

[0103] Where desired, a given DNA may include flanking sequences that are homologous to genomic regions of the cell, e.g., to promote genomic integration of the cargo nucleic acid. When present, such sequences may vary in length, ranging in some instances from 30 to 5,000 nt, such as 50 to 1000 nt. While the sequences of such regions may vary depending on the genomic integration location of interest, examples of such sequences include, but are not limited to: actin, ADA, albumin, a-globin, β-globin, CD2, CD3, CD5, CD7, CCR5, Ela, IL2RG, Insl, Ins2, NCF1, p50, p65, PF4, PGC-γ, PTEN, TERT, TRAC, UBC, and VWF, and the like.

[0104] In embodiments where the DNA is to be integrated into the genome of a target cell in process mediated by a gene editing system, e.g., as described below, the DNA may be serve as a donor DNA or donor template in such a system. Site-directed polypeptides, such as a DNA endonuclease, can introduce double-strand breaks or single-strand breaks in nucleic acids, e.g., genomic DNA. The double-strand break can stimulate a cell's endogenous DNA-repair pathways (e.g., homology-dependent repair (HDR) or non-homologous end joining or alternative non-homologous end joining (A-NHEJ) or microhomology- mediated end joining (MMEJ). NHEJ can repair cleaved target nucleic acid without the need for a homologous template. This can sometimes result in small deletions or insertions (indels) in the target nucleic acid at the site of cleavage, and can lead to disruption or alteration of gene expression. HDR, which is also known as homologous recombination (HR) can occur when a homologous repair template, or donor, is available. The homologous donor template has sequences that are homologous to sequences flanking the target nucleic acid cleavage site. The sister chromatid is generally used by the cell as the repair template. However, for the purposes of genome editing, the repair template is often supplied as an exogenous nucleic acid, such as a plasmid, duplex oligonucleotide, single-strand oligonucleotide, double-stranded oligonucleotide, or viral nucleic acid. With exogenous donor templates, it is common to introduce an additional nucleic acid sequence (such as a transgene) or modification (such as a single or multiple base change or a deletion) between the flanking regions of homology so that the additional or altered nucleic acid sequence also becomes incorporated into the target locus. MMEJ results in a genetic outcome that is similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ makes use of homologous sequences of a few base pairs flanking the cleavage site to drive a favored end-joining DNA repair outcome. In some instances, it can be possible to predict likely repair outcomes based on analysis of potential microhomologies in the nuclease target regions.

[0105] Thus, in some cases, homologous recombination is used to insert an exogenous polynucleotide sequence into the target nucleic acid cleavage site. An exogenous polynucleotide sequence is termed a donor polynucleotide (or donor DNA or donor sequence or polynucleotide donor template) herein, and may in embodiments of the present invention be a DNA or component thereof, e.g., cargo nucleic acid component of a DNA. In some embodiments, the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide is inserted into the target nucleic acid cleavage site. In some embodiments, the donor polynucleotide is an exogenous polynucleotide sequence, i.e., a sequence that does not naturally occur at the target nucleic acid cleavage site.

[0106] When an exogenous DNA molecule is supplied in sufficient concentr ation inside the nucleus of a cell in which the double strand break occurs, the exogenous DNA can be inserted at the double strand break during the NHEJ repair process and thus become a permanent addition to the genome. These exogenous DNA molecules are referred to as donor templates in some embodiments. If the donor template contains a coding sequence for a gene-of-interest optionally together with relevant regulatory sequences such as promoters, enhancers, polyA sequences and / or splice acceptor sequences (also referred to herein as a "donor cassette"), the gene of interest can be expressed from the integrated copy in the genome resulting in permanent expression for the life of the cell. Moreover, the integrated copy of the donor DNA template can be transmitted to the daughter cells when the cell divides.

[0107] In the presence of sufficient concentrations of a donor DNA template that contains flanking DNA sequences with homology to the DNA sequence either side of the double strand break (referred to as homology arms), the donor DNA template can be integrated via the HDR pathway. The homology arms act as substrates for homologous recombination between the donor template and the sequences either side of the double strand break. This can result in an error free insertion of the donor template in which the sequences either side of the double strand break are not altered from that in the un-modified genome.

[0108] Supplied donors for editing by HDR vary markedly but generally contain the intended sequence with small or large flanking homology arms to allow annealing to the genomic DNA. The homology regions flanking the introduced genetic changes can be 30 bp or smaller, or as large as a multi-kilobase cassette that can contain promoters, cDNAs, etc. Both single-stranded and double-stranded oligonucleotide donors can be used. These oligonucleotides range in size from less than 100 nt to over many kb, though longer ssDNA can also be generated and used. Double-stranded donors are often used, including PCR amplicons, plasmids, and mini-circles.

[0109] In some embodiments, an exogenous sequence, e.g., cargo nucleic acid, that is intended to be inserted into a genome is a gene-of-interest (GOI) or functional derivative thereof. The exogenous gene can include a nucleotide sequence encoding a GOI product, e.g., GOI protein, or functional derivative thereof. The functional derivative of a GOI can include a nucleic acid sequence encoding a functional derivative of a GOI protein that has a substantial activity of a wildtype GOI protein such as. the wildtype human GOI protein, e.g., at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 100% of the activity that the wildtype GOI protein exhibits. In some embodiments, the functional derivative of a GOI protein can have at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% amino acid sequence identity to the GOI protein, e.g., the wildtype GOI protein. In some embodiments, one having ordinary skill in the art can use a number of methods known in the field to test the functionality or activity of a compound, e.g., peptide or protein. The functional derivative of the GOI protein can also include any fragment of the wildtype GOI protein or fragment of a modified GOI protein that has conservative modification on one or more of amino acid residues in the full length, wildtype GOI protein. Thus, in some embodiments, the functional derivative of a nucleic acid sequence of a GOI can have at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% nucleic acid sequence identity to the GOI, e g. the wildtype GOI.

[0110] In some embodiments where the insertion of a GOI or functional derivative thereof is concerned, a cDNA of GOI or functional derivative thereof can be inserted into a genome of a patient having defective GOI or its regulatory sequences. In such a case, a donor DNA or donor template can be an expression cassette or vector construct having the sequence encoding GOI or functional derivative thereof, e.g., cDNA sequence. In some embodiments, the expression vector contains a sequence encoding a modified GOI protein, which is described elsewhere in the disclosures, can be used. In some embodiments, according to any of the donor templates described herein comprising a donor cassette, the donor cassette is flanked on one or both sides by a gRNA target site. For example, such a donor template may comprise a donor cassette with a gRNA target site 5' of the donor cassette and / or a gRNA target site 3' of the donor cassette. In some embodiments, the donor template comprises a donor cassette with a gRNA target site 5' of the donor cassette. In some embodiments, the donor template comprises a donor cassette with a gRNA target site 3' of the donor cassette. In some embodiments, the donor template comprises a donor cassette with a gRNA target site 5' of the donor cassette and a gRNA target site 3' of the donor cassette. In some embodiments, the donor template comprises a donor cassette with a gRNA target site 5' of the donor cassette and a gRNA target site 3' of the donor cassette, and the two gRNA target sites comprise the same sequence. In some embodiments, the donor template comprises at least one gRNA target site, and the at least one gRNA target site in the donor template comprises the same sequence as a gRNA target site in a target locus into which the donor cassette of the donor template is to be integrated. In some embodiments, the donor template comprises at least one gRNA target site, and the at least one gRNA target site in the donor template comprises the reverse complement of a gRNA tar get site in a target locus into which the donor cassette of the donor template is to be integrated. In some embodiments, the donor template comprises at least one gRNA target site, and the at least one gRNA target site in the donor template does not comprise the same sequence as a gRNA target site in a target locus into which the donor cassette of the donor template is to be integrated. In some embodiments, the donor template comprises a donor cassette with a gRNA target site 5' of the donor cassette and a gRNA target site 3' of the donor cassette, and the two gRNA target sites in the donor template comprises the same sequence as a gRNA target site in a target locus into which the donor cassette of the donor template is to be integrated. In some embodiments, the donor template comprises a donor cassette with a gRNA target site 5' of the donor cassette and a gRNA target site 3' of the donor cassette, and the two gRNA target sites in the donor template comprises the reverse complement of a gRNA target site in a target locus into which the donor cassette of the donor template is to be integrated. In some embodiments, the donor template comprises a donor cassette with a gRNA target site 5' of the donor cassette and a gRNA target site 3' of the donor cassette, and the two gRNA target sites in the donor template do not comprise the same sequence as a gRNA target site in a target locus into which the donor cassette of the donor template is to be integrated.

[0111] A given DNA may or may not be configured to be maintained in a bacterium, as desired. For example, in some instances a DNA is configured to be maintained in a bacterium. In such instances, the DNA may include a bacterial origin of replication and a selection element, e.g., a plasmid or a nanoplasmid. In other instances, a DNA may be configured to not be maintained in a bacterium. In such instances, the structure of the DNA may vary, wherein examples of such structures include minicircle DNA (mcDNA), a linear DNA, a covalently closed DNA (doggybone DNA, or “dbDNA”, ministring DNA, etc.), double stranded linear DNA comprising terminal repeat sequences at both ends, a 3DNA structure, etc., and the like.

[0112] Nuclear Targeted Deoxyribonucleic Acids (NTDNAs) and Nuclear Targeting Factors

[0113] Nuclear Targeted Deoxyribonucleic Acids (NTDNAs). In some embodiments, the DNA, e.g., as described above, is a component of a Nuclear Targeted Deoxyribonucleic Acid (NTDNA). NTDNAs finding use in embodiments of the invention are described in PCT Application Serial No. PCT / US2023 / 035932 filed on October 25, 2023, the disclosure of which is herein incorporated by reference. NTDNAs employed in methods of the invention comprise both a DNA nuclear targeting sequence (DTS) and a DNA, e.g., as described above, which may be referred to as a "cargo nucleic acid sequence" in the context of a NTDNA. where the cargo nucleic acid is heterologous to the DTS. As such, the NTDNAs include a DTS domain and a cargo nucleic acid domain that is heterologous to the DTS. By "heterologous" to the DTS it is meant that the cargo nucleic acid is not naturally associated with the DTS, e.g., is not part of the same gene as the DTS in nature. NTDNAs employed in methods of the invention comprise both a DNA nuclear targeting sequence (DTS) and a cargo nucleic acid sequence that is heterologous to the DTS. As such, the NTDNAs include a DTS domain and a cargo nucleic acid domain. Each of these domains is now described further in greater detail.

[0114] A DTS refers to a nucleotide sequence that mediates the translocation of a polynucleotide that comprises it into the nucleus of a cell. Without wishing to be bound by theory, it is believed that DTSs leverage the movement of nuclear-acting DNA binding proteins as they move from the cytoplasm into the nucleus. These nuclear-acting DNA binding proteins act like nuclear targeting factors for DNA, binding to sequences on the DNA and dragging the DNA into the nucleus as the DNA binding protein translocates into the nucleus. Accordingly, the nuclear-acting DNA binding proteins that are leveraged are referred to herein as nuclear targeting factors (NTFs), and the DNA sequences to which they bind are referred to herein as nuclear targeting factor binding sites (NTFBSs, or more simply, TFBSs).

[0115] In some embodiments, the DTS comprises a TFBS that is bound by a nuclear targeting factor that is provided to the cell as a protein or an mRNA encoding a protein. As will be appreciated by one of ordinary skill in the art, any protein that binds to DNA and that traffics to the nucleus when delivered to the cytoplasm can be provided to the cell to mediate nuclear translocation. Thus, for example, any of the naturally occurring proteins described in Tables 1 and 2 may be exogenously provided. As another example, a protein that is not native to the cell, i.e., that is heterologous to the cell, may be provided. Such a protein may be naturally occurring or engineered. Examples include any of the proteins listed in Table 1. Exemplary proteins of each class and the DNA sequences to which they bind are well known in the art and include those described in greater detail below.

[0116] Table 1. Classes of nuclear targeting factors that may be co-delivered with NTDNA to achieve DNA nuclear transport.

[0117] In some embodiments, the TFBS is a binding sequence for a Tet Repressor (TetR) protein, for example, a TetO sequence such as for example, In some embodiments, the DTS comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the TetR TFBS. In certain embodiments, the DTS comprises 7 copies of the TetR TFBS. In certain embodiments, the DTS comprises 10 or more copies of the TetR TFBS. In some embodiments, the DTS comprises a sequence having 80% identity or more to a sequence listed in Table 2, for example, 85% or 90% identity or more, in some cases 95% identity or more, e.g., 96%, 97%, 98%, or 99% identity to a sequence in Table 2. In some instances, the DTS binding sequence is identical to a sequence in Table 2. In some instances, the DTS consists essential of a sequence in Table 2. In certain embodiments, the DTS comprises a tetracycline response element (TRE), as known in the art. In certain embodiments, the DTS consists essentially of a TRE.

[0118] Table 2. Examples of DTSs that comprise a TetR binding sequence (e.g., TetO) in varying numbers and sequences.

[0119] In some embodiments, the TFBS is a binding sequence for the DNA binding domain of a gene editing system, e.g., as described herein or as known in the art, e.g., the guide RNA of a Cas nuclease, the zinc finger domain of a zinc finger nuclease, the TALE DNA binding domain of a TALEN. In some embodiments, the TFBS is a binding sequence for a zinc-finger containing protein ("ZF protein”). As will be appreciated by the ordinarily skilled artisan, any ZF protein and its cognate ZF binding sequence may be used as a nuclear targeting factor (NTF) and cognate TFBS in the compositions and methods of the present disclosure. In some embodiments the DTS comprises a ZF-responsive TFBS having a sequence identity of 90% or more to In some embodiments, the TFBS is a binding sequence for a TAL effector DNA-binding domain- containing protein (“TALE protein”). A TALE is a protein of 32 fixed amino acids and 2 variable residues, the 2 residues being engineered to recognize specific nucleotides (e.g., NN for G, NI for A, HD for C, etc.). As will be appreciated by the ordinarily skilled artisan, any TALE protein and its cognate TALE binding sequence may be used in the compositions and methods of the present disclosure, such examples being found in, e.g., Li et al. 2011 (Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Research, Volume 39, Issue 14, pp 6315-6325) and Kim et. al. 2013 (A library of TAL effector nucleases spanning the human genome. Nature Biotechnology volume 31, pages 251-258 (2013). In some embodiments, the TALE TFBS has 90% identity or more to a sequence is selected from the group consisting of

[0120] In some embodiments, the TFBS is a binding sequence for a GAL4 protein. In some such embodiments, the GAL4 TFBS comprises the sequence CGG-Nn-CCG, for example, In some embodiments, the DTS comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the GAL4 TFBS. In certain embodiments, the DTS comprises 5 copies of the GAL4

[0121] TFBS. In certain embodiments, the DTS comprises 7 copies of the GAL4 TFBS. In certain embodiments, the DTS comprises 10 or more copies of the GAL4 TFBS. Tn certain embodiments, the DTS comprises an upstream activation sequence (UAS) for the native GAL4 protein, as known in the art. In certain embodiments, the DTS consists essentially of a UAS. In some embodiments, the DTS comprises a sequence having 80% identity or more to a sequence listed in Table 3, for example, 85% or 90% identity or more, in some cases 95% identity or more, e.g., 96%, 97%, 98%, or 99% identity to a sequence in Table 3. In some instances, the DTS is identical to a sequence in Table 3.

[0122] Table 3. Examples of DTSs comprising a GAL4 TFBS (e.g., UAS)

[0123] In some embodiments, the TFBS is a binding sequence for an Arc protein, where Arc is a bacteriophage regulatory protein. In some such embodiments, the Arc TFBS comprises the sequence for example, In some embodiments, the Arc TFBS comprises a sequence having 80%, 85%, 90% identity or more to In some embodiments, the DTS comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the Arc TFBS. In certain embodiments, the DTS comprises 5 copies of the Arc TFBS. In certain embodiments, the DTS comprises 7 copies of the Arc TFBS. In certain embodiments, the DTS comprises 10 or more copies of the Arc TFBS. In some embodiments, the DTS comprises a sequence having 80%, 85%, or 90% identity or more to the sequence , in some cases 95% identity or more to this sequence, in certain cases sharing 100% identity with this sequence. In some embodiments, the TFBS is a binding sequence for a Mnt protein, where Mnt is a bacteriophage regulatory protein. In some such embodiments, the Mnt TFBS comprises the sequence for example, In some embodiments, the Mnt TFBS comprises a sequence having 80%, 85%, 90% identity or more to In some embodiments, the DTS comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the Mnt TFBS. In certain embodiments, the DTS comprises 5 copies of the Mnt TFBS. In certain embodiments, the DTS comprises 7 copies of the Mnt TFBS. In certain embodiments, the DTS comprises 10 or more copies of the Mnt TFBS. In some embodiments, the DTS comprises a sequence having 80%, 85%, or 90% identity or more to the sequence ill some cases 95% identity or more to this sequence, in certain cases sharing 100% identity with this sequence.

