WO2022079054A1 - Replacement of rag1 for use in therapy - Google Patents

Replacement of rag1 for use in therapy Download PDF

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Publication number
WO2022079054A1
WO2022079054A1 PCT/EP2021/078222 EP2021078222W WO2022079054A1 WO 2022079054 A1 WO2022079054 A1 WO 2022079054A1 EP 2021078222 W EP2021078222 W EP 2021078222W WO 2022079054 A1 WO2022079054 A1 WO 2022079054A1
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identity
region
cells
homologous
chr
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English (en)
French (fr)
Inventor
Anna Villa
Pietro Genovese
Luigi Naldini
Nicolo SACCHETTI
Maria Carmina CASTIELLO
Samuele FERRARI
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Fondazione Telethon
Ospedale San Raffaele SRL
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Fondazione Telethon
Ospedale San Raffaele SRL
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Priority claimed from GBGB2016139.4A external-priority patent/GB202016139D0/en
Priority claimed from AU2021202657A external-priority patent/AU2021202657A1/en
Priority to CA3195268A priority Critical patent/CA3195268A1/en
Application filed by Fondazione Telethon, Ospedale San Raffaele SRL filed Critical Fondazione Telethon
Priority to CN202180082483.2A priority patent/CN116635523A/zh
Priority to JP2023521740A priority patent/JP2023544633A/ja
Priority to IL302031A priority patent/IL302031A/en
Priority to EP21798950.8A priority patent/EP4225900A1/en
Priority to AU2021359781A priority patent/AU2021359781A1/en
Priority to US18/030,711 priority patent/US20230365996A1/en
Publication of WO2022079054A1 publication Critical patent/WO2022079054A1/en
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    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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Definitions

  • the present invention relates to methods for gene-editing cells to introduce a RAG1 polypeptide, for example as a treatment for severe combined immunodeficiency.
  • the present invention also relates to polynucleotides, vectors, guide RNAs, kits, compositions, and gene editing systems for use in said methods.
  • the present invention also relates to genomes and cells obtained or obtainable by said methods.
  • the RAG1 and RAG2 proteins initiate V(D)J recombination, allowing generation of a diverse repertoire of T and B cells (Teng G, Schatz DG. Advances in Immunology. 2015;128:1-39).
  • RAG mutations in humans cause a broad spectrum of phenotypes, including T B' SCID, Omenn syndrome (OS), atypical SCID (AS) and combined immunodeficiency with granuloma/autoimmunity (CID-G/AI) (Notarangelo LD, et al. Nat Rev Immunol. 2016;16(4):234-246).
  • Hematopoietic stem cell transplantation is the mainstay for severe forms of RAG1 deficiency, including T B' SCID, OS and AS with an overall survival of -80% after transplantation from donors other than matched siblings (Haddad E, et al. Blood. 2018;132(17):1737-49).
  • overall survival rate is lower in non-matched-sibling donors and a high rate of graft failure and poor T and B cell immune reconstitution are observed in the absence of myeloablative or reduced intensity conditioning.
  • donor type and conditioning other factors associated with worse outcomes after HSCT include age (>3.5 months of life) and infections at the time of transplantation.
  • HSCs gene-corrected hematopoietic stem cells
  • the present inventors have developed a gene editing strategy to correct mutations in the RAG1 gene by targeting the genomic region located at the 5’ of the second exon, which contains the entire coding sequence of the gene.
  • the present inventors have designed and selected a panel of CRISPR-Cas9 nucleases and identified specific sites in non-repeated regions of the first intron of the human RAG1 gene.
  • the present inventors have identified guide RNAs and optimal conditions for the delivery of the CRISPR-Cas9 nuclease ribonucleoprotein complexes.
  • the present inventors have developed a donor DNA carrying the human RAG1 cDNA.
  • the gene editing strategy allows a high level of activity (measured as frequency of NHEJ- mutagenesis) and targeting efficiency (measured as GFP expression), both in a surrogate cell line deficient in RAG1 expression and expressing a recombination cassette, and in humans CD34+ HSCs obtained from mobilized peripheral blood (mPB). High editing efficiencies were reached in mobilized peripheral blood (mPB) CD34 + cells using the gene editing strategy.
  • the present invention provides a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region.
  • the present invention provides a polynucleotide comprising from 5’ to 3’: a first homology region, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region.
  • the first homology region is homologous to a first region of the RAG1 intron 1 and the second homology region is homologous to a second region of the RAG1 intron 1 ; or (ii) the first homology region is homologous to a first region of the RAG1 intron 1 or the RAG1 exon 2 and the second homology region is homologous to a second region of the RAG1 exon 2.
  • the first homology region is homologous to a first region of the RAG1 intron 1 and the second homology region is homologous to a second region of the RAG1 intron
  • the first homology region is homologous to a first region of the RAG1 intron 1 and the second homology region is homologous to a second region of the RAG1 exon
  • the first homology region is homologous to a first region of the RAG1 exon 2 and the second homology region is homologous to a second region of the RAG1 exon 2.
  • the first homology region is homologous to a region upstream of chr 11 : 36569295 and the second homology region is homologous to a region downstream of chr 11 : 36569298;
  • the first homology region is homologous to a region upstream of chr 11 : 36573790 and the second homology region is homologous to a region downstream of chr 11 : 36573793;
  • the first homology region is homologous to a region upstream of chr 11 : 36573641 and the second homology region is homologous to a region downstream of chr 11 : 36573644;
  • the first homology region is homologous to a region upstream of chr 11 : 36573351 and the second homology region is homologous to a region downstream of chr 11 : 36573354;
  • the first homology region is homologous to a region upstream of chr 11 : 36569080 and the second homology region is homologous to a region downstream of chr 11 : 36569083;
  • the first homology region is homologous to a region upstream of chr 11 : 36572472 and the second homology region is homologous to a region downstream of chr 11 : 36572475;
  • the first homology region is homologous to a region upstream of chr 11 : 36571458 and the second homology region is homologous to a region downstream of chr 11 : 36571461 ;
  • the first homology region is homologous to a region upstream of chr 11 : 36571366 and the second homology region is homologous to a region downstream of chr 11 : 36571369;
  • the first homology region is homologous to a region upstream of chr 11 : 36572859 and the second homology region is homologous to a region downstream of chr 11 : 36572862;
  • the first homology region is homologous to a region upstream of chr 11 : 36571457 and the second homology region is homologous to a region downstream of chr 11 : 36571460;
  • the first homology region is homologous to a region upstream of chr 11 : 36569351 and the second homology region is homologous to a region downstream of chr 11 : 36569354; or
  • the first homology region is homologous to a region upstream of chr 11 : 36572375 and the second homology region is homologous to a region downstream of chr 11 : 36572378.
  • the first homology region is homologous to a region upstream of chr 11 : 36569295 and the second homology region is homologous to a region downstream of chr 11 : 36569298;
  • the first homology region is homologous to a region upstream of chr 11 : 36573351 and the second homology region is homologous to a region downstream of chr 11 : 36573354;
  • the first homology region is homologous to a region upstream of chr 11 : 36571366 and the second homology region is homologous to a region downstream of chr 11 : 36571369.
  • the first homology region is homologous to a region upstream of chr 11 : 36569295 and the second homology region is homologous to a region downstream of chr 11 : 36569298. In some embodiments, the first homology region is homologous to a region upstream of chr 11 : 36573790 and the second homology region is homologous to a region downstream of chr 11 : 36573793.
  • the first homology region is homologous to a region upstream of chr 11 : 36573641 and the second homology region is homologous to a region downstream of chr 11 : 36573644.
  • the first homology region is homologous to a region upstream of chr 11 : 36573351 and the second homology region is homologous to a region downstream of chr 11 : 36573354.
  • the first homology region is homologous to a region upstream of chr 11 : 36569080 and the second homology region is homologous to a region downstream of chr 11 : 36569083.
  • the first homology region is homologous to a region upstream of chr 11 : 36572472 and the second homology region is homologous to a region downstream of chr 11 : 36572475.
  • the first homology region is homologous to a region upstream of chr 11 : 36571458 and the second homology region is homologous to a region downstream of chr 11 : 36571461.
  • the first homology region is homologous to a region upstream of chr 11 : 36571366 and the second homology region is homologous to a region downstream of chr 11 : 36571369.
  • the first homology region is homologous to a region upstream of chr 11 : 36572859 and the second homology region is homologous to a region downstream of chr 11 : 36572862.
  • the first homology region is homologous to a region upstream of chr 11 : 36571457 and the second homology region is homologous to a region downstream of chr 11 : 36571460.
  • the first homology region is homologous to a region upstream of chr 11 : 36569351 and the second homology region is homologous to a region downstream of chr 11 : 36569354. In some embodiments, the first homology region is homologous to a region upstream of chr 11 : 36572375 and the second homology region is homologous to a region downstream of chr 11 : 36572378.
  • the first homology region is homologous to a region comprising chr 11 : 36569245-chr 11 : 36569294 and/or the second homology region is homologous to a region comprising chr 11 : 36569299-chr 11 : 36569348.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity to SEQ ID NO: 7 and/or the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity to SEQ ID NO: 19.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity to SEQ ID NO: 31 , or a fragment thereof and/or the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity to SEQ ID NO: 32, or a fragment thereof.
  • the first and second homology regions are each 50-1000bp in length, 100-500 bp in length, or 200-400 bp in length.
