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Compositions for enhancing targeted gene editing and methods of use thereof — Yale University (US11136597B2)

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patent, google patents, intellectual property, US11136597B2, Yale University, W. Mark Saltzman, en, 2021

ABSTRACT

Abstract

Compositions and methods for enhancing targeted gene editing and methods of use thereof are disclosed. In the most preferred embodiments, gene editing is carried out utilizing a gene editing composition such as triplex-forming oligonucleotides, CRISPR, zinc finger nucleases, TALENS, or others, in combination with a gene modification potentiating agent such as stem cell factor (SCF), a CHK1 or ATR inhibitor, or a combination thereof. A particular preferred gene editing composition is triplex-forming peptide nucleic acids (PNAs) substituted at the Î³ position for increased DNA binding affinity. Nanoparticle compositions for intracellular delivery of the gene editing composition are also provided and particular advantageous for use with in vivo applications.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Ser. No. 62/295,789 filed Feb. 16, 2016 and which is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under AI112443 awarded by National Institutes of Health and under 1012467 awarded by National Science Foundation. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted as a text file named âYU_6876_5_ST25.txt,â created on Nov. 3, 2020, and having a size of 93,350 bytes is hereby incorporated by reference pursuant to 37 C.F.R Â§ 1.52(e)(5).

FIELD OF THE INVENTION

The field of the invention is generally related to gene editing technology used in combination with a gene modification potentiating agent, and compositions and methods of use thereof for ex vivo and in vivo gene editing.

BACKGROUND OF THE INVENTION

Gene editing in hematopoietic stem/progenitor cells (HSPCs) provides an attractive strategy for treatment of inherited disorders such as 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)). Recent excitement has focused on 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.

One alternative is triplex-forming peptide nucleic acid (PNA) oligomers designed to bind site-specifically to genomic DNA via strand invasion and formation of PNA/DNA/PNA triplexes via both Watson-Crick and Hoogsteen binding) with a displaced DNA strand (Egholm, et al., Nature (London), 365:566-568 (1993); Nielsen, et al., Science (Washington, D.C., 1883-), 254:1497-1500 (1991); Faruqi, et al., Proc Natl Acad Sci USA, 95:1398-1403 (1998)). PNAs have a charge-neutral peptide-like backbone and nucleobases enabling hybridization with DNA and RNA with high affinity. PNA/DNA/PNA triplexes 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 containing the desired sequence modification (Rogers, et al., Proc. Natl. Acad. Sci. USA, 99:16695-16700 (2002)).

PNA-induced genome modification is believed to be mediated in part by the nucleotide excision repair (NER) and homology-dependent repair (HDR) pathways (Rogers, et al., Proc. Natl. Acad. Sci. USA, 99:16695-16700 (2002); Chin, et al., Molecular Carcinogenesis, 48:389-399 (2009)). Both NER and HDR are high fidelity pathways, and the PNAs lack any intrinsic nuclease activity. Together these features may account for the very low frequencies of off-target genotoxicity seen with PNA-mediated gene editing compared to nuclease based approaches (McNeer, et al., Gene Therapy, 20:658-669 (2013); Schleifinan, et al., Chem. Biol . (Cambridge, Mass., U.S.), 18:1189-1198 (2011); Schleifman, et al., Mol. Ther.âNucleic Acids, 2:e135 (2013)). Tail-clamp PNAs (tcPNAs) with an extended Watson-Crick binding domain can enhance gene editing in human hematopoietic cells with increased efficiency and specificity (Schleifman, et al., Chem. Biol . (Cambridge, Mass., U.S.), 18:1189-1198 (2011)) and that polymer nanoparticles (NPs) can effectively deliver these molecules into human HSPCs both ex vivo and in vivo in a humanized mouse model (McNeer, et al., Gene Therapy, 20:658-669 (2013); Bahal, et al., Curr. Gene Ther., 14:331-342 (2014)).

Nonetheless, compositions and methods for improved gene editing are needed.

It is an object of the invention to provide potentiating agents that increase gene modification induced or enhanced by gene editing technology.

It is another object of the invention to provide triplex forming molecules with enhanced DNA binding.

It is a further object of the invention to provide gene modification formulations that achieve therapeutically significant target site modification with reduced low off-target modification.

SUMMARY OF THE INVENTION

Highly elevated levels of gene editing in hematopoietic stem/progenitor cells are achieved using triplex-forming peptide nucleic acids (PNAs) substituted at the Î³ position for increased DNA binding affinity in combination with stimulation of the stem cell factor (SCF)/c-Kit pathway. The SCF/c-Kit pathway is believed to boost DNA repair gene expression and homology-dependent repair activity as evidence shows that stimulation is correlated with elevated DNA repair, specifically increased HDR activity and increased levels of HDR gene expression, including BRCA2 and Rad51. In a mouse model of human Î²-thalassemia, injection with SCF plus nanoparticles containing Î³PNAs and donor DNAs yielded amelioration of the disease phenotype, with clinically relevant Î²-globin gene correction frequencies (4% in bone marrow) and extremely low off-target effects. The mice showed alleviation of anemia with sustained elevation of blood hemoglobin levels into the normal range, reduced reticulocyte counts, and reversal of splenomegaly.

Compositions and methods for enhancing targeted gene editing and methods of use thereof are disclosed. In the most preferred embodiments, gene editing is carried out utilizing a gene editing composition such as triplex-forming oligonucleotides, CRISPR, zinc finger nucleases, TALENS, or others, in combination with a gene modification potentiating agent such as SCF, a CHK1 or ATR inhibitor, a DNA polymerase alpha inhibitor, a heat shock protein 90 inhibitor (HSP90i) or a combination thereof. A particularly preferred gene editing composition is triplex-forming peptide nucleic acids (PNAs) substituted at the Î³ position for increased DNA binding affinity. Nanoparticle compositions for intracellular delivery of the gene editing composition are also provided and particularly advantageous for use with in vivo applications.

For example, 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 selected from the group consisting of tyrosine kinase C-kit ligands, ATR-Chk1 cell cycle checkpoint pathway inhibitors, DNA polymerase alpha inhibitors, and heat shock protein 90 inhibitors (HSP90i), and (ii) a gene editing technology that can induce genomic modification of the cell selected from the group consisting of triplex forming molecules, pseudocomplementary oligonucleotides, a CRISPR system, zinc finger nucleases (ZFN), and transcription activator-like effector nucleases (TALEN); wherein genomic modification occurs at a higher frequency in a population of cells contacted with both (i) and (ii), then in an equivalent population contacted with (ii) in the absence of (i). 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.