[0124] In some embodiments, the TFBS is a binding sequence for a purine synthesis repressor (PurR) protein. In some such embodiments, the PurR TFBS comprises a sequence having 80%, 85%, 90% identity or more to , in some cases 95% identity or more to this sequence, in certain cases sharing 100% identity with this sequence. In some embodiments, the DTS comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the PurR TFBS. In certain embodiments, the DTS comprises 5 copies of the PurR TFBS. In certain embodiments, the DTS comprises 7 copies of the PurR TFBS. In certain embodiments, the DTS comprises 10 or more copies of the PurR TFBS. I In some embodiments, the DTS comprises a sequence having 80%, 85%, or 90% identity or more to the sequence in some cases 95% identity or more to this sequence, in certain cases sharing 100% identity with this sequence.

[0125] In some embodiments, the TFBS is a binding sequence for a Bac434 protein, where Bac434 is a bacteriophage regulatory protein. In some such embodiments, the Bac434 TFBS comprises a sequence that has 80%, 85%, 90% identity or more to in some cases 95% identity or more to , or in certain cases sharing 100% identity with , or In some embodiments, the DTS comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the Bac434 TFBS. In certain embodiments, the DTS comprises 5 copies of the Bac434 TFBS. In certain embodiments, the DTS comprises 7 copies of the Bac434 TFBS. In certain embodiments, the DTS comprises 10 or more copies of the Bac434 TFBS. In certain embodiments, the DTS comprises a sequence having 80%, 85%, or 90% identity or more to a sequence listed in Table 4, for example, 85% or 90% identity or more, in some cases 95% identity or more, e.g., 96%, 97%, 98%, or 99% identity to a sequence in Table 4. In some instances, the DTS is identical to a sequence in Table 4. Table 4. Examples of DTSs comprising a Bac434 TFBS

[0126] In some embodiments, the TFBS is a binding sequence for a GCN4 protein. In some such embodiments, the GCN4 TFBS comprises a sequence having 80%, 85%, 90% identity or more to TGACTC, in some cases 95% identity or more to TGACTC, in certain cases 100% identity with TGACTC, for example, AGTGACTCATT. In some embodiments, the DTS comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the GCN4 TFBS. In certain embodiments, the DTS comprises 5 copies of the GCN4 TFBS. In certain embodiments, the DTS comprises 7 copies of the GCN4 TFBS. In certain embodiments, the DTS comprises 10 or more copies of the GCN4 TFBS. In some embodiments, the DTS comprises a sequence having 80%, 85%, or 90% identity or more to the sequence , in some cases 95% identity or more to this sequence, in certain cases sharing 100% identity with this sequence.

[0127] In some embodiments, the TFBS is a binding sequence for a Lactose Modulator (LacI) protein, also referred to herein as a Lactose Repressor (LacR) protein, for example, a LacO sequence, e.g., In some such embodiments, the LacR TFBS comprises a sequence having 80%, 85%, 90% identity or more to In certain embodiments, the DTS comprises a lactose operon (LacO), as is known in the art. In certain embodiments, the DTS consists essentially of a LacO. In some embodiments, the DTS comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the LacR TFBS. In certain embodiments, the DTS comprises 5 copies of the LacR TFBS. In certain embodiments, the DTS comprises 7 copies of the LacR TFBS. In certain embodiments, the DTS comprises 10 or more copies of the LacR TFBS. In some embodiments, the DTS comprises a sequence that has 80%, 85%, or 90% identity or more to the sequence in some cases 95% identity or more to this sequence, in certain cases sharing 100% identity with this sequence.

[0128] In some embodiments, the TFBS is a binding sequence for an endonuclease, e.g., an I-Scel D44A protein. In some embodiments the DTS comprises a TFBS having 90% identity or more to the sequence

[0129] It will be appreciated by one of ordinary skill in the art that DNA binding proteins can tolerate some degree of nucleotide substitution in their binding sequences and that the TFBSs provided herein are but examples of sequences that may be used. In some embodiments, the TFBS shares 80% identity or more with a sequence disclosed herein, for example, 85%, 90%, 95% identity or more, e.g., 96%, 97%, 98%, or 99% identity or more, in certain instances 100% identity to the sequence. Publicly available databases such as Uniprot, Cis-BP, Transfac, and GrassiusX can be consulted to identify which nucleotides can be varied and which should be conserved in designing DTSs for use in the inventions of the present disclosure.

[0130] A given DTS may include 2 or more different TFBSs (i.e., TFBSs that differ from each other by nucleotide sequence and are therefore distinct), such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 different TFBSs. A given DTS may include 1 or more copies of the same TFBS, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, copies of the same TFBS, where in some instances the number of identical TFBS copies does not exceed 10.

[0131] The length of a given DTS may vary depending on the number of TFBSs comprised by it, the lengths of the TFBS sequence to which each nuclear targeting factor binds, and the number nucleotides between TFBSs (the spacer sequence). Where two or more TFBSs (either the same or different) are present in a given DTS, the distance between any two TFBSs may vary as desired, ranging in some instances from 5 to 100 bp, such as 10 to 75 bp, including 15 to 50 bp. For example, the TFBSs may be separated from one another by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 or more nts, for example, 0-5 nts, 6-10 nts, 11-15 nts, 16-20 nts, 21-25 nts, 26-30 nts, 31-35 nts, 36-40 nts, in some instances 40 - 50 nts. In some instances, the length of a given DTS ranges from 10 to 500 nt, such as 100 to 300 nt and including 150 to 200 nt.

[0132] A given NTDNA may include a single DTS or a plurality of DTSs, as desired. Where a given NTDNA includes a plurality of DTSs, the disparate DTSs may be the same or different. As such, a given NTDNA may include 2 or more different DTSs (i.e., DTSs that differ from each other by nucleotide sequence and are therefore distinct).

[0133] The DTS may be incorporated in the NTDNA in any of a variety of positions. For example, the DTS may be placed 5' of the promoter, 3’ of the promoter and 5’ of the expression cassette, within an intron of the expression cassette, or 3' of the expression cassette. In some instances, the DTSs is placed 5’ of the promoter. In some embodiments, the DTS is placed 3’ of the expression cassette.

[0134] Nuclear targeting factors. As summarized above, in some embodiments, in addition to the DNA and the agent that modulates BAF, a cell may also be contacted with a nuclear targeting factor or an mRNA that encodes the nuclear targeting factor to which the DTS binds to mediate nuclear entry of the DNA. Without wishing to be bound by theory, it is believed that the exogenously provided nuclear' targeting factor is translated from the mRNA and translocates from the cytosol to the nucleus, and in doing so brings the NTDNA associated with it (via the binding interaction between the DTS and the nuclear targeting factor) into the nucleus. In embodiments, the nuclear targeting factor will comprise a nuclear localization sequence (NLS) or a fragment thereof, as described herein or known in the art. In some instances, the NLS will be native, or endogenous, to the nuclear targeting factor. In other instances, a DNA binding protein will be engineered to comprise an NLS so as to create the nuclear targeting factor. In such instances, the NLS will be heterologous to the protein. If engineered into the NTF, the heterologous NLS may be engineered to be anywhere within the NTF. In some instances, the heterologous NLS is engineered to be within the center of the protein, i.e. the NLS is flanked on both N and C termini by 20 or more amino acids endogenous to the DNA binding protein. In some instances, the heterologous NLS is engineered to be at the N terminus. In some instances, the heterologous NLS is engineered to be at the C terminus. In some instances, the heterologous NLS is engineered to be at both the N terminus and the C terminus. When engineered to reside at a terminus, the NLS is typically engineered to be within 0-20 amino acids of the terminus, in some instances, within 0-10 amino acids of the terminus, in certain instances within 0-5 amino acids of the terminus, in some such cases, at the terminus. Typically, the NLS is engineered to be at a site that is distinct from the protein domain that mediates binding to the DNA. In some instances, the NLS may be flanked by one or more amino acids, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some instances, the nuclear targeting factor will comprise one NLS, in other instances multiple NLSs. In some instances, in which multiple NLSs are employed, the same NLS is employed multiple times. In other instances, in which multiple NLSs are employed, different NLSs are used. Often, when multiple NLSs are employed, they will be separated by a linker, e.g., a 6xK linker. Exemplary NLSs and their origins are provided in Table 5. Table 5. Exemplary NLS sequences

[0135] In some embodiments, the nuclear targeting factor comprises a nuclear export signal. In some embodiments, the NES is NMD3 ribosome export adaptor. In some such embodiments, the NES comprises a sequence having 85% identity or more to In some such embodiments, the NES comprises a sequence having 85% identity or more to

[0136] In some embodiments, the nuclear targeting factor comprises a tag, a linker or other sequence that may be used, e.g., to preserve the structure of an appended NLS or NES, to purify protein in recombinant protein production, to visualize the protein in cells, etc. Examples of tags or linkers include but are not limited to CS3, HIS, Flag, GGS, RIGID, SpyTag, Strep tags, etc. Exemplary linkers are provided in Table 6.

[0137] Table 6. Exemplary linkers.

[0138] Any method for making mRNA may be employed to generate the subject mRNA encoding the NTF. As one example, mRNA may be chemically synthesized using solid-phase methods. As another example, mRNA may be synthesized from a DNA template by an in vitro transcription (IVT) reaction, in which a DNA template comprising a promoter sequence for an RNA polymerase operably linked to a sequence encoding the target mRNA (in this instance, the nuclear targeting factor) is contacted with the RNA polymerase, which binds the template at the promoter region and starts the RNA synthesis. As yet another example, the mRNA may be synthesized in vivo and purified from the biological source. The mRNA may comprise naturally occurring ribonucleotides and / or chemically modified ribonucleotides. Typically, some chemically modified nucleotides may be included to reduce the immunogenicity of the mRNA, for example, N1-methylpseudouridine, 2-thiouridine (s2U), 5 -methylcytidine (m5C), N6- methyladenosine (m6A), 2’-O-methyluridine (Um), 2’-O-methylcytidine (Cm), 2’-O-methyladenosine (Am), and 2’-O-methylguanosine (Gm), N4-Acetyl-cytidine 5-triphosphate (AC4C). The mRNA may comprise a 5’ cap or analog thereof, e.g., an m7GpppG cap, anti-reverse cap analog (ARCA), a two- headed cap, an S cap, a 2S cap, and the like. The mRNA will typically comprise a 5’ untranslated region (UTR). The mRNA will typically comprise a 3’ UTR, e.g., as described in Table 7. The mRNA may comprise a tail modification, e.g., a ribose modified adenosine, 8-azaadenosine, cordycepin, and the like.

[0139]

[0140] For example, when the DTS comprises a binding sequence for a Tet Repressor (TetR) protein, e.g., a or a variant thereof, in addition to contacting the cell with the DNA and the BAF modulator, the cell may also be contacted with an mRNA encoding a TetR protein. In some embodiments, the TetR protein has been engineered to comprise an NLS. In some embodiments, the NLS is an NLS listed in Table 5. In some certain embodiments, the NLS is the NLS of SV40 Large T antigen or an optimized variant thereof, the NLS of the influenza A nuclear protein 1NF-A, the NLS of c-myc, or a fusion of the c-myc NLS to the influenza A nuclear protein INF-A NLS. In some embodiments, the NLS is located proximal to the N terminus. In other embodiments, the NLS is located proximal to the C-terminus. In some embodiments, the NLS is fused to the TetR protein with a linker.

[0141] In some embodiments, the TetR protein comprises a sequence having a sequence identity of 90% or more, in some instances 92%, 93%, 94%, 95% or more, in certain instances 96%, 97%, 98%, 99% identity or more, e.g., is 100% identical to the wild type Escherichia coli transcriptional regulator TetR protein sequence:

[0142] In some embodiments, the TetR protein is encoded by a polynucleotide comprising a sequence having a sequence identity of 80% or more to a sequence listed in Table 8, for example 85%, 90%, or 95% sequence identity or more, in some instances 96%, 97%, 98%, or 99% sequence identity, in some cases 100% sequence identity across the length of a sequence in Table 8. In some embodiments, the polynucleotide sequence has been codon optimized for expression in a particular host cell, e.g., codon optimized for expression in human cells or mouse cells.

[0143] Table 8. RNA polynucleotide sequences for expression of TetR protein

[0144] As another example, when the DTS comprises a binding sequence for a ZF protein, in addition to contacting the cell with the DNA and the BAF modulator, the cell may also be contacted with an mRNA encoding a ZF protein. For example, when the DTS comprises the sequence AAACTGCAAAAG or a variant thereof, the cell may also be contacted with an mRNA encoding the ZF protein ZF-CCR5. In some embodiments, the ZF protein has been engineered to comprise an NLS. In some embodiments, the NLS is proximal to the N terminus. In other embodiments, the NLS is proximal to the C-terminus. In s(me embodiments, the ZF protein comprises an NLS at both the N terminus and the C terminus. In some embodiments, the NLS is fused to the ZF protein with a linker.

[0145] In some embodiments, the ZF protein comprises a sequence having a sequence identity of 90% or more, in some instances 92%, 93%, 94%, 95% or more, in certain instances 96%, 97%, 98%, 99% identity or more, e.g. is 100% identical to the ZF-CCR5 fusion protein: , for example, the Variant

[0146] As another example, when the DTS comprises a binding sequence for a TALE protein, e.g., or or variants thereof, in addition to contacting the cell with the DNA and the BAF modulator, the cell may also be contacted with an mRNA encoding a TALE protein. In some embodiments, the TALE protein has been engineered to comprise an NLS. In some embodiments, the NLS is proximal to the N terminus. In other embodiments, the NLS is proximal to the C-terminus. In some embodiments, the TALE protein comprises an NLS at both the N terminus and the C terminus. In some embodiments, the NLS is fused to the TALE protein with a linker.

[0147] In some embodiments, the TALE protein comprises a sequence having a sequence identity of 90% or more, in some instances 92%, 93%, 94%, 95% or more, in certain instances 96%, 97%, 98%, 99% identity or more, e.g., is 100% identical to , for example . Other exemplary TALEs and methods for engineering them may be found in Li ct a. 2011 (supra) and Kim ct al. 2011 (supra).

[0148] As another example, when the DTS comprises the binding sequence for a GAL4 protein, for example CGG-N11-CCG or a variant thereof, in addition to contacting the cell with the DNA and the BAF modulator, the cell may also be contacted with an mRNA encoding a GAL4 protein. In some embodiments, the GAL4 protein has been engineered to comprise an NLS. In some embodiments, the NLS is proximal to the N terminus. In other embodiments, the NLS is proximal to the C-terminus. In some embodiments, the GAL4 protein is engineered to comprise an NLS at both the N-terminus and the C-terminus. In some embodiments, the NLS is fused to the GAL4 protein with a linker. In some embodiments, the NLS is selected from those listed in Table 5. In some certain embodiments, the NLS is the NLS of SV40 Large T antigen or an optimized variant thereof, the NLS of the influenza A nuclear protein INF-A, the NLS of c-myc, or a fusion of the c-myc NLS to the influenza A nuclear protein INF-A NLS.

[0149] In some embodiments, the GAL4 protein comprises a sequence having a sequence identity of 90% or more, in some instances 92%, 93%, 94%, 95% or more, in certain instances 96%, 97%, 98%, 99% identity or more, e.g., is 100% identical to wild type Saccharomyces cerevisiae GAL4 protein In some cases, the GAL4 protein comprises a sequence having a sequence identity of 90% or more, in some instances 92%, 93%, 94%, 95% or more, in certain instances 96%, 97%, 98%, 99% identity or more, e.g., is 100% identical to:

[0150] In some embodiments, the GAL4 protein is encoded by a polynucleotide comprises a sequence having a sequence identity of 80% or more to a sequence listed in Table 9, for example 85%, 90%, or 95% sequence identity or more, in some instances 96%, 97%, 98%, or 99% sequence identity, in some cases 100% sequence identity across the length of a sequence in Table 9. In some embodiments, the GAL4 polynucleotide has been codon optimized for expression in a particular host cell, e.g., codon optimized for expression in human or mouse cells.

[0151] Table 9. RNA polynucleotide sequences encoding GAL4 protein.

