Compositions and methods for delivery of nucleic acids to cells

Abstract

To provide compositions and methods for delivery of nucleic acids to cells.SOLUTION: Compositions and methods of use thereof for delivering nucleic acid cargo into cells are provided. The compositions typically include: (a) a 3E10 monoclonal antibody or an antigen binding, cell-penetrating fragment thereof; a monovalent, divalent or multivalent single-chain variable fragment (scFv); or a diabody; or a humanized form or variant thereof; and (b) nucleic acid cargo including, for example, a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof. Elements (a) and (b) are typically non-covalently linked to form a complex.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 944,281, filed on December 5, 2019, entitled "Compositions And Methods For Delivery Of Nucleic Acids To Cells"; International Application No. PCT / US2019 / 048953, filed on August 30, 2019, entitled "Compositions And Methods For Enhancing Donor Oligonucleotide - Based Gene Editing"; and International Application No. PCT / US2019 / 048962, filed on August 30, 2019, entitled "Compositions And Methods For Enhancing Triplex And Nuclease - Based Gene Editing". U.S. Provisional Patent Application No. 62 / 944,281, International Application No. PCT / US2019 / 048953, U.S. Provisional Patent Application No. 62 / 725,920, International Application No. PCT / US2019 / 048962, and U.S. Provisional Patent Application No. 62 / 725,852 are hereby specifically incorporated by reference in their entireties. Description of Research and Development Sponsored by the Federal Government

[0002] This invention was made with government support under Grant No. CA197574 awarded by the National Institutes of Health. The government has certain rights in this invention. Reference to Sequence Listing

[0003] The sequence listing, submitted as a text file named "YU_7503_3_ST25" created on August 31, 2020, and having a size of 154,701 bytes, is hereby incorporated by reference into this specification in accordance with 37 C.F.R.§1.52(e)(5) of the United States Patent Law Regulations. Field of the Invention

[0004] The present invention generally relates to the field of intracellular delivery of nucleic acids for uses including, but not limited to, gene therapy and gene editing in vitro, ex vivo, and in vivo. BACKGROUND OF THE INVENTION

[0005] Background of the Invention Gene therapy includes a wide variety of uses, from gene replacement or knockdown for genetic or acquired diseases such as cancer to vaccination. Viral vectors and synthetic liposomes have emerged as the media of choice for many current uses, but both have limitations and risks including production complexity, limited packaging capacity, and unfavorable immunological characteristics, which limit the use of gene therapy and prevent the possibility of preventive gene therapy (Seow and Wood, Mol Ther. 17(5):767 - 777(2009)). In vivo uptake and distribution of nucleotides have been observed in cells and tissues (Huang et al., FEBS Lett., 558(1 - 3):69 - 73(2004)). Furthermore, for example, Nyce et al. showed that when antisense oligodeoxynucleotides (ODNs) are inhaled, they bind to endogenous surfactants (lipids produced by lung cells) and are taken up by lung cells without the need for additional carrier lipids (Nyce et al., Nature, 385:721 - 725(1997)), and small nucleic acids are taken up by T24 bladder cancer tissue culture cells (Ma et al., Antisense Nucleic Acid Drug Dev., 8:415 - 426(1998)), so there is still a need to improve nucleic acid transfection technology, especially for in vivo uses. AAV9 is still a commonly used viral vector in humans and was discovered in 2003 (Robbins, "Gene therapy pioneer says the field is behind - and that delivery technology is embarrassing", Stat, November 2019). PRIOR ART DOCUMENTS NON - PATENT LITERATURE

[0006] [Non-Patent Document 1] Seow and Wood, Mol Ther. 17(5):767-777(2009) [Non-Patent Document 2] Huang et al., FEBS Lett., 558(1-3):69-73(2004) [Non-Patent Document 3] Nyce et al., Nature, 385:721-725(1997) [Non-Patent Document 4] Ma et al., Antisense Nucleic Acid Drug Dev., 8:415-426(1998) [Summary of the Invention] [Means for Solving the Problems]

[0007] Accordingly, an object of the present invention is to provide a composition for improving the delivery of nucleic acids to cells and a method for using the same. Summary of the Invention

[0008] A composition for delivering a nucleic acid cargo into a cell and a method for using the same are provided. The composition generally comprises (a) a 3E10 monoclonal antibody or a cell membrane-permeable fragment thereof; a monovalent, divalent, or multivalent single-chain variable fragment (scFv); or a diabody; or a humanized form or variant thereof, and (b) a nucleic acid cargo comprising, for example, a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof. Elements (a) and (b) generally non-covalently associate to form a complex. In addition to DNA, 3E10 is thought to bind to RNA, PNA, and other nucleic acids.

[0009] Exemplary 3E10 antibodies, as well as fragments and fusion proteins thereof, include (i) a combination of any one CDR of SEQ ID NOs: 1-6, 12, 13, 46-48, or 50-52 and any one CDR of SEQ ID NOs: 7-11, 14, or 53-58; (ii) a combination of a first, second, and third heavy chain CDR selected from SEQ ID NOs: 15-23, 42, and 43 and a first, second, and third light chain CDR selected from SEQ ID NOs: 24-30, 44, and 45; (iii) a humanized form of (i) or (ii); (iv) a combination of a heavy chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1 or 2 and a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NOs: 7 or 8; (v) a humanized form of (iv); or (vi) a combination of a heavy chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 3-6, 46-48, or 50-52 and a light chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 9-11 or 53-58.

[0010] In some embodiments, the antibody, as well as fragments and fusion proteins thereof, is a CDR1 heavy chain variant having an amino acid residue corresponding to D31 or N31 of the 3E10 heavy chain amino acid sequence or a CDR thereof substituted with arginine (R) or lysine (L).

[0011] In some embodiments, the antibody, as well as fragments and fusion proteins thereof, includes the nucleic acid binding pocket of SEQ ID NO: 92 or 93, or a variant thereof having the same or improved ability to bind to a nucleic acid, such as DNA, RNA, or a combination thereof.

[0012] Also provided are binding proteins themselves that contain a CDR1 heavy chain variant having an amino acid residue corresponding to D31 or N31 of the 3E10 heavy chain amino acid sequence, or that CDR1 substituted with arginine (R) or lysine (L), as well as binding proteins themselves having a variant thereof that has the same or improved ability to bind to the nucleic acid binding pocket of SEQ ID NO: 92 or 93, or to a nucleic acid, such as DNA, RNA, or a combination thereof.

[0013] In some embodiments, the antibody or fragment or fusion protein can be bispecific and can include, for example, a binding sequence that targets a cell type, tissue, or organ of interest.

[0014] The nucleic acid cargo can be composed of DNA, RNA, modified nucleic acids including, but not limited to, PNA, or combinations thereof. The nucleic acid cargo is typically a functional cargo, such as a functional nucleic acid (e.g., inhibitory RNA), mRNA, or a vector, such as an expression vector. The nucleic acid cargo containing a vector can include a nucleic acid sequence encoding a polypeptide of interest operably linked to an expression control sequence. The vector can be, for example, a plasmid. Typically, the cargo does not randomly shear or fragment genomic DNA, for example.

[0015] In some embodiments, the cargo includes or consists of a nucleic acid encoding a Cas endonuclease, gRNA, or a combination thereof. In some embodiments, the cargo includes or consists of a nucleic acid encoding a chimeric antigen receptor polypeptide. In some embodiments, the cargo is a functional nucleic acid, such as an antisense molecule, siRNA, microRNA (miRNA), aptamer, ribozyme, RNAi, or an external guide sequence, or a nucleic acid construct encoding the same.

[0016] The cargo can comprise, or consist of, a plurality of single nucleic acid molecules, or a plurality of two, three, four, five, six, seven, eight, nine, ten, or more different nucleic acid molecules. In some embodiments, the nucleic acid molecules of the cargo comprise, or consist of, nucleic acid molecules having a nucleic acid base length between about 1 and about 25,000. The cargo can be single-stranded nucleic acid, double-stranded nucleic acid, or a combination thereof.

[0017] Also provided is a pharmaceutical composition comprising this complex and a pharmaceutically acceptable excipient. In some embodiments, the complex is encapsulated in polymeric nanoparticles. The targeting moiety, cell membrane permeable peptide, or a combination thereof can be associated, linked, conjugated, or otherwise attached to the nanoparticles directly or indirectly.

[0018] Also provided is a method of delivering nucleic acid cargo into a cell by contacting the cell with an effective amount of the complex, either alone or encapsulated in nanoparticles. The contacting can be carried out in vitro, ex vivo, or in vivo. In some embodiments, an effective amount of the ex vivo treated cells is administered to a subject in need thereof, for example, in an effective amount to treat one or more symptoms of a disease or disorder.

[0019] In some embodiments, the contacting is carried out in vivo after administration to a subject in need thereof. The subject can have a disease or disorder such as a genetic disorder or cancer. The composition can be administered to the subject in an effective amount to reduce one or more symptoms of the subject's disease or disorder, for example, by injection or infusion.

[0020] Also provided are uses of the composition and method, which include, but are not limited to, gene therapy and the manufacture / formation / treatment of CAR T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

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Mode for Carrying Out the Invention

[0035] Detailed Description of the Invention I. Definitions As used herein, the terms "single-chain Fv" or "scFv" mean, when used herein, a single-chain variable fragment comprising a light-chain variable region (VL) and a heavy-chain variable region (VH) linked by a linker that enables the scFv to form a structure desirable for antigen binding (i.e., for the VH and VL of a single polypeptide chain to bind to each other to form an Fv). The VL and VH regions may be derived from a parent antibody or may be chemically or recombinantly synthesized.

[0036] As used herein, the term "variable region" is intended to distinguish such domains of immunoglobulins from domains widely shared by antibodies (such as the antibody Fc domain). The variable region includes "hypervariable regions" whose residues are involved in antigen binding. The hypervariable regions include the amino acid residues of "complementary determining regions" or "CDRs" (i.e., approximately residues 24 - 34 (L1), 50 - 56 (L2), and 89 - 97 (L3) of the light-chain variable domain and approximately residues 27 - 35 (H1), 50 - 65 (H2), and 95 - 102 (H3) of the heavy-chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or the residues of "hypervariable loops" (i.e., residues 26 - 32 (L1), 50 - 52 (L2), and 91 - 96 (L3) of the light-chain variable domain and residues 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3) of the heavy-chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901 - 917).

[0037] As used herein, the term "framework region" or "FR" residue refers to variable domain residues other than hypervariable region residues as defined herein.

[0038] As used herein, the term "antibody" refers to natural or synthetic antibodies that bind to a target antigen. This term includes polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term "antibody" includes binding proteins, fragments, and polymers of those immunoglobulin molecules, as well as human or humanized versions of immunoglobulin molecules that bind to a target antigen.

[0039] As used herein, the term "cell membrane permeable antibody" refers to an immunoglobulin protein, fragment, variant, or fusion protein based thereon that is transported into the cytoplasm and / or nucleus of living mammalian cells. A "cell membrane permeable anti-DNA antibody" specifically binds to DNA (e.g., single-stranded and / or double-stranded DNA). In some embodiments, the antibody is transported into the cytoplasm of the cell without the aid of a carrier or conjugate. In other embodiments, the antibody is conjugated to a cell membrane permeable moiety such as a cell membrane permeable peptide. In some embodiments, the cell membrane permeable antibody is transported into the nucleus with or without a carrier or conjugate.

[0040] In addition to intact immunoglobulin molecules, fragments, binding proteins, and polymers of immunoglobulin molecules, chimeric antibodies containing sequences of more than one species, class, subclass of immunoglobulin, such as human or humanized antibodies, and recombinant proteins containing at least the idiotypes of immunoglobulins that specifically bind to DNA are also included in the term "antibody". Antibodies can be tested for their desired activity using the in vitro assays described herein or by similar methods, and then their in vivo therapeutic activity can be tested according to known clinical trial methods.

[0041] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains important characteristics. A typical variant of a polypeptide has an amino acid sequence that differs from another reference polypeptide. Generally, the sequences of the reference polypeptide and the variant are overall very similar and the differences are limited such that they are identical in many regions. The variant and the reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). The substituted or inserted amino acid residues may or may not be those encoded by the genetic code. Variants of polypeptides may be naturally occurring, such as allelic variants, or may be variants not known to occur in nature.

[0042] Modifications and changes can be made to the structure of the polypeptides of the present disclosure to obtain molecules that still have similar characteristics (e.g., conservative amino acid substitutions) as the polypeptides. For example, certain amino acids can be replaced with other amino acids within the sequence without apparently losing activity. Since the biological functional activity of a polypeptide is defined by its ability to interact and its properties, certain amino acid sequence substitutions can be made within the polypeptide sequence and yet a polypeptide with similar activity can be obtained.

[0043] When making such changes, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobicity index of amino acids in conferring interactive biological functions to polypeptides is generally understood in the art. It is known that certain amino acids can be substituted with other amino acids having similar hydrophobicity indices or scores, and yet polypeptides having similar biological activities can be obtained. Each amino acid is assigned a hydrophobicity index based on its hydrophobic and charge characteristics. These indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0044] The relative hydrophobicity characteristics of amino acids are thought to define the secondary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that substituting one amino acid with another having a similar hydrophobicity index can result in a functionally equivalent polypeptide. For such changes, substitutions of amino acids with hydrophobicity indices within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0045] Similar amino acid substitutions can also be made on the basis of hydrophilicity, especially when the biological function created thereby is such that the equivalent polypeptide or peptide is intended for use in immunological embodiments. The following hydrophilicity values are assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that substituting one amino acid with another having a similar hydrophilicity value can result in a biologically equivalent, especially immunologically equivalent, polypeptide. For such changes, substitutions of amino acids with hydrophilicity values within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0046] As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into account the various characteristics described above are well known to those of skill in the art and include the following (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Accordingly, embodiments of the present disclosure contemplate functional or biological equivalents of polypeptides as described above. In particular, embodiments of a polypeptide can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to the polypeptide of interest.

[0047] As used herein, the term “percent sequence identity (%)” is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to the nucleotides or amino acids in a reference nucleic acid or amino acid sequence after aligning the sequences and introducing gaps, if necessary, to maximize sequence identity percentage. Alignment for purposes of determining percent sequence identity can be achieved in various ways within the skill in the art using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including the algorithm to be used in order to achieve the maximal alignment over the full length of the sequences being compared, can be determined by known methods.

[0048] For the purposes of this specification, the percent sequence identity of a given nucleotide or amino acid sequence C to a given nucleic acid sequence D (or, equivalently, a given sequence C that has or contains a specified percent sequence identity to a given nucleic acid sequence D) is calculated as follows: 100 × fraction W / Z, where W is the number of nucleotides or amino acids that the sequence alignment program has scored as identical matches in the alignment of C and D for that program, and Z is the total number of nucleotides or amino acids in D. It will be understood that if the length of sequence C is not equal to the length of sequence D, the percentage of sequence identity of C to D will not be equal to the percentage of sequence identity of D to C.

[0049] As used herein, the term "specifically binds" refers to an antibody that binds to its cognate antigen (e.g., DNA), but does not significantly bind to other antigens. Specific binding of an antibody to its target under such conditions requires selection of the antibody for its specificity to the target. A variety of immunoassay formats may be used to select antibodies that specifically immunoreact with a particular protein. For example, solid phase ELISA immunoassays are routinely used to select monoclonal antibodies that specifically immunoreact with a protein. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York. Preferably, the antibody has an affinity constant (Ka) for its second molecule of about 10 5 mol -1 greater than (e.g., 10 6 mol -1 、10 7 mol -1 、10 8 mol -1 、10 9 mol -1 、10 10 mol -1 、10 11mol -1 、 and 10 12 mol -1 or more) antigens "specifically binds to".

[0050] As used herein, the term "monoclonal antibody" or "MAb" refers to a substantially homogeneous population of antibodies, i.e., a population of antibodies where the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in minor amounts of antibody molecules.

[0051] As used herein, the term "subject" means any individual that is the target of administration. The subject can be a vertebrate, e.g., a mammal. Thus, the subject can be a human. This term does not denote a particular age or sex.

[0052] As used herein, the term "effective amount" means that the amount of the composition being used is sufficient to effect amelioration of one or more causes or symptoms of a disease or disorder. Such amelioration only requires reduction or modification and not necessarily elimination. The exact dosage will vary depending on a variety of factors such as variables that depend on the subject (e.g., age, health of the immune system, etc.), the disease or disorder being treated, as well as the pharmacokinetics of the drug being administered and the route of administration.

[0053] As used herein, the term "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable. That is, the material can be administered to a subject without causing any undesirable biological effects and without interacting in a harmful manner with any of the other components of the pharmaceutical composition in which it is contained.

[0054] As used herein, the terms "carrier" or "excipient" refer to organic or inorganic constituents in a formulation in which one or more active ingredients are combined, and denote natural or synthetic inert ingredients. As is well known to those skilled in the art, carriers or excipients would be naturally selected to minimize the degradation of active ingredients and minimize harmful side effects in a subject.

[0055] As used herein, the term "treating" refers to the medical management of a patient with the goal of curing, alleviating, stabilizing, or preventing a disease, condition, or disorder. This term includes active treatment, i.e., treatment particularly directed to the improvement of a disease, condition, or disorder, and causal treatment, i.e., treatment directed to the removal of the cause of the related disease, condition, or disorder. Moreover, this term includes palliative treatment, i.e., treatment designed for the relief of symptoms rather than the cure of a disease, condition, or disorder; prophylactic treatment, i.e., treatment directed to minimizing or partially or completely suppressing the onset of a related disease, condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy directed to the improvement of a related disease, condition, or disorder.

[0056] As used herein, a "targeting moiety" is a substance that can direct particles or molecules to receptor sites on selected cell or tissue types, can function as an adhesion molecule, or can function to bind or attach another molecule. As used herein, "directing" refers to preferentially attaching a molecule to a selected cell or tissue type. This can be used to direct cell materials, molecules, or drugs, as described below.

[0057] As used herein, the terms "suppress" or "reduce" mean to decrease an activity, response, symptom, disease, or other biological parameter. This can include, but is not limited to, complete ablation of the activity, response, symptom, or disease. It can also include, for example, a 10% decrease in an activity, response, symptom, or disease compared to a native or control level. Thus, this decrease can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount in between, decrease compared to a native or control level.

[0058] As used herein, a "fusion protein" refers to a polypeptide formed by linking two or more polypeptides via a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. A fusion protein may be formed by chemical coupling of the constituent polypeptides or may be expressed as a single polypeptide from a nucleic acid sequence encoding a single contiguous fusion protein. A single-chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques of molecular biology to ligate two genes in-frame into a single nucleic acid sequence and then expressing that nucleic acid in a suitable host cell under conditions in which the fusion protein is produced.

[0059] The recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0060] The use of the term "about" is intended to describe values that are above or below the stated value by approximately + / - 10%; in other embodiments, the value may vary above or below the stated value by approximately + / - 5%; in other embodiments, the value may vary above or below the stated value by approximately + / - 2%; in other embodiments, the value may vary above or below the stated value by approximately + / - 1%. The above ranges are intended to be made clear by the context and no further limitations are implied.

[0061] All methods described herein can be performed in any suitable order, unless otherwise indicated or clearly contradicted by the context. The use of any example, or exemplary language (e.g., "such as") described herein is merely intended to better illustrate the embodiments and does not limit the scope of the embodiments unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention. II. Compositions

[0062] The 3E10 antibody has been found to be useful for delivering nucleic acids across the cell membrane into the cytoplasm and nucleus. Accordingly, compositions and methods for enhancing the delivery of nucleic acid constructs using 3E10 are provided. Typically, an effective amount of the 3E10 antibody is contacted with the nucleic acid desired to be delivered to the cell. Typically, the contact is for a time sufficient for the 3E10 and the nucleic acid cargo to form a complex. The complex is contacted with the cell for a time sufficient for the nucleic acid cargo to be delivered to the cell. The cargo may accumulate in larger amounts, higher quality (e.g., intact, functional, etc.), or at a faster rate, or a combination thereof, than when the cell is contacted with the nucleic acid cargo in the absence of the antibody. Since the antibody functions as a delivery means, the delivery system is typically non-viral. A. 3E10 Antibody

[0063] Although generally referred to herein as "3E10" or "3E10 antibody", antigen-binding fragments, variants, and fragments and binding proteins containing fusion proteins such as scFv, di-scFv, tr-scFv, and other single-chain variable fragments, as well as other cell membrane-permeable nucleic acid transport molecules disclosed herein, are also explicitly provided for use in the compositions and methods disclosed herein. Thus, antibodies and other binding proteins are also referred to herein as cell membrane-permeable.

[0064] In preferred embodiments, the 3E10 antibody is transported into the cytoplasm and / or nucleus of cells without the aid of a carrier or conjugate. For example, the monoclonal antibody 3E10 and its active fragments, which are transported into the nuclei of mammalian cells in vivo without cytotoxic effects, are disclosed in U.S. Pat. Nos. 4,812,397 and 7,189,396 to Richard Weisbart.

[0065] In some embodiments, this antibody may bind to and / or inhibit Rad51. See, for example, the antibodies described in Turchick et al., Nucleic Acids Res., 45(20):11782-11799 (2017), WO2020 / 047344, and WO2020 / 047353. Each of these is specifically incorporated herein by reference in its entirety.

[0066] Antibodies that can be used in the compositions and methods include intact immunoglobulins of any class (i.e., intact antibodies), fragments thereof, and synthetic proteins that include at least the antigen-binding variable domains of the antibodies. The variable domains have different sequences for each antibody and are used for the binding and specificity of each particular antibody to a particular antigen. However, the variability is not usually uniformly distributed throughout the entire variable domain of the antibody. It is usually concentrated in three segments called the complementarity-determining regions (CDRs) or hypervariable regions of both the variable domains of the light and heavy chains. The more highly conserved portions of the variable domain are called the framework (FR). The variable domains of the native heavy and light chains each contain four FR regions, which mainly adopt a β-sheet structure and are connected by three CDRs. These CDRs form loops that connect the β-sheet structures and, in some cases, form part of the β-sheet structure. The CDRs of each chain are held in close proximity by the FR regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody. Therefore, an antibody usually contains at least the CDRs necessary to maintain DNA binding and / or to interfere with DNA repair.

[0067] The 3E10 antibody is usually a monoclonal 3E10, or a variant, derivative, fragment, fusion, or humanized form thereof, that binds to one or more epitopes that are the same as or different from 3E10.

[0068] The deposit of the hybridoma cell line that produces the monoclonal antibody 3E10, in accordance with the Budapest Treaty, was received on September 6, 2000, and was accepted by the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209, USA, and was assigned the patent deposit number PTA-2439.

[0069] Therefore, this antibody may have the same or different epitope specificities as monoclonal antibody 3E10 produced by hybridoma with ATCC number PTA2439. This antibody can have the paratope of monoclonal antibody 3E10. The antibody can be a single-chain variable fragment of 3E10, or a variant thereof, such as a conservative variant. For example, this antibody can be a single-chain variable fragment (3E10 Fv) of 3E10, or a variant thereof. 1.3E10 Sequence

[0070] The amino acid sequence of monoclonal antibody 3E10 is known in the art. For example, the sequences of the 3E10 heavy and light chains are provided below. Here, a single underline indicates the CDR regions identified according to the Kabat system, the italicized sequences of SEQ ID NOs: 12-14 indicate the variable regions, and a double underline indicates the signal peptide. CDRs according to the IMGT system are also provided. a.3E10 Heavy Chain

[0071] ]>In some embodiments, the heavy chain variable region of 3E10 is:

Chemical Formula

[0072] In some embodiments, the 3E10 heavy chain is represented as follows

Chemical Formula

Chemical Formula

[0073] Variants of the 3E10 antibody that incorporate mutations into the wild-type sequence are also known in the art, for example, as disclosed in Zack et al., J. Immunol., 157(5):2082-8(1996). For example, amino acid position 31 in the heavy chain variable region of 3E10 has been determined to influence the ability of the antibody and its fragments to penetrate the nucleus and bind to DNA (bold in SEQ ID NOs: 1, 2, and 13). The D31N mutation in CDR1 (bold in SEQ ID NOs: 2 and 13) penetrates the nucleus and binds to DNA with much higher efficiency than the original antibody (Zack et al., Immunology and Cell Biology, 72:513-520(1994), Weisbart et al., J. Autoimmun., 11, 539-546(1998); Weisbart, Int. J. Oncol., 25, 1867-1873(2004)). In some embodiments, the antibody has a D31N substitution.

[0074] In some embodiments, the amino acid sequence of a preferred variant of the heavy chain variable region of 3E10 is

Chemical formula

[0075] In some embodiments, the 3E10 heavy chain is represented as follows

Chemical formula

[0076] In some embodiments, the C-terminal serine of SEQ ID NO: 1 or 2 is absent from the 3E10 heavy chain variable region or is substituted, for example, with alanine.

[0077] The complementarity-determining regions (CDRs) identified by Kabat are underlined above, and include CDR H1.1 (original sequence): DYGMH (SEQ ID NO: 15); CDR H1.2 (including the D31N mutation): NYGMH (SEQ ID NO: 16); CDR H2.1: YISSGSSTIYYADTVKG (SEQ ID NO: 17); CDR H3.1: RGLLLDY (SEQ ID NO: 18) are included.

[0078] Variants of Kabat's CDR H2.1 include YISSGSSTIYYADSVKG (SEQ ID NO: 19 ) and YISSSSSTIYYADSVKG (SEQ ID NO: 42).

[0079] Additionally or alternatively, the heavy chain complementarity determining regions (CDRs) can be defined according to the IMGT system. The complementarity determining regions (CDRs) identified by the IMGT system include CDR H1.3 (original sequence): GFTFSDYG (SEQ ID NO: 20); CDR H1.4 (including the D31N mutation): GFTFSNYG (SEQ ID NO: 21); CDR H2.2: ISSGSSTI (SEQ ID NO: 22) and variant ISSSSSTI (SEQ ID NO: 43); CDR H3.2: ARRGLLLDY (SEQ ID NO: 23). b. 3E10 light chain In some embodiments, the light chain variable region of 3E10 is

[0080]

Chemical formula

[0081] The amino acid sequence of the light chain variable region of 3E10 is

Chemical formula

[0082] In some embodiments, the 3E10 light chain is represented as follows.

Chemical formula

Chemical formula

[0083] Other 3E10 light chain sequences are known in the art. See, for example, Zack et al., J. Immunol., 15; 154(4):1987-94(1995); GENBANK: L16981.1 - Mouse Ig rearranged L-chain gene, partial cds; GenBank: AAA65681.1 - immunoglobulin light chain, partial [Mus musculus]).

[0084] The complementarity-determining regions (CDRs) identified by Kabat are underlined and include CDR L1.1: RASKSVSTSSYSYMH (SEQ ID NO: 24); CDR L2.1 : YASYLES (SEQ ID NO: 25); CDR L3.1: QHSREFPWT (SEQ ID NO: 26).