  • the nucleotide sequence encoding a RAG1 polypeptide comprises or consists of a nucleotide sequence encoding an amino acid sequence that has at least 70% identity to SEQ ID NO: 4 or SEQ ID NO: 5.
  • the nucleotide sequence encoding a RAG1 polypeptide comprises or consists of a nucleotide sequence that has at least 70% identity to SEQ ID NO: 6.
  • the splice acceptor site comprises or consists of a nucleotide sequence that has at least 70% identity to SEQ ID NO: 33.
  • the nucleotide sequence encoding a RAG1 polypeptide is operably linked to a polyadenylation sequence, optionally wherein the polyadenylation sequence is a bGH polyadenylation sequence.
  • the nucleotide sequence encoding a RAG1 polypeptide is operably linked to a polyadenylation sequence comprising or consisting of a nucleotide sequence that has at least 70% identity to SEQ ID NO: 35.
  • the nucleotide sequence encoding a RAG1 polypeptide is operably linked a Kozak sequence, optionally wherein the Kozak sequence comprises or consists of a nucleotide sequence that has at least 70% identity to SEQ ID NO: 36.
  • the polynucleotide comprises or consists of a nucleotide sequence that has at least 70% identity to SEQ ID NO: 39.
  • the present invention provides a vector comprising the polynucleotide of the invention.
  • the vector is a viral vector, optionally an adeno-associated viral (AAV) vector such as an AAV6 vector.
  • the vector is a lentiviral vector, such as an integration-defective lentiviral vector (IDLV).
  • the present invention provides a guide RNA comprising or consisting of a nucleotide sequence that has at least 90% identity to any of SEQ ID NOs: 41-52.
  • the present invention provides a guide RNA comprising or consisting of a nucleotide sequence that has at least 90% identity to any of SEQ ID NOs: 53-55.
  • the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 41. In preferred embodiments, the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 53. In some embodiments, the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 42. In some embodiments, the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 43. In some embodiments, the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 44.
  • the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 45. In some embodiments, the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 46. In some embodiments, the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 47. In some embodiments, the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 48. In some embodiments, the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 49.
  • the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 50. In some embodiments, the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 51. In some embodiments, the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 52. In some embodiments, the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 54. In some embodiments, the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity to SEQ ID NO: 55.
  • the guide RNA from one to five of the terminal nucleotides at 5’ end and/or 3’ end of the guide RNA are chemically modified to enhance stability, optionally wherein three terminal nucleotides at 5’ end and/or 3’ end if the guide RNA are chemically modified to enhance stability, optionally wherein the chemical modification is modification with 2'-O-methyl 3'phosphorothioate.
  • the present invention provides a kit comprising the polynucleotide or the vector of the invention.
  • the present invention provides a composition comprising the polynucleotide or the vector of the invention.
  • the present invention provides a gene-editing system comprising the polynucleotide or the vector of the invention.
  • the kit, composition, or gene-editing system further comprises a guide RNA of the invention. In some embodiments, the kit, composition, or gene-editing system further comprises a RNA-guided nuclease, optionally wherein the RNA-guided nuclease is a Cas9 endonuclease
  • the present invention provides for use of the polynucleotide, the vector, the kit, the composition, or the gene-editing system, for gene editing a cell or a population of cells.
  • the use is ex vivo or in vitro use.
  • the present invention provides a genome comprising the polynucleotide of the invention.
  • the present invention provides a genome comprising a splice acceptor sequence and a nucleotide sequence encoding a RAG1 polypeptide located in the RAG1 intron 1 or RAG1 exon 2.
  • the splice acceptor sequence and the nucleotide sequence encoding RAG1 are located in the RAG1 intron 1.
  • the splice acceptor sequence and the nucleotide sequence encoding RAG1 replace chr 11 : 36569295 to chr 11 : 36569298.
  • the present invention provides a cell comprising the polynucleotide, the vector, or the genome of the invention.
  • the present invention provides a population of cells comprising one or more cells of the present invention.
  • the present invention provides a method of gene editing a population of cells comprising delivering the polynucleotide or the vector of the invention to a population of cells to obtain a population of gene-edited cells.
  • the method is an ex vivo or in vitro method.
  • the present invention provides a method of treating immunodeficiency in a subject in need thereof, comprising delivering the polynucleotide or the vector of the invention to a population of cells to obtain a population of gene-edited cells and administering the population of gene-edited cells to the subject.
  • the present invention provides a population of gene-edited cells obtainable by the method of the invention.
  • the present invention provides the polynucleotide, the vector, the guide RNA, the kit, the composition, or the gene-editing system, for use in treating immunodeficiency in a subject.
  • the present invention provides a method of treating a subject comprising administering a cell, a population of cells, or a population of gene edited cells of the present invention to the subject.
  • the present invention provides a method of treating immunodeficiency in a subject in need thereof comprising administering a cell, a population of cells, or a population of gene edited cells of the present invention to the subject.
  • the present invention provides a cell, a population of cells, or a population of gene edited cells of the present invention for use as a medicament. In another aspect, the present invention provides a cell, a population of cells, or a population of gene edited cells of the present invention for use in treating immunodeficiency in a subject.
  • VCN Vector Copy Number
  • A) RAG1 gene expression measured by RT-qPCR, represented as fold change vs RAG1 expression in 293T cell line, actin was used as normalizer; B) Schematic representation of different SA_GFP DNA donor tested; C) Schematic representation of the splicing mechanism with SA_GFP_SD donor; D) Percentage of targeted cells measured by flow cytometry as GFP+ cells, 7 days after transfection; E) GFP expression levels measured as Mean Fluorescence Intensity (MFI) gating on GFP+ events; F) Representative FlowJo plots; Oneway ANOVA, Geisser-Greenhouse correction for multiple comparison, n 3. P values: * ⁇ 0.05; ** ⁇ 0.005; *** ⁇ 0.0005; **** ⁇ 0.0001. Mean ⁇ SD are shown.
  • D-E) Plots show the coverage of on-target reads (chromosome 11) of guide 9 (D) and guide 7 (E) and off-target reads identified for guide 7 by relaxed constraints (chromosome 20 and 9).
  • One-way ANOVA, Geisser-Greenhouse correction for multiple comparison, n 3. P values: * ⁇ 0.05; ** ⁇ 0.005; *** ⁇ 0.0005; **** ⁇ 0.0001.
  • E, G, I Targeted cells among the B-cell, T-cell and Myeloidcell compartment in PB measured as GFP + cells in the hCD19 + gate (E), hCD3 + gate (G) and hCD13 + gate (I), respectively;
  • L Frequency of hCD34 + cells measured by flow cytometry among hCD45 + cells in the bone marrow;
  • M Frequency of targeted cells measured by flow cytometry as GFP + cells among hCD34 + cells in the bone marrow;
  • N Frequency of GFP + expressing cells measured by flow cytometry, among different T-cell development stages in the thymus (according to the expression of hCD4 and hCD8), in the peripheral blood and in the spleen (according to the expression of hCD3, hCD4 and hCD8), 17 weeks after transplant. Mann-Whitney test at 17 weeks after transplant.
  • Group size: SA_GFP n 5;
  • PGK_GFP n
  • A) Schematic representation of the corrective donor; B) Schematic representation of the experimental protocol; C) Percentages of targeted cells measured by ddPCR on sorted hCD34 + cell subpopulation according to the expression of hCD133 and hCD90 at day 4, telomerase genomic region was used as normalizer; D) Total number of cells at day 4 represented as fold increase compared day 0. N 3.
  • Figure 10 Multiparametric analysis of hMPB-CD34 + cells from HD and RAG1 -patient before and after gene editing manipulation.
  • Graphs show 20 subtypes analyzed in the Lineage negative (Lin ) CD34 + gate including: Hematopoietic Stem cells (HSC), Multipotent Progenitors (MPP), Multi-Lymphoid Progenitors (MLP), Early T Progenitors (ETP), B and NK cell precursors (Pre-B/NK), common myeloid progenitors (CMP), granulocyte-monocyte progenitors (GMP), megakaryoerythroid progenitors (MEP), megakaryocyte progenitors (MKp) and erythroid progenitors (EP).
  • HSC Hematopoietic Stem cells
  • MPP Multipotent Progenitors
  • MLP Multi-Lymphoid Progenitors
  • ETP Early T Progenitors
  • B and NK cell precursors Pre-B/NK
  • CMP common myeloid progenitors
  • GMP granulocyte-monocyte progenitors
  • MEP
  • HA_L left homology arm
  • HA_R right homology arm
  • SA splice acceptor
  • SD splice donor
  • BGHpA bovine growth hormone poly A
  • WPRE Woodchuck hepatitis virus post-transcriptional regulatory element
  • IRES the internal ribosome entry site sequence
  • PEST proline (P), glutamic acid (E), serine (S), and threonine
  • T threonine
  • B schematic representation of the experimental protocol.
  • D) Modulation of GFP expression in serum starved cells is shown as ratio of GFP MFI of starved cells (- FBS) and GFP MFI of not starved cells (+ FBS) (1 experiment representative of 3).
  • Graphs show 20 subtypes analyzed in the Lineage negative (Lin ) CD34 + gate including: Hematopoietic Stem cells (HSC), Multipotent Progenitors (MPP), MultiLymphoid Progenitors (MLP), Early T Progenitors (ETP), B and NK cell precursors (Pre-B/NK), common myeloid progenitors (CMP), granulocyte-monocyte progenitors (GMP), megakaryoerythroid progenitors (MEP), megakaryocyte progenitors (MKp) and erythroid progenitors (EP).
