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WO2022115878A1 - Édition génique médiée par crispr/cas de cellules souches humaines - Google Patents

Édition génique médiée par crispr/cas de cellules souches humaines Download PDF

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WO2022115878A1
WO2022115878A1 PCT/US2021/072642 US2021072642W WO2022115878A1 WO 2022115878 A1 WO2022115878 A1 WO 2022115878A1 US 2021072642 W US2021072642 W US 2021072642W WO 2022115878 A1 WO2022115878 A1 WO 2022115878A1
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cell
peptide
locus
cells
sgrna
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Ron BAIK
Daniel P. DEVER
Matthew H. PORTEUS
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • a new class of medicines through DNA editing has been revolutionized by the development and advancement of CRISPR systems (1).
  • a targeted break on both strands of DNA (a double-stranded break (DSB))
  • the lesion on DNA is repaired in one of two primary ways: ligation of the two ends together by nonhomologous end-joining (NHEJ) and microhomology-mediated end joining (MMEJ), or the homology-directed repair (HDR) pathway.
  • NHEJ and MMEJ are used in all cells to repair spontaneous breaks and can result in insertions or deletions (indels) of one to several bases at the site of the break.
  • HDR the molecular homologous recombination machinery is used and results in precise changes to the DNA.
  • HDR requires a homologous donor DNA to template the precise changes that are made in the genomic DNA (1, 2).
  • HSCs hematopoietic stem and progenitor cells
  • IPCs induced pluripotent stem cells
  • MSCs mesenchymal stromal cells
  • HSCs have the ability to repopulate an entire hematopoietic system and thus strategies aimed at developing cell-based therapies involving genome editing for various hematological diseases such as sickle cell disease, p-thalassemia, and X-linked severe combined immunodeficiency are progressing towards clinical trials.
  • adeno-associated viral vectors of serotype 6 can efficiently deliver single-stranded DNA cargos to serve as a gene-targeting donor template (3-6).
  • rAAV6 recombinant adeno-associated viral vectors of serotype 6
  • current xenograft studies support the idea that HSCs are more resistant to HDR-mediated editing, perhaps one mechanistic explanation for the observation that HDR-edited cells engraft less efficiently following transplantation in immunodeficient mice. Reductions in HDR frequency during long-term engraftment have been observed previously and therefore remains a major impediment to bringing HDR-mediated therapies to clinic (3, 7-9).
  • One of the key questions in the metabolism of DSBs is how a cell chooses to repair the break.
  • a key step is how the end is processed.
  • the NHEJ pathway is activated if proteins such as 53BP1 bind the end and the Ku70/Ku80 dimer is recruited to bind the end.
  • end resection to generate 3’ single strand tails is a key early step in activating the recombination pathway of repair and is facilitated by the protein CtIP.
  • One of the mechanisms by which 53BP1 biases repair towards NHEJ is by inhibiting binding of BRCAl, a protein required for homologous recombination (10).
  • i53 an engineered ubiquitin variant called i53 delivered by either plasmid transfection or AAV delivery could increase the frequency of Cas9 mediated HDR in human cancer cell lines.
  • i53 was shown to inhibit accumulation of 53BP1 at DSBs and was thus thought to block NHEJ and promote breaks being repaired by alternative mechanisms such as HDR (11).
  • Such an approach would face important limitations in primary cells, however, as the transfection of naked DNA plasmids into primary human cells results in the induction of a toxic Type I interferon response, and the kinetics of expression via AAV transduction might not be effective.
  • the present disclosure provides a method of genetically modifying a primary human cell, the method comprising: (i) introducing into the cell an RNA-guided nuclease and a single guide RNA (sgRNA) targeting a genetic locus of interest; (ii) introducing a homologous donor template into the cell, wherein the homologous donor template comprises a nucleotide sequence that is homologous to the locus of interest; and (iii) introducing a purified i53 peptide into the cell; wherein the sgRNA directs the RNA-guided nuclease to the locus of interest, the RNA-guided nuclease cleaves the locus at the target sequence of the sgRNA, and the homologous donor template is integrated at the site of the cleaved locus by homology directed repair (HDR).
  • HDR homology directed repair
  • the primary human cell is a cell selected from the group consisting of a CD34 + hematopoietic stem and progenitor cell (HSPC), a T cell, a mesenchymal stem cell (MSC), an airway basal stem cell, and an induced pluripotent stem cell (IPSC).
  • HSPC hematopoietic stem and progenitor cell
  • T cell hematopoietic stem and progenitor cell
  • MSC mesenchymal stem cell
  • IPC induced pluripotent stem cell
  • the locus of interest is a gene selected from the group consisting of Hemoglobin Subunit Beta (HBB), C-C Motif Chemokine Receptor 5 (CCR5), Interleukin 2 Receptor Subunit Gamma (IL2RG), Hemoglobin Subunit Alpha 1 (HBA1), or Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).
  • HBB Hemoglobin Subunit Beta
  • CCR5 C-C Motif Chemokine Receptor 5
  • IL2RG Interleukin 2 Receptor Subunit Gamma
  • HBA1 Hemoglobin Subunit Alpha 1
  • CFTR Cystic Fibrosis Transmembrane Conductance Regulator
  • the sgRNA comprises 2'-O-methyl-3'-phosphorothioate (MS) modifications at one or more nucleotides.
  • the 2'-O-methyl-3'-phosphorothioate (MS) modifications are present at the three terminal nucle
  • the sgRNA and RNA-guided nuclease are introduced into the cell as a ribonucleoprotein (RNP).
  • RNP ribonucleoprotein
  • the i53 peptide is introduced into the cell by electroporation.
  • the i53 peptide and the RNP are introduced together into the cell.
  • the level of the i53 peptide in the cell four hours after electroporation is less than 0.1% of the level in the cell immediately after electroporation.
  • the amino acid sequence of the i53 peptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO:1 or SEQ ID NO:2.
  • the i53 peptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2.
  • the i53 peptide is recombinant.
  • the homologous repair template is introduced into the cell using an adeno-associated virus serotype 6 (AAV6) vector.
  • AAV6 vector is transduced into the cell at a multiplicity of infection (MOI) of less than about 2500, 1250, or 625.
  • the MOI is about 625.
  • the concentration of the i53 peptide used for electroporation is about 1-2 mg/ml. In some embodiments, the concentration of the i53 peptide is about 1.5 mg/ml.
  • the frequency of HDR at the locus of interest in the cell is higher than the frequency in an equivalent cell in the presence of the sgRNA, RNA-guided nuclease, and homologous donor template, but in the absence of the i53 peptide. In some embodiments, the frequency of HDR at the locus of interest in the cell is at least about 10%, 20%, 30%, 40%, or more higher than the frequency in an equivalent cell in the presence of the sgRNA, RNA-guided nuclease, and homologous donor template, but in the absence of the i53 peptide.
  • the frequency of indels at the locus of interest in the cell is lower than the frequency in an equivalent cell in the presence of the sgRNA, RNA-guided nuclease, and homologous donor template, but in the absence of the i53 peptide. In some embodiments, the frequency of indels at the locus of interest in the cell is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more lower than the frequency in an equivalent cell in the presence of the sgRNA, RNA-guided nuclease, and homologous donor template, but in the absence of the i53 peptide.
  • the method further comprises introducing a second sgRNA into the cell targeting a second genetic locus, and introducing a second homologous donor template comprising a nucleotide sequence that is homologous to the second genetic locus, wherein the second sgRNA directs the RNA-guided nuclease to the second genetic locus, the RNA-guided nuclease cleaves the second genetic locus at the target sequence of the second sgRNA, and the second homologous donor template is integrated at the site of the cleaved second genetic locus by HDR.
  • the frequency of HDR is higher at both the locus of interest and at the second genetic locus in the presence of the i53 peptide than in the absence of the i53 peptide. In some embodiments, the frequency of indels is lower at both the locus of interest and at the second genetic locus in the presence of the i53 peptide than in the absence of the i53 peptide.
  • the present disclosure provides a method of treating a genetic disorder in a human subject in need thereof, the method comprising: isolating a primary cell from the subject; genetically modifying the primary cell using the method of any of the herein-described methods, wherein the integration of the homologous donor template at the locus of interest in the cell corrects a mutation at the locus or leads to the expression of a therapeutic protein in the cell that is absent or deficient in the subject; and reintroducing the genetically modified cell into the subject.
  • the genetic disorder is a disorder selected from the group consisting of p-thalassemia, sickle cell disease (SCD), severe combined immunodeficiency (SCID), mucopolysaccharidosis type 1, Gaucher disease, Cystic Fibrosis, Krabbe disease, and X-linked chronic granulomatous disease (X-CGD).
  • SCD sickle cell disease
  • SCID severe combined immunodeficiency
  • mucopolysaccharidosis type 1 Gaucher disease
  • Cystic Fibrosis Cystic Fibrosis
  • Krabbe disease X-linked chronic granulomatous disease
  • FIGS. 1A-1B Cas9-RNP and AAV6-mediated targeting in different CD34 + donors.