[0085] Variants of Kabat's CDR L1.1 include RASKSVSTSSYSYLA (SEQ ID NO: 27) and RASKTVSTSSYSYMH (SEQ ID NO: 44).

[0086] A variant of Kabat's CDR L2.1 is YASYLQS (SEQ ID NO: 28).

[0087] Additionally or alternatively, the heavy chain complementarity-determining regions (CDRs) can be defined according to the IMGT system. The complementarity-determining regions (CDRs) identified by the IMGT system include CDR L1.2 KSVSTSSYSY (SEQ ID NO: 29) and variant KTVSTSSYSY (SEQ ID NO: 45); CDR L2.2: YAS (SEQ ID NO: 30); CDR L3.2: QHSREFPWT (SEQ ID NO: 26).

[0088] In some embodiments, the C-terminus of the sequence of SEQ ID NO: 7 or 8 further includes the arginine of the 3E10 light chain variable region. 2. Humanized 3E10

[0089] In some embodiments, the antibody is a humanized antibody. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are usually obtained from the "import" variable domain. Antibody humanization techniques generally involve the use of recombinant DNA techniques to manipulate the DNA sequence encoding one or more polypeptide chains of the antibody molecule.

[0090] Exemplary 3E10 humanized sequences are discussed in WO2015 / 106290, WO2016 / 033324, WO2019 / 018426, and WO / 2019 / 018428 and are provided below. a. Humanized 3E10 heavy chain variable region

[0091] In some embodiments, the humanized 3E10 heavy chain variable domain includes the following.

Chemical formula

Chemical formula

[0092] In some embodiments, the humanized 3E10 light chain variable domain includes the following

Chemical formula

Chemical formula

[0093] The disclosed compositions and methods typically utilize antibodies that maintain the ability to penetrate cells and, optionally, the nucleus.

[0094] The mechanisms of intracellularization by autoantibodies are diverse. Some are taken up by cells through electrostatic interactions or endocytosis via FcR, while others utilize mechanisms based on binding to myosin or calreticulin on the cell surface and then undergo endocytosis (Ying-Chyi et al., Eur J Immunol 38, 3178-3190 (2008), Yanase et al., J Clin Invest 100, 25-31 (1997)). 3E10 penetrates cells by an Fc-independent mechanism (as demonstrated by the ability of the 3E10 fragment lacking Fc to penetrate cells) and is associated with the presence of the nucleoside transporter ENT2 (Weisbart et al., Sci Rep 5:12022.doi:10.1038 / srep12022. (2015), Zack et al., J Immunol 157, 2082-2088 (1996), Hansen et al., J Biol Chem 282, 20790-20793 (2007)). Thus, in some embodiments, the antibodies utilized in the disclosed compositions and methods are antibodies that penetrate cells by an Fc-independent mechanism and are associated with the presence of the nucleoside transporter ENT2.

[0095] Mutations in 3E10 that interfere with the ability to bind DNA may prevent the antibody from penetrating the nucleus. Thus, typically, the disclosed antibody variants and humanized forms maintain the ability to bind nucleic acids, particularly DNA. Moreover, 3E10 scFv has previously been shown to be permeable to live cells and nuclei in an ENT2-dependent manner, with uptake efficiency impaired in ENT2-deficient cells (Hansen, et al., J.Biol.Chem.282, 20790-20793 (2007)). Thus, in some embodiments, the disclosed antibody variants and humanized forms maintain the ability to penetrate the cell nucleus in an ENT-dependent, preferably ENT2-dependent manner.

[0096] As discussed in International Publication No. WO 2019 / 152806 and International Publication No. WO 2019 / 152808, some humanized 3E10 variants were found to penetrate the cell nucleus more efficiently than the original mouse 3E10 (D31N) di-scFv, while other variants were found to have lost the ability to penetrate the nucleus. In particular, variants 10 and 13 penetrated the nucleus very well compared to the mouse antibody.

[0097] A potential bipartite nuclear localization signal (NLS) has been identified in humanized 3E10 VL and may be included in some or all of the following sequences:

Chem.

[0098] An exemplary consensus NLS is

Chem.

[0099] Thus, in some embodiments, particularly where nuclear import is important, the disclosed antibodies may include any one of the sequences of SEQ ID NOs: 88 - 91, or fragments and variants thereof (e.g., having 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% amino acid sequence identity to any one of SEQ ID NOs: 88 - 91) that are capable of translocating into the nucleus of a cell.

[0100] The presence of the NLS indicates that 3E10 may pass through the nuclear envelope via the nuclear import pathway. In some embodiments, the NLS improves import by interacting with one or more members of the import pathway. Thus, in some embodiments, the NLS can bind to importin-β, the importin-β / importin-α heterodimer, or a combination thereof. 3. Nucleic Acid Binding

[0101] The disclosed compositions and methods typically utilize antibodies that maintain the ability to bind to nucleic acids such as DNA, RNA, or combinations thereof.

[0102] The following examples show the molecular modeling of 3E10 and additional 3E10 variants. Molecular modeling of 3E10 (Pymol) revealed a putative nucleic acid binding pocket (NAB1) (see, e.g., FIGS. 14A and 14B), which is underlined in the following sequences. WT heavy chain scFv sequence [Chemical formula] Light chain scFv sequence [Chemical formula]

[0103] In some embodiments, the disclosed antibodies include some or all of the underlined NAB1 sequence. In some embodiments, the antibodies include variant sequences with an altered ability to bind nucleic acids. In some embodiments, mutations in NAB1 (e.g., substitutions, insertions, and / or deletions) improve the binding of the antibody to nucleic acids such as DNA, RNA, or a combination thereof. In some embodiments, the mutations are conservative substitutions. In some embodiments, the mutations increase the cationic charge of the NAB1 pocket.

[0104] As discussed and illustrated herein, mutating the aspartic acid at residue 31 of CDR1 to asparagine increased the cationic charge of this residue and enhanced nucleic acid binding and delivery in vivo (3E10-D31N).

[0105] Additional exemplary variants include the mutation of the aspartic acid at residue 31 of CDR1 to arginine (3E10-D31R) (modeling indicates an increase in cationic charge), or the mutation to lysine (3E10-D31K) (modeling indicates a change in the orientation of the charge). Thus, in some embodiments, the 3E10 binding protein includes the substitution D31R or D31K.

[0106] All sequences disclosed herein having a residue corresponding to D31 or N31 of 3E10 are explicitly disclosed with their D31R or D31K or N31R or N31K substitutions.

[0107] Molecular modeling of 3E10 (Pymol) revealed a putative nucleic acid binding pocket (NAB1) (Figures 14A - 14B). Mutating the aspartic acid at residue 31 of CDR1 to asparagine increased the cationic charge of this residue and enhanced nucleic acid binding and delivery in vivo (3E10-D31N).

[0108] Mutating the aspartic acid at residue 31 of CDR1 to arginine (3E10-D31R) further increased the cationic charge, and mutating it to lysine (3E10-D31K) changed the direction of the charge (Figure 14A).

[0109] [[ID=2,0]]The NAB1 amino acids predicted from molecular modeling are underlined in the heavy and light chain sequences above. Figure 14B is an explanatory diagram showing the molecular modeling of 3E10-scFv (Pymol) with NAB1 amino acid residues indicated by dotted dots. 4. Fragments, Variants, and Fusion Proteins

[0110] An anti-DNA antibody can be composed of an antibody fragment or a fusion protein comprising a variable heavy chain and / or a variable light chain amino acid sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of the variable heavy chain and / or light chain of 3E10 or its humanized form (e.g., any of SEQ ID NOs: 1-11 or 46-58, or the heavy chain and / or light chain of any of SEQ ID NOs: 12-14).

[0111] An anti-DNA antibody can be composed of an antibody fragment or a fusion protein comprising one or more CDRs with an amino acid sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of one or more CDRs of 3E10, or its variant or humanized form (e.g., any one or more CDRs of SEQ ID NOs: 1-11 or 46-58, or SEQ ID NOs: 12-14, or SEQ ID NOs: 15-30 or 42-45). The percent identity of two amino acid sequences can be determined by BLAST protein comparison. In some embodiments, the antibody comprises one, two, three, four, five, or all six of the above-described preferred variable domain CDRs.

[0112] Preferably, the antibody comprises a combination of one of each of heavy chain CDR1, CDR2, and CDR3 and one of each of light chain CDR1, CDR2, and CDR3.

[0113] The predicted complementarity determining regions (CDRs) of the 3E10 light chain variable array are provided above. GENBANK: AAA65681.1 - immunoglobulin light chain, partial [Mus musculus] and GenBank: L34051.1 - Mouse Ig rearranged kappa-chain See also the mRNA V-region. The predicted complementarity determining regions (CDRs) of the 3E10 heavy chain variable array are provided above. For example, Zack et al., Immunology and Cell Biology, 72:513 - 520 (1994), GenBank Accession number AAA65679.1. Zach et al., J. Immunol. 154(4), 1987 - 1994 (1995) and GenBank: L16982.1 - Mouse Ig reagrranged H-chain See also the gene, partial cds.

[0114] Thus, in some embodiments, the cell membrane permeable antibody comprises the CDRs of SEQ ID NO: 1 or 2, or the entire heavy and light chain variable regions, or the heavy chain region of SEQ ID NO: 12 or 13; or a humanized form thereof, and the light chain region of SEQ ID NO: 7 or 8, or SEQ ID NO: 14; or a humanized form thereof. In some embodiments, the cell membrane permeable antibody comprises the CDRs of SEQ ID NO: 3, 4, 5, or 6, or the entire heavy and light chain variable regions, in combination with SEQ ID NO: 9, 10, or 11. In some embodiments, the cell membrane permeable antibody comprises any one CDR of SEQ ID NOs: 46 - 48 or 50 - 52, or the entire heavy and light chain variable regions, in combination with any one of SEQ ID NOs: 53 - 58.

[0115] All sequences disclosed herein having a residue corresponding to D31 or N31 of 3E10 are explicitly disclosed with their D31R or D31K or N31R or N31K substitutions. Thus, in some embodiments, the 3E10-binding protein is a variant of any of the foregoing or following sequences in which the amino acid residue corresponding to residue 31 of the 3E10 heavy chain is substituted with arginine (R) or lysine (K).

[0116] Also included are fragments of antibodies having biological activity. The fragments include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is not substantially altered or impaired compared to the unmodified antibody or antibody fragment, regardless of whether it is bound to other sequences.

[0117] The techniques can also be adapted for the production of single-chain antibodies specific for the antigenic proteins of the present disclosure. Methods for the production of single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can be created by fusing the variable domains of the heavy and light chains using a short peptide linker, thereby reconstructing the antigen-binding site on a single molecule. Single-chain antibody variable fragments (scFvs), in which the C-terminus of one variable domain is linked to the N-terminus of the other variable domain via a 15- to 25-amino acid peptide or linker, have been developed without significantly disrupting antigen binding or binding specificity. The linker is selected such that the heavy and light chains can bind in an appropriate steric orientation.

[0118] Anti-DNA antibodies can be modified to enhance their therapeutic potential. For example, in some embodiments, a cell membrane-permeable anti-DNA antibody is conjugated to another antibody specific for a second therapeutic target in the cytoplasm and / or nucleus of a target cell. For example, the cell membrane-permeable anti-DNA antibody can be a fusion protein comprising the 3E10 Fv and a single-chain variable fragment of a monoclonal antibody that specifically binds to the second therapeutic target. In other embodiments, the cell membrane-permeable anti-DNA antibody is a bispecific antibody having the first heavy and first light chains of 3E10 and the second heavy and second light chains of a monoclonal antibody that specifically binds to the second therapeutic target.

[0119] Bispecific antibodies and other binding proteins having the first heavy and first light chains of 3E10 and the second heavy and second light chains of a monoclonal antibody that specifically binds to a second target have been discussed in Weisbart et al., Mol. Cancer Ther., 11(10):2169-73 (2012), and Weisbart et al., Int. J. Oncology, 25:1113-8 (2004), and U.S. Patent Application No. 2013 / 0266570, which are specifically incorporated by reference in their entirety. In some embodiments, the second target is specific for a target cell type, tissue, organ, etc. Thus, the second heavy and second light chains can serve as targeting moieties to target the complex to the target cell type, tissue, organ. In some embodiments, the second heavy and second light chains target hematopoietic stem cells, CD34 + cells, T cells, or any other preferred cell type, for example, by targeting a receptor or ligand expressed on the preferred cell type. In some embodiments, the second heavy and second light chains target the thymus, spleen, or cancer cells.

[0120] In some embodiments, particularly embodiments for targeting T cells in vivo, such as CAR In embodiments for producing T cells in vivo, immune cell or T cell markers such as CD3, CD7, or CD8 can be targeted. For example, both anti-CD8 antibodies and anti-CD3 Fab fragments have been used to target T cells in vivo (Pfeiffer et al., EMBO Mol Med., 10(11)(2018). pii: e9158. doi: 10.15252 / emmm.201809158., Smith et al., Nat Nanotechnol., 12(8):813-820(2017). doi: 10.1038 / nnano.2017.57). Thus, in some embodiments, the 3E10 antibody or antigen-binding fragment or fusion protein is a bispecific antibody that can specifically bind in part to CD3, CD7, CD8, or another immune cell (e.g., T cell) marker, or a marker of a specific tissue such as the thymus, spleen, or liver.

[0121] A bivalent single-chain variable fragment (di-scFv) can be engineered by linking two scFvs. This can be accomplished by generating a single peptide chain with two VH regions and two VL regions to produce a tandem scFv. Also, the scFv is too short to fold the two variable regions together and can also be designed using a linker peptide (about 5 amino acids) that dimerizes the scFv. This type is known as a diabody. Diabodies have been shown to have a dissociation constant up to 40-fold lower than the corresponding scFv, which means they have a very high affinity for the target. Even shorter linkers (1 or 2 amino acids) cause the formation of trimers (triabodies or tribodies). Tetrabodies have also been made. These show an even higher affinity for their target than diabodies. In some embodiments, the anti-DNA antibody may comprise two or more linked single-chain variable fragments of 3E10 (e.g., 3E10 di-scFv, 3E10 tri-scFv), or a conservative variant thereof. In some embodiments, the anti-DNA antibody is a diabody or a tribody (e.g., 3E10 diabody, 3E10 tribody). The sequences of the single and two or more linked single-chain variable fragments of 3E10 are described in WO2017 / 218825 and WO2016 / 033321.

[0122] The function of an antibody can be enhanced by conjugating the antibody or a fragment thereof to a therapeutic agent. Such conjugation of the antibody or fragment to the therapeutic agent can be achieved by creating an immune complex comprising the antibody or antibody fragment and the therapeutic agent, or by creating a fusion protein, or by conjugating the antibody or fragment to a nucleic acid such as DNA or RNA (e.g., siRNA).

[0123] A recombinant fusion protein is a protein produced by genetic engineering of a fusion gene. This typically involves removing the stop codon from the cDNA sequence encoding the first protein and adding in-frame the cDNA sequence of the second protein by ligation or overlap extension PCR. The DNA sequence is then expressed in cells as a single protein. This protein can be engineered to include the entire sequences of both original proteins or just parts of either. When the two entities are proteins, a linker (or "spacer") peptide is often added, increasing the likelihood that the proteins will fold independently and behave as expected.

[0124] In some embodiments, the cell membrane-permeable antibody is modified to alter its half-life. In some embodiments, it is desirable to increase the half-life of the antibody so that the antibody persists in circulation or at the site of treatment for an extended period. For example, it may be desirable to maintain the antibody titer in circulation or at a site being treated for an extended period. In other embodiments, the half-life of the anti-DNA antibody is shortened to reduce potential side effects. Antibody fragments such as 3E10Fv may have a shorter half-life than full-length antibodies. Other methods of altering the half-life are known and can be used in the methods described. For example, the antibody can be engineered using an Fc variant that extends the half-life, using, for example, the Xtend™ Antibody Half-Life Extension Technology (Xencor, Monrovia, CA). a. Linker

[0125] As used herein, the term "linker" includes, but is not limited to, peptide linkers. Peptide linkers can be of any size as long as they do not interfere with the binding of epitopes by the variable regions. In some embodiments, the linker includes one or more glycine and / or serine amino acid residues. The C-terminus of one variable domain of a monovalent single-chain antibody variable fragment (scFv) is typically linked to the N-terminus of the other variable domain via a peptide or linker of 15 to 25 amino acids. The linker is selected such that the heavy and light chains can bind in the appropriate steric orientation. Linkers for diabodies, triabodies, etc. typically include shorter linkers than those of monovalent scFvs as described above. Di-, tri-, and other multivalent scFvs typically include three or more linkers. The linkers may be the same or different in length and / or amino acid composition. Therefore, the number of linkers, the composition of one or more linkers, and the length of one or more linkers can be determined based on the desired valency of the scFv, as is known in the art. One or more linkers can enable or facilitate the formation of di-, tri-, and other multivalent scFvs.

[0126] For example, the linker can include 4 to 8 amino acids. In certain embodiments, the linker includes the amino acid sequence GQSSRSS (SEQ ID NO: 31). In another embodiment the linker includes 15 to 20 amino acids, for example, 18 amino acids. In certain embodiments, the linker includes the amino acid sequence GQSSRSSSGGGSSGGGGS (SEQ ID NO: 32). Other flexible linkers include, but are not limited to, the amino acid sequences Gly-Ser, Gly-Ser-Gly-Ser (SEQ ID NO: 33), Ala-Ser, Gly-Gly-Gly-Ser (SEQ ID NO: 34), (Gly4-Ser)2 (SEQ ID NO: 35) and (Gly4-Ser)4 (SEQ ID NO: 36), and (Gly-Gly-Gly-Gly-Ser)3 (SEQ ID NO: 37).

[0127] As other exemplary linkers, for example, RADAAPGGGGSGGGGSGGGGS (SEQ ID NO: 59) and ASTKGPSVFPLAPLESSGS (SEQ ID NO: 60) can be mentioned. b. Exemplary anti-DNA scFv sequences

[0128] Exemplary mouse 3E10 scFv sequences including mono-, di-, and tri-scFv are disclosed in WO2016 / 033321, WO2017 / 218825, WO2019 / 018426, and WO2019 / 018428 and are provided below. Cell membrane-permeable antibodies for use in the disclosed compositions and methods include exemplary scFv, as well as fragments and variants thereof.

[0129] The amino acid sequence of scFv 3E10 (D31N) is as follows:

Chemical formula

[0130] Annotation of the ScFv protein domain with reference to SEQ ID NO: 38 ● The AGIH sequence increases solubility (amino acids 1-4 of SEQ ID NO: 38) ● Vk variable region (amino acids 5-115 of SEQ ID NO: 38) ● The first 6aa of the light chain CH1 (amino acids 116-121 of SEQ ID NO: 38) ● (GGGGS)3 (SEQ ID NO: 37) linker (amino acids 122-136 of SEQ ID NO: 38) ● VH variable region (amino acids 137-252 of SEQ ID NO: 38) ● Myc tag (amino acids 253-268 of SEQ ID NO: 38) ● His6 tag (amino acids 269-274 of SEQ ID NO: 38) The amino acid sequence of 3E10 di-scFv (D31N)

[0131] di-scFv 3E10 (D31N) contains the heavy and light chain variable regions of 3E10 doubled, and is a di-single chain variable fragment in which the aspartic acid at position 31 of the heavy chain is mutated to asparagine. The amino acid sequence of di-scFv 3E10 (D31N) is as follows:

Chemical formula

[0132] Annotation of the di-ScFv protein domain with reference to SEQ ID NO: 39 ● The AGIH sequence enhances solubility (amino acids 1-4 of SEQ ID NO: 39) ● Vk variable region (amino acids 5-115 of SEQ ID NO: 39) ● The first (6aa) of the light chain CH1 (amino acids 116-121 of SEQ ID NO: 39) ● (GGGGS)3 (SEQ ID NO: 37) linker (amino acids 122-136 of SEQ ID NO: 39) ● VH variable region (amino acids 137-252 of SEQ ID NO: 39) ● Linker between Fv fragments consisting of the first 13 amino acids of human IgG CH1 (amino acids 253-265 of SEQ ID NO: 39) ● Swivel sequence (amino acids 266-271 of SEQ ID NO: 39) ● Vk variable region (amino acids 272-382 of SEQ ID NO: 39) ● The first (6aa) of the light chain CH1 (amino acids 383-388 of SEQ ID NO: 39) ● (GGGGS)3 (SEQ ID NO: 37) linker (amino acids 389-403 of SEQ ID NO: 39) ● VH variable region (amino acids 404-519 of SEQ ID NO: 39) ● Myc tag (amino acids 520-535 of SEQ ID NO: 39) ● His6 tag (amino acids 536-541 of SEQ ID NO: 39) Amino acid sequence of tri-scFv

[0133] Tri-scFv 3E10(D31N) is a tri-single-chain variable fragment that contains three times the heavy and light chain variable regions of 310E, with the aspartic acid at position 31 of the heavy chain mutated to asparagine. The amino acid sequence of tri-scFv 3E10(D31N) is as follows:

Chemical formula

Chemical formula

[0134] Annotation of the tri-ScFv protein domain with reference to SEQ ID NO: 40 ● The AGIH sequence enhances solubility (amino acids 1-4 of SEQ ID NO: 40) ● Vk variable region (amino acids 5-115 of SEQ ID NO: 40) ● The first (6aa) of the light chain CH1 (amino acids 116-121 of SEQ ID NO: 40) ● (GGGGS)3 (SEQ ID NO: 37) linker (amino acids 122-136 of SEQ ID NO: 40) ● VH variable region (amino acids 137-252 of SEQ ID NO: 40) ● Linker between Fv fragments consisting of the first 13 amino acids of human IgG CH1 (amino acids 253-265 of SEQ ID NO: 40) ● Swivel sequence (amino acids 266-271 of SEQ ID NO: 40) ● Vk variable region (amino acids 272-382 of SEQ ID NO: 40) ● The first (6aa) of the light chain CH1 (amino acids 383-388 of SEQ ID NO: 40) ● (GGGGS)3 (SEQ ID NO: 37) linker (amino acids 389-403 of SEQ ID NO: 40) ● VH variable region (amino acids 404-519 of SEQ ID NO: 40) ● Human IgG C H Linker between Fv fragments consisting of the first 13 amino acids of 1 (amino acids 520-532 of SEQ ID NO: 40) ● Swivel sequence (amino acids 533-538 of SEQ ID NO: 40) ● Vk variable region (amino acids 539 - 649 of SEQ ID NO: 40) ● First (6aa) of light chain CH1 (amino acids 650 - 655 of SEQ ID NO: 40) ● (GGGGS)3 (SEQ ID NO: 37) linker (amino acids 656 - 670 of SEQ ID NO: 40) ● VH variable region (amino acids 671 - 786 of SEQ ID NO: 40) ● Myc tag (amino acids 787 - 802 of SEQ ID NO: 40) ● His6 tag (amino acids 803 - 808 of SEQ ID NO: 40)

[0135] WO2016 / 033321 and Noble et al., Cancer Research, 75(11):2285 - 2291 (2015) have shown that di - scFv and tri - scFv have several improvements and additional activities compared to their monovalent counterparts. Sub - sequences corresponding to the different domains of the exemplary fusion proteins are also provided above. Those skilled in the art will understand that the exemplary fusion proteins or their domains can be used to construct the fusion proteins discussed in more detail above. For example, in some embodiments, di - scFv includes a first scFv comprising a Vk variable region (e.g., amino acids 5 - 115 of SEQ ID NO: 39, or a functional variant or fragment thereof) linked to a VH variable domain (e.g., amino acids 137 - 252 of SEQ ID NO: 39, or a functional variant or fragment thereof), linked to a second scFv comprising a Vk variable region (e.g., amino acids 272 - 382 of SEQ ID NO: 39, or a functional variant or fragment thereof) linked to a VH variable domain (e.g., amino acids 404 - 519 of SEQ ID NO: 39, or a functional variant or fragment thereof). In some embodiments, tri - scFv includes a di - scFv linked to a third scFv domain comprising a Vk variable region (e.g., amino acids 539 - 649 of SEQ ID NO: 40, or a functional variant or fragment thereof) linked to a VH variable domain (e.g., amino acids 671 - 786 of SEQ ID NO: 40, or a functional variant or fragment thereof).

[0136] The Vk variable region can be linked to the VH variable domain, for example, by using a linker (e.g., (GGGGS)3 (SEQ ID NO: 37)) alone or in combination with (6aa) of the light chain CH1 (amino acids 116-121 of SEQ ID NO: 39). Other suitable linkers have been discussed above and are known in the art. The scFv can be linked by using a linker (e.g., the first 13 amino acids (253-265) of human IgG CH1 of SEQ ID NO: 39) alone or in combination with a swivel sequence (e.g., amino acids 266-271 of SEQ ID NO: 39). Other suitable linkers have been discussed above and are known in the art.

[0137] Therefore, the di-scFv can contain amino acids 5-519 of SEQ ID NO: 39. The tri-scFv can contain amino acids 5-786 of SEQ ID NO: 40. In some embodiments, the fusion protein contains additional domains. For example, in some embodiments, the fusion protein contains a sequence that enhances solubility (e.g., amino acids 1-4 of SEQ ID NO: 39). Therefore, in some embodiments, the di-scFv can contain amino acids 1-519 of SEQ ID NO: 39. The tri-scFv can contain amino acids 1-786 of SEQ ID NO: 40. In some embodiments, the fusion protein contains one or more domains that enhance the purification, isolation, capture, identification, separation, etc. of the fusion protein. Exemplary domains include, for example, a Myc tag (e.g., amino acids 520-535 of SEQ ID NO: 39) and / or a His tag (e.g., amino acids 536-541 of SEQ ID NO: 39). Therefore, in some embodiments, the di-scFv can contain the amino acid sequence of SEQ ID NO: 39. The tri-scFv can contain the amino acid sequence of SEQ ID NO: 40. Other replaceable domains and additional domains have been discussed in more detail above.

[0138] Exemplary 3E10 humanized Fv sequences are discussed in WO2016 / 033324:

Chemical Structure

[0139] Exemplary 3E10 humanized di-scFv sequences are considered in WO2019 / 018426 and WO2019 / 018428 and include the following: [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] c. Additional sequences

[0140] Additional sequences that may be used in the construction of anti-DNA antigen binding proteins, antibodies, fragments, and fusion proteins include, but are not limited to, the following [Chemical formula] [Chemical formula] [Chemical formula] B. Cargo

[0141] As used in the methods provided herein, 3E10 is typically contacted with cells as a complex with nucleic acid cargo. The interaction between the antibody or binding protein and the nucleic acid cargo is non-covalent.

[0142] The nucleic acid cargo can be single-stranded or double-stranded. The nucleic acid cargo can be, or can contain, DNA, RNA, nucleic acid analogs, or combinations thereof. As will be discussed in more detail below, nucleic acid analogs can be modified in the base moiety, sugar moiety, or phosphate backbone. Such modifications can, for example, improve the stability, hybridization, or solubility of the nucleic acid.