  • HSC Hematopoietic Stem cells
  • MPP Multipotent Progenitors
  • MLP MultiLymphoid Progenitors
  • ETP Early T Progenitors
  • B and NK cell precursors Pre-B/NK
  • CMP common myeloid progenitors
  • GMP granulocyte-monocyte progenitors
  • MEP megak
  • ATO artificial thymic organoid
  • UT Untreated cells
  • HDR enhancers B) total number of cells harvested from ATOs 4 weeks after ATO seeding.
  • C) HDR efficiency is shown as percentages of edited alleles measured by ddPCR in bulk differentiated T cells 4 weeks after ATO seeding.
  • D) HDR efficiency is measured as percentage of GFP+ cells within distinct T cell subpopulation by flow cytometry 4 weeks after ATO seeding.
  • HA homology arm
  • SA splice acceptor
  • SD splice donor
  • coRAGI CDS codon optimized RAG1 coding sequence
  • BGHpA bovine growth hormone poly A
  • Ex. exon
  • gRNA guide RNA
  • 3’IITR 3’ untranslated region
  • HDR homology directed repair.
  • C Kinetics of cell growth in untreated (UT) cr edited HSPC according to the indicated donors, doses and days after gene editing (GE).
  • D Colony forming unit (CFU) assay was performed on untreated or edited HSPC by counting the number of red (erythroid), white (myeloid) and mixed colonies at microscope 14 days after the plating.
  • E Distribution of the CD34+ cell subpopulations and CD34- cells measured by flow cytometry based on the expression of hCD133 and hCD90 analysed 4 days after the editing.
  • F Representative plots of the T cell differentiation stages analysed by flow cytometry 7 weeks after ATO seeding.
  • G HDR efficiency is measured as proportion of edited alleles in bulk, CD4+ CD8+ double positive (DP) cells and CD4- CD8- double negative (DN) cells by flow cytometry 6 weeks after ATO seeding.
  • nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • All recited genomic locations are based on human genome assembly GRCh38.p13 (GCF_000001405.39).
  • GCF_000001405.39 human genome assembly GRCh38.p13
  • One of skill in the art will be able to identify the corresponding genome locations in alternative genome assemblies and convert the recited genomic location accordingly.
  • RAG1 is located at chr 11 : 36510353 to 36579762 in assembly GRCh38.p13 and at chr 11 : 36532053 to 36601312 in assembly GRCh37.p13.
  • Recombination activating gene 1 (RAG1)
  • the present invention relates to methods for gene-editing cells to introduce a RAG1 polypeptide, for example as a treatment for severe combined immunodeficiency.
  • the present invention also relates to polynucleotides, vectors, guide RNAs, kits, compositions, and gene editing systems for use in said methods, and genomes and cells obtained or obtainable by said methods.
  • RAG1 is the abbreviated name of the polypeptide encoded by recombination activating gene 1 and is also known as RAG-1 , RNF74, and recombination activating 1.
  • RAG1 is the catalytic component of the RAG complex, a multiprotein complex that mediates the DNA cleavage phase during V(D)J recombination.
  • V(D)J recombination assembles a diverse repertoire of immunoglobulin and T-cell receptor genes in developing B and T- lymphocytes through rearrangement of different V (variable), in some cases D (diversity), and J (joining) gene segments.
  • RAG1 mediates the DNA-binding to the conserved recombination signal sequences (RSS) and catalyses the DNA cleavage activities by introducing a double-strand break between the RSS and the adjacent coding segment.
  • RSS conserved recombination signal sequences
  • RAG2 is not a catalytic component but is required for all known catalytic activities.
  • a “RAG1 polypeptide” is a polypeptide having RAG1 activity, for example a polypeptide which is able to form a RAG complex, mediate DNA-binding to the RSS, and introduce a doublestrand break between the RSS and the adjacent coding segment.
  • a RAG1 polypeptide may have the same or similar activity to a wild-type RAG1 , e.g.
  • RAG1 polypeptide may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% of the activity of a wild-type RAG1 polypeptide.
  • the RAG1 polypeptide may be a fragment of RAG1 and/or a RAG1 variant.
  • a “fragment of RAG1” may refer to a portion or region of a full-length RAG1 polypeptide that has the same of similar activity as a full-length RAG1 polypeptide, i.e. the fragment may be a functional fragment.
  • the fragment may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of a full-length RAG1 polypeptide.
  • a person skilled in the art would be able to generate fragments based on the known structural and functional features of RAG1. These are described, for instance, in Arbuckle, J.L., et al., 2011. BMC biochemistry, 12(1), p.23; Ru, H., et al., 2015. Cell, 163(5), pp.1138-1152; and Kim, M.S., et al., 2015. Nature, 518(7540), pp.507-511.
  • Core RAG1 consists of multiple structural domains, termed the nonamer binding domain (NBD; residues 389-464), the central domain (residues 528-760), and the C-terminal domain (residues 761-980) domains.
  • NBD nonamer binding domain
  • core RAG1 contains the essential acidic active site residues (Arbuckle, J.L., et al., 2011. BMC biochemistry, 12(1), p.23).
  • a fragment of RAG1 comprises the nonamer binding domain, the central domain, and/or the C-terminal domain.
  • a “RAG1 variant” may include an amino acid sequence or a nucleotide sequence which may be at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, optionally at least 95% or at least 97% or at least 99% identical to a wild-type RAG1 polypeptide.
  • RAG1 variants may have the same or similar activity to a wild-type RAG1 polypeptide, e.g.
  • RAG1 may have at least at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% of the activity of a wild-type RAG1 polypeptide.
  • a person skilled in the art would be able to generate RAG1 variants based on the known structural and functional features of RAG1 and/or using conservative substitutions.
  • the gene encoding RAG1 (NCBI gene ID: 5896) is located in the human genome at chr 11 : 36510353 to 36579762.
  • Transcript variant 1 (NM_000448) has two exons and one intron.
  • the region of the RAG1 gene corresponding to the first exon of transcript variant 1 is called the “RAG1 exon 1”
  • the region of the RAG1 gene corresponding to the intron of transcript variant 1 is called the “RAG1 intron 1”
  • the region of the RAG1 gene corresponding to the second exon (which encodes a RAG1 polypeptide) is called the “RAG1 exon 2”.
  • the RAG1 exon 1 is from chr 11 : 36568006 to chr 11 : 36568122; the RAG1 intron 1 is from chr 11 : 36568123 to chr 11 : 36573290; and/or the RAG1 exon 2 is from chr 11 : 36573291 to chr 11 : 36579762.
  • the RAG1 exon 1 consists of the nucleotide sequence of SEQ ID NO: 1 , or variants thereof; the RAG1 intron 1 consists of the nucleotide sequence of SEQ ID NO: 2, or variants thereof; and/or the RAG1 exon 2 consists of the nucleotide sequence of SEQ ID NO: 3, or variants thereof.
  • RAG1 exon 1 (SEQ ID NO: 1) agaaacaagagggcaaggagagagcagagaacacactttgccttctttggtattgagtaatatcaaccaaattgc agacatctcaacactttggccaggcagcctgctgagcaag
  • RAG1 intron 1 (SEQ ID NO: 2) gtaacactcatacttttcatgccttgagccaaaatatttattacatttttatgtttctaactagaagtgcttgagcttttttccttcc aggtgatgaggggatggaatgagcaaagctacatcaattttttttttaatgtatgaaaataaaaaggtacaagaggccc aagtttagggccactgaaggttcatagaaagatgcaaaatatctgaattactataaatgaatgctattgtcagaggaaa ggtttaaggaggtgcttcttgaatgaatgtgtgtacaaatcagcagaaggtaaggtgtgtgagactcttggaaatga
  • RAG1 exon 2 (SEQ ID NO: 3) gtacctcagccagcATGGCAGCCTCTTTCCCACCCACCTTGGGACTCAGTTCTGCCCC AGATGAAATTCAGCACCCACATATTAAATTTTCAGAATGGAAATTTAAGCTGTTC CGGGTGAGATCCTTTGAAAAGACACCTGAAGAAGCTCAAAAGGAAAAGAAGGAT TCCTTTGAGGGGAAACCCTCTCTGGAGCAATCTCCAGCAGTCCTGGACAAGGC TGATGGTCAGAAGCCAGTCCCAACTCAGCCATTGTTAAAAGCCCACCCTAAGTT TTCAAAGAAATTTCACGACAACGAGAAAGCAAGAGGCAAAGCGATCCATCAAGC CAACCTTCGACATCTCTGCCGCATCTGTGGGAATTCTTTTAGAGCTGATGAGCA CAACAGGAGATATCCAGTCCATGGTCCTGTGGATGGTAAAACCCTAGGCCTTTT ACGAAAGAAGGAAAAGAGCTACTTCCTGGCCGGACCTCATT
  • RAG1 exon 2 SEQ ID NO: 3
  • upper case letters indicate a nucleotide sequence which encodes a RAG1 polypeptide.
  • the RAG1 polypeptide may be a human RAG1 polypeptide.
  • the RAG1 polypeptide may comprise or consist of a polypeptide sequence of UniProtKB accession P15918, or a fragment or variant thereof.
  • the RAG1 polypeptide comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 4 or a fragment thereof.