A preferred C-kit ligand is a stem cell factor protein or fragment thereof sufficient to cause dimerization of C-kit and activate its tyrosine kinase activity. In some embodiments, the C-kit ligand is a nucleic acid such as an mRNA or an expression vector encoding a stem cell factor protein or fragment thereof sufficient to cause dimerization of C-kit and activate its tyrosine kinase activity.

ATR-Chk1 cell cycle checkpoint pathway inhibitors are typically small molecules though they can also be inhibitory nucleic acids such as siRNA that target and reduce expression a gene in the pathway. Inhibitors include, for example, AZD7762, SCH900776/MK-8776, IC83/LY2603618, LY2606368, GDC-0425, PF-00477736, XL844, CEP-3891, SAR-020106, CCT-244747, Arry-575, SB218075, Schisandrin B, NU6027, NVP-BEZ235, VE-821, VE-822 (VX-970), AZ20, AZD6738, MIRIN, KU5593, VE-821, NU7441, LCA, and L189.

In some embodiments, the cell's genome has a mutation underlying a disease or disorder, for example a genetic disorder such as hemophilia, globinopathies, cystic fibrosis, xeroderma pigmentosum, muscular dystrophy, and lysosomal storage diseases. 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.

The contacting of the compositions with the cell can occur ex vivo. In some embodiments, the ex vivo-treated cells are hematopoietic stem cells. The modified cells can be administered to a subject in need thereof in an effective amount to treat one or more symptoms of a disease or disorder such as hemophilia, a globinopathy, cystic fibrosis, xeroderma pigmentosum, muscular dystrophy, a lysosomal storage disease, or HIV.

In vivo applications are also provided. For example in some embodiments, the potentiating agent, gene editing technology and optionally the donor oligonucleotide are administered to a subject in need thereof. 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, for example, hemophilia, a globinopathy, xeroderma pigmentosum, a lysosomal storage disease, or HIV 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. In preferred embodiments, the nanoparticles include poly(lactic-co-glycolic acid) (PLGA) alone or in a blend with poly(beta-amino) esters (PBAEs). In particular embodiments, the nanoparticles include a blend of PLGA and PBAE having between about 10 and about 20 percent PBAE (wt %). In preferred embodiments, the nanoparticles are prepared by double emulsion. 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, a 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. In particularly preferred embodiments, a cell penetrating peptide including the sequence GALFLGFLGAAGSTMGAWS QPKKKRKV (SEQ ID NO:12) (MPG (Synthetic chimera: SV40 Lg T. Ant.+HIV gb41 coat)) is conjugated to the surface of the nanoparticles.

Improved DNA-binding triplex forming molecules are also provided. The triplex forming molecules can be utilized in all manners of gene modification including those methods both with and without a potentiating agent. The triplex forming composition typically includes a Hoogsteen binding peptide nucleic acid (PNA) segment and a Watson-Crick binding PNA segment collectively totaling no more than about 50 nucleobases in length, wherein the two segments can bind or hybridize to a target region having a polypurine stretch in a cell's genome to induce strand invasion, displacement, and formation of a triple-stranded molecule among the two PNA segments and the polypurine stretch. The Hoogsteen binding segment binds to the target duplex by Hoogsteen binding for a length of at least five nucleobases, and the Watson-Crick binding segment typically binds to the target duplex by Watson-Crick binding for a length of least five nucleobases.

In preferred embodiments, one or more of the PNA monomers are Î³PNA. The side chain at the Î³ position of the Î³PNA monomer(s) can be, for example, the side chain of an amino acid selected from the group consisting of alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, and the derivatives thereof. In some embodiments, the side chain at the Î³ position of the Î³PNA monomer(s) is a diethylene glycol (âminiPEGâ). In some embodiments, all of the peptide nucleic acid monomers in the Hoogsteen-binding portion only, all of the peptide nucleic acid monomers in the Watson-Crick-binding portion only, or all of the peptide nucleic monomers in the PNA oligomer are Î³PNA monomers. In some embodiments, alternating residues in the Hoogsteen-binding portion only, the Watson-Crick-binding portion only, or across the entire PNA are PNA and Î³PNA. Specific exemplary sequences are provided below.

In some embodiments, one or more of the cytosines is replaced with a clamp-G (9-(2-guanidinoethoxy) phenoxazine). In preferred embodiments, the Hoogsteen binding segment includes one or more chemically modified cytosines selected from the group consisting of pseudocytosine, pseudoisocytosine, and 5-methylcytosine. The Watson-Crick binding segment preferably includes a tail sequence of up to fifteen nucleobases that binds to the target duplex by Watson-Crick binding outside of the triplex. In preferred embodiments, the two segments are linked by a linker, for example, between 1 and 10 units of 8-amino-3,6-dioxaoctanoic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic showing a strategy for targeted correction of a Î²-globin gene IVS2-654 (CâT) mutation in Î²-globin/GFP transgenic mice using triplex-forming tail clamp PNAs (tcPNAs) and donor DNAs. FIG. 1B is an illustration showing tcPNA and Î³tcPNA oligomers (SEQ ID NOS:33-35, 162, and 158, respectively) designed to bind to the homopurine regions within intron 2 of the human Î²-globin gene in the vicinity of the thalassemia-associated mutation IVS2-654 (CâT), and a scrambled control sequence (SEQ ID NO:158). FIG. 1C is the chemical structures of DNA, unmodified PNA and miniPEG gamma PNA ( MP Î³PNA) units. FIG. 1D is a bar graph showing gene correction of the IVS2-654 (CâT) mutation within the Î²-globin/GFP fusion gene in mouse bone marrow cells treated ex vivo with blank NPs and NPs containing donor DNA (SEQ ID NO:65) alone or in combination with tcPNA3 (SEQ ID NO:35), tcPNA2 (SEQ ID NO:34), or tcPNA1 (SEQ ID NO:33). The % GFP+ cells among mouse bone marrow cells was determined by flow cytometry and indicates successful gene editing. Data are shown as meanÂ±s.e., n=3; statistical analysis was performed with student's t-test, asterisk, p<0.05. FIG. 1E is a line graph showing release of total nucleic acids (PNAs in combination with donor DNA (SEQ ID NO:65): Î³tcPNA4 (SEQ ID NO:162), tcPNA1 (SEQ ID NO:33), tcPNA2 (SEQ ID NO:34), tcPNA3 (SEQ ID NO:35) or Î³tcPNA4-Scr (SEQ ID NO:158); or DNA donor (SEQ ID NO:65) alone) from PLGA nanoparticles during incubation at 37Â° C. in PBS. At 64 hrs, the residual nucleic acid in the NP pellet was extracted and the total nucleic acid load was calculated as a sum of absorbance obtained from the pellet and supernatant. FIG. 1F is a bar graph showing % GFP+ cells determined by flow cytometry among mouse bone marrow cells (from Î²-globin/GFP transgenic mice) after ex vivo treatment with PLGA NPs containing tcPNA1 (SEQ ID NO:33), Î³tcPNA4 (SEQ ID NO:162), or Î³tcPNA4-Scr (SEQ ID NO:158) plus donor DNAs (SEQ ID NO:65). Replicates and statistics as above for FIG. 1D . FIG. 1G is a bar graph showing mouse total bone marrow cells were treated with either blank NPs or NPs containing Î³tcPNA4 (SEQ ID NO:162) an

