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Compositions and methods for enhancing triplex and nuclease-based gene editing — Yale University (US20260048118A1)

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patent, google patents, intellectual property, US20260048118A1, Yale University, Elias Quijano, en, 2026

ABSTRACT

Abstract

Compositions for improved gene editing and methods of use thereof are disclosed. In a preferred method, gene editing involves use of a cell-penetrating anti-DNA antibody, such as 3E10, as a potentiating agent to enhance gene editing by nucleases and triplex forming oligonucleotides. Genomic modification occurs at a higher frequency when cells are contacted with the potentiating agent and nuclease or triplex forming oligonucleotide, as compared to the absence of the potentiating agent. The methods are suitable for both ex vivo and in vivo approaches to gene editing and are useful for treating a subject with a genetic disease or disorder. Nanoparticle compositions for intracellular delivery of the gene editing compositions are provided and are particularly advantageous for use with in vivo applications.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No. 17/272,151, which is the U.S. National Phase of International Application No. PCT/US2019/048962, filed on Aug. 30, 2019, which claims the priority benefit of U.S. Provisional Application No. 62/725,852, filed Aug. 31, 2018, each of which is specifically incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under CA197574 and CA168733 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO THE SEQUENCE LISTING

The content of the electronically submitted sequence listing (Name: 2681_1230003_Sequencelisting_ST26; Size: 96,521 bytes; and Date of Creation: Nov. 3, 2025) is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention is generally related to the field of gene editing technology, and more particularly to methods of using cell-penetrating antibodies to improve triplex-forming oligonucleotide- and nuclease-mediated gene editing.

BACKGROUND OF THE INVENTION

Gene editing provides an attractive strategy for treatment of inherited genetic disorders such as, for example, sickle cell anemia and Î²-thalassemia. Genes can be selectively edited by several methods, including targeted nucleases such as zinc finger nucleases (ZFNs) (Haendel, et al., Gene Ther., 11:28-37 (2011)) and CRISPRs (Yin, et al., Nat. Biotechnol., 32:551-553 (2014)), short fragment homologous recombination (SFHR) (Goncz, et al., Oligonucleotides, 16:213-224 (2006)), or triplex-forming oligonucleotides (TFOs) (Vasquez, et al., Science, 290:530-533 (2000)). It is generally thought that a DNA break in a target gene is needed for high efficiency gene editing with a donor DNA. Hence, there has been widespread focus on targeted nucleases such as CRISPR/Cas9 technology because of its ease of use and facile reagent design (Doudna, et al., Science, 346:1258096 (2014)). However, like ZFNs, the CRISPR approach introduces an active nuclease into cells, which can lead to off-target cleavage in the genome (Cradick, et al., Nucleic Acids Res., 41:9584-9592 (2013)), a problem that so far has not been eliminated.

Alternatives have been developed such as triplex-forming peptide nucleic acid (PNA) oligomers which recruit the cell's endogenous DNA repair systems to initiate site-specific modification of the genome when single-stranded âdonor DNAsâ are co-delivered as templates (Rogers, et al., Proc. Natl. Acad. Sci. USA, 99:16695-16700 (2002)).

Historically however, the efficiency of gene modification could be low, especially in the context of CRISPR/Cas-mediated editing in primary stem cells. For example, in an attempt to correct the CFTR locus in cystic fibrosis patient derived stem cells, approximately 0.3% of treated organoids (3 to 6/1400) had the desired modification (Schwank, et al., Cell Stem Cell., 13:653-658 (2013)).

Accordingly, there remains a need for compositions and methods for improved gene editing.

It is therefore an object of the invention to provide gene editing potentiating agents and methods for achieving an increased frequency of gene modification.

It is another object of the invention to provide methods for achieving on-target modification with reduced or low off-target modification.

It is a further object of the invention to provide compositions and methods for gene modification that improve one or more symptoms of a disease or disorder in a subject.

SUMMARY OF THE INVENTION

Compositions for enhancing targeted gene editing and methods of use thereof are disclosed. Disclosed are methods of gene editing utilizing a gene editing composition such as triplex-forming oligonucleotides, CRISPR, zinc finger nucleases, TALENS, or others, in combination with a gene editing potentiating agent such as a cell-penetrating anti-DNA antibody.

An exemplary method of modifying the genome of a cell can include contacting the cell with an effective amount of (i) a gene editing potentiating agent, and (ii) a gene editing technology that can induce genomic modification of the cell (e.g., triplex-forming molecules, pseudocomplementary oligonucleotides, a CRISPR system, zinc finger nucleases (ZFN), and transcription activator-like effector nucleases (TALEN)). In the foregoing method, genomic modification occurs at a higher frequency in a population of cells contacted with both (i) and (ii), than in an equivalent population contacted with (ii) in the absence of (i). Preferred gene editing technologies include a triplex forming molecule, such as a peptide nucleic acid (PNA), and a CRISPR system such as CRISPR/Cas9 D10A nickase.