  • the RAG1 polypeptide comprises or consists of an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 4 or a fragment thereof.
  • the RAG1 polypeptide comprises or consists of SEQ ID NO: 4 or a fragment thereof.
  • RAG1 polypeptide isoform 1 UniProtKB accession P15918 (SEQ ID NO: 4)
  • the RAG1 polypeptide comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 5 or a fragment thereof.
  • the RAG1 polypeptide comprises or consists of an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 5 or a fragment thereof.
  • the RAG1 polypeptide comprises or consists of SEQ ID NO: 5 or a fragment thereof.
  • RAG1 polypeptide isoform 2 UniProtKB accession P15918 (SEQ ID NO: 5)
  • RAG1 polynucleotides
  • the nucleotide sequence encoding a RAG1 polypeptide may be codon-optimised.
  • the nucleotide sequence encoding a RAG1 polypeptide may be codon optimised for expression in a human cell.
  • Codon usage tables are known in the art for mammalian cells (e.g. humans), as well as for a variety of other organisms.
  • the nucleotide sequence encoding a RAG1 polypeptide comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 6 or a fragment thereof.
  • the nucleotide sequence encoding a RAG1 polypeptide comprises or consists of a nucleotide sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 6 or a fragment thereof.
  • the nucleotide sequence encoding a RAG1 polypeptide comprises or consists of the nucleotide sequence SEQ ID NO: 6 or a fragment thereof.
  • nucleotide sequence encoding a RAG1 polypeptide (SEQ ID NO: 6) atggccgcctccttcccacctacccttggattgtcctccgcccctgacgaaattcaacatccccacatcaaattctcgga gtggaagttcaagctctttcgcgtgcgctcgttcgaaaagacccccgaggaagcccaaaaggagaagaaagactc attcgaaggaaacccagcctcgaacagtccccggccgtctggacaaggccgacgggcagaagcctgtgccga cccagccgctgctgaaagcacccgaaattctccaagaagtttcacgagaagtttgcga
  • the present invention provides a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region.
  • the polynucleotide may be an isolated polynucleotide.
  • the polynucleotide may be a DNA molecule, e.g. a double-stranded DNA molecule.
  • the polynucleotide of the invention may be limited to a size suitable to be inserted into a vector (e.g. an adeno-associated viral (AAV) vector, such as AAV6).
  • a vector e.g. an adeno-associated viral (AAV) vector, such as AAV6
  • the polynucleotide of the invention may be 5.0 kb or less, 4.9 kb or less, 4.8 kb or less, 4.7 kb or less, 4.6 kb or less, 4.5 kb or less, 4.4 kb or less, 4.3 kb or less, 4.2 kb or less, 4.1 kb or less, 4.0 kb or less in total size.
  • the polynucleotide of the invention is 4.1 kb or less or 4.0 kb or less in size.
  • the present invention provides a genome comprising a splice acceptor sequence and a nucleotide sequence encoding a RAG1 polypeptide.
  • the genome may comprise the polynucleotide of the present invention.
  • the genome may be an isolated genome.
  • the genome may be a mammalian genome, e.g. a human genome.
  • a “homology region” is a nucleotide sequence which is located upstream or downstream of a nucleotide sequence to be inserted (a “nucleotide sequence insert” e.g. a splice acceptor sequence and a nucleotide sequence encoding a RAG1 polypeptide).
  • the polynucleotide of the present invention comprises two homology regions, one upstream of the nucleotide sequence insert (the “first homology region”) and one downstream of the nucleotide insert (the “second homology region”).
  • Each “homology region” is designed such that the nucleotide sequence insert can be introduced into a genome at a site of a double strand break (DSB) by homology-directed repair (HDR).
  • HDR homology-directed repair
  • One of skill in the art will be able to design homology arms depending on the desired insertion site (i.e. the site of the DSB) (see e.g. Ran, F.A., et al., 2013. Nature protocols, 8(11), pp.2281-2308).
  • Each “homology region” is homologous to a region either side of the DSB.
  • the first homology region may be homologous to a region upstream of the DSB and the second homology region may be homologous to a region downstream of the DSB.
  • the term “homologous” means that the nucleotide sequences are similar or identical.
  • the nucleotide sequences may be at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical.
  • upstream and downstream both refer to relative positions in DNA or RNA.
  • Each strand of DNA or RNA has a 5’ end and a 3’ end and, by convention, “upstream” and “downstream” relate to the 5' to 3' direction respectively in which RNA transcription takes place.
  • upstream is toward the 5' end of the coding strand for the gene in question (e.g. RAG1) and downstream is toward the 3' end of the coding strand for the gene in question (e.g. RAG1).
  • the homology regions may be any length suitable for HDR.
  • the homology regions may be the same or different lengths.
  • the homology regions are each independently 50-1000 bp in length, 100-500 bp in length, or 200-400 bp in length.
  • the first homology may be 50-1000 bp in length and homologous to a region upstream of a DSB and the second homology region may be 50-1000 bp in length and homologous to a region downstream of the DSB.
  • the first homology region is homologous to a first region of the RAG1 intron 1 and the second homology region is homologous to a second region of the RAG1 intron 1 ;
  • the first homology region is homologous to a first region of the RAG1 intron 1 or the RAG1 exon 2 and the second homology region is homologous to a second region of the RAG1 exon 2.
  • the first homology region is homologous to a first region of the RAG1 intron 1 and the second homology region is homologous to a second region of the RAG1 intron 1.
  • the first homology region is homologous to a region upstream of chr 11 : 36569295 and the second homology region is homologous to a region downstream of chr 11 : 36569298;
  • the first homology region is homologous to a region upstream of chr 11 : 36573790 and the second homology region is homologous to a region downstream of chr 11 : 36573793;
  • the first homology region is homologous to a region upstream of chr 11 : 36573641 and the second homology region is homologous to a region downstream of chr 11 : 36573644;
  • the first homology region is homologous to a region upstream of chr 11 : 36573351 and the second homology region is homologous to a region downstream of chr 11 : 36573354;
  • the first homology region is homologous to a region upstream of chr 11 : 36569080 and the second homology region is homologous to a region downstream of chr 11 : 36569083;
  • the first homology region is homologous to a region upstream of chr 11 : 36572472 and the second homology region is homologous to a region downstream of chr 11 : 36572475;
  • the first homology region is homologous to a region upstream of chr 11 : 36571458 and the second homology region is homologous to a region downstream of chr 11 : 36571461 ;
  • the first homology region is homologous to a region upstream of chr 11 : 36571366 and the second homology region is homologous to a region downstream of chr 11 : 36571369;
  • the first homology region is homologous to a region upstream of chr 11 : 36572859 and the second homology region is homologous to a region downstream of chr 11 : 36572862;
  • the first homology region is homologous to a region upstream of chr 11 : 36571457 and the second homology region is homologous to a region downstream of chr 11 : 36571460;
  • the first homology region is homologous to a region upstream of chr 11 : 36569351 and the second homology region is homologous to a region downstream of chr 11 : 36569354; or
  • the first homology region is homologous to a region upstream of chr 11 : 36572375 and the second homology region is homologous to a region downstream of chr 11 : 36572378.
  • the first homology region is homologous to a region upstream of chr 11 : 36569295 and the second homology region is homologous to a region downstream of chr 11 : 36569298;
  • the first homology region is homologous to a region upstream of chr 11 : 36573351 and the second homology region is homologous to a region downstream of chr 11 : 36573354;
  • the first homology region is homologous to a region upstream of chr 11 : 36571366 and the second homology region is homologous to a region downstream of chr 11 : 36571369.
  • the first homology region is homologous to a region upstream of chr 11 : 36569295 and the second homology region is homologous to a region downstream of chr 11 : 36569298.
  • the first homology region is homologous to a region comprising chr 11 : 36569245- 36569294 and the second homology region is homologous to a region comprising chr 11 : 36569299-36569348;
  • the first homology region is homologous to a region comprising chr 11 : 36573740- 36573789 and the second homology region is homologous to a region comprising chr 11 : 36573794-36573843;
  • the first homology region is homologous to a region comprising chr 11 : 36573591- 36573640 and the second homology region is homologous to a region comprising chr 11 : 36573645-36573694;
  • the first homology region is homologous to a region comprising chr 11 : 36573301- 36573350 and the second homology region is homologous to a region comprising chr 11 : 36573355-36573404;
  • the first homology region is homologous to a region comprising chr 11 : 36569030- 36569079 and the second homology region is homologous to a region comprising chr 11 : 36569084-36569133;
  • the first homology region is homologous to a region comprising chr 11 : 36572422- 36572471 and the second homology region is homologous to a region comprising chr 11 : 36572476-36572525;
  • the first homology region is homologous to a region comprising chr 11 : 36571408- 36571457 and the second homology region is homologous to a region comprising chr 11 : 36571462-36571511 ;
  • the first homology region is homologous to a region comprising chr 11 : 36571316- 36571365 and the second homology region is homologous to a region comprising chr 11 : 36571370-36571419;
  • the first homology region is homologous to a region comprising chr 11 : 36572809- 36572858 and the second homology region is homologous to a region comprising chr 11 : 36572863-36572912;
  • the first homology region is homologous to a region comprising chr 11 : 36571407- 36571456 and the second homology region is homologous to a region comprising chr 11 : 36571461-36571510;
  • the first homology region is homologous to a region comprising chr 11 : 36569301- 36569350 and the second homology region is homologous to a region comprising chr 11 : 36569355-36569404; or
  • the first homology region is homologous to a region comprising chr 11 : 36572325- 36572374 and the second homology region is homologous to a region comprising chr 11 : 36572379-36572428.