[0152] As another example, when the DTS comprises a binding sequence for an Arc protein, e.g., or a variant thereof, in addition to contacting the cell with the DNA and the BAF modulator, the cell may also be contacted with an mRNA encoding an Arc protein. In some embodiments, the Arc protein has been engineered to comprise an NLS. In some embodiments, the NLS is proximal to the N terminus. In other embodiments, the NLS is proximal to the C-terminus. In some embodiments, the Arc protein is engineered to comprise an NLS at both the N- terminus and the C-terminus. In some embodiments, the NLS is fused to the Arc protein with a linker. In some embodiments, the NLS is selected from those listed in Table 5. In some certain embodiments, the NLS is the NLS of SV40 Large T antigen or an optimized variant thereof, the NLS of the influenza A nuclear protein INF-A, the NLS of c-myc, or a fusion of the c-myc NLS to the influenza A nuclear protein INF-A NLS.

[0153] In some embodiments, the Arc protein comprises a sequence having a sequence identity of 90% or more, in some instances 92%, 93%, 94%, 95% or more, in certain instances 96%, 97%, 98%, 99% identity or more, e.g., is 100% identical to the sequence encoding the Salmonella phage Arc-like repressor: For example, the Arc protein may be the stl 1 variant Or a protein having a sequence identity of 90% or more, in some instances 92%, 93%, 94%, 95% or more, in certain instances 96%, 97%, 98%, 99% identity or more to the stl l variant. In some embodiments, the Arc protein is encoded by a polynucleotide comprising a sequence having a sequence identity of 80% or more, for example 85%, 90%, or 95% sequence identity or more, in some instances 96%, 97%, 98%, or 99% sequence identity, in some cases 100% sequence identity to the Salmonella phage Arc-like repressor wild type mRNA sequence: . In some embodiments, the Arc protein is encoded by a polynucleotide comprises a sequence having a sequence identity of 80% or more, for example 85%, 90%, or 95% sequence identity or more, in some instances 96%, 97%, 98%, or 99% sequence identity, in some cases 100% sequence identity to the sequence encoding the stl 1 Arc variant: . In some instances, the polynucleotide has been codon optimized for expression in a particular host cell, c.g., codon optimized for expression in human cells.

[0154] As another example, when the DTS comprises the binding sequence for a Mnt protein, e.g., or a variant thereof, for example , in addition to contacting the cell with the DNA and the BAF modulator, the cell may also be contacted with an mRNA encoding a Mnt protein. In some embodiments, the Mnt protein has been engineered to comprise an NLS. In some embodiments, the NLS is proximal to the N terminus. In other embodiments, the NLS is proximal to the C-terminus. In some embodiments, the Mnt protein is engineered to comprise an NLS at both the N-terminus and the C- terminus. In some embodiments, the NLS is fused to the Mnt protein with a linker. In some embodiments, the NLS is selected from those listed in Table 5. In some certain embodiments, the NLS is the NLS of SV40 Large T antigen or an optimized variant thereof, the NLS of the influenza A nuclear protein INF-A, the NLS of c-myc, or a fusion of the c-myc NLS to the influenza A nuclear protein INF-A NLS.

[0155] In some embodiments, the Mnt protein comprises a sequence having a sequence identity of 90% or more, in some instances 92%, 93%, 94%, 95% or more, in certain instances 96%, 97%, 98%, 99% identity or more, e.g., is 100% identical to native wild-type Mnt:

[0156] In some embodiments, the Mnt protein is encoded by a polynucleotide comprising a sequence having a sequence identity of 80% or more, for example 85%, 90%, or 95% sequence identity or more, in some instances 96%, 97%, 98%, or 99% sequence identity, in some cases 100% sequence identity across the sequence: In some embodiments, the Mnt polynucleotide has been codon optimized for expression in a particular host cell, e.g., codon optimized for expression in human or mouse cells.

[0157] As another example, when the DTS comprises the binding sequence for a PurR protein, e.g., or a variant thereof, in addition to contacting the cell with the DNA and the BAF modulator, the cell may also be contacted with an mRNA encoding a PurR protein. In some embodiments, the PurR protein has been engineered to comprise an NLS. In some embodiments, the NLS is proximal to the N terminus. In other embodiments, the NLS is proximal to the C-terminus. In some embodiments, the PurR protein is engineered to comprise an NLS at both the N-terminus and the C- terminus. In some embodiments, the NLS is fused to the PurR protein with a linker. In some embodiments, the NLS is selected from those listed in Table 5.

[0158] In some embodiments, the PurR protein comprises a sequence having a sequence identity of 90% or more, in some instances 92%, 93%, 94%, 95% or more, in certain instances 96%, 97%, 98%, 99% identity or more, e.g. is 100% identical to wild-type Escherichia coli HTH-type transcriptional repressor PurR:

[0159] In some embodiments, the PurR protein is encoded by a polynucleotide comprising a sequence having a sequence identity of 80% or more, for example 85%, 90%, or 95% or more, in some instances 96%. 97%. 98%. or 99% identity, in some cases 100% sequence identity across the length of the sequence: . In some embodiments, the polynucleotide encoding the PurR protein has been codon optimized for expression in a particular host cell, e.g., codon optimized for expression in human or mouse cells.

[0160] As another example, when the DTS comprises the binding sequence for a Bac434 protein, e.g., or a variant thereof, in addition to contacting the cell with the DNA and the BAF modulator, the cell may also be contacted with an mRNA encoding a Bac434 protein. In some embodiments, the Bac434 protein has been engineered to comprise an NLS. In some embodiments, the NLS is proximal to the N terminus. In other embodiments, the NLS is proximal to the C-terminus. In some embodiments, the Bac434 protein is engineered to comprise an NLS at both the N- terminus and the C-terminus. In some embodiments, the NLS is fused to the Bac434 protein with a linker. In some embodiments, the NLS is selected from those listed in Table 5.

[0161] In some embodiments, the Bac434 protein comprises a sequence having a sequence identity of 90% or more, in some instances 92%, 93%, 94%, 95% or more, in certain instances 96%, 97%, 98%, 99% identity or more, e.g., is 100% identical to the wild type Escherichia coli Phage 434 Repressor protein CL MSI SSRVKSKRIQLGLNQAELAQKVGTTQQSIEQLENGKTKRPRFLPELASALGVSVDWLLNGTSDSNVRFVGHVEP KGKYPLI SMVRAGSWCEA ( SEQ ID NO : 479 ) .

[0162] In some embodiments, the Bac434 protein is encoded by a polynucleotide comprising a sequence having a sequences identity of 80% or more, for example 85%, 90%, or 95% sequence identity or more, in some instances 96%, 97%, 98%, or 99% sequence identity, in some cases 100% sequence identity to: In some embodiments, the polynucleotide sequence encoding the Bac434 protein has been codon optimized for expression in a particular host cell, e.g., codon optimized for expression in human or mouse cells.

[0163] As another example, when the DTS comprises the binding sequence for a GCN4 protein, e.g., AGTGACTCATT or a variant thereof, in addition to contacting the cell with the DNA and the BAF modulator, the cell may also be contacted with an mRNA encoding a GCN4 protein. In some embodiments, the GCN4 protein has been engineered to comprise an NLS. In some embodiments, the NLS is proximal to the N terminus. In other embodiments, the NLS is proximal to the C-terminus. In some embodiments, the GCN4 protein is engineered to comprise an NLS at both the N-terminus and the C-terminus. In some embodiments, the NLS is fused to the GCN4 protein with a linker. In some embodiments, the NLS is selected from those listed in Table 5.

[0164] In some embodiments, the GCN4 protein comprises a sequence having a sequence identity of 90% or more, in some instances 92%, 93%, 94%, 95% or more, in certain instances 96%, 97%, 98%, 99% identity or more, e.g. is 100% identical to the wild type sequence for Saccharomyces cerevisiae GCN4:

[0165] In some embodiments, the GCN4 protein is encoded by a polynucleotide comprising a sequence having a sequences identity of 80% or more, for example 85%, 90%, or 95% sequence identity or more, in some instances 96%, 97%, 98%, or 99% sequence identity, in some cases 100% sequence identity to . In some embodiments, the GCN polynucleotide has been codon optimized for expression in a particular host cell, e.g., codon optimized for expression in human or mouse cells.

[0166] As another example, when the DTS comprises the binding sequence for a Lac Repressor protein (“LacR”), e.g. or a variant thereof, in addition to contacting the cell with the DNA and the BAF modulator, the cell may also be contacted with an mRNA encoding a LacR protein. In some embodiments, the LacR protein has been engineered to comprise an NLS. In some embodiments, the NLS is proximal to the N terminus. In other embodiments, the NLS is proximal to the C-terminus. In some embodiments, the LacR protein is engineered to comprise an NLS at both the N- terminus and the C-terminus. In some embodiments, the NLS is fused to the LacR protein with a linker. In some embodiments, the NLS is selected from those listed in Table 5.

[0167] In some embodiments, the LacR protein comprises a sequence having a sequence identity of 90% or more, in some instances 92%, 93%, 94%, 95% or more, in certain instances 96%, 97%, 98%, 99% identity or more, e.g. is 100% identical to the wild type sequence for the Escherichia coli DNA-binding transcriptional repressor LacI:

[0168] In some embodiments, the LacR protein is encoded by a polynucleotide comprising a sequence having a sequence identity of 80% or more, for example 85%, 90%, or 95% or more, in some instances 96%, 97%, 98%, or 99% identity, in some cases 100% sequence identity across the length of the sequence: In some embodiments, the polynucleotide encoding the LacR protein has been codon optimized for expression in a particular host cell, e.g., codon optimized for expression in human or mouse cells.

[0169] As another example, when the DTS comprises the binding sequence for a LScel D44A protein, e.g., or a variant thereof, in addition to contacting the cell with the DNA and the BAF modulator, the cell may also be contacted with an mRNA encoding a LScel D44A protein. In some embodiments, the LScel protein has been engineered to comprise an NLS. In some embodiments, the NLS is proximal to the N terminus. In other embodiments, the NLS is proximal to the C-terminus. In some embodiments, the I-Scel protein is engineered to comprise an NLS at both the N-terminus and the C-terminus. In some embodiments, the NLS is fused to the I-Scel protein with a linker.

[0170] In some embodiments, the I-Scel protein comprises a sequence having a sequence identity of 90% or more, in some instances 92%, 93%, 94%, 95% or more, in certain instances 96%, 97%, 98%, 99% identity or more, e.g. is 100% identical to e.g. a sequence having 85% identity more to:

[0171] As another example, when the TFBS is the binding sequence for a gene editing system, e.g. the guide RNA for a Cas nuclease, the zinc finger of a zinc finger nuclease, the TALE of a TALEN, e.g. as described below and as known in the art, in addition to contacting the cell with the DNA and the BAF modulator, the cell may also be contacted with an mRNA encoding the Cas nuclease, the ZF nuclease, or the TALEN. In some certain embodiments, the nuclease of the gene editing system is catalytically active. Put another way, the nuclease is able to modify DNA, for example to nick it, to create a double-stranded break, to replace one or more nucleotides within it. In other certain embodiments, the gene editing system is catalytically attenuated, e.g., its activity is reduced 50% or more, in some instances 60%, 70%, 80%, 90% or more, in some cases 95% or more, e.g., the catalytic activity has been abrogated.

[0172] Delivery Compositions and Methods of Delivery

[0173] Where desired, the DNA and / or BAF modulator and other agents as disclosed herein may be present in a delivery composition that includes the DNA and / or BAF modulator and a cytosolic delivery vehicle component, e.g., a delivery vehicle component that mediates entry of the DNA and / or BAF modulator into the cytosol from an extracellular location. Typically, the cytosolic delivery vehicle is a non-naturally occurring vehicle, i.e. a non-viral delivery vehicle. The DNA and BAF modulator may be present in the same cytosolic delivery vehicle or present in different cytosolic delivery vehicles, i.e., a first cytosolic delivery vehicle for the DNA and a second cytosolic delivery vehicle for the BAF modulator. When present in separate cytosolic deliver vehicles, the DNA and BAF modulator may be contacted with the cell simultaneously or sequentially, where when the DNA and BAF modulator are contacted sequentially with the cell, the DNA may be contacted with the cell prior to the BAF modulator or the BAF modulator may be contacted with the cell prior to the DNA. In some aspects, the methods provided herein comprise delivering a DNA and BAF modulator to a target cell.

[0174] Methods of delivery of nucleic acids can include lipofection, nucleofection, microinjection, biolistics, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355 and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofcctin™). Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration), as indicated above.

[0175] Various techniques and methods are known in the art for delivering nucleic acids to cells. For example, a DNA and / or BAF modulator can be delivered to a cell by conjugating the nucleic acid with a ligand that is internalized by the cell. For example, the ligand can bind a receptor on the cell surface and internalized via endocytosis. The ligand can be covalently linked to a nucleotide in the nucleic acid. Exemplary conjugates for delivering nucleic acids into a cell are described, example, in WO2015 / 006740, WO2014 / 025805, WO2012 / 037254, WO2009 / 082606, WO2009 / 073809, WO2009 / 018332, WO2006 / 112872, WO2004 / 090108, WO2004 / 091515 and WO2017 / 177326.

[0176] DNAs and / or BAF modulators, e.g., as described herein, can also be delivered to a cell by transfection. Useful transfection methods include, but are not limited to, lipid-mediated transfection, cationic polymer-mediated transfection, or calcium phosphate precipitation. Transfection reagents are well known in the art and include, but are not limited to, TurboFect Transfection Reagent (Thermo Fisher Scientific), Pro-Ject Reagent (Thermo Fisher Scientific), TRANSPASS™ P Protein Transfection Reagent (New England Biolabs), CHARIOT™ Protein Delivery Reagent (Active Motif), PROTEOJUICE™ Protein Transfection Reagent (EMD Millipore), 293fectin, LIPOFECT AMINE™ 2000, LIPOFECT AMINE™ 3000 (Thermo Fisher Scientific), LIPOFECT AMINE™ (Thermo Fisher Scientific), LIPOFECTIN™ (Thermo Fisher Scientific), DMRIE-C, CELLFECTIN™ (Thermo Fisher Scientific), OLIGOFECT AMINE™ (Thermo Fisher Scientific), LIPOFECTACE™, FUGENE™ (Roche, Basel, Switzerland), FUGENE™ HD (Roche), TRANSFECTAM™ (Transfectam, Promega, Madison, Wis.), TFX-10™ (Promega), TFX-20™ (Promega), TFX-50™ (Promega), TRANSFECTIN™ (BioRad, Hercules, Calif.), SILENTFECT™ (Bio-Rad), Effectene™ (Qiagen, Valencia, Calif.), DC-chol (Avanti Polar Lipids), GENEPORTER™ (Gene Therapy Systems, San Diego, Calif.), DHARMAFECT 1™ (Dharmacon, Lafayette, Colo.), DHARMAFECT 2™ (Dharmacon), DHARMAFECT 3™ (Dharmacon), DHARMAFECT 4™ (Dharmacon), ESCORT™ III (Sigma, St. Louis, Mo.), and ESCORT™ IV (Sigma Chemical Co.). Nucleic acids, such as DNA, can also be delivered to a cell via microfluidics methods, e.g., such as those known to those of skill in the art.

[0177] Methods of non-viral delivery of nucleic acids in vivo or ex vivo include electroporation, lipofection (see, U.S. Pat. Nos. 5,049,386; 4,946,787 and commercially available reagents such as Transfectam™ and Lipofectin™), microinjection, biolistics, LNPs, virosomes, liposomes (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et ah, Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787), immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids.

[0178] For example, DNAs and / or BAF modulators can be formulated into lipid nanoparticles (LNPs), lipidoids, liposomes, lipoplexes, or core-shell nanoparticles. Delivery reagents such as liposomes, nanocapsules, microparticles, microspheres, lipid nanoparticles, vesicles, and the like, can be used for the intr oduction of the compositions of the present disclosure into suitable host cells. In particular, the nucleic acids can be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticle, a gold particle, or the like. Such formulations can be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids disclosed herein.

[0179] Various delivery methods known in the art or modifications thereof can be used to deliver DNAs and / or BAF modulators as described herein in vitro or in vivo. For example, in some embodiments, DNAs and / or BAF modulators are delivered by making transient penetration in cell membrane by mechanical, electrical, ultrasonic, hydrodynamic, or laser-based energy so that DNA entrance into the targeted cells is facilitated. For example, a DNA and / or BAF modulator can be delivered by transiently disrupting cell membrane by squeezing the cell through a size-restricted channel or by other means known in the art. In some cases, a DNA and / or BAF modulator alone is directly injected as naked DNA into skin, thymus, cardiac muscle, skeletal muscle, or liver cells.

[0180] In some cases, a DNA and / or BAF modulator is delivered by gene gun. Gold or tungsten spherical particles (1-3 pm diameter) coated with DNA can be accelerated to high speed by pressurized gas to penetrate into target tissue cells.