A preferred gene editing potentiating agent is a cell-penetrating anti-DNA antibody which is transported into the cytoplasm and/or nucleus of the cell without the aid of a carrier or conjugate. In some embodiments, the cell-penetrating anti-DNA antibody is isolated or derived from a subject with systemic lupus erythematous or an animal model thereof (such as a mouse or rabbit). In a preferred embodiment, the cell-penetrating anti-DNA antibody is the monoclonal anti-DNA antibody 3E10, or a variant, fragment (e.g., cell-penetrating fragment), or humanized form thereof that binds the same epitope(s) as 3E10. A particularly preferred variant is a 3E10 variant incorporating a D31N substitution in the heavy chain. The cell-penetrating anti-DNA antibody may have the same or different epitope specificity as monoclonal antibody 3E10 produced by ATCC No. PTA 2439 hybridoma.

In some embodiments, the antibody has

(i) the CDRs of any one of SEQ ID NO: 1-6, 12, or 13 in combination with the CDRs of any one of SEQ ID NO:7-11, or 15; (ii) first, second, and third heavy chain CDRs selected from SEQ ID NOS: 15-23 in combination with first, second and third light chain CDRs selected from SEQ ID NOS: 24-30; (iii) humanized forms of (i) or (ii); (iv) a heavy chain comprising an amino acid sequence comprising at least 85% sequence identity to any one of SEQ ID NO:1 or 2 in combination with a light chain comprising an amino acid sequence comprising 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 comprising at least 85% sequence identity to any one of SEQ ID NO:3-6 in combination with a light chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:9-11.

Preferably, the antibody can bind directly to RAD51. In some embodiments, the anti-DNA antibody has the paratope of monoclonal antibody 3E10. The anti-DNA antibody may be a single chain variable fragment of an anti-DNA antibody, or conservative variant thereof. For example, the anti-DNA antibody can be a monovalent, divalent, or multivalent single chain variable fragment of 3E10 (3E10 Fv), or a variant, for example a conservative variant, thereof. In some embodiments, the anti-DNA antibody is a monovalent, divalent, or multivalent single chain variable fragment of 3E10 (3E10 Fv) incorporating a D31N substitution in the heavy chain.

The method can further include contacting the cells with a donor oligonucleotide including, for example, a sequence that corrects or induces a mutation(s) in the cell's genome by insertion or recombination of the donor induced or enhanced by the gene editing technology. The donor oligonucleotide (e.g., DNA) may be single stranded or double stranded. Preferably, the donor oligonucleotide is single stranded DNA. The potentiating agent, gene editing technology, and/or donor oligonucleotide can be contacted with the cell in any order.

In some embodiments, the cell's genome has a mutation underlying a disease or disorder, for example a genetic disorder such as hemophilia, muscular dystrophy, globinopathies, cystic fibrosis, xeroderma pigmentosum, lysosomal storage diseases, immune deficiency syndromes such as X-linked severe combined immunodeficiency and ADA deficiency, tyrosinemia, Fanconi anemia, the red cell disorder spherocytosis, alpha-1-anti-trypsin deficiency, Wilson's disease, Leber's hereditary optic neuropathy, or chronic granulomatous disorder. The globinopathy can be sickle cell anemia or beta-thalassemia. The lysosomal storage disease can be Gaucher's disease, Fabry disease, or Hurler syndrome. In some embodiments, the method induces a mutation that reduces HIV infection, for example, by reducing an activity of a cell surface receptor that facilitates entry of HIV into the cell.

In some embodiments, the cells (e.g., hematopoietic stem cells) are contacted ex vivo and the cells may further be administered to a subject in need thereof. The cells may be administered to the subject in an effective amount to treat one or more symptoms of a disease or disorder.

In other embodiments, the cells are contacted in vivo following administration of the potentiating agent, gene editing technology, and optionally the donor oligonucleotide to a subject. Each of the foregoing can be in the same or different pharmaceutical compositions and can be administered to the subject in any order. In preferred embodiments, the compositions induce or enhance in vivo gene modification in an effective amount to reduce one or more symptoms of the disease or disorder in the subject.

Any of the disclosed compositions including potentiating agent, gene editing technology, and/or donor oligonucleotide can be packaged together or separately in nanoparticles. The nanoparticles may be formed from polyhydroxy acids. In preferred embodiments, the nanoparticles include poly(lactic-co-glycolic acid) (PLGA) alone or in a blend with poly(beta-amino) esters (PBAEs). The nanoparticles may be prepared by double emulsion or nanoprecipitation. In some embodiments, the gene editing technology, the donor oligonucleotide or a combination thereof are complexed with a polycation prior to preparation of the nanoparticles.