[0143] The nucleic acid cargo is typically functional in the sense that it is, or encodes, an agent that is biologically active once delivered to a cell. Exemplary cargo includes, although more details are provided below, for example, mRNA or DNA encoding a polypeptide of interest (such as expression constructs and vectors), inhibitory nucleic acids such as siRNA, or nucleic acids encoding inhibitory nucleic acids (such as expression constructs and vectors).

[0144] The disclosed compositions can contain multiple single nucleic acid cargo molecules. In some embodiments, the composition contains multiple multiplicities (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of different nucleic acid molecules.

[0145] In some embodiments, the length of the cargo molecule is 0.001, 0.01, 1, 10, 100, 1,000, 10,000, and / or 100,000 kilobases.

[0146] In some embodiments, for example, the cargo may be between 0.001 kb and 100 kb, or between 0.001 kb and 50 kb, or between 0.001 kb and 25 kb, or between 0.001 kb and 12.5 kb, or between 0.001 kb and 10 kb, or between 0.001 kb and 8 kb, or between 0.001 kb and 5 kb, or between 0.001 kb and 2.5 kb, or between 0.001 kb and 1 kb, or between 0.01 kb and 100 kb, or between 0.01 kb and 50 kb, or between 0.01 kb and 25 kb, or between 0.01 kb and 12.5 kb, or between 0.01 kb and 10 kb, or between 0.01 kb and 8 kb, or between 0.01 kb and 5 kb, or between 0.01 kb and 2.5 kb, or between 0.01 kb and 1 kb, or between 0.1 kb and 100 kb, or between 0.1 kb and 50 kb, or between 0.1 kb and 25 kb, or between 0.1 kb and 12.5 kb, or between 0.1 kb and 10 kb, or between 0.1 kb and 8 kb, or between 0.1 kb and 5 kb, or between 0.1 kb and 2.5 kb, or between 0.1 kb and 1 kb, or between 1 kb and 100 kb, or between 1 kb and 50 kb, or between 1 kb and 25 kb, or between 1 kb and 12.5 kb, or between 1 kb and 10 kb, or between 1 kb and 8 kb, or between 1 kb and 5 kb, or between 1 kb and 2.5 kb (including both ends).

[0147] In some embodiments, for example, the cargo may be between 0.2 kb and 10 kb, or between 0.2 kb and 5 kb, or between 0.2 kb and 2.5 kb, or between 0.2 kb and 1 kb, or between 0.2 kb and 0.5 kb, or between 0.2 kb and 0.25 kb, or between 0.5 kb and 10 kb, or between 0.5 kb and 5 kb, or between 1 kb and 5 kb, or between 1 kb and 3 kb, or between 2 kb and 10 kb, or between 3 kb and 5 kb.

[0148] For certain applications, the nucleic acid cargo may be one or more discrete lengths, including at either end, within the aforementioned ranges, and it will be understood that each specific value is explicitly disclosed. For example, the size can be as small as a single nucleotide or nucleobase. In an exemplary application, the cargo is a cyclic dinucleotide such as cGAMP and is a STING agonist. In other embodiments, the cargo is a short oligomer. For example, a short oligomer of about 8mers can be used for antisense or splicing switching. Slightly longer ones (e.g., 18 - 20mers) can be used for gene editing. 1. Form of the Cargo

[0149] The nucleic acid cargo is a nucleic acid and can be an isolated nucleic acid composition. As used herein, "isolated nucleic acid" refers to a nucleic acid that has been separated from other nucleic acid molecules present in the mammalian genome, which typically includes nucleic acids flanking one or both sides of the nucleic acid of the mammalian genome. The term "isolated" as used herein with respect to nucleic acids also includes combinations with non - naturally occurring nucleic acid sequences, because such non - naturally occurring sequences do not exist in nature and have no directly adjacent sequences in a naturally occurring genome.

[0150] An isolated nucleic acid can be, for example, a DNA molecule if, in a naturally occurring genome, one of the nucleic acid sequences that is normally found immediately adjacent to the DNA molecule is removed or absent. Thus, isolated nucleic acids include, but are not limited to, DNA molecules that exist as separate molecules independent of other sequences (e.g., chemically synthesized nucleic acids, or cDNA or genomic DNA fragments generated by PCR or restriction endonuclease treatment), as well as recombinant DNA incorporated into vectors, self-replicating plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, or herpesviruses), or prokaryotic or eukaryotic genomic DNA. Furthermore, isolated nucleic acids can include engineered nucleic acids such as recombinant DNA molecules that are part of hybrid or fusion nucleic acids. For example, a nucleic acid present among hundreds to millions of other nucleic acids in a cDNA library or genomic library, or a gel slice containing a genomic DNA restriction digest, is not considered an isolated nucleic acid.

[0151] Nucleic acid sequences encoding polypeptides include genomic sequences. Also disclosed are mRNA / cDNA sequences with exons removed. Other nucleic acid sequences encoding polypeptides, such as the amino acid sequences identified above, and polypeptides containing fragments and variants thereof are also disclosed. The nucleic acid encoding the polypeptide may be optimized for expression in a selected expression host. Codons may be replaced with alternative codons that encode the same amino acid to account for differences in codon usage frequency between the organism from which the nucleic acid sequence is derived and the expression host. Thus, the nucleic acid may be synthesized using codons preferred by the expression host.

[0152] The nucleic acid can be in a sense or antisense orientation, or can be complementary, for example, to a reference sequence encoding a polypeptide. a. Vector

[0153] The cargo can be a vector, for example, a vector encoding one or more polypeptides and / or one or more functional nucleic acids. Nucleic acids such as those described above can be inserted into a vector for expression in a cell. As used herein, a "vector" is a replicon, such as a plasmid, phage, virus, or cosmid, into which another DNA segment can be inserted so that the inserted segment is replicated, and can be an expression vector. An "expression vector" is a vector containing one or more expression control sequences, and an "expression control sequence" is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0154] The nucleic acid in the vector can be operably linked to one or more expression control sequences. For example, the control sequences can be incorporated into a gene construct such that the expression control sequences effectively control the expression of the coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence consisting of a region of a DNA molecule that is usually within 100 nucleotides upstream (generally near the start site of RNA polymerase II) of the point where transcription begins. To place a coding sequence under the control of a promoter, the translation start site of the polypeptide's translation reading frame needs to be located 1 to about 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site and can also be located downstream of the transcription start site. A coding sequence is "operably linked" and "under the control" of an expression control sequence in a cell when RNA polymerase can transcribe the coding sequence into mRNA, which can then be translated into the protein encoded by the coding sequence.

[0155] Suitable expression vectors include, but are not limited to, for example, plasmids, cosmids, and viral vectors derived from bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus, and adeno-associated virus. Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), Invitrogen Life Technologies (Carlsbad, Calif.), and the like.

[0156] In some embodiments, the cargo is delivered into the cell and remains episomal. In some embodiments, the cargo is introduced into the host cell and integrated into the host cell's genome. As will be discussed in more detail below, this composition can be used in methods of gene therapy. Methods of gene therapy can include introducing into a cell a polynucleotide that modifies the genotype of the cell. Introduction of the polynucleotide can modify, replace, or alter an endogenous gene via gene recombination. The methods can include introduction of an entire replacement copy of a defective gene, a heterologous gene, or a small nucleic acid molecule such as an oligonucleotide. For example, a modified gene can be introduced at a non-specific location within the genome of the host.

[0157] In some embodiments, the cargo is a vector. Methods for constructing expression vectors containing gene sequences and appropriate transcriptional and translational control elements are well known in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. Expression vectors generally contain regulatory sequences and necessary elements for the translation and / or transcription of the inserted coding sequence, which can be, for example, the polynucleotide of interest. The coding sequence can be operably linked to a promoter and / or enhancer that serves to control the expression of the desired gene product. Promoters used in biotechnology come in various types depending on the type of control of the intended gene expression. They can generally be classified into constitutive promoters, tissue-specific or developmental stage-specific promoters, inducible promoters, and synthetic promoters.

[0158] For example, in some embodiments, the polynucleotide of interest is operably linked to a promoter or other regulatory element known in the art. Thus, the cargo can be a vector such as an expression vector. Manipulation of polynucleotides for expression in prokaryotic or eukaryotic systems may be carried out by techniques generally known to those skilled in the art of recombinant expression. Expression vectors usually contain one of the disclosed compositions under the control of one or more promoters. To place a coding sequence "under the control" of a promoter, the 5' end of the translation start site of the reading frame is generally placed between about 1 to 50 nucleotides "downstream" (i.e., 3') of the generally selected promoter. An "upstream" promoter stimulates the transcription of the inserted DNA and promotes the expression of the encoded recombinant protein or functional nucleic acid. This is the meaning of "recombinant expression" in the context used herein.

[0159] To achieve the expression of proteins or peptides or functional nucleic acids in a variety of host expression systems, many standard techniques are available for constructing expression vectors containing appropriate nucleic acids and transcriptional / translational control sequences.

[0160] Expression vectors for use in mammalian cells usually contain an origin of replication (if necessary), a promoter located upstream of the gene to be expressed, and the necessary ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. The origin of replication may be provided by constructing the vector to include an exogenous origin such as may be derived from SV40 or other virus (e.g., polyoma, adenovirus, VSV, BPV) sources, or it may be provided by the chromosomal replication machinery of the host cell. When the vector is integrated into the host cell chromosome, the latter is often sufficient.

[0161] The promoter may be derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). Additionally, if such control sequences are compatible with the host cell line, it is often possible and may be desirable to utilize the promoter or control sequences normally associated with the desired gene sequence.

[0162] A number of virus-based expression systems can be utilized. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40 (SV40). The early and late promoters of the SV40 virus are useful because both are readily obtainable from the virus as fragments containing the origin of replication of the SV40 virus. Smaller or larger SV40 fragments may also be used if they contain a sequence of approximately 250 bp extending from the HindIII site located at the origin of virus replication towards the BglI site.

[0163] When using an adenovirus as an expression vector, the coding sequence may be linked to an adenovirus transcription / translation control complex, such as a late promoter and a tripartite leader sequence. This chimeric gene may then be inserted into the adenovirus genome by in vitro or in vivo recombination. By inserting the viral genome into a non-essential region (e.g., region E1 or E3), a protein can be expressed in the infected host and a viable recombinant virus can be obtained.

[0164] For efficient translation of the disclosed compositions, certain initiation signals may also be required. These signals include the ATG initiation codon and adjacent sequences. It may be necessary to further provide an exogenous translation control signal containing the ATG initiation codon. Those skilled in the art can readily determine this necessity and provide the required signals. It is well known that the initiation codon must be in-frame (or in-phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and initiation codons can be of diverse natural and synthetic origins. The expression efficiency can be enhanced by including appropriate transcription enhancer elements or transcription terminators.

[0165] In eukaryotic expression, if the original cloned segment does not contain a polyadenylation site, it may also generally be desirable to incorporate an appropriate polyadenylation site into the transcription unit. Typically, the polyA addition site is located about 30 to 2000 nucleotides "downstream" of the protein termination site at a position before transcription termination.

[0166] For the long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express constructs encoding proteins can be engineered. Instead of using an expression vector containing a viral origin of replication, host cells can be transformed with a vector controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, the engineered cells can be grown in a concentrated medium for 1-2 days and then switched to a selection medium for growth. The selectable marker of the recombinant plasmid confers resistance to the selection, and the cells can stably integrate the plasmid into the chromosome, grow to form lesions, and then be cloned and grown into cell lines. b.mRNA

[0167] The cargo can be mRNA.

[0168] Chemical structures having the ability to promote stability and / or translation efficiency may also be used. For example, RNA can have a 5’UTR and a 3’UTR. The length of the 3’UTR can be, for example, greater than 100 nucleotides. In some embodiments, the 3’UTR sequence is between 100 and 5000 nucleotides. In some embodiments, the 5’UTR is 0 to 3000 nucleotides in length. The lengths of the 5’ and 3’UTR sequences added to the coding region can be varied by various methods including, but not limited to, the design of primers for PCR that anneal to various regions of the UTRs. Using this approach, one of ordinary skill in the art can vary the lengths of the 5’ and 3’UTRs necessary to achieve optimal translation efficiency after delivery of the transcribed RNA.

[0169] The 5’ and 3’ UTRs can be the endogenous 5’ and 3’ UTRs that naturally exist in the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into forward and reverse primers or by other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for altering RNA stability and / or translation efficiency. For example, AU-rich elements in 3’ UTR sequences are known to be able to reduce mRNA stability. Therefore, based on the properties of UTRs well-known in the art, the 3’ UTR can be selected or designed to enhance the stability of the transcribed RNA.

[0170] In some embodiments, the 5’ UTR contains the Kozak sequence of an endogenous gene. Alternatively, when adding a 5’ UTR that is not endogenous to the gene of interest by PCR as described above, the consensus Kozak sequence can also be redesigned by adding the 5’ UTR sequence. The Kozak sequence can enhance the translation efficiency of some RNA transcripts, but it does not seem to be necessary for all RNAs to enable efficient translation. The requirements for the Kozak sequence of many mRNAs are known in the art. In other embodiments, the 5’ UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3’ or 5’ UTR to prevent exonucleolytic degradation of the mRNA.

[0171] In some embodiments, the mRNA has a 5’-end cap, a 3’ poly(a) tail, or a combination thereof, which determine ribosome binding, translation initiation, and mRNA stability in cells.

[0172] The 5’ cap confers stability to the RNA molecule. The 5’ cap is, for example, m 7 G(5’)ppp(5’)G, m 7It may be a G(5’)ppp(5’)A, G(5’)ppp(5’)G or G(5’)ppp(5’)A cap analog, all of which are commercially available. The 5’ cap may be an anti-reverse cap analog (ARCA) (Stepinski et al., RNA, 7:1468-95 (2001)) or other suitable analog. The 5’ cap can be incorporated using techniques known in the art (Cougot et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski et al., RNA, 7:1468-95 (2001); Elango et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0173] The RNA can also contain an internal ribosome entry site (IRES) sequence within the sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to the mRNA and promotes the start of translation.

[0174] Generally, the length of the poly(A) tail has a positive correlation with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is adenosine between 100 and 5000.

[0175] The polyA segment can be prepared during PCR by using a reverse primer containing a polyT tail such as a 100T tail (the size can be, for example, 50 - 5000T), or after PCR, by any other method including, but not limited to, DNA ligation or in vitro recombination. The poly(A) tail also provides stability to the RNA and reduces RNA degradation. The poly(A) tail of the RNA can be extended additionally or alternatively after in vitro transcription using a poly(A) polymerase such as Escherichia coli polyA polymerase (E-PAP).

[0176] Furthermore, attaching different chemical groups to the 3' end can enhance the stability of mRNA. Such attachments can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further enhance the stability of RNA. Suitable ATP analogs include, but are not limited to, cordycepin and 8-azoadenosine. 2. Sequence of the Cargo a. Polypeptide of Interest

[0177] The cargo can encode one or more proteins. The cargo can be a polynucleotide that can be monocistronic or polycistronic. In some embodiments, the polynucleotide is multigenic. The polynucleotide can be, for example, an expression construct such as mRNA or a vector.

[0178] The cargo can encode one or more polypeptides of interest. The polypeptide can be any polypeptide. For example, the polypeptide encoded by the polynucleotide can be a polypeptide that provides a therapeutic or prophylactic effect to an organism, or a polypeptide that can be used to diagnose a disease or disorder in an organism. For example, in the treatment of cancer, autoimmune disorders, parasites, viruses, bacteria, fungi, or other infections, the one or more polynucleotides to be expressed can encode a polypeptide that functions as a ligand or receptor for cells of the immune system, or can function to stimulate or suppress the immune system of the organism.

[0179] In some embodiments, the polynucleotide replenishes or replaces a polynucleotide that is deficient in an organism.

[0180] In certain embodiments, the polynucleotide encodes dystrophin, utrophin, or a combination thereof. Such compositions may be administered in an effective amount to treat subjects with dystrophy, particularly muscular dystrophy, such as Duchenne muscular dystrophy.

[0181] In another specific embodiment, the polynucleotide encodes an antigen, for example, an antigen that can be utilized in vaccine formulations and related methods. In certain embodiments, the polynucleotide encodes one or more viral antigens, for example, one or more SARS-CoV-2 antigens. Accordingly, compositions and methods of use thereof for protection and treatment against the SARS-CoV-2 virus and related viral infections and diseases (including COVID-19) are provided.

[0182] In some embodiments, the polynucleotide comprises a selectable marker, for example, a selectable marker effective in eukaryotic cells such as a drug resistance selectable marker. This selectable marker gene can encode a factor necessary for the survival or growth of transformed host cells grown in a selective medium. Typical selectable genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, kanamycin, gentamicin, zeocin, or tetracycline, proteins that complement auxotrophic deficiencies, or proteins that supply essential nutrients not provided by the medium.

[0183] In some embodiments, the polynucleotide comprises a reporter gene. A reporter gene is typically a gene that is not normally present or expressed in the host cell. A reporter gene typically encodes a protein that provides a phenotypic change or enzymatic property. Examples of such genes are described in Weising et al., Ann. Rev. Genetics, 22, 421 (1988). Preferred reporter genes include the glucuronidase (GUS) gene and the GFP gene. b. Functional nucleic acids

[0184] The cargo can be a functional nucleic acid or can encode a functional nucleic acid. A functional nucleic acid is a nucleic acid molecule having a specific function, such as binding to a target molecule or catalyzing a specific reaction. As will be discussed in more detail below, functional nucleic acid molecules can be classified into the following non-limiting categories: antisense molecules, siRNA, miRNA, aptamers, ribozymes, RNAi, and external guide sequences, and cyclic dinucleotides. Functional nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of specific activities possessed by target molecules, or functional nucleic acid molecules can have novel activities independent of other molecules.

[0185] Functional nucleic acid molecules can interact with macromolecules such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functional nucleic acids can interact with the mRNA or genomic DNA of a target polypeptide, or they can interact with the polypeptide itself. Functional nucleic acids are often designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, the specific recognition between a functional nucleic acid molecule and a target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of a tertiary structure that allows specific recognition to occur.

[0186] Therefore, the composition can include one or more functional nucleic acids designed to reduce the expression of a gene or its gene product. For example, a functional nucleic acid or polypeptide can be designed to target and reduce or suppress the expression or translation of mRNA, or to reduce or suppress the expression of a protein, reduce its activity, or increase its degradation. In some embodiments, the composition includes a vector suitable for in vivo expression of the functional nucleic acid. i. Antisense

[0187] The functional nucleic acid can be an antisense molecule or can encode an antisense molecule. Antisense molecules are designed to interact with target nucleic acid molecules through either canonical or non-canonical base pairing. The interaction between the antisense molecule and the target molecule is designed to promote the destruction of the target molecule, for example, by RNase H-mediated RNA-DNA hybrid degradation. Alternatively, the antisense molecule is designed to interrupt the processing functions that would normally occur in the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. There are numerous methods for optimizing antisense efficiency by finding the most accessible region of the target molecule. Exemplary methods include in vitro selection experiments and DNA modification studies using DMS and DEPC. Antisense molecules can be designed to disrupt the target molecule's normal functions, such as transcription and replication. -6 , 10 -8 , 10 -10 , or 10 -12 The following dissociation constants (K d ) to bind to the target molecule. ii.RNA interference

[0188] In some embodiments, the functional nucleic acid induces gene silencing by RNA interference. Gene expression can also be effectively silenced in a very specific way by RNA interference (RNAi). This silencing was first observed by the addition of double-stranded RNA (dsRNA) (Fire et al., (1998) Nature, 391:806-11; Napoli et al., (1990) Plant Cell 2:279-89; Hannon, (2002) Nature, 418:244-51). When dsRNA enters the cell, it is cleaved by Dicer, an RNase III-like enzyme, into short interfering double-stranded RNAs (siRNAs) 21-23 nucleotides in length, containing a 2-nucleotide overhang at the 3' end (Elbashir et al., (2001) Genes Dev., 15:188-200; Bernstein et al., (2001) Nature, 409:363-6; Hammond et al., (2000) Nature, 404:293-6). At an ATP-dependent step, the siRNA is generally integrated into a multi-subunit protein complex known as the RNAi-induced silencing complex (RISC), which guides the siRNA to the target RNA sequence (Nykanen et al., (2001) Cell, 107:309-21). At some point the siRNA duplex unwinds, and the antisense strand remains bound to RISC and is thought to direct the degradation of the complementary mRNA sequence by a combination of endonucleases and exonucleases (Martinez et al., (2002) Cell, 110:563-74). However, the effects of iRNA or siRNA or their use effects are not limited to any kind of mechanism.

[0189] Small interfering RNA (siRNA) is a double-stranded RNA that can induce sequence-specific post-transcriptional gene silencing, thereby reducing or even suppressing gene expression. In one example, siRNA causes specific degradation of homologous RNA molecules such as mRNA within the region of sequence identity between both the siRNA and the target RNA. For example, WO02 / 44321 discloses siRNAs capable of sequence-specific degradation of target mRNA when forming base pairs with 3' overhang ends, and the methods for making these siRNAs are incorporated herein by reference.

[0190] Sequence-specific gene silencing can be achieved in mammalian cells using synthetic small double-stranded RNAs that mimic siRNAs generated by the enzyme Dicer (Elbashir et al., (2001) Nature, 411:494 - 498) (Ui-Tei et al., (2000) FEBS Lett 479:79 - 82). siRNAs can be chemically or synthetically produced in vitro or can result from small double-stranded hairpin-like RNAs (shRNAs) that are processed intracellularly into siRNAs. Synthetic siRNAs are generally designed using algorithms and conventional DNA / RNA synthesizers. Suppliers include Ambion (Austin, Texas), ChemGenes (Ashland, Massachusetts), Dharmacon (Lafayette, Colorado), Glen Research (Sterling, Virginia), MWB Biotech (Ebersberg, Germany), Proligo (Boulder, Colorado), and Qiagen (Venlo, Netherlands). Also, siRNAs can be synthesized in vitro using kits such as Ambion's SILENCER® siRNA Construction Kit.

[0191] The generation of siRNA from vectors is more commonly achieved by transcription of short hairpin RNase (shRNA). Kits for producing vectors containing shRNA are available, such as Imgenex's GENESUPPRESSOR™ construction kit and Invitrogen's BLOCK-IT™ inducible RNAi plasmids and lentiviral vectors.

[0192] In some embodiments, the functional nucleic acid is an siRNA, shRNA, miRNA. In some embodiments, the composition comprises a vector that expresses the functional nucleic acid. iii. Aptamers

[0193] The functional nucleic acid can be an aptamer or can encode an aptamer. Aptamers are molecules that interact with target molecules, preferably in a specific manner. Typically, aptamers are small nucleic acids ranging from 15 to 50 bases in length that fold into defined secondary and tertiary structures such as stem-loops or G-quartets. Aptamers can bind not only small molecules such as ATP and theophylline, but also large molecules such as reverse transcriptase and thrombin. Aptamers are molecules that bind to K+ from target molecules. d is 10 -12 Aptamers can bind very tightly to molecules with a molecular weight of less than 10 M. -6 , 10 -8 , 10 -10 , or 10 -12 Less than K d Aptamers can bind to target molecules with extremely high specificity. For example, aptamers have been isolated that have a binding affinity between a target molecule and another molecule that differs at only one position on the molecule that is more than 10,000-fold different. Aptamers bind to target molecules with a K d However, the K d Preferably, it is at least 10, 100, 1000, 10,000, or 100,000 times lower than When comparing molecules such as polypeptides, the background molecule is preferably a different polypeptide. iv. Ribozymes

[0194] Functional nucleic acids can be ribozymes or can encode ribozymes. A ribozyme is a nucleic acid molecule that can catalyze a chemical reaction intramolecularly or intermolecularly. Ribozymes preferably catalyze intermolecular reactions. There are many types of ribozymes that catalyze nuclease or nucleic acid polymerase-type reactions, which are based on ribozymes existing in natural systems, such as hammerhead ribozymes. There are also some ribozymes that do not exist in natural systems but have been engineered to newly catalyze specific reactions. Preferred ribozymes cleave RNA or DNA substrates, more preferably RNA substrates. Ribozymes usually cleave nucleic acid substrates by recognizing and binding to a target substrate followed by cleavage. This recognition is often mainly based on standard or non-standard base pair interactions. Since the recognition of the target substrate is based on the target substrate sequence, this property makes ribozymes particularly suitable candidates for target-specific cleavage of nucleic acids. v. External guide sequences

[0195] Functional nucleic acids can be external guide sequences or can encode external guide sequences. An external guide sequence (EGS) is a molecule that binds to a target nucleic acid molecule to form a complex, and the RNase P recognizes this complex and then cleaves the target molecule. EGS can be designed to specifically target a selected RNA molecule. RNase P helps in the processing of transfer RNA (tRNA) within cells. Bacterial RNase P can be supplemented to cleave virtually any RNA sequence by using an EGS that mimics the natural tRNA substrate for the target RNA:EGS complex. Similarly, eukaryotic EGS / RNase P-directed RNA cleavage can be utilized to cleave desired targets within eukaryotic cells. Representative examples of methods for preparing and using EGS molecules that facilitate cleavage of a variety of different target molecules are known in the art.

[0196] Methods for making and using vectors for in vivo expression of functional nucleic acids such as antisense oligonucleotides, siRNA, shRNA, miRNA, EGS, ribozymes, and aptamers are known in the art. vi. cyclic dinucleotide

[0197] The functional nucleic acid can be a cyclic dinucleotide or can encode a cyclic dinucleotide. Cyclic dinucleotides directly bind to the STING adapter protein and produce IFN-β (Zhang et al., Mol Cell., 51(2):226-35(2013). doi:10.1016 / j.molcel.2013.05.022.). Several standard and non-standard dinucleotides are known in the art, including but not limited to 2’3’-cGAMP, 2’3’-cGAMP, 3’3’-cGAMP, c-di-AMP, c-di-GMP, cAIMP(CL592), cAIMP Difluor(CL614), cAIM(PS)2 Difluor(Rp / Sp)(CL656), 2’2’-cGAMP, 2’3’-cGAM(PS)2(Rp / Sp), 3’3’-cGAMP fluorinated, c-di-AMP fluorinated, 2’3’-c-di-AMP, 2’3’-c-di-AM(PS)2(Rp,Rp), 2’3’-c-di-AM(PS)2(Rp,Rp), c-di-GMP fluorinated, 2’3’-c-di-GMP, c-di-IMP, DMXAA. vii. immunostimulatory oligonucleotide

[0198] In some embodiments, the functional nucleic acid can be an oligonucleotide ligand or can encode an oligonucleotide ligand. Examples include but are not limited to pattern recognition receptor (PRR) ligands.