  • the first homology region is homologous to a region comprising chr 11 : 36569245- 36569294 and the second homology region is homologous to a region comprising chr 11 : 36569299-36569348;
  • the first homology region is homologous to a region comprising chr 11 : 36573301- 36573350 and the second homology region is homologous to a region comprising chr 11 : 36573355-36573404; or
  • the first homology region is homologous to a region comprising chr 11 : 36571316- 36571365 and the second homology region is homologous to a region comprising chr 11 : 36571370-36571419.
  • the first homology region is homologous to a region comprising chr 11 : 36569245-36569294 and the second homology region is homologous to a region comprising chr 11 : 36569299-36569348.
  • the first homology region is homologous to a region comprising chr 11 : 36573740-36573789 and the second homology region is homologous to a region comprising chr 11 : 36573794-36573843.
  • the first homology region is homologous to a region comprising chr 11 : 36573591-36573640 and the second homology region is homologous to a region comprising chr 11 : 36573645-36573694.
  • the first homology region is homologous to a region comprising chr 11 : 36573301-36573350 and the second homology region is homologous to a region comprising chr 11 : 36573355-36573404.
  • the first homology region is homologous to a region comprising chr 11 : 36569030-36569079 and the second homology region is homologous to a region comprising chr 11 : 36569084-36569133.
  • the first homology region is homologous to a region comprising chr 11 : 36572422-36572471 and the second homology region is homologous to a region comprising chr 11 : 36572476-36572525.
  • the first homology region is homologous to a region comprising chr 11 : 36571408-36571457 and the second homology region is homologous to a region comprising chr 11 : 36571462-36571511.
  • the first homology region is homologous to a region comprising chr 11 : 36571316-36571365 and the second homology region is homologous to a region comprising chr 11 : 36571370-36571419.
  • the first homology region is homologous to a region comprising chr 11 : 36572809-36572858 and the second homology region is homologous to a region comprising chr 11 : 36572863-36572912.
  • the first homology region is homologous to a region comprising chr 11 : 36571407-36571456 and the second homology region is homologous to a region comprising chr 11 : 36571461-36571510.
  • the first homology region is homologous to a region comprising chr 11 : 36569301-36569350 and the second homology region is homologous to a region comprising chr 11 : 36569355-36569404.
  • the first homology region is homologous to a region comprising chr 11 : 36572325-36572374 and the second homology region is homologous to a region comprising chr 11 : 36572379-36572428.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to any of SEQ ID NOs: 7-18 and/or the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to any of SEQ ID NOs: 19-30.
  • the first and second homology regions comprise or consist of nucleotide sequences that have at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to first and second homology regions in the same row of Table 1.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to any of SEQ ID NOs: 7-18 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to the corresponding nucleotide sequence in Table 1 (i.e. SEQ ID NOs: 19-30).
  • Table 1 i.e. SEQ ID NOs: 19-30.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 7 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 19;
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 8 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 20;
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 9 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 21 ;
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 10 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 22;
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 11 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 23;
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 12 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 24;
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 13 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 25;
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 14 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 26;
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 15 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 27;
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 16 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 28;
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 17 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 29; or
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 18 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 30.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 7 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 19;
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 10 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 22; or
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 14 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 26.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 7 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 19.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 8 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 20.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 9 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 21.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 10 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 22.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 11 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 23.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 12 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 24.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 13 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 25.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 14 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 26.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 15 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 27.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 16 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 28.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 17 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 29.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 18 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 30.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 98% identity to SEQ ID NO: 7 and the second homology region comprises or consists of a nucleotide sequence that has at least 98% identity to SEQ ID NO: 19.
  • the first homology region comprises or consists of the nucleotide sequence of SEQ ID NO: 7 and the second homology region comprises or consists of the nucleotide sequence of SEQ ID NO: 19.
  • the 3’ terminal sequence of the first homology region consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to any of SEQ ID NOs: 7-18 and/or the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to any of SEQ ID NOs: 19-30.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to any of SEQ ID NOs: 7-18 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to the corresponding nucleotide sequence in Table 1 (i.e. SEQ ID NOs: 19-30).
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 7 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 19;
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 8 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 20;
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 9 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 21 ;
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 10 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 22;
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 11 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 23;
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 12 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 24;
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 13 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 25;
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 14 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 26;
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 15 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 27;
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 16 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 28;
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 17 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 29; or
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 18 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 30.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 7 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 19;
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 10 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 22; or
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 14 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 26.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 7 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 19.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 8 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 20.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 9 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 21.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 10 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 22.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 11 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 23.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 12 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 24.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 13 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 25.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 14 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 26.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 15 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 27.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 16 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 28.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 17 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 29.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 18 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 30.
  • the 3’ terminal sequence of the first homology region comprises or consists of a nucleotide sequence that has at least 98% identity to SEQ ID NO: 7 and the 5’ terminal sequence of the second homology region comprises or consists of a nucleotide sequence that has at least 98% identity to SEQ ID NO: 19.
  • the 3’ terminal sequence of the first homology region comprises or consists of the nucleotide sequence of SEQ ID NO: 7 and the 5’ terminal sequence of the second homology region comprises or consists of the nucleotide sequence of SEQ ID NO: 19.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 31 , or a fragment thereof; and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 32, or a fragment thereof.
  • the fragments are at least 50 bp in length, for example 50-250 bp or 100-200 bp in length.
  • the first homology region comprises or consists of a nucleotide sequence that has at least 98% identity to SEQ ID NO: 31 , or a fragment thereof; and the second homology region comprises or consists of a nucleotide sequence that has at least 98% identity to SEQ ID NO: 32, or a fragment thereof.
  • the first homology region comprises or consists of the nucleotide of SEQ ID NO: 31 , or a fragment thereof
  • the second homology region comprises or consists of the nucleotide sequence of SEQ ID NO: 32, or a fragment thereof.
  • Illustrative first homology region for guide RNA 9 (SEQ ID NO: 31) tgagcacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac ggggtgtatgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgcaccctggcctc ctgaactaatgatatcactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaat ctgtgctgtggagggaggcacgctgtagctctgatgtcagatggcaatgt
  • Illustrative second homology region for guide RNA 9 (SEQ ID NO: 32) atggcag
  • the site of the double-strand break can be introduced specifically by any suitable technique, for example using a CRISPR/Cas9 system and the guide RNAs disclosed herein.
  • the DSB is introduced into the RAG1 intron 1 or RAG1 exon 2.
  • a DSB may be introduced at any of the sites recited in Table 2 below.
  • a DSB is introduced into the RAG1 intron 1.
  • each homology region is homologous to a fragment of the RAG1 intron 1 and/or RAG1 exon 2 either side of the DSB.
  • the first homology region may be homologous to a region in the RAG1 intron 1 and/or RAG1 exon 2 upstream of the DSB and the second homology region may be homologous to a region downstream of the DSB.
  • the nucleotide sequence insert e.g.
  • a splice acceptor sequence and a nucleotide sequence encoding a RAG1 polypeptide may be introduced at the DSB site by homology-directed repair (HDR).
  • HDR homology-directed repair
  • the nucleotide insert e.g. a splice acceptor sequence and a nucleotide sequence encoding a RAG1 polypeptide
  • nucleotide sequence insert may consist of the region of the polynucleotide flanked by the first homology region and the second homology region.
  • the nucleotide sequence insert may comprise a splice acceptor sequence and a nucleotide sequence encoding a RAG1 polypeptide.
  • the nucleotide sequence insert may be introduced into a genome at any of the sites recited in Table 2 above.
  • the genome of the present invention may comprise the nucleotide sequence insert at any of the sites recited in Table 2 above.
  • nucleotide sequence insert is introduced:
  • the nucleotide sequence insert is introduced between chr 11 : 36569296 and 36569297.
  • the genome of the present invention comprises a nucleotide sequence comprising a splice acceptor sequence and a nucleotide sequence encoding a RAG1 polypeptide, which is introduced:
  • the genome of the present invention comprises a nucleotide sequence comprising a splice acceptor sequence and a nucleotide sequence encoding a RAG1 polypeptide, which is introduced between chr 11 : 36569296 and 36569297.
  • the nucleotide sequence insert may replace any of the regions recited in Table 3 below.
  • the genome of the present invention may comprise the nucleotide sequence insert replacing any of the regions recited in Table 3.
  • nucleotide sequence insert replaces:
  • the nucleotide sequence insert replaces chr 11 : 36569295 to 36569298.
  • the genome of the present invention comprises a nucleotide sequence comprising a splice acceptor sequence and a nucleotide sequence encoding a RAG1 polypeptide, which replaces:
  • the genome of the present invention comprises a nucleotide sequence comprising a splice acceptor sequence and a nucleotide sequence encoding a RAG1 polypeptide, which replaces chr 11 : 36569295 to 36569298.
  • RNA splicing is a form of RNA processing in which a newly made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). During splicing, introns (non-coding regions) are removed and exons (coding regions) are joined together.
  • pre-mRNA precursor messenger RNA
  • mRNA mature messenger RNA
  • a donor site (5' end of the intron), a branch site (near the 3' end of the intron) and an acceptor site (3' end of the intron) are required for splicing.