  • FIGS. 2A-2F Cas9-RNP and AAV6-mediated targeting of human primary stem cells using i53 recombinant peptide.
  • FIG. 2A Schematic of DNA repair pathways (NHEJ and HDR) illustrating transient inhibition of NHEJ by i53 peptide.
  • FIG. 2B CD34 + HSPCs were electroporated with Cas9-RNP and AAV6 and with or without i53 peptide. HDR- mediated outcomes were assessed by ddPCR. Data from n ⁇ 4 independent biological replicates with mean ⁇ SD graphed, unless indicated otherwise.
  • FIG. 2C Indel rates were determined PCR amplicon analyses through ICE or TIDE.
  • FIG. 2D HDR rates in airway stem cells were determined by ICE or TIDER analyses. HDR in MSCs were determined by the read out of GFP expressing cells via flow cytometry. Data from n ⁇ 3 biological replicates with meaniSD graphed.
  • FIGS. 3A-3B show the distribution of indels identified with or without i53.
  • FIGS. 4A-4G Optimizing the use of i53 peptide for targeting HBB locus in CD34 + HSPCs.
  • FIG. 4A Experimental layout for targeting CD34 + HSPCs at HBB locus using i53 peptide.
  • FIG. 4B Heatmap illustrating HDR rates in response to various doses of AAV6 and i53 peptide.
  • CD34 + HSPCs were electroporated with Cas9-RNP, 625 MOI to 5000 MOIs of AAV6 and 0 ⁇ g/ml to 1500 ⁇ g/ml of i53 peptide.
  • HDR-mediated outcomes were assessed by ddPCR Data from n ⁇ 2 independent biological replicates with mean values graphed.
  • FIG. 4C Indel rates were determined by ddPCR or ICE. Data from n ⁇ 3 independent biological replicates with meantSD graphed.
  • FIG. 4D Edited HSPCs were plated on methylcellulose and scored as CFU-E, BFU-E, CFU-GM, or CFU-GEMM based on morphology 14 days after plating. Data from n ⁇ 4 independent biological replicates with meantSD graphed. *:p ⁇ 0.05 by unpaired t-test.
  • FIG. 4E X-Y linear correlation between HDR frequency and % colonies formed on methocult.
  • FIG. 4F Allele spectra and corresponding percentages of alleles generated by ICE following editing with Cas9-RNP.
  • FIG. 4G Representative FACS plots of biallelic targeting using HBB-mCheny and HBB-GFP encoding AAV donors.
  • FIGS. 5A-5C Determining the dosage of i53 peptide for targeting HBB locus in CD34 + HSPCs.
  • FIG. 5A CD34 + HSPCs were electroporated with Cas9-RNP, 625 MOI of AAV6 and 0 ⁇ g/ml to 5000 ⁇ g/ml of i53 peptide.
  • HDR-mediated outcomes were assessed by ddPCR Data from n ⁇ 3 independent biological replicates with mean values graphed. Indel rates were determined by ddPCR or ICE. Data from n ⁇ 3 independent biological replicates with meantSD graphed.
  • FIG. 5A CD34 + HSPCs were electroporated with Cas9-RNP, 625 MOI of AAV6 and 0 ⁇ g/ml to 5000 ⁇ g/ml of i53 peptide.
  • HDR-mediated outcomes were assessed by ddPCR Data from n ⁇ 3 independent biological replicates with mean values graphed. Indel rates were determined by
  • FIGS. 6A-6G Determining the toxicity of the use of i53 peptide during genome editing in CD34 + HSPCs.
  • FIG. 5C Edited HSPCs were plated on methylcellulose and scored as CFU-E, BFU- E, CFU-GM, or CFU-GEMM based on morphology 14 days after plating. Data from n ⁇ 4 independent biological replicates with mean ⁇ SD graphed. *:p ⁇ 0.05 by impaired t-test.
  • FIGS. 6A-6G Determining the toxicity of the use of i53 peptide during genome editing in CD34 + HSPCs.
  • FIG. 6A Immunoblot showing the expression of His-i53 peptide in CD34 + cells at Ohr, Ltd, 2hrs, 3hrs, 4hrs, 24hrs and 48hrs post-electroporation. GAPDH served as a loading control.
  • FIG. 6B HSPCs were electroporated with Cas9-RNP and AAV with or without i53 peptide. Cells were collected at time points 0, 2, 4, 6, 8, 24, 48, and 72 hours post-electroporation and HDR and indel rates were analyzed by ddPCR
  • FIG. 6D and FIG. 6E Expression of p21 assessed by ddPCR.
  • FIG. 6F HSPCs were edited with indicated MOI of AAV6 and with or without i53 peptide. Expression of p21 assessed by ddPCR.
  • FIG. 6G Measuring translocation after HBB and AAVS1 di-genic targeting.
  • FIGS. 7A-7C Determining the kinetics of i53 peptide during genome editing in CD34 + HSPCs.
  • FIG. 7A Immunoblot showing the expression of His-i53 peptide in CD34 + cells at Ohr, 4hrs, 24hrs and 48hrs post-electroporation. GAPDH served as a loading control.
  • FIG. 7B Quantification of the immunoblot in FIG 7A.
  • FIG. 7C Allele spectra and corresponding percentages of alleles generated by ICE following editing with Cas9-RNP.
  • FIGS. 8A-8G HBB-gene targeted CD34 + HSPCs display improved long-term and multi-lineage reconstitution in NSG mice.
  • FIG. 8A Experimental layout.
  • FIG. 8B HBB gene editing outcomes in CD34 + HSPCs in vitro. Data from two biological donors (donors A and B).
  • FIG. 8C Human engraftment (14 weeks post-transplantation) in NSG mice from all experimental groups. Data for donor A and donor B represented in a separate panel. Median values reported.
  • FIG. 8D Percentage of human cells representing B cells (yellow circle), myeloid cells (red square) and other cells (brown triangle). Bars represent median.
  • FIG. 8A Experimental layout.
  • FIG. 8B HBB gene editing outcomes in CD34 + HSPCs in vitro. Data from two biological donors (donors A and B).
  • FIG. 8C Human engraftment (14 weeks post-transplantation) in NSG mice from all experimental groups. Data for donor A and donor
  • FIG. 8E Percentage of HDR alleles in the human cells in the bone marrow of NSG mice. Median values reported.
  • FIG. 8F Percentage of HDR alleles in the human B cells in the bone marrow of NSG mice.
  • FIG. 8G Percentage of HDR alleles in the human myeloid cells in the bone marrow of NSG mice.
  • FIG. 9 Analysis of human CD34 + HSPC engraftment in NSG mice. DETAILED DESCRIPTION
  • the present disclosure provides methods for improving the efficiency of homology directed repair (HDR)-mediated modification of genomic sequences in primary cells.
  • the methods involve the introduction into cells of single guide RNAs (sgRNAs), RNA-guided nucleases (e.g., Cas9), homologous repair templates, and recombinant i53 polypeptide.
  • sgRNAs single guide RNAs
  • Cas9 RNA-guided nucleases
  • homologous repair templates e.g., Cas9
  • recombinant i53 polypeptide e.g., to integrate cDNAs encoding functional proteins into cells to correct or compensate for mutations in cells from a subject with a genetic disorder, or to modify endogenous genomic sequences for any purpose using HDR.
  • the sgRNA and nuclease are delivered to cells as ribonucleoprotein (RNP) complexes, and both the RNPs and i53 peptide are delivered together by electroporation, followed by the transduction of the homologous repair template using an AAV6 viral vector.
  • RNP ribonucleoprotein
  • the introduction of the i53 peptide transiently increases the rate of HDR and reduces non-homologous end-joining (NHEJ) in the primary cells, and also permits the use of lower amounts of donor template (e.g., reduced MOIs when using viral vectors such as AAV6) than is possible in the absence of i53 peptide, while still achieving high levels of HDR in the cells and high levels of engrafhnent in vivo.
  • This system can be used to modify any human cell, and in particular embodiments CD34 + HSPCs are used.
  • any reference to “about X” specifically indicates at least the values X, 0.8X, 0.8 IX, 0.82X, 0.83X, 0.84X, 0.85X, 0.86X, 0.87X, 0.88X, 0.89X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, LUX, 1.12X, 1.13X, 1.14X, 1.15X, 1.16X, 1.17X, 1.18X, 1.19X, and 1.2X.
  • “about X” is intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.”
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • gene means the segment of DNA involved in producing a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
  • a “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • the promoter can be a heterologous promoter.
  • i53 or “i53 peptide” is a peptide variant of ubiquitin that can specifically inhibit 53BP1 (see, e.g., UniProt Ref. Q12888; NCBI ID Gene ID 7158). 53BP1 binds to double stranded breaks in the DNA and promotes non-homologous end-joining (NHEJ).
  • the i53 peptide used in the present methods comprises or consists of the sequence of SEQ ID NO: 1 or SEQ ID NO:2, or a derivative, variant, and/or fragment thereof that maintains 53BPl-inhibiting activity.
  • i53 can be, e.g., about 74 amino acids in length (see, e.g., SEQ ID NO:1), or longer, e.g., if the peptide contains a tag such as a His or FLAG tag (see, e.g., SEQ ID NO:2).