[0181] In some embodiments, electroporation is used to deliver DNA and / or an BAF modulator to a target cell. Electroporation causes temporary destabilization of the cell membrane target cell tissue by insertion of a pair of electrodes into the tissue so that DNA molecules in the surrounding media of the destabilized membrane would be able to penetrate into cytoplasm and nucleoplasm of the cell. Electroporation has been used in vivo for many types of tissues, such as skin, lung, and muscle.

[0182] In some cases, DNA and / or an BAF modulator is delivered by hydrodynamic injection, which is a simple and highly efficient method for direct intracellular delivery of any water-soluble compounds and particles into internal organs and skeletal muscle in an entire limb.

[0183] In some cases, DNAs and / or BAF modulators are delivered by ultrasound by making nanoscopic pores in membrane to facilitate intracellular delivery of DNA particles into cells of internal organs or tumors, so the size and concentration of plasmid DNA have great role in efficiency of the system. In some cases, DNAs and / or BAF modulators are delivered by magnetofection by using magnetic fields to concentrate particles containing nucleic acid into the target cells.

[0184] In some cases, chemical delivery systems can be used, for example, by using nanomeric complexes, which include compaction of negatively charged nucleic acid by polycationic nanomeric particles, belonging to cationic liposome / micelle or cationic polymers. Cationic lipids used for the delivery method includes, but not limited to monovalent cationic lipids, polyvalent cationic lipids, guanidine containing compounds, cholesterol derivative compounds, cationic polymers, (e.g., poly(ethylenimine), poly-L-lysine, protamine, other cationic polymers), and lipid-polymer hybrid.

[0185] DNAs and / or BAF modulators, e.g., as described herein, can also be administered directly to an organism for transduction of cells in vivo. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.

[0186] Compositions comprising a DNA and / or BAF modulator, e.g., as described herein, and a cytosolic delivery vehicle are specifically contemplated herein. In some embodiments, a DNA and / or BAF modulator is formulated with a lipid delivery system, for example, LNPs as described herein. In some embodiments, such compositions are administered by any route desired by a skilled practitioner. The compositions may be administered to a subject by different routes including orally, parenterally, subretinally, intravitreally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenous, intra-arterial, intraperitoneal, intracranially, subcutaneous, intramuscular, intranasal, intrathecal, and intraarticular or combinations thereof. For veterinary use, the composition may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian may readily determine the dosing regimen and route of administration that is most appropriate for a particular animal. The compositions may be administered by traditional syringes, needleless injection devices, “microprojectile bombardment gene guns”, or other physical methods such as electroporation (“EP”), hydrodynamic methods or ultrasound.

[0187] Microparticle / Nanoparticles. In some embodiments, a DNA and / or BAF modulator as described herein is delivered by a nanoparticle. One example of a nanoparticle that finds use is a lipid nanoparticle (LNP). Generally, LNPs of the present disclosure may be composed of nucleic acid (e.g., DNA and / or BAF modulator) molecules, one or more ionizable or cationic lipids (or salts thereof), one or more non- ionic or neutral lipids (e.g., a phospholipid), a molecule that prevents aggregation (e.g., PEG or a PEG- lipid conjugate), and optionally a sterol (e.g., cholesterol). For example, lipid nanoparticles may comprise an ionizable amino lipid (e.g., heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin- MC3-DMA, a phosphatidylcholine (l,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol and a coat lipid (polyethylene glycol-dimyristolglycerol, PEG-DMG), for example as disclosed by Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3): 498-507.

[0188] In some embodiments, a lipid nanoparticle has a mean diameter between about 10 and about 1000 nm. In some embodiments, a lipid nanoparticle has a diameter that is less than 300 nm. In some embodiments, a lipid nanoparticle has a diameter between about 10 and about 300 nm. In some embodiments, a lipid nanoparticle has a diameter that is less than 200 nm. In some embodiments, a lipid nanoparticle has a diameter between about 25 and about 200 nm. In some embodiments, a lipid nanoparticle preparation (e.g., composition comprising a plurality of lipid nanoparticles) has a size distribution in which the mean size (e.g., diameter) is about 70 nm to about 200 nm, and more typically the mean size is about 100 nm or less.

[0189] In some aspects, the disclosure provides for a lipid nanoparticle comprising a DNA and / or BAF modulator as described herein and an ionizable lipid. The ionizable lipid is typically employed to condense the nucleic acid cargo, e.g., DNA at low pH and to drive membrane association and fusogenicity. Generally, ionizable lipids are lipids comprising at least one amino group that is positively charged or becomes protonated under acidic conditions, for example at pH of 6.5 or lower. Ionizable lipids are also referred to as cationic lipids herein. Exemplary ionizable lipids are described in International PCT patent applications PCT / US2023 / 079923 and PCT / US2023 / 76457 and International PCT publications WO2015 / 095340, WO2015 / 199952, WO2018 / 011633, WO2017 / 049245, W 02015 / 061467, WO2012 / 040184, WO2012 / 000104, WO2015 / 074085, WO2016 / 081029, WO2017 / 004143, WO2017 / 075531, WO2017 / 117528, WO2011 / 022460, WO2013 / 148541, WO2013 / 116126, WO2011 / 153120, WO2012 / 044638, WO2012 / 054365, WO2011 / 090965, WO2013 / 016058, WO2012 / 162210, WO2008 / 042973, WO2010 / 129709, WO2010 / 144740, WO2012 / 099755, WO2013 / 049328, WO2013 / 086322, WO2013 / 086373, WO2011 / 071860, WO2009 / 132131, WO2010 / 048536, WO2010 / 088537, WO2010 / 054401, WO2010 / 054406, WO2010 / 054405, WO2010 / 054384, WO2012 / 016184, WO2009 / 086558, WO2010 / 042877, WO2011 / 000106, WO2011 / 000107, WO2005 / 120152, WO2011 / 141705, WO2013 / 126803, WO2006 / 007712, WO2011 / 038160, WO2005 / 121348, WO2011 / 066651, WO2009 / 127060, WO2011 / 141704, WO2006 / 069782, WO2012 / 031043, WO2013 / 006825, WO2013 / 033563, WO2013 / 089151, WO2017 / 099823, WO2015 / 095346, and WO2013 / 086354, and US patent publications US2016 / 0311759, US2015 / 0376115, US2016 / 0151284, US2017 / 0210697, US2015 / 0140070, US2013 / 0178541, US2013 / 0303587, US2015 / 0141678, US2015 / 0239926, US2016 / 0376224, US2017 / 0119904, US2012 / 0149894, US2015 / 0057373, US2013 / 0090372, US2013 / 0274523, US2013 / 0274504, US2013 / 0274504, US2009 / 0023673, US2012 / 0128760, US2010 / 0324120, US2014 / 0200257, US2015 / 0203446, US2018 / 0005363, US2014 / 0308304, US2013 / 0338210, US2012 / 0101148, US2012 / 0027796, US2012 / 0058144, US2013 / 0323269, US2011 / 0117125, US2011 / 0256175, US2012 / 0202871, US2011 / 0076335, US2006 / 0083780, US2013 / 0123338, US2015 / 0064242, US2006 / 0051405, US2013 / 0065939, US2006 / 0008910, US 2003 / 0022649, US2010 / 0130588, U52013 / 0116307, US2010 / 0062967, US2013 / 0202684, US2014 / 0141070, US2014 / 0255472, US2014 / 0039032, US2018 / 0028664, U52016 / 0317458, and US2013 / 0195920.

[0190] Various LNP formulations known in the art can be used to deliver DNAs and / or BAF modulators as described herein. For example, various LNP formulations and delivery methods using lipid nanoparticles are described in U.S. Pat. Nos. 9,404,127; 9,006,417; 9,518,272; and US Patent Application No. 63 / 415,229. Such particles can be prepared by high energy mixing of ethanolic lipids with aqueous DNA and / or BAF modulator at low pH which protonates the ionizable lipid and provides favorable energetics for nucleic acid / lipid association and nucleation of particles. The particles can be further stabilized through aqueous dilution and removal of the organic solvent. The particles can be concentr ated to the desired level.

[0191] Another example of a nanoparticle that finds use in the delivery of the subject DNAs and / or BAF modulators is a metal nanoparticle. In some embodiments, a DNA and / or BAF modulator as described herein is delivered by a gold nanoparticle. Generally, a nucleic acid can be covalently bound to a gold nanoparticle or non-covalently bound to a gold nanoparticle (e.g., bound by a charge-charge interaction), for example as described by Ding et al. (2014). Gold Nanoparticles for Nucleic Acid Delivery. Mol. Ther. 22(6); 1075-1083. In some embodiments, gold nanoparticle-nucleic acid conjugates are produced using methods described, for example, in U.S. Pat. No. 6,812,334.

[0192] In some embodiments, DNAs and / or BAF modulators described herein can be readily formulated in high concentrations of chitosan-nucleic acid polyplex compositions and administered orally in DNA enteric coated pills described in U.S. Pat. Nos. 8,846,102; 9,404,088; and 9,850,323, each of which is incorporated herein by its entirety. Exosomes. In some embodiments, a DNA and / or BAF modulator as described herein is delivered by being packaged in an exosome. Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane. Their surface consists of a lipid bilayer from the donor cell’s cell membrane, they contain cytosol from the cell that produced the exosome, and exhibit membrane proteins from the parental cell on the surface. Exosomes are produced by various cell types including epithelial cells, B and T lymphocytes, mast cells (MC) as well as dendritic cells (DC). Some embodiments, exosomes with a diameter between 10 nm and 1 pm, between 20 nm and 500 nm, between 30 nm and 250 nm, between 50 nm and 100 nm are envisioned for use. Exosomes can be isolated for delivery to target cells using either their donor cells or by introducing specific nucleic acids into them. Various approaches known in the art can be used to produce exosomes containing capsid-free AAV vectors of the present invention.

[0193] Conjugates. In some embodiments, a DNA and / or BAF modulator as described herein as disclosed herein is conjugated (e.g., covalently bound to an agent that increases cellular uptake. An “agent that increases cellular uptake” is a molecule that facilitates transport of a nucleic acid across a lipid membrane. For example, a nucleic acid can be conjugated to a lipophilic compound (e.g., cholesterol, tocopherol, etc.), a cell penetrating peptide (CPP) (e.g., penetratin, TAT, SynlB, etc.), and polyamines (e.g., spermine). Further examples of agents that increase cellular uptake are disclosed, for example, in Winkler (2013). Oligonucleotide conjugates for therapeutic applications. Ther. Deliv. 4(7); 791-809.

[0194] In some embodiments, a DNA and / or BAF modulator as described herein as disclosed herein is conjugated to a polymer (e.g., a polymeric molecule) or a folate molecule (e.g., folic acid molecule). Generally, delivery of nucleic acids conjugated to polymers is known in the ait, for example as described in WO2000 / 34343 and WO2008 / 022309. In some embodiments, a DNA and / or BAF modulator as disclosed herein is conjugated to a poly(amide) polymer, for example as described by U.S. Pat. No. 8,987,377. In some embodiments, a nucleic acid described by the disclosure is conjugated to a folic acid molecule as described in U.S. Pat. No. 8,507,455. In some embodiments, a DNA and / or BAF modulator as described herein as disclosed herein is conjugated to a carbohydrate, for example as described in U.S. Pat. No. 8,450,467.

[0195] Nanocapsules. Alternatively, nanocapsule formulations of a DNA and / or BAF modulator can be used. Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.

[0196] Liposomes. A DNA and / or BAF modulator as described herein can be added to liposomes for delivery to a cell or target organ in a subject. Liposomes are vesicles that possess at least one lipid bilayer and an aqueous core. Liposomes are typically used as carriers for drug / therapeutic delivery in the context of pharmaceutical development. They work by fusing with a cellular membrane and repositioning its lipid structure to deliver a drug or active pharmaceutical ingredient (API). Liposome compositions for such delivery are composed of phospholipids, especially compounds having a phosphatidylcholine group, however these compositions may also include other lipids.

[0197] The formation and use of liposomes is generally known to those of skill in the art. Liposomes have been developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

[0198] Also provided herein are cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.

[0199] Target Cells

[0200] Cells employed in embodiments of the invention, referred to herein as “target cells” may vary. It should be appreciated that the target cell may be of any origin, for example from an organism. In some embodiments, the target cell is a mammalian cell. Some non-limiting examples of a mammalian cell include, without limitation, a mouse cell, a rat cell, hamster cell, a rodent cell, and a nonhuman primate cell. In some embodiments, the target cell is a human cell. It should also be appreciated that the target cell may be of any cell type. For example, the target cell may be a stem cell, which may include embryonic stem cells, induced pluripotent stem cells (iPS cells), fetal stem cells, cord blood stem cells, or adult stem cells (i.e., tissue specific stem cells). In other cases, the target cell may be any differentiated cell type found in a subject. Cells of interest include both dividing cells and non-dividing cells. Examples of specific target cells of interest include, but are not limited to: hepatocytes, stellate cells, T lymphocytes, B lymphocytes, NK cells, skeletal muscle cells, cardiomyocytes, neurons, astrocytes, oligodendrocytes, dendritic cells, skin cells, photoreceptors, RPE cells, radial glia, etc.

[0201] In some embodiments, the target cell is a cell in vitro, and the method includes contacting the cell in vitro. In some embodiments, the target cell is a cell in a subject, and the methods include administering the DNA and the agent that modulates BAF activity (where either or both may be present in the same or different suitable delivery vehicle, as desired) to the subject. In some embodiments, the subject is a mammalian subject, for example, a rodent, a mouse, a rat, a hamster, or a non-human primate. In some embodiments, the subject is a human subject.

[0202] The target cell may be contacting simultaneously or sequentially with the DNA and the agent that modulates BAF, as desired. In some instances, the target cell is contacted simultaneously with the DNA and the agent that modulates BAF. As such, the DNA and the agent that modulates BAF are contacted at the same time with the target cells. In other instances, the DNA and the agent that modulates BAF are sequentially contacted with the cell. For example, the target cell may be contacted with the DNA before being contacted with the agent that modulates BAF. Alternatively, the target cell may be contacted with the DNA after being contacted with the agent that modulates BAF.

[0203] In embodiments of the invention in which a nuclear targeting factor is provided to the cell, the target cell may be contacted with the DNA and the BAF modulator simultaneously or sequentially with an mRNA encoding the NTF. In some instances, the target cell is contacted simultaneously with the DNA, the agent that modulates BAF, and the NTF. In certain such embodiments, the DNA, the agent that modulates BAF, and the NTF are provided as a single formulation in a delivery vehicle (as described in greater detail below) that is administered to the target cell. In other embodiments, the DNA and agent that modulates BAF are provided as a separate formulation from the NTF that are administered simultaneously to the cell, i.e., as an admixture, or sequentially. In other embodiments, the DNA and the NTF are provided as a separate formulation from the BAF modulator that are administered simultaneously to the cell, i.e., as an admixture, or sequentially. In yet other embodiments, all of the elements are formulated individually, i.e. the DNA is provided as a first formulation, the BAF modulator is provided as a second formulation, and the NTF is provided as a third formulation, where all may be administered simultaneously to the cell, i.e., as an admixture, or sequentially. Preferably, the DNA, the agent that modulates BAF, and the NTF are prepared as a single formulation in the delivery vehicle for contacting with the cell.

[0204] Genomic Integration

[0205] In some instances, the DNA and agent that modulates BAF are contacted with a target cell in conjunction with a gene editing system, e.g., that is configured to provide genomic integration of the cargo nucleic acid component of the DNA. In such embodiments, as the DNA is contacted with the target cell in conjunction with a gene editing system, it may be contacted with the target cell simultaneously or sequentially with the gene editing system. In some instances, one or more components of a gene editing system may be present with the DNA in a delivery composition, such as a cytosolic delivery composition (e.g., LNP), such as described in greater detail below. Gene editing systems that may be employed in such embodiments may vary, as desired. In some embodiments, the employed gene editing system is configured to genomically integrate the DNA (or desired component thereof, e.g., coding sequence, expression cassette, etc., such as described above) into a specific safe harbor location in the genome, for example to a genomic location within or near an endogenous albumin locus. Generally, one skilled in the art will understand that a safe harbor locus is a location within a genome that can be used for integrating exogenous nucleic acids, where the addition of exogenous nucleic acids into the safe har bor locus does not cause significant effect on the growth of the host cell by the addition of the nucleic acids alone. In some embodiments, the cargo nucleic acid may be inserted into the specific safe harbor location in the genome that may either utilize the promoter found at that safe harbor locus, or allow the expressional regulation of a coding sequence of the cargo nucleic acid by an exogenous promoter that is fused to the cargo nucleic acid coding sequence prior to insertion.

