WO2023034704A1 - Produits et procédés pour annoter une fonction génique à l'aide de cellules non cancéreuses humaines haploïdes localement - Google Patents

Produits et procédés pour annoter une fonction génique à l'aide de cellules non cancéreuses humaines haploïdes localement Download PDF

Info

Publication number
WO2023034704A1
WO2023034704A1 PCT/US2022/075365 US2022075365W WO2023034704A1 WO 2023034704 A1 WO2023034704 A1 WO 2023034704A1 US 2022075365 W US2022075365 W US 2022075365W WO 2023034704 A1 WO2023034704 A1 WO 2023034704A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
gene
variant
cells
brca2
Prior art date
Application number
PCT/US2022/075365
Other languages
English (en)
Inventor
Dirk Friedrich HOCKEMEYER
Hanqin LI
Rebecca Marguerite BARTKE
Original Assignee
The Regents Of The University Of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Publication of WO2023034704A1 publication Critical patent/WO2023034704A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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 [CRISPRs]
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts
    • 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
    • C12N2503/00—Use of cells in diagnostics
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present disclosure relates to products and methods for functionally annotating genes using locally haploid, human non-cancer cells, and for determining if gene variants are clinically actionable.
  • Genetic variation is a key determinant of disease risk and can significantly impact diagnosis, prognosis and treatment outcome from patient to patient. Gaining a detailed understanding of how specific genetic variants contribute to a disease would improve patient specific clinical decisions as well as guide the development of new drugs and interventions. Thus methods to determine whether a genetic variant in an actionable gene (one that would alter clinical management) is pathogenic are needed.
  • a clinically relevant platform would allow for the assessment of a clinical genetic variant in a genetically-controlled and disease-relevant primary cell system through comparison of the phenotypic impact of a specific gene mutation to its isogenic counterpart without the mutation. For example, in the case of familial forms of BRCA deficiency, such a system could evaluate the allele specific drug sensitivity of a cell that underwent loss of heterozygosity of BRCA compared to the patients heterozygous noncancer tissue [Smith et al., Nat Genetics, 2:128-131 (1992), Futreal et al., Science, 266:120- 122 (1994)].
  • Global haploid cancer and stem cells have a strong intrinsic tendency to spontaneously endoreduplicate and re-gain diploidy [Carette et al., Nature, 477: 340-343 (2011); Sagi et al., Nature, 532: 107-111 (2016); Sagi et al., Nature Protocols, 11 : 2274- 2286 (2016); Findley etal., Nature, 562: 217-222 (2018)] reflecting their unstable genome and a non-physiological cellular state.
  • undefined alterations compensate for estimated three thousand haplo-insufficient genes in the human genome as well as the deleterious effects of recessive alleles uncovered in the haploid setting.
  • the disclosure provides products and methods for the functional annotation of gene variants in multiple human primary cell types. Classifying gene variants allows determination of, for example, drug sensitivity for inhibitors, resulting in clinically actionable patient specific information.
  • a highly efficient method to generate locally haploid cells (loHAPs) from human embryonic stem cells (hESCs) and human induced pluripotent stem cell (hiPSCs), collectively referred to herein as hPSCs.
  • hESCs human embryonic stem cells
  • hiPSCs human induced pluripotent stem cell
  • loHAPs one allele of the genomic region of interest is deleted and genetic variants are introduced on the remaining allele, creating a haploid setting where mutations can be directly functionally tested.
  • the disclosure provides locally haploid, human non-cancer cells (“loHAP cells”), wherein the cell is haploid with respect to an actionable gene.
  • the cell can be a primary cell such as a breast epithelium cell, an intestinal epithelium cell, a fibroblast, a stem cell, a glial cell, a neuron, a neural precursor cell, or a muscle cell.
  • the primary cell can be optionally immortalized, for example, by the overexpression of TERT or the catalytic component of the enzyme telomerase.
  • the stem cell can be, for example, an embryonic stem cell; a hematopoietic stem cell; a tissue-specific stem cell including a pulmonary stem cells, hepatic stem cell, skin stem cell; a neural stem cell; a mesenchymal stem cell; or an induced pluripotent stem cell.
  • the actionable gene is, for example, BRCA1 , BRCA2, TERT, POUF5, a gene of the HOXA cluster, or a gene of the FADS cluster.
  • the disclosure provides locally haploid, human non-cancer cells with a variant haploid actionable gene allele (a “variant loHAP cell”).
  • the variant haploid actionable gene can comprise mutations such as one or more insertions, one or more deletions, one or more insertions and deletions, and/or one or more substitutions as compared to a reference non-variant gene.
  • the variant haploid actionable gene can comprise an insertion, deletion or substitution in, for example, an intron sequence, an exon sequence, a promoter sequence, a terminator sequence, a translational regulatory sequence, an enhancer sequence, a silencer sequence, an insulator sequence, a boundary element, a replication origin, a matrix attachment site or a locus control region.
  • the variant actionable gene can be, for example, a variant BRCA1 or BRCA2 gene.
  • the variant BRCA2 gene can comprise, for example, one or more mutations in one or more of exon 2, 11 , 13, 15, 18, 23 and 27.
  • the disclosure provides methods of generating locally haploid, human non-cancer cells comprising deleting one allele of an actionable gene in a cell and isolating the resulting cell.
  • the deletion of one allele of an actionable gene can be generated, for example, using a targeted nuclease system such as a CRISPR/Cas system, a ZFN, or a TALEN.
  • An exemplary method comprises the steps of: a) growing cells comprising an actionable gene in culture medium supplemented with Y27632; b) nucleofecting the cells with RNPs comprising CRISPR/Cas machinery and sgRNA targeting a region 50-200 nucleotides downstream of a variant SNP in the actionable gene; c) seeding the cells in a 96-well plate at a density of 10, 30, 100 or 300 cells/well and growing the cells for up to 14 days; d) splitting the cells into replicates such that one replicate is a reference set and one replicate is subjected to DNA extractions; e) performing a PCR reaction to amplify the region comprising the desired allele deletion or a region comprising a variant SNP within the desired allele deletion, wherein the PCR primers comprise a multiplexed next generation sequencing (NGS) barcode attachment site; and f) subjecting the amplicons from e) to NGS such that wells comprising cells comprising the deletion junction or only one
  • the cells of the method can be, for example, a primary cell such as a breast epithelium cell, an intestinal epithelium cell, a fibroblast, a stem cell, a glial cell, a neuron, a neural precursor cell, or a muscle cell.
  • the primary cell can be optionally immortalized by the overexpression of TERT or the catalytic component of the enzyme telomerase.
  • the stem cell can be, for example, an embryonic stem cell; a hematopoietic stem cell; a tissuespecific stem cell including a pulmonary stem cells, hepatic stem cell, skin stem cell; a neural stem cell; a mesenchymal stem cell; or an induced pluripotent stem cell.
  • the actionable gene can be, for example, BRCA1 , BRCA2, TERT, POUF5, a gene of the HOXA cluster, or a gene of the FADS cluster.