[0199] Examples of PRRs include the Toll-like family of signaling molecules that play a role in initiating the innate immune response and also influence later, more antigen-specific adaptive immune responses. Thus, this oligonucleotide can serve as a ligand for Toll-like family signaling molecules such as Toll-like receptor 9 (TLR9).

[0200] For example, unmethylated CpG sites can be detected by TLR9 in human plasmacytoid dendritic cells and B cells (Zaida et al., Infection and Immunity, 76(5):2123-2129, (2008)). Thus, the oligonucleotide sequence can contain one or more unmethylated cytosine-guanine (CG or CpG, used interchangeably) dinucleotide motifs. "p" refers to the phosphodiester backbone of DNA, but in some embodiments, oligonucleotides containing CG can have a modified backbone, such as a phosphorothioate (PS) backbone.

[0201] In some embodiments, the oligonucleotide can contain more than one CG dinucleotide that are either adjacent or separated by one or more intervening nucleotides. One or more CpG motifs can be present within the oligonucleotide sequence. A number of nucleotide sequences vary the number and position of one or more CG dinucleotides, as well as the exact base sequence adjacent to the CG dimer, to stimulate TLR9.

[0202] Generally, CG ODNs are classified based on their sequences, secondary structures, and effects on human peripheral blood mononuclear cells (PBMCs). The five classes are class A (type D), class B (type K), class C, class P, and class S (Vollmer, J & Krieg, AM, Advanced drug delivery reviews 61(3):195-204(2009), which is incorporated herein by reference). CG ODNs can stimulate the production of type I interferons (such as IFNα) and induce the maturation of dendritic cells (DCs). Some classes of ODNs are also potent activators of natural killer (NK) cells via indirect cytokine signaling. Some classes are potent stimulants of human B cell and monocyte maturation (Weiner, GL, PNAS USA 94(20):10833-7(1997); Dalpke, AH, Immunology 106(1):102-12(2002); Hartmann, G, J of Immun.164(3):1617-2(2000), which are each incorporated herein by reference).

[0203] Other PRR Toll-like receptors include TLR3 and TLR7, which recognize double-stranded RNA, single-stranded RNA, and short double-stranded RNA, respectively, as well as retinoic acid-inducible gene I (RIG-I)-like receptors, namely RIG-I and melanoma differentiation-associated gene 5 (MDA5), which are best known as cytoplasmic RNA-sensing receptors.

[0204] RIG-I (retinoic acid-inducible protein 1, also known as Ddx58) and MDA-5 (melanoma differentiation-associated gene 5, also known as Ifih1 or Helicard) are cytoplasmic RNA helicases belonging to the RIG-I-like receptor (RLR) family and are important for the host antiviral response.

[0205] RIG-I and MDA-5 sense double-stranded RNA (dsRNA), which is an RNA virus replication intermediate, and transmit signals via the mitochondrial antiviral signaling protein MAVS (also known as IPS-1, VISA, or Cardif), resulting in the production of type I interferons (IFN-α, IFN-β).

[0206] RIG-I detects viral RNAs that exhibit two important features that facilitate discrimination from self-RNA: an uncapped 5'-diphosphate / triphosphate terminus and a short blunt-ended double-stranded portion. The characteristics of the MDA-5 physiological ligand have not yet been fully elucidated. However, RIG-I and MDA-5 have been shown to exhibit different dependencies on dsRNA length: RIG-I selectively binds to short dsRNA, and MDA-5 selectively binds to long dsRNA. Consistent with this, RIG-I and MDA-5 bind to poly(I:C), a synthetic dsRNA analog, with different length preferences.

[0207] Under certain circumstances, RIG-I can also indirectly sense dsDNA. Viral dsDNA can be transcribed by RNA polymerase III into dsRNA with a 5'-triphosphate moiety. Thus, poly(dA:dT), a synthetic analog of type B DNA, constitutes another ligand for RIG-I.

[0208] Exemplary RIG-I ligands include, but are not limited to, 5'ppp-dsRNA, which is a specific agonist of RIG-I; 3p-hpRNA, which is a specific agonist of RIG-I; poly(I:C) / LyoVec complexes recognized by RIG-I and / or MDA-5 depending on the size of poly(I:C); poly(dA:dT) / LyoVec complexes indirectly recognized by RIG-I.

[0209] In some embodiments, the oligonucleotide comprises a functional ligand of TLR3, TLR7, TLR8, TLR9, or RIG-I-like receptors, or combinations thereof.

[0210] Examples of immunostimulatory oligonucleotides, and methods of making them, are known in the art and are commercially available. See, for example, Bodera, P. Recent Pat Inflamm Allergy Drug Discov. 5(1):87-93(2011), which is incorporated herein by reference. 3. Composition of Cargo

[0211] The disclosed nucleic acid cargo can be, or can include, DNA or RNA nucleotides that typically include a heterocyclic base (nucleobase), a sugar moiety attached to the heterocyclic base, and a phosphate moiety that esterifies a hydroxyl functionality of the sugar moiety. The major naturally occurring nucleotides include uracil, thymine, cytosine, adenine, and guanine as heterocyclic bases, and ribose or deoxyribose sugars linked by phosphodiester bonds.

[0212] In some embodiments, the cargo comprises, or consists of, nucleotide analogs that are chemically modified to improve stability, half-life, or specificity or affinity for a target receptor as compared to their DNA or RNA counterparts. Chemical modifications include chemical modifications of the nucleobase, sugar moiety, nucleotide linkage, or combinations thereof. As used herein, "modified nucleotide" or "chemically modified nucleotide" defines a nucleotide having a chemical modification of one or more of the components of the heterocyclic base, sugar moiety, or phosphate moiety. In some embodiments, the charge of the modified nucleotide is reduced as compared to DNA or RNA of the same nucleobase sequence. For example, an oligonucleotide can have a low negative charge, no charge, or a positive charge.

[0213] Typically, nucleoside analogs support a base capable of hydrogen bonding by Watson-Crick base pairing to a standard polynucleotide base, and the analog backbone presents the base in a manner that allows such hydrogen bonding in a sequence-specific fashion between the oligonucleotide analog molecule and the bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). In some embodiments, the analog has a substantially uncharged phosphorus-containing backbone. a. Heterocyclic base

[0214] The major naturally occurring nucleotides include uracil, thymine, cytosine, adenine, and guanine as heterocyclic bases. The cargo can include chemical modifications to those nucleobase components. Chemical modification of a heterocyclic base or heterocyclic base analog may be effective to enhance the binding affinity or stability upon binding to a target sequence. Chemically modified heterocyclic bases include, but are not limited to, inosine, 5-(1-propynyl)uracil (pU), 5-(1-propynyl)cytosine (pC), 5-methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5 and 2-amino-5-(2'-deoxy-β-D-ribofuranosyl)pyridine (2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine derivatives. b. Sugar modification

[0215] The cargo can also include nucleotides modified with a sugar moiety or a sugar moiety analog. Modifications of the sugar moiety include, but are not limited to, 2'-O-aminoethoxy, 2'-O-amonioethyl (2'-OAE), 2'-O-methoxy, 2'-O-methyl, 2-guanidinoethyl (2'-OGE), 2'-O,4'-C-methylene (LNA), 2'-O-(methoxyethyl) (2'-OME) and 2'-O-(N-(methyl)acetamide) (2'-OMA). Substitution of the 2'-O-aminoethyl sugar moiety is particularly preferred because it is protonated at neutral pH and thus suppresses the charge repulsion between the TFO and the target duplex. This modification stabilizes the C3'-endo conformation of ribose or deoxyribose and also forms a crosslink with the i-1 phosphate of the purine strand of the duplex.

[0216] In some embodiments, the nucleic acid is a morpholino oligonucleotide. Morpholino oligonucleotides typically contain a purine or pyrimidine base pairing moiety effective to bind to a base in a polynucleotide by base-specific hydrogen bonding, and are further composed of two morpholino monomers, which are linked by a phosphorus-containing bond 1 to 3 atoms in length that links the morpholino nitrogen of one monomer to the 5' exocyclic carbon of the adjacent monomer. The purine or pyrimidine base pairing moiety is typically adenine, cytosine, guanine, uracil, or thymine. The synthesis, structure, and binding properties of morpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337.

[0217] Important properties of morpholino-based subunits typically include the ability to be linked in oligomeric form by bonds of a stable uncharged backbone; the ability of the polymer formed, even if it is a short oligomer of about 10 to 14 bases, to have a high T mThe ability to support nucleotide bases (e.g., adenine, cytosine, guanine, thymidine, uracil or inosine) so as to be capable of hybridizing with a complementary base target nucleic acid containing the target RNA; the ability of the oligomer to be actively transported into mammalian cells; and the ability of the oligomer:RNA heteroduplex to resist RNase degradation.

[0218] In some embodiments, the oligonucleotide uses morpholino-based subunits having base pairing moieties linked by non-charged linkages as described above. c. Inter-nucleotide linkage

[0219] Oligonucleotides are linked by inter-nucleotide linkages, which refer to chemical bonds between two nucleoside moieties. Modifications to the phosphate backbone of DNA or RNA oligonucleotides can increase the binding affinity or stability of the oligonucleotide or decrease its susceptibility to oligonucleotide nuclease digestion. Cationic modifications, including but not limited to diethylethylenediamide (DEED) or dimethylaminopropylamine (DMAP), may be particularly useful as they reduce the electrostatic repulsion between the oligonucleotide and the target. Modifications to the phosphate backbone may also include substituting one of the non-bridging oxygens of the phosphodiester bond with a sulfur atom. This substitution creates phosphorothioate inter-nucleoside linkages instead of phosphodiester bonds. Oligonucleotides containing phosphorothioate inter-nucleoside linkages have been shown to be more stable in vivo.

[0220] Examples of modified nucleotides with reduced charge include modified nucleotide linkages such as phosphate analogs having achiral and uncharged subunit linkages (e.g., Sterchak, E.P. et al., J. Org. Chem., 52:4202, (1987)) as described above, and uncharged morpholino-based polymers having achiral subunit linkages (see, e.g., U.S. Patent No. 5,034,506). Some nucleotide linkage analogs include morphidates, acetals, and polyamide-linked heterocycles.

[0221] In another embodiment, the cargo is composed of locked nucleic acids. Locked nucleic acids (LNA) are modified RNA nucleotides (see, e.g., Braasch et al., Chem. Biol., 8(1):1-7 (2001)). LNA forms more stable hybrids with DNA than DNA / DNA hybrids, which have properties similar to peptide nucleic acid (PNA) / DNA hybrids. Therefore, LNA can be used in the same way as PNA molecules. The binding efficiency of LNA can be increased, in some embodiments, by adding a positive charge thereto. Commercial nucleic acid synthesizers and standard phosphoramidite chemistry are used for the preparation of LNA.

[0222] In some embodiments, the cargo is composed of peptide nucleic acids. Peptide nucleic acids (PNA) are synthetic DNA mimetics in which the entire phosphate backbone of the oligonucleotide is replaced by a repeating unit of N-(2-aminoethyl)-glycine, and the phosphodiester bond is typically replaced by a peptide bond. Various heterocyclic bases are linked to the backbone by methylene carbonyl bonds. PNA maintains the spacing of heterocyclic bases similar to conventional DNA oligonucleotides but is an achiral, neutrally charged molecule. Peptide nucleic acids are composed of peptide nucleic acid monomers.

[0223] Other backbone modifications include variations and modifications of peptides and amino acids. Thus, the backbone components of oligonucleotides such as PNA may be peptide bonds or, alternatively, non-peptide peptide bonds. Examples include acetyl caps, amino spacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein as the O-linker), amino acids such as lysine (particularly useful when a positive charge is desired in PNA), and the like. Methods for the chemical construction of PNA are well known. See, for example, U.S. Patent Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571, and 5,786,571.

[0224] The cargo may include one or more terminal residues or modifications at either or both ends, as necessary, to enhance stability and / or the affinity of the oligonucleotide for its target. Commonly used positively charged moieties include the amino acids lysine and arginine, although other positively charged moieties may also be useful. The cargo may further be modified to be end-capped using propylamine groups to prevent degradation. Procedures for capping 3' or 5' oligonucleotides are well known in the art.

[0225] In some embodiments, the nucleic acid can be single-stranded or double-stranded. C. Pharmaceutical Compositions

[0226] The composition can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0227] A composition comprising a nucleic acid cargo complexed with the 3E10 antibody is preferably used for therapeutic purposes in combination with a suitable pharmaceutical carrier. Such compositions include an effective amount of the composition and a pharmaceutically acceptable carrier or excipient.

[0228] The composition may be a formulation for topical, local or systemic administration with a suitable pharmaceutical carrier. Remington’s Pharmaceutical Sciences, 15th Edition (Mark Publishing Company, 1975) by E.W. Martin discloses typical carriers and methods of preparation. Also, the complex may be encapsulated in suitable biocompatible particles formed of biodegradable or non-biodegradable polymers or proteins or liposomes for targeting cells. Such systems are well known to those skilled in the art. In some embodiments, the complex is encapsulated in nanoparticles.

[0229] The injectable formulation may be presented in unit dosage form, for example, in an ampoule or a multi-dose container, with a preservative added as required. The composition can take the form of a sterile aqueous or non-aqueous solution, suspension and emulsion, and in certain embodiments, can be isotonic with the blood of the subject. Examples of non-aqueous solvents are vegetable oils such as polypropylene glycol, polyethylene glycol, olive oil, sesame oil, coconut oil, peanut oil, mineral oil, injectable organic esters such as ethyl oleate, or fixed oils containing synthetic mono- or di-glycerides. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions containing saline or buffered media. Parenteral vehicles include sodium chloride solution, 1,3-butanediol, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous vehicles include liquids and nutrient supplements, and electrolyte supplements (such as those based on Ringer's dextrose). The material may be a solution, emulsion, or suspension (for example, incorporated into particles, liposomes, or cells). Usually, an appropriate amount of pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Trehalose may usually be added to the pharmaceutical composition in an amount of 1-5%. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5.

[0230] The pharmaceutical composition may include a carrier, a thickening agent, a diluent, a buffer, a preservative, and a surfactant. Carrier formulations can be found in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. One of ordinary skill in the art can readily determine various parameters for preparing and formulating the composition without undue experimentation.

[0231] The composition can also be made into an aerosol formulation (i.e., “sprayed”) for administration via inhalation, either alone or in combination with other suitable ingredients. The aerosol formulation can be placed in a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen, and air. In the case of administration by inhalation, the compound is delivered in a form that presents an aerosol spray from a pressurized pack or nebulizer using a suitable propellant.

[0232] In some embodiments, it includes a pharmaceutically acceptable carrier and compounding ingredients such as salts, carriers, buffers, emulsifiers, diluents, excipients, chelating agents, preservatives, solubilizers, or stabilizers.

[0233] Nucleic acids may be conjugated with cholesterol and lipophilic groups such as lauric acid and lithocholic acid derivatives having a C32 functional group to improve cellular uptake. For example, cholesterol has been demonstrated in vitro (Lorenz et al., Bioorg. Med. Chem. Lett., 14(19):4975-4977(2004)) and in vivo (Soutschek et al., Nature, 432(7014):173-178(2004)) to enhance siRNA uptake and serum stability. Furthermore, it has been shown that steroid-conjugated oligonucleotides bind to different lipoproteins in the bloodstream such as LDL, protecting integrity and promoting biodistribution (Rump et al., Biochem. Pharmacol., 59(11):1407-1416(2000)). Other groups that can bind to or be conjugated to the above nucleic acids to increase cellular uptake include acridine derivatives; cross-linking agents such as psoralen derivatives, azidophenacyl, proflavine, and azidoproflavine; artificial endonucleases; metal complexes such as EDTA-Fe(II) and porphyrin-Fe(II); alkylating moieties; nucleases such as alkaline phosphatase; terminal transferase; abzymes; cholesteryl moieties; lipophilic carriers; peptide conjugates; long-chain alcohols; phosphate esters; radioactive markers; non-radioactive markers; carbohydrates; and polylysine or other polyamines. U.S. Patent No. 6,919,208 to Levy et al. also describes methods for enhancing delivery. These pharmaceutical formulations may be manufactured by methods known per se, for example, by conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or lyophilization processes.

[0234] Additional carriers include sustained release preparations such as the semipermeable matrix of a solid hydrophobic polymer containing the complex, the matrix being in the form of shaped particles such as films, liposomes, or microparticles. Administration may involve implantable drug delivery systems such as microspheres, hydrogels, polymer reservoirs, cholesterol matrices, polymer systems such as matrix erosion and / or diffusion systems, and non-polymer systems. Administration by inhalation involves administering the composition and aerosol in the inhaler, alone or attached to a carrier that can be absorbed. In the case of systemic administration, the composition may preferably be encapsulated in liposomes.

[0235] The composition may be delivered using invasive devices such as vascular or urethral catheters, and further using intervention devices such as stents or stent grafts having drug delivery capabilities and configured as expansion devices, in a manner that enables tissue-specific uptake of the drug and / or nucleotide delivery system.

[0236] The formulation may be delivered using bioerodible implants, by diffusion or by degradation of the polymer matrix. In certain embodiments, administration of the formulation may be designed to provide continuous exposure to the composition over a specific period, such as several hours, days, weeks, months, or years. This may be achieved, for example, by repeated administration of the formulation or by a sustained release or controlled release delivery system that delivers the composition over a long period without repeated administration.

[0237] Other suitable delivery systems include timed-release, delayed-release, sustained-release, or controlled-release delivery systems. Such systems can often avoid repeated dosing and improve convenience for both the subject and the physician. Many types of release delivery systems are available and are known to those of skill in the art. They include, for example, systems based on polymers such as polylactic acid and / or polyglycolic acid, polyanhydrides, polycaprolactone, copolioxalate, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and / or combinations thereof. Microcapsules of the aforementioned polymers containing nucleic acids are described, for example, in U.S. Patent No. 5,075,109. Other examples include lipid-based non-polymeric systems containing sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di- and triglycerides; hydrogel release systems; liposome-based systems; phospholipid-based systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; or partially fused implants. The formulation can be, for example, microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric systems. In some embodiments, the system can enable sustained or controlled release of the composition, for example, through control of the diffusion or erosion / degradation rate of a formulation containing the complex.

[0238] The complex contains a nucleic acid cargo and an antibody, and their compositions can be formulated for pulmonary or mucosal administration. Administration can include delivery of the composition to the lungs, nose, oral (sublingual, buccal), vaginal, or rectal mucosa. As used herein, the term aerosol refers to a preparation of a fine mist of particles that can be in solution or suspension, whether generated using a propellant or not. Aerosols can be made using standard techniques such as sonication or high-pressure treatment.

[0239] In the case of administration via the upper respiratory tract, the formulation can be formulated as a solution, for example in water or (buffered or unbuffered) isotonic saline, or as a suspension, and in the case of nasal administration as droplets or a spray. Preferably, such a solution or suspension is isotonic with nasal secretions and has approximately the same pH, for example in the range of about pH 4.0 to about pH 7.4, or pH 6.0 to pH 7.0. The buffer should be physiologically compatible, and by way of example only, a phosphate buffer can be mentioned.

[0240] The complex can be delivered to the target cells using a particle delivery vehicle. Nanoparticles generally refer to particles in the range from 500 nm to less than 0.5 nm, preferably having a diameter between 50 and 500 nm, more preferably having a diameter between 50 and 300 nm. The cellular internalization of polymeric particles is highly dependent on their size, and the polymeric nanoparticles of nanoparticles are internalized into cells with much higher efficiency than the polymeric particles of microparticles. For example, Desai et al. demonstrated that nanoparticles with a diameter of 100 nm were taken up approximately 2.5 times more by cultured Caco-2 cells compared to microparticles with a diameter of 1 μm (Desai et al., Pharm. Res., 14: 1568-73 (1997)). Nanoparticles also have a high ability to diffuse deep into tissues in vivo.

[0241] In some embodiments, the delivery vehicle is a dendrimer.

[0242] Examples of preferred biodegradable polymers include synthetic polymers that degrade by hydrolysis, such as poly(hydroxy acids), for example, polymers and copolymers of lactic acid and glycolic acid, other degradable polyesters, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butic acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoate), poly(lactide-co-caprolactone), and poly(amine-co-ester) polymers, such as those described in Zhou et al., Nature Materials, 11:82-90 (2012) and International Publication No. WO 2013 / 082529, U.S. Patent Application Publication No. 2014 / 0342003, and PCT / US2015 / 061375.

[0243] In some embodiments, particularly those for targeting T cells in vivo, such as CAR In embodiments for producing T cells in vivo, immune cell or T cell markers such as CD3, CD7, or CD8, or markers of target tissues such as the liver can be targeted. For example, both anti-CD8 antibodies and anti-CD3 Fab fragments have been used to target T cells in vivo (Pfeiffer et al., EMBO Mol Med., 10(11)(2018). pii: e9158. doi: 10.15252 / emmm.201809158., Smith et al., Nat Nanotechnol., 12(8):813-820(2017). doi: 10.1038 / nnano.2017.57). Thus, in some embodiments, the particle or other delivery vehicle includes a targeting moiety specific for CD3, CD7, CD8, or another immune cell (e.g., T cell) marker, or a marker specific for a particular tissue such as the thymus, spleen, or liver. This binding moiety can be, for example, an antibody or an antigen-binding fragment thereof.

[0244] The targeting moiety can be directly or indirectly associated, linked, conjugated, or otherwise attached to the nanoparticles or other delivery vehicles. The targeting molecule can be a protein, peptide, nucleic acid molecule, saccharide, or polysaccharide that binds to a receptor or other molecule on the surface of the targeted cell. The degree of specificity and binding strength of the attachment to the graft can be adjusted by the choice of the targeting molecule.

[0245] Examples of moieties include, for example, targeting moieties that provide for the delivery of molecules to specific cells, such as antibodies to hematopoietic stem cells, CD34 + cells, T cells, or other preferred cell types, as well as receptors and ligands expressed on the preferred cell types. Preferably, this moiety targets hematopoietic stem cells. Examples of molecules that target the extracellular matrix ("ECM") include glycosaminoglycans ("GAG") and collagen. In one embodiment, the outer surface of the polymeric particle may be modified to enhance the ability of the particle to interact with the selected cell or tissue. The above method in which an adapter element conjugated to the targeting molecule is inserted into the particle is preferred. However, in another embodiment, the outer surface of a polymeric microparticle or nanoparticle having a carboxy terminus may be linked to a targeting molecule having a free amine terminus.

[0246] Other useful ligands that bind to polymeric micro- and nanoparticles include pathogen-associated molecular patterns (PAMPs). PAMPs can target Toll-like receptors (TLRs) on the surface of cells or tissues, or transmit signals inside cells or tissues, thereby increasing uptake. PAMPs that are bound to or co-encapsulated in the particle surface can include: unmethylated CpG DNA (bacteria), double-stranded RNA (viruses), lipopolysaccharide (bacteria), peptidoglycan (bacteria), lipoarabinomannin (bacteria), zymosan (yeast), mycoplasma lipoproteins such as MALP-2 (bacteria), flagellin (bacteria), poly(inosinic-cytidylic) acid (bacteria), lipoteichoic acid (bacteria) or imidazoquinoline (synthetic).

[0247] In another embodiment, the outer surface of the particle can be treated with mannosamine, thereby mannoseylating the outer surface of the particle. This treatment allows the particle to bind to target cells or tissues via mannose receptors on the surface of antigen-presenting cells. Alternatively, surface conjugation with immunoglobulin molecules including Fc moieties (targeting Fc receptors), heat shock protein moieties (HSP receptors), phosphatidylserine (scavenger receptors), and lipopolysaccharide (LPS) are additional receptor targets on cells or tissues.

[0248] Lectins that can covalently bind to microparticles and nanoparticles and target them specifically to mucin and mucosal cell layers.

[0249] The choice of targeting moiety will depend on the method of administration of the nanoparticle composition and the cells or tissues to be targeted. Targeting molecules can generally increase the binding affinity of the particle for cells or tissues, or target the nanoparticle to specific tissues within an organ or specific cell types within a tissue. In some embodiments, the targeting moiety targets the thymus, spleen, or cancer cells.

[0250] Covalently attaching any of the natural components of mucin in pure or partially purified form to the particles reduces the surface tension at the bead-intestine interface and increases the solubility of the beads in the mucin layer. Attachment of polyamino acids containing extra pendant carboxylic acid side groups, such as polyaspartic acid and polyglutamic acid, increases bioadhesion. Using polyamino acids in the molecular weight range of 15,000 to 50,000 kDa gives chains of 120 to 425 amino acid residues attached to the surface of the particles. The polyamino chains enhance bioadhesion by entanglement of the chains of the mucin chains and by increasing the charge of the carboxylic acid. III. Method of Use

[0251] A method is provided for enhancing the delivery of nucleic acid constructs using 3E10. Generally, an effective amount of the 3E10 antibody is first contacted with a nucleic acid cargo whose delivery to cells is desired. For example, the nucleic acid cargo and the antibody can be mixed in solution for a time sufficient for the nucleic acid cargo and the antibody to form a complex. Next, the mixture is contacted with the cells. In other embodiments, the cargo and the antibody are added to a solution containing or otherwise bathing the cells to form the complex in the presence of the cells. The complex can be contacted with the cells in vitro, ex vivo, or in vivo. Thus, in some embodiments, the solution of the complex is added to cells in culture or injected into an animal to be treated.

[0252] This antibody is thought to help deliver nucleic acids into the cell nucleus and then alter the function of the RAD51 pathway that promotes gene editing by donor DNA. There are no sequence restrictions on the design of the nucleic acid cargo for this approach. The treatment can be, for example, administration to a subject in need thereof by simple IV administration of a mixture of the antibody and the nucleic acid cargo.

[0253] The compositions and methods can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid constructs formed from RNA, DNA, PNA, or other modified nucleic acids, or combinations thereof.

[0254] An effective amount or a therapeutically effective amount of the composition can be an amount sufficient to treat, suppress, or alleviate one or more symptoms of a disease or disorder, or an amount sufficient to provide a desired pharmacological and / or physiological effect, e.g., reduction, suppression, or reversal of one or more pathophysiological mechanisms underlying the disease or disorder.

[0255] Also, the effective amount can be an amount effective to increase the rate, amount, and / or quality of nucleic acid cargo delivery as compared to administration of the cargo in the absence of the antibody. The formulation of the composition is made to be compatible with the mode of administration. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Thus, there are a wide variety of suitable formulations of pharmaceutical compositions containing the complex. The exact dosage will vary depending on various factors such as variables that depend on the subject (e.g., age, immune system health, clinical symptoms, etc.).

[0256] The composition can be administered to or otherwise contacted with the target cells once, twice, or three times a day; once, twice, three times, four times, five times, six times, or seven times a week; once, twice, three times, four times, five times, six times, seven times, or eight times a month. For example, in some embodiments, the composition is administered every two or three days, or on average about two to about four times a week. Thus, in some embodiments, the composition is administered as part of a dosing regimen that includes two or more separate treatments.