  • the i53 peptide comprises a His tag and is 85 amino acids in length (approx. 9.6 kDa).
  • i53 peptides that comprise the amino acid sequence of SEQ ID NO: 1 and that also contain a tag such as a His tag (as shown, e.g., in SEQ ID NO:2) or a FLAG tag can be used.
  • the i53 peptide comprises an amino acid sequence that comprises 1, 2, 3 or 4 amino acid substitutions relative to SEQ ID NO: 1 or SEQ ID NO:2.
  • An "expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell.
  • An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment.
  • an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter.
  • the promoter can be a heterologous promoter.
  • a “heterologous promoter” refers to a promoter that would not be so operably linked to the same polynucleotide as found in a product of nature (e.g., in a wild-type organism).
  • a first polynucleotide or polypeptide is "heterologous" to an organism or a second polynucleotide or polypeptide sequence if the first polynucleotide or polypeptide originates from a foreign species compared to the organism or second polynucleotide or polypeptide, or, if from the same species, is modified from its original form.
  • a promoter when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence).
  • Polypeptide “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including foil-length proteins, wherein the amino acid residues are linked by covalent peptide bonds. [0036] The terms “expression” and “expressed” refer to the production of a transcriptional and/or translational product, e.g., of an introduced cDNA or encoded protein.
  • the term refers to the production of a transcriptional and/or translational product encoded by a gene or a portion thereof.
  • the level of expression of a DNA molecule in a cell may be assessed on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell.
  • “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a poly-peptide also describes every- possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. In some cases, conservatively modified variants of a protein can have an increased stability, assembly, or activity as described herein. [0039] The following eight groups each contain amino acids that are conservative substitutions for one another:
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • amino acid residues are numbered according to their relative positions from the left most residue, which is numbered 1, in an unmodified wild- type polypeptide sequence.
  • the terms “identical” or percent “identity,” in the context of describing two or more polynucleotide or amino acid sequences, refer to two or more sequences or specified subsequences that are the same. Two sequences that are “substantially identical” have at least 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection where a specific region is not designated.
  • polynucleotide sequences tills definition also refers to the complement of a test sequence.
  • amino acid sequences in some cases, the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST 2.0 algorithm and the default parameters discussed below are used.
  • a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • CRISPR-Cas refers to a class of bacterial systems for defense against foreign nucleic acids.
  • CRISPR-Cas systems are found in a wide range of bacterial and archaeal organisms.
  • CRISPR-Cas systems fall into two classes with six types, I, II, III, IV, V, and VI as well as many sub-types, with Class 1 including types I and III CRISPR systems, and Class 2 including types II, IV, V and VI; Class 1 subtypes include subtypes I-A to I-F, for example.
  • Endogenous CRISPR-Cas systems include a CRISPR locus containing repeat clusters separated by non-repeating spacer sequences that correspond to sequences from viruses and other mobile genetic elements, and Cas proteins that carry out multiple functions including spacer acquisition, RNA processing from the CRISPR locus, target identification, and cleavage.
  • Cas proteins that carry out multiple functions including spacer acquisition, RNA processing from the CRISPR locus, target identification, and cleavage.
  • these activities are effected by multiple Cas proteins, with Cas3 providing the endonuclease activity, whereas in class 2 systems they are all carried out by a single Cas, Cas9.
  • a “homologous repair template” or “homologous donor template” refers to a polynucleotide sequence that can be used to repair a double stranded break (DSB) in the DNA, e.g., a CRISPR/Cas9-mediated break at a locus targeted by a herein-described sgRNA as induced using the herein-described methods and compositions.
  • the homologous repair template comprises homology to the genomic sequence surrounding the DSB, i.e., comprising target locus homology arms as described herein.
  • two distinct homologous regions are present on the template, with each region comprising at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or more nucleotides or more of homology with the corresponding genomic sequence.
  • the templates comprise two homology arms comprising about 500 nucleotides of homology extending from either site of the sgRNA target site.
  • the repair template can be present in any form, e.g., on a plasmid that is introduced into the cell, as a free-floating doubled stranded DNA template (e.g., a template that is liberated from a plasmid in the cell), or as single-stranded DNA.
  • the template is present within a viral vector, e.g., an adeno- associated viral vector such as AAV6.
  • the templates of the disclosure a codon-optimized, e.g., full-length, codon-optimized cDNAs, as well as, typically, a polyadenylation signal such as from bovine growth hormone or rabbit beta-globin.
  • the cDNA comprises a promoter, operably linked to the cDNA.
  • the template comprises a sequence other than a cDNA, e.g., a sequence designed to correct a specific mutation in a genomic locus, or to introduce a specific deletion or insertion into a locus.
  • the process of repairing a double-stranded break using a homologous donor template is referred to as Homology Directed Repair (HDR).
  • HDR Homology Directed Repair
  • homologous recombination refers to insertion of a nucleotide sequence during repair of double-strand breaks in DNA via homology-directed repair (HDR) mechanisms.
  • HDR homology-directed repair
  • This process uses a “donor template” or “homologous repair template” with homology to nucleotide sequence in the region of the break as a template for repairing a double-strand break.
  • the presence of a double-stranded break facilitates integration of the donor sequence.
  • the donor sequence may be physically integrated or used as a template for repair of the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence.
  • This process is used by a number of different gene editing platforms that create the double-strand break, such as meganucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas9 gene editing systems.
  • meganucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas9 gene editing systems.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • HR involves double-stranded breaks induced by CRISPR-Cas9.
  • the present disclosure provides methods for improving the efficiency of genomic editing through homology-directed repair (HDR), e.g., for editing genomic sequences or integrating cDNAs into endogenous loci in cells, through the administration of purified i53 peptide to the cells.
  • HDR homology-directed repair
  • the present methods and compositions allow genomic editing to be performed with higher rates of HDR and with lower rates of non-homologous end-joining (NHEJ) and, as a result, of insertions and deletions (indels).
  • the methods allow for high levels of HDR and cell engraftment to be achieved with lower levels of administered donor templates, e.g., using lower multiplicities of infection (MOI) when donor templates are introduced using viral vectors such as adeno-associated viral vectors (AAV) such as AAV6.
  • MOI multiplicities of infection
  • AAV adeno-associated viral vectors
  • the effects observed using i53 peptides in cells is transient, allowing HDR to be achieved without introducing longer-term genomic instability as might be observed, e.g. using nucleic acids encoding i53.
  • the cells are primary human cells, including stem cells such as CD34+ hematopoietic stem and progenitor cells (HSPCs) or hematopoietic stem cells (HSCs).
  • stem cells such as CD34+ hematopoietic stem and progenitor cells (HSPCs) or hematopoietic stem cells (HSCs).
  • HSPCs hematopoietic stem and progenitor cells
  • HSCs hematopoietic stem cells
  • cells from a subject are modified using the methods described herein and then reintroduced into the subject.
  • the cells can be taken from a subject with a genetic condition and the methods used to integrate a functional cDNA into the genome of the cells, wherein the expression of the cDNA in the modified cells in vivo restores protein activity that is missing or deficient in the subject or is otherwise beneficial to the subject.
  • the present disclosure is based in part on the identification that purified i53 peptide, e.g., purified recombinant i53 peptide, can effectively and safely increase HDR, decrease NHEJ, and decrease indels, when introduced together with a guide RNA and RNA-guided nuclease such as Cas9, and with a homologous donor template.
  • a guide RNA and RNA-guided nuclease such as Cas9
  • the guide RNA and RNA-guided nuclease are introduced as a ribonucleoprotein (RNP), for example by electroporation.
  • the i53 peptide is introduced together with the RNP.
  • the i53 peptide used in the present methods comprises (or consists of) the sequence of SEQ ID NO: 1 or SEQ ID NO:2, or comprises (or consists of) the sequence of SEQ ID NO: 1 or SEQ ID NO:2 with 1, 2, 3, 4 or more amino acid substitutions (e.g., conservative amino acid substitutions).
  • the i53 sequence can also be found, e.g., at ww , w.addgene.org/92170/sequences/, which is herein incorporated by reference in its entirety.
  • the i53 peptide used in the present methods comprises (or consists of) an i53 amino acid sequence as disclosed in www.addgene.org/92170/sequences/, or comprises (or consists of) an i53 amino acid sequence as disclosed in www.addgene.org/92170/sequences/ with 1, 2, 3, 4, or more amino acid substitutions (e.g., a conservative amino acid substitution).
  • the i53 peptide used in the present methods is a derivative, variant, or fragment of SEQ ID NO: 1 or SEQ ID NO:2 or an i53 amino acid sequence as disclosed in www.addgene.org/92170/sequences/ that comprises 53BPl-inhibiting activity.
  • the i53 peptide is about 74 amino acids long. In some embodiments, the i53 peptide is shorter than 74 amino acids long, e.g., 50, 55, 60, 65, 70, 71, 72, or 73 amino acids long. In some embodiments, the i53 peptide is 75 amino acids long or longer, e.g., 76, 77, 78, 79, 80, 85, 90, 95, 100 or more amino acids. In some embodiments, the i53 peptide comprises additional elements such as a label such as a His tag or a FLAG tag. In some embodiments, the i53 peptide comprises a His tag and is 85 amino acids long.