  • the disclosure provides methods of determining the effect of genetic variation in an actionable gene in a locally haploid, human non-cancer cell comprising: making a mutation in the haploid allele of the actionable gene in the cell, and determining the activity of the variant haploid actionable gene allele of the variant cell.
  • the mutation in the haploid allele of the actionable gene can be made, for example, using a targeted nuclease system, a base editor system, a prime editor system, a transposon-based system, an HDR dependent system or an HDR independent system.
  • the mutation in the haploid allele of the actionable gene can be one or more insertions, deletions or nucleotide substitutions.
  • the activity of the variant haploid actionable gene can be determined, for example, by treating the variant cells with a compound and assaying cell response.
  • Variant cell response can be analyzed, for example, by assaying RNA expression, protein expression, reporter expression, a gain of function as compared to a reference cell, a loss of function as compared to a reference cell, change in a biological assay or change in cell growth.
  • the change in a biological assay can be, for example, an increase in sensitivity to a PARP inhibitor.
  • Variant cell response can be assayed phenotypically through use of biochemical, molecular biological or image-based assays.
  • assays that can be used in the method include phenotypic measurements such as antibody-based staining for HDR foci and other downstream assays including NGS coupled with imaging, in situ hybridization, enzymatic colorimetric techniques and the like.
  • Variant cells can be analyzed in a pool with other variant cells or as a separate isolate.
  • the variant cells of the method can be generated from, for example, a primary cell such as a breast epithelium cell, an intestinal epithelium cell, a fibroblast, a stem cell, a glial cell, a neuron, a neural precursor cell, or a muscle cell.
  • the primary cell can be optionally immortalized by the overexpression of TERT or the catalytic component of the enzyme telomerase.
  • the stem cell can be, for example, an embryonic stem cell; a hematopoietic stem cell; a tissue-specific stem cell including a pulmonary stem cells, hepatic stem cell, skin stem cell; a neural stem cell; a mesenchymal stem cell; or an induced pluripotent stem cell.
  • the actionable gene can be, for example, BRCA1 , BRCA2, TERT, POUF5, a gene of the HOXA cluster, or a gene of the FADS cluster.
  • the actionable gene can be, for example, a variant BRCA1 or BRCA2 gene.
  • the variant BRCA2 gene can comprise, for example, one or more mutations in one or more of exon 2, 11 , 13, 15, 18, 23 and 27.
  • the disclosure also provides variant cells generated by the methods.
  • the disclosure provides methods of determining the effect of genetic variation in an actionable gene in a population of locally haploid, human non-cancer cells comprising: making mutations in the haploid allele of an actionable gene in the cells to generate a population of variant cells with variant haploid alleles, and determining the activity of the drug on the population of variant cells based on changes in the relative frequencies of the variant haploid alleles in the population.
  • the locally haploid, non-cancerous human cell can be generated from a primary cell such as a breast epithelium cell, an intestinal epithelium cell, a fibroblast, a stem cell, a glial cell, a neuron, a neural precursor cell, or a muscle cell.
  • the primary cell can be optionally immortalized by the overexpression of TERT or the catalytic component of the enzyme telomerase.
  • a stem cell can be, for example, an embryonic stem cell; a hematopoietic stem cell; a tissue-specific stem cell including a pulmonary stem cells, hepatic stem cell, skin stem cell; a neural stem cell; a mesenchymal stem cell; or an induced pluripotent stem cell.
  • the actionable gene can be, for example, BRCA1 , BRCA2, TERT, POUF5, a gene of the HOXA cluster, or a gene of the FADS cluster.
  • the actionable gene can be, for example, a variant BRCA1 or BRCA2 gene.
  • the variant BRCA2 gene can comprise, for example, one or more mutations in one or more of exon 2, 11 , 13, 15, 18, 23 and 27.
  • the disclosure also provides variant cells generated by the methods.
  • the disclosure provides methods of treatment of a patient wherein the method of treatment is determined by the effect of genetic variation in an actionable gene in a variant locally haploid, non-cancer human derived from the patient by a method described herein, for example, a method of the preceding two paragraphs.
  • the patient has, for example, cancer (such as breast cancer, ovarian cancer or prostate cancer), a cardiovascular disease, a central nervous system disease, a metabolic disease or an autoimmune disease.
  • an actionable gene includes, but is not limited to, a change in the transcription start site, splice site(s), or one or more amino acids in a protein encoded by the actionable gene.
  • Figure 1 Design and gene editing pipeline of loHAPs.
  • 1a General design of loHAPs.
  • 1b Specific design of loHAPs for each gene of interest, with deletion size specified.
  • 1c A scalable high-throughput gene editing pipeline combining limit dilution into 96-well right after nucleofection and multiplex NGS genotyping.
  • Figure 2 Generation and characterization of loHAPs.
  • 2a Detection of junctions of designed deletion for all 6 genes in bulk after CRISPR machinery delivered as RNP.
  • 2b Allelic profiles of the 96-well plates from which the loHAPs of BRCA1 , BRCA2 and POU5F1 were isolated. Wells which did not yield enough reads for accurate allele calling are uncolored.
  • 2c Allelic profiles of the final established loHAPs of BRCA1 , BRCA2 and POU5F1 comparing to the parental diploid cells.
  • Figure 3 Pluripotency of the BRCA2 loHAP hiPSC and hESCs. Immunostaining for pluripotency marker SSEA4 and OCT4 as well as histochemistry staining for the pluripotency marker alkaline phosphatase (AP). Scale bar, 20pm.
  • FIG. 4 Annotating functional protein domains in BRCA2 using CRISPR- mediated mutagenesis in BRCA2 loHAPs.
  • 4a Schematic representation of annotating genetic variants using loHAPs.
  • a gene of interest e.g., BRCA2
  • the remaining allele is mutagenized to identify alleles that are permissive and stably maintained in the cell pool.
  • the pool of cells is challenged (e.g., with a PARP inhibitors in BRCA2 loHAPs) to identify variants sensitive to the treatment.
  • 4b Schematic representation of the BRCA2 locus and the BRCA2 protein domain structure.
  • 4e Heatmap indicating the allele frequency changes, Iog2 (day14/day7), of individual frame-shift mutation in 3 biological replicates.
  • 4f Quantification of categorized allele frequencies as in c for an sgRNA targeting exon 2 assayed in diploid and BRCA2 loHAPs hESCs at day 7, 14 and 21 after editing.
  • 4g Quantification of the relative depletion of frame-shift mutation in exon 2 comparing samples collected at day 7 and 14 in diploid and BRCA2 loHAPs hESCs.
  • Whiskers defined by Tukey’s method 4h, Quantification of categorized allele frequencies as in c for an sgRNA targeting exon 2 assayed in diploid and BRCA2 loHAPs hPSCs-derived fibroblasts at day 7, 14 after editing. 4i, Quantification of the relative depletion of frame-shift mutation in exon 2 comparing samples collected at day 7 and 14 in diploid and BRCA2 loHAPs hPSCs-derived fibroblasts. Whiskers defined by Tukey’s method. 4j, Quantification of categorized allele frequencies as in 4c for sgRNAs targeting exon 27 assayed in diploid and BRCA2 loHAPs hiPSCs at day 7, 14 and 21 after editing. 4k, The top 10 alleles survived 3-weeks post mutagenesis, frameshift mutations in grey.
  • Figure 5 Essentiality assay of the C-terminus of BRCA2 using CRISPR-mediated mutagenesis in loHAPs hESCs and fibroblasts. Quantification of categorized allele frequencies (unedited, in-frame and frame-shift mutations) in exon 27 assayed in diploid and BRCA2 loHAPs hESCs and fibroblasts at day 7, 14 and 21 after editing.