[0257] Dosing regimens include maintenance regimens in which the dosage does not change between two or more administrations, escalating regimens in which the dosage increases between two or more administrations, tapering regimens in which the dosage decreases between two or more administrations, or combinations thereof.

[0258] In some embodiments, the initial dose may be a low dose. The dose increase can continue until a satisfactory biochemical or clinical response is reached. The clinical response varies depending on the disease or disorder being treated and / or the desired outcome. In some embodiments, the dose can be increased until a therapeutic effect is confirmed, preferably without inducing unacceptable toxicity, or until a tolerable high dose. Next, the dose can be maintained or gradually reduced to a maintenance dose. These methods can be used to standardize, optimize, or customize the dosing level, frequency, or duration of treatment.

[0259] Generally, prior to administration, particularly in the case of in vivo administration, the antibody and nucleic acid are mixed at room temperature for a period of time. In some embodiments, the time for complex formation ranges from, for example, 1 minute to 30 minutes, or 10 minutes to 20 minutes (including both ends), and the preferred complex formation time is about 15 minutes. The dose of the antibody can range from 0.0001 mg to 1 mg (including both ends), and the preferred dose is about 0.1 mg. The dose of the nucleic acid can range from 0.001 μg to 100 μg (including both ends), and the preferred dose is 10 μg. The following in vivo data (e.g., Figure 6B) was generated using 0.1 mg of 3E10 and 10 μg of mRNA with complex formation for 15 minutes.

[0260] The following examples may show that while DNA cargo is more commonly delivered to multiple tissues and often not limited to tumors, RNA delivery is often more selective for tumor tissue. Thus, in some embodiments, RNA cargo can be selectively delivered to cancer cells or other tumor tissues (e.g., alone). In some embodiments, when a broader distribution of RNA cargo is desired, RNA can be mixed with DNA (e.g., carrier DNA) to facilitate delivery to non-cancer / tumor tissues. Carrier DNA can be, for example, plasmid DNA or low molecular weight DNA (e.g., derived from salmon sperm). In some embodiments, carrier DNA is non-coding DNA. Carrier DNA can be single-stranded, double-stranded, or a combination thereof. In some embodiments, carrier DNA is composed of nucleic acids having a length of 1 to 10, 1 to 100, 1 to 1,000, or 1 to 10,000 nucleotides, or any sub-range or integer thereof, or a combination thereof. Typically, carrier DNA is not conjugated or covalently bound to an antibody. Typically, carrier DNA is co-incubated with the cargo nucleic acid (e.g., RNA) and an antibody and co-delivered as a complex with them. In some embodiments, carrier DNA is non-coding DNA. A. Methods in vitro and ex vivo

[0261] For methods in vitro and ex vivo, cells are typically contacted with the composition during culture. For methods ex vivo, cells can be isolated from a subject and contacted with the composition ex vivo to generate cells containing one or more cargo nucleic acids. In preferred embodiments, the cells are isolated from the subject to be treated or a syngeneic host. Target cells can be removed from the subject prior to contact with the composition. B. Methods in vivo

[0262] In some embodiments, in vivo delivery of nucleic acid cargo to cells is used for gene editing and / or treatment of a subject's disease or disorder. Typically, a composition comprising an antibody-nucleic acid cargo can be administered directly to a subject for in vivo therapy.

[0263] Generally, methods of administering compounds including antibodies, oligonucleotides, and related molecules are well known in the art. In particular, the routes of administration already used for nucleic acid therapies provide preferred routes of administration and formulations for the above donor oligonucleotides, along with the currently used formulations. Preferably, the composition is injected or infused into an animal.

[0264] The composition can be administered by many routes including, but not limited to, intravenous, intraperitoneal, intraamniotic, intramuscular, subcutaneous, or topical (sublingual, rectal, intranasal, pulmonary, rectal mucosa, and vaginal), and oral (sublingual, buccal).

[0265] In some embodiments, the composition is formulated for pulmonary delivery, such as intranasal administration or oral inhalation. Administration of the formulation may be achieved by any acceptable method that enables the complexes to reach their target. The administration may be local (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the symptoms being treated. Compositions and methods for in vivo delivery are also contemplated in WO2017 / 143042.

[0266] This method may also include administering an effective amount of the antibody-nucleic acid complex composition in vivo to an embryo or fetus, or its pregnant mother. In some methods, the composition is delivered in utero by injecting and / or infusing the composition into a vein or artery, such as the umbilical vein or umbilical cord vein, or into the amniotic sac of the embryo or fetus. See, for example, Ricciardi et al., Nat Commun. 2018 Jun 26;9(1):2481.doi:10.1038 / s41467-018-04894-2, and WO2018 / 187493. C. Use

[0267] Nucleic acid cargos encoding a polypeptide of interest or functional nucleic acids, such as mRNA, functional nucleic acids, DNA expression constructs, vectors, etc., can be delivered to cells using the 3E10 antibody for the expression or suppression of polypeptides within the cells. The compositions and methods can be used for a variety of different applications. Non-limiting examples include delivery of CRISPR and gRNA expression vectors + / - edited DNA, large DNA (plasmids and expression vectors), gene replacement and gene therapy, such as for generating CAR-T cells in vivo or ex vivo, and for purifying the production of CAR-T cells in vivo or ex vivo, delivery of siRNA, delivery of mRNA, etc. Exemplary applications related to gene therapy / gene editing and immunomodulation, particularly chimeric antigen receptor T cell production, are considered below. 1. Gene Therapy and Editing

[0268] In some embodiments, the composition is used for gene editing. For example, this method can be particularly useful for treating genetic deficiencies, disorders, and diseases resulting from mutations in a single gene, such as for correcting genetic deficiencies, disorders, and diseases resulting from point mutations. If the target gene contains a mutation that causes a genetic disorder, this method can be used for mutagenic repair that can normally restore the DNA sequence of the target gene. The target sequence can be within the coding DNA sequence of the gene or within an intron. Also, the target sequence can be within a DNA sequence that regulates the expression of the target gene, including a promoter sequence or an enhancer sequence.

[0269] In the methods herein, cells contacted with the complex can be administered to a subject. The subject can have a disease or disorder such as hemophilia, muscular dystrophy, globinopathy, cystic fibrosis, xeroderma pigmentosum, or lysosomal storage disease. In such embodiments, gene modification, gene replacement, gene addition, or combinations thereof can occur in an effective amount to alleviate one or more symptoms of the subject's disease or disorder.

[0270] In some embodiments, the DNA cargo comprises a nucleic acid encoding a nuclease, a donor oligonucleotide or a nucleic acid encoding a donor oligonucleotide, or a combination thereof. a. Gene editing nuclease

[0271] The nucleic acid cargo includes those encoding one or more elements that induce single-stranded or double-stranded breaks in the genome of the target cell, and, optionally, other elements, preferably donor oligonucleotides, and / or those encoding in combination with other elements of the system such as, particularly in the case of CRISPR / Cas, gRNA. The composition can be used, for example, to reduce the expression of a target gene or otherwise modify it. i. Strand break-inducing element CRISPR / Cas

[0272] In some embodiments, the nucleic acid cargo comprises one or more elements of a CRISPR / Cas-mediated genome editing composition, a nucleic acid encoding one or more elements of a CRISPR / Cas-mediated genome editing composition, or a combination thereof. As used herein, a CRISPR / Cas-mediated genome editing composition refers to the elements of the CRISPR system necessary to perform CRISPR / Cas-mediated genome editing in a mammalian subject. As will be discussed in more detail below, a CRISPR / Cas-mediated genome editing composition typically includes one or more nucleic acids encoding crRNA, tracrRNA (or its chimera also called guide RNA or single guide RNA), and a Cas enzyme such as Cas9. A CRISPR / Cas-mediated genome editing composition can optionally include a donor polynucleotide that can recombine into the genome of the target cell at or adjacent to the target site (e.g., the single-stranded or double-stranded break site induced by Cas9).

[0273] The CRISPR / Cas system has been adapted for use as a gene editing (silencing, enhancing, or altering of a specific gene) for use in eukaryotes (see, for example, Cong, Science, 15:339(6121):819-823(2013) and Jinek et al., Science, 337(6096):816-21(2012)). By transfecting cells with the necessary elements including the cas gene and specially designed CRISPRs, the genome of an organism can be cleaved and modified at any desired position. Methods for preparing compositions for use in genome editing using the CRISPR / Cas system are described in detail in WO2013 / 176772 and WO2014 / 018423, which are hereby specifically incorporated by reference in their entirety.

[0274] The delivery methods disclosed herein are suitable for use with numerous variations of the CRISPR / Cas system.

[0275] Generally, the "CRISPR system" refers to the collective term for transcripts and other elements that are involved in the expression of or direct the activity of CRISPR associated ("Cas") genes, and includes sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or the active portion of tracrRNA), tracr-mate sequences (including "direct repeats" and the processed direct repeat portion of tracrRNA in the context of an endogenous CRISPR system), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), or other sequences and transcripts derived from the CRISPR locus. One or more tracr mate sequences (e.g., direct repeat-spacer-direct repeat) operably linked to a guide sequence may be referred to as pre-crRNA (pre-CRISPR RNA) prior to processing or crRNA after processing by a nuclease.

[0276] As will be discussed in more detail below, in some embodiments, the tracrRNA and crRNA are linked to form a chimeric crRNA-tracrRNA hybrid, in which the mature crRNA is fused to a partial tracrRNA via a synthetic stem loop, mimicking the native crRNA:tracrRNA duplex as described in Cong, Science, 15:339(6121):819-823(2013) and Jinek et al., Science, 337(6096):816-21(2012). A single fusion crRNA-tracrRNA construct is also referred to herein as a guide RNA or gRNA (or single guide RNA (sgRNA)). Within the sgRNA, the crRNA portion can be identified as the "target sequence" and the tracrRNA is often referred to as the "scaffold".

[0277] In some embodiments, one or more elements of the CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a particular organism, such as Streptococcus pyogenes, that contains an endogenous CRISPR system.

[0278] Generally, a CRISPR system is characterized by an element (also referred to as a protospacer in the context of an endogenous CRISPR system) that promotes the formation of a CRISPR complex at the site of a target sequence. In the context of CRISPR complex formation, a "target sequence" refers to a sequence that is designed such that the guide sequence has complementarity, where hybridization of the target sequence and the guide sequence promotes the formation of the CRISPR complex. The target sequence can be any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located within the nucleus or cytoplasm of a cell.

[0279] In a target nucleic acid, each protospacer is associated with a protospacer adjacent motif (PAM) whose recognition is specific to an individual CRISPR system. In the CRISPR / Cas system of Streptococcus pyogenes, the PAM is the nucleotide sequence NGG. In the CRISPR / Cas system of Streptococcus thermophiles, the PAM is the nucleotide sequence NNAGAAW. The tracrRNA duplex directs Cas to a DNA target consisting of the protospacer and the required PAM through heteroduplex formation between the spacer region of the crRNA and the protospacer DNA.

[0280] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (including a guide sequence that hybridizes to a target sequence and forms a complex with one or more Cas moieties) results in cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs therefrom). Also, all or part of the tracr sequence can form part of the CRISPR complex, such as by hybridization to all or part of a tracr mate sequence operably linked to the guide sequence.

[0281] Once a desired DNA target sequence is identified, many resources are available to assist an expert in determining a suitable target site. For example, numerous public resources are available to assist an expert in selecting a target site and designing an associated sgRNA that affects nicking or double-strand breakage at that site, including a list of approximately 190,000 bioinformatically generated potential sgRNAs that target more than 40% of human exons. See also crispr.u-psud.fr / , a tool designed to help scientists discover CRISPR target sites in a wide range of species and generate appropriate crRNA sequences.

[0282] In some embodiments, one or more vectors that drive the expression of one or more elements of the CRISPR system are introduced into a target cell such that the expression of the elements of the CRISPR system directs the formation of CRISPR complexes at one or more target sites. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in a single vector with one or more additional vectors to provide components of the CRISPR system not included in the first vector. The CRISPR system elements combined in a single vector can be arranged in any suitable orientation, such as one element located 5’ (“upstream”) or 3’ (“downstream”) relative to a second element. The coding sequence of one element can be located on the same or opposite strand of the coding sequence of the second element and can be oriented in the same or opposite direction. In some embodiments, a single promoter drives the expression of a transcript encoding the CRISPR enzyme, and one or more guide sequences, the tracr mate sequence (operably linked to the guide sequence if necessary), and the tracr sequence embedded in one or more intron sequences (e.g., each in a different intron, two or more sequences in at least one intron, or all sequences in a single intron). In some embodiments, the CRISPR enzyme, the guide sequence, the tracr mate sequence, and the tracr sequence are operably linked to the same promoter and are expressed from the same promoter.

[0283] In some embodiments, the vector contains one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site"). In some embodiments, one or more insertion sites (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and / or downstream of one or more array elements of one or more vectors. In some embodiments, the vector contains an insertion site upstream of the tracr mate sequence and, optionally, downstream of a regulatory element operably linked to the tracr mate sequence, such that upon expression after the guide sequence is inserted into the insertion site, the guide sequence directs sequence-specific binding of the CRISPR complex to a target sequence in a eukaryotic cell. In some embodiments, the vector contains two or more insertion sites, each insertion site being located between two tracr mate sequences such that insertion of a guide sequence is enabled at each site. In such an arrangement, two or more guide sequences can include two or more copies of a single guide sequence, two or more different guide sequences, or combinations thereof. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences in a cell. For example, a single vector can contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more guide sequences. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more such guide sequence-containing vectors are provided and can be delivered to cells as needed.

[0284] In some embodiments, the vector comprises a regulatory element operably linked to an enzyme-encoding sequence encoding a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified forms thereof. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.

[0285] In some embodiments, the vector encodes a CRISPR enzyme that is mutated with respect to the corresponding wild-type enzyme such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, the substitution of aspartate to alanine (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves one strand). Examples of other mutations that convert Cas9 to a nickase include, but are not limited to, H840A, N854A, and N863A. As a further example, two or more catalytic domains (RuvC I, RuvC II, and RuvC III) of Cas9 can be mutated to generate a mutant Cas9 that substantially lacks all DNA cleavage activity. In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to generate a Cas9 enzyme that substantially lacks all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity if the DNA cleavage activity of the mutant enzyme is about 25%, 10%, 5%, 1%, 0.1%, less than 0.01%, or lower than its non-mutated form.

[0286] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is a codon optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell can be of or derived from a particular organism, including but not limited to mammals such as humans, mice, rats, rabbits, dogs, or non-human primates. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a target host cell by replacing at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with a codon that is more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species exhibit particular biases for particular codons of a particular amino acid. Codon bias (differences in codon usage frequency among organisms) often correlates with the translational efficiency of messenger RNA (mRNA), which is thought to depend, among other things, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules.

[0287] The predominance of a selected tRNA intracellularly generally reflects the codons most frequently used in peptide synthesis. Thus, based on codon optimization, genes can be adjusted for optimal gene expression in a given organism. Codon usage frequency tables are readily available, for example, in the "Codon Usage Frequency Database", and these tables can be adapted in many ways. See Nakamura, Y. et al., Nucl. Acids Res., 28:292 (2000). Computer algorithms for optimizing codons for a particular sequence for expression in a particular host cell, such as Gene Forge (Aptagen; Jacobus, PA), are also available. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in the sequence encoding the CRISPR enzyme match the codons most frequently used for a particular amino acid.

[0288] In some embodiments, the vector encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs). If more than one NLS is present, each may be selected independently of the others such that one NLS is present in more than one copy and / or is present in combination with one or more other NLSs present in one or more copies. In some embodiments, the NLS is considered to be near the N-terminus or C-terminus if the closest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 amino acids, or more amino acids, along the polypeptide chain from the N-terminus or C-terminus.

[0289] Generally, one or more NLSs have sufficient strength to drive the accumulation of a detectable amount of the CRISPR enzyme in the nucleus of a eukaryotic cell. Generally, the strength of the nuclear localization activity can be derived from the number of NLSs in the CRISPR enzyme, the particular one or more NLSs used, or a combination of these factors.

[0290] Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the CRISPR enzyme such that the intracellular location, e.g., the location of the nucleus, can be visualized in combination with means for detecting the location, e.g., staining specific for the nucleus such as DAPI. The cell nucleus may be isolated from the cell and its contents analyzed by any suitable process for detecting proteins, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may be determined indirectly, such as by assays for the effect on CRISPR complex formation (e.g., assays for DNA cleavage or mutation at the target sequence, or assays for changes in gene expression activity affected by CRISPR complex formation and / or CRISPR enzyme activity), compared to a control not exposed to the CRISPR enzyme or complex, or a control exposed to a CRISPR enzyme lacking one or more NLSs.

[0291] In some embodiments, one or more elements of the CRISPR system are under the control of an inducible promoter, which may include inducible Cas such as Cas9.

[0292] Cong, Science, 15:339(6121):819 - 823(2013) reported that heterologous expression of Cas9, tracrRNA, pre - crRNA (or Cas9 and sgRNA) can achieve targeted cleavage of mammalian chromosomes. Thus, the CRISPR system utilized in the methods disclosed herein, and thus the cargo nucleic acid, can include one or more vectors that include elements of a CRISPR system encoding an element of a CRISPR system that can be operably linked to a first regulatory element in a chimeric RNA (chiRNA) polynucleotide sequence of the CRISPR / Cas system, where the polynucleotide sequence includes (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence; and, optionally, a second regulatory element operably linked to an enzyme - coding sequence encoding a CRISPR enzyme that can include at least one or more nuclear localization sequences. Elements (a), (b), and (c) can be arranged in the 5' to 3' direction, and components I and II are located on the same or different vectors of the system. When transcribed, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence directs sequence - specific binding of the CRISPR complex to the target sequence. And the CRISPR complex can include (1) a guide sequence hybridized to the target sequence and (2) a CRISPR enzyme complexed with a tracr mate sequence hybridized to the tracr sequence, and the enzyme - coding sequence encoding the CRISPR enzyme further encodes a heterologous functional domain. In some embodiments, one or more vectors also encode a suitable Cas enzyme, such as Cas9. Different genetic elements may be under the control of the same promoter or different promoters.

[0293] Details may vary depending on the various CRISPR systems designed, but the overall methodology is similar. For experts interested in targeting a DNA sequence (identified using one of many available online tools) using CRISPR technology, a short DNA fragment containing the target sequence can be inserted into a guide RNA expression plasmid. The sgRNA expression plasmid contains the target sequence (about 20 nucleotides), one form of the tracrRNA sequence (scaffold), as well as a suitable promoter and elements necessary for proper processing in eukaryotic cells. Such vectors are commercially available (e.g., see Addgene). Many of the systems rely on custom complementary oligos that anneal to form double-stranded DNA and are then cloned into the sgRNA expression plasmid. Co-expression of the sgRNA and the appropriate Cas enzyme from the same or separate plasmids in transfected cells results in single-stranded or double-stranded breaks at the desired target site (depending on the activity of the Cas enzyme). ii. Zinc finger nuclease

[0294] In some embodiments, the element that induces single-stranded or double-stranded breaks in the genome of the target cell is one or more nucleic acid constructs encoding a zinc finger nuclease (ZFN). Thus, the nucleic acid cargo can encode a ZFN.

[0295] ZFNs are typically fusion proteins that contain a DNA-binding domain derived from zinc finger proteins linked to a cleavage domain. The most common cleavage domain is FokI of the IIS type enzymes. FokI catalyzes double-strand breaks in DNA nine nucleotides from the recognition site on one strand and 13 nucleotides from the recognition site on the other strand. See, for example, U.S. Patent Nos. 5,356,802; 5,436,150 and 5,487,994; and Li et al., Proc., Natl. Acad. Sci. USA 89(1992):4275-4279; Li et al., Proc. Natl. Acad. Sci. USA, 90:2764-2768(1993); Kim et al., Proc. Natl. Acad. Sci. USA. 91:883-887(1994a); Kim et al., J. Biol. Chem. 269:31,978-31,982(1994b). One or more of these enzymes (or enzymatic functional fragments thereof) can be used as a source of the cleavage domain.

[0296] In principle, DNA-binding domains that can be designed to target a desired genomic location can be tandem arrays of Cys2His2 zinc fingers, each of which generally recognizes 3-4 nucleotides of a target DNA sequence. The Cys2His2 domain has the general structure: Phe (sometimes Tyr)-Cys-(2-4 amino acids)-Cys-(3 amino acids)-Phe (sometimes Tyr)-(5 amino acids)-Leu-(2 amino acids)-His-(3 amino acids)-His. By linking multiple fingers (the number varies: 3-6 fingers per monomer have been used in published studies), ZFN pairs can be designed to bind to genomic sequences 18-36 nucleotides in length.

[0297] Engineering approaches include, but are not limited to, rational design and various types of empirical selection approaches. Rational design includes, for example, the use of databases containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or quadruplet nucleotide sequence is associated with the amino acid sequence of one or more zinc fingers that bind to a specific triplet or quadruplet sequence. See, for example, U.S. Patent Nos. 6,140,081; 6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997; 7,067,617; U.S. Patent Application Publication Nos. 2002 / 0165356; 2004 / 0197892; 2007 / 0154989; 2007 / 0213269; and International Patent Application Publication Nos. WO98 / 53059 and WO2003 / 016496. iii. Transcription activator-like effector nuclease

[0298] In some embodiments, the element that induces a single-stranded or double-stranded break in the genome of the target cell is one or more nucleic acid constructs encoding a transcription activator-like effector nuclease (TALEN). Thus, the nucleic acid cargo can encode a TALEN.

[0299] TALENs have an overall structure similar to ZFNs, but the main difference is that the DNA-binding domain is derived from the TAL effector protein, a transcription factor of plant pathogens. The DNA-binding domain of TALENs is a tandem array of amino acid repeats, each approximately 34 residues in length. The repeats are very similar to each other and usually differ mainly at two positions (amino acids 12 and 13, the repeat-variable diresidue, or RVD for short). Each RVD specifies preferential binding to one of the four possible nucleotides. That is, each TALEN repeat binds to a single base pair, although the NN RVD means binding to both guanine and adenine. The DNA binding of TAL effectors is less mechanistically understood compared to that of zinc finger proteins, but their seemingly simple code may prove very useful for the design of engineered nucleases. TALENs also cleave as dimers and have relatively long target sequences (the shortest reported so far binds 13 nucleotides per monomer) and do not seem to be as strict as ZFNs with respect to the length of the spacer between binding sites. Monomeric and dimeric TALENs can contain more than 10, more than 14, more than 20, or more than 24 repeats.

[0300] Methods for engineering TALs to bind to specific nucleic acids are described in Cermak et al., Nucl. Acids Res. 1-11 (2011). US Patent Application Publication No. 2011 / 0145940 discloses TAL effectors and methods for using them to modify DNA. Miller et al., Nature Biotechnol 29:143 (2011) reported generating TALENs for site-specific nuclease constructs by linking TAL truncation variants to the catalytic domain of FokI nuclease. The resulting TALENs were shown to induce gene modification in immortalized human cells. General design principles for the TALE binding domain can be found, for example, in WO2011 / 072246. b. Donor polynucleotide

[0301] The nuclease activity of the genome editing system described herein cleaves the target DNA to generate single-stranded or double-stranded breaks in the target DNA. The double-stranded breaks can be repaired by the cell in either of two ways: non-homologous end joining and homology-directed repair. In non-homologous end joining (NHEJ), the double-stranded breaks are repaired by directly ligating the broken ends to each other. As a result, no new nucleic acid material is inserted at the site, but some nucleic acid material is lost, which may result in deletions. In homology-directed repair (HDR), a donor polynucleotide having homology to the cleaved target DNA sequence is used as a template for repair of the cleaved target DNA sequence, and as a result, genetic information is transferred from the donor polynucleotide to the target DNA. Therefore, new nucleic acid material can be inserted / copied at that site.

[0302] Accordingly, in some embodiments, the nucleic acid cargo is or comprises a donor polynucleotide. Modification of the target DNA by NHEJ and / or homology-directed repair can be used to induce gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc.

[0303] Thus, by using DNA cleavage by a genome editing composition to cleave a target DNA sequence, nucleic acid material can be deleted from the target DNA sequence and cells can be enabled to repair the sequence in the absence of an exogenously provided donor polynucleotide. Alternatively, if the genome editing composition comprises a donor polynucleotide sequence comprising at least a segment having homology to the target DNA sequence, these methods can be used for the addition of nucleic acid material to the target DNA sequence, i.e., insertion or substitution (e.g., "knock-in" of a nucleic acid encoding a protein, siRNA, miRNA, etc.), addition of a tag (e.g., 6xHis, fluorescent protein (e.g., green fluorescent protein; yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), addition of a regulatory sequence to a gene (e.g., promoter, polyadenylation signal, internal ribosome entry sequence (IRES), 2A peptide, start codon, stop codon, splice signal, localization signal, etc.), modification of a nucleic acid sequence (e.g., introduction of a mutation), and the like. Thus, this composition can be used, for example, in site-specific, i.e., "targeted", methods such as those used in gene therapy, e.g., gene knockout, gene knock-in, gene editing, gene labeling, etc., to modify DNA.

[0304] In applications where it is desirable to insert a polynucleotide sequence into a target DNA sequence, a polynucleotide containing the donor sequence to be inserted is also provided to the cell. A "donor sequence" or "donor polynucleotide" or "donor oligonucleotide" means a nucleic acid sequence that is inserted at the cleavage site. The donor polynucleotide typically has sufficient homology with the genomic sequence at the cleavage site, for example, 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequence adjacent to the cleavage site, for example, within about 50 bases, for example, within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or directly adjacent to the cleavage site of the cleavage site, and supports homology-directed repair between the genomic sequences having homology. The donor sequence is usually not identical to the genomic sequence it replaces. Rather, the donor sequence may contain at least one or more single-base changes, insertions, deletions, inversions, or rearrangements relative to the genomic sequence, as long as there is sufficient homology to support homology-directed repair. In some embodiments, the donor sequence contains non-homologous sequences flanked by two regions of homology such that non-homologous sequences are inserted into the target region by homology-directed repair between the target DNA region and the two flanking sequences. 2. Immunomodulation a. CAR T cells

[0305] The disclosed compositions and methods are particularly useful in situations where lymphocytes expressing chimeric immunoreceptors (CIRs), particularly chimeric antigen receptors (CARs), are prepared. Artificial immunoreceptors (also known as chimeric T cell receptors, chimeric immunoreceptors, chimeric antigen receptors (CARs), and chimeric immunoreceptors (CIRs) and referred to herein) are engineered receptors that confer selected specificities to cells. As will be discussed in more detail below, cells modified according to the methods discussed can be utilized in a variety of immunotherapies for the treatment of cancer, infectious diseases, inflammation, and autoimmune diseases.

[0306] In particularly preferred embodiments, mRNA or DNA encoding a chimeric antigen receptor cargo is delivered to immune cells such as lymphocytes.