Functional molecules such as targeting moieties, cell penetrating peptides, or a combination thereof can be associated with, linked, conjugated, or otherwise attached directly or indirectly to the potentiating agent, the gene editing technology, the nanoparticle, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A is a bar graph showing PNA/DNA mediated gene correction of the IVS2-654 (C->T) mutation within the Î²-globin/GFP fusion gene in MEFs treated with Rad51 siRNA or 3E10. FIGS. 1 B and 1 C are box plots showing the frequency of in vivo gene editing in bone marrow-( 1 B) and spleen-derived ( 1 C) CD117+ cells from Î²-globin/GFP transgenic mice treated with 3E10.

FIG. 2 is a bar graph showing the percentage of gene editing following treatment of MEFs from Townes mice with PNA/DNA-containing nanoparticles with or without the 3E10 antibody.

FIG. 3 A is a schematic representation of binding site positions of tcPNAs 1, 2, and 3 targeting the beta globin gene in the vicinity of the SCD mutation. FIG. 3 B is a bar graph showing the percentage of gene editing in bone marrow cells from Townes mice treated with tcPNA2A/donor DNA-containing nanoparticles with or without the 3E10 antibody.

FIG. 4 is a box plot showing the percentage of gene editing in bone marrow cells following in vivo treatment of Townes mice with PNA/donor DNA-containing nanoparticles with or without the 3E10 antibody.

FIG. 5 is a bar graph showing the percentage of gene editing in SC-1 cells treated with PNA/DNA-containing nanoparticles with or without the 3E10 antibody.

FIGS. 6 A and 6 B are bar graphs showing the percentage of Cas9-mediated gene editing in K562 BFP/GFP reporter cells treated with or without the 3E10 antibody in the presence of CRISPR/Cas9 WT ( 6 A) and CRISPR/Cas9 D10A nickase ( 6 B).

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein, the term âsingle chain Fvâ or âscFvâ as used herein means a single chain variable fragment that includes a light chain variable region (VL) and a heavy chain variable region (VH) in a single polypeptide chain joined by a linker which enables the scFv to form the desired structure for antigen binding (i.e., for the VH and VL of the single polypeptide chain to associate with one another to form a Fv). The VL and VH regions may be derived from the parent antibody or may be chemically or recombinantly synthesized.

As used herein, the term âvariable regionâ is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain). The variable region includes a âhypervariable regionâ whose residues are responsible for antigen binding. The hypervariable region includes amino acid residues from a âComplementarity Determining Regionâ or âCDRâ (i.e., typically at approximately residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (H1), 50-65 (H2) and 95-102 (H3) in 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 those residues from a âhypervariable loopâ (i.e., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917).

As used herein, the term âFramework Regionâ or âFRâ residues are those variable domain residues other than the hypervariable region residues as herein defined.

As used herein, the term âantibodyâ refers to natural or synthetic antibodies that bind a target antigen. The term includes polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term âantibodiesâ are binding proteins, fragments, and polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that bind the target antigen.

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CROSS-REFERENCE TO RELATED APPLICATION

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under CA197574 and CA168733 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO THE SEQUENCE LISTING

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Accordingly, there remains a need for compositions and methods for improved gene editing.

It is therefore an object of the invention to provide gene editing potentiating agents and methods for achieving an increased frequency of gene modification.

It is another object of the invention to provide methods for achieving on-target modification with reduced or low off-target modification.

It is a further object of the invention to provide compositions and methods for gene modification that improve one or more symptoms of a disease or disorder in a subject.

SUMMARY OF THE INVENTION

In some embodiments, the antibody has

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 2 is a bar graph showing the percentage of gene editing following treatment of MEFs from Townes mice with PNA/DNA-containing nanoparticles with or without the 3E10 antibody.

FIG. 5 is a bar graph showing the percentage of gene editing in SC-1 cells treated with PNA/DNA-containing nanoparticles with or without the 3E10 antibody.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein, the term âFramework Regionâ or âFRâ residues are those variable domain residues other than the hypervariable region residues as herein defined.

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

In addition to intact immunoglobulin molecules, also included in the term âantibodiesâ are fragments, binding proteins, and polymers of immunoglobulin molecules, chimeric antibodies containing sequences from more than one species, class, or subclass of immunoglobulin, such as human or humanized antibodies, and recombinant proteins containing a least the idiotype of an immunoglobulin that specifically binds DNA. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic activities are tested according to known clinical testing methods.