  • FIG. 6 Using HDR mediated genome engineering in BRCA2 loHAPs to assess functionality of designer mutations.
  • 6a HDR-based editing strategy to introduce designer mutations in exon 11 of BRCA2. Shown is the targeted sequence with the PAM shaded in grey. Indicated below are the two HDR single-stranded repair oligos specifying the mutations that result in either synonymous, or non-sense mutations.
  • 6b Quantification of categorized allele frequencies (unedited, in-frame, HDR-synonymous mutation and frame-shift mutations) in exon 11 assayed in diploid and BRCA2 loHAPs hiPSCs at day 7 and 14 after editing.
  • 6c Analysis as in 6b, using the HDR template introducing a non-sense mutation.
  • 6d HDR-based editing strategy to perform alanine scanning in BRCA2. Shown is the targeted sequence within exon 2 with the PAM shaded in grey. Indicated below are examples for the HDR single stranded repair oligos, specifying the mutations that result in alanine replacements. The start codon is indicated in grey.
  • 6e Quantification of categorized allele frequencies (unedited, in-frame, HDR alanine substitution and frame-shift mutations) in exon 2 assayed in diploid and BRCA2 loHAPs hiPSCs at day 7 and 14 after editing.
  • 6f Analysis as in e, using diploid and BRCA2 loHAPs hESCs.
  • 6g Shown is a representative allele spectrum for an exon 2 Alanine scanning experiment in BRCA2 loHAPs hiPSCs with annotations of amino acid or nucleotide changes on the right.
  • 6h Quantification of individual alanine mutations in diploid and BRCA2 loHAPs hiPSCs at day 7 and day 14 after editing. For reference a dashed line indicates an allele frequency of 0.5%, a frequency that can be robustly quantified by NGS in our analysis
  • Figure 7 Using BRCA2 loHAPs to identify hypomorphic BRCA2 mutations that sensitize cells to radiation and standard of care cancer PARP inhibitors.
  • 7a Schematic of the experimental design to identify hypomorphic BRCA2 mutations.
  • Cells (diploid or BRCA2 loHAPs) are mutagenized using editing strategies outlined in Figures 4-6. After allele stabilization (3-4 weeks after editing) cells are exposed to 2 Gy of gamma- radiation, 1 pM or 2 pM of Niraparib or Olaparib or remain untreated. Changes in allele frequency are quantified one week after treatment starts.
  • Figure 8 Dose-response curve of irradiation and PARPi on hPSCs.
  • 8a Dose-response of irradiation on hPSCs at 0, 0.2, 0.5, 1 , 2 and 5 Gy.
  • 8b Dose-response of Niraparib on hPSCs at 0, 0.5, 1 , 2 or 5pM.
  • 8c Dose-response of Olaparib on hPSCs at 0, 0.5, 1 , 2 or 5pM.
  • Figure 9 Introducing clinically observed BRCA2 mutations in loHAPs hPSCs.
  • 9a Shown is a representative allele spectrum for an experiment introducing clinically observed BRCA2 exon 2 mutations in BRCA2 loHAPs hiPSCs at Day 7, with annotations of the ClinVar-documented amino acid changes on the right.
  • 9b Shown is a representative allele spectrum as in 9a in BRCA2 loHAPs hESCs.
  • Figure 10 Schematic outlining a proposed application for loHAPs. Rapid assessment of drug efficacy in multiple cell types in the context of patient specific mutations to guide therapeutic decisions.
  • FIG. 11 Alanine mutagenesis in BRCA2 loHAPs hESCs and hiPSCs. Shown is a representative allele spectrum for an exon 2 Alanine scanning experiment in BRCA2 loHAPs hESCs with annotations of amino acid or nucleotide changes on the right.
  • Figure 12 Using HDR mediated genome engineering in BRCA2 loHAPs to assess functionality of designer mutations.
  • 12a Allele frequency changes overtime of mutations generated in BRCA2 exon 2 alanine scanning in BRCA2 LoHAPs hPSCs, represented as Iog2 (day 14/day7), circle and Iog2 (day 21/day7), triangle.
  • 12b Normalized allele frequency changes upon treatment of PARP inhibitor, Niraparib, of BRCA2 exon 2 alanine mutations in BRCA2 LoHAPs hPSCs, represented as Iog2 (drug/mock). 1 pM, circle; 2pM, triangle.
  • Figure 13 Schematics of editing strategy for generating G2508S, K2729N and Y3035S. Shown is the targeted sequence with the PAM shaded in grey. Indicated below are HDR single stranded repair oligos.
  • Figure 14 Using HDR mediated genome engineering in BRCA2 loHAPs to assess functionality of designer mutations.
  • 14a Normalized allele frequency changes overtime of G2508, K2729N and Y3035S in BRCA2 loHAPs hPSCs, with WT and one frameshift mutation at each locus as controls, represented as Iog2 (day 14/day7), circle and Iog2 (day 21/day7), triangle.
  • 14b Normalized allele frequency changes upon treatment of PARP inhibitor, Niraparib, of G2508, K2729N and Y3035S in BRCA2 loHAPs hPSCs, represented as Iog2 (drug/mock). 1 pM, circle; 2pM, triangle.
  • Figure 15 Allele frequency changes overtime of mutations generated around G2508, K2729 and Y3035.
  • 15a Allele frequency changes overtime of mutations generated around G2508 in BRCA2 loHAP hPSCs. Represented as Iog2 (day 14/day7), circle and Iog2 (day 21/day7), triangle.
  • 15b Same as 15a, for K2729.
  • 15c Same as 15a, for Y3035
  • Figure 16 Using BRCA2 loHAPs to identify functionally critical amino acids by CRISPR-mediated deletion tiling in BRCA2 exon 2.
  • 16a Schematic of editing strategy in the deletion tiling experiments on BRCA2 exon 2. Grey boxes indicate protospacers.
  • 16b The full deletion profile generated and heatmaps of allele frequency changes overtime of each mutation, with deletion category labels to the left and sample labels at the bottom (circle, day 7; triangle, day 14; rectangle, day 21 ). Allele frequencies of each mutation in each replicate were normalized to the corresponding day 7 frequencies. Similar data was obtained when plotting in frame and frameshift mutations generated by insertions ( Figure 19a).
  • 16c As in 16b, but now only showing A2, A4, A5 alleles (frameshift mutations), and A3, A6, A9 alleles (in frame mutations).
  • 16d Categorized allele frequency changes at day 21 comparing to day 7, presented as Iog2(day 21/day7).
  • 16e Functional score of each amino acid in BRCA2 exon 2 calculated by a linear regression model.
  • 16f Categorized allele frequency changes of F11 , F12 deletions at day 21 comparing to day 7, presented as Iog2(day 21/day7).
  • 16g Normalized allele frequency changes of amionacid F11 , F12 deletions at day 21 comparing to day 7, presented as Iog2(day 21/day7), in a targeted validation experiment, with allele category labels to the right.
  • Figure 17 A representative deletion profile of lentiviral sgRNA library-based deletion tiling in BRCA2 exon 2.
  • Figure 18 Using BRCA2 loHAPs to identify functionally critical amino acids by CRISPR-mediated mutation tiling in exon 27.
  • 18a Schematic of editing strategy in the deletion tiling experiments on BRCA2 exon 27. Grey boxes indicate protospacers. conserveed amino acids across species are highlighted in the multiple sequence alignment for the RAD51 binding region.
  • 18b The full deletion profile generated and heatmaps of allele frequency changes overtime of each mutation, with deletion category labels to the left and sample labels at the bottom (circle, day 7; triangle, day 14; rectangle, day 21). Allele frequencies of each mutation in each replicate were normalized to the corresponding day 7 allele frequencies. Similar data was obtained when plotting in frame and frameshift mutations generated by insertions ( Figure 19b).
  • 18c The deletion profile of A2, A4, A5 frameshift mutations and heatmaps of allele frequency changes upon irradiation or PARP inhibition of each mutation, with deletion category labels to the left and sample labels at the bottom (circle, mock; triangle, irradiation; rectangle, Niraparib; diamond, Olaparib). Allele frequencies of each mutation were normalized to the corresponding frequencies in the mock condition. 18d, Categorized allele frequency changes upon irradiation comparing to the mock condition, presented as Iog2 (irradiation/mock). The x axis labels shown the position of the last intact amino acid. 18e, As in 18d, shown PARP inhibition with Niraparib. 18f, As in 18d, shown PARP inhibition with Olaparib.