[0307] The cargo can be delivered to immune cells in vivo, ex vivo, or in vitro. In a preferred embodiment, the cargo is mRNA, which can enable one or more of cost reduction, ease of manufacture, and reduction of side effects (e.g., cytokine storm, neurotoxicity, graft-versus-host disease, etc.). In certain embodiments, immune cells (e.g., T cells) are collected from a subject in need of CAR T cell therapy, and mRNA encoding one or more CAR T cell constructs is delivered to the collected cells using the compositions and methods disclosed herein, and the cells are returned to the subject. In some embodiments, the process from initially collecting the cells to returning them to the subject is one week or less, e.g., 1, 2, 3, 4, 5, 6, or 7 days. In certain embodiments, the process from initially collecting the cells to returning them to the subject is 1 or 2 days, or less than 1 day, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours.

[0308] Strategies for the design and development of chimeric antigen receptors are outlined in Dotti et al., Immunol Rev. 2014 January; 257(1):. doi:10.1111 / imr.12131 (35 pages), which is specifically incorporated herein by reference in its entirety, as well as Dotti, Molecular Therapy, 22(5):899 - 890 (2014), Karlsson et al., Cancer Gene Therapy, 20:386 - 93 (2013), Charo et al., Cancer Res., 65(5):2001 - 8 (2005), Jensen et al., Immunol Rev., 257(1):127 - 144 (2014), Eaton et al., Gene Therapy, 9:527 - 35 (2002), Barrett et al., Annu Rev Med., 65:333 - 347 (2014), Cartellieri et al., Journal of Biomedicine and Biotechnology, Volume 2010, Article ID 956304, 13 pages doi:10.1155 / 2010 / 956304; and U.S. Patent Application Publication Nos. 2015 / 0017120, 2015 / 0283178, 2015 / 0290244, 2014 / 0050709, and 2013 / 0071414.

[0309] CAR combines the antigen - binding properties of monoclonal antibodies with the lytic ability and self - renewal of T cells and has several advantages compared to conventional T cells (Ramos and Dotti, Expert Opin Biol Ther., 11:855 - 873 (2011), Curran et al., J Gene Med., 14:405 - 415 (2012), Maher, ISRN Oncol. 2012:278093 (2012)). CAR - T cells recognize and kill cancer cells independently of the major histocompatibility complex (MHC). Thus, the recognition of target cells is not affected by some mechanisms by which tumors evade MHC - restricted T - cell recognition, such as down - regulation of human leukocyte antigen (HLA) class I molecules and defective antigen processing.

[0310] Chimeric immune receptors were first developed in the 1980s and originally contained the variable (antigen-binding) region of a monoclonal antibody and the constant regions of the T cell receptor (TCR) α and β chains (Kuwana et al., Biochem Biophys Res Commun., 149:960-968 (1987)). In 1993, this design was modified to include an ectodomain, transmembrane domain, and endodomain with a signaling domain derived from CD3-ζ, from the antigen-binding regions of both the heavy and light chains of a monoclonal antibody, a single-chain variable fragment (scFv). Subsequent CARs generally follow a similar structural design with a co-stimulatory signaling endodomain. Thus, the CAR constructs utilized in the methods herein can include antigen-binding domains or ectodomains, hinge domains, transmembrane domains, endodomains, and combinations thereof.

[0311] In some embodiments, the ectodomain is a scFv. The affinity of the scFv predicts CAR function (Hudecek et al., Clin Cancer Res., 19(12):3153-64(2013), Chmielewski et al., J Immunol., 173:7647-7653(2004)). Antigen binding and subsequent activation can also be modified by adding a flexible linker sequence to the CAR, which allows for the expression of two distinct scFvs that can recognize two different antigens (Grada et al., Mol Ther Nucleic Acids, 2:e105(2013)) (also called tandem CAR (TanCAR)). Tandem CARS may be highly effective in killing cancers with low antigen expression individually and may reduce the risk of tumor immune escape by a single antigen-loss variant. Other ectodomains include IL13Rα2 (Kahlon et al., Cancer Res., 64:9160-9166(2004), Brown et al., Clin Cancer Res., 18(8):2199-209(2012), Kong et al., Clin Cancer Res., 18:5949-5960(2012), NKG2D-ligands and CD70 receptors, peptide ligands (e.g., T1E peptide ligand), and so-called "universal ectodomains" (e.g., an avidin ectodomain designed to recognize a target contacted with a biotinylated monoclonal antibody, or a FITC-specific scFv designed to recognize a target contacted with a FITC-labeled monoclonal antibody (Zhang et al., Blood, 106:1544-1551(2005), Barber et al., Exp Hematol., 36:1318-1328(2008), Shaffer et al., Blood, 117:4304-4314(2011), Davies et al., Mol Med., 18:565-576(2012), Urbanska et al., Cancer Res., 72:1844-1852(2012), Tamada et al., Clin Cancer Res., 18:6436-6445(2012))).

[0312] In some embodiments, the CAR includes a hinge region. While the ectodomain is important for the specificity of the CAR, the sequence connecting the ectodomain and the transmembrane domain (hinge region) can also affect the function of CAR-T cells by creating differences in the length and flexibility of the CAR. The hinge can include, for example, the CH2CH3 hinge derived from an immunoglobulin such as IgG1, or a fragment thereof. For example, Hudecek et al. (Hudecek et al., Clin Cancer Res., 19(12):3153-64(2013)) compared the effects of the CH2-CH3 hinge [229 amino acids (AA)], the CH3 hinge (119 AA), and the short hinge (12 AA) on the effector function of T cells expressing a third-generation ROR1-specific CAR, and found that T cells expressing the "short hinge" CAR had excellent antitumor activity. On the other hand, other researchers found that the CH2-CH3 hinge impaired epitope recognition of a first-generation CD30-specific CAR (Hombach et al., Gene Ther., 7:1067-1075(2000)).

[0313] Between the hinge (or the ectodomain if there is no hinge domain) and the signaling endodomain, there is usually a transmembrane domain, most commonly derived from the CD3-ζ, CD4, CD8, or CD28 molecule. Similar to the hinge, the transmembrane domain can also affect CAR-T cell effector function.

[0314] When the antigen is recognized, the CAR endodomain transmits activation and costimulatory signals to the T cell. Activation of the T cell depends on phosphorylation of the cytoplasmic CD3-ζ domain of the TCR complex by immunoreceptor tyrosine-based activation motifs (ITAMs) present in the cytoplasmic domain (Irving et al., Cell, 64:891-901(1991)). Most of the CAR endodomains include an activation domain derived from CD3-ζ, but some can also include ITAM-containing domains such as the Fc receptor of the IgE-γ domain (Haynes et al., J Immunol., 166:182-187(2001)).

[0315] The target specificity of cells expressing a CAR is determined by the antigen recognized by the antibody / ectodomain. The disclosed compositions and methods can be used to generate constructs that target any antigen, and cells that express such constructs. In the context of immunotherapy, particularly cancer immunotherapy, numerous antigens, and ectodomains suitable for targeting them are well known. Unlike native TCRs, the majority of scFv-based CARs recognize target antigens expressed on the cell surface, rather than internal antigens processed and presented by the cell's MHC, although CARs can recognize structures other than protein epitopes including carbohydrates and glycolipids (Dotti et al., Immunol Rev. 2014 January;257(1):. doi:10.1111 / imr.12131 (35 pages)), and thus have the advantage over conventional TCRs of an increased pool of potential target antigens. Preferred targets include antigens that are expressed only in cancer cells or the surrounding stroma (Cheever et al., Clin Cancer Res., 15:5323-5337 (2009)), such as the splice variant of EGFR specific to glioma cells (EGFRvIII) (Sampson et al., Semin Immunol., 20(5):267-75 (2008)). However, human antigens meet this requirement, and the majority of target antigens are expressed at low levels in normal cells (e.g., GD2, CAIX, HER2), and / or expressed in a lineage-restricted manner (e.g., CD19, CD20).

[0316] Preferred targets, and CARs that target them, are known in the art (see, e.g., Dotti et al., Immunol Rev. 2014 January; 257(1):. doi:10.1111 / imr.12131 (35 pages)). For example, CAR targets for hematological malignancies include, but are not limited to, CD19 (e.g., B cells) (Savoldo et al., J Clin Invest., 121:1822-1826 (2011), Cooper et al., Blood, 105:1622-1631 (2005); Jensen et al., Biol Blood Marrow Transplant (2010), Kochenderfer et al., Blood, 119:2709-2720 (2012), Brentjens et al., Molecular Therapy, 17:S157 (2009), Brentjens et al., Nat Med., 9:279-286 (2003), Brentjens et al., Blood, 118:4817-4828 (2011), Porter et al., N Engl J Med., 365:725-733 (2011), Kalos et al., Sci Transl Med., 3:95ra73 (2011), Brentjens et al., Sci Transl Med., 5:177ra38 (2013), Grupp et al., N Engl J Med (2013)); CD20 (e.g., B cells) (Jensen et al., Biol Blood Marrow Transplant (2010), Till et al., Blood, 112:2261-2271 (2008), Wang et al., Hum Gene Ther., 18:712-725 (2007), Wang et al., Mol Ther., 9:577-586 (2004), Jensen et al., Biol Blood Marrow Transplant, 4:75-83 (1998)); CD22 (e.g., B cells) ((Haso et al., Blood, 121:1165-1174 (2013)); CD30 (e.g., B cells) (Di Stasi et al., Blood, 113:6392-6402 (2009), Savoldo et al., Blood, 110:2620-2630 (2007), Hombach et al., Cancer Res., 58:1116-1119(1998));); CD33 (e.g., bone marrow) ((Finney et al., J Immunol., 161:2791-2797(1998)); CD70 (e.g., B cells / T cells) (Shaffer et al., Blood, 117:4304-4314(2011)); CD123 (e.g., bone marrow) (Tettamanti et al., Br J Haematol., 161:389-401(2013)); Kappa (e.g., B cells) (Vera et al., Blood, 108:3890-3897(2006)); Lewis Y (e.g., bone marrow) (Peinert et al., Gene Ther., 17:678-686(2010), Ritchie et al., Mol Ther. (2013)); NKG2D ligand (e.g., bone marrow) (Barber et al., Exp Hematol., 36:1318~1328(2008), Lehner et al., PLoS One., 7:e31210(2012), Song et al., Hum Gene Ther., 24:295~305(2013), Spear et al., J Immunol. 188:6389~6398(2012)); ROR1 (e.g., B cells) (Hudecek et al., Clin Cancer Res. (2013)) can be mentioned.

[0317] CAR targets for solid tumors include, but are not limited to, B7H3 (e.g., sarcoma, glioma) (Cheung et al., Hybrid Hybridomics, 22:209-218 (2003)); CAIX (e.g., kidney) (Lamers et al., J Clin Oncol., 24:e20-e22. (2006)), Weijtens et al., Int J Cancer, 77:181-187 (1998)); CD44 v6 / v7 (e.g., cervix) (Hekele et al., Int J Cancer, 68:232-238 (1996)), Dall et al., Cancer Immunol Immunother, 54:51-60 (2005); CD171 (e.g., neuroblastoma) (Park et al., Mol Ther., 15:825-833 (2007)); CEA (e.g., colon) (Nolan et al., Clin Cancer Res., 5:3928-3941 (1999)); EGFRvIII (e.g., glioma) (Bullain et al., J Neurooncol. (2009), Morgan et al., Hum Gene Ther., 23:1043-1053 (2012)); EGP2 (e.g., carcinoma) (Meier et al., Magn Reson Med., 65:756-763 (2011), Ren-Heidenreich et al., Cancer Immunol Immunother., 51:417-423 (2002)); EGP40 (e.g., colon) (Daly et al., Cancer Gene Ther., 7:284-291 (2000); EphA2 (e.g., glioma, lung) (Chow et al., Mol Ther., 21:629-637 (2013)); ErbB2 (HER2) (e.g., breast, lung, prostate, glioma) (Zhao et al., J Immunol., 183:5563-5574 (2009), Morgan et al., Mol Ther., 18:843-851 (2010), Pinthus et al., 114:1774-1781 (2004), Teng et al., Hum Gene Ther., 15:699-708 (2004), Stancovski et al., J Immunol., 151:6577-6582 (1993), Ahmed et al., Mol Ther., 17:1779-1787 (2009), Ahmed et al., Clin Cancer Res., 16:474 - 485 (2010), Moritz et al., Proc Natl Acad Sci U.S.A., 91:4318 - 4322 (1994)); ErbB receptor family (e.g., breast, lung, prostate, glioma) (Davies et al., Mol Med., 18:565 - 576 (2012)); ErbB3 / 4 (e.g., breast, ovary) (Muniappan et al., Cancer. Gene Ther., 7: 128 - 134 (2000), Altenschmidt et al., Clin Cancer Res., 2: 1001 - 1008 (1996)); HLA - A1 / MAGE1 (e.g., melanoma) (Willemsen et al., Gene Ther., 8: 1601 - 1608 (2001), Willemsen et al., J Immunol., 174: 7853 - 7858 (2005)); HLA - A2 / NY - ESO - 1 (e.g., sarcoma, melanoma) (Schuberth et al., Gene Ther., 20: 386 - 395 (2013)); FR - α (e.g., ovary) (Hwu et al., J Exp Med., 178: 361 - 366 (1993), Kershaw et al., Nat Biotechnol., 20: 1221 - 1227 (2002), Kershaw et al., Clin Cancer Res., 12: 6106 - 6115 (2006), Hwu et al., Cancer Res., 55: 3369 - 3373 (1995)); FAP (e.g., cancer - associated fibroblasts) (Kakarla et al., Mol Ther. (2013)); FAR (e.g., rhabdomyosarcoma) (Gattenlohner et al., Cancer Res., 66: 24 - 28 (2006)); GD2 (e.g., neuroblastoma, sarcoma, melanoma) (Pule et al., Nat Med., 14: 1264 - 1270 (2008), Louis et al., Blood, 118: 6050 - 6056 (2011), Rossig et al., Int J Cancer., 94: 228 - 236 (2001)); GD3 (e.g., melanoma, lung cancer) (Yun et al., Neoplasia., 2: 449 - 459 (2000)); HMW - MAA (e.g., melanoma) (Burns et al., Cancer Res., 70: 3027 - 3033 (2010)); IL11Rα (e.g., osteosarcoma) (Huang et al., Cancer Res., 72: 271 - 281 (2012)); IL13Rα2 (e.g., glioma) (Kahlon et al., Cancer Res., 64: 9160 - 9166 (2004), Brown et al., Clin Cancer Res. (2012), Kong et al., Clin Cancer Res., 18: 5949 - 5960 (2012), Yaghoubi et al., Nat Clin Pract Oncol., 6: 53 - 58 (2009)); Lewis Y (e.g., breast / ovary / pancreas) (Peinert et al., Gene Ther., 17: 678 - 686 (2010), Westwood et al., Proc Natl Acad Sci U.S.A., 102: 19051 - 19056 (2005), Mezzanzanica et al., Cancer Gene Ther., 5: 401 - 407 (1998)); Mesothelin (e.g., mesothelioma, breast, pancreas) (Lanitis et al., Mol Ther., 20: 633 - 643 (2012), Moon et al., Clin Cancer Res., 17: 4719 - 4730 (2011)); Mue1 (e.g., ovary, breast, prostate) (Wilkie et al., J Immunol., 180: 4901 - 4909 (2008)); NCAM (e.g., neuroblastoma, colorectal) (Gilham et al., J Immunother., 25: 139 - 151 (2002)); NKG2D ligand (e.g., ovary, sacoma) (Barber et al., Exp Hematol., 36: 1318 - 1328 (2008), Lehner et al., PLoS One, 7: e31210 (2012), Song et al., Gene Ther., 24: 295 - 305 (2013), Spear et al., J Immunol., 188: 6389 - 6398 (2012)); PSCA (e.g., prostate, pancreas) (Morgenroth et al., Prostate, 67: 1121 - 1131 (2007), Katari et al., HPB, 13: 643 - 650 (2011)); PSMA (e.g., prostate) (Maher et al., Nat Biotechnol., 20: 70 - 75 (2002), Gong et al., Neoplasia., 1: 123 - 127 (1999)); TAG72 (e.g., colon) (Hombach et al., Gastroenterology, 113: 1163 - 1170 (1997), McGuinness et al., Hum Gene Ther., 10: 165 - 173 (1999)); VEGFR - 2 (e.g., tumor vasculature) (J Clin Invest.,120:3953-3968(2010), Niederman et al., Proc Natl Acad Sci U.S.A., 99:7009-7014(2002)) can be cited. b. Metabolic stability

[0318] In some embodiments, the metabolic stability of a cell (e.g., a CAR cell) is improved by the cell's ability to produce growth factors, which is limited in vivo. In some embodiments, a nucleic acid cargo encoding an anti-apoptotic factor such as BCL-XL is transiently delivered to the cell. The large B-cell lymphoma (Bcl-XL, or BCL2-like 1 isoform 1) is a mitochondrial transmembrane protein. This is a member of the Bcl-2 family of proteins and acts as a survival-promoting protein in the intrinsic apoptosis pathway by preventing the release of mitochondrial contents such as cytochrome c that leads to caspase activation. Both the amino acid sequence and the nucleic acid sequence encoding BCL-XL are known in the art and include, for example, UniProtKB-Q07817 (B2CL1_HUMAN), isoform Bcl-X(L) (identifier: Q07817-1) (amino acid sequence); ENA|U72398|U72398.1 Human Bcl-x beta (bcl-x) gene, complete cds (genomic nucleic acid sequence); ENA|Z23115|Z23115.1 H. sapiens bcl-XL mRNA (mRNA / cDNA nucleic acid sequence).

[0319] In some embodiments, the nucleic cargo encodes a growth-inducing factor such as IL-2. Both the amino acid sequence and the nucleic acid sequence encoding IL-2 are known in the art, and include, for example, UniProtKB-P60568 (IL2_HUMAN) (amino acid sequence); ENA|X00695|X00695.1 Human Interleukin-2 (IL-2) gene and 5’-flanking region (gene nucleic acid sequence); and ENA|V00564|V00564.1 Human mRNA encoding Interleukin-2 (IL-2) (mRNA / cDNA nucleic acid sequence).

[0320] However, the production of secreted IL-2 can also have undesirable side effects, such as stimulating the growth of lymphoma and Treg cells and impairing the formation of memory T cells (Zhang et al., Nature Medicine, 11:1238-1243 (2005)). Furthermore, the use of IL-2 in patients treated with tumor-infiltrating lymphocytes (TIL) increased toxicity (Heemskerk et al., Human Gene Therapy, 19:496-510 (2008)). To avoid this possibility, in addition to or instead of IL-2, the nucleic cargo can encode a chimeric γc cytokine receptor (CγCR), such as one composed of interleukin-7 (IL-7) linked to IL-7Rα / CD127, which confers cell-intrinsic STAT5 cytokine signaling independent of exogenous cytokines (Hunter et al., Molecular Immunology, 56:1-11 (2013)). This design is modular and can enhance Shc activity by exchanging the cytoplasmic chain of IL-2Rβ / CD122 with the cytoplasmic chain of IL-7Rα / CD127. The construct mimics wild-type IL-2 signaling in human CD8+ T cells (Hunter et al., Molecular Immunology, 56:1-11 (2013)), and thus should act similarly to IL-2 mRNA without undesirable side effects.

[0321] Furthermore, alternatively, the cell viability can also be maintained in a native state using other anti-apoptotic molecules and cytokines. Exemplary factors include, but are not limited to, the following:

[0322] Myeloid cell leukemia 1 (MCL-1) (e.g., UniProtKB-Q07820 (MCL1_HUMAN) (amino acid sequence); ENA|AF147742|AF147742.1 Homo sapiens myeloid cell differentiation protein (MCL1) gene, promoter and complete cds (genomic nucleic acid sequence); The anti-apoptotic factor ENA|AF118124|AF118124.1 Homo sapiens myeloid cell leukemia sequence1 (MCL1) mRNA, complete cds. (mRNA / cDNA nucleic acid sequence));

[0323] IL-7 (e.g., UniProtKB-P13232 (IL7_HUMAN) (amino acid sequence); ENA|EF064721|EF064721.1 Homo sapiens interleukin7 (IL7) gene, complete cds. (genomic nucleic acid sequence); ENA|J04156|J04156.1 Human interleukin7 (IL-7) mRNA, complete cds. (mRNA / cDNA nucleic acid sequence), and

[0324] IL-15 (e.g., UniProtKB - P40933 (IL15_HUMAN) (amino acid sequence); ENA|X91233|X91233.1 H.sapiens IL15 gene (genomic nucleic acid sequence); ENA|U14407|U14407.1 Human interleukin15 (IL15) mRNA, complete cds. (mRNA / cDNA nucleic acid sequence) that promotes the survival of T and NK cells) (Opferman et al., Nature, 426:671 - 676 (2003); Meazza et al., Journal of Biomedicine & Biotechnology, 861920, doi:10.1155 / 2011 / 861920 (2011); Michaud et al., Journal of Immunotherapy, 33:382 - 390 (2010)). These cytokine mRNAs can be used independently or in combination with BCL - XL, IL - 2, and / or CγCR mRNA. Thus, in some embodiments, mRNA encoding MCL - 1, IL - 7, IL - 15, or combinations thereof is delivered to the cells. c. Inhibitory CAR (iCAR)

[0325] In some embodiments, T - cell therapy is delivered to CAR cells that have shown long - term efficacy and the potential for cure in the treatment of some cancers, but their use is limited by damage to non - cancerous tissues associated with graft - versus - host disease after donor lymphocyte infusion. Any of the disclosed compositions and methods can be used in combination with non - specific immunosuppression (e.g., high - dose corticosteroid therapy that exerts a cytostatic or cytotoxic effect on T cells to suppress the immune response), irreversible T - cell depletion (e.g., so - called suicide gene engineering strategies), or combinations thereof. However, in some preferred embodiments, off - target effects are reduced by introducing a construct encoding an inhibitory chimeric antigen receptor (iCAR) into the CAR cells. T cells that are specific for both tumor tissue and off - target tissue can be restricted to the tumor only by using the antigen - specific iCAR introduced into the T cells to protect off - target tissue (Fedorov et al., Science Translational Medicine, 5:215ra172 (2013)). iCAR contains a surface antigen recognition domain combined with a strong acute inhibitory signaling domain and can limit T cell responsiveness despite co - binding of activating receptors (e.g., CAR). In a preferred embodiment, iCAR is a single - chain variable fragment (scFv) specific for an inhibitory antigen fused to the signaling domain of an immunosuppressive receptor (e.g., CTLA - 4, PD - 1, LAG - 3, 2B4 (CD244), BTLA (CD272), KIR, TIM - 3, TGFβ receptor dominant - negative analog, etc.) via a transmembrane region that specifically inhibits T cell function upon antigen recognition. When CAR - expressing cells encounter cells that do not express the inhibitory antigen (e.g., cancer cells), T cells transduced with iCAR can initiate a CAR - induced response against the CAR - target antigen. DNA iCAR using an scFv specific for PSMA with the inhibitory signaling domain of either CTLA - 4 or PD - 1 has been discussed in (Fedorov et al., Science Translational Medicine, 5:215ra172 (2013)).

[0326] Design considerations include the fact that PD - 1 is a more potent inhibitor than CTLA - 4, CTLA - 4 showed cytoplasmic localization unless the Y165G mutant was used, and the finding that the iCAR expression level is important.

[0327] iCAR can be designed against cell - type - specific surface molecules. In some embodiments, iCAR is designed to prevent the reactivity of T cells, NK cells, or other immune cells against a particular tissue or cell type. d. Reduction of endogenous inhibitory signaling

[0328] In some embodiments, a cell is contacted with a nucleic acid cargo that reprograms the cell to prevent the expression of one or more antigens. For example, in some embodiments, the nucleic acid cargo is or encodes an interfering RNA that prevents the expression of mRNA encoding an antigen such as CTLA-4 or PD-1. This method can be used to prepare universal donor cells. The RNA used to alter the expression of allogeneic antigens may be used alone or in combination with RNA that causes dedifferentiation of the target cells.

[0329] In the above section, compositions and methods are provided that utilize inhibitory signaling domains from, for example, CTLA-4 or PD-1 in artificial iCARs to limit on-target / off-tumor cytotoxicity. Additionally or alternatively, the expression of endogenous inhibitory signaling in CAR cells can be decreased to render the CAR cells resistant to inhibitory signals in the hostile tumor microenvironment, thereby overall enhancing the effector efficiency of the CAR cells on tumors.

[0330] CTLA-4 and PD-1 suppress T cells at various stages of activation and function. CTLA-4 regulates the T cell response to self-antigens. This is because knockout mice spontaneously develop organ damage due to highly activated tissue-infiltrating T cells without exposure to specific antigens (Tivol et al., Immunity, 3:541-547 (1995); Waterhouse et al., Science, 270:985-988 (1995)). Interestingly, conditional knockout of CTLA-4 in Treg cells recapitulates the global knockout, indicating that it functions normally within Tregs (Wing et al., Science, 322:271-275 (2008)). In contrast, PD-L1 knockout mice are prone to autoimmunity, but do not naturally have massive infiltration of inflammatory cells into normal organs, indicating that its main physiological function is to mediate negative feedback control of ongoing tissue inflammation in an inducible manner (Dong et al., Immunity, 20:327-336 (2004)). Indeed, according to the "adaptive resistance" hypothesis, most tumors upregulate PD-L1 in response to IFNγ, an important cytokine released by effector T cells including CAR T cells (Greenwald et al., Annu Rev Immunol, 23:515-548 (2005); Carreno et al., Annu Rev Immunol, 20:29-53 (2002); Chen et al., The Journal of Clinical Investigation, 125:3384-3391 (2015); Keir et al., Annu Rev Immunol, 26:677-704 (2008); Pentcheva-Hoang et al., Immunological Reviews, 229:67-87 (2009)).Next, PD-L1 delivers inhibitory signals to T cells, reducing their proliferation, cytokine, and perforin production (Butte et al., Immunity, 27:111-122 (2007); Chen et al., Immunology, 4:336-347 (2004); Park et al., Blood, 116:1291-1298 (2010); Wherry et al., Nat Immunol, 12:492-499 (2011); Zou et al., Immunology, 8:467-477 (2008)). Furthermore, reverse signaling from T cells through B7-H1 in cancer cells induces an anti-apoptotic effect that counteracts Fas-L signaling (Azuma et al., Blood, 111:3635-3643 (2008)). Azuma et al., Blood, 111:3635-3643 (2008).