As used herein, the term âvariantâ refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

Modifications and changes can be made in the structure of the polypeptides of in disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.

In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those 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 (â0.4); threonine (â0.7); serine (â0.8); tryptophan (â0.9); tyrosine (â1.3); proline (â1.6); histidine (â3.2); glutamate (â3.5); glutamine (â3.5); aspartate (â3.5); asparagine (â3.5); lysine (â3.9); and arginine (â4.5).

It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant 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 an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within Â±2 is preferred, those within Â±1 are particularly preferred, and those within Â±0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0Â±1); glutamate (+3.0Â±1); serine (+0.3); asparagine (+0.2); glutamnine (+0.2); glycine (0); proline (â0.5Â±1); threonine (â0.4); alanine (â0.5); histidine (â0.5); cysteine (â1.0); methionine (â1.3); valine (â1.5); leucine (â1.8); isoleucine (â1.8); tyrosine (â2.3); phenylalanine (â2.5); tryptophan (â3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within Â±2 is preferred, those within Â±1 are particularly preferred, and those within Â±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (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). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide of interest.

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

For purposes herein, the % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given sequence C that has or includes a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:

100 times the fraction W/Z,

where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program's alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C.

As used herein, the term âspecifically bindsâ refers to the binding of an antibody to its cognate antigen (for example, DNA) while not significantly binding to other antigens. Specific binding of an antibody to a target under such conditions requires the antibody be selected for its specificity to the target. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Preferably, an antibody âspecifically bindsâ to an antigen with an affinity constant (Ka) greater than about 10 5 mol â1 (e.g., 10 6 mol â1 , 10 7 mol â1 , 10 8 mol â1 , 10 9 mol â1 , 10 10 mol â1 , 10 11 mol â1 , and 10 12 mol â1 or more) with that second molecule.

As used herein, the term âmonoclonal antibodyâ or âMAbâ refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.

As used herein a âgene editing potentiating factorâ or âgene editing potentiating agentâ or âpotentiating factor or âpotentiating agentâ refers to a compound that increases the efficacy of editing (e.g., mutation, including insertion, deletion, substitution, etc.) of a gene, genome, or other nucleic acid by a gene editing technology relative to use of the gene editing technology in the absence of the compound.

As used herein, the term âsubjectâ means any individual who is the target of administration. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human. The term does not denote a particular age or sex.

As used herein, the terms âeffective amountâ or âtherapeutically effective amountâ means that the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder being treated, as well as the route of administration and the pharmacokinetics of the agent being administered.

As used herein, the term âpharmaceutically acceptableâ refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

As used herein, the term âcarrierâ or âexcipientâ refers to an organic or inorganic ingredient, natural or synthetic inactive ingredient in a formulation, with which one or more active ingredients are combined. The carrier or excipient would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

As used herein, the term âtreatâ refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

As used herein, âtargeting moietyâ is a substance which can direct a nanoparticle to a receptor site on a selected cell or tissue type, can serve as an attachment molecule, or serve to couple or attach another molecule. As used herein, âdirectâ refers to causing a molecule to preferentially attach to a selected cell or tissue type. This can be used to direct cellular materials, molecules, or drugs, as discussed below.

As used herein, the term âinhibitâ or âreduceâ means to decrease an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

As used herein, a âfusion proteinâ refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from a nucleic acid sequence encoding the 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 in molecular biology to join the two genes in frame into a single nucleic acid sequence, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.

As used herein, the term âsmall moleculeâ as used herein, generally refers to an organic molecule that is less than about 2000 g/mol in molecular weight, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. Small molecules are non-polymeric and/or non-oligomeric.

Recitation of ranges of values herein are 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.

Use of the term âaboutâ is intended to describe values either above or below the stated value in a range of approx. +/â10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/â5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/â2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/â1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., âsuch asâ) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the embodiments unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

II. Gene Editing Potentiating Agents

Several methods have been developed to mediate gene editing. These methods include the use of Zinc Finger Nucleases, Talens, Meganucleases, CRISPR/Cas9, and triplex-forming Peptide Nucleic Acids (PNAs) (Maeder, et al., Mol. Ther., 24 (3): 430-46 (2016); Quijano, et al., Yale J. Biol. Med., 90 (4): 583-598 (2017)). These approaches either make a direct cut at the target site DNA (nucleases), or they bind to the target gene and trigger the cells endogenous repair pathways (e.g., PNAs), which secondarily leads to strand breaks. Common among these methods, the gene editing information is carried by single-stranded or double-stranded oligonucleotides, or donor DNAs, that are co-administered to the cell or animal with the nuclease or the PNA. It is generally thought that a DNA strand break in the target site is needed to enable high efficiency gene editing with a donor DNA.