  • Figure 19 Insertion profile in CRISPR tiling experiments.
  • 19a The full insertion profile generated in exon 2 CRISPR tiling experiments and heatmaps of allele frequency changes overtime of each mutation, with insertion category labels to the left and sample labels at the bottom (circle, day 7; triangle, day 14; rectangle, day 21 ). Allele frequencies of each mutation in each replicate were normalized to the corresponding day 7 frequencies.
  • 19b As in 19a, shown exon 27 CRISPR tiling experiments.
  • the disclosure provides a local haploid (loHAP), human non-cancer cell system in which one allele of a genomic region of interest is deleted and genetic variants are introduced on the remaining allele (a loHAP system).
  • a loHAP system the biological impact of any genetic variant within a region of interest can be directly assessed in genetically-defined, primary human cells and/or their differentiated derivatives.
  • Panels of genetic variants are introduced, for example, by targeted nuclease-based [e.g., CRISPR/Cas, Zinc finger nuclease, transcription activator-like effector nuclease (TALEN)], base-editor, prime-editor, transposon-based, HDR or HDR-independent mutagenesis, or through use of other methods known in the art for introduction of mutations in a nucleic acid sequence.
  • the resulting variant loHAP cells are then arrayed out and analyzed as isogenic cell clones.
  • cells are maintained as a pooled variant cell libraries that differ exclusively at haploid locus of interest.
  • Phenotypes of arrayed cells are measured by biochemical or molecular biological assays on individual cell lines carrying specific mutations. Assays that can be used in the method include phenotypic measurements such as antibody-based staining for HDR foci and other downstream assays including NGS coupled with imaging, in situ hybridization, enzymatic colorimetric techniques and the like. Alternatively, pooled variant cell libraries are screened for a phenotype of interest (e.g., sensitivity to a specific drug) using the changes in the allele frequency of genetic variants present in the pool as a readout. This can be done by comparing the alleles present in treated and non-treated cell populations.
  • a phenotype of interest e.g., sensitivity to a specific drug
  • Changes in the allele frequency can be used to quantify the functionality of a variant under a treatment regime. Moreover, the changes in allele frequency can be used to screen for drugs that alter the gene(s) encoded in the modified genomic region.
  • the haploid variants can be linked to a reporter function allowing the monitoring of allele expression in response to an applied condition (e.g., drug substance, growth condition and the like).
  • the loHAP system allows a direct correlation between genotype and phenotype. This significantly increases the sensitivity of the phenotypic evaluation of a specific mutation and increases the number of mutations that can be interrogated in a given screen.
  • the loHAP system can functionally screen thousands of genetic variants. It can be used to screen, for example, for functional protein domains, regulatory genetic elements and disease-causing SNPs. Specific screening examples include, but are not limited to, screening for transcription start sites, splice site(s), and/or an amino acid change or changes in an encoded gene product.
  • this loHAP system can be employed to screen for genetic variants responsive or resistant to existing drugs, and also to screen for novel drug candidates.
  • loHAP cells can be locally haploid, human embryonic stem cells (hESCs).
  • loHAP cells can be locally haploid, induced pluripotent stem cells (iPSCs).
  • hESCs and IPSCs are collectively referred to herein as human pluripotent stem cells (hPSCs).
  • loHAP cells can be locally haploid hPSCs.
  • loHAP cells can be fibroblasts differentiated from an hESC.
  • loHAP cells can also be made from other primary cell types including, but not limited to, muscle cells, fibroblasts, neurons, glial cells, intestinal cells, cells present in ocular tissue (e.g., retinal pigmented epithelium, retinal ganglion cells, photoreceptor cells), blood cells (e.g., T cells, B cells, NK cells, platelets), hepatic cells, kidney cells, stem cells including hematopoietic stem cells, tissue specific stem cells (e.g., pulmonary stem cells, hepatic stem cells), embryonic stem cells, mesenchymal stem cells, neural stem cells, skin stem cells and induced pluripotent stem cells.
  • ocular tissue e.g., retinal pigmented epithelium, retinal ganglion cells, photoreceptor cells
  • blood cells e.g., T cells, B cells, NK cells, platelets
  • hepatic cells e.g., kidney cells, stem cells including hematopoietic stem cells,
  • loHAP cells are not cancer cells or derived from cancer cells (in other words, loHAP cells are non-cancer cells). loHAP cells can be immortalized by the overexpression of TERT or the catalytic component of the enzyme telomerase in the loHAP cells.
  • loHAP cells are cells rendered locally haploid by laboratory deletion of one allele of at least one actionable gene.
  • An “actionable gene” herein is a gene for which gene variants are of clinical significance for a patient.
  • variant refers to a polynucleotide or polypeptide having a sequence substantially similar to a reference polynucleotide or polypeptide.
  • a variant can have deletions, substitutions, additions of one or more nucleotides at the 5' end, 3' end, and/or one or more internal sites as compared to a reference polynucleotide.
  • a variant nucleotide or polypeptide sequence may have one or more nucleotide or amino acid substitutions as compared to a reference polynucleotide or polypeptide.
  • variants and/or differences in sequences between a variant and the reference polynucleotide can be detected using conventional techniques known in the art, for example polymerase chain reaction (PCR) and hybridization techniques.
  • variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis.
  • a variant of a polynucleotide including, but not limited to, a DNA, can have at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88% about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the reference polynucleotide as determined by sequence alignment programs known by skilled artisans.
  • a variant can have deletions, substitutions, additions of one or more amino acids in comparison to the reference polypeptide.
  • a variant of a polypeptide can have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88% about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the reference polypeptide as determined by sequence alignment programs known by skilled artisans.
  • a loHAP cell may comprise locally haploid regions comprising one, two, three, four, five, six, seven, eight, nine, ten or more actionable genes. In some instances, loHAP systems are used to investigate intragenic interactions between one or more actionable genes.
  • a loHAP actionable gene can be the Breast cancer gene 1 (BRCA1) or Breast cancer gene 2 (BRCA2) gene.
  • a loHAP actionable gene can be telomerase reverse transcriptase gene (TERT), POU class 5 homeobox 1 gene (POU5F1), Homeobox A (HOXA) cluster, or the fatty acid desaturase (FADS) cluster.
  • TERT telomerase reverse transcriptase gene
  • POU5F1 POU class 5 homeobox 1 gene
  • HOXA Homeobox A
  • FADS fatty acid desaturase
  • the disclosure provides an exemplary BRCA2 gene loHAP.
  • a variant loHAP cell can be used to determine the effect of genetic variation in the haploid allele of its actionable gene.
  • a mutation can be introduced into the allele and its effect determined.
  • the effect of a single mutation in a pool of variant loHAP cells containing a single mutation in the allele can be determined.