[0206] Gene editing can be conducted using nucleases engineered to target specific sequences. To date there are four major types of nucleases: meganucleases and their derivatives, zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and CRISPR-Cas9 nuclease systems. The nuclease platforms vary in difficulty of design, targeting density and mode of action, particularly as the specificity of ZFNs and TALENs is through protein-DNA interactions, while RNA-DNA interactions primarily guide Cas9. Cas9 cleavage also requires an adjacent motif, the PAM, which differs between different CRISPR systems. Cas9 from Streptococcus pyogenes cleaves using a NRG PAM, CR1SPR from Neisseria meningitidis can cleave at sites with PAMs including NNNNGATT, NNNNNGTTT and NNNNGCTT. A number of other Cas9 orthologs target protospacer adjacent to alternative PAMs. CRISPR endonucleases, such as Cas9, can be used in various embodiments of the methods of the disclosure. However, the teachings described herein, such as therapeutic target sites, could be applied to other forms of endonucleases, such as ZFNs, TALENs, HEs, or MegaTALs, or using combinations of nucleases. These different systems are now described in further detail.

[0207] A given DNA delivery cytosolic delivery composition may include one or more elements of a given gene editing system. Gene editing system employed in embodiments of the invention may include a number of different elements, such as nucleic acid elements, e.g., genome-targeting nucleic acids or Guide RNAs, nucleic acids, e.g., mRNAs encoding endonucleases; polypeptide components, e.g., endonucleases, etc. These various components are now reviewed in greater detail in conjunction with the description of representative endonuclease based genomic integration systems.

[0208] In some embodiments, the methods of genome edition and compositions therefore use a nucleic acid sequence (or oligonucleotide) encoding a site-directed polypeptide or DNA endonuclease. The nucleic acid sequence encoding the site-directed polypeptide can be DNA or RNA. If the nucleic acid sequence encoding the site-directed polypeptide is RNA, it can be covalently linked to a gRNA sequence or exist as a separate sequence. In some embodiments, a peptide sequence of the site-directed polypeptide or DNA endonuclease can be used instead of the nucleic acid sequence thereof.

[0209] The modifications of the target DNA due to NHEJ and / or HDR can lead to, for example, mutations, deletions, alterations, integrations, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocations and / or gene mutation. The process of integrating non-native nucleic acid, e.g., an exogenously provided DNA, into genomic DNA is an example of genome editing. A site-directed polypeptide is a nuclease used in genome editing to cleave DNA. The site-directed can be administered to a cell or a patient as either: one or more polypeptides, or one or more mRNAs encoding the polypeptide. In some embodiments, a site-directed polypeptide has a plurality of nucleic acid-cleaving (i.e., nuclease) domains. Two or more nucleic acid-cleaving domains can be linked together via a linker. In some embodiments, the linker has a flexible linker. Linkers can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or more amino acids in length.

[0210] CRISPR. In the context of a CRISPR / Cas or CRISPR / Cpfl system, the site-directed polypeptide can bind to a guide RNA that, in turn, specifies the site in the target DNA to which the polypeptide is directed. In some embodiments of CRISPR / Cas or CRISPR / Cpfl systems herein, the site-directed polypeptide is an endonuclease, such as a DNA endonuclease.

[0211] Naturally-occurring wild- type Cas9 enzymes have two nuclease domains, a HNH nuclease domain and a RuvC domain. Herein, the "Cas9" refers to both naturally-occurring and recombinant Cas9s. Cas9 enzymes contemplated herein have a HNH or HNH-like nuclease domain, and / or a RuvC or RuvC-like nuclease domain.

[0212] HNH or HNH-like domains have a McrA-like fold. HNH or HNH-like domains has two antiparallel p-strands and an a-helix. HNH or HNH-like domains has a metal binding site (e.g., a divalent cation binding site). HNH or HNH-like domains can cleave one strand of a target nucleic acid (e.g., the complementary strand of the crRNA targeted strand).

[0213] RuvC or RuvC-like domains have an RNaseH or RNaseH-like fold. RuvC / RNaseH domains are involved in a diverse set of nucleic acid-based functions including acting on both RNA and DNA. The RNaseH domain has 5 P-strands surrounded by a plurality of a-helices. RuvC / RNaseH or RuvC / RNaseH- like domains have a metal binding site (e.g., a divalent cation binding site). RuvC / RNaseH or RuvC / RNaseH-like domains can cleave one strand of a target nucleic acid (e.g., the non-complementary strand of a double-stranded target DNA).

[0214] In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity to a wild-type exemplary site-directed polypeptide [e.g., Cas9 from S. pyogenes, US2014 / 0068797 Sequence ID No. 8 or Sapranauskas et al., Nucleic Acids Res, 39(21): 9275-9282 (2011)], and various other site-directed polypeptides). In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity to the nuclease domain of a wild-type exemplary site- directed polypeptide (e.g., Cas9 from S. pyogenes, supra). In some embodiments, a site-directed polypeptide has at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids. In some embodiments, a site-directed polypeptide has at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site- directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids. In some embodiments, a site-directed polypeptide has at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g.. Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a HNH nuclease domain of the site-directed polypeptide. In some embodiments, a site-directed polypeptide has at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a HNH nuclease domain of the site-directed polypeptide. In some embodiments, a site-directed polypeptide has at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a RuvC nuclease domain of the site-directed polypeptide. In some embodiments, a site-directed polypeptide has at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site -directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a RuvC nuclease domain of the site-directed polypeptide.

[0215] In some embodiments, the site-directed polypeptide has a modified form of a wild-type exemplary site-directed polypeptide. The modified form of the wild-type exemplary site-directed polypeptide has a mutation that reduces the nucleic acid-cleaving activity of the site-directed polypeptide. In some embodiments, the modified form of the wild-type exemplary site-directed polypeptide has less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity of the wild- type exemplary site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra). The modified form of the site-directed polypeptide can have no substantial nucleic acid-cleaving activity. When a site-directed polypeptide is a modified form that has no substantial nucleic acid-cleaving activity, it is referred to herein as "enzymatically inactive."

[0216] In some embodiments, the modified form of the site-directed polypeptide has a mutation such that it can induce a single-strand break (SSB) on a target nucleic acid (e.g., by cutting only one of the sugar- phosphate backbones of a double-strand target nucleic acid). In some embodiments, the mutation results in less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity in one or more of the plurality of nucleic acid-cleaving domains of the wild-type site directed polypeptide (e.g., Cas9 from S. pyogenes, supra). In some embodiments, the mutation results in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the complementary strand of the target nucleic acid, but reducing its ability to cleave the non-complementary strand of the target nucleic acid. In some embodiments, the mutation results in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the non-complementary strand of the target nucleic acid, but reducing its ability to cleave the complementary strand of the target nucleic acid. For example, residues in the wild-type exemplary S. pyogenes Cas9 polypeptide, such as AsplO, His840, Asn854 and Asn856, are mutated to inactivate one or more of the plurality of nucleic acid-cleaving domains (e.g., nuclease domains). In some embodiments, the residues to be mutated correspond to residues AsplO, His840, Asn854 and Asn856 in the wild-type exemplary S. pyogenes Cas9 polypeptide (e.g., as determined by sequence and / or structural alignment). Non-limiting examples of mutations include D10A, H840A, N854A or N856A. One skilled in the art will recognize that mutations other than alanine substitutions are suitable.

[0217] In some embodiments, a D10A mutation is combined with one or more of H840A, N854A, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity. In some embodiments, a H840A mutation is combined with one or more of D10A, N854A, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity. In some embodiments, a N854A mutation is combined with one or more of H840A, D10A, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity. In some embodiments, a N856A mutation is combined with one or more of H840A, N854A, or D10A mutations to produce a site- directed polypeptide substantially lacking DNA cleavage activity. Site-directed polypeptides that have one substantially inactive nuclease domain are referred to as "nickases".

[0218] In some embodiments, variants of RNA-guided endonucleases, for example Cas9, can be used to increase the specificity of CRISPR-mediated genome editing. Wild type Cas9 is generally guided by a single guide RNA designed to hybridize with a specified about.20 nucleotide sequence in the target sequence (such as an endogenous genomic locus). However, several mismatches can be tolerated between the guide RNA and the target locus, effectively reducing the length of required homology in the target site to, for example, as little as 13 nt of homology, and thereby resulting in elevated potential for binding and double-strand nucleic acid cleavage by the CRISPR / Cas9 complex elsewhere in the target genome— also known as off-target cleavage. Because nickase variants of Cas9 each only cut one strand, in order to create a double-strand break it is necessary for a pair of nickases to bind in close proximity and on opposite strands of the target nucleic acid, thereby creating a pair of nicks, which is the equivalent of a double-strand break. This requires that two separate guide RNAs— one for each nickase— must bind in close proximity and on opposite strands of the target nucleic acid. This requirement essentially doubles the minimum length of homology needed for the double-strand break to occur, thereby reducing the likelihood that a double-strand cleavage event will occur elsewhere in the genome, where the two guide RNA sites— if they exist-are unlikely to be sufficiently close to each other to enable the double-strand break to form. As described in the art, nickases can also be used to promote HDR versus NHEJ. HDR can be used to introduce selected changes into target sites in the genome through the use of specific donor sequences that effectively mediate the desired changes. Descriptions of various CRISPR / Cas systems for use in gene editing can be found, e.g., in international patent application publication number WO2013 / 176772, and in Nature Biotechnology 32, 347-355 (2014), and references cited therein.

[0219] In some embodiments, the site-directed polypeptide (e.g., variant, mutated, enzymatically inactive and / or conditionally enzymatically inactive site -directed polypeptide) targets nucleic acid. In some embodiments, the site-directed polypeptide (e.g., variant, mutated, enzymatically inactive and / or conditionally enzymatically inactive endoribonuclease) targets DNA. In some embodiments, the site- directed polypeptide (e.g., variant, mutated, enzymatically inactive and / or conditionally enzymatically inactive endoribonuclease) targets RNA.

[0220] In some embodiments, the site-directed polypeptide has one or more non-native sequences (e.g., the site-directed polypeptide is a fusion protein). In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), a nucleic acid binding domain, and two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain). In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain). In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), and two nucleic acid cleaving domains, wherein one or both of the nucleic acid cleaving domains have at least 50% amino acid identity to a nuclease domain from Cas9 from a bacterium (e.g., S. pyogenes). In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain), and non-native sequence (for example, a nuclear localization signal) or a linker linking the site-directed polypeptide to a non-native sequence. In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain), wherein the site-directed polypeptide has a mutation in one or both of the nucleic acid cleaving domains that reduces the cleaving activity of the nuclease domains by at least 50%. In some embodiments, the site-directed polypeptide has an amino acid sequence having at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain), wherein one of the nuclease domains has mutation of aspartic acid 10, and / or wherein one of the nuclease domains has mutation of histidine 840, and wherein the mutation reduces the cleaving activity of the nuclease domain(s) by at least 50%.

[0221] In some embodiments, the one or more site -directed polypeptides, e.g., DNA endonucleases, include two nickases that together effect one double-strand break at a specific locus in the genome, or four nickases that together effect two double-strand breaks at specific loci in the genome. Alternatively, one site -directed polypeptide, e.g., DNA endonuclease, affects one double-strand break at a specific locus in the genome.

[0222] In some embodiments, a polynucleotide encoding a site-directed polypeptide can be used to edit genome. In some of such embodiments, the polynucleotide encoding a site-directed polypeptide is codon- optimized according to methods standard in the art for expression in the cell containing the target DNA of interest. For example, if the intended target nucleic acid is in a human cell, a human codon-optimized polynucleotide encoding Cas9 is contemplated for use for producing the Cas9 polypeptide.

[0223] A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic locus can be found in the genomes of many prokaryotes (e.g., bacteria and archaea). In prokaryotes, the CRISPR locus encodes products that function as a type of immune system to help defend the prokaryotes against foreign invaders, such as vims and phage. There are three stages of CRISPR locus function: integration of new sequences into the CRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreign invader nucleic acid. Five types of CRISPR systems (e.g., Type I, Type II, Type 111, Type U, and Type V) have been identified.

[0224] A CRISPR locus includes a number of short repeating sequences referred to as "repeats." When expressed, the repeats can form secondary hairpin structures (e.g., hairpins) and / or have unstructured single-stranded sequences. The repeats usually occur in clusters and frequently diverge between species. The repeats are regularly interspaced with unique intervening sequences referred to as "spacers," resulting in a repeat-spacer-repeat locus architecture. The spacers are identical to or have high homology with known foreign invader sequences. A spacer-repeat unit encodes a crisprRNA (crRNA), which is processed into a mature form of the spacer-repeat unit. A crRNA has a "seed" or spacer sequence that is involved in targeting a target nucleic acid (in the naturally occurring form in prokaryotes, the spacer sequence targets the foreign invader nucleic acid). A spacer sequence is located at the 5' or 3' end of the crRNA.

[0225] A CRISPR locus also has polynucleotide sequences encoding CRISPR Associated (Cas) genes. Cas genes encode endonucleases involved in the biogenesis and the interference stages of crRNA function in prokaryotes. Some Cas genes have homologous secondary and / or tertiary structures. crRNA biogenesis in a Type II CRISPR system in nature requires a trans-activating CRISPR RNA (tracrRNA). The tracrRNA is modified by endogenous RNaselll, and then hybridizes to a crRNA repeat in the pre-crRNA array. Endogenous RNaselll is recruited to cleave the pre-crRNA. Cleaved crRNAs are subjected to exoribonuclease trimming to produce the mature crRNA form (e.g., 5' trimming). The tracrRNA remains hybridized to the crRNA, and the tracrRNA and the crRNA associate with a site-directed polypeptide (e.g., Cas9). The crRNA of the crRNA-tracrRNA-Cas9 complex guides the complex to a target nucleic acid to which the crRNA can hybridize. Hybridization of the crRNA to the target nucleic acid activates Cas9 for targeted nucleic acid cleavage. The target nucleic acid in a Type II CRISPR system is referred to as a protospacer adjacent motif (PAM). In nature, the PAM is essential to facilitate binding of a site -directed polypeptide (e.g., Cas9) to the target nucleic acid. Type II systems (also referred to as Nmeni or CASS4) are further subdivided into Type II-A (CASS4) and II-B (CASS4a). Jinek et al., Science, 337(6096):816-821 (2012) showed that the CRISPR / Cas9 system is useful for RNA- programmable genome editing, and international patent application publication number WO 2013 / 176772 provides numerous examples and applications of the CRISPR / Cas endonuclease system for site-specific gene editing.

[0226] Type V CRISPR systems have several important differences from Type II systems. For example, Cpfl is a single RNA-guided endonuclease that, in contrast to Type II systems, lacks tracrRNA. In fact, Cpfl -associated CRISPR arrays are processed into mature crRNAS without the requirement of an additional trans-activating tracrRNA. The Type V CRISPR array is processed into short mature crRNAs of 42-44 nucleotides in length, with each mature crR A beginning with 19 nucleotides of direct repeat followed by 23-25 nucleotides of spacer sequence. In contrast, mature crRNAs in Type II systems start with 20-24 nucleotides of spacer sequence followed by about 22 nucleotides of direct repeat. Also, Cpfl utilizes a T-rich protospacer- adjacent motif such that Cpfl -crRNA complexes efficiently cleave target DNA preceded by a short T-rich PAM, which is in contrast to the G-rich PAM following the target DNA for Type II systems. Thus, Type V systems cleave at a point that is distant from the PAM, while Type II systems cleave at a point that is adjacent to the PAM. In addition, in contrast to Type II systems, Cpfl cleaves DNA via a staggered DNA double-stranded break with a 4 or 5 nucleotide 5' overhang. Type II systems cleave via a blunt double-stranded break. Similar to Type II systems, Cpfl contains a predicted RuvC-like endonuclease domain, but lacks a second HNH endonuclease domain, which is in contrast to Type II systems.

[0227] Exemplary CRISPR / Cas polypeptides include the Cas9 polypeptides in FIG. 1 of Fonfara et al., Nucleic Acids Research, 42: 2577-2590 (2014). The CRISPR / Cas gene naming system has undergone extensive rewriting since the Cas genes were discovered.

[0228] A genome-targeting nucleic acid interacts with a site-directed polypeptide (e.g., a nucleic acid- guided nuclease such as Cas9), thereby forming a complex. The genome-targeting nucleic acid (e.g., gRNA, such as described in greater detail below) guides the site-directed polypeptide to a target nucleic acid.

[0229] In some embodiments the site-directed polypeptide and genome-targeting nucleic acid can each be administered separately to a cell or a patient. On the other hand, in some other embodiments the site- directed polypeptide can be pre-complexed with one or more guide RNAs, or one or more crRNA together with a tracrRNA. The pre-complexed material can then be administered to a cell or a patient. Such pre-complexed material is known as a ribonucleoprotein particle (RNP).