  • the present i53 peptides can comprise non-natural or non-proteinogenic amino acids, such as chemical mimetics of corresponding naturally occurring amino acids, non-standard amino acids such as D-amino acids, ⁇ -alanine, GABA, ornithine, citrulline, hydroxyproline, norleucine, 3-nitrotyrosine, nitroarginine, naphtylalanine, Abu, DAB, methionine sulfoxide, methionine sulfone, and more generally, P-amino acids (i.e., ⁇ 3 and ⁇ 2), homo-amino acids, beta-homo-amino acids, proline and pyruvic acid derivatives, 3- substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, N-methyl amino acids, alpha-methyl amino acids, ACHC, peptoids, and others.
  • non-standard amino acids
  • the i53 peptide is produced recombinantly and purified for use in the present methods.
  • the synthesis of i53 for use in the present methods can be accomplished using standard molecular biology methods.
  • the nucleotide sequences encoding i53 can be synthesized using standard methods and cloned into a suitable expression vector, e.g., the His-tag expression vector pET30(a)+.
  • Recombinant i53 can then be expressed in suitable cells, e.g., E. coli, and purified, and the protein concentrations and purities determined by, e.g., BCA assay and SDS-PAGE, respectively.
  • nucleic acids sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences.
  • kb kilobases
  • bp base pairs
  • proteins sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
  • Oligonucleotides that are not commercially available can be chemically synthesized, e.g., according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Lett. 22 1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al., Nucleic Acids Res. 12: 6159-6168 (1984). Purification of oligonucleotides is performed using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255: 137-149 (1983).
  • sequence of a polynucleotide encoding an i53 peptide can be verified after cloning or subcloning using, e.g., the drain termination method for sequencing double- stranded templates of Wallace et al, Gene 16: 21-26 (1981).
  • Nucleotide sequences encoding i53 peptides can be determined based on their encoded amino acid sequences (e.g., as shown in SEQ ID NO: 1 or SEQ ID NO:2, at www.addgene.org/92170/sequences/, and as described, e.g., in Canny et al. (2016) and in US Patent App. Pub. No.
  • Nucleotide sequences encoding i53 peptide can also be found, e.g., at www.addgene.org/92170/sequences/.
  • Nucleic acid sequences encoding i53 can be isolated using standard cloning techniques such as polymerase chain reaction (PCR). Most commonly used techniques for this purpose are described in standard texts, e.g., Sambrook and Russell, supra.
  • the coding sequence can be modified as appropriate (e.g., adding a coding sequence for a heterologous tag, such as an affinity tag, for example, 6 x His tag or GST tag) and then be subcloned into a vector, for instance, an expression vector, so that recombinant i53 can be produced from the resulting construct, for example, after transfection and culturing host cells under conditions permitting recombinant protein expression directed by a promoter operably linked to the coding sequence.
  • a heterologous tag such as an affinity tag, for example, 6 x His tag or GST tag
  • the polynucleotide sequence encoding an i53 peptide can be further altered to coincide with the preferred codon usage of a particular host.
  • the preferred codon usage of one strain of bacterial cells can be used to derive a polynucleotide that encodes an i53 peptide and includes the codons favored by this strain.
  • the frequency of preferred codon usage exhibited by a host cell can be calculated by averaging frequency of preferred codon usage in a large number of genes expressed by the host cell (e.g., calculation service is available from web site of the Kazusa DNA Research Institute, Japan). This analysis is preferably limited to genes that are highly expressed by the host cell.
  • a polynucleotide encoding the peptide can be subcloned into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator and a ribosome binding site for translational initiation.
  • Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook and Russell, supra, and Ausubel et al., supra.
  • Bacterial expression systems for expressing a recombinant polypeptide are available in, e.g., E. coli, Bacillus sp., Salmonella, and Caulobacter. Kits for such expression systems are commercially available.
  • the eukaryotic expression vector is an adenoviral vector, an adeno-associated vector, or a retroviral vector.
  • the promoter used to direct expression of a heterologous nucleic acid depends on the particular application.
  • the promoter is optionally positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • the promoter is an IPTG-inducible promoter.
  • the expression vector typically includes a transcription unit or expression cassette that contains all the additional elements required for the expression of the peptide in host cells.
  • a typical expression cassette thus contains a promoter operably linked to the coding sequence and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.
  • the nucleic acid sequence encoding the peptide is typically linked to a cleavable signal peptide sequence to promote secretion of the recombinant peptide by the transformed cell.
  • signal peptides include, among others, the signal peptides from tissue plasminogen activator, insulin, and neuron growth factor, and juvenile hormone esterase of Heliothis virescens.
  • Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
  • the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
  • the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
  • the particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, pET30(a)+, and fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., His, FLAG, or c-myc.
  • Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr vims.
  • exemplary eukaryotic vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • Some expression systems have markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase.
  • markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase.
  • high yield expression systems not involving gene amplification are also suitable, such as a baculovirus vector in insect cells, with a polynucleotide sequence encoding the peptide under the direction of the polyhedrin promoter or other strong baculovirus promoters.
  • the elements that are typically included in expression vectors also include a replicon that functions in E. colt, a gene encoding a protein that provides antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences.
  • the particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable.
  • the prokaryotic sequences are optionally chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary. Similar to antibiotic resistance selection markers, metabolic selection markers based on known metabolic pathways may also be used as a means for selecting transformed host cells.
  • the expression vector further comprises a sequence encoding a secretion signal, such as the E. coli OppA (Periplasmic Oligopeptide Binding Protein) secretion signal or a modified version thereof, which is directly connected to 5' of the coding sequence of the protein to be expressed.
  • a secretion signal such as the E. coli OppA (Periplasmic Oligopeptide Binding Protein) secretion signal or a modified version thereof, which is directly connected to 5' of the coding sequence of the protein to be expressed.
  • This signal sequence directs the recombinant protein produced in cytoplasm through the cell membrane into the periplasmic space.
  • the expression vector may further comprise a coding sequence for signal peptidase 1, which is capable of enzymatically cleaving the signal sequence when the recombinant protein is entering the periplasmic space.
  • Standard transfection methods are used to produce bacterial, mammalian, yeast, insect, or plant cell lines that express large quantities of a recombinant polypeptide (e.g., i53 peptide), which are then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264: 17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J. Bact. 132: 349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101: 347-362 (Wu etal., eds, 1983).
  • a recombinant polypeptide e.g., i53 peptide
  • Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell (see, e.g., Sambrook and Russell, supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the recombinant polypeptide.
  • the transfected cells are cultured under conditions favoring expression of the peptide.
  • the cells are then screened for the expression of the recombinant peptide, which is subsequently recovered from the culture using standard techniques (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et al., supra; and Sambrook and Russell, supra).
  • gene expression can be detected at the nucleic acid level.
  • a variety of methods of specific DNA and RNA measurement using nucleic acid hybridization techniques are commonly used (e.g., Sambrook and Russell, supra). Some methods involve an electrophoretic separation (e.g., Southern blot for detecting DNA and northern blot for detecting RNA), but detection of DNA or RNA can be carried out without electrophoresis as well (such as by dot blot).
  • the presence of nucleic acid encoding an i53 peptide in transfected cells can also be detected by PCR or RT-PCR using sequence-specific primers.
  • gene expression can be detected at the polypeptide level.
  • Various immunological assays are routinely used by those skilled in the art to measure the level of a gene product, particularly using polyclonal or monoclonal antibodies that react specifically with an i53 peptide (e.g., Harlow and Lane, Antibodies, A Laboratory Manual, Chapter 14, Cold Spring Harbor, 1988; Kohler and Milstein, Nature, 256: 495-497 (1975)).
  • Such techniques require antibody preparation by selecting antibodies with high specificity against the peptide.
  • the methods of raising polyclonal and monoclonal antibodies are well established and their descriptions can be found in the literature, see, e.g., Harlow and Lane, supra, Kohler and Milstein, Eur. J. Immunol., 6: 511-519 (1976).
  • polypeptides such as the i53 peptide are produced recombinantly by transformed bacteria in large amounts, typically after promoter induction, although expression can be constitutive, the polypeptides may form insoluble aggregates.
  • purification of protein inclusion bodies typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells, e.g., by incubation in a buffer of about 100-150 ⁇ g/ml lysozyme and 0.1% Nonidet P40, a non-ionic detergent.
  • the cell suspension can be ground using a Polytron grinder (Brinkman Instruments, Westbury, NY). Alternatively, the cells can be sonicated on ice. Alternate methods of lysing bacteria are described in Ausubel et al. and Sambrook and Russell, both supra, and will be apparent to those of skill in the art.
  • the cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer which does not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the wash step to remove as much cellular debris as possible.
  • the remaining pellet of inclusion bodies may be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl).
  • an appropriate buffer e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl.