  • the effect(s) of multiple mutations in a pool of variant loHAP cells containing different mutations in the allele can be determined.
  • a mutation in a loHAP actionable gene allele can be a deletion of a nucleotide or nucleotides in the allele.
  • a mutation can be an insertion of a nucleotide or nucleotides.
  • a mutation can be a substitution of a nucleotide or nucleotides.
  • a variant loHAP actionable gene allele can have a mutation found in nature.
  • a variant loHAP actionable gene allele can have a mutation made by any gene editing technique that introduces a mutation in the haploid actionable gene allele. Mutations may be introduced by any method known in the art including, but not limited to, targeted mutagenesis using a targeted nuclease (e.g., a CRISPR/Cas system including systems based on Cas9, Cas12, Cas 13, Cas14, Cas X, Cas Y and Cas Z; ZFN; TALEN; MegaTALI or meganuclease), or through use of a base editor or a prime editor.
  • CRISPR/Cas9 can be used to make a mutation by causing a targeted cleavage followed by error-prone non- homologous end joining.
  • a mutation can be made using CRISPR/Cas9 and a template for homology directed repair.
  • a variant loHAP actionable gene allele can be a BRCA2 gene allele and the BRCA2 gene allele can have a mutation in one or more of exon 2, exon 11 , exon 13, exon 15, exon 18, exon 23 and exon 27.
  • a variant loHAP actionable gene allele can be a BRCA2 gene allele and the BRCA2 gene allele can have a mutation in exon 2.
  • a variant loHAP actionable gene allele can be a BRCA2 gene allele and the BRCA2 gene allele can have a mutation in exon 11 .
  • a variant loHAP actionable gene allele can be a BRCA2 gene allele and the BRCA2 gene allele can have a mutation in exon 13.
  • a variant loHAP actionable gene allele can be a BRCA2 gene allele and the BRCA2 gene allele can have a mutation in exon 15.
  • a variant loHAP actionable gene allele can be a BRCA2 gene allele and the BRCA2 gene allele can have a mutation in exon 18.
  • a variant loHAP actionable gene allele can be a BRCA2 gene allele and the BRCA2 gene allele can have a mutation in exon 23.
  • a variant loHAP actionable gene allele can be a BRCA2 gene allele and the BRCA2 gene allele can have a mutation in exon 27.
  • a variant loHAP actionable gene can be a pathogenic gene variant.
  • gene variants can be BRCA2 variants that are associated with breast cancer.
  • gene variants can be BRCA2 variants that are associated with ovarian cancer.
  • gene variants can be BRCA2 variants that are associated with pancreatic cancer.
  • gene variants can be BRCA2 variants that are associated with prostate cancer.
  • gene variants can be BRCA2 variants that are associated with colorectal cancer.
  • gene variants can BRCA2 variants that are associated with melanoma.
  • a loHAP actionable gene variant can be a gene variant that renders a cell with the gene variant sensitive to a medical treatment.
  • a gene variant can render a cell sensitive to a drug.
  • a gene variant can render a cell sensitive to a chemotherapeutic drug.
  • a gene variant can render a loHAP with a mutation in a BRCA2 haploid allele sensitive to a Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi).
  • PARPi Poly (ADP-ribose) polymerase
  • Exemplary PARPi inhibitors are Olaparib (Lynparza), Rucaparib (Rubraca), Niraparib (Zejula), and Talazoparib (Talzenna).
  • a loHAP system can be utilized to screen potential drug molecules for effects in different diseases.
  • the system can be used to study and/or identify specific targets of interest in disease states such as cancer (e.g., breast cancer, ovarian cancer and prostate cancer), metabolic disease, obesity, cardiovascular disease, diabetes, neurological disease, autoimmune disease, infectious disease, trinucleotide repeat disorders and hematological disorders.
  • cancer e.g., breast cancer, ovarian cancer and prostate cancer
  • the disclosure provides methods of generating a loHAP cell.
  • a method of generating a loHAP comprises: deleting one allele of a BRCA2 gene in a hPSC and isolating the resulting loHAP or pool of loHAPs.
  • the deletion of the allele of an actionable gene, such as a BRCA2 gene, in a cell can be carried out by any gene editing technique that accomplishes the deletion.
  • Successful deletions can be accomplished by delivery of Cas9 and two or multiple sgRNAs whose targets flank the genomic regions of deletion.
  • Cas9 and sgRNAs can be nucleofected into cells as ribonucleoprotein (RNP) complex using techniques known in the art.
  • sgRNA can have a, for example, a criteria of specificity score >70 [Hsu et al., Nature biotechnology, 31: 827-832 (2013)].
  • Successful introductions of desired deletions can be assayed by standard techniques including sequencing.
  • Other potential methods that can be used to generate a loHAP include delivery of the targeted nucleases by delivery of one or more plasmids encoding the nuclease; use of electroporation to introduce the RNP or delivery plasmid; or use of gel electrophoretic genotyping.
  • the deletion can be made using CRISPR/Cas9.
  • the deletions can be made, for example, by contacting a target cell with one or more RNPs comprising a CRISPR/Cas system comprising two or more guide RNAs.
  • the RNPs can be introduced into the target cells by, for example, nucleofection.
  • RNP treated cells are seeded by limited dilution into multi-well plates. The plates are incubated at an optimal growth temperature. The plates are then split into two plates creating duplicate cultures. One plate is reserved to maintain the cells while the other is used for genotyping by multiplexed next generation sequencing (NGS) of PCR amplicons generated in the regions comprising the desired deletions.
  • NGS next generation sequencing
  • Analysis of the sequencing data confirms the loss of specific sequences and/or SNPs within the deleted regions. Sequencing of the deletion junctions also confirms the exact size and placement of the introduced deletion.
  • the disclosure provides methods of determining the effect of genetic variation in an actionable gene in a cell.
  • the disclosure provides a method comprising: making a mutation in the haploid allele of an actionable gene in a loHAP cell, isolating the variant loHAP cells with the variant haploid allele and determining the activity of the variant haploid actionable gene allele of the variant loHAP cell.
  • a method of determining the effect of genetic variation in a BRCA2 gene in a cell comprises: making a mutation in the haploid allele of the BRCA2 gene in the loHAP cell, isolating the variant loHAP cells with the variant haploid allele and determining the BRCA2 tumor suppressor activity of the loHAP cells.
  • a method of determining the effect of genetic variation in a BRCA2 gene in a cell comprises: making a mutation in the haploid allele of the BRCA2 gene in the loHAP cell, isolating the variant loHAP cells with the variant haploid allele and determining the PARPi sensitivity of the variant loHAP cells.
  • a method of determining the effect of genetic variation in an actionable gene in a population of cells comprises: making mutations in the remaining haploid allele of the actionable gene in a loHAP cell to generate a population of variant loHAP cells with variant haploid alleles, and determining the activity of the drug on the population of variant loHAP cells based on changes in the relative frequencies of the variant haploid alleles in the population.
  • the population of variant cells can comprise different types of mutations within the actionable gene.
  • the population of variant cells can comprise mutations in more than one actionable gene.
  • the disclosure provides methods of treatment of a patient wherein the method of treatment is determined by the effect of genetic variation in an actionable gene allele derived from the patient in a loHAP system.