[0230] Genome-Targeting Nucleic Acid or Guide RNA. Genomic editing components may include a genome-targeting nucleic acid that can direct the activities of an associated polypeptide (e.g., a site- directed polypeptide or DNA endonuclease) to a specific target sequence within a target nucleic acid. In some embodiments, the genome-targeting nucleic acid is an RNA. A genome-targeting RNA is referred to as a "guide RNA" or "gRNA" herein. A guide RNA has at least a spacer sequence that hybridizes to a target nucleic acid sequence of interest and a CR1SPR repeat sequence. In Type II systems, the gRNA also has a second RNA called the tracrRNA sequence. In the Type II guide RNA (gRNA), the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. In the Type V guide RNA (gRNA), the crRNA forms a duplex. In both systems, the duplex binds a site-directed polypeptide such that the guide RNA and site-direct polypeptide form a complex. The genome-targeting nucleic acid provides target specificity to the complex by virtue of its association with the site-directed polypeptide. The genome-targeting nucleic acid thus directs the activity of the site-directed polypeptide.

[0231] In some embodiments, the genome-targeting nucleic acid is a double-molecule guide RNA. In some embodiments, the genome-targeting nucleic acid is a single-molecule guide RNA. A double- molecule guide RNA has two strands of RNA. The first strand has in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence and a minimum CRISPR repeat sequence. The second strand has a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3' tracrRNA sequence and an optional tracrRNA extension sequence. A single-molecule guide RNA (sgRNA) in a Type II system has, in the 5’ to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3' tracrRNA sequence and an optional tracrRNA extension sequence. The optional tracrRNA extension may have elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker links the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension has one or more hairpins. A single-molecule guide RNA (sgRNA) in a Type V system has, in the 5' to 3’ direction, a minimum CRISPR repeat sequence and a spacer sequence.

[0232] By way of illustration, guide RNAs used in the CRISPR / Cas / Cpf 1 system, or other smaller RNAs can be readily synthesized by chemical means as illustrated below and described in the art. While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high performance liquid chromatography (HPLC), which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides. One approach used for generating RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding a Cas9 or Cpfl endonuclease, are more readily generated enzymatically. Various types of RNA modifications can be introduced during or after chemical synthesis and / or enzymatic generation of RNAs, e.g., modifications that enhance stability, reduce the likelihood or degree of innate immune response, and / or enhance other attributes, as described in the art.

[0233] In some embodiments of genome-targeting nucleic acids, a spacer extension sequence can modify activity, provide stability and / or provide a location for modifications of a genome-targeting nucleic acid. A spacer extension sequence can modify on- or off-target activity or specificity. In some embodiments, a spacer extension sequence is provided. A spacer extension sequence can have a length of more than 1, 5, 10, 15. 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, or 7000 or more nucleotides. A spacer extension sequence can have a length of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, or 7000 or more nucleotides. A spacer extension sequence can have a length of less than 1, 5, 10, 15, 20. 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280. 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, 7000 or more nucleotides. In some embodiments, a spacer extension sequence is less than 10 nucleotides in length. In some embodiments, a spacer extension sequence is between 10-30 nucleotides in length. In some embodiments, a spacer extension sequence is between 30-70 nucleotides in length.

[0234] In some embodiments, the spacer extension sequence has another moiety (e.g., a stability control sequence, an endoribonuclease binding sequence, a ribozyme). In some embodiments, the moiety decreases or increases the stability of a nucleic acid targeting nucleic acid. In some embodiments, the moiety is a transcriptional terminator segment (i.e., a transcription termination sequence). In some embodiments, the moiety functions in a eukaryotic cell. In some embodiments, the moiety functions in a prokaryotic cell. In some embodiments, the moiety functions in both eukaryotic and prokaryotic cells. Non-limiting examples of suitable moieties include: a 5' cap (e.g., a 7-methylguanylate cap (m7 G)), a riboswitch sequence (e.g., to allow for regulated stability and / or regulated accessibility by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets the RNA to a subccllular location (e.g., nucleus, mitochondria, chloroplasts, and the like), a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.), and / or a modification or sequence that provides a binding site for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and the like).

[0235] The spacer sequence hybridizes to a sequence in a target nucleic acid of interest. The spacer of a genome-targeting nucleic acid interacts with a target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing). The nucleotide sequence of the spacer thus varies depending on the sequence of the target nucleic acid of interest.

[0236] In a CRISPR / Cas system herein, the spacer sequence is designed to hybridize to a target nucleic acid that is located 5' of a PAM of the Cas9 enzyme used in the system. The spacer can perfectly match the target sequence or can have mismatches. Each Cas9 enzyme has a particular PAM sequence that it recognizes in a target DNA. Tor example, S. pyogenes recognizes in a target nucleic acid a PAM that has the sequence 5'-NRG-3', where R has either A or G, where N is any nucleotide and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence.

[0237] In some embodiments, the target nucleic acid sequence has 20 nucleotides. In some embodiments, the target nucleic acid has less than 20 nucleotides. In some embodiments, the target nucleic acid has more than 20 nucleotides. In some embodiments, the target nucleic acid has at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid has at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid sequence has 20 bases immediately 5' of the first nucleotide of the PAM. For example, in a sequence having 5'-NNNNNNNNNNNNNNNNNNNNNRG-3', the target nucleic acid has the sequence that corresponds to the Ns, wherein N is any nucleotide, and the underlined NRG sequence (R is G or A) is the Streptococcus pyogenes Cas9 PAM. In some embodiments, the PAM sequence used in the compositions and methods of the present disclosure as a sequence recognized by S.p. Cas9 is NGG.

[0238] In some embodiments, the spacer sequence that hybridizes to the target nucleic acid has a length of at least about 6 nucleotides (nt). The spacer sequence can be at least about 6 nt, about 10 nt, about 15 nt, about 18 nt, about 19 nt, about 20 nt, about 25 nt, about 30 nt, about 35 nt or about 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50 nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, from about 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt to about 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about 45 nt, from about 10 nt to about 40 nt, from about 10 nt to about 35 nt, from about 10 nt to about 30 nt, from about 10 nt to about 25 nt, from about 10 nt to about 20 nt, from about 10 nt to about 19 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt. In some embodiments, the spacer sequence has 20 nucleotides. In some embodiments, the spacer has 19 nucleotides. In some embodiments, the spacer has 18 nucleotides. In some embodiments, the spacer has 17 nucleotides. In some embodiments, the spacer has 16 nucleotides. In some embodiments, the spacer has 15 nucleotides.

[0239] In some embodiments, the percent complementarity between the spacer sequence and the target nucleic acid is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. In some embodiments, the percent complementarity between the spacer sequence and the target nucleic acid is at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, at most about 97%, at most about 98%, at most about 99%, or 100%. In some embodiments, the percent complementarity between the spacer sequence and the target nucleic acid is 100% over the six contiguous 5'-most nucleotides of the target sequence of the complementary strand of the target nucleic acid. In some embodiments, the percent complementarity between the spacer sequence and the target nucleic acid is at least 60% over about 20 contiguous nucleotides. In some embodiments, the length of the spacer sequence and the target nucleic acid can differ by 1 to 6 nucleotides, which can be thought of as a bulge or bulges.

[0240] In some embodiments, the spacer sequence is designed or chosen using a computer program. The computer program can use variables, such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, % GC, frequency of genomic occurrence (e.g., of sequences that are identical or are similar but vary in one or more spots as a result of mismatch, insertion or deletion), methylation status, presence of SNPs, and the like.

[0241] Zinc Finger Nucleases. Zinc finger nucleases (ZFNs) are modular proteins having an engineered zinc finger DNA binding domain linked to the catalytic domain of the type II endonuclease Fokl. Because FokI functions only as a dimer, a pair of ZFNs must be engineered to bind to cognate target "half-site" sequences on opposite DNA strands and with precise spacing between them to enable the catalytically active Fokl dimer to form. Upon dimerization of the Fokl domain, which itself has no sequence specificity per se, a DNA double-strand break is generated between the ZFN half-sites as the initiating step in genome editing. The DNA binding domain of each ZFN generally has 3-6 zinc fingers of the abundant Cys2-His2 architecture, with each finger primarily recognizing a triplet of nucleotides on one strand of the target DNA sequence, although cross-strand interaction with a fourth nucleotide also can be important. Alteration of the amino acids of a finger in positions that make key contacts with the DNA alters the sequence specificity of a given finger. Thus, a four-finger zinc finger protein will selectively recognize a 12 bp target sequence, where the target sequence is a composite of the triplet preferences contributed by each finger, although triplet preference can be influenced to varying degrees by neighboring fingers. An important aspect of ZFNs is that they can be readily re-targeted to almost any genomic address simply by modifying individual fingers, although considerable expertise is required to do this well. In most applications of ZFNs, proteins of 4-6 fingers are used, recognizing 12-18 bp respectively. Hence, a pair of ZFNs will generally recognize a combined target sequence of 24-36 bp, not including the 5-7 bp spacer between half-sites. The binding sites can be separated further with larger spacers, including 15-17 bp. A target sequence of this length is likely to be unique in the human genome, assuming repetitive sequences or gene homologs are excluded during the design process. Nevertheless, the ZFN protein-DNA interactions are not absolute in their specificity so off-target binding and cleavage events do occur, either as a heterodimer between the two ZFNs, or as a homodimer of one or the other of the ZFNs. The latter possibility has been effectively eliminated by engineering the dimerization interface of the FokI domain to create "plus" and "minus" variants, also known as obligate heterodimer variants, which can only dimerize with each other, and not with themselves. Forcing the obligate heterodimer prevents formation of the homodimer. This has greatly enhanced specificity of ZFNs, as well as any other nuclease that adopts these FokI variants.

[0242] A variety of ZFN-based systems have been described in the ait, modifications thereof are regularly reported, and numerous references describe rules and parameters that are used to guide the design of ZFNs; see, e.g., Segal et al., Proc Natl Acad Sci USA 96(6):2758-63 (1999); Dreier B et al., J Mol Biol. 303(4):489-502 (2000); Liu Q et al., J Biol Chem. 277(6):3850-6 (2002); Dreier et al., J Biol Chem 280(42):35588-97 (2005); and Dreier et al., J Biol Chem. 276(31):29466-78 (2001).

[0243] Transcription Activator-Like Effector Nucleases (TALENs). TALENs represent another format of modular nucleases whereby, as with ZFNs, an engineered DNA binding domain is linked to the FokI nuclease domain, and a pair of TALENs operate in tandem to achieve targeted DNA cleavage. The major difference from ZFNs is the nature of the DNA binding domain and the associated target DNA sequence recognition properties. The TALEN DNA binding domain derives from TALE proteins, which were originally described in the plant bacterial pathogen Xanthomonas sp. TALEs have tandem arrays of 33-35 amino acid repeats, with each repeat recognizing a single base pair in the target DNA sequence that is generally up to 20 bp in length, giving a total target sequence length of up to 40 bp. Nucleotide specificity of each repeat is determined by the repeat variable diresidue (RVD), which includes just two amino acids at positions 12 and 13. The bases guanine, adenine, cytosine and thymine are predominantly recognized by the four RVDs: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly, respectively. This constitutes a much simpler recognition code than for zinc fingers, and thus represents an advantage over the latter for nuclease design. Nevertheless, as with ZFNs, the protein-DNA interactions of TALENs are not absolute in their specificity, and TALENs have also benefitted from the use of obligate heterodimer variants of the FokI domain to reduce off-target activity.

[0244] Additional variants of the FokI domain have been created that are deactivated in their catalytic function. If one half of either a TALEN or a ZFN pair contains an inactive FokI domain, then only single- strand DNA cleavage (nicking) will occur at the target site, rather than a DSB. The outcome is comparable to the use of CRISPR / Cas9 / Cpfl "nickase" mutants in which one of the Cas9 cleavage domains has been deactivated. DNA nicks can be used to drive genome editing by HDR, but at lower efficiency than with a DSB. The main benefit is that off-target nicks are quickly and accurately repaired, unlike the DSB, which is prone to NHEJ-mediated mis-repair.

[0245] A variety of TALEN-based systems have been described in the art, and modifications thereof are regularly reported; see, e.g., Boch, Science 326(5959): 1509-12 (2009); Mak et aL, Science 335(6069):716-9 (2012); and Moscou et al., Science 326(5959): 1501 (2009). The use of TALENs based on the "Golden Gate" platform, or cloning scheme, has been described by multiple groups; see, e.g., Cermak et al., Nucleic Acids Res. 39(12):e82 (2011); Li et al.. Nucleic Acids Res. 39(14):6315-25 (2011); Weber et al., PLoS One. 6(2):el6765 (2011); Wang et al., J Genet Genomics 41(6):339-47, Epub 2014 Can 17 (2014); and Cermak T et al., Methods Mol Biol. 1239:133-59 (2015).

[0246] Homing Endonucleases. Homing endonucleases (HEs) are sequence-specific endonucleases that have long recognition sequences (14-44 base pairs) and cleave DNA with high specificity-often at sites unique in the genome. There are at least six known families of HEs as classified by their structure, including LAGLIDADG, GIY-YIG, His-Cis box, H-N-H, PD-(D / E)xK, and Vsr-like that are derived from a broad range of hosts, including eukarya, protists, bacteria, archaea, cyanobacteria and phage. As with ZFNs and TALENs, HEs can be used to create a DSB at a target locus as the initial step in genome editing. In addition, some natural and engineered HEs cut only a single strand of DNA, thereby functioning as site-specific nickases. The large target sequence of HEs and the specificity that they offer have made them attractive candidates to create site-specific DSBs.

[0247] A variety of HE-based systems have been described in the art, and modifications thereof are regularly reported; see, e.g., the reviews by Steentoft et al., Glycobiology 24(8):663-80 (2014); Belfort and Bonocora, Methods Mol Biol. 1123:1-26 (2014); Hafez and Hausncr, Genome 55(8):553-69 (2012); and references cited therein. MegaTAL / Tev-mTALEN / MegaTev. As further examples of hybrid nucleases, the MegaTAL platform and Tev-mTALEN platform use a fusion of TALE DNA binding domains and catalytically active HEs, taking advantage of both the tunable DNA binding and specificity of the TALE, as well as the cleavage sequence specificity of the HE; see, e.g., Boissel et al., NAR 42: 2591-2601 (2014); Kleinstiver et al., G3 4:1155-65 (2014); and Boissel and Scharenberg, Methods Mol. Biol. 1239: 171-96 (2015).

[0248] In a further variation, the MegaTev architecture is the fusion of a meganuclease (Mega) with the nuclease domain derived from the GIY-YIG homing endonuclease I-TevI (Tev). The two active sites are positioned about 30 bp apart on a DNA substrate and generate two DSBs with non-compatible cohesive ends; see, e.g., Wolfs et al., NAR 42, 8816-29 (2014). It is anticipated that other combinations of existing nuclease-based approaches will evolve and be useful in achieving the targeted genome modifications described herein. dCas9-Fokl or dCpfl -Fokl and Other Nucleases. Combining the structural and functional properties of the nuclease platforms described above offers a further approach to genome editing that can potentially overcome some of the inherent deficiencies. As an example, the CRISPR genome editing system generally uses a single Cas9 endonuclease to create a DSB. The specificity of targeting is driven by a 20 or 22 nucleotide sequence in the guide RNA that undergoes Watson-Crick base-pairing with the target DNA (plus an additional 2 bases in the adjacent NAG or NGG PAM sequence in the case of Cas9 from S. pyogenes). Such a sequence is long enough to be unique in the human genome, however, the specificity of the RNA / DNA interaction is not absolute, with significant promiscuity sometimes tolerated, particularly in the 5' half of the target sequence, effectively reducing the number of bases that drive specificity. One solution to this has been to completely deactivate the Cas9 or Cpfl catalytic functionretaining only the RNA-guided DNA binding function— and instead fusing a Fokl domain to the deactivated Cas9; see, e.g., Tsai et al., Nature Biotech 32: 569-76 (2014); and Guilinger et al., Nature Biotech. 32: 577-82 (2014). Because Fokl must dimerize to become catalytically active, two guide RNAs are required to tether two Fokl fusions in close proximity to form the dimer and cleave DNA. This essentially doubles the number of bases in the combined target sites, thereby increasing the stringency of targeting by CRISPR-based systems.

[0249] As further example, fusion of the TALE DNA binding domain to a catalytically active HE, such as LTevI, takes advantage of both the tunable DNA binding and specificity of the TALE, as well as the cleavage sequence specificity of LTevI, with the expectation that off-target cleavage can be further reduced.

[0250] Additional details regarding gene editing systems that find use in embodiments of the invention may be found in United States Published Patent Application Publication No. 20210348159. Additional components

[0251] In some embodiments, a delivery composition may include a DNA and / or BAF modulator, as well as one or more additional components. In some instances, the one or more additional components may be one or more additional components of a gene editing system, e.g., as described above, which one or more additional components mediate genomic integration of the DNA. As such, a DNA lipid nanoparticle may further include one or more of: an agent that modulates BAF, a guide RNA, an endonuclease, or a nucleic acid coding sequence therefore, e.g., RNA (such as mRNA) or DNA, etc.