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Ser. No. 62/295,789 filed Feb. 16, 2016 and which is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

REFERENCE TO SEQUENCE LISTING

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Nonetheless, compositions and methods for improved gene editing are needed.

It is an object of the invention to provide potentiating agents that increase gene modification induced or enhanced by gene editing technology.

It is another object of the invention to provide triplex forming molecules with enhanced DNA binding.

It is a further object of the invention to provide gene modification formulations that achieve therapeutically significant target site modification with reduced low off-target modification.

SUMMARY OF THE INVENTION

Compositions and methods for enhancing targeted gene editing and methods of use thereof are disclosed. In the most preferred embodiments, gene editing is carried out utilizing a gene editing composition such as triplex-forming oligonucleotides, CRISPR, zinc finger nucleases, TALENS, or others, in combination with a gene modification potentiating agent such as SCF, a CHK1 or ATR inhibitor, a DNA polymerase alpha inhibitor, a heat shock protein 90 inhibitor (HSP90i) or a combination thereof. A particularly preferred gene editing composition is triplex-forming peptide nucleic acids (PNAs) substituted at the Î³ position for increased DNA binding affinity. Nanoparticle compositions for intracellular delivery of the gene editing composition are also provided and particularly advantageous for use with in vivo applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 2A is a bar graph showing % GFP expression in treated mouse bone marrow cells based on selected hematopoietic cell surface markers. Total bone marrow was treated with NPs containing either tcPNA1/donor DNA (SEQ ID NOS:33 and 65) or Î³tcPNA4/donor DNA (SEQ ID NOS:162 and 65), and then the cells were stained using antibodies specific for the indicated markers and assayed by flow cytometry for marker and GFP expression. Data are shown as meanÂ±s.e., n=3; statistical analysis was performed with student's t-test, asterisk, p<0.05. FIG. 2B is a bar graph showing % GFP expressing CD117 (c-Kit+) cells after ex vivo treatment with NPs carrying Î³tcPNAs and donor DNAs (SEQ ID NOS:162 and 65) versus with blank NPs. Data are shown as meanÂ±s.e., n=3; statistical analysis was performed with student's t-test, asterisk, p<0.05. FIG. 2C is a bar graph showing % GFP expressing CD117+ cells from Î²-globin/GFP transgenic mice after ex vivo treatment with NPs containing Î³tcPNA4/donor DNA (SEQ ID NOS:162 and 65) with or without prior treatment with the c-Kit ligand, SCF. Data are shown as meanÂ±s.e., n=3; statistical analysis was performed with student's t-test, asterisk, p<0.05. FIG. 2D is a bar graph showing % GFP expressing CD117+ cells isolated from Î²-globin/GFP transgenic mice after ex vivo treatment with NPs containing Î³tcPNA4/donor DNA (SEQ ID NOS:162 and 65) in the presence or absence of selected c-Kit pathway kinase inhibitors: dasatinib (inhibits c-Kit), MEK162 (inhibits mitogen/extracellular signal-regulated kinase, MEK) and BKM120 (inhibits phosphatidylinositol-3-kinase, PI3K). Data are shown as meanÂ±s.e., n=3; statistical analysis was performed with student's t-test, asterisk, p<0.05. FIGS. 2E and 2F are bar graphs showing qPCR determination of mRNA expression levels of BRCA2 (2E) and Rad51 (2I) in CD117- and CD117+ cells. FIG. 2G is a heat map showing up-regulated genes involved in DNA repair pathways in CD117+ cells with or without treatment with SCF; rows are clustered by Euclidean distance measure. FIG. 2H is a bar graph showing the results of a gene assay for homology-dependent repair (HDR) activity in the presence or absence of selected c-Kit pathway kinase inhibitors: dasatinib (inhibits c-Kit), MEK162 (inhibits mitogen/extracellular signal-regulated kinase, MEK) and BKM120 (inhibits phosphatidylinositol-3-kinase, PI3K). Inset shows a diagram of the luciferase reporter gene assay for repair of a nuclease-induced double-strand break by homology-dependent repair (HDR). Luciferase expression occurs only after homologous recombination and is scored as % reactivation of the DSB-damaged plasmid, normalized to a transfection control. FIG. 2I is a bar graph showing the results of an HDR assay in CD117+ cells with or without the addition of SCF. Data are shown as meanÂ±s.e., n=3; statistical analysis was performed with student's t-test, asterisk, p<0.05. FIG. 2J is a bar graph showing the results of an HDR assay in DLD-1 cells either proficient or deficient in the homology dependent repair factor BRCA2 as a validation of the assay. Data are shown as meanÂ±s.e., n=3; statistical analysis was performed with student's t-test, asterisk, p<0.05.

FIGS. 3A and 3B are dot plots showing frequencies of gene editing (GFP expression) in bone marrow ( 3 A) and spleen ( 3 B) cells from Î²-globin/GFP transgenic mice (6 mice per group) injected or not (as indicated) with 15.6 Î¼g of SCF i.p. followed by a single treatment of 4 mg of NPs injected intravenously. Each group received either blank NPs or NPs containing Î³tcPNA4 and donor DNA (SEQ ID NOS:162 and 65), with or without SCF and were harvested and analysed two days later. Each data point represents analysis of cells from a single mouse. Statistical analyses were performed using student's t-test: asterisk, p<0.05. FIG. 3C is a bar graph showing the results of deep-sequencing analysis to quantify the frequency of targeted gene editing (% modification frequency IVS2-654 (TâC)) in vivo in CD117+ cells from bone marrow and spleen of Î²-globin/GFP mice treated as described for FIGS. 3A and 3B . Error bars indicate standard error of proportions.