  • the splice donor site includes an almost invariant sequence Gil at the 5' end of the intron, within a larger, less highly conserved region.
  • the splice acceptor site at the 3' end of the intron terminates the intron with an almost invariant AG sequence.
  • Upstream (5'-ward) from the AG there is a region high in pyrimidines (C and U), or polypyrimidine tract. Further upstream from the polypyrimidine tract is the branchpoint.
  • a “splice acceptor sequence” is a nucleotide sequence which can function as an acceptor site at the 3’ end of the intron. Consensus sequences and frequencies of human splice site regions are described in Ma, S.L., et al., 2015. PLoS One, 10(6), p.e0130729.
  • the splice acceptor sequence may comprise the nucleotide sequence (Y) n NYAG, where n is 10-20, or a variant with at least 90% or at least 95% sequence identity.
  • the splice acceptor sequence may comprise the sequence (Y) n NCAG, where n is 10-20, or a variant with at least 90% or at least 95% sequence identity.
  • the splice acceptor sequence comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 33 or a fragment thereof.
  • the splice acceptor sequence comprises or consists of a nucleotide sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 33 or a fragment thereof.
  • the splice acceptor sequence comprises or consists of the nucleotide sequence SEQ ID NO: 33 or a fragment thereof.
  • Exemplary splice acceptor sequence (SEQ ID NO: 33) ctgacctcttctcttcctcccacag
  • the polynucleotide of the invention may comprise a splice donor sequence.
  • the genome may comprise a splice donor sequence in the RAG1 intron 1.
  • the splice donor sequence nucleotide sequence is 3’ of the nucleotide sequence encoding a RAG1 polypeptide.
  • the splice donor sequence may be used to provide an mRNA comprising the RAG1 polypeptide and RAG1 exon 2.
  • a “splice donor sequence” is a nucleotide sequence which can function as a donor site at the 5’ end of the intron. Consensus sequences and frequencies of human splice site regions are describe in Ma, S.L., et al., 2015. PLoS One, 10(6), p.e0130729.
  • the splice donor sequence comprises or consists of a nucleotide sequence which is at least 85% identical to SEQ ID NO: 34 or a fragment thereof. In some embodiments of the invention, the splice donor sequence comprises or consists of the nucleotide sequence SEQ ID NO: 34 or a fragment thereof.
  • Exemplary splice donor sequence (SEQ ID NO: 34) aggtaagt
  • the polynucleotide of the invention does not comprise a splice donor sequence.
  • the polynucleotide of the invention may comprise one or more regulatory elements which may act pre- or post-transcriptionally.
  • the nucleotide sequence encoding a RAG1 polypeptide is operably linked to one or more regulatory elements which may act pre- or post- transcriptionally.
  • the one or more regulatory elements may facilitate expression of the RAG1 polypeptide in the cells of the invention.
  • a “regulatory element” is any nucleotide sequence which facilitates expression of a polypeptide, e.g. acts to increase expression of a transcript or to enhance mRNA stability. Suitable regulatory elements include for example promoters, enhancer elements, post- transcriptional regulatory elements and polyadenylation sites.
  • the polynucleotide of the invention may comprise a polyadenylation sequence.
  • the nucleotide sequence encoding a RAG1 polypeptide is operably linked to a polyadenylation sequence.
  • the polyadenylation sequence may improve gene expression.
  • Suitable polyadenylation sequences will be well known to those of skill in the art. Suitable polyadenylation sequences include a bovine growth hormone (BGH) polyadenylation sequence or an early SV40 polyadenylation signal. In some embodiments of the invention, the polyadenylation sequence is a BGH polyadenylation sequence. In some embodiments of the invention, the polyadenylation sequence comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 35, 62 or 65 or a fragment thereof.
  • BGH bovine growth hormone
  • the polyadenylation sequence comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 35, 62 or 65 or a fragment thereof.
  • the polyadenylation sequence comprises or consists of a nucleotide sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 35, 62 or 65 or a fragment thereof.
  • the polyadenylation sequence comprises or consists of the nucleotide sequence SEQ ID NO: 35, 62 or 65 or a fragment thereof.
  • Exemplary BGH polyadenylation sequence SEQ ID NO: 35
  • Exemplary BGH polyadenylation sequence (SEQ ID NO: 62)
  • Exemplary BGH polyadenylation sequence (SEQ ID NO: 65) ctgtgccttctagttgccagccatctgttgtttgccctccccgtgccttccttgaccctggaaggtgccactcccactgtc ctttcctaataaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggggggggcaggac agcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggtgggctctatggggggtgggtctatgg
  • the polynucleotide of the invention may comprise a Kozak sequence.
  • the nucleotide sequence encoding a RAG1 polypeptide is operably linked to a Kozak sequence.
  • a Kozak sequence may be inserted before the start codon of the RAG1 polypeptide to improve the initiation of translation.
  • the Kozak sequence comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 36 or a fragment thereof.
  • the Kozak sequence comprises or consists of a nucleotide sequence which is at least 80%, or at least 90% identical to SEQ ID NO: 36 or a fragment thereof.
  • the Kozak sequence comprises or consists of the nucleotide sequence SEQ ID NO: 36 or a fragment thereof.
  • the polynucleotide of the invention may comprise a post-transcriptional regulatory element.
  • the nucleotide sequence encoding a RAG1 polypeptide is operably linked to a post- transcriptional regulatory element.
  • the post-transcriptional regulatory element may improve gene expression.
  • Suitable post-transcriptional regulatory elements will be well known to those of skill in the art.
  • the polynucleotide of the invention may comprise a Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element (WPRE).
  • WPRE Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element
  • the nucleotide sequence encoding a RAG1 polypeptide is operably linked to a WPRE.
  • the WPRE comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 37 or a fragment thereof.
  • the WPRE comprises or consists of a nucleotide sequence which is at least 80%, or at least 90% identical to SEQ ID NO: 37 or a fragment thereof.
  • the WPRE comprises or consists of the nucleotide sequence SEQ ID NO: 37 or a fragment thereof.
  • Exemplary WPRE (SEQ ID NO: 37) aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgct g ctttaatg cctttgtatcatg ctattg ctttcccgtatg gctttcattttctcctctttg tataaatcctg gttgctgtctctttatg ag gagttgtggcccgttgtcaggcaacgtggcgtggtgtgtgcactgtgttttgctgacgcaacccccactggttggggcattgc caccacctgtcagctccctttccgggacttttcgc
  • the polynucleotide of the invention may comprise an endogenous RAG1 3’IITR.
  • the nucleotide sequence encoding a RAG1 polypeptide is operably linked to an endogenous RAG1 3’IITR.
  • the RAG1 3’IITR comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 38 or a fragment thereof.
  • the RAG1 3’IITR comprises or consists of a nucleotide sequence which is at least 80%, or at least 90% identical to SEQ ID NO: 38 or a fragment thereof.
  • the RAG1 3’IITR comprises or consists of the nucleotide sequence SEQ ID NO: 38 or a fragment thereof.
  • Exemplary RAG 1 3’UTR (SEQ ID NO: 38) gtagggcaaccacttatgagttggtttttgcaattgagtttccctctgggttgcattgagggcttctctagcaccctttactg ctgtgtatggggcttcaccatccaagaggtggtaggttggagtaagatgctacagatgctctcaagtcaggaataga actgatgagctgattgcttgaggcttttagtgagttccgaaaagcaacaggaaaatcagttatctgaaagctcagtaa ctcagaacaggagtaactgcaggggaccagagatgagcaaagatctgtgtgtggggagctgtcatgtaaatcaa agcca
  • the RAG1 polypeptide is not operably linked to a RAG1 3’IITR.
  • the polynucleotide of the invention may comprise a further coding sequence.
  • the polynucleotide of the invention may comprise an internal ribosome entry site sequence (IRES).
  • IRES may increase or allow expression of the further coding sequence.
  • the IRES may be operably linked to the further coding sequence.
  • the IRES comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 63 or a fragment thereof.
  • the IRES comprises or consists of a nucleotide sequence which is at least 80%, or at least 90% identical to SEQ ID NO: 63 or a fragment thereof.
  • the IRES comprises or consists of the nucleotide sequence SEQ ID NO: 63 or a fragment thereof.
  • IRES gaattaactcgaggaattccgCccctctccctccccccccctaacgttactggccgaagccgcttggaataaggccg gtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttg acgcattctaggggtctttccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgtgaaggaagcagttcctctg gaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccccccccccta
  • the further coding sequence may encode a selector, for example a NGFR receptor, e.g. a low affinity NGFR, such as a C-terminal truncated low affinity NGFR.
  • the selector may be used for enrichment of cells.
  • the NGFR-encoding sequence comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 64 or a fragment thereof.
  • the NGFR-encoding sequence comprises or consists of a nucleotide sequence which is at least 80%, or at least 90% identical to SEQ ID NO: 64 or a fragment thereof.
  • the NGFR-encoding sequence comprises or consists of the nucleotide sequence SEQ ID NO: 64 or a fragment thereof.
  • Exemplary A/GFR-encoding sequence (SEQ ID NO: 64) atgggagctggtgctaccggcagagctatggatggacctagactgctgctcctgctgctgctgctcggagtttctcttggcgg agccaaagaggcctgtcctaccggcctgtatacacactctggcgagtgctgcaaggcctgcaatcttggagaaggcg tggcacagccttgcggcgctaatcagacagtgtgcgagccttgctggacagcgtgacctttagcgacgtggtgtctgc caccgagccatgcaagccttgtaccgagtgtgtgggcctgcagagcatgtctgccccttgtggaagc
  • the PEST-encoding sequence comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 66 or a fragment thereof.