In early work with DNA triplex-forming oligonucleotides (TFOs), it was observed that RAD51, a factor implicated in homology search and strand invasion in homology-directed repair processes, was required for TFO-induced gene editing (Bahal, et al., Nat. Commun., 7:13304 (2016)). It has now been discovered that RAD51 is, in contrast, not required for PNA-mediated gene editing (through experiments using co-delivered PNAs/donor DNAs in combination with anti-RAD51 siRNAs). Moreover, it has been discovered that knockdown of RAD51 actually boosts the efficiency of editing, as measured by allele-specific PCR.

The experiments described in the Examples also show that 3E10, a cell-penetrating anti-DNA antibody that binds to and inhibits RAD51, stimulates gene editing by PNAs/donor DNAs in mouse and human cells in culture, and in mice in vivo. 3E10 is also shown to enhance gene editing by the D10A nickase version of CRISPR/Cas9 in combination with a donor DNA.

Accordingly, compositions and methods of increasing the efficacy of a gene editing technology, such as, a triplex-forming PNA and donor DNA (optionally in a nanoparticle composition), or a CRISPR/Cas9 system (e.g., CRISPR/Cas9 D10A nickase) and donor DNA are provided. The disclosed methods typically include contacting cells with both a potentiating agent and a gene editing technology. Exemplary potentiating agents and gene editing technologies are provided. The potentiating agent and gene editing technology can be part of the same or different compositions.

In some embodiments, potentiating agents can engage one or more endogenous high fidelity DNA repair pathways, or inhibit/modulate error prone (i.e. low fidelity) DNA repair pathways. Potentiating agents include, for example, modulators of DNA damage and/or DNA repair factors, modulators of homologous recombination factors, cell adhesion modulators, cell cycle modulators, cell proliferation modulators, and stem cell mobilizers. The potentiating factor may modulate (e.g., alter, inhibit, promote, compete with) one or more endogenous high fidelity DNA repair pathways or inhibit/modulate error prone (i.e. low fidelity) DNA repair pathways. In preferred embodiments, the potentiating factor may be an inhibitor of a DNA damage, DNA repair, or homologous recombination factor. In more preferred embodiments, the potentiating factor may be an inhibitor of RAD51.

For example, an inhibitor of a DNA damage and/or DNA repair factor may be used as a potentiating agent. An inhibitor of a homologous recombination factor may be used as a potentiating agent.

Cells repair DNA breaks mainly through endogenous non-homologous end joining (NHEJ) DNA-repair, the predominant but error-prone pathway that can introduce or delete nucleotides at the DNA-break region. NHEJ is therefore amenable to permanent silencing of target genes. Alternatively, cells can also repair double-strand breaks by homology-directed repair (HDR), a more accurate mechanism involving homologous recombination in the presence of a template DNA strand. Typically, targeted genome editing is directed to correction of a mutated sequence in a genome by replacing the mutated sequence with a corrective sequence provided by a template/donor DNA. As such, there is ongoing effort in the field to identify and utilize mechanisms that favor homologous recombination of a template/donor DNA to enhance efficiency of targeted genome editing. Modulating the expression and/or activity of factors involved in DNA repair is a promising approach to enhance precision genome engineering.

The term âDNA repairâ refers to a collection of processes by which a cell identifies and corrects damage to DNA molecules. Single-strand defects are repaired by base excision repair (BER), nucleotide excision repair (NER), or mismatch repair (MMR). Double-strand breaks are repaired by non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or homologous recombination. After DNA damage, cell cycle checkpoints are activated, which pause the cell cycle to give the cell time to repair the damage before continuing to divide. Checkpoint mediator proteins include BRCA1, MDC1, 53BP1, p53, ATM, ATR, CHK1, CHK2, and p21. Accordingly, a factor involved in any of the above-mentioned processes, including BER, NER, MMR, NHEJ, MMEJ, homologous recombination, or DNA synthesis and the like, may be described as a DNA damage and/or DNA repair factor.

Non-limiting examples of DNA damage, DNA repair, DNA synthesis, or homologous recombination factors include XRCC1, ADPRT (PARP-1), ADPRTL2, (PARP-2), POLYMERASE BETA, CTPS, MLH1, MSH2, FANCD2, PMS2, p53, p21, PTEN, RPA, RPAI, RPA2, RPA3, XPD, ERCC1, XPF, MMS19, RAD51, RAD51B, RAD51C, RAD51D, DMC1, XRCCR, XRCC3, BRCA1, BRCA2, PALB2, RAD52, RAD54, RAD50, MREU, NB51, WRN, BLM, KU70, KU80, ATM, ATR CPIK1, CHK2, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, RAD1, and RAD9. In a preferred embodiment, the DNA damage factor or DNA repair factor is RAD51.