FIGS. 4A-4C are line graphs showing blood hemoglobin levels (g/dl) of thalassemic mice treated with blank NPs, SCF plus scrambled Î³tcPNA4-Scr/donor DNA (SEQ ID NOS:158 and 65) NPs, or with SCF plus Î³tcPNA4/donor DNA (SEQ ID NOS:162 and 65) NPs performed at the indicated times after treatment. Each line represents an individual mouse followed over time. FIG. 4D is a bar graph showing reticulocyte counts (% of total RBCs) calculated in blood smears from thalassemic mice treated with either blank NPs or with NPs containing Î³tcPNA4/donor DNA (SEQ ID NOS:162 and 65) plus SCF on days 0 and 36 post treatment. FIG. 4E is a bar graphs showing the % gene modification (TâC) as determined by deep-sequencing analysis of genomic DNA from bone marrow cells after treatment of thalassemic mice with either blank NPs or with NPs containing and Î³tcPNA4/donor DNA (SEQ ID NOS:162 and 65) plus SCF.

FIG. 5A is a flow diagram illustrating a GFP/beta globin gene correction assay. FIG. 5B is a bar graph showing gene correction of cells treated with nanoparticles containing tcPNA1 (SEQ ID NO:191) and donor DNA (SEQ ID NO:65) alone, or in combination with an ataxia telangiectasia and Rad3-related protein (ATR) pathway inhibitor (MIRIN, KU5593, VE-821, NU7441, LCA, or L189). FIG. 5C is a bar graph showing gene correction of cells treated with nanoparticles containing tcPNA1 (SEQ ID NO:191) and donor DNA (SEQ ID NO:65) alone, or in combination with a Checkpoint Kinase 1 inhibitor (Chk1i) (SB218075), a DNA polymerase alpha inhibitor (Aphi) (aphidicolin) or a polyADP ribose polymerase (PARPi) (AZD-2281 (olaparib)). FIG. 5D is a bar graph showing gene correction of control (blank), and cells treated with nanoparticles containing tcPNA1 (SEQ ID NO:191) and donor DNA (SEQ ID NO:76) alone, or in combination with a heat shock protein 90 inhibitor (HSP90i) (STA-9090 (ganetespib)).

FIG. 6A is an illustration of a Sickle Cell Disease mutation (GAGâGTG) in the human beta globin gene, relative to the ATG transcriptional start site and exemplary tcPNAs. FIG. 6B shows the sequences of exemplary PNAs: tcPNA1: lys-lys-lys-JJTJTTJ-OOO-CTTCTCCAAAGGAGT-lys-lys-lys (SEQ ID NO:66); tcPNA2: lys-lys-lys-TTJJTJT-OOO-TCTCCTTAAACCTGT-lys-lys-lys (SEQ ID NO:67); and tcPNA3: lys-lys-lys-TJTJTTJT-OOO-TCTTCTCTGTCTCCAC-lys-lys-lys (SEQ ID NO:68). FIG. 6C shows the sequence of a DNA donor (SEQ ID NO:64).

FIG. 7A is a bar graph showing the results of a MQAE (N-(Ethoxycarbonylmethyl)-6-Methoxyquinolinium Bromide) assay (delta(AFU)/(delta(Time (sec)) measuring chloride flux for negative control CFBE cells; CFBE cells treated with blank nanoparticles, PNA2: lys-lys-lys-TJTJJTTT-OOO-TTTCCTCTATGGGTAAG-lys-lys-lys (SEQ ID NO:93)-loaded nanoparticles, PNA2 (SEQ ID NO:93)-loaded nanoparticles with an MPG peptide, Î³PNA2 lys-lys-lys- T J T JJ T T T -OOO-TTTCCTCTATGGGTAAG-lys-lys-lys (SEQ ID NO:69)-loaded nanoparticles; and untreated positive control wildtype 16HBE14o-cells. FIG. 7B is a dot pot showing nasal potential difference (NPD) (pretreatment, after treatment with Î³PNA2 (SEQ ID NO:69)-loaded nanoparticles, and after treatment with blank nanoparticles) measured using a non-invasive assay used to detect chloride potential differences in vivo.

FIG. 8A is an illustration of a mutation (GâA) in the CFTR gene (W1282X) relative to three exemplary tcPNAs. FIG. 8B provides the sequences of the tcPNAs: CF-1236 lys-lys-lys-JTTJJTJTTT-OOO-TTTCTCCTTCAGTGTTCA-lys-lys-lys (SEQ ID NO:169), CF-1314 lys-lys-lys-TTTTJJT-OOO-TCCTTTTGCTCACCTGTGGT-lys-lys-lys (SEQ ID NO:170), and CF-1329: lys-lys-lys-TJTTTTTTJJ-OOO-CCTTTTTTCTGGCTAAGT-lys-lys-lys (SEQ ID NO:171). FIG. 8C provides the sequence of an exemplary donor DNA: T(s)C(s)T(s)TGGGATTCAATAAC C TTGCA G ACAGTGGAGGAAGGCCTT TGG C GTGATACCACAGG-(s)T(s)G(s) (SEQ ID NO:109).

FIG. 9A is an illustration of a mutation (GâT) in the CFTR gene (G542X) relative to three exemplary tcPNAs. FIG. 9B provides the sequences of the tcPNAs: CF-302 lys-lys-lys-TJTTTTT-OOO-TTTTTCTGTAATTTTTAA-lys-lys-lys (SEQ ID NO:172), CF-529 lys-lys-lys-TJTJTTTJT-OOO-TCTTTCTCTGCAAACTT-lys-lys-lys (SEQ ID NO:173), and CF-586 lys-lys-lys-TTTJTTT-OOO-TTTCTTTAAGAACGAGCA-lys-lys-lys (SEQ ID NO:174). FIG. 9C provides the sequence of an exemplary donor DNA: T(s)C(s)C(s)-AAGTTTGCAGAGAAAGA T AATATAGT C CTT G GAGAAGG A GGAATCA C C CTGAGTGGA-G(s)G(s)T(s) (SEQ ID NO:124).

FIG. 10A is an illustration of Strategy for targeted correction of a Î²-globin gene containing SCD mutation (AâT) mutation and tcPNAs designed to bind to homopurine regions near the mutation. FIGS. 10B-10C are bar graphs showing hydrodynamic diameter of formulated PLGA nanoparticles measured using dynamic light scattering in PBS buffer ( FIG. 10B ) and zeta potential of formulated PLGA nanoparticles ( FIG. 10C ). Data in both graphs are presented as meanÂ±s.e.m., n=3. FIGS. 10D-10E are bar graphs showing the results of deep-sequencing analysis to quantify the frequency of targeted gene editing in vivo in bone marrow cells of Berkley âBerkâ mice ( FIG. 10D ) and Townes mice ( FIG. 10E ). Error bars indicate standard error of proportions.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein, âaffinity tagsâ are defined herein as molecular species which form highly specific, non-covalent, physiochemical interactions with defined binding partners. Affinity tags which form highly specific, non-covalent, physiochemical interactions with one another are defined herein as âcomplementaryâ.