  • the PEST-encoding sequence comprises or consists of a nucleotide sequence which is at least 80%, or at least 90% identical to SEQ ID NO: 66 or a fragment thereof.
  • the PEST-encoding sequence comprises or consists of the nucleotide sequence SEQ ID NO: 66 or a fragment thereof.
  • Exemplary PEST-encoding sequence (SEQ ID NO: 66) atgaggaccgaggccccgagggcaccgagagcgagatggagacccccagcgccatcaacggcaaccccagc tggcac
  • nucleotide sequence encoding a RAG1 polypeptide is operably linked to a promoter and/or enhancer element.
  • a “promoter” is a region of DNA that leads to initiation of transcription of a gene. Promoters are located near the transcription start sites of genes, upstream on the DNA (towards the 5' region of the sense strand). Any suitable promoter may be used, the selection of which may be readily made by the skilled person.
  • Enhancers are cis-acting. They can be located up to 1 Mbp (1 ,000,000 bp) away from the gene, upstream or downstream from the start site. Any suitable enhancer may be used, the selection of which may be readily made by the skilled person.
  • Transcription of the nucleotide sequence encoding a RAG1 polypeptide may be driven by an endogenous promoter.
  • the polynucleotide of the present invention is inserted into the RAG1 intron 1 , transcription of the nucleotide sequence encoding a RAG1 polypeptide may be driven by the endogenous RAG1 promoter.
  • the polynucleotide of the invention does not comprise a promoter and/or enhancer element.
  • the genome of the invention does not comprise a promoter and/or enhancer element (e.g. an exogenous promoter and/or enhancer element) in the RAG1 intron 1.
  • the polynucleotide of the invention comprises, essentially consists of, or consists of from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, a polyadenylation sequence and a second homology region.
  • the polynucleotide of the invention comprises, essentially consists of, or consists of from 5’ to 3’: a first homology region, a splice acceptor sequence, a kozak sequence, a nucleotide sequence encoding a RAG1 polypeptide, a polyadenylation sequence and a second homology region.
  • the polynucleotide of the invention comprises, essentially consists of, or consists of from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, a WPRE, a polyadenylation sequence and a second homology region.
  • the polynucleotide of the invention comprises, essentially consists of, or consists of from 5’ to 3’: a first homology region, a splice acceptor sequence, a kozak sequence, a nucleotide sequence encoding a RAG1 polypeptide, a WPRE, a polyadenylation sequence and a second homology region.
  • the polynucleotide of the invention comprises, essentially consists of, or consists of from 5’ to 3’: a first homology region, a splice acceptor sequence, a kozak sequence, a nucleotide sequence encoding a RAG1 polypeptide, a 3’ UTR, a polyadenylation sequence and a second homology region.
  • the polynucleotide of the invention comprises, essentially consists of, or consists of from 5’ to 3’: a first homology region, a splice acceptor sequence, a kozak sequence, a nucleotide sequence encoding a RAG1 polypeptide, an IRES, a nucleotide sequence encoding a selector (e.g. NGFR), a polyadenylation sequence and a second homology region.
  • a selector e.g. NGFR
  • the polynucleotide of the invention comprises, essentially consists of, or consists of from 5’ to 3’: a first homology region, a splice acceptor sequence, a kozak sequence, a nucleotide sequence encoding a RAG1 polypeptide, an IRES, a nucleotide sequence encoding a destabilisation domain (e.g. a PEST sequence), a splice donor sequence, and a second homology region.
  • the polynucleotide of the invention comprises, essentially consists of, or consists of from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, a splice donor sequence and a second homology region.
  • the polynucleotide of the invention comprises or consists of a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 39.
  • the polynucleotide of the invention comprises or consists of the nucleotide sequence of SEQ ID NO: 39.
  • the genome of the invention comprises a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 39.
  • the genome of the invention comprises the nucleotide sequence of SEQ ID NO: 39.
  • the genome of the invention comprises a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to nucleotides 297-3687 of SEQ ID NO: 39 or nucleotides 291- 3693 of SEQ ID NO: 39.
  • the genome of the invention comprises the nucleotide sequence of nucleotides 297-3687 of SEQ ID NO: 39 or nucleotides 291-3693 of SEQ ID NO: 39.
  • Exemplary polynucleotide (SEQ ID NO: 39) tgagcacacacagttattacttggaaattgtgtacagactaagttgaagatgttaggagggaagattgtgggccaagtaac ggggtgtatgtgtgtgggtatagggtgggcagctgggatggaaatggggggctgctgctgctgctgcaccctggcctc ctgaactaatgatatcactcaccagaaactactgttcctgcactgtccaagccaccccaaactagtttgtcaaaatgaat ctgtgctgtggagggaggcacgctgtagctctgatgtcagatggcaatgtgaattcctgacctcttctctctcccac aggcc
  • the genome of the invention comprises a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 40.
  • the genome of the invention comprises the nucleotide sequence of SEQ ID NO: 40.
  • nucleotide sequence insert (SEQ ID NO: 40) gaattcctgacctcttctcttcctcccacaggccgccaccaccatggccgcctccttccacctacccttggattgtcctccgcc cctgacgaaattcaacatccccacatcaaattctcggagtggaagttcaagctctttcgcgtgcgttcgaaaaga ccccgaggaagcccaaaggagaagaaagactcattcgaaggaaaacccagcctcgaacagtccccggcgt cgt cgt cgt cgt cgt cgaaggagaagaaagactcattcgaaggaaaacccagcctcgaacagtcccgacccag
  • the invention also encompasses variants, derivatives, and fragments thereof.
  • a “variant” of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one of its endogenous functions.
  • a variant of RAG1 may retain the ability to form a RAG complex, mediate DNA-binding to the RSS, and introduce a double-strand break between the RSS and the adjacent coding segment.
  • a variant sequence can be obtained by addition, deletion, substitution, modification, replacement and/or variation of at least one residue present in the naturally occurring polypeptide or polynucleotide.
  • derivative as used herein in relation to proteins or polypeptides of the invention includes any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence, providing that the resultant protein or polypeptide retains at least one of its endogenous functions.
  • a derivative of RAG1 may retain the ability to form a RAG complex, mediate DNA-binding to the RSS, and introduce a double-strand break between the RSS and the adjacent coding segment.
  • amino acid substitutions may be made, for example from 1 , 2 or 3, to 10 or 20 substitutions, provided that the modified sequence retains the required activity or ability.
  • Amino acid substitutions may include the use of non-naturally occurring analogues.
  • Proteins used in the invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues as long as the endogenous function is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.
  • a variant may have a certain identity with the wild type amino acid sequence or the wild type nucleotide sequence.
  • a variant sequence is taken to include an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, suitably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence.
  • a variant can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express in terms of sequence identity.
  • a variant sequence is taken to include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, suitably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence.
  • a variant can also be considered in terms of similarity, in the context of the present invention it is preferred to express it in terms of sequence identity.
  • reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to.
  • Sequence identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent identity between two or more sequences.
  • Percent identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs).
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • the percent sequence identity may be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to.
  • “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full- length polypeptide or polynucleotide.
  • Such variants, derivatives, and fragments may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis.
  • synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made.
  • the flanking regions will contain convenient restriction sites corresponding to sites in the naturally- occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut.
  • the DNA is then expressed in accordance with the invention to make the encoded protein.
  • the present invention provides a vector comprising the polynucleotide of the invention.
  • the vector may be suitable for editing a genome using the polynucleotide of the invention.
  • the vector may be used to deliver the polynucleotide into the cell.
  • the nucleotide sequence insert can be introduced into a genome at a site of a double strand break (DSB) by homology-directed repair (HDR).
  • DLB double strand break
  • HDR homology-directed repair
  • the vector of the present invention may be capable of transducing mammalian cells, for example human cells.
  • the vector of the present invention is capable of transducing HSCs, HPCs, and/or LPCs.
  • the vector of the present invention is capable of transducing CD34+ cells.
  • the vector of the present invention is capable of transducing NALM6, K562, and/or other human cell lines (e.g. Molt4, LI937, etc.).
  • the vector of the present invention is capable of transducing T cells.
  • the vector of the present invention is a viral vector.
  • the vector of the invention may be an adeno-associated viral (AAV) vector, although it is contemplated that other viral vectors may be used e.g. lentiviral vectors (e.g. IDLV vectors), or single or double stranded DNA.
  • AAV adeno-associated viral
  • the vector of the present invention may be in the form of a viral vector particle.
  • the viral vector of the present invention is in the form of an AAV vector particle.
  • the viral vector of the present invention is in the form of a lentiviral vector particle, for example an IDLV vector particle.
  • Methods of preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art. Suitable methods are described in Ayuso, E., et al., 2010. Current gene therapy, 10(6), pp.423-436, Merten, O.W., et al., 2016. Molecular Therapy-Methods & Clinical Development, 3, p.16017; and Nadeau, I. and Kamen, A., 2003. Biotechnology advances, 20(7-8), pp.475-489.
  • AAV Adeno-associated viral
  • the vector of the present invention may be an adeno-associated viral (AAV) vector.