[0331] Considering the upregulation of B7-H1 by cancer cells and its association with cancer progression and poor clinical outcomes (Flies et al., Journal of Immunotherapy, 30:251-260 (2007); Nishimura et al., Immunity, 11:141-151 (1999); Wang et al., Curr Top Microbiol Immunol, 344:245-267 (2011)), antibodies that antagonize the PD-1 and CTLA-4 pathways have shown dramatic efficacy in solid tumors, particularly melanoma, and the combination of these two shows even more activity. The anti-CTLA-4 antibody ipilimumab improves the overall survival of metastatic melanoma mainly by increasing T cell infiltration into tumors and the CD8+:Treg ratio within tumors through the suppression of Treg cells (Hamid et al., J Transl Med, 9:204(2011); Ribas et al., Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 15:6267 - 6276(2009); Twyman-Saint et al., Nature, 520:373 - 377(2015)). The anti-PD-1 antibody nivolumab has an overall response rate of 30 - 40% in metastatic melanoma (Robert et al., The New England Journal of Medicine, 372:320 - 330(2015); Topalian et al., J Clin Oncol, 32:1020 - 1030(2014)), and similar findings have been seen in early clinical trials of other solid tumors including metastatic renal cancer, non-small cell lung cancer and recurrent Hodgkin lymphoma (Ansell et al., The New England Journal of Medicine, 372:311 - 319(2015); Brahmer et al., J Clin Oncol, 28:3167 - 3175(2010); Topalian et al., The New England Journal of Medicine, 366:2443 - 2454(2012)). Since resistance to anti-CTLA-4 antibody in mouse melanoma models is due to upregulation of PD-L181, the combination of both ipilimumab and nivolumab shows further efficacy in both mouse models and human patients (Larkin et al., The New England Journal of Medicine, 373:23 - 34(2015); Spranger et al., J Immunother Cancer, 2, 3, doi:10.1186 / 2051 - 1426 - 2 - 3(2014); Yu et al., Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 16:6019 - 6028(2010)). Considering the importance of the checkpoint inhibitory pathway, PD-1 / CTLA-4 inhibition is thought to release the brake, and chimeric antigen receptors are thought to step on the accelerator pedal.Importantly, transient delivery can only be used to temporarily release the brakes so that these cells do not cause future autoimmune diseases. i. CRISPRi

[0332] To avoid permanent genome modification and inactivation of inhibitory signals such as PD-1 and CTLA-4, the dCAS9 CRISPRi system (Larson et al., Nat Protoc, 8:2180 - 2196 (2013)) can be utilized. Nucleic acids encoding enzymatically inactive dCAS9-KRAB-repressor domains, fusion proteins, and inhibitory signaling proteins (e.g., CTLA-4, PD-1, LAG-3, 2B4 (CD244), BTLA (CD272), KIR, TIM-3, TGFβ receptor dominant negative analogs, etc.) can be co-delivered to CAR cells. One or more sgRNAs can be utilized. The sgRNAs can be designed to target the proximal promoter region and the coding region (non-template strand). In another approach, the single-component Cpf1 CRISPR system, which is a small molecule RNA for electroporation-induced expression, is utilized (Zetsche et al., Cell, doi:10.1016 / j.cell.2015.09.038 (2015)). Any of the aforementioned RNA components can be encoded in a DNA expression construct such as a vector, e.g., a plasmid. Thus, either RNA, DNA, or a combination thereof can function as nucleic acid cargo.

[0333] Extensive inhibition of CTLA-4 by ipilimumab causes autoimmune sequelae, but these side effects are thought to be reduced by limiting the loss to CAR cells and the transient nature of mRNA delivery. The inhibitory function will eventually recover. ii. Inhibitory RNA

[0334] Nucleic acid cargos that can be delivered to cells are functional nucleic acids or polypeptides designed to target and reduce or suppress the expression or translation of inhibitory signaling molecule mRNAs, or to reduce or suppress the expression of inhibitory signaling molecule proteins, decrease their activity, or increase their degradation, or can encode the same. Suitable techniques include, but are not limited to, antisense molecules, siRNA, miRNA, aptamers, ribozymes, triple helix-forming molecules, RNAi, etc. In some embodiments, the mRNA encodes an antagonist polypeptide that reduces inhibitory signaling.

[0335] In some embodiments, cargos that are functional RNAs or encode the same that are suitable for reducing or silencing the expression of CTLA-4, PD-1, LAG-3, 2B4 (CD244), BTLA (CD272), KIR, TIM-3, TGFβ receptor dominant negative analogs, etc., alone or in combination, can be delivered to cells.

[0336] In some embodiments, the cargo is an RNA or DNA that encodes a polypeptide that functions as an antagonist or other negative regulator or inhibitor of CTLA-4, PD-1, LAG-3, 2B4 (CD244), BTLA (CD272), KIR, TIM-3, TGFβ receptor dominant negative analog, or another protein in the immunosuppressive pathway, which reduces bioavailability. The protein can be paracrine, endocrine, or autocrine. It can regulate the cell intracellularly. It can be secreted and regulate the expressing cell and / or other (e.g., neighboring) cells. It can be a transmembrane protein that regulates the expressing cell and / or other cells. The protein can be a fusion protein, e.g., an Ig fusion protein. e. Apoptosis promoting factors

[0337] Compositions and methods for activating and reactivating the apoptosis pathway are also provided. In some embodiments, the nucleic acid is, or encodes, a factor or agent that activates, reactivates, or otherwise enhances or increases the endogenous apoptosis pathway. Preferably, this factor activates, reactivates, or otherwise enhances the endogenous apoptosis pathway in cancer (e.g., tumor) cells, and more preferably is specific for or targeted to cancer cells.

[0338] In some embodiments, cells are more resistant or less sensitive to induced apoptosis than untreated cells after delivery of anti-apoptosis factors or growth promoting factors, such as those discussed above or known in the art. Apoptosis promoting factors can, for example, induce or increase apoptosis in untreated cells compared to treated T cells, and are preferably selective for cancer cells. The regimen provides a two-pronged attack on cancer cells, one a cellular attack and the other a molecular attack.

[0339] The endogenous apoptosis pathway can be activated, reactivated, or otherwise enhanced by targeting members of the BCL-2 family. BCL-2 family members are classified into three subgroups based on function and Bcl-2 homology (BH) domains: multi-domain anti-apoptotic (e.g., BCL-2 or BCL-XL), multi-domain pro-apoptotic (e.g., BAX and BAK), and BH3-only pro-apoptotic (e.g., BIM) proteins. Members of the BH3-only subgroup, such as BIM, function as sentinels of death located throughout the cell, primed to transmit various physiological and pathological signals of cellular injury to the central apoptotic machinery located in the mitochondria (Danial et al., Cell, 116:205-219 (2004)).

[0340] In some embodiments, the apoptosis promoting factor is an apoptosis promoting BH3 mimetic. Various apoptosis promoting BH3 mimetics can simulate the native apoptosis promoting activity of BIM and provide the function of manipulating multiple points in the apoptosis pathway. For example, BIM SAHB (stabilized α helix of the BCL-2 domain), ABT-737, and ABT-199 are apoptosis promoting BH3 mimetics designed by structural studies of the interaction between the apoptosis promoting BH3-only helical domain and the hydrophobic groove formed by the convergence of the BH1, BH2, and BH3 domains of anti-apoptotic proteins (Oltersdorf et al., Nature, 435:677-681 (2005)). D. Target cells

[0341] In some embodiments, one or more specific cell types or tissues are targets of the disclosed complexes. The target cells can be in vitro, ex vivo, or within a subject (i.e., in vivo). The uses contemplated herein can be performed in vitro, ex vivo, or in vivo. In the case of ex vivo uses, cells can be collected or isolated and treated in culture. Cells treated ex vivo can be administered to a subject in need thereof in a therapeutically effective amount. In the case of in vivo uses, the cargo can be delivered passively to the target cells, for example, based on circulation of the composition, local delivery, etc., or can be actively targeted, for example, using additional cell, tissue, organ-specific targeting moieties. Thus, in some embodiments, the cargo is delivered to the target cells excluding other cells. In some embodiments, the cargo is delivered to the target cells and non-target cells.

[0342] The target cells can be selected by one skilled in the art based on the desired treatment and therapy, as well as the intended effect of the nucleic acid cargo. For example, if the nucleic acid cargo is intended to induce cell death, the target cells can be cancer cells. If the nucleic acid cargo is intended to induce genomic changes, the target cells can be stem cells. If the nucleic acid cargo encodes a chimeric antigen receptor, the target cells can be immune cells.

[0343] The 3E10 scFv has previously been shown to be permeable to the cell nucleus in an ENT2-dependent manner, and its nuclear uptake efficiency is greatly impaired in ENT2-deficient cells (Hansen et al., J Biol Chem 282, 20790-20793 (2007)). ENT2 (SLC29A2) is a sodium-independent transporter involved in the transport of purine and pyrimidine nucleosides and nucleobases, and is less sensitive to nitrobenzylmercaptopurine riboside (NBMPR) than ENT1.

[0344] In some embodiments, the target cells express ENT2 on their plasma membrane, their nuclear membrane, or both. Although the expression of ENT2 is relatively widely distributed, the amount varies depending on the tissue and cell type. This has been confirmed in the brain, heart, placenta, thymus, pancreas, prostate, and kidney (Griffiths et al., Biochem J, 1997. 328 (Pt 3): p. 739-43, Crawford et al., J Biol Chem, 1998. 273 (9): p. 5288-93). Compared with other transporters, ENT2 is one of the ones with the highest mRNA expression in skeletal muscle (Baldwin et al., Pflugers Arch, 2004. 447 (5): p. 735-43, Govindarajan et al., Am J Physiol Regul Integr Comp Physiol, 2007. 293 (5): p. R1809-22). Thus, in some embodiments, the target cells are the brain, heart, placenta, thymus, pancreas, prostate, kidney, or skeletal muscle. Because ENT2 is highly expressed in skeletal muscle, the disclosed compositions and methods can be particularly effective in delivering nucleic acid cargo to these cells, and / or a higher level of cargo can be delivered to these cells compared to other cells that express lower levels of ENT2.

[0345] Additional non-limiting exemplary target cells are discussed below. 1. Progenitor cells and stem cells

[0346] The cells can be hematopoietic progenitor cells or stem cells. In some embodiments, particularly those related to gene editing and gene therapy, the target cells are CD34 + hematopoietic stem cells. Hematopoietic stem cells (HSCs), such as CD34+ cells, are pluripotent stem cells that give rise to all blood cell types, including red blood cells.

[0347] Stem cells can be isolated and concentrated by those skilled in the art. Methods for such isolation and concentration of CD34 + and other cells are known in the art and are disclosed, for example, in U.S. Patent Nos. 4,965,204; 4,714,680; 5,061,620; 5,643,741; 5,677,136; 5,716,827; 5,750,397 and 5,759,793. As used herein, in the context of a concentrated composition of hematopoietic progenitor cells and stem cells, "concentrated" indicates that the proportion of the desired elements (e.g., hematopoietic progenitor cells and stem cells) is higher than the proportion found in the natural source of the cells. The composition of the cells may be concentrated by at least one order of magnitude, preferably two or three orders of magnitude, more preferably 10, 100, 200 or 1000 times, compared to the natural source of the cells.

[0348] In humans, CD34 + cells can be recovered from umbilical cord blood, bone marrow or blood after cytokine mobilization, which is brought about by injecting a hematopoietic growth factor, such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), etc., subcutaneously or intravenously into the donor in an amount sufficient to cause the migration of hematopoietic stem cells from the bone marrow cavity to the peripheral circulation. Initially, bone marrow cells may be obtained from any suitable source of bone marrow, such as the tibia, femur, spine, and other bone cavities. To separate the bone marrow, the bone may be rinsed with an appropriate solution, which generally consists of a balanced salt solution conveniently supplemented with fetal bovine serum or other naturally occurring factors, together with a low concentration (about 5 - 25 mM) of an acceptable buffer. Convenient buffers include Hepes, phosphate buffer, lactate buffer, etc.

[0349] Cells can be selected by positive and negative selection techniques. Cells can be selected using methods known to those skilled in the art, using commercially available antibodies that bind to hematopoietic progenitor or stem cell surface antigens, such as CD34. For example, the antibody can be conjugated to magnetic beads and immunogenic procedures utilized to recover the desired cell type. Other techniques include the use of fluorescence-activated cell sorting (FACS). The CD34 antigen is found on progenitor cells within the hematopoietic system of non-leukemic individuals and is expressed on a cell population recognized (i.e., expressing the CD34 antigen) by the monoclonal antibody MY-10, and can be used for the isolation of stem cells for bone marrow transplantation. MY-10, which was deposited as HB-8483 at the American Type Culture Collection (Rockville, Maryland), is commercially available as anti-HPCA1. Additionally, negative selection of "committed" cells differentiated from human bone marrow can be utilized to select against virtually any desired cell marker. For example, progenitor or stem cells, most preferably CD34 + Cells are CD3 - CD7 - CD8 - CD10 - CD14 - CD15 - CD19 - CD20 - CD33 - Class II HLA + and Thy-1 + can be characterized as being any of these.

[0350] Once progenitor or stem cells are isolated, they can be expanded by growing them in any suitable medium. For example, progenitor or stem cells can be grown in a conditioned medium derived from stromal cells, such as that obtained from bone marrow or liver associated with factor secretion, or in a medium containing cell surface factors that support stem cell growth. Stromal cells can be removed from hematopoietic cells using appropriate monoclonal antibodies to remove unwanted cells.

[0351] An isolated cell is contacted ex vivo with a complex of an antibody and a nucleic acid cargo. The cell that has received the cargo may be referred to as a modified cell. The solution of the complex may simply be added to the cells in culture. It may be desirable to synchronize the cells in the S phase. For example, methods for synchronizing cultured cells by double thymidine block are known in the art (Zielke et al., Methods Cell Biol., 8:107-121 (1974)).

[0352] The modified cells can be maintained or grown in culture prior to administration to a subject. Culture conditions are generally known in the art depending on the cell type. In particular, conditions for maintaining CD34 + have been well studied and several suitable methods are available. A common approach to ex vivo expansion of pluripotent hematopoietic cells is to culture purified progenitor or stem cells in the presence of early-acting cytokines such as interleukin-3. Also, including a combination of thrombopoietin (TPO), stem cell factor (SCF), and flt3 ligand (Flt-3L; i.e., the ligand of the flt3 gene product) in a nutrient medium for maintaining hematopoietic progenitor cells ex vivo has been shown to be useful for growing primitive (i.e., relatively undifferentiated) human hematopoietic progenitor cells in vitro and for their ability to engraft in SCID-hu mice (Luens et al., 1998, Blood 91:1206-·1215). In other known methods, cells can be maintained ex vivo (e.g., for several minutes, hours, or 3, 6, 9, 13, or more days) in a nutrient medium containing mouse prolactin-like protein E (mPLP-E) or mouse prolactin-like protein F (mPIP-F; collectively mPLP-E / IF) (U.S. Patent No. 6,261,841). It will be understood that other suitable cell culture and growth methods can be used as well. The cells can also be grown in serum-free medium as described in U.S. Patent No. 5,945,337.

[0353] In another embodiment, the modified hematopoietic stem cells are differentiated into CD4 cells ex vivo using a specific combination of interleukin and growth factor before administration to a subject using methods well known in the art. + The cells may be grown ex vivo in large quantities, preferably at least 5-fold, more preferably at least 10-fold, even more preferably at least 20-fold, compared to the original population of isolated hematopoietic stem cells.

[0354] In another embodiment, the cells can be dedifferentiated somatic cells. The somatic cells can be reprogrammed to become pluripotent stem-like cells that can be induced to become hematopoietic progenitor cells. The hematopoietic progenitor cells can then be treated with a composition as described above for CD34 cells. Representative somatic cells that can be reprogrammed include, but are not limited to, fibroblasts, adipocytes, and myocytes. Hematopoietic progenitor cells from induced stem-like cells have been successfully developed in mice (Hanna, J. et al., Science, 318:1920-1923 (2007)). + To generate hematopoietic progenitor cells from induced stem-like cells, somatic cells are harvested from a host. In a preferred embodiment, the somatic cells are autologous fibroblasts. The cells are cultured and transduced with vectors encoding the Oct4, Sox2, Klf4, and c-Myc transcription factors. The transduced cells are cultured and screened for embryonic stem (ES) cell markers including, but not limited to, AP, SSEA1, and Nanog, as well as the morphology of ES cells. The transduced ES cells are cultured and induced to generate induced stem-like cells. The cells are then screened for CD41 and c-kit markers (early hematopoietic progenitor cell markers), as well as markers of myeloid and erythroid differentiation.

[0355]

[0356] ​Next, modified hematopoietic stem cells or modified cells such as, for example, induced hematopoietic progenitor cells are introduced into a subject. Delivery of the cells can be effected using a variety of methods, most preferably including intravenous administration by injection, as well as direct depot injection into the periosteum, bone marrow and / or subcutaneous sites.

[0357] The subject to which the modified cells are administered may be treated for bone marrow conditioning to enhance engraftment of the cells. The recipient may be treated to enhance engraftment using radiation or chemotherapy treatment prior to administration of the cells. Once administered, the cells generally require a period of time to engraft. It typically takes several weeks to several months to achieve significant engraftment of hematopoietic stem cells or progenitor cells.

[0358] A high engraftment rate of modified hematopoietic stem cells may not be necessary to achieve a significant prophylactic or therapeutic effect. The engrafted cells are thought to proliferate over time after engraftment to increase the proportion of modified cells. In some cases, engraftment of modified hematopoietic stem cells necessary to obtain a prophylactic or therapeutic effect is thought to be a very small or low proportion.

[0359] In a preferred embodiment, the cells administered to the subject are autologous, e.g., derived from the subject or isogeneic. 2\. Embryo

[0360] In some embodiments, the compositions and methods can be used to deliver cargo to embryonic cells in vitro. This method generally involves contacting an embryo in vitro with an effective amount of antibody-cargo DNA to improve transduction of the cargo into the embryo. The embryo may be a single cell zygote, but treatment of embryos having 2, 4, 8, or 16 cells, including male and female gametes before and during fertilization, as well as zygotes, morulas and blastomeres, is also provided. In some embodiments, the embryo is contacted with the composition on days 0-6 of culture during or after in vitro fertilization.

[0361] Contact may be by adding the composition to a liquid medium in which the embryo is immersed. For example, the composition can be pipetted directly into the embryo's medium and then taken up by the embryos. 3. Immune cells

[0362] In some embodiments, the target cells are one or more types of immune cells. For example, for immunomodulation and CAR-based therapies, various types of cells can be utilized or targeted. Preferred targeted / engineered T cells can vary depending on the tumor and adoptive therapy goals. Effector T cells, which secrete high levels of effector cytokines and are generally preferred for effectively killing tumor targets in vitro (Barrett et al., Annu Rev Med., 65:333-347 (2014)), two complementary lymphocyte populations with strong cytotoxicity via CAR are CD3-CD56+ NK cells and CD3+CD8+ T cells. Using CD8+ T cells and CD4+ helper T cells increases the presence of inhibitory T-reg cells and weakens the cytotoxicity of CD8+ T cells. Since reprogrammed CD8+ T cells are pre-activated to act directly on tumor cells without the need for activation in lymph nodes, the support of CD4+ T cells is not essential.

[0363] Furthermore, the injection of naive T cells (Rosenberg et al., Adv. Cancer Res., 25:323-388 (1977)), central memory T cells (T CM cells) (Berger et al., J. Clin. Invest., 118:294-305 (2008)), Th17 cells (Paulos et al., Sci. Transl. Med., 2:55-78 (2010)), and T stem memory cells (Gattinoni et al., Nat. Med., 17:1290-1297 (2012)) all have certain advantages for specific applications, for example, due to their high replicative capacity. Tumor-infiltrating lymphocytes (TIL) also have certain advantages due to their antigen specificity and can be used in the delivery strategies disclosed herein.

[0364] Sometimes referred to as CAR cells, CAR immune cells, and CART cells (or CAR T cells), the CARs and other delivery strategies disclosed herein can be implemented in different types of immune cells, including but not limited to those considered herein (e.g., lymphocytes, natural killer cells, dendritic cells, B cells, antigen-presenting cells, macrophages, etc.) and those described elsewhere (e.g., see Barrett et al., Annu Rev Med., 65:333-347 (2014)). 4. Cancer Cells and Tumors

[0365] In some embodiments, the target cells are cancer cells. In such embodiments, methods of treatment that may be useful in the context of cancer, including tumor treatment, are provided. The following examples may show that while DNA cargo is more generally delivered to multiple tissues and often not limited to tumors, RNA delivery is often more selective for tumor tissue. Thus, in some embodiments, when cancer cells are the target cells, the cargo may be composed of RNA (e.g., RNA only).

[0366] Cargo that can be delivered to cancer cells includes, but is not limited to, constructs for the expression of one or more apoptosis-promoting factors, immunogenic factors, or tumor suppressors; gene editing compositions, inhibitory nucleic acids that target oncogenes; and other strategies considered herein and elsewhere. In some embodiments, the cargo is an mRNA encoding an apoptosis-promoting factor or an immunogenic factor that increases the immune response to the cell. In other embodiments, the cargo is an siRNA that reduces the expression of an oncogene or other cancer-causing transcript.

[0367] In mature animals, normally, the balance between cell regeneration and cell death is maintained in most organs and tissues. Various types of mature cells in the body have a certain lifespan. When these cells die, new cells are generated through the proliferation and differentiation of various types of stem cells. Under normal circumstances, the production of new cells is regulated so that the number of specific types of cells is kept constant. However, occasionally, cells that no longer respond to normal growth control mechanisms occur. These cells give rise to clones of cells, which can grow to a significant size and produce a tumor or neoplasm. A tumor that cannot grow indefinitely and does not extensively infiltrate healthy surrounding tissue is benign. A tumor that continues to grow and becomes increasingly invasive is malignant. The term cancer specifically refers to malignant tumors. In addition to uncontrolled growth, malignant tumors exhibit metastasis. In this process, small clusters of cancerous cells break away from the tumor, invade blood vessels and lymphatic vessels, are carried to other tissues, and continue to grow there. Thus, a primary tumor at one site can cause secondary tumors at other sites.

[0368] The compositions and methods described herein may be useful for treating subjects having a benign or malignant tumor by delaying or inhibiting the growth of the target tumor, reducing the growth or size of the tumor, suppressing or reducing tumor metastasis, and / or suppressing or reducing symptoms associated with the development or growth of the tumor.

[0369] Malignant tumors that can be treated are classified herein according to the embryonic origin of the tissue from which the tumor derives. Carcinomas are tumors that arise from endodermal or ectodermal tissues such as the skin or the inner lining of internal organs and glands. The disclosed compositions are particularly effective in treating carcinomas. Sarcomas, which are less frequent, are derived from mesodermal connective tissues such as bone, fat, and cartilage. Leukemias and lymphomas are malignant tumors of hematopoietic cells in the bone marrow. Leukemias tend to grow as single cells, and lymphomas tend to grow as tumor masses. Malignant tumors can appear in many organs or tissues of the body and establish cancer.

[0370] The types of cancer that can be treated with the provided compositions and methods include, but are not limited to, cancers such as multiple myeloma, adenocarcinoma, and sarcoma of bone, bladder, brain, breast, cervix, colorectal, esophagus, kidney, liver, lung, nasopharyngeal, pancreas, prostate, skin, stomach, and uterus. In some embodiments, the disclosed compositions are used to treat multiple cancer types simultaneously. The compositions can also be used to treat metastases or tumors at multiple sites.

[0371] The disclosed compositions and methods can be further understood through the following numbered paragraphs.

[0372] 1. (a) 3E10 monoclonal antibody, its cell membrane permeable fragment; monovalent, divalent, or multivalent single-chain variable fragment (scFv); or diabody; or its humanized form or variant, and (b) a nucleic acid cargo comprising a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof A composition comprising or consisting of.

[0373] 2. (a) is (i) a combination of any one CDR of SEQ ID NOs: 1 - 6, 12, 13, 46 - 48, or 50 - 52 and any one CDR of SEQ ID NOs: 7 - 11, 14, or 53 - 58; (ii) a combination of the first, second, and third heavy chain CDRs selected from any of SEQ ID NOs: 15 - 23, 42, or 43 and the first, second, and third light chain CDRs selected from any of SEQ ID NOs: 24 - 30, 44, or 45; (iii) a humanized form of (i) or (ii); (iv) a combination of a heavy chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1 or 2 and a light chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 7 or 8; (v) a humanized form or (iv); or (vi) a combination of a heavy chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 3-6, 46-48, or 50-52 and a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NOs: 9-11 or 53-58 The composition according to paragraph 1, comprising

[0374] 3. The composition according to paragraph 1 or 2, wherein (a) comprises the same or different epitope specificities as the monoclonal antibody 3E10 produced by the hybridoma with ATCC deposit number PTA2439.

[0375] 4. The composition according to any one of paragraphs 1-3, wherein (a) is a recombinant antibody having the paratope of the monoclonal antibody 3E10.

[0376] 5. (a) The following (i) A combination of any one CDR of SEQ ID NOs: 1-6, 12, 13, 46-48, or 50-52 and any one CDR of SEQ ID NOs: 7-11, 14, or 53-58; (ii) A combination of the first, second, and third heavy chain CDRs selected from SEQ ID NOs: 15-23, 42, or 43 and the first, second, and third light chain CDRs selected from SEQ ID NOs: 24-30, 44, or 45; (iii) A humanized form of (i) or (ii); (iv) A combination of a heavy chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1 or 2 and a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NOs: 7 or 8; (v) A humanized form or (iv); or (vi) A combination of a heavy chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 3-6, 46-48, or 50-52 and a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NOs: 9-11 or 53-58, A binding protein comprising, (b) A nucleic acid cargo comprising a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof A composition comprising or consisting of.

[0377] 6. The composition according to any one of paragraphs 1 to 5, wherein (a) is bispecific.

[0378] 7. The composition according to paragraph 6, wherein (a) targets a cell type of interest.

[0379] 8. The composition according to any one of paragraphs 1 to 7, wherein (a) and (b) are non-covalently linked.

[0380] 9. The composition according to any one of paragraphs 1 to 8, wherein (a) and (b) form a complex.

[0381] 10. The composition according to any one of paragraphs 1 to 9, wherein (b) comprises DNA, RNA, PNA or other modified nucleic acids, or nucleic acid analogs, or a combination thereof.

[0382] 11. The composition according to any one of paragraphs 1 to 10, wherein (b) comprises mRNA.

[0383] 12. The composition according to any one of paragraphs 1 to 11, wherein (b) comprises a vector.

[0384] 13. The composition according to paragraph 12, wherein the vector comprises a nucleic acid sequence encoding a polypeptide of interest operably linked to an expression control sequence.

[0385] 14. The composition according to paragraph 13, wherein the vector is a plasmid.

[0386] 15. The composition according to any one of paragraphs 1 to 14, wherein (b) comprises a nucleic acid encoding a Cas endonuclease, a gRNA, or a combination thereof.

[0387] 16. The composition according to any one of paragraphs 1 to 15, wherein (b) comprises a nucleic acid encoding a chimeric antigen receptor polypeptide.