RAD51 recombinase, an ortholog of E. coli RecA, is a key protein in homologous recombination in mammalian cells. RAD51 promotes the repair of double-strand breaks, the most harmful type of DNA lesion. Double-strand breaks can be induced by various chemical agents and ionizing radiation, and are also formed during the repair of inter-strand crosslinks. Once double-strand breaks are formed, they are processed first by exonucleases to generate extensive 3â² single-stranded DNA (ssDNA) tails (Cejka et al., Nature., 467 (7311): 112-16 (2010); Mimitou & Symington, DNA Repair., 8 (9): 983-95 (2009)). These tracks of ssDNA rapidly become coated by single strand DNA-binding protein, RPA, which is ultimately displaced from the ssDNA by RAD51. RAD51 has ATP-dependent DNA binding activity, and so binds the ssDNA tails, and multimerizes to form helical nucleoprotein filaments that promote search for homologous dsDNA sequences (Kowalczykowski, Nature., 453 (7194): 463-6 (2008)). The ability of RAD51 to displace RPA on ssDNA in cells requires several mediator proteins, which include BRCA2, RAD52, the RAD51 paralog complexes, and other proteins (Thompson & Schild, Mutat Res., 477:131-53 (2001)). Once homologous dsDNA sequences are found, RAD51 promotes DNA strand exchange between the ssDNA that resides within the filament and homologous dsDNA, i.e., an invasion of ssDNA into homologous DNA duplex that results in the displacement of the identical ssDNA from the duplex and formation of a joint molecule. Joint molecules, key intermediates of DSB repair, provide both the template and the primer for DNA repair synthesis that is required for double-strand break repair (Paques & Haber, Microbiol. Mol. Biol. Rev., 63 (2): 349-404 (1999)).

By promoting DNA strand exchange, RAD51 plays a key role in homologous recombination. The protein is evolutionarily conserved from bacteriophages to mammals. In all organisms, RAD51 orthologs play an important role in DNA repair and homologous recombination (Krough & Symington, Annu. Rev. Genet., 38:233-71 (2004); Helleday et al., DNA Repair., 6 (7): 923-35 (2007); Huang et al., Proc. Natl. Acad. Sci. USA., 93 (10): 4827-32 (1996)).

In preferred embodiments, the potentiating agent is one that antagonizes or reduces expression and/or activity of RAD51, XRCC4, or a combination thereof. For example, in some embodiments, the potentiating agent is a RAD51 and/or XRCC4 inhibitor. Non-limiting examples of potentiating agents include, ribozymes, triplex-forming molecules, siRNAs, shRNAs, miRNAs, aptamers, antisense oligonucleotides, small molecules, and antibodies.

Methods for designing and producing any of the foregoing factors are well-known in the art and can be used. For example, predesigned anti-RAD51 siRNAs are commercially available through Dharmacon (as described in the Examples) and may be used as potentiating agents. Likewise, anti-XRCC4 siRNAs, shRNAs and miRNAs are known in the art and are readily available. Further, small molecule inhibitors of XRCC4 and RAD51 are known in the art (e.g., Jekimovs, et al., Front. Oncol., 4:86 (2014)) and can be used as potentiating agents in accordance with the disclosed methods.

In some embodiments, the potentiating agent is a cell-penetrating antibody. Although the cell-penetrating molecules are generally referred to herein as âcell-penetrating antibodies,â it will be appreciated that fragments and binding proteins, including antigen-binding fragments, variants, and fusion proteins such as scFv, di-scFv, tri-scFv, and other single chain variable fragments, and other cell-penetrating molecules disclosed herein are encompassed by the phrase and also expressly provided for use in compositions and methods disclosed herein.

Cell-penetrating antibodies for use in the compositions and methods may be anti-DNA antibodies. The cell-penetrating antibody may bind single stranded DNA and/or double stranded DNA. The cell-penetrating antibody may be an anti-RNA antibody (e.g., the antibody specifically binds RNA).

Autoantibodies to double-stranded deoxyribonucleic acid (dsDNA) are frequently identified in the serum of patients with systemic lupus erythematosus (SLE) and are often implicated in disease pathogenesis. Therefore, in some embodiments, cell-penetrating antibodies (e.g., cell-penetrating anti-DNA antibodies) can be derived or isolated from patients with SLE or animal models of SLE.

In preferred embodiments, the anti-DNA antibodies are monoclonal antibodies, or antigen binding fragments or variants thereof. In some embodiments, the anti-DNA antibodies are conjugated to a cell-penetrating moiety, such as a cell penetrating peptide to facilitate entry into the cell and transport to the cytoplasm and/or nucleus. Examples of cell penetrating peptides include, but are not limited to, Polyarginine (e.g., R 9 ), Antennapedia sequences, TAT, HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II, Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (Bis-Guanidinium-Spermidine-Cholesterol, and BGTC (Bis-Guanidinium-Tren-Cholesterol). In other embodiments, the antibody is modified using TransMabsâ¢ technology (InNexus Biotech., Inc., Vancouver, BC).