  • Other appropriate buffers will be apparent to those of skill in the art.
  • the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties).
  • a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor or a combination of solvents each having one of these properties.
  • the proteins that farmed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer.
  • Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M), formamide (at least about 80%, vohime/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M).
  • Some solvents that are capable of solubilizing aggregate-forming proteins may be inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity.
  • SDS sodium dodecyl sulfate
  • 70% formic acid may be inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity.
  • guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re- formation of the immunologically and/or biologically active protein of interest.
  • the protein can be separated from other bacterial proteins by standard separation techniques.
  • purifying recombinant polypeptides from bacterial inclusion body see, e.g., Patra et al., Protein Expression and Purification 18: 182- 190 (2000).
  • recombinant polypeptides e.g., i53 peptide
  • the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art (see e.g., Ausubel et al, supra).
  • the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose.
  • the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO» and kept in an ice bath for approximately 10 minutes.
  • the cell suspension is centrifuged and the supernatant decanted and saved.
  • the recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art. Protein Separation Techniques for Purification
  • a recombinant polypeptide When a recombinant polypeptide is expressed in host cells in a soluble form, its purification can follow a standard protein purification procedure as described herein. Such standard purification procedures are also suitable for purifying a polypeptide obtained from chemical synthesis.
  • an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest.
  • the preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfete concentrations. A typical protocol is to add saturated ammonium sulfete to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%.
  • a protein of greater and lesser size can be isolated using ultrafiltration through membranes of different pore sizes (for example, Amicon or Millipore membranes).
  • the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of a protein of interest, e.g., an i53 peptide.
  • the retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest.
  • the recombinant protein will pass through the membrane into the filtrate.
  • the filtrate can then be chromatographed as described below.
  • Proteins of interest can also be separated from other proteins on the basis of their size, net surface charge, hydrophobicity, or affinity for ligands.
  • antibodies raised against i53 can be conjugated to column matrices and the corresponding peptide immunopurified. All of these methods are well known in the art. It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).
  • the i53 peptide can also be synthesized chemically using peptide synthesis or other protocols well known in the art.
  • i53 peptides may be synthesized by solid-phase peptide synthesis methods using procedures similar to those described by Merrifield et al, J. Am. Chem. Soc., 85:2149-2156 (1963); Barany and Merrifield, Solid-Phase Peptide Synthesis, in The Peptides: Analysis, Synthesis, Biology Gross and Meienhofer (eds.), Academic Press, N.Y., vol. 2, pp. 3-284 (1980); and Stewart et al., Solid Phase Peptide Synthesis 2nd ed., Pierce Chem. Co., Rockford, Ill. (1984).
  • N-a-protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C- terminal and to a solid support, i.e., polystyrene beads.
  • the peptides are synthesized by linking an amino group of an N-a-deprotected amino acid to an a-carboxy group of an N-a- protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide. The attachment of a free amino group to the activated carboxyl leads to peptide bond formation.
  • the most commonly used N-a-protecting groups include Boc, which is acid labile, and Fmoc, which is base labile.
  • the purified i53 can be introduced into cells in any of a number of ways, e.g., by electroporation, microinjection, lipofection, electroporation, nanoparticle bombardment, the use of cell-penetrating peptide (CPP) tags, and the like.
  • the i53 peptide is introduced into cells by electroporation.
  • the i53 is introduced by electroporation together with RNPs comprising an sgRNA and RNA-guided nuclease.
  • the i53 can be introduced into cells at any suitable concentration, i.e., a concentration sufficient to increase HDR in the cell and decrease NHEJ, indels, etc.
  • concentration i.e., a concentration sufficient to increase HDR in the cell and decrease NHEJ, indels, etc.
  • concentration will depend upon the cell type, the targeted locus, the nature of genetic modification desired, and other factors known to one of skill in the art.
  • the effect of i53 peptide is concentration dependent, and HDR in HSPCs, for example, increases in a dose dependent manner.
  • the i53 peptide is present at a concentration of from 10 ⁇ g/ml to 10 mg/ml, from 100 ⁇ g/ml to 5 mg/ml, from 250 ⁇ g/ml to 2.5 mg/ml, or at about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5 or more mg/ml.
  • the i53 peptide is introduced at about 1.5 mg/ml.
  • the i53 peptide introduced into cells is transient.
  • the transient nature of purified i53 peptide in cells is advantageous in that it transiently promotes HDR upon introduction of the peptide into the cells, but does not persist long enough to promote longer-term instability in the cells.
  • sgRNAs single guide RNAs
  • sgRNAs interact with a site-directed nuclease such as Cas9 and specifically bind to or hybridize to a target nucleic acid within the genome of a cell, such that the sgRNA and the site-directed nuclease co-localize to the target nucleic acid in the genome of the cell.
  • the sgRNAs as used herein comprise a targeting sequence comprising homology (or complementarity) to a target DNA sequence, and a constant region that mediates binding to Cas9 or another RNA-guided nuclease.
  • the sgRNA can target any sequence within the target gene adjacent to a PAM sequence.
  • the sgRNAs used in the present methods and compositions can target any locus that is to be modified or edited.
  • the target gene or locus is a safe harbor locus such as CCR5 or a locus associated with a genetic disorder, such as sickle cell disease, ⁇ -thalassemia, X-linked severe combined immunodeficiency (e.g., SCID-X1), X-linked chronic granulomatous disease (X- CGD), cystic fibrosis, lysosomal storage disorders such as mucopolysaccharidosis type 1, Gaucher’s disease, or Krabbe disease, and others, and the methods are used to correct a mutated copy of the gene in a patient.
  • a non-limiting list of genes that can be targeted or introduced using the present methods includes HBB, CYBB, CCR5, IL2RG, HBA1, HBA2, CFTR, and others.
  • the targeting sequence of the sgRNAs may be, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or 15-25, 18-22, or 19-21 nucleotides in length, and shares homology with a targeted genomic sequence, in particular at a position adjacent to a CRISPR PAM sequence.
  • the sgRNA targeting sequence is designed to be homologous to the target DNA, i.e., to share the same sequence with the non-bound strand of the DNA template or to be complementary to the strand of the template DNA that is bound by the sgRNA.
  • the homology or complementarity of the targeting sequence can be perfect (i.e., sharing 100% homology or 100% complementarity to the target DNA sequence) or the targeting sequence can be substantially homologous (i.e., having less than 100% homology or complementarity, e.g., with 1-4 mismatches with the target DNA sequence).
  • Each sgRNA also includes a constant region that interacts with or binds to the site- directed nuclease, e.g., Cas9.
  • the constant region of an sgRNA can be from about 70 to 250 nucleotides in length, or about 75-100 nucleotides in length, 75-85 nucleotides in length, or about 80-90 nucleotides in length, or 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides in length.
  • the overall length of the sgRNA can be, e.g., from about 80-300 nucleotides in length, or about 80-150 nucleotides in length, or about 80-120 nucleotides in length, or about 90-110 nucleotides in length, or, e.g, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 nucleotides in length.
  • crRNAs two-piece gRNAs
  • crtracrRNAs two-piece gRNAs
  • the target sequence is located near the translational start site of the gene, such that the full-length cDNA can be expressed under the control of the endogenous promoter.
  • the target sequence can be elsewhere in a gene or locus, e.g., to modify the sequence at the site of a mutation, to introduce a regulatory element, to introduce a deletion to remove protein function, to introduce an expression cassette comprising a coding sequence operably linked to a promoter, etc. It will be understood that the present methods can be used to enhance the rate of HDR for any purpose, and using sgRNAs targeting any part of a gene or genome.
  • the sgRNAs comprise one or more modified nucleotides.
  • the polynucleotide sequences of the sgRNAs may also comprise RNA analogs, derivatives, or combinations thereof.
  • the probes can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone (e.g., phosphorothioates).
  • the sgRNAs comprise 3’ phosphorothiate intemucleotide linkages, 2’-O- methyl-3 ’-phosphoacetate modifications, 2 ’-fluoro-pyrimidines, S-constrained ethyl sugar modifications, or others, at one or more nucleotides.
  • the sgRNAs comprise 2'-O-methyl-3'-phosphorothioate (MS) modifications at one or more nucleotides (see, e.g., Hendel et al. (2015) Nat. Biotech. 33(9):985-989, the entire disclosure of which is herein incorporated by reference).
  • the 2'-O-methyl-3'- phosphorothioate (MS) modifications are at the three terminal nucleotides of the 5' and 3' ends of the sgRNA.
  • the sgRNAs can be obtained in any of a number of ways.
  • primers can be synthesized in the laboratory using an oligo synthesizer, e.g., as sold by Applied Biosystems, Biolytic Lab Performance, Sierra Biosystems, or others.
  • primers and probes with any desired sequence and/or modification can be readily ordered from any of a large number of suppliers, e.g., ThermoFisher, Biolytic, IDT, Sigma-Aldritch, GeneScript, etc.
  • the sgRNAs are used together with an RNA-guided nuclease, e.g. a CRISPR-Cas nuclease.