  • Mutations in the BRCA2 gene are associated with sporadic and familial cancer, cause genomic instability, and sensitize cancer cells to PARP inhibition.
  • the present disclosure provides an exemplary human primary cell system to annotate variants in BRCA2 and to test the variants’ sensitivities to PARP inhibition.
  • LoHAP cells that lack one copy of BRCA2 were engineered from hPSCs or fibroblasts.
  • VUS uncertain significance
  • BRCA variants have previously been introduced into cancer cells using non-homologous end joining (NHEJ), homology directed repair (HDR) [Findlay et a!., Nature, 562:217-222 (2016), or base editing-mediated mutagenesis [Hanna et a!., Cell, 184:1064-1080 (2021 )].
  • NHEJ non-homologous end joining
  • HDR homology directed repair
  • a key limitation of these previous experiments towards their clinical translation concerns their use of cancer cell lines which inevitably harbor uncharacterized mutations in the DNA damage and repair pathways. These cell line-specific idiosyncrasies alter cellular responses to BRCA perturbation and complicate interpretation and generalization of the results.
  • a clinically relevant platform would allow for the assessment of a clinical BRCA variant in a genetically-controlled and disease-relevant primary cell system through comparison of the phenotypic impact of a specific BRCA mutation to its isogenic counterpart without the mutation.
  • haploid cells have a strong intrinsic tendency to spontaneously endoreduplicate and re-gain diploidy reflecting their instable genome and a non-physiological cellular state.
  • the BRCA2 gene comprises twenty-seven exons and encodes a full-length BRCA2 protein of 3418 amino acids with previously annotated protein domains of distinct functions [Esashi etal., Nature, 434: 598-604 (2005); Roy et a!., Nat Rev Cancer, 12: 68-78 (2011)] ( Figure 4b). Out of the ⁇ 10,000 BRCA2 variants in ClinVar, more than 5500 are listed as VUS which causes difficulties in their functional and therapeutic interpretation.
  • loHAP system was also used to functionally annotate BRCA2 variants in cell types differentiated from hPSCs. Indels were introduced in exon 2 of fibroblasts differentiated from either diploid or BRCA2 loHAPs. Frameshift mutations in exon 2 of BRCA2 were depleted more slowly in hPSC-derived fibroblasts than in hPSCs ( Figure 4h). Importantly, again, variants were more effectively annotated in variant loHAP cells than their diploid counterpart ( Figure 4i). Together this data demonstrates that loHAP systems can also facilitate the annotation of variants in differentiated cell types.
  • a challenge in the clinical interpretation of BRCA2 variants is to identify hypomorphic mutations that, while retaining some BRCA2 function, are still sensitive to PARP inhibition.
  • loHAP systems were tested for functionally annotating such hypomorphic alleles.
  • frameshift mutations were generated in exon 27, the last coding exon of BRCA2, where hypomorphic alleles have been identified in genetically- engineered mouse models and VUS are prevalent in patients.
  • Frameshift mutations were more tolerated in exon 27 and the enrichment for the unedited allele and in-frame mutations was reduced.
  • the unedited and in-frame alleles increased to less than 50% of all alleles in the diploid cells after 3 weeks ( Figure 4j, Figure 5).
  • the disclosure provides an efficient gene editing platform to generate, clonally isolate and genotype genome-engineered hPSCs at speed and scale (Figure 1c). It allows the efficient generation of locally haploid, non-cancer cells, loHAP cells, in which large genomic regions of interest excised from a single allele, from hPSCs.
  • loHAP cells only one allele of gene of interest is present thus the allele frequencies in a cell population report on the fitness of mutations in response to an environmental or genetic perturbation, while the main challenge of inherent genetic and cellular abnormality associated with the use haploid cancer cells [Carette et al., Science, 326: 1231-1235 (2009)] and global haploid human pluripotent stem cells [Sage et al., Nature, 532: 107-111 (2016)] is overcome.
  • the disclosure provides proof of concept for functional annotation of mutations introduced on the remaining gene copy in loHAP cells.
  • In-frame, frameshift, and designer mutations can be functionally annotated more effectively and accurately in variant loHAP cells as compared to their diploid counterpart.
  • sensitizing alleles in the remaining copy of the BRCA2 gene can be screened for response to drug treatments and ionizing irradiation by measuring changes in the relative allele frequencies.
  • This functional genomic approach in loHAP systems presents two key technical advances.
  • Example 1 describes the hPSC editing methodology that allows for the efficient excision of an entire gene of interest and generation of clonal loHAP cells.
  • Example 2 demonstrates the power of variant loHAP cells to annotate gene function.
  • Example 3 demonstrates the power of variant loHAP cells to assay drug sensitivity.
  • Example 4 demonstrates using loHAPs for comprehensive characterization of protein domains
  • RNAs used to generate loHAP cells were selected with criteria of specificity score [Hsu et al., Nature biotechnology, 31: 827-832 (2013)] >70 from a region 50-200bp downstream of the TES using the CRISPR Targets track [Haeussler etal., Genome biology, 17: 148 (2016) in UCSC genome browser.
  • Chemically-modified sgRNAs were purchased from Synthego.
  • Cas9 protein was bought from the QB3 MacroLab, UC Berkeley.
  • ribonucleoprotein 300 pmol sgRNA and 80 pmol Cas9 were mixed in nuclease-free water to a final volume 1 Opl, then incubated at room temperature for 10min before nucleofection.
  • hPSCs A total of 300 pmol sgRNAs targeting sites flanking the intended deletion region were delivered to hPSCs as RNPs by nucleofection.
  • hPSCs cultured on MEF were detached from feeder cells by treating with 1 mg/ml collagenase IV and 0.5U/ml dispase for 20 min. Detached colonies were washed with DMEM/F12 once then dissociated to single cells using 1x accutase (SCR005, MilliporeSigma) for 5 min at 37°C followed by one wash with DMEM/F12 followed by a wash with phosphate buffered saline (PBS).
  • 1x accutase SCR005, MilliporeSigma
  • hPSCs were pelleted and then resuspended in 20pl Lonza P3 primary nucleofection reagent, mixed with pre-assembled Cas9/sgRNA RNP with or without 100pmol ssODN HDR donor, then nucleofected using program CA137 on Lonza nucleofector 4D. Nucleofection of hPSC-derived fibroblasts followed the same protocol except cells were dissociated with 0.25% trypsin.
  • the cell lysates from the 96-well plates pre-loaded with lysate buffer were incubated at 50°C overnight then heated to 95°C for 10 min to inactivate proteinase K.
  • a region locating within the desired deletion harboring a single-nucleotide polymorphism (SNP) in the hPSC cell line used were identified from whole genome sequencing data, and amplified from 2pl cell lysis from each well with primers contains NGS barcode attachment sites (GCTCTTCCGATCT) (SEQ ID NO: 1) and Titan DNA polymerase.
  • Amplicons were then purified with automated SPRI beads purification in UC Berkeley DNA Sequencing Facility, and i5/i7 barcoded, pooled and sequenced on 150PE iSeq or 300PE MiSeq in the NGS core facility at the Innovative Genomics Institute (IGI).
  • NGS data was analyzed by CRISPResso2 [Clement et al., Nature Biotechnology, 37: 224-226 (2019)] to identify wells only containing one allele at the SNP.
  • cell clones were genotyped by deletion detecting PCR in which the deletion junctions were amplified using a pair of primers flanking the deletion region and resolved by agarose electrophoresis and Sanger sequencing. Cells in those identified wells were then subcloned by low density seeding, manually picking and genotyping to establish the loHAP lines.