[0388] 17. The composition according to any one of paragraphs 1 to 16, wherein (b) comprises a functional nucleic acid.

[0389] 18. The composition according to any one of paragraphs 1 to 17, wherein (b) comprises a nucleic acid encoding a functional nucleic acid.

[0390] 19. The composition according to paragraph 17 or 18, wherein the functional nucleic acid is an antisense molecule, siRNA, miRNA, aptamer, ribozyme, RNAi, or an external guide sequence.

[0391] 20. The composition according to any one of paragraphs 1 to 19, wherein (b) comprises a plurality of single nucleic acid molecules.

[0392] 21. The composition according to any one of paragraphs 1 to 19, wherein (b) comprises a plurality of two, three, four, five, six, seven, eight, nine, ten, or more different nucleic acid molecules.

[0393] 22. The composition according to any one of paragraphs 1 to 21, wherein (b) comprises or consists of a nucleic acid molecule having a length between about 1 and 25,000 nucleobases.

[0394] 23. The composition according to any one of paragraphs 1 to 22, wherein (b) comprises or consists of single-stranded nucleic acid, double-stranded nucleic acid, or a combination thereof.

[0395] 24. The composition according to any one of paragraphs 1 to 23, further comprising carrier DNA.

[0396] 25. The composition according to paragraph 24, wherein the carrier DNA is non-coding DNA.

[0397] 26. The composition according to paragraph 24 or 25, wherein (b) is composed of RNA.

[0398] 27. A pharmaceutical composition comprising the composition according to any one of paragraphs 1 to 26 and a pharmaceutically acceptable excipient.

[0399] 28. The composition according to paragraph 27, further comprising polymer nanoparticles encapsulating the complex of (a) and (b).

[0400] 29. The composition according to paragraph 28, wherein a targeting moiety, a cell membrane permeable peptide, or a combination thereof is directly or indirectly associated, linked, conjugated, or otherwise bound to the nanoparticles.

[0401] 30. A method for delivering nucleic acid cargo to a cell, comprising contacting the cell with an effective amount of the composition according to any one of paragraphs 1 to 29.

[0402] 31. The method according to paragraph 30, wherein the contacting is performed ex vivo.

[0403] 32. The method according to paragraph 31, wherein the cell is a hematopoietic stem cell or a T cell.

[0404] 33. The method according to any one of paragraphs 30 to 32, further comprising administering the cell to a subject in need thereof.

[0405] 34. The method according to paragraph 33, wherein the cell is administered to the subject in an effective amount to treat one or more symptoms of a disease or disorder.

[0406] 35. The method according to paragraph 30, wherein the contacting is performed in vivo after administration to a subject in need thereof.

[0407] 36. The method according to any one of paragraphs 33 to 35, wherein the subject has a disease or disorder.

[0408] 37. The method according to paragraph 36, wherein the disease or disorder is a genetic disorder, cancer, or an infectious disease or infectious disorder.

[0409] 38. The method according to paragraph 36 or 37, wherein (b) is delivered to the cells of the subject in an amount effective to reduce one or more symptoms of the disease or disorder of the subject.

[0410] 39. A method of preparing a composition according to any one of paragraphs 1 to 29, comprising incubating and / or mixing (a) and (b) at an effective time and suitable temperature to form a complex of (a) and (b) prior to contact with the cells.

[0411] 40. A method of preparing a composition according to any one of paragraphs 1 to 29, comprising incubating and / or mixing (a) and (b) for about 1 minute to about 30 minutes, about 10 minutes to about 20 minutes, or about 15 minutes, optionally at room temperature or 37°C.

[0412] 41. A composition or method according to any one of paragraphs 1 to 40, wherein the 3E10 monoclonal antibody, its cell membrane permeable fragment; monovalent, bivalent, or multivalent single-chain variable fragment (scFv); or diabody; or its humanized form or variant comprises a nucleic acid binding pocket of SEQ ID NO: 92 or 93 or a variant thereof having the same or improved ability to bind to nucleic acids.

[0413] 42. A composition or method according to any one of paragraphs 1 to 41, wherein the amino acid residue corresponding to D31 or N31 of the heavy chain amino acid sequence or its CDR is substituted with R.

[0414] 43. A composition or method according to any one of paragraphs 1 to 42, wherein the amino acid residue corresponding to D31 or N31 of the heavy chain amino acid sequence or its CDR is substituted with L.

[0415] 44. (i) A variant of any one of CDRs with SEQ ID NOs: 1-6, 12, 13, 46-48, or 50-52, in combination with a CDR selected from any one of SEQ ID NOs: 7-11, 14, or 53-58; (ii) A variant of the first heavy-chain CDR in combination with the second and third heavy-chain CDRs selected from SEQ ID NOs: 15-23, 42, or 43, and a combination with the first, second, and third light-chain CDRs selected from SEQ ID NOs: 24-30, 44, or 45; (iii) A humanized form of (i) or (ii); (iv) A combination of a heavy chain comprising an amino acid sequence having at least 85% sequence identity to either SEQ ID NO: 1 or 2, and a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 7 or 8; (v) A humanized form or (iv); or (vi) A combination of a heavy chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 3-6, 46-48, or 50-52, and a light chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 9-11 or 53-58 comprising, a binding protein in which the amino acid residue corresponding to D31 or N31 is substituted with R or L.

[0416] 45. The binding protein according to paragraph 44, comprising a nucleic acid-binding pocket of SEQ ID NO: 92 or 93, or a variant thereof having the same or improved ability to bind nucleic acids.

Examples

[0417] For the following experiments, except where stated to be the D31N variant (e.g., Example 4), the standard 3E10 sequence was used. Both the standard 3E10 and the D31N variant were used as full-length antibodies.

[0418] Example 1: 3E10 increases the cellular uptake of PNA after 1 hour. Materials and Methods Only PNA (1 nmol) (MW = 9984.39; 29 nucleotides in length), or PNA complexed with 3E10 (0.75 mg), was mixed at room temperature for 5 minutes. Next, 200,000 K562 cells were added to a suspension of 3E10 or only PNA in serum-free medium. Additional serum-free medium was added to bring the final volume to 500 μl. After incubation with the cells at 37 °C for 1 hour, the cells were centrifuged, washed three times with PBS, and then analyzed by flow cytometry. The PNA was labeled by binding to the fluorescent dye tetramethylrhodamine (TAMRA). Results

[0419] The results are shown in the flow cytometry dot plots (Figures 1A - 1C). The % uptake was quantified (Figure 1D).

[0420] The results show that the uptake of PNA increases when mixed with 3E10.

[0421] Example 2: 3E10 increases the cellular uptake of PNA after 24 hours. Materials and Methods Only PNA (1 nmol) (MW = 9984.39; 29 nucleotides in length), or PNA complexed with 3E10 (0.75 mg), was mixed at room temperature for 5 minutes. Next, 200,000 K562 cells were added to a suspension of 3E10 or only PNA in serum-free medium. Additional serum-free medium was added to bring the final volume to 500 μl. After incubation with the cells at 37 °C for 24 hours, the cells were centrifuged, washed three times with PBS, and then analyzed by flow cytometry.

[0422] 20,000 U2OS cells were seeded onto an 8-well chamber slide and allowed to adhere for 24 hours. The cells were then treated with PNA alone (1 nmole) or PNA complexed with 3E10 (10 uM). After incubation at 37 °C for 24 hours, the PNA or PNA mixed with 3E10 was washed with PBS and then fixed and nuclear stained. Thereafter, PNA uptake was quantified by flow cytometry and imaged using a fluorescence microscope. The PNA was labeled by conjugation with the fluorescent dye tetramethylrhodamine (TAMRA). Results

[0423] The results are shown in the dot plots of flow cytometry (Figs. 2A - 2C). The % uptake was quantified (Fig. 2D).

[0424] The results show that the uptake of PNA increases when mixed with 3E10.

[0425] The fluorescence microscope showed the co - localization of nuclear DNA (blue DAPI) and PNA (red Tamra) by the production of a distinct pink coloration.

[0426] Example 3: 3E10 increases the cellular uptake of siRNA after 24 hours. Materials and Methods Labeled siRNA (by attachment to fluorescein amidite, FAM) (1 nmole) or siRNA complexed with 3E10 (0.75 mg) was mixed at room temperature for 5 minutes. Next, 200,000 K562 cells were added to a suspension of 3E10 or siRNA alone in serum - free medium. Additional serum - free medium was added to bring the final volume to 500 ul. After incubation with the cells at 37 °C for 24 hours, the cells were centrifuged, washed 3 times with PBS, and then analyzed by flow cytometry. Results

[0427] The results are shown in the dot plots of flow cytometry (Figs. 3A - 3C). The % uptake was quantified (Fig. 3D).

[0428] The results show that when mixed with 3E10, the cellular uptake of siRNA increases.

[0429] Example 4: 3E10 increases the cellular uptake of mRNA after 24 hours. Materials and Methods Labeled mRNA only (2 μg) attached to cyanine 5 (Cy5), or labeled mRNA (2.5, 5, and 10 μM) complexed with 3E10, were mixed at room temperature for 5 minutes. The suspension of 3E10 and mRNA, or the suspension of mRNA only, was added to 200,000 K562 cells in serum-free medium. Additional serum-free medium was added to bring the final volume to 500 μl. After incubation with the cells at 37 °C for 24 hours, the cells were centrifuged, washed three times with PBS, and then analyzed by flow cytometry. Results

[0430] The results are shown in the dot plots of flow cytometry (Figures 4A - 4H). The % uptake was quantified (Figure 4I).

[0431] The results show that when mixed with 3E10, the uptake of mRNA increases.

[0432] Note that delivery of mRNA by the D31N variant of 3E10 resulted in the highest level of cellular uptake of mRNA.

[0433] The fluorescence microscope showed the expression of functional GFP in U2OS cells after translation of the same Cy5-labeled mRNA encoding the green fluorescent protein (GFP) reporter.

[0434] Example 5: 3E10 increases the cellular uptake of mRNA after 1 hour. Materials and Methods Labeled mRNA (Cy5) (2 μg) or labeled mRNA (0.1 - 10 μM) complexed with the D31N variant of 3E10 was mixed at room temperature for 5 minutes. The suspension of 3E10 and mRNA, or the suspension of mRNA alone, was added to 200,000 K562 cells in serum-free medium. Additional serum-free medium was added to make the final volume 500 μl. After incubation with the cells at 37°C for 1 hour, the cells were centrifuged, washed three times with PBS, and then analyzed by flow cytometry. Results

[0435] The results are shown in the dot plots of flow cytometry (Figures 5A - 5H). The % uptake was quantified (Figure 5I).

[0436] Example 6: 3E10 increases cellular uptake of plasmid DNA. Materials and Methods GFP reporter plasmid DNA (250 μg) was complexed with 3E10 (10 μM) at room temperature for 5 minutes. The suspension of 3E10 and plasmid DNA, or the suspension of plasmid DNA alone, was added to 200,000 K562 cells in serum-free medium. Additional serum-free medium was added to make the final volume 500 μl. After incubation with the cells at 37°C for 24 hours, the cells were centrifuged and washed three times with PBS. 72 hours after the first treatment, the cells were imaged and analyzed for GFP expression. Results

[0437] The results, as measured by green fluorescence, showed that when 3E10 was combined with plasmid DNA, the GFP plasmid was firmly taken up by the cells, indicating the uptake and functional expression of the GFP construct. When only plasmid DNA was used, neither uptake nor green fluorescence was observed. (Figure 6).

[0438] Example 7: 3E10 mediates mRNA delivery in vivo Materials and Methods 10 μg of mRNA encoding GFP was mixed with 0.1 mg of 3E10 at room temperature for 15 minutes. The mRNA that formed a complex with 3E10 was systemically injected into BALB / c mice bearing 100 mm 3 EMT6 flank tumors. Twenty hours after treatment, tumors were harvested and mRNA expression (GFP) was analyzed using IVIS imaging. Results

[0439] Delivery of mRNA mediated by 3E10 resulted in significantly higher levels of GFP expression in tumors compared to freely injected mRNA, which showed no GFP expression in tumors. No detectable expression of GFP was seen in normal tissues, including the liver, spleen, heart, and kidneys, examined in any of the treatments. These results indicate efficient delivery of mRNA to tumors with functional translation and expression.

[0440] Example 8: 3E10 Mediates siRNA Delivery In Vivo Materials and Methods 40 μg of fluorescently labeled siRNA was mixed with increasing doses of 3E10 (0.25, 0.5, and 1 mg) at room temperature for 15 minutes. The siRNA that formed a complex with 3E10 was systemically injected into BALB / c mice bearing 100 mm 3 EMT6 flank tumors. Twenty hours after treatment, tumors were harvested and siRNA delivery was analyzed using IVIS imaging.

[0441] 40 μg of fluorescently labeled siRNA was mixed with 1 mg of 3E10 or the D31N variant of 0.1 mg of 3E10 at room temperature for 15 minutes. The siRNA that formed a complex with 3E10 was systemically injected into BALB / c mice bearing 100 mm 3 EMT6 flank tumors. Twenty hours after treatment, tumors were harvested and siRNA delivery was analyzed using IVIS imaging. Results

[0442] As shown in Figure 7A, increasing the dose of 3E10 increased the accumulation of siRNA in tumors.

[0443] As shown in Figure 7B, a low dose of 1 / 10 of D31N 3E10 resulted in a similar level of siRNA delivery as 3E10.

[0444] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications cited herein and the materials to which they are cited are specifically incorporated by reference.

[0445] Example 9: Carrier DNA Enhances mRNA in Non-Tumor Tissues Materials and Methods 2 μg of fluorescently labeled mRNA and 20 μg of 3E10-D31N were mixed with or without carrier DNA (5 μg) at room temperature for 15 minutes. The mRNA complexed with 3E10 was injected into the fetus at E15.5. 24 - 48 hours after treatment, the fetuses were harvested and mRNA delivery was analyzed using IVIS imaging. Results

[0446] In the absence of carrier DNA, 3E10-D31N complexed with mRNA was rapidly excreted from the fetus within 24 hours. However, by adding carrier DNA, detectable mRNA signals were obtained in multiple tissues of the fetus at 48 hours.

[0447] The above examples can show that while the delivery of DNA cargo is more generally to multiple tissues and is often not limited to tumors, RNA delivery is often more selective for tumor tissues. Example 10: 3E10(D31N) Complexed with mRNA and Carrier DNA Result in Sustained Levels of Protein Expression. Materials and Methods

[0448] 10 μg of luciferase mRNA and 10 μg of single-stranded carrier DNA (60 nts) were mixed with 100 μg of 3E10 (WT) or 3E10 (D31N) at room temperature for 15 minutes. The mRNA complexed with 3E10 was intramuscularly injected (IM) into the right quadriceps muscle of each mouse. Luciferase expression was monitored over 6 days. Results

[0449] As shown in Figure 8, administration of 3E10 (D31N) complexed with mRNA and carrier DNA resulted in sustained levels of luciferase expression, while 3E10 (WT) complexed with mRNA and carrier DNA could not generate a detectable signal above background.

[0450] Example 11: In vivo distribution of 3E10 administered by IV injection. The distribution of IV-injected 3E10 into muscle was examined. Mice were intravenously injected with 200 μg of 3E10, WT or D31N labeled with VivoTag680 (Perkin Elmer). Four hours after injection, the muscle was harvested and imaged by IVIS (Perkin Elmer) (Figures 9A and 9B). Quantification of the IVIS images showed that 3E10-D31N achieved a higher distribution into muscle compared to 3E10-WT (Figure 9C).

[0451] The dose-dependent in vivo distribution of 3E10-D31N into tissues was examined. Mice were intravenously injected with 100 μg or 200 μg of 3E10-D31N labeled with VivoTag680 (Perkin Elmer). Twenty-four hours after injection, the tissues were harvested and imaged by IVIS (Perkin Elmer). Quantification of the tissue distribution showed a dose-dependent 2-fold increase in accumulation into muscle and no proportional increase in multiple tissues including the liver (Figure 10).

[0452] Distribution to tumors of 3E10. Mice with syngeneic colon tumors (CT26) in the flanks were intravenously injected with 200 μg of 3E10, WT or D31N labeled with VivoTag680 (Perkin Elmer). Twenty-four hours after injection, the tumors were harvested and imaged by IVIS (Perkin Elmer) (Figures 11A - 11B). Quantification of tissue distribution showed that 3E10 - D31N had greater accumulation in tumors compared to 3E10 - WT (Figure 11C).

[0453] The distribution of ssDNA non-covalently bound to 3E10 was examined. Mice with syngeneic colon tumors (CT26) in the flanks were intravenously injected with 200 μg of 3E10, WT or D31N mixed with 40 μg of labeled ssDNA (IR680). Twenty-four hours after injection, the tumors were harvested and imaged by IVIS (Perkin Elmer) (Figures 12A - 12C). Quantification of tissue distribution showed that delivery of ssDNA by 3E10 - D31N resulted in higher tumor accumulation compared to 3E10 - WT (Figure 12D).

[0454] Example 12: Delivery of RIG-I ligands mediated by 3E10, and stimulation of RIG-I activity. Materials and methods RIG-I reporter cells (HEK-Lucia RIG-I, Invivogen) were seeded at 50,000 cells per well and treated with RIG-I ligand (1 μg) or ligand complexed with 3E10 - D31N (20 μg). This assay uses a cell line with a luciferase reporter that is activated in the presence of interferon induction. Results

[0455] In all cases, the RIG-I ligand alone did not stimulate IFN-γ secretion. However, delivery of the RIG-ligand by 3E10 - D31N stimulated more IFN-γ secretion than the control, and the highest secretion was observed with both low molecular weight and high molecular weight (LMW and HMW) poly(I:C).

[0456] Example 13: Molecular modeling of 3E10 and its engineered variants. WT heavy chain scFv sequence

Chem.

Chem.

[0457] Molecular modeling of 3E10 (Pymol) revealed a putative nucleic acid binding pocket (NAB1) (Figures 14A - 14B). Mutation of the aspartic acid at residue 31 of CDR1 to asparagine increased the cationic charge of this residue and enhanced nucleic acid binding and delivery in vivo (3E10 - D31N).

[0458] Mutation of the aspartic acid at residue 31 of CDR1 to arginine (3E10 - D31R) further increased the cationic charge, and mutation to lysine (3E10 - D31K) changed the direction of the charge (Figure 14A).

[0459] The NAB1 amino acids predicted from molecular modeling are underlined in the heavy and light chain sequences above. Figure 14B is an explanatory diagram showing the molecular modeling of 3E10 - scFv (Pymol) with NAB1 amino acid residues indicated as dotted dots.

[0460] One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. The present invention provides, for example, the following items. (Item 1) (a) A 3E10 monoclonal antibody, its cell - membrane - permeable fragment; a monovalent, bivalent, or multivalent single - chain variable fragment (scFv); or a diabody; or a humanized form or variant thereof, and (b) A nucleic acid cargo comprising a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof A composition comprising or consisting of (Item 2) (a) is (i) Any one of CDRs of SEQ ID NOs: 1-6, 12, 13, 46-48, or 50-52, combined with any one of the CDRs of SEQ ID NOs: 7-11, 14, or 53-58 (ii) The first, second, and third heavy chain CDRs selected from any of SEQ ID NOs: 15-23, 42, or 43, combined with the first, second, and third light chain CDRs selected from any of SEQ ID NOs: 24-30, 44, or 45 (iii) A humanized form of (i) or (ii) (iv) A heavy chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1 or 2, combined with a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 7 or 8 (v) A humanized form or (iv); or (vi) A heavy chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 3-6, 46-48, or 50-52, combined with a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NOs: 9-11 or 53-58 The composition according to item 1, comprising (Item 3) (a) The composition according to item 1 or 2, comprising an epitope specificity the same as or different from that of the monoclonal antibody 3E10 produced by the hybridoma with ATCC deposit number PTA2439 (Item 4) (a) The composition according to any one of items 1-3, wherein (a) is a recombinant antibody having the paratope of the monoclonal antibody 3E10 (Item 5) (a) The following (i) Any one of SEQ ID NOs: 1-6, 12, 13, 46-48, or 50-52, in combination with any one of the CDRs of SEQ ID NOs: 7-11, 14, or 53-58 CDR; (ii) The first, second, and third heavy chain CDRs selected from SEQ ID NOs: 15-23, 42, or 43, in combination with the first, second, and third light chain CDRs selected from SEQ ID NOs: 24-30, 44, or 45; (iii) A humanized form of (i) or (ii); (iv) A heavy chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1 or 2, in combination with a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 7 or 8; (v) A humanized form or (iv); or (vi) A heavy chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 3-6, 46-48, or 50-52, in combination with a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NOs: 9-11 or 53-58 A binding protein comprising; (b) A nucleic acid cargo comprising a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof A composition comprising. (Item 6) (a) The composition according to any one of Items 1-5, wherein (a) is bispecific. (Item 7) (a) The composition according to Item 6, wherein (a) targets a cell type of interest. (Item 8) (a) The composition according to any one of Items 1-7, wherein (a) and (b) are non-covalently linked. (Item 9) (a) The composition according to any one of Items 1-8, wherein (a) and (b) are complexed. (Item 10) The composition according to any one of items 1 to 9, wherein (b) comprises DNA, RNA, PNA or other modified nucleic acids, or nucleic acid analogs, or a combination thereof. (Item 11) The composition according to any one of items 1 to 10, wherein (b) comprises mRNA. (Item 12) The composition according to any one of items 1 to 11, wherein (b) comprises a vector. (Item 13) The composition according to item 12, wherein the vector comprises a nucleic acid sequence encoding a polypeptide of interest operably linked to an expression control sequence. (Item 14) The composition according to item 13, wherein the vector is a plasmid. (Item 15) The composition according to any one of items 1 to 14, wherein (b) comprises a nucleic acid encoding a Cas endonuclease, gRNA, or a combination thereof. (Item 16) The composition according to any one of items 1 to 15, wherein (b) comprises a nucleic acid encoding a chimeric antigen receptor polypeptide. (Item 17) The composition according to any one of items 1 to 16, wherein (b) comprises a functional nucleic acid. (Item 18) The composition according to any one of items 1 to 17, wherein (b) comprises a nucleic acid encoding a functional nucleic acid. (Item 19) The composition according to item 17 or 18, wherein the functional nucleic acid is an antisense molecule, siRNA, miRNA, aptamer, ribozyme, RNAi, or an external guide sequence. (Item 20) The composition according to any one of items 1 to 19, wherein (b) comprises a plurality of single nucleic acid molecules. (Item 21) The composition according to any one of items 1 to 19, wherein (b) comprises a plurality of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid molecules. (Item 22) The composition according to any one of items 1 to 21, wherein (b) comprises or consists of a nucleic acid molecule having a length between about 1 and 25,000 nucleobases. (Item 23) The composition according to any one of items 1 to 22, wherein (b) comprises or consists of single-stranded nucleic acid, double-stranded nucleic acid, or a combination thereof. (Item 24) The composition according to any one of items 1 to 23, further comprising carrier DNA. (Item 25) The composition according to item 24, wherein the carrier DNA is non-coding DNA. (Item 26) The composition according to item 24 or 25, wherein (b) is composed of RNA. (Item 27) A pharmaceutical composition comprising the composition according to any one of items 1 to 26 and a pharmaceutically acceptable excipient. (Item 28) The composition according to item 27, further comprising polymer nanoparticles encapsulating the complex of (a) and (b). (Item 29) The composition according to item 28, wherein a targeting moiety, a cell membrane-permeable peptide, or a combination thereof is directly or indirectly associated, linked, conjugated, or otherwise bound to the nanoparticles. (Item 30) A method for delivering a nucleic acid cargo to a cell, the method comprising contacting the cell with an effective amount of the composition according to any one of items 1 to 29. (Item 31) The method according to item 30, wherein the contacting is performed ex vivo. (Item 32) The method according to item 31, wherein the cell is a hematopoietic stem cell or a T cell. (Item 33) The method according to any one of items 30 to 32, further comprising administering the cell to a subject in need thereof. (Item 34) The method according to item 33, wherein the cells are administered to the subject in an amount effective to treat one or more symptoms of a disease or disorder. (Item 35) The method according to item 30, wherein the contact is performed in vivo after administration to a subject in need thereof. (Item 36) The method according to any one of items 33 to 35, wherein the subject has a disease or disorder. (Item 37) The method according to item 36, wherein the disease or disorder is a genetic disorder, cancer, or an infectious disease or infection. (Item 38) (b) is delivered to the cells of the subject in an amount effective to reduce one or more symptoms of the disease or disorder of the subject, according to the method of item 36 or 37. (Item 39) A method of preparing a composition according to any one of items 1 to 29, comprising incubating and / or mixing (a) and (b) at an effective time and suitable temperature to form a complex of (a) and (b) prior to contact with the cells. (Item 40) A method of preparing a composition according to any one of items 1 to 29, comprising incubating and / or mixing (a) and (b) for about 1 minute to about 30 minutes, about 10 minutes to about 20 minutes, or about 15 minutes, optionally at room temperature or 37 °C. (Item 41) A composition or method according to any one of items 1 to 40, comprising a 3E10 monoclonal antibody, a cell membrane permeable fragment thereof; a monovalent, divalent, or multivalent single chain variable fragment (scFv); or a diabody; or a humanized form or variant thereof having the same or improved ability to bind to the nucleic acid binding pocket of SEQ ID NO: 92 or 93 or a variant thereof that binds to nucleic acid. (Item 42) A composition or method according to any one of items 1 to 41, wherein the amino acid residue corresponding to D31 or N31 of the heavy chain amino acid sequence or its CDR is substituted with R. (Item 43) The composition or method according to any one of items 1 to 42, wherein the amino acid residue corresponding to D31 or N31 of the heavy chain amino acid sequence or its CDR is substituted with L. (Item 44) (i) A variant of any one of CDRs of SEQ ID NOs: 1 to 6, 12, 13, 46 to 48, or 50 to 52, combined with any one of the CDRs of SEQ ID NOs: 7 to 11, 14, or 53 to 58; (ii) A variant of the first heavy chain CDR, combined with the second and third heavy chain CDRs selected from SEQ ID NOs: 15 to 23, 42, or 43, and combined with the first, second, and third light chain CDRs selected from SEQ ID NOs: 24 to 30, 44, or 45; (iii) A humanized form of (i) or (ii); (iv) A heavy chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1 or 2, combined with a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 7 or 8; (v) A humanized form or (iv); or (vi) A heavy chain comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 3 to 6, 46 to 48, or 50 to 52, combined with a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NOs: 9 to 11 or 53 to 58 comprising A binding protein, wherein the amino acid residue corresponding to D31 or N31 is substituted with R or L. (Item 45) The binding protein according to item 44, comprising the nucleic acid binding pocket of SEQ ID NO: 92 or 93, or a variant thereof having the same or improved ability to bind nucleic acids.

Claims

【Claim 1】 The invention described in the specification.

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