As used herein, âcoupling agentsâ are defined herein as molecular entities which associate with polymeric nanoparticles and provide substrates that facilitate the modular assembly and disassembly of functional elements onto the nanoparticle. Coupling agents can be conjugated to affinity tags. Affinity tags allow for flexible assembly and disassembly of functional elements which are conjugated to affinity tags that form highly specific, noncovalent, physiochemical interactions with affinity tags conjugated to adaptor elements. Coupling agents can also be covalently coupled to functional elements in the absence of affinity tags.

As used herein, the term âisolatedâ describes a compound of interest (e.g., either a polynucleotide or a polypeptide) that is in an environment different from that in which the compound naturally occurs, e.g., separated from its natural milieu such as by concentrating a peptide to a concentration at which it is not found in nature. âIsolatedâ is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified.

As used herein with respect to nucleic acids, the term âisolatedâ includes any non-naturally-occurring nucleic acid sequence, since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.

As used herein, the term âhost cellâ refers to prokaryotic and eukaryotic cells into which a nucleic acid can be introduced.

As used herein, âtransformedâ and âtransfectedâ encompass the introduction of a nucleic acid into a cell by one of a number of techniques known in the art.

As used herein, the phrase that a molecule âspecifically bindsâ to a target refers to a binding reaction which is determinative of the presence of the molecule in the presence of a heterogeneous population of other biologics. Thus, under designated immunoassay conditions, a specified molecule binds preferentially to a particular target and does not bind in a significant amount to other biologics present in the sample. 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. Specific binding between two entities means an affinity of at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M â1 . Affinities greater than 10 8 M â1 are preferred.

As used herein, âtargeting moleculeâ 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 terms âantibodyâ or âimmunoglobulinâ are used to include intact antibodies and binding fragments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to an antigen fragment including separate heavy chains, light chains Fab, Fabâ² F(abâ²)2, Fabc, and Fv. Fragments are produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins. The term âantibodyâ also includes one or more immunoglobulin chains that are chemically conjugated to, or expressed as, fusion proteins with other proteins. The term âantibodyâ also includes a bispecific antibody. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fabâ² fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny, et al., J. Immunol., 148, 1547-1553 (1992). As used herein, the terms âepitopeâ or âantigenic determinantâ refer to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10, amino acids, in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen. T-cells recognize continuous epitopes of about nine amino acids for CD8 cells or about 13-15 amino acids for CD4 cells. T cells that recognize the epitope can be identified by in vitro assays that measure antigen-dependent proliferation, as determined by 3 H-thymidine incorporation by primed T cells in response to an epitope (Burke, et al., J. Inf. Dis., 170:1110-19 (1994)), by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges, et al., J. Immunol., 156, 3901-3910) or by cytokine secretion.

As used herein, the term âsmall molecule,â 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.

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.

As used herein, the term âpharmaceutically acceptableâ means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.

As used herein, the terms âeffective amountâ or âtherapeutically effective amountâ means a dosage sufficient to alleviate one or more symptoms of a disorder, disease, or condition being treated, or to otherwise provide a desired pharmacologic and/or physiologic effect. 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 âpreventionâ or âpreventingâ means to administer a composition to a subject or a system at risk for or having a predisposition for one or more symptom caused by a disease or disorder to cause cessation of a particular symptom of the disease or disorder, a reduction or prevention of one or more symptoms of the disease or disorder, a reduction in the severity of the disease or disorder, the complete ablation of the disease or disorder, stabilization or delay of the development or progression of the disease or disorder.

II. Gene Editing Potentiating Factors

It has been discovered that certain potentiating factors can be used to increase the efficacy of gene editing technologies. Gene expression profiling on SCF-treated CD117+ cells versus untreated CD117+ cells discussed in the Examples below showed additional up-regulation of numerous DNA repair genes including RAD51 and BRCA2. These results and others discussed below indicate that a functional c-Kit signaling pathway mediates increased HDR and promotes gene editing, rather than CD117 simply being a phenotypic marker. When CD117+ cells were treated with SCF, expression of these DNA repair genes was increased even more, correlating with a further increase in gene editing.

Accordingly, compositions and methods of increasing the efficacy of gene editing technology are provided. As used herein a âgene editing potentiating factorâ or âgene editing potentiating agentâ or âpotentiating factor or âpotentiating agentâ refers 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. Preferred gene editing technologies suitable for use alone or more preferably in combination with the disclosed potentiating factors are discussed in more detail below. In certain preferred embodiments, the gene editing technology is a triplex-forming Î³PNA and donor DNA, optionally, but preferably in a nanoparticle composition.

Potentiating factors include, for example, DNA damage or repair-stimulating or -potentiating factors. Preferably the factor is one that engages one or more endogenous high fidelity DNA repair pathways. In some embodiments, the factor is one that increases expression of Rad51, BRCA2, or a combination thereof.

As discussed in more detail below, the preferred methods typically include contacting cells with an effective amount of a gene editing potentiating factor. The contacting can occur ex vivo, for example isolated cells, or in vivo following, for example, administration of the potentiating factor to a subject.

A. C-Kit Ligands

In some embodiments, the factor is an activator of the receptor tyrosine kinase c-Kit. CD117 (also known as mast/stem cell growth factor receptor or proto-oncogene c-Kit protein) is a receptor tyrosine kinase expressed on the surface of hematopoietic stem and progenitor cells as well as other cell types. Stem cell factor (SCF), the ligand for c-Kit, causes dimerization of the receptor and activates its tyrosine kinase activity to trigger downstream signaling pathways that can impact survival, proliferation, and differentiation. SCF and c-Kit are reviewed in Lennartsson and Ronnstrand, Physiological Reviews, 92(4):1619-1649 (2012)).

The human SCF gene encodes for a 273 amino acid transmembrane protein, which contains a 25 amino acid N-terminal signal sequence, a 189 amino acid extracellular domain, a 23 amino acid transmembrane domain, and a 36 amino acid cytoplasmic domain. A canonical human SCF amino acid sequence is:

(SEQ ID NO: 1, UniProtKB-P21583 (SCF_HUMAN))

MKKTQTWILTCIYLQLLLFNPLVKT EGICRNRVTNNNKDVTKLVANLPK

DYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNY

SIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSI

DAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVA ASSLRNDSS

SSNRKAKNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKR

QPSLTRAVENIQINEEDNEISMLQEKEREFQEV.