  • a CRISPR-Cas nuclease can be used in the method, i.e., a CRISPR-Cas nuclease capable of interacting with a guide RNA and cleaving the DNA at the target site as defined by the guide RNA.
  • the nuclease is Cas9 or Cpfl.
  • the nuclease is Cas9.
  • the Cas9 or other nuclease used in the present methods can be from any source, so long that it is capable of binding to an sgRNA of the present disclosure and being guided to and cleaving the specific sequence targeted by the targeting sequence of the sgRNA.
  • the Cas9 is from Streptococcus pyogenes.
  • a high fidelity Cas9 nuclease is used.
  • CRISPR/Cas or CRISPR/Cpfl systems that target and cleave DNA at a locus of interest.
  • An exemplary CRISPR/Cas system comprises (a) a Cas (e.g., Cas9) or Cpfl polypeptide or a nucleic acid encoding said polypeptide, (b) an sgRNA that hybridizes specifically to the locus of interest, or a nucleic acid encoding said guide RNA, (c) a donor template as described herein, and (d) an i53 peptide.
  • the CRISPR/Cas system comprises an RNP comprising an sgRNA targeting the locus of interest and a Cas protein such as Cas9.
  • CRISPR/Cas9 which is a type II CRISPR/Cas system
  • CRISPR/Cas9 platform which is a type II CRISPR/Cas system
  • alternative systems exist including type I CRISPR/Cas systems, type III CRISPR/Cas systems, and type V CRISPR/Cas systems.
  • Various CRISPR/Cas9 systems have been disclosed, including Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Campylobacter jejuni Cas9 (CjCas9) and Neisseria cinerea Cas9 (NcCas9) to name a few.
  • Cas system alternatives include the Francisella novicida Cpfl (FnCpfl), Acidaminococcus sp. Cpfl (AsCpfl), and Lachnospiraceae bacterium ND2006 Cpfl (LbCpfl) systems. Any of the above CRISPR systems may be used to induce a single or double stranded break at the locus of interest to carry out the methods disclosed herein.
  • FnCpfl Francisella novicida Cpfl
  • AsCpfl Acidaminococcus sp. Cpfl
  • LbCpfl Lachnospiraceae bacterium ND2006 Cpfl
  • the sgRNA and nuclease can be introduced into a cell using any suitable method, e.g., by introducing one or more polynucleotides encoding the sgRNA and the nuclease into the cell, e.g., using a vector such as a viral vector or delivered as naked DNA or RNA, such that the sgRNA and nuclease are expressed in the cell.
  • a vector such as a viral vector or delivered as naked DNA or RNA
  • one or more polynucleotides encoding the sgRNA, the nuclease or a combination thereof are included in an expression cassette.
  • the sgRNA, the nuclease, or both sgRNA and nuclease are expressed in the cell from an expression cassette.
  • the sgRNA, the nuclease, or both sgRNA and nuclease are expressed in the cell under the control of a heterologous promoter.
  • one or more polynucleotides encoding the sgRNA and the nuclease are operatively linked to a heterologous promoter.
  • the sgRNA and nuclease are assembled into ribonucleoproteins (RNPs) prior to delivery to the cells.
  • the RNPs can be introduced into the cell using any suitable method, e.g., microinjection, electroporation, or other chemical transfection (e.g., lipid vesicles, osmocytosis, soluporation or other permeabilization techniques, etc.) or physical transfection methods (e.g., mechanical transfection, membrane disruption or permeabilization, etc.).
  • the RNPs are introduced into the cell by electroporation.
  • transgenes including large transgenes, capable of expressing functional proteins, including enzymes, cytokines, antibodies, and cell surface receptors are known in the art (See, e.g. Bak and Porteus, Cell Rep. 2017 Jul 18; 20(3): 750- 756 (integration of EGFR); Kanojia et al., Stem Cells. 2015 Oct;33(10):2985-94 (expression of anti-Her2 antibody); Eyquem et al., Nature.
  • the homologous repair template used in the present methods can be any template used for genomic editing purposes, e.g., to integrate a cDNA or other sequence into a corresponding endogenous locus or a safe harbor locus, to introduce a deletion, insertion, or sequence modification into a targeted genomic locus, or for any other method wherein a genomic locus is cleaved using an sgRNA and RNA-guided nuclease such as Cas9, and the cleaved sequence is modified via HDR using a homologous donor template .
  • an sgRNA and RNA-guided nuclease such as Cas9
  • the methods are used to introduce a cDNA into a targeted genomic locus.
  • the methods can be used to integrate a cDNA such as a functional, codon-optimized cDNA into the genome of cells of a subject with a genetic disorder caused by a deficit or absence in the protein encoded by the cDNA, or a genetic or other disorder that can be treated or ameliorated in any way by the expression of the cDNA.
  • the cDNA is integrated, e.g., at the translational start site of the endogenous locus, such that the cDNA is expressed under the control of the endogenous promoter and other regulatory elements.
  • the template comprises a promoter, operably linked to the cDNA, e.g., when the cDNA is integrated in a safe harbor locus such as the C-C chemokine receptor type 5 (CCR5) locus.
  • CCR5 C-C chemokine receptor type 5
  • any promoter that can induce expression of the therapeutic protein in the modified cells can be used, including endogenous and heterologous promoters, inducible promoters, constitutive promoters, cell-specific promoters, and others.
  • the promoter is the phosphoglycerate kinase (PGK) promoter, the spleen focus-forming virus (SFFV) promoter, or the CD68 promoter.
  • PGK phosphoglycerate kinase
  • SFFV spleen
  • the transgene in addition to the promoter, is optionally linked to one or more regulatory elements such as enhancers or post-transcriptional regulatory- sequences.
  • regulatory elements such as enhancers or post-transcriptional regulatory- sequences.
  • miRNA miRNA
  • the expression control sequence functions to express the therapeutic transgene following the same expression pattern as in normal individuals (physiological expression) (See Toscano et al., Gene Therapy (2011) 18, 117-127 (2011), incorporated herein by reference in its entirety for its references to promoters and regulatory sequences).
  • the cDNA in the homologous repair template is codon- optimized, e.g., comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or more homology to the wild-type cDNA sequence, or to a fiagment thereof.
  • the template further comprises a polyA sequence or signal, e.g., a bovine growth hormone polyA sequence or a rabbit beta-globin polyA sequence, at the 3’ end of the cDNA.
  • a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element is included within the 3’UTR of the template, e.g., between the 3’ end of the cDNA coding sequence and the 5’ end of the polyA sequence, so as to increase the expression of the cDNA.
  • Any suitable WPRE sequence can be used; See, e.g., Zufferey et al. (1999) J. Virol.
  • the cDNA (or cDNA and polyA signal) is flanked in the template by homology regions corresponding to the targeted locus.
  • an exemplary template can comprise, in linear order: a first genomic homology region, an optional promoter, a cDNA, a polyA sequence, and a second genomic homology region, where the first and second homology regions are homologous to the genomic sequences extending in either direction from the sgRNA target site.
  • the homology regions can be of any size, e.g., 100-1000 bp, 300-800 bp, 400-600 bp, or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more base pairs.
  • any suitable method can be used to introduce the polynucleotide, or donor construct, into the primary cells.
  • the polynucleotide is introduced using a recombinant adeno-associated viral vector, e.g., rAAV6.
  • the donor template is single stranded, double stranded, a plasmid or a DNA fragment.
  • plasmids comprise elements necessary for replication, including a promoter and optionally a 3’ UTR.
  • vectors comprising (a) one or more nucleotide sequences homologous to the locus of interest, and (b) a cDNA as described herein.
  • the vector can be a viral vector, such as a retroviral, lentiviral (both integration competent and integration defective lentiviral vectors), adenoviral, adeno-associated viral or herpes simplex viral vector.
  • Viral vectors may further comprise genes necessary for replication of the viral vector.
  • the targeting construct comprises: (1) a viral vector backbone, e.g. an AAV backbone, to generate virus; (2) arms of homology to the target site of at least 200 bp but ideally at least 400 bp on each side to assure high levels of reproducible targeting to the site (see, Porteus, Annual Review of Pharmacology and Toxicology, Vol. 56:163-190 (2016); which is hereby incorporated by reference in its entirety); (3) a cDNA encoding a functional protein and capable of expressing the functional protein, optionally a promoter, a polyA sequence, and optionally a WPRE element; and optionally (4) an additional marker gene to allow for enrichment and/or monitoring of the modified host cells.
  • a viral vector backbone e.g. an AAV backbone
  • the primary AAV serotype is AAV6.
  • the vector, e.g., rAAV6 vector, comprising the donor template is from about 1 -2 kb, 2-3 kb, 3-4 kb, 4-5 kb, 5-6 kb, 6-7 kb, 7-8 kb, or larger.
  • viral vectors e.g., AAV6 vector
  • MOI multiplicity of infection
  • the viral vector is introduced at an MOI of less than about 2500, e.g., about 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 850, 800, 750, 700, 675, 650, 625, 600, 550, 500, 450, 400, or less.
  • the viral vector is introduced at an MOI of about 625 in the presence of the i53 peptide.