In preferred embodiments, the anti-DNA antibody is transported into the cytoplasm and/or nucleus of the cells without the aid of a carrier or conjugate. For example, the monoclonal antibody 3E10 and active fragments thereof that are transported in vivo to the nucleus of mammalian cells without cytotoxic effect are disclosed in U.S. Pat. Nos. 4,812,397 and 7,189,396 to Richard Weisbart. Briefly, the antibodies may be prepared by fusing spleen cells from a host having elevated serum levels of anti-DNA antibodies (e.g., MRL/1pr mice) with myeloma cells in accordance with known techniques or by transforming the spleen cells with an appropriate transforming vector to immortalize the cells. The cells may be cultured in a selective medium and screened to select antibodies that bind DNA.

In some embodiments, the cell-penetrating antibody may bind and/or inhibit Rad51. See for example, the cell-penetrating antibody described in Turchick, et al., Nucleic Acids Res., 45 (20): 11782-11799 (2017).

Antibodies that can be used in the compositions and methods include whole immunoglobulin (i.e., an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen binding variable domain of an antibody. The variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies. Therefore, the antibodies typically contain at least the CDRs necessary to maintain DNA binding and/or interfere with DNA repair.

A. 3E10 Sequences

In some embodiments, the cell-penetrating anti-DNA antibody is the monoclonal anti-DNA antibody 3E10, or a variant, derivative, fragment, or humanized form thereof that binds the same or different epitope(s) as 3E10. Thus, the cell-penetrating anti-DNA antibody may have the same or different epitope specificity as monoclonal antibody 3E10 produced by ATCC No. PTA 2439 hybridoma. The anti-DNA antibody can have the paratope of monoclonal antibody 3E10. The anti-DNA antibody can be a single chain variable fragment of an anti-DNA antibody, or conservative variant thereof. For example, the anti-DNA antibody can be a single chain variable fragment of 3E10 (3E10 Fv), or a variant thereof.

Amino acid sequences of monoclonal antibody 3E10 are known in the art. For example, sequences of the 3E10 heavy and light chains are provided below, where single underlining indicates the CDR regions identified according to the Kabat system, and in SEQ ID NOS: 12-14 italics indicates the variable regions and double underlining indicates the signal peptide. CDRs according to the IMGT system are also provided.

1. 3E10 Heavy Chain

In some embodiments, a heavy chain variable region of 3E10 is:

EVQLVESGGGLVKPGGSRKLSCAASGFTFS D YGMH WVRQAPEKGLEWVA

YISSGSSTIYYADTVKG RFTISRDNAKNTLFLQMTSLRSEDTAMYYCAR

RGLLLDY WGQGTTLTVSSâ

(SEQâIDâNO:â1;âZack,âetâal.,

ImmunologyâandâCellâBiology ,â72:513-520â(1994);

GenBank:âL16981.1-MouseâIgârearrangedâL-chain

gene,âpartialâcds;âandâGenBank:âAAA65679.1-

immunoglobulinâheavyâchain,âpartialâ[ Mus

musculus ]).

In some embodiments, a 3E10 heavy chain is expressed as

(3E10âWTâHeavyâChain;âSEQâIDâNO:â12)

MGWSCIILFLVATATGVHS

EVQLVESGGGLVKPGGSRKLSCAASGFTFS

GMH

WVRQAPERGLEWVA

YISSGSSTIYYADTVKG

RFTISRDNAKNTL

FLQMTSLRSEDTAMYYCAR

RGLLLDY

WGQGTTLTVS AASTKGPSVFPLA

PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP

<td class="desc

CLAIMS

Claims ( 29 )

1 . A composition comprising

a gene editing technology selected from the group consisting of triplex-forming molecules, pseudocomplementary oligonucleotides, a CRISPR system, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and intron encoded meganucleases, and a potentiating agent that reduces one or more DNA repair pathways and increases genomic editing by the gene editing technology compared to the gene editing technology alone.

2 . The composition of claim 1 , wherein the potentiating agent is a cell-penetrating antibody, fragment or humanized variant thereof.

3 . The composition of claim 2 , wherein the cell-penetrating antibody is an anti-DNA antibody and inhibits RAD51.

4 . The composition of claim 3 , wherein the cell-penetrating antibody comprises a 3E10 monoclonal antibody or a cell-penetrating fragment thereof; a monovalent, divalent, or multivalent single chain variable fragment (scFv); or a diabody; or humanized form or variant thereof.