The secreted soluble form of SCF is generated by proteolytic processing of the membrane-anchored precursor. A cleaved, secreted soluble form of human SCF is underlined in SEQ ID NO:1, which corresponds to SEQ ID NO:2 without the N-terminal methionine.

MEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISE

MVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKD

LKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPE

KDSRVSVTKPFMLPPVA (SEQ ID NO: 2, Preprotech

Recombinant Human SCF Catalog Number: 300-07).

Murine and rat SCF are fully active on human cells. A canonical mouse SCF amino acid sequence is:

(SEQ ID NO: 3, UniProtKB-P20826 (SCF_MOUSE))

MKKTQTWIITCIYLQLLLENPLVKT KEICGNPVTDNVKDITKLVANLPND

YMITLNYVAGMDVLPSHCWLRDMVIQLSLSLTTLLDKFSNISEGLSNYSI

IDKLGKIVDDLVLCMEENAPKNIKESPKRPETRSFTPEEFFSIFNRSIDA

FKDFMVASDTSDCVLSSTLGPEKDSRVSVTKPFMLPPVA ASSLRNDSSSS

NRKAAKAPEDSGLQWTAMALPALISLVIGFAFGALYWKKKQSSLTRAVEN

IQINEEDNEISMLQQKEREFQEV.

A cleaved, secreted soluble form of mouse SCF is underlined in SEQ ID NO:3, which corresponds to SEQ ID NO:4 without the N-terminal methionine.

MKEICGNPVTDNVKDITKLVANLPNDYMITLNYVAGMDVLPSHCWLRD

MVIQLSLSLTTLLDKFSNISEGLSNYSIIDKLGKIVDDLVLCMEENAPKN

IKESPKRPETRSFTPEEFFSIFNRSIDAFKDFMVASDTSDCVLSSTLGPE

KDSRVSVTKPFMLPPVA (SEQ ID NO: 4, Preprotech

Recombinant Murine SCF Catalog Number: 250-03)

A canonical mouse SCF amino acid sequence is:

(SEQ ID NO: 5, UniProtKB-P21581 (SCF_RAT))

MKKTQTWIITCIYLQLLLFNPLVKT QEICRNPVTDNVKDITKLVANLPND

YMITLNYVAGMDVLPSHCWLRDMVTHLSVSLTTLLDKFSNISEGLSNYS

IIDKLGKIVDDLVACMEENAPKNVKESLKKPETRNFTPEEFFSIFNRSID

AFKDFMVASDTSDCVLSSTLGPEKDSRVSVTKPFMLPPVA ASSLRNDSSS

SNRKAAKSPEDPGLQWTAMALPALISLVIGFAFGALYWKKKQSSLTRAV

ENIQINEEDNEISMLQQKEREFQEV.

A cleaved, secreted soluble form of rat SCF is underlined in SEQ ID NO:5, which corresponds to SEQ ID NO:6 without the N-terminal methionine.

MQEICRNPVTDNVKDITKLVANLPNDYMITLNYVAGMDVLPSHCWLRD

MVTHLSVSLTTLLDKFSNISEGLSNYSIIDKLGKIVDDLVACMEENAPKN

VKESLKKPETRNFTPEEFFSIFNRSIDAFKDFMVASDTSDCVLSSTLGPE

KDSRVSVTKPFMLPPVA (SEQ ID NO: 6, Shenandoah

Biotechnology, Inc., Recombinant Rat SCF (Stem

Cell Factor) Catalog Number: 300-32).

In some embodiments, the factor is a SCF such as any of SEQ ID NO:1-6, with or without the N-terminal methionine, or a functional fragment thereof, or a variant thereof with at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more sequence identity to any one of SEQ ID NO:1-6.

It will be appreciated that SCF can be administered to cells or a subject as SCF protein, or as a nucleic acid encoding SCF (transcribed RNA, DNA, DNA in an expression vector). Accordingly, nucleic acid sequences, including RNA (e.g., mRNA) and DNA sequences, encoding SEQ ID NOS:1-6 are also provided, both alone and inserted into expression cassettes and vectors. For example, a sequence encoding SCF can be incorporated into an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote.

The observed effect of SCF indicates that other cytokines or growth factors including, but not limited to, erythropoietin, GM-CSF, EGF (especially for epithelial cells; lung epithelia for cystic fibrosis), hepatocyte growth factor etc., could similarly serve to boost gene editing potential in bone marrow cells or in other tissues. In some embodiments, gene editing is enhanced in specific cell types using cytokines targeted to these cell types.

B. Replication Modulators

In some embodiments, the potentiating factor is a replication modulator that can, for example, manipulate replication progression and/or replication forks. For example, the ATR-Chk1 cell cycle checkpoint pathway has numerous roles in protecting cells from DNA damage and stalled replication, one of the most prominent being control of the cell cycle and prevention of premature entry into mitosis (Thompson and Eastman, Br J Clin Pharmacol., 76(3): 358-369 (2013), Smith, et al., Adv Cancer Res., 108:73-112 (2010)). However, Chk1 also contributes to the stabilization of stalled replication forks, the control of replication origin firing and replication fork progression, and homologous recombination. DNA polymerase alpha also known as Pol Î± is an enzyme com

CLAIMS

Claims ( 30 )

We claim:

1. A method of modifying the genomes of CD117+ cells comprising contacting the CD117+ cells with an effective amount of

(i) a gene editing potentiating agent selected from the group consisting of receptor tyrosine kinase C-kit ligands, ATR-Chk1 cell cycle checkpoint pathway inhibitors, and heat shock protein 90 inhibitors (HSP90i), and

(ii) a gene editing technology that can induce genomic modification through a mechanism comprising a DNA repair pathway endogenous to the CD117+ cells,

to modify the genomes of the CD117+ cells contacted with both (i) and (ii) at a higher frequency than an equivalent population of cells contacted with (ii) in the absence of (i).

2. The method of claim 1 further comprising contacting the cells with a donor oligonucleotide comprising a sequence that corrects a mutation(s) in the cells' genomes by insertion or recombination induced or enhanced by the gene editing technology.

3. The method of claim 2 , wherein the cells' genomes have a mutation underlying a disease or disorder.

4. The method of claim 3 , wherein the disease is a globinopathy.

5. The method of claim 3 , wherein the disease is a lysosomal storage disease.

6. The method of claim 1 further comprising contacting the cells with a donor oligonucleotide comprising a sequence that induces a mutation(s) in the cells' genomes by insertion or recombination induced or enhanced by the gene editing technology.