  • the viral vector is administered in the presence of the i53 peptide at an MOI that is 1-fold, 2-fold, 3-fold, 4-fold, or more lower than a standard or recommended MOI in the absence of the i53 peptide.
  • Suitable marker genes are known in the art and include Myc, HA, FLAG, GFP, truncated NGFR, truncated EGFR, truncated CD20, truncated CD 19, as well as antibiotic resistance genes.
  • the homologous repair template and/or vector e.g., AAV6
  • the inserted construct can also include other safety switches, such as a standard suicide gene into the locus (e.g. iCasp9) in circumstances where rapid removal of cells might be required due to acute toxicity.
  • a standard suicide gene into the locus e.g. iCasp9
  • the present disclosure provides a robust safety switch so that any engineered cell transplanted into a body can be eliminated, e.g.. by removal of an auxotrophic factor. This is especially important if the engineered cell has transformed into a cancerous cell.
  • the present methods allow for the efficient integration of the donor template at the endogenous locus of interest.
  • the present methods allow for the insertion of the donor template in 20%, 25%, 30%, 35%, 40%, or more cells, e.g., cells fiom an individual with a condition to be treated using the present methods and/or compositions.
  • the methods also allow for high levels of expression of protein in cells, e.g., cells fiom an individual with an integrated cDNA as described herein, e.g., levels of expression that are at least about 70%, 75%, 80%, 85%, 90%, 95%, or more relative to the expression in healthy control cells.
  • Animal cells mammalian cells, preferably human cells, modified ex vivo, in vitro, or in vivo are contemplated. Also included are cells of other primates; mammals, including commercially relevant mammals, such as cattle, pigs, horses, sheep, cats, dogs, mice, rats; birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
  • the cells are human cells, e.g., human cells from a subject with a genetic disorder or condition.
  • the cells used in the present methods are primary cells, i.e., cells taken directly from a living tissue (e.g., biopsy, blood sample, etc.).
  • the cell is an embryonic stem cell, a stem cell, a progenitor cell, a pluripotent stem cell, an induced pluripotent stem cell (iPSC), a somatic stem cell, a differentiated cell, a mesenchymal stem cell or a mesenchymal stromal cell, an airway basal stem cell, a neural stem cell, a hematopoietic stem cell or a hematopoietic progenitor cell, an adipose stem cell, a keratinocyte, a skeletal stem cell, a muscle stem cell, a fibroblast, an NK cell, a B-cell, a T cell, or a peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • the cells are CD34 + hematopoietic stem and progenitor cells (HSPCs), e.g., cord blood-derived (CB), adult peripheral blood-derived (PB), or bone marrow derived HSPCs.
  • HSPCs can be isolated from a subject, e.g., by collecting mobilized peripheral blood and then enriching the HSPCs using the CD34 marker.
  • the cells to be modified are preferably derived from the subject’s own cells.
  • the mammalian cells are autologous cells from the subject to be treated with the modified cells.
  • the cells are allogeneic, i.e., isolated from an HLA-matched or HLA-compatible, or otherwise suitable, donor.
  • cells are harvested from the subject and modified according to the methods disclosed herein, which can include selecting certain cell types, optionally expanding the cells and optionally culturing the cells, and which can additionally include selecting cells that contain a transgene integrated into the targeted locus.
  • such modified cells are then reintroduced into the subject.
  • nuclease systems comprising introducing into a mammalian cell: (a) an RNP comprising a Cas nuclease such as Cas9 and an sgRNA specific to a locus of interest, (b) an i53 peptide, and (c) a homologous donor template or vector as described herein.
  • Each component can be introduced into the cell directly or can be expressed in the cell by introducing a nucleic acid encoding the components of said one or more nuclease systems.
  • the present methods target integration of a functional cDNA at the corresponding endogenous locus or at a safe harbor locus in a host cell ex vivo.
  • the methods target the modification of a genomic sequence, e.g., the alteration of a genomic sequence, or the introduction of a deletion or insertion, at an endogenous locus.
  • Such methods can further comprise (a) optionally expanding said cells, and/or (b) optionally culturing the cells.
  • the nuclease can produce one or more single stranded breaks within the locus of interest, or a double stranded break within the locus of interest.
  • the locus is modified by homologous recombination with said donor template or vector to result in insertion of the transgene into the locus.
  • the methods can further comprise (c) selecting cells that contain the transgene integrated into the locus of interest.
  • the activity of i53 peptide and/or the efficacy of the present methods can be assessed in any of a number of ways.
  • the activity of i53 peptide can be assessed by measuring the rate of HDR in cells such as CD34 + HSPCs, e.g., the rate of integration of a cDNA at genomic loci such as HBB, CCR5, 1L2RG, HBA1, or CFTR when an i53 peptide is introduced together with an sgRNA, RNA-guided nuclease, and homologous donor template.
  • the rate of HDR in such cells is increased by at least about 10%, 20%, 30%, 40%, 50%, or more relative to the rate in equivalent cells but in the absence of i53.
  • the activity of i53 peptide can be assessed by measuring the rate of NHEJ or indels in cells such as CD34 + HSPCs.
  • the rate of indels is decreased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more relative to the rate in equivalent cells in the absence of i53 peptide.
  • the activity of i53 peptide is assessed by determining the MOI for a viral vector comprising a homologous donor template that is required to achieve a given level of HDR
  • the presence of i53 can allow a decrease in the MOI used of, e.g., 1-fold, 2- fold, 3-fold, 4-fold, or more, while still maintaining similar rates of HDR as compared to in an equivalent cell in the absence of i53.
  • the activity of i53 peptide can be assessed by determining, e.g., the ability of modified cells to achieve a given rate of engrafhent in animal models.
  • the presence of i53 can allow the use of an MOI that is, e.g., 1-fold, 2-fold, 3-fold, 4-fold, or more lower than the MOI needed in the absence of i53 peptide, to achieve a given rate of engrafhnent.
  • the activity of i53 can also be assessed in cells by examining, e.g., the activity of 53BP1, such as the binding of 53BP1 to the ends of double-stranded DNA breaks. 8.
  • a plurality of modified cells can be reintroduced into the subject, such that they can repopulate and differentiate, and due to the expression of the integrated cDNA (or other genetic modification), can improve one or more abnormalities or symptoms in the subject with the genetic disorder.
  • the cells are expanded, selected, and/or induced to undergo differentiation, prior to reintroduction into the subject.
  • the modified host cells of the present disclosure included in the pharmaceutical compositions described above may be administered by any delivery route, systemic delivery or local delivery, which results in a therapeutically effective outcome.
  • these include, but are not limited to, enteral, gastroenteral, epidural, oral, transdermal, intracerebral, intracerebroventricular, epicutaneous, intradermal, subcutaneous, nasal, intravenous, intra- arterial, intramuscular, intracardiac, intraosseous, intrathecal, intraparenchymal, intraperitoneal, intravesical, intravitreal, intracavemous), interstitial, intra-abdominal, intralymphatic, intramedullary, intrapulmonary, intraspinal, intrasynovial, intrathecal, intratubular, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, soft tissue, and topical.
  • the cells are administered intravenously.
  • a subject will undergo a conditioning regime before cell transplantation.
  • a conditioning regime may involve administration of cytotoxic agents.
  • the conditioning regime may also include immunosuppression, antibodies, and irradiation.
  • conditioning regimens include antibody-mediated conditioning (see, e.g., Czechowicz et al., 318(5854) Science 1296-9 (2007); Palchaudari et al., 34(7) Nature Biotechnology 738-745 (2016); Chhabra et al, 10:8(351) Science Translational Medicine 351ral05 (2016)) and CAR T-mediated conditioning (see, e.g., Arai et al., 26(5) Molecular Therapy 1181-1197 (2016); each of which is hereby incorporated by reference in its entirety).
  • conditioning needs to be used to create space in the brain for microglia derived from engineered hematopoietic stem cells (HSCs) to migrate in to deliver the protein of interest (as in recent gene therapy trials for ALD and MLD).
  • the conditioning regimen is also designed to create niche “space” to allow the transplanted cells to have a place in the body to engraft and proliferate.
  • the conditioning regimen creates niche space in the bone marrow for the transplanted HSCs to engraft. Without a conditioning regimen, the transplanted HSCs cannot engraft.
  • the present disclosure additionally provides methods of administering modified host cells in accordance with the disclosure to a subject in need thereof.
  • Pharmaceutical compositions including the modified host cell may be administered to a subject using any amount and any route of administration effective for preventing, treating, or managing the condition in question. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
  • the subject may be a human, a mammal, or an animal.
  • the specific therapeutically or prophylactically effective dose level for any particular individual will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific payload employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration; the duration of the treatment; drugs used in combination or coincidental with the specific modified host cell employed; and like factors well known in the medical arts.
  • modified host cell pharmaceutical compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from, e.g., about 1 x 10 4 to 1 x 10 5 , 1 x 10 5 to 1 x 10 6 , 1 x 10 6 to 1 x 10 7 , or more modified cells to the subject, or any amount sufficient to obtain the desired therapeutic or prophylactic, effect.