[0252] The one or more additional compounds can be a therapeutic agent. The therapeutic agent can be selected from any class suitable for the therapeutic objective. In other words, the therapeutic agent can be selected from any class suitable for the therapeutic objective. In other words, the therapeutic agent can be selected according to the treatment objective and biological action desired. For example, if the DNA within the LNP is useful for treating cancer, the additional compound can be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate). In another example, if the LNP containing the DNA is useful for treating an infection, the additional compound can be an antimicrobial agent (e.g., an antibiotic or antiviral compound). In yet another example, if the LNP containing the DNA is useful for heating an immune disease or disorder, the additional compound can be a compound that modulates an immune response (e.g., an immunosuppressant, immunostimulatory compound, or compound modulating one or more specific immune pathways). In some embodiments, different cocktails of different lipid nanoparticles containing different compounds, such as a DNA encoding a different protein or a different compound, such as a therapeutic may be used in the compositions and methods of the invention. In some embodiments, the additional compound is an immune modulating agent. For example, the additional compound is an immunosuppressant. In some embodiments, the additional compound is immune stimulatory agent.

[0253] COMPOSITIONS

[0254] Also provided are compositions that find use in practicing embodiments of the invention. Compositions of the invention include those having a DNA and / or BAF modulator, e.g., as described, where the DNA and / or BAF modulator may be present in combination with one or more additional components, such as but not limited to, components of a gene editing system, e.g., as described above, such as a gRNA, endonuclease, or nucleic acid encoding the same, etc.

[0255] In some embodiments, a composition can have a DNA and / or BAF modulator cytosolic delivery vehicle, e.g., as described above, such as liposome or a lipid nanoparticle or other non-naturally occurring delivery vehicle. Therefore, in some embodiments, any compounds (e.g., a DNA endonuclease or a nucleic acid encoding thereof, gRNA and DNA and / or BAF modulator) of the composition can be formulated in a liposome or lipid nanoparticle. In some embodiments, one or more such compounds are associated with a liposome or lipid nanoparticle via a covalent bond or non-covalent bond. In some embodiments, any of the compounds can be separately or together contained in a liposome or lipid nanoparticle. Therefore, in some embodiments, each of a DNA endonuclease or a nucleic acid encoding thereof, gRNA and DNA and / or BAF modulator (donor template) is separately formulated in a liposome or lipid nanoparticle. In some embodiments, a DNA endonuclease is formulated in a liposome or lipid nanoparticle with gRNA. In some embodiments, a DNA endonuclease or a nucleic acid encoding thereof, gRNA and donor template are formulated in a liposome or lipid nanoparticle together.

[0256] In some embodiments, a composition described above further has one or more additional reagents, where such additional reagents are selected from a buffer, a buffer for introducing a polypeptide or polynucleotide into a cell, a wash buffer, a control reagent, a control vector, a control RNA polynucleotide, a reagent for in vitro production of the polypeptide from DNA, adaptors for sequencing and the like. A buffer can be a stabilization buffer, a reconstituting buffer, a diluting buffer, or the like. In some embodiments, a composition can also include one or more components that can be used to facilitate or enhance the on-target binding or the cleavage of DNA by the endonuclease, or improve the specificity of targeting.

[0257] Also provided herein is a pharmaceutical composition comprising the delivery vehicle - encapsulated DNA and / or BAF modulator and a pharmaceutically acceptable carrier or excipient. In some aspects, the disclosure provides for a lipid nanoparticle formulation further comprising one or more pharmaceutical excipients. In some embodiments, the lipid nanoparticle formulation further comprises sucrose, tris, trehalose and / or glycine.

[0258] In some embodiments, any components of a composition are formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending upon the particular mode of administration and dosage form. In some embodiments, guide RNA compositions are generally formulated to achieve a physiologically compatible pH, and range from a pH of about 3 to a pH of about 11, about pH 3 to about pH 7, depending on the formulation and route of administration. In some embodiments, the pH is adjusted to a range from about pH 5.0 to about pH 8. In some embodiments, the composition has a therapeutically effective amount of at least one compound as described herein, together with one or more pharmaceutically acceptable excipients. Optionally, the composition can have a combination of the compounds described herein, or can include a second active ingredient useful in the treatment or prevention of bacterial growth (for example and without limitation, anti-bacterial or anti- microbial agents), or can include a combination of reagents of the disclosure. In some embodiments, gRNAs are formulated with other one or more oligonucleotides, e.g., a nucleic acid encoding DNA endonuclease and / or a donor template. Alternatively, a nucleic acid encoding DNA endonuclease and a donor template, separately or in combination with other oligonucleotides, are formulated with the method described above for gRNA formulation.

[0259] Suitable excipients can include, for example, carrier molecules that include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Other exemplary excipients include antioxidants (for example and without limitation, ascorbic acid), chelating agents (for example and without limitation, EDTA), carbohydrates (for example and without limitation, dextrin, hydroxyalkylcellulose, and hydroxy alkylmethylcellulose), stearic acid, liquids (for example and without limitation, oils, water, saline, glycerol and ethanol), wetting or emulsifying agents, pH buffering substances, and the like.

[0260] In some embodiments, a composition refers to a therapeutic composition having therapeutic cells, e.g., modified via methods of the invention such as described herein, that are used in an ex vivo treatment method. In some embodiments, therapeutic compositions contain a physiologically tolerable carrier together with the cell composition, and optionally at least one additional bioactive agent as described herein, dissolved or dispersed therein as an active ingredient. In some embodiments, the therapeutic composition is not substantially immunogenic when administered to a mammal or human patient for therapeutic purposes, unless so desired. In general, the genetically-modified, therapeutic cells described herein are administered as a suspension with a pharmaceutically acceptable carrier. One of skill in the art will recognize that a pharmaceutically acceptable carrier to be used in a cell composition will not include buffers, compounds, cryopreservation agents, preservatives, or other agents in amounts that substantially interfere with the viability of the cells to be delivered to the subject. A formulation having cells can include, e.g., osmotic buffers that permit cell membrane integrity to be maintained, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration. Such formulations and suspensions are known to those of skill in the art and / or can be adapted for use with the progenitor cells, as described herein, using routine experimentation. In some embodiments, a cell composition can also be emulsified or presented as a liposome composition, provided that the emulsification procedure does not adversely affect cell viability. The cells and any other active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient, and in amounts suitable for use in the therapeutic methods described herein. Additional agents included in a cell composition can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active compound used in the cell compositions that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.

[0261] KITS

[0262] Aspects of the present disclosure also include kits. Some aspects of this disclosure provide kits that include a DNA and / or BAF modulator, e.g., as described above. Kits of the invention may further include one or more additional components, e.g., an inducing agent (e.g., as described above), a gene editing system or components thereof, e.g., as described above, etc. In some instances, the various components of a given kit may be combined into a single composition, e.g., with a cytosolic delivery vehicle, as a pharmaceutical composition, etc., such as described above.

[0263] In some instances, kits may include an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is a compound of the invention. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate- buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. Components of the kits may be present in separate containers, or multiple components may be present in a single container. In addition to the above-mentioned components, a subject kit may further include instructions for using the components of the kit, e.g., to practice the subject methods. The instructions are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, Hard Disk Drive (HDD), portable flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0264] UTILITY

[0265] The subject methods and compositions, e.g., as described above, can be used in any application where nuclear delivery of a DNA is desired. Applications of interest include both research and therapeutic applications. Applications of interest include, but are not limited to: research applications, diagnostic applications and therapeutic applications. In some instances, nucleic acids that may be introduced into a nucleus via methods of the invention include those encoding research proteins, diagnostic proteins and therapeutic proteins.

[0266] Research proteins are proteins whose activity finds use in a research protocol. As such, research proteins are proteins that are employed in an experimental procedure. The research protein may be any protein that has such utility, where in some instances the research protein is a protein domain that is also provided in research protocols by expressing it in a cell from an encoding vector. Examples of specific types of research proteins include, but are not limited to: transcription modulators of inducible expression systems, members of signal production systems, e.g., enzymes and substrates thereof, hormones, prohormones, proteases, enzyme activity modulators, perturbimers and peptide aptamers, antibodies, modulators of protein-protein interactions, genomic modification proteins, such as CRE recombinase, meganucleases, Zine-finger nucleases, CRISPR / Cas-9 nuclease, TAL effector nucleases, etc., cellular reprogramming proteins, such as Oct 3 / 4, Sox2, Klf4, c-Myc, Nanog, Lin-28, etc., and the like.

[0267] Diagnostic proteins are proteins whose activity finds use in a diagnostic protocol. As such, diagnostic proteins are proteins that are employed in a diagnostic procedure. The diagnostic protein may be any protein that has such utility. Examples of specific types of diagnostic proteins include, but are not limited to: members of signal production systems, e.g., enzymes and substrates thereof, labeled binding members, e.g., labeled antibodies and binding fragments thereof, peptide aptamers and the like.

[0268] Proteins of interest further include therapeutic proteins. Therapeutic proteins are proteins that provide a therapeutic benefit to a patient, and include secreted proteins, transmembrane proteins, and intracellularly acting proteins. As will be appreciated by one of ordinary skill in the art, cargo nucleic acids that encode any protein that is associated with a liver disease or that, upon secretion from the liver, find use in treating another organ in the body, may be delivered using the subject compositions and methods.

[0269] Target cells to which nucleic acids may be delivered in accordance with the invention may vary widely. Target cells of interest include, but are not limited to: cell lines, HeLa, HEK, CHO, 293 and the like, Mouse embryonic stem cells, human stem cells, mesenchymal stem cells, primary cells, tissue samples and the like. Some non-limiting examples of a mammalian cell include, without limitation, a mouse cell, a rat cell, hamster cell, a rodent cell, and a nonhuman primate cell. In some embodiments, the target cell is a human cell. It should also be appreciated that the target cell may be of any cell type. For example, the target cell may be a stem cell, which may include embryonic stem cells, induced pluripotent stem cells (iPS cells), fetal stem cells, cord blood stem cells, or adult stem cells (i.e., tissue specific stem cells). In other cases, the target cell may be any differentiated cell type found in a subject. Cells of interest include both dividing cells and non-dividing cells. Examples of specific target cells of interest include, but are not limited to: hepatocytes, stellate cells, T lymphocytes, B lymphocytes, NK cells, skeletal muscle cells, cardiomyocytes, neurons, astrocytes, oligodendrocytes, dendritic cells, skin cells, etc.

[0270] In some instances, the application of interest is a therapeutic application, for example, in the treatment of a disease. For example, the compositions and methods of the present application may be used to deliver a nucleic acid sequence to the nucleus of a cell to complement a genetic deficiency. As one nonlimiting example, compositions of the present application may be used in the treatment of a genetic deficiency that impacts the function of hepatocytes, or in the treatment of a genetic deficiency elsewhere in the body that can be remedied by leveraging hepatocytes as a biofactory to secrete the deficient protein.

[0271] The following example(s) is / are offered by way of illustration and not by way of limitation.

[0272] EXAMPLES

[0273] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0274] Exogenous DNA becomes associated with nuclear envelope proteins upon entry into the cytoplasm of cells. To assess the extent to which DNA is able to access the nucleus in nondividing cells, growth-arrested HepG2 cells (Fig. 1 -3) and primary hepatocytes (data not shown) were transfected with EdU-labeled doggybone DNA (i.e., doggybone DNA with 5-ethynyl-2’ -deoxyuridine (EdU) incorporated to enable conjugation of a fluorophore during the staining protocol) and analyzed by confocal microscopy 16 hours later. As shown in Fig. 1A and at higher magnification in Fig. 1A’, the EdU-labeled DNA was observed to form puncta throughout the cytoplasm of the cell. To better understand what was driving the formation of these puncta, the cells were incubated with a panel of antibodies that are specific for proteins previously reported to associate with DNA and assessed by immunofluorescence. Two proteins were observed to colocalize to the DNA puncta: Barrier-to-autointegration factor (BAF) and emerin. BAF was observed to reside primarily in the nucleus of untransfected HepG2 controls (Fig.lC and 1C’), and colocalized with DNA in the cytoplasm of EdU-DNA transfected cells (Fig. IB and IB’). Similarly, emerin was observed to reside primarily at the nuclear membrane in untransfected (Fig.2A, B), mock transfected (Fig.2C), and mRNA-transfected (Fig. 2D) cells, but colocalized with the EdU-labeled DNA in DNA-transfected cells (Fig. IE, IF). Confocal microscopy revealed emerin (Fig. 3A and 3C, red) enveloping the EdU-DNA (Fig.3B and 3C, yellow).

[0275] Exogenous mRNAs encoding BAF mutant proteins enhance expression of co-delivered DNA. BAF and emerin are two proteins that have been reported to be involved in the formation of the nuclear envelope around chromosomal DNA following mitosis. BAF is known in the art to bind chromatin and help it to anchor to the nuclear envelope. During mitosis, phosphorylation by vaccinia-related kinase 1 (VRK1) or 2 (VRK2) causes BAF to release chromosomal DNA, allowing the nuclear envelope to disassemble. BAF dephosphorylation by protein phosphatase 2A (PP2A) at the end of mitosis allows BAF to recapture the DNA and, working in concert with LEM-domain containing proteins LAP2, emerin and MANI, mediate its packaging within a new nuclear envelope. BAF has also been reported to recognize and bind foreign (viral and plasmid) DNA immediately following entry into the cytoplasm, nucleating the formation of a nuclear envelope-like structure around the DNA so as to sequester the DNA for degradation.

[0276] To determine if inhibiting BAF could render more DNA available to translocate to the nucleus, we attempted to create a BAF-deficient cell line by CRISPR-directed knockout; however, this deficiency was lethal, indicating that at least some amount of BAF is required for cell viability. In an alternative strategy, preparations of lipid nanoparticles (LNPs) were co-formulated with a nanoplasmid DNA (npDNA) encoding a human Factor IX (hFIX) expression cassette and an mRNA encoding a BAF mutant comprising a L58R mutation, which is predicted to disrupt BAF’s binding to LEM-domain containing proteins and the formation / stability of the DNA-BAF-LEM complex. Four different LNP formulations were generated following methods as described in PCT Application Serial No. PCT / US2023 / 079923 filed on November 15, 2023, the disclosure of which is herein incorporated by reference. Each formulation comprised a different weight ratio of DNA to BAF - 1:2, 1:1, 1:0.33, and 1:0. The LNP formulations were administered to cohorts of wild type mice by i.v. injection at a dose of 0.3 mg / kg DNA (resulting in doses of 0.3mg / kg DNA / 0.6 mg / kg mRNA [Fig.4, group 1]; 0.3 mg / kg DNA / 0.3 mg / kg mRNA [Fig.4, group 2] ; 0.3 mg / kg DNA / 0.1 mg / kg mRNA [Fig.4, group 3]; and 0.3 mg / kg DNA / 0.03 mg / kg mRNA [Fig.4, group 4, respectively), and the amount of hFIX in the plasma of the treated mice was detected by ELISA 7 days later. In these experiments and below, the concentration of hFIX in plasma is reported relative to the amount of hFIX in plasma of normal human samples, i.e. “% normal hFIX levels”. Given that the average concentration of hFIX in pooled normal human samples is 5 ug / ml, 100% normal human Factor IX levels is approximately 5 ug / ml.

[0277] Expecting that the mRNA encoding the BAF modulator would take time to be translated while BAF and emerin would colocalize with DNA and begin to sequester it within a nuclear envelope-type structure immediately upon entry of the DNA into the cell, we were surprised to detect levels of hFIX in the plasma of mice injected with LNPs comprising DNA+BAF modulator that were 4-to 150-fold greater than in mice injected with LNPs comprising only the npDNA (Fig.4, groups 1-4 versus DNA only group 6). Expression levels appeared to be dependent on the dose of BAF modulator delivered.

[0278] To determine if these expression levels could be increased further by actively driving nuclear translocation of the available DNA, an LNP was formulated comprising a npDNA encoding the human Factor IX (hFIX) expression cassette and comprising a UAS DNA targeting sequence (DTS) as disclosed in PCT / US2023 / 035932; an mRNA encoding the BAF-L58R protein; and an mRNA encoding a GAL4 nuclear translocating factor (NTF), i.e. a GAL4 protein fused to an NLS at its C terminus, as disclosed in PCT / US2023 / 035932, in a weight ratio of 1 : 1 :2 respectively. Administration of this LNP at a dose of 0.3 mg / kg DNA / 0.3 mg / kg BAF modulator mRNA / 0.6 mg / kg GAL4 mRNA yielded amounts of hFIX protein in plasma at day 7 that were 200-fold greater than the amounts achieved by the LNP comprising the npDNA alone (Fig.4, group 5 versus DNA only group 6), and 4-fold greater than the LNP comprising the npDNA + BAF modulator (Fig.4, group 5 versus group 2).