  • FIG. 1 b shows the allele deletions made for the TERT gene, POU5F1 gene, BRCA1 gene, BRCA2 gene, HOXA cluster and FADS cluster, with the deletion size indicated for each.
  • Figure 2a shows the deletions generated were those expected to be generated by the CRISPR machinery delivered as RNPs. From left to right, junctions formed by BRCA2, HOXA, POU5F1 , BRCA1 , TERT and FADS deletion upon delivery of pairs of sgRNAs as RNP, with unedited cells as negative controls (-)
  • Figure 2b shows allelic profiles of the 96-well plates for the loHAP cells of BRCA1 , BRCA2 and POU5F1 .
  • wells that did not yield enough reads are uncolored.
  • Figure 2c shows DNA sequences of alleles of the starting diploid hPSCs and the final established BRCA1 , BRCA2 and POU5F1 loHAP cells.
  • the pluripotency of the loHAP cells was established by immunostaining for pluripotency marker genes SSEA4, OCT4 and histochemistry staining for the hPSCs marker alkaline phosphatase.
  • pluripotency marker genes SSEA4, OCT4 and histochemistry staining for the hPSCs marker alkaline phosphatase For immunofluorescent staining, cells were fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 in PBS for 30 min at RT, blocked in 3% BSA in PBS for 60 min at RT, then incubated in primary antibody (PCRP-POU5F1-1 D2, MC-813-70 (SSEA-4), Developmental Studies Hybridoma Bank) overnight at 4°C, secondary antibody (Thermo Fisher A11001) for 2 hours at RT.
  • Variant BRCA2 loHAP cells were used to functionally annotate the BRCA2 gene.
  • the BRCA2 gene comprises twenty-seven exons and encodes a full length BRCA2 protein of 3418 amino acids that fold into protein domains with distinct functions [Esashi et al., Nature, 434: 598-604 (2005) and Roy et aL, Nature Reviews, Cancer, 12: 68-78 (2011)].
  • Figure 4b shows schematic representations of the BRCA2 locus and the BRCA2 protein domain structure.
  • hPSC-derived fibroblasts were differentiated from hPSCs by harvesting hPSCs colonies with 1 mg/ml collagenase IV, washing three times with 5% FCS in DMEM by gravitational settling, then culturing in suspension to form embryonic bodies (EBs) in KSR medium (hPSC medium w/o bFGF) in ultralow attachment 6 well plates.
  • EBs embryonic bodies
  • fibroblast medium (15% FBS in DMEM, 1x NEAA, 1x penicillin/streptomycin, 1x glutamine).
  • EBs were harvested and seeded onto 0.2% gelatin-coated 10 cm dishes in fibroblast medium with medium change weekly until fibroblast morphology established. Human fibroblasts were then passaged with 0.25% trypsin every week with 1 :3 splitting ratio until used in the experiment. Table 4
  • Figure 4c-j shows quantification of categorized allele frequencies (unedited, inframe and frame-shift mutations) in exons 2, 11 , 13 and 27 assayed in diploid and BRCA2 variant loHAP cells using the primers shown in Table 5 below at days 7, 14 and 21 after editing.
  • ssODN singlestranded donor oligonucleotides
  • loHAP systems were used to identify and annotate hypomorphic BRCA2 alleles that are sensitive to irradiation or PARP inhibition ( Figure 7a).
  • loHAPs can be used to nominate hypomorphic alleles that are sensitive to PARPi and that the relative response to the drug treatment reflects the level of defect that a mutation imposes on their biological activity, in this BRCA2 to mediate HR. This quantitative information gained from loHAP cells is expected to be useful to inform clinical decisions on the therapeutic strategy.
  • loHAPs for comprehensive characterization of protein domains
  • BRCA2 was mutagenized using every possible sgRNA (as determined by the spCAS9 NGG protospacer adjacent motif) in exon 2 ( Figure 16a). Two approaches were taken: (1) delivery of the editing components as RNPs and (2) infection of cells with a lentiviral library expressing that sgRNA followed by CAS9-protein transfection.
  • oligonucleotides to clone lentiviral sgRNA expression vectors targeting exon 2 and 27 were purchased as oligo pools (IDT) cloned into digested pJR104 BstXI/Blp1 (R0113S & R0585, NEB) double (a gift from Jonathan Weissman) using NEBuilder Hi Fi DNA Assembly kit (E2627, NEB). Library complexity and quality was confirmed using next generation sequencing (each sgRNA had a coverage > 100x.
  • 10 pg sgRNA library plasmids were transfected into one -8 million HEK293T cells together with 7.5 pg psPAX2 (#12260, Addgene) and 2.5 pg pMD2.G (#12259, addgene) using LipofectamineTM 2000 (11668019, Thermo Fisher). Lentivirus were collected daily, filtered through 0.45pm filter and frozen at -80°C. for the lentivirus-based mutagenesis, human PSCs were detached from MEFs by collagenase IV and dissociated to single cells by accutase as described above.
  • -5x10 4 cells were either irradiated in suspension with the indicated dose of gamma irradiation and seeded back onto MEF, or seeded and then exposed to PARP inhibitor (Niraparib or Olaparib) using the indicated concentrations. Untreated samples with the same number of cells were seeded in parallel as negative controls. Cells were cultured for one week before genomic DNA was isolated. The mutagenized genomic regions in BRCA2 were then amplified by PCR, NGS-sequenced and analyzed as described above. Alleles with a frequency greater than 0.5% in at least one sample of a given cell line were considered as robustly detected by NGS sequencing and included in the statistical analysis.
  • Drug sensitivity screening was started at day 21 and performed as described above with exception that at least 0.3 million cells were used in each replicate, and cells were collected at day 28. At least 2pg phenol-chloroform extracted genomic DNA of each sample was used as template in PCR amplifying the mutagenized region, then samples passed through the NGS pipeline as described above and sequenced at depth >1 million per sample on PE250 NovaSeq. Raw fastq files were firstly analyzed using CRISPResso to generate allele frequency table which then further analyzed using customized python and R scripts. Interestingly, this approach also identified the two phenyl alanine residues F 11 and F12 as critical residues in BRAC2.
  • the disclosure provides an optimized procedure to clonally isolate cells with large regions of the genome excised from one allele to generate locally haploid non-cancer cells, loHAPs.
  • BRCA2 gene shown here to be essential in hPSCs
  • proof of concept is provided for the functional annotation of mutations introduced on the remaining copy of BRCA2 in variant loHAP cells.
  • inframe, frameshift, and designer mutations generated by a template process rather than by random NHEJ can all be functionally annotated more effectively and accurately as compared to their diploid counterpart.
  • sensitizing alleles in the remaining copy of BRCA2 can be screened in response to drug treatments and irradiation by measuring changes in the relative allele frequencies.
  • the functional genomic approach taken here in loHAP systems encompasses key technical advances: it provides a platform for the functional annotation of variants in a pooled format in multiple human primary cell types and it provides for genes such as BRCA2 an approach to evaluate if VUS could be clinically actionable with PARPi.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Transplantation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Une variation génétique est un déterminant clé d'un risque de maladie et peut considérablement impacter un diagnostic, un pronostic et les résultats d'un traitement d'un patient à un autre patient. La présente invention concerne des produits et des procédés pour annoter de manière fonctionnelle des gènes à l'aide de cellules humaines non cancéreuses haploïdes locales, et pour déterminer si des variants de gènes sont cliniquement exploitables.
PCT/US2022/075365 2021-08-31 2022-08-23 Produits et procédés pour annoter une fonction génique à l'aide de cellules non cancéreuses humaines haploïdes localement WO2023034704A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163239255P 2021-08-31 2021-08-31
US63/239,255 2021-08-31