7. The method of claim 2 , wherein the cells are hematopoietic stem cells.

8. The method of claim 2 , wherein the contacting occurs in vivo following administration of (i), (ii), and the donor oligonucleotide to a subject in need thereof.

9. The method of claim 8 , wherein the subject has a disease or disorder.

10. The method of claim 8 , wherein (i), (ii), the donor oligonucleotide or a combination thereof are packaged together or separately in nanoparticles.

11. The method of claim 10 , wherein the nanoparticles comprise poly(lactic-co-glycolic acid) (PLGA).

12. The method of claim 1 , wherein the gene editing potentiating agent is a receptor tyrosine kinase C-kit ligand.

13. A method of modifying the genomes of CD117+ cells comprising contacting the CD117+ cells with an effective amount of

(ii) a triplex forming composition comprising a peptide nucleic acid (PNA), wherein one or more of the PNA monomers is a Î³PNA,

to modify the genomes of the cells contacted with both (i) and (ii) at a higher frequency than an equivalent population of cells contacted with (ii) in the absence of (i).

14. An isolated population of cells treated according to the method of claim 2 .

15. The method of claim 1 , wherein the gene editing technology is a triplex forming molecule.

16. The method of claim 15 , wherein the triplex forming molecule comprises a peptide nucleic acid (PNA) comprising:

(a) a Hoogsteen binding PNA segment;

(b) a Watson-Crick binding PNA segment; and

17. The method of claim 16 , wherein (a) and (b) are linked by a linker.

18. The method of claim 16 , wherein the PNA comprises a polyethylene glycol moiety.

19. The method of claim 16 , wherein the triplex forming molecule is a tail-clamp PNA.

20. The method of claim 16 , wherein the Hoogsteen binding segment comprises a chemically modified cytosine.

21. The method of claim 15 , wherein the cells comprise a genome encoding a human beta-globin gene,

the triplex forming molecule forms a triplex at the cells' genomic beta-globin locus and,

the triplex forming molecule comprises a Hoogsteen binding peptide nucleic acid (PNA) segment and a Watson-Crick binding PNA segment, wherein

(i) the Hoogsteen binding segment comprises the sequence JTTTJTTTJTJT (SEQ ID NO:30) and the Watson-Crick binding segment comprises the sequence TCTCTTTCTTTC (SEQ ID NO:22) or TCTCTTTCTTTCAGGGCA (SEQ ID NO:23);

(ii) the Hoogsteen binding segment comprises the sequence TTTTJJJ (SEQ ID NO:31) and the Watson-Crick binding segment comprises the sequence CCCTTTT (SEQ ID NO:25) or CCCTTTTGCTAATCATGT (SEQ ID NO:26);

(iii) the Hoogsteen binding segment comprises the sequence TTTJTJJ (SEQ ID NO:32) and the Watson-Crick binding segment comprises the sequence CCTCTTT (SEQ ID NO:28) or CCTCTTTGCACCATTCT (SEQ ID NO:29);

(iv) the Hoogsteen binding segment comprises the sequence TJTTTTJTTJ (SEQ ID NO:36) and the Watson-Crick binding segment comprises the sequence CTTCTTTTCT (SEQ ID NO:37);

(v) the Hoogsteen binding segment comprises the sequence TTJTTJTTTJ (SEQ ID NO:38) and the Watson-Crick binding segment comprises the sequence CTTTCTTCTT (SEQ ID NO:39);

(vi) the Hoogsteen binding segment comprises the sequence JJJTJJTTJT (SEQ ID NO:40) and the Watson-Crick binding segment comprises TCTTCCTCCC (SEQ ID NO:41);

(vii) the Hoogsteen binding segment comprises the sequence JJTJTTJ (SEQ ID NO:56) and the Watson-Crick binding segment comprises the sequence CTTCTCC (SEQ ID NO:46) or CTTCTCCAAAGGAGT (SEQ ID NO:47) or CTTCTCCACAGGAGTCAG (SEQ ID NO:48) or CTTCTCCACAGGAGTCAGGTGC (SEQ ID NO:205);

(viii) the Hoogsteen binding segment comprises the sequence TTJJTJT (SEQ ID NO:214) and the Watson-Crick binding segment comprises the sequence TCTCCTT (SEQ ID NO:50) or TCTCCTTAAACCTGT (SEQ ID NO:51) or TCTCCTTAAACCTGTCTT (SEQ ID NO:212); or

(ix) the Hoogsteen binding segment comprises the sequence TJTJTTJT (SEQ ID NO:215) and the Watson-Crick binding segment comprises the sequence TCTTCTCT (SEQ ID NO:53) or TCTTCTCTGTCTCCAC (SEQ ID NO:54) or TCTTCTCTGTCTCCACAT (SEQ ID NO:55);

wherein âJâ is pseudoisocytosine, and

wherein the two segments are optionally linked by a linker.

22. The method of 21 , wherein the triplex forming molecule comprises a sequence selected from:

(i)

(SEQ ID NO: 33)

lys-lys-lys-JTTTJTTTJTJT-OOO-T C T C T T T C T T T C A G G G C A -

lys-lys-lys;

(ii)

(SEQ ID NO: 34)

lys-lys-lys-TTTTJJJ-OOO-C C C T T T T G C T A A T C A T G T -lys-

lys-lys;

(iii)

(SEQ ID NO: 35)

lys-lys-lys-TTTJTJJ-OOO-C C T C T T T G C A C C A T T C T-lys-

lys-lys,

(iv)

(SEQ ID NO: 42)

lys-lys-lys-TJTTTTJTTJ-OOO-C T T C T T T T C T -lys-lys-lys

(IVS2-24);

(v)

(SEQ ID NO: 43)

lys-lys-lys-TTJTTJTTTJ-OOO-C T T T C T T C T T -lys-lys-lys

(IVS2-512);

(vi)

(SEQ ID NO: 44)

lys-lys-lys-JJJTJJTTJT-OOO-T C T T C C T C C C -lys-lys-lys

(IVS2-830);

(vii)

(SEQ ID NO: 160)

lys-lys-lys-JJTJTTJ-OOO-C T T C T C C A A A G G A G T-lys-lys-

lys;

(viii)

(SEQ ID NO: 57)

lys-lys-lys-TTJJTJT-OOO-T C T C C T T A A A C C T G T-lys-lys-

lys;

(ix)