  • the vector is an AAV6 vector.
  • the vector of the present invention may be in the form of an AAV vector particle.
  • the vector is in the form of an AAV6 vector particle.
  • the AAV vector or AAV vector particle may comprise an AAV genome or a fragment or derivative thereof.
  • An AAV genome is a polynucleotide sequence, which may encode functions needed for production of an AAV particle. These functions include those operating in the replication and packaging cycle of AAV in a host cell, including encapsidation of the AAV genome into an AAV particle.
  • Naturally occurring AAVs are replication-deficient and rely on the provision of helper functions in trans for completion of a replication and packaging cycle. Accordingly, the AAV genome of the AAV vector of the invention is typically replicationdeficient.
  • the AAV genome may be in single-stranded form, either positive or negative-sense, or alternatively in double-stranded form.
  • the use of a double-stranded form allows bypass of the DNA replication step in the target cell and so can accelerate transgene expression.
  • AAVs occurring in nature may be classified according to various biological systems.
  • the AAV genome may be from any naturally derived serotype, isolate or clade of AAV.
  • AAV may be referred to in terms of their serotype.
  • a serotype corresponds to a variant subspecies of AAV which, owing to its profile of expression of capsid surface antigens, has a distinctive reactivity which can be used to distinguish it from other variant subspecies.
  • an AAV vector particle having a particular AAV serotype does not efficiently crossreact with neutralising antibodies specific for any other AAV serotype.
  • AAV serotypes include AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11.
  • the AAV vector of the invention may be an AAV6 serotype.
  • AAV may also be referred to in terms of clades or clones. This refers to the phylogenetic relationship of naturally derived AAVs, and typically to a phylogenetic group of AAVs which can be traced back to a common ancestor, and includes all descendants thereof. Additionally, AAVs may be referred to in terms of a specific isolate, i.e. a genetic isolate of a specific AAV found in nature. The term genetic isolate describes a population of AAVs which has undergone limited genetic mixing with other naturally occurring AAVs, thereby defining a recognisably distinct population at a genetic level.
  • the AAV genome of a naturally derived serotype, isolate or clade of AAV comprises at least one inverted terminal repeat sequence (ITR).
  • ITR sequence acts in cis to provide a functional origin of replication and allows for integration and excision of the vector from the genome of a cell.
  • ITRs may be the only sequences required in cis next to the therapeutic gene.
  • one or more ITR sequences flank the polynucleotide of the invention.
  • the AAV genome may also comprise packaging genes, such as rep and/or cap genes which encode packaging functions for an AAV particle.
  • a promoter may be operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19 and p40 promoters. For example, the p5 and p19 promoters are generally used to express the rep gene, while the p40 promoter is generally used to express the cap gene.
  • the rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof.
  • the cap gene encodes one or more capsid proteins such as VP1 , VP2 and VP3 or variants thereof.
  • the AAV genome may be the full genome of a naturally occurring AAV.
  • a vector comprising a full AAV genome may be used to prepare an AAV vector or vector particle.
  • the AAV genome is derivatised for the purpose of administration to patients. Such derivatisation is standard in the art and the invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art.
  • the AAV genome may be a derivative of any naturally occurring AAV.
  • the AAV genome is a derivative of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11.
  • the AAV genome is a derivative of AAV6.
  • Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a transgene from an AAV vector of the invention in vivo.
  • a derivative will include at least one inverted terminal repeat sequence (ITR), optionally more than one ITR, such as two ITRs or more.
  • ITRs may be derived from AAV genomes having different serotypes, or may be a chimeric or mutant ITR.
  • a suitable mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication of the genome to generate a single-stranded genome which contains both coding and complementary sequences, i.e. a self-complementary AAV genome. This allows for bypass of DNA replication in the target cell, and so enables accelerated transgene expression.
  • the AAV genome may comprise one or more ITR sequences from any naturally derived serotype, isolate or clade of AAV or a variant thereof.
  • the AAV genome may comprise at least one, such as two, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITRs, or variants thereof.
  • the one or more ITRs may flank the nucleotide sequence of the invention at either end.
  • the inclusion of one or more ITRs is can aid concatamer formation of the AAV vector in the nucleus of a host cell, for example following the conversion of single-stranded vector DNA into doublestranded DNA by the action of host cell DNA polymerases.
  • the formation of such episomal concatamers protects the AAV vector during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo.
  • ITR elements will be the only sequences retained from the native AAV genome in the derivative.
  • a derivative may not include the rep and/or cap genes of the native genome and any other sequences of the native genome. This may reduce the possibility of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the transgene.
  • derivatives may additionally include one or more rep and/or cap genes or other viral sequences of an AAV genome.
  • Naturally occurring AAV integrates with a high frequency at a specific site on human chromosome 19, and shows a negligible frequency of random integration, such that retention of an integrative capacity in the AAV vector may be tolerated in a therapeutic setting.
  • the invention additionally encompasses the provision of sequences of an AAV genome in a different order and configuration to that of a native AAV genome.
  • the invention also encompasses the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus.
  • Such chimeric genes may be composed of sequences from two or more related viral proteins of different viral species.
  • the AAV vector particle may be encapsidated by capsid proteins.
  • the AAV vector particles may be transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype.
  • the AAV vector particle also includes mosaic forms wherein a mixture of unmodified capsid proteins from two or more different serotypes makes up the viral capsid.
  • the AAV vector particle also includes chemically modified forms bearing ligands adsorbed to the capsid surface.
  • ligands may include antibodies for targeting a particular cell surface receptor.
  • a derivative comprises capsid proteins i.e. VP1 , VP2 and/or VP3
  • the derivative may be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAVs.
  • the invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones, or isolates of AAV within the same vector (i.e. a pseudotyped vector).
  • the AAV vector may be in the form of a pseudotyped AAV vector particle.
  • Chimeric, shuffled or capsid-modified derivatives will be typically selected to provide one or more desired functionalities for the AAV vector.
  • these derivatives may display increased efficiency of gene delivery and/or decreased immunogenicity (humoral or cellular) compared to an AAV vector comprising a naturally occurring AAV genome.
  • Increased efficiency of gene delivery may be effected by improved receptor or co-receptor binding at the cell surface, improved internalisation, improved trafficking within the cell and into the nucleus, improved uncoating of the viral particle and improved conversion of a single-stranded genome to double-stranded form.
  • Chimeric capsid proteins include those generated by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This may be performed for example by a marker rescue approach in which non-infectious capsid sequences of one serotype are co-transfected with capsid sequences of a different serotype, and directed selection is used to select for capsid sequences having desired properties.
  • the capsid sequences of the different serotypes can be altered by homologous recombination within the cell to produce novel chimeric capsid proteins.
  • Chimeric capsid proteins also include those generated by engineering of capsid protein sequences to transfer specific capsid protein domains, surface loops or specific amino acid residues between two or more capsid proteins, for example between two or more capsid proteins of different serotypes.
  • Hybrid AAV capsid genes can be created by randomly fragmenting the sequences of related AAV genes e.g. those encoding capsid proteins of multiple different serotypes and then subsequently reassembling the fragments in a self-priming polymerase reaction, which may also cause crossovers in regions of sequence homology.
  • a library of hybrid AAV genes created in this way by shuffling the capsid genes of several serotypes can be screened to identify viral clones having a desired functionality.
  • error prone PCR may be used to randomly mutate AAV capsid genes to create a diverse library of variants which may then be selected for a desired property.
  • capsid genes may also be genetically modified to introduce specific deletions, substitutions or insertions with respect to the native wild-type sequence.
  • capsid genes may be modified by the insertion of a sequence of an unrelated protein or peptide within an open reading frame of a capsid coding sequence, or at the N- and/or C-terminus of a capsid coding sequence.
  • the unrelated protein or peptide may advantageously be one which acts as a ligand for a particular cell type, thereby conferring improved binding to a target cell or improving the specificity of targeting of the vector to a particular cell population.
  • the unrelated protein may also be one which assists purification of the viral particle as part of the production process, i.e. an epitope or affinity tag.
  • the site of insertion will typically be selected so as not to interfere with other functions of the viral particle e.g. internalisation, trafficking of the viral particle.
  • the capsid protein may be an artificial or mutant capsid protein.
  • artificial capsid as used herein means that the capsid particle comprises an amino acid sequence which does not occur in nature or which comprises an amino acid sequence which has been engineered (e.g. modified) from a naturally occurring capsid amino acid sequence.
  • the artificial capsid protein comprises a mutation or a variation in the amino acid sequence compared to the sequence of the parent capsid from which it is derived where the artificial capsid amino acid sequence and the parent capsid amino acid sequences are aligned.
  • the AAV vector particle may comprise an AAV6 capsid protein.
  • the vector of the present invention may be a retroviral vector or a lentiviral vector.
  • the vector of the present invention may be a retroviral vector particle or a lentiviral vector particle.
  • a retroviral vector may be derived from or may be derivable from any suitable retrovirus.
  • retroviruses include murine leukaemia virus (MLV), human T-cell leukaemia virus (HTLV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29) and avian erythroblastosis virus (AEV).
  • MMV murine leukaemia virus
  • HTLV human T-cell leukaemia virus
  • MMTV mouse mammary tumour virus
  • RSV Rous sarcoma virus
  • Fujinami sarcoma virus FuSV
  • Retroviruses may be broadly divided into two categories, “simple” and “complex