[0279] The aforementioned study was performed with an mRNA encoding a mouse BAF-L58R protein. To determine if a human BAF protein comprising the L58R substitution would behave similarly, the UAS-hFIX-npDNA, the GAL4 mRNA, and either the mRNA encoding the mouse BAF-L58R protein used previously or an mRNA encoding a human BAF-L58R protein were coformulated into LNPs in a 1:1:2 weight ratio and administered to mice by i.v. bolus injection at a dose of 0.3 mg / kg DNA / 0.3 mg / kg BAF modulator mRNA / 0.6 mg / kg GAL4 mRNA. The human BAF-L58R appeared to be approximately 50% as efficient as the mouse BAF-L58R at facilitating DNA uptake and translocation by the nuclear translocating factor in mouse tissue (Fig.5, group 2 versus group 1), suggesting some species selectivity.

[0280] To determine how the kinetics of the BAF modulator impacted DNA delivery and expression, an mRNA was designed encoding a mouse BAF-L58R protein modified to comprise an alternative UTR known to reduce the half-life of the mRNA (UTR38, disclosed above). The UAS-hFIX-npDNA, the GAL4 mRNA, and the mRNA encoding the BAF-L58R with UTR38 were co-formulated into LNPs in a 1:1:2 weight ratio and administered to mice by i.v. bolus injection at a dose of 0.3 mg / kg DNA / 0.3 mg / kg BAF modulator mRNA / 0.6 mg / kg GAL4 mRNA. Decreasing the stability of the BAF modulator mRNA yielded a 2-fold increase in expression from the npDNA expression cassette (Fig.5, group 3) over the unmodified mouse BAF-L58R protein (Fig.5, group 1).

[0281] To determine if forced nuclear localization of the BAF-L58R protein could further enhance delivery of DNA to the nucleus, e.g. as a nuclear translocating factor, an mRNA was designed to encode a protein that comprised the mouse BAF-L58R protein with an NLS at the C-terminus. The UAS-hFlX- npDNA, the mRNA encoding the BAF modulator (i.e. mBAF-L58R or mBAF-L58R-C’NLS), and an mRNA encoding GAL4 were co-formulated into LNPs in a 1:0.3:1 weight ratio and administered to mice by i.v. bolus injection at a dose of 0.3 mg / kg DNA / 0.1 mg / kg BAF modulator mRNA 10.3 mg / kg GAL4 mRNA. Inclusion of an NLS enhanced DNA delivery 80-fold (Fig.6, group 2).

[0282] Exogenous mRNAs encoding wild type BAF enhance expression of co-delivered DNA. To determine the effect that an excess amount of wild type BAF protein might have on the sequestration of DNA in the cytoplasm, mRNA encoding wild type mouse BAF protein was co-formulated in LNPs with UAS-hFIX-npDNA and GAL4 mRNA. The nucleic acid species were formulated in a weight ratio of 1:1:1 [DNA : wt BAF mRNA : GAL4 mRNA], and administered to wild type mice by i.v. injection at a dose of 0.3 mg / kg DNA / 0.3 mg / kg wt BAF mRNA / 0.3 mg / kg GAL4 mRNA. Levels of hFIX in plasma were detected at day 7 post-dosing by ELISA (Fig. 7). hFIX levels were augmented by exogenously added wild type BAF 160-fold or more over DNA alone (81% normal human levels with Fig. 7 group 1 versus 0.5% normal human levels for DNA alone historical controls).

[0283] Exogenous mRNAs encoding BAF kinases enhance expression of co-delivered DNA. BAF engagement with DNA is modulated by phosphorylation at BAF’s N-tcrminus, with phosphorylation of residues Threonine 2, Threonine 3, and Serine 4 triggering BAF release of DNA. To determine the effect that a co-delivered BAF kinase would have on BAF’s ability to mediate DNA sequestration, mRNAs encoding the vaccinia B 1 protein Ankara strain and the vaccinia B 1 protein WR strain were co- formulated in LNPs with UAS-hFIX-npDNA and GAL4 mRNA. An LNP co-formulated with UAS- hFIX-npDNA and mRNAs encoding GAL4 and mouse BAF-L58R was also prepared as a control. As before, the nucleic acid species were formulated in a weight ratio of 1:1:2 [DNA : BAF modulator (kinase or BAF-L58R mutant) mRNA : GAL4 mRNA], and administered to wild type mice at a dose of 0.3 mg / kg DNA / 0.3 mg / kg BAF modulator mRNA / 0.6 mg / kg GAL4 mRNA. Levels of hFIX in plasma were detected at day 3 and day 7 post-dosing by ELISA. Surprisingly, hFIX levels were augmented 3000-fold or more over DNA alone by the mRNA encoding the Ankara strain Bl protein (1661% normal human levels with Fig. 8 group 1 versus 0.5% normal human levels for DNA alone historical controls) and 600-fold or more over DNA alone by the mRNA encoding the Western Reserve strain Bl protein (310% normal human level with Fig. 8, group 2 versus 0.5% normal human levels for DNA alone historical controls). Interestingly, relative to the BAF-L58R control (Fig. 8, group 3), a 40-fold boost and 8-fold boost in expression were observed with the Ankara and Western Reserve Bl proteins, respectively, suggesting that BAF kinases may be acting on other proteins in addition to BAF to facilitate DNA delivery to the nucleus or that BAF kinases are more efficient modulators of endogenous BAF than BAF mutant proteins.

[0284] BAF inhibition increases the number of cells expressing protein from the co-delivered DNA. Increases in hFIX levels in plasma following exposure of a tissue to an agent that modulates BAF could be due to the few cells that expressed protein in the absence of agent expressing more protein in the presence of the agent, more cells in the tissue expressing protein, or both. To determine whether the increase in hFIX expression in the mice receiving LNPs comprising BAF modulator was due to an increase in the total number of cells expressing protein, an increase in the amount of protein expressed per cell, or both, immunohistochemistry for hFIX protein was performed on sections of liver from mice dosed with PBS (Fig.9A, 27 days post-dosing) or with LNPs formulated with UAS-hFIX-npDNA (Fig.9B, 27 days post-dosing), with UAS-hFIX-npDNA + GAL4 mRNA (Fig.9C, 27 days post-dosing), or with UAS- hFIX-npDNA + GAL4 mRNA + BAF-L58R mRNA (Fig.9D, 15 days post dosing). As illustrated in Figure 8, it appears that both the number of cells and the amount of protein within each cell increases when mRNA encoding a BAF modulator is included in the formulation, with 15% of cells expressing hFIX in mice receiving the BAF modulator versus 0.5% of cells receiving DNA alone and 4% of cells receiving DNA and the mRNA encoding the nuclear translocating protein GAL4. Additional exemplary embodiments of proteins that modulate BAF and the mRNAs that encode them are provided in Table 10.

[0285] BAF inhibition results in better FIX expression across species, from mice to rats to NHPs. LNP formulations were generated following methods as described in PCT Application Serial No. PCT / US2023 / 079923 filed on November 15, 2023, the disclosure of which is herein incorporated by reference. LNP formulations comprising 0.3 mg / kg FIX npDNA (comprising the hFIX transgene operably linked to a promoter but no BAF modulator DTS sequence) and 0.2 mg / kg of mRNA encoding a mutant human BAF protein of the present disclosure fused to an NLS were intravenously administered to cohorts of wild type mice, rats, or NHPs. Levels of hFIX protein in plasma were detected from day 3 to day 14 post-dosing by ELISA. In mice, delivery of the npDNA + BAF mRNA LNP resulted in hFIX levels over 100% of normal human levels, which persisted over 14 days. In rats, delivery of the npDNA + BAF mRNA LNP resulted in hFIX levels of approximately 100% of normal human levels, which persisted over 14 days. In NHPs, delivery of the npDNA + BAF mRNA LNP resulted in hFIX levels of approximately 5% of normal human levels, which persisted over 14 days.

[0286] BAF inhibition results in better FIX expression in human cells in vitro. Primary human hepatocytes (PHHs) were plated in 384-well collagen-coated tissue culture plates. LNP formulations comprising 40 ng GFP npDNA and 20 ng mRNA encoding BAF variants of the present disclosure were added to the PHHs in OptiMEM with 45 ug / mL ApoE. GFP fluorescence intensity was measured in each well 72 h post-transfection. BAF variant 3 produced the highest increase in GFP expression in human cells, approximately 5-fold higher than the GFP signal observed in untreated cells (Fig. 11).

[0287] Table 10. Exemplary embodiments of agents that modulate BAF In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0288] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g.. “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the ai t will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g. , “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0289] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0290] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub- ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub- ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0291] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0292] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.c., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0293] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

1. WHAT IS CLAIMED IS:

1. A composition for the delivery of DNA to the nucleus of a cell, the composition comprising: a cytosolic delivery vehicle, a DNA, and an agent that modulates the protein Barrier to Autointegration Factor (BAF).

2. The composition according to claim 1, wherein the agent is a protein comprising an amino acid sequence having a sequence identity of 90% or more to wild type BAF, or an mRNA encoding the protein comprising an amino acid sequence having a sequence identity of 90% or more to wild type BAF.

3. The composition according to claim 2, wherein the protein comprises a mutation relative to wild type human BAF selected from the group consisting of T2D, T3D, S4D, A12T, E28D, G31S, K33R, E36D, R37K, L58R, A71K, and G79R.

4. The composition according to claim 2, wherein the protein comprises a heterologous UTR.

5. The composition according to claim 1, wherein the agent is a BAF kinase or an mRNA encoding a BAF kinase.

6. The composition according to claim 5, wherein the BAF kinase comprises an amino acid sequence having a sequence identity of 90% or more to vaccinia virus Ankara strain Bl protein.

7. The composition according to claim 6, wherein the BAF kinase comprises an amino acid sequence having a sequence identity of 90% or more to human VRK1, VRK1-X1, human VRK2A, human VRK2B, or human VRK3.

8. The composition according to claim 2, wherein the agent is a siRNA that is specific for BAF.

9. The composition according to claim 2, wherein the agent is a small molecule.

10. The composition according to claim 9, wherein the small molecule is selected from the group consisting of obtusilactone B, Mahubanolide, kotomolide B, epilitsenolide D2, and rabeprazole.

11. The composition according to claim 1, wherein the agent comprises a LEM domain or an mRNA encoding a LEM domain.

12. The composition according to claim 11, wherein the LEM domain comprises an animo acid sequence having a sequence identity of 90% or more to a sequence selected from the group consisting of: a. an emerin LEM domainb. a LAP2-beta LEM domain:c. a MANI LEM domain:

13. The composition according to claim 1, wherein the agent does not comprise a LEM domain or a nucleic acid encoding a LEM domain.

14. The composition according to any one of claims 1-13, wherein the cytosolic delivery vehicle comprises a non- viral delivery vehicle.

15. The composition according to claim 14, wherein the non-viral delivery vehicle is a nanoparticle.

16. The composition according to claim 15, wherein the nanoparticle is a lipid nanoparticle (LNP).

17. The composition according to claim 14, wherein the non-viral delivery vehicle is a vesicle.

18. The composition according to claim 17, wherein the vesicle is selected from the group consisting of an exosome, an extracellular vesicle, and a micelle.

19. The composition according to any one of claims 1-18, wherein the composition comprises a single cytosolic delivery vehicle comprising both the DNA and the agent.

20. The composition according to any one of claims 1-18, wherein the composition comprises a first cytosolic delivery vehicle comprising the DNA and a second cytosolic delivery vehicle comprising the agent.

21. The composition according to any one of claims 1-20, wherein the composition further comprises an mRNA encoding a nuclear targeting factor (NTF), and the DNA comprises a DNA nuclear targeting sequence (DTS).

22. The composition according to claim 21, wherein: a. the NTF is a Gal4 NTF and the DTS is a Gal4 binding sequence; or b. the NTF is a TetR NTF and the DTS is a TetR binding sequence.

23. The composition according to any one of claims 1-22, wherein the composition comprises a nuclease, an integrase or a transposase.

24. The composition according to claim 23, wherein the composition encodes a nuclease and the composition comprises a guide RNA (gRNA).

25. The composition according to any one of claims 1-24, wherein the DNA is 100 - 15,000 bp.

26. The composition according to any one of claims 1-25, wherein the DNA comprises a coding sequence.

27. The composition according to claim 26, wherein the DNA comprises an expression cassette comprising a promoter operably linked to the coding sequence.

28. The composition according to claims 26 or claim 27, wherein the DNA comprises sequences flanking the coding sequence of claim 26 or the expression cassette of claim 27 that are homologous to genomic sequences of the cell for which the DNA is configured to be used.

29. A method comprising contacting a cell with a DNA and an agent that modulates the activity of the protein Barrier to Autointegration Factor (BAF).

30. The method according to claim 29, wherein the agent is a protein comprising an amino acid sequence having a sequence identity of 90% or more to wild type BAF, or an mRNA encoding the protein comprising an amino acid sequence having a sequence identity of 90% or more to wild type BAF.

31. The method according to claim 30, wherein the protein comprises a mutation relative to wild type human BAF selected from the group consisting of T2D, T3D, S4D, A12T, E28D, G31S, K33R, E36D, R37K,L58R, A71K, and G79R.

32. The method according to claim 30, wherein the protein comprises a heterologous UTR.

33. The method according to claim 29, wherein the agent is a BAF kinase or an mRNA encoding a BAF kinase.

34. The method according to claim 33, wherein the BAF kinase comprises an amino acid sequence having a sequence identity of 90% or more to the vaccinia virus Ankara strain B 1 protein.

35. The method according to claim 33, wherein the BAF kinase comprises a sequence having a sequence identity of 90% or more to human VRK1, VRK1-X1, human VRK2A, human VRK2B, or human VRK3.

36. The method according to claim 29, wherein the agent is a siRNA that is specific for BAF.

37. The method according to claim 29, wherein the agent is a small molecule.

38. The method according to claim 37, wherein the small molecule is selected from the group consisting of obtusilactone B, Mahubanolide, kotomolide B, epilitsenolide D2, and rabeprazole.

39. The method according to claim 29, wherein the agent comprises a LEM domain or an mRNA encoding a LEM domain.

40. The method according to claim 39, wherein the LEM domain comprises a sequence having a sequence identity of 90% or more to a sequence selected from the group consisting of:a. an emerin LEM domainMDNYADLSDTELTTLLRRYNIPHGPVVGSTRRLYEKKIFEYETQ; b. a LAP2-beta LEM domain:DLDVTELTNEDLLDQLVKYGVNPGPIVGTTRKLYEKKLLKLREQ; and c. a MANI LEM domain:ASAPQQLSDEELFSQLRRYGLSPGPVTESTRPVYLKKLKKLREE.

41. The method according to claim 29, wherein the agent does not comprise a LEM domain or a nucleic acid encoding a LEM domain.

42. The method according to any one of claims 29-41, wherein the cell is simultaneously or sequentially contacted with the DNA and the agent.

43. The method according to any one of claims 29-42, wherein the DNA and the agent are present in a cytosolic delivery vehicle.

44. The method according to claim 43, wherein the DNA and the agent are present in the same cytosolic delivery vehicle.

45. The method according to claim 43, wherein the DNA and the agent are present in different cytosolic delivery vehicles.

46. The method according to any one of claims 43-45, wherein the cytosolic delivery vehicle comprises a nanoparticle.

47. The method according to claim 46, wherein the nanoparticle is a lipid nanoparticle.

48. The method according to any one of claims 44-45, wherein the cytosolic delivery vehicle comprises a vesicle.

49. The method according to claim 48, wherein the vesicle is selected from the group consisting of an exosome, an extracellular vesicle, and a micelle.

50. The method according to any one of claims 29-42, wherein the method comprises electroporating the cell.

51. The method according to any one of claims 29-50, wherein the cell is in vivo.

52. The method according to claim 51 , wherein the method comprises orally, parenterally, subretinally, intravitreally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenously, intra-arterially, intraperitoneally, intracranially, subcutaneously, intramuscularly, intranasally, intrathecally and / or intraarticularly delivering the DNA and the agent.

53. The method according to any one of claims 29-52, wherein the cell is ex vivo.

54. The method according to any one of claims 29-53, wherein the cell is selected from the group consisting of hepatocytes, stellate cells, T lymphocytes, B lymphocytes, NK cells, hematopoietic stem cells, skeletal muscle cells, cardiomyocytes, neurons, astrocytes, oligodendrocytes, dendritic cells and skin cells.

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