(SEQ ID NO: 213)

lys-lys-lys-TTJJTJT-OOO-T C T C C T T A A A C C T G T C T T -lys-

lys-lys

(x)

(SEQ ID NO: 58)

lys-lys-lys-TJTJTTJT-OOO-T C T T C T C T G T C T C C A C -lys-

lys-lys (tc816);

(xi)

(SEQ ID NO: 59)

lys-lys-lys-JJTJTTJ-OOO-C T T C T C C A C A G G A G T C A G -lys-

lys-lys;

(xii)

(SEQ ID NO: 59)

lys-lys-lys-JJTJTTJ-OOO- C T T C T C C A C A G G A G T C A G-lys-

lys-lys (SCD-tcPNA 1A);

(xiii)

(SEQ ID NO: 59)

lys-lys-lys-JJTJTTJ-OOO- CTTCTCCACAGGAGTCAG -lys-

lys-lys (SCD-tcPNA 1B);

(xiv)

(SEQ ID NO: 59)

lys-lys-lys-JJ T J TT J-OOO- CTTCTCCACAGGAGTCAG -lys-

lys-lys (SCD-tcPNA 1C);

(xv)

(SEQ ID NO: 209)

lys-lys-lys-JJTJTTJ-OOO- C T T C T C C A C A G G A G T C A G G T G C-

lys-lys-lys (SCD-tcPNA 1D);

(xvi)

(SEQ ID NO: 209)

lys-lys-lys-JJTJTTJ-OOO- CTTCTCCACAGGAGTCAGGTGC -

lys-lys-lys (SCD-tcPNA 1E);

(xvii)

(SEQ ID NO: 209)

lys-lys-lys-JJ T J TT J-OOO- CTTCTCCACAGGAGTCAGGTGC -

lys-lys-lys (SCD-tcPNA 1F);

(xviii)

(SEQ ID NO: 60)

lys-lys-lys-TJTJTTJT-OOO-T C T T C T C T G T C T C C A C A T -lys-

lys-lys;

wherein each âlysâ is the amino acid lysine, each âJâ is pseudoisocytosine, each â0â is selected from 8-amino-3,6-dioxaoctanoic acid, 6-aminohexanoic acid, and 8-amino-2,6,10-trioxaoctanoic acid, and the bolded and underlined residues are miniPEG-containing Î³PNA residues.

23. The method of claim 15 , wherein the cells comprise a genome encoding a human cystic fibrosis transmembrane conductance regulator (CFTR) gene,

the triplex forming molecule forms a triplex at the genomic locus of the CFTR gene and

the triplex forming molecule comprises a Hoogsteen binding peptide nucleic acid (PNA) segment and a Watson-Crick binding PNA segment, wherein

(i) the Hoogsteen binding segment comprises the sequence JTTJJTJTTT (SEQ ID NO:106) and the Watson-Crick binding segment comprises the sequence TTTCTCCTTC (SEQ ID NO:98) or TTTCTCCTTCAGTGTTCA (SEQ ID NO:99);

(ii) the Hoogsteen binding segment comprises the sequence TTTTJJT (SEQ ID NO:107) and the Watson-Crick binding segment comprises the sequence TCCTTTT (SEQ ID NO:101) or TCCTTTTGCTCACCTGTGGT (SEQ ID NO:102);

(iii) the Hoogsteen binding segment comprises the sequence TJTTTTTTJJ (SEQ ID NO:108) and the Watson-Crick binding segment comprises the sequence CCTTTTTTCT (SEQ ID NO:104) or CCTTTTTTCTGGCTAAGT (SEQ ID NO:105);

(iv) the Hoogsteen binding segment comprises the sequence TJTTTTT (SEQ ID NO:118) Watson-Crick binding segment comprises the sequence TTTTTCT (SEQ ID NO:111) or TTTTTCTGTAATTTTTAA (SEQ ID NO:112);

(v) the Hoogsteen binding segment comprises the sequence TJTJTTTJT (SEQ ID NO:119) and the Watson-Crick binding segment comprises the sequence TCTTTCTCT (SEQ ID NO:114) or TCTTTCTCTGCAAACTT (SEQ ID NO:115); or

(vi) the Hoogsteen binding segment comprises the sequence TTTJTTT (SEQ ID NO:120) and the Watson-Crick binding segment comprises the sequence TTTCTTT (SEQ ID NO:116) or TTTCTTTAAGAACGAGCA (SEQ ID NO:117);

wherein âJâ is pseudoisocytosine, and

wherein the two segments are optionally linked by a linker.

24. The method of claim 23 , wherein the triplex forming molecule comprises a sequence selected from:

(i)

lys-lys-lys-JTTJJTJTTT-OOO-TTTCTCCTTCAGTGTTCA-lys-lys-lys;

(SEQâIDâNO:â155)

(ii)

lys-lys-lys-TTTTJJT-OOO-TCCTTTTGCTCACCTGTGGT-lys-lys-lys;

(SEQâIDâNO:â156)

(iii)

lys-lys-lys-TJTTTTTTJJ-OOO-CCTTTTTTCTGGCTAAGT-lys-lys-lys;

(SEQâIDâNO:â157)

(iv)

lys-lys-lys-TJTTTTT-OOO-TTTTTCTGTAATTTTTAA-lys-lys-lys;

(SEQâIDâNO:â121)

(v)

lys-lys-lys-TJTJTTTJT-OOO-TCTTTCTCTGCAAACTT-lys-lys-lys;

(SEQâIDâNO:â122)

(vi)

lys-lys-lys-TTTJTTT-OOO-TTTCTTTAAGAACGAGCA-lys-lys-lys;âand

(SEQâIDâNO:â123)

(vii)

lys-lys-lys-TJTJJTTT-OOO-TTTCCTCTATGGGTAAG-lys-lys-lys

(SEQâIDâNO:â93)

25. The method of claim 3 , wherein the disease is cystic fibrosis.

26. The method of claim 1 , wherein DNA repair pathway is the homology-dependent repair (HDR) pathway.

27. The method of claim 1 , wherein the gene editing technology comprises an enzyme that induces a single or double strand break in cells' genomes.

28. The method of claim 27 , wherein the enzyme is a Cas endonuclease, zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), or intron encoded meganuclease.

29. The method of claim 13 , further comprising contacting the cells with a donor oligonucleotide comprising a sequence that introduces or corrects a mutation(s) in the cells' genomes by insertion or recombination induced or enhanced by the triplex forming composition.

30. The method of claim 13 , wherein the gene editing potentiating agent is a receptor tyrosine kinase C-kit ligand.

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