  • the desired dosage of the modified host cells of the present disclosure may be administered one time or multiple times.
  • delivery of the modified host cell to a subject provides a therapeutic effect for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years.
  • the modified host cells may be used in combination with one or more other therapeutic, prophylactic, research or diagnostic agents, or medical procedures, either sequentially or concurrently.
  • each agent w-ill be administered at a dose and/or on a time schedule determined forthat agent.
  • i53 recombinant peptide is an effective method of increasing the frequency of HDR genome editing at a variety of loci in human HSPCs and also increases HDR in a variety- of therapeutically relevant human primary- cell types including T-cells, MSCs, airway stem cells (basal cells) and IPSCs.
  • the inhibition is transient because the peptide is rapidly degraded and thus this approach could become an important method to increase the frequency- of HDR for therapeutic purposes.
  • i53 peptide also reduces the formation of INDELs and enables the multiplicity of infection (MOI) of AAV6 needed to achieve high frequencies of gene targeting to be significantly reduced (4- fold).
  • CD34 + cells based on previously defined cell surface markers (CD34, CD38, CD90, and CD45RA) for progenitor cells (CD38 + ; CD34 + and CD38 + ), multi-potent progenitor cells (MPP; CD34*, CD38-, CD90-, CD45RA-), hematopoietic stem cells (HSCs; CD34 + , CD38-, CD90 + , CD45RA-) and long-term multi-potent progenitor cells (LMPP, CD34 + , CD38-, CD90-, CD45RA + ).
  • MPP multi-potent progenitor cells
  • HSCs hematopoietic stem cells
  • LMPP long-term multi-potent progenitor cells
  • the cells were electroporated with Cas9 RNP using previously described chemically modified sgRNAs (15) either with or without i53 recombinant protein and transduced with adeno-associated virus type 6 (AAV6) homologous donor templates (5).
  • AAV6 adeno-associated virus type 6
  • the HBB gene with the R2 guide is a good candidate to extend the evaluation for the efficacy and safety of using i53 recombinant peptide because the HBB gene is of immediate clinical relevance. It is also a gene target in which increasing HDR and decreasing indels could both have clinical benefit.
  • i53 peptide concentration dependent as HDR in HSPCs increased in a dose dependent manner: ⁇ 10% increase at 250 ⁇ g/ml; 46% increase at 1500 ⁇ g/ml by approximately 46% and 38% increase at 5000 ⁇ g/ml (FIG. 5A).
  • i53 peptide concentrations up to 5000 ⁇ g/ml did not result in notable decrease in viability of HSPCs and dosage of i53 peptide beyond 1500 ⁇ g/ml did not result in any additional improvement in HDR and we therefore used 1500 ⁇ g/ml of peptide in subsequent experiments unless noted otherwise.
  • HSPCs transduced with AAV6 were plated to semisolid methylcellulose media, which supports the growth of multiple progenitor cells (myeloid: CFU-GM; erythroid: BFU-E and CFU-E; and mixed myeloid and erythroid: CFU-GEMM). After 14 days of plating, we observed that only approximately 42.7% of HSPCs transduced with 5000 MOI of AAV6 are capable of forming colonies but with lower MOIs of 2500, 1250, and 625, approximately 65.6%, 85.0% and 90.5% of HSPCs, respectively, were able to form colonies (FIG. 5C).
  • i53 peptide we used i53 peptide to determine if it could facilitate using lower MOI’s without compromising the high frequencies of HDR in HSPCs.
  • One of the positive features of using i53 peptide is to reduce the amount of AAV loaded onto the cells without compromising HDR frequency.
  • AAV AAV-dose dependent manner
  • the CFU assay gives a measure of HSPC function.
  • To evaluate potential HSPC and HSC function we performed transplantation experiments in NSG mice (FIG. 8A). We evaluated the efficiency of engrafting HSPCs targeted with low and high MOIs (625 MOI for low vs 2500 MOI for high) of AAV6 with i53 peptide, treatment.
  • mPB frozen mobilized peripheral blood
  • NOD non-obese diabetic
  • SJD severe combined immunodeficiency
  • 53BP1 a DNA damage response sensor
  • Escribano-Diaz, C. et al. A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCAl-CtIP controls DNA repair pathway choice. Mol. Cell 49, 872-883 (2013).
  • a method of genetically modifying a primary human cell comprising:
  • RNA-guided nuclease and a single guide RNA (sgRNA) targeting a genetic locus of interest introducing into the cell an RNA-guided nuclease and a single guide RNA (sgRNA) targeting a genetic locus of interest;
  • sgRNA directs the RNA-guided nuclease to the locus of interest, the RNA-guided nuclease cleaves the locus at the target sequence of the sgRNA, and the homologous donor template is integrated at the site of the cleaved locus by homology directed repair (HDR).
  • HDR homology directed repair
  • the primary human cell is a cell selected from the group consisting of a CD34+ hematopoietic stem and progenitor cell (HSPC), a T cell, a mesenchymal stem cell (MSC), an airway basal stem cell, and an induced pluripotent stem cell (IPSC).
  • HSPC hematopoietic stem and progenitor cell
  • T cell hematopoietic stem and progenitor cell
  • MSC mesenchymal stem cell
  • IPC induced pluripotent stem cell
  • locus of interest is a gene selected from the group consisting of Hemoglobin Subunit Beta (HBB), C-C Motif Chemokine Receptor 5 (CCR5), Interleukin 2 Receptor Subunit Gamma (IL2RG), Hemoglobin Subunit Alpha 1 (HBA1), and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).
  • HBB Hemoglobin Subunit Beta
  • CCR5 C-C Motif Chemokine Receptor 5
  • IL2RG Interleukin 2 Receptor Subunit Gamma
  • HBA1 Hemoglobin Subunit Alpha 1
  • CFTR Cystic Fibrosis Transmembrane Conductance Regulator
  • sgRNA comprises 2'-O-methyl-3'-phosphorothioate (MS) modifications at one or more nucleotides.
  • RNA-guided nuclease are introduced into the cell as a ribonucleoprotein (RNP).
  • RNP ribonucleoprotein
  • amino acid sequence of the i53 peptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 1 or SEQ ID NO:2.
  • a method of treating a genetic disorder in a human subject in need thereof comprising: isolating a primary cell from the subject; genetically modifying the primary cell using the method of any one of claims 1 to 26, wherein the integration of the homologous donor template at the locus of interest in the cell corrects a mutation at the locus or leads to the expression of a therapeutic protein in the cell that is absent or deficient in the subject; and reintroducing the genetically modified cell into the subject.
  • the genetic disorder is a disorder selected from the group consisting of ⁇ -thalassemia, sickle cell disease (SCD), severe combined immunodeficiency (SCID), mucopolysaccharidosis type 1, Cystic Fibrosis, Gaucher disease, Krabbe disease, and X-linked chronic granulomatous disease (X-CGD).
  • SCD sickle cell disease
  • SCID severe combined immunodeficiency
  • X-CGD X-linked chronic granulomatous disease

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Abstract

La présente divulgation concerne des méthodes d'amélioration de la vitesse de réparation dirigée par homologie (HDR) pendant une édition génomique dans des cellules primaires.
PCT/US2021/072642 2020-11-30 2021-11-30 Édition génique médiée par crispr/cas de cellules souches humaines Ceased WO2022115878A1 (fr)

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Cited By (2)

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WO2024040059A1 (fr) * 2022-08-19 2024-02-22 Integrated Dna Technologies, Inc. Variant d'ubiquitine à haute affinité pour la liaison à 53bp1 réduisant la quantité de vaa nécessaire pour obtenir des taux élevés de hdr
WO2025198596A1 (fr) * 2024-03-22 2025-09-25 Integrated Dna Technologies, Inc. Utilisation d'un variant d'ubiquitine humaine se liant à 53bp1 pour améliorer les taux de hdr dans de multiples types de cellules

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US20180051281A1 (en) * 2014-12-03 2018-02-22 Agilent Technologies, Inc. Guide rna with chemical modifications
US20190010196A1 (en) * 2016-02-01 2019-01-10 The Governing Council Of The University Of Toronto Ubiquitin variants and uses therof as 53bp1 inhibitors
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SWEENEY ET AL.: "Correction of X-CGD patient HSPCs by targeted CYBB cDNA insertion using CRISPR/Cas9 with 53BP1 inhibition for enhanced homology-directed repair", GENE THERAPY, vol. 28, no. 6, 21 March 2021 (2021-03-21), pages 373 - 390, XP037488715, DOI: 10.1038/s41434-021-00251-z *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024040059A1 (fr) * 2022-08-19 2024-02-22 Integrated Dna Technologies, Inc. Variant d'ubiquitine à haute affinité pour la liaison à 53bp1 réduisant la quantité de vaa nécessaire pour obtenir des taux élevés de hdr
WO2025198596A1 (fr) * 2024-03-22 2025-09-25 Integrated Dna Technologies, Inc. Utilisation d'un variant d'ubiquitine humaine se liant à 53bp1 pour améliorer les taux de hdr dans de multiples types de cellules

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