Publications (1)

Publication Number Publication Date
WO2023034704A1 true WO2023034704A1 (fr) 2023-03-09

Family

ID=85411577

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/075365 WO2023034704A1 (fr) 2021-08-31 2022-08-23 Produits et procédés pour annoter une fonction génique à l'aide de cellules non cancéreuses humaines haploïdes localement

Country Status (1)

Country Link
WO (1) WO2023034704A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018039145A1 (fr) * 2016-08-20 2018-03-01 Avellino Lab Usa, Inc. Arn guide unique, systèmes crispr/cas9 et leurs procédés d'utilisation
US20200270632A1 (en) * 2017-09-15 2020-08-27 The Board Of Trustees Of The Leland Stanford Junior University Multiplex production and barcoding of genetically engineered cells
WO2021168146A1 (fr) * 2020-02-18 2021-08-26 Tempus Labs, Inc. Procédés et systèmes de dosage de biopsie de liquide

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018039145A1 (fr) * 2016-08-20 2018-03-01 Avellino Lab Usa, Inc. Arn guide unique, systèmes crispr/cas9 et leurs procédés d'utilisation
US20200270632A1 (en) * 2017-09-15 2020-08-27 The Board Of Trustees Of The Leland Stanford Junior University Multiplex production and barcoding of genetically engineered cells
WO2021168146A1 (fr) * 2020-02-18 2021-08-26 Tempus Labs, Inc. Procédés et systèmes de dosage de biopsie de liquide

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEN ALICE, CACA: "PARP inhibitors: its role in treatment of cancer", ATZHENG -CHINESE JOURNAL OF CANCER, GAI KAN BIANJIBU, GUANGZHOU, CN, 5 July 2011 (2011-07-05), CN , pages 463 - 471, XP055824005, ISSN: 1000-467X, DOI: 10.5732/cjc.011.10111 *
TARO TSUJIMURA, OSAMU TAKASE, MASAHIRO YOSHIKAWA, ETSUKO SANO, MATSUHIKO HAYASHI, TSUYOSHI TAKATO, ATSUSHI TOYODA, HIDEYUKI OKANO,: "Control of directionality of chromatin folding for the inter- and intra-domain contacts at the Tfap2c–Bmp7 locus", EPIGENETICS & CHROMATIN, vol. 11, no. 1, 1 December 2018 (2018-12-01), XP055688331, DOI: 10.1186/s13072-018-0221-1 *
WANG LIMING, ELLIS MATHEW J., FIELDS TIMOTHY A., HOWELL DAVID N., SPURNEY ROBERT F.: "Beneficial Effects of the Rho Kinase Inhibitor Y27632 in Murine Puromycin Aminonucleoside Nephrosis", KIDNEY AND BLOOD PRESSURE RESEARCH, BASEL, CH, vol. 31, no. 2, 1 January 2008 (2008-01-01), CH , pages 111 - 121, XP009544257, ISSN: 1420-4096, DOI: 10.1159/000121531 *
ZHANG HANWEN, ZHANG SIWEI: "CRISPR/Cas9-mediated Precise SNP Editing in Human iPSC Lines", BIO-PROTOCOL, BIO-PROTOCOL.ORG, SUNNYVALE, CA, USA, vol. 11, no. 12, 1 January 2021 (2021-01-01), Sunnyvale, CA, USA , XP093043810, ISSN: 2331-8325, DOI: 10.21769/BioProtoc.4051 *

Similar Documents

Publication Publication Date Title
Giuliano et al. Generating single cell–derived knockout clones in mammalian cells with CRISPR/Cas9
Van Groningen et al. Neuroblastoma is composed of two super-enhancer-associated differentiation states
Kearns et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion
Ludlow et al. NOVA1 regulates hTERT splicing and cell growth in non-small cell lung cancer
Horikawa et al. Δ133p53 represses p53-inducible senescence genes and enhances the generation of human induced pluripotent stem cells
Hodel et al. POLE mutation spectra are shaped by the mutant allele identity, its abundance, and mismatch repair status
Marchetto et al. Differential L1 regulation in pluripotent stem cells of humans and apes
US20220229044A1 (en) In situ cell screening methods and systems
Zhang et al. KHDC3L mutation causes recurrent pregnancy loss by inducing genomic instability of human early embryonic cells
Plebani et al. Long-range transcriptome sequencing reveals cancer cell growth regulatory chimeric mRNA
CN108359692B (zh) 一种特异性靶向hDGKθ基因的荧光素酶报告系统
Mahajan et al. Genomic comparison of early-passage conditionally reprogrammed breast cancer cells to their corresponding primary tumors
Kim et al. Concurrent progress of reprogramming and gene correction to overcome therapeutic limitation of mutant ALK2-iPSC
Delhermite et al. Systematic mapping of rRNA 2’-O methylation during frog development and involvement of the methyltransferase Fibrillarin in eye and craniofacial development in Xenopus laevis
Li et al. Functional annotation of variants of the BRCA2 gene via locally haploid human pluripotent stem cells
Vanoli et al. Generation of human embryonic stem cell models to exploit the EWSR1-CREB fusion promiscuity as a common pathway of transformation in human tumors
Ravindranathan et al. Preliminary development of an assay for detection of TERT expression, telomere length, and telomere elongation in single cells
Wang et al. Genome-wide CRISPR-Cas9 screening and identification of potential genes promoting prostate cancer growth and metastasis
Deyle et al. Lack of genotoxicity due to foamy virus vector integration in human iPSCs
WO2023034704A1 (fr) Produits et procédés pour annoter une fonction génique à l'aide de cellules non cancéreuses humaines haploïdes localement
Li et al. The role of CYP3A4 mRNA transcript with shortened 3′-untranslated region in hepatocyte differentiation, liver development, and response to drug induction
US11293065B2 (en) Compositions and methods for the quality control of stem cell preparations
Tatarakis et al. Requirements for establishment and epigenetic stability of mammalian heterochromatin
Sebeson et al. Differential nucleosome occupancies across Oct4-Sox2 binding sites in murine embryonic stem cells
Yadav et al. Microhomology-mediated endjoining repair mechanism enables rapid and effective indel generations in stem cells

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22865685

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22865685

Country of ref document: EP

Kind code of ref document: A1