5 . The composition of claim 4 , comprising

(i) the CDRs of any one of SEQ ID NO:1-6, 12, or 13 in combination with the CDRs of any one of SEQ ID NO:7-11, or 15; (ii) first, second, and third heavy chain CDRs selected from SEQ ID NO [S]: 15-23 in combination with first, second and third light chain CDRs selected from SEQ ID NO [S]: 24-30; (iii) a humanized forms of (i) or (ii); (iv) a heavy chain comprising an amino acid sequence comprising at least 85% sequence identity to any one of SEQ ID NO:1 or 2 in combination with a light chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:7 or 8; (v) a humanized form of (iv); or (vi) a heavy chain comprising an amino acid sequence comprising at least 85% sequence identity to any one of SEQ ID NO:3-6 in combination with a light chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:9-11.

6 - 9 . (canceled)

10 . The composition of claim 1 , further comprising a donor oligonucleotide that induces a mutation(s) in the cell's genome by insertion or recombination induced or enhanced by the gene editing technology.

11 - 19 . (canceled)

20 . A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable excipient.

21 . (canceled)

22 . (canceled)

23 . A method of modifying the genome of a cell comprising contacting the cell with a gene editing technology selected from the group consisting of triplex-forming molecules, pseudocomplementary oligonucleotides, a CRISPR system, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and intron encoded meganucleases, and

a potentiating agent that reduces one or more DNA repair pathways and increases genomic editing by the gene editing technology compared to the gene editing technology alone.

24 . The method of claim 23 , wherein the gene editing technology and potentiating agent are part of different compositions.

25 - 34 . (canceled)

35 . The method of claim 23 , wherein the cell's genome has a mutation underlying a disease or disorder selected from the group consisting of hemophilia, muscular dystrophy, globinopathies, cystic fibrosis, xeroderma pigmentosum, and lysosomal storage diseases, immune deficiency syndromes such as X-linked severe combined immunodeficiency and ADA deficiency, tyrosinemia, Fanconi anemia, the red cell disorder spherocytosis, alpha-1-anti-trypsin deficiency, Wilson's disease, Leber's hereditary optic neuropathy, and chronic granulomatous disorder.

36 . The method of claim 35 , wherein the mutation is in a gene encoding coagulation factor VIII, coagulation factor IX, dystrophin, beta-globin, CFTR, XPC, XPD, DNA polymerase eta, Fanconi anemia genes A through L, SPTA1 and other spectrin genes, ANK1 gene, SERPINA1 gene, ATP7B gene, interleukin 2 receptor gamma (IL2RG) gene, ADA gene, FAH gene, and genes linked to chronic granulomatous disease including the CYBA, CYBB, NCF1, NCF2, or NCF4 genes.

37 . (canceled)

38 . The method of claim 23 , wherein the contacting occurs ex vivo.

39 . The method of claim 38 , wherein the cell is a hematopoietic stem cell.

40 . The method of claim 23 , further comprising administering a plurality of the cells to a subject in need thereof.

41 . The method of claim 40 , wherein the cells are administered to the subject in an effective amount to treat one or more symptoms of a disease or disorder.

42 . The method of claim 23 , wherein the contacting occurs in vivo following administration to a subject in need thereof.

43 . The method of claim 42 , wherein the subject has a disease or disorder selected from the group consisting of hemophilia, muscular dystrophy, globinopathies, cystic fibrosis, xeroderma pigmentosum, and lysosomal storage diseases, immune deficiency syndromes such as X-linked severe combined immunodeficiency and ADA deficiency, tyrosinemia, Fanconi anemia, the red cell disorder spherocytosis, alpha-1-anti-trypsin deficiency, Wilson's disease, Leber's hereditary optic neuropathy, and chronic granulomatous disorder.

44 . The method of claim 43 , wherein gene modification occurs in an effective amount to reduce one or more symptoms of the disease or disorder in the subject.

45 . The method of claim 23 , wherein the gene editing technology, potentiating agent, and optional donor oligonucleotide are encapsulated in nanoparticles together or separately.

46 . (canceled)

47 . (canceled)

48 . The method of claim 45 , wherein a targeting moiety, a cell penetrating peptide, or a combination thereof is associated with, linked, conjugated, or otherwise attached directly or indirectly to the nanoparticle.

49 - 54 . (canceled)

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2021-05-27

CA3111186A1

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2020-03-05

US20230277658A1

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AU2019328326A1

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2021-03-18

BR112021003823A2

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2021-03-30

EP3844275A1

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2021-07-07

Compositions and methods for enhancing triplex and nuclease-based gene editing — Yale University (US20260048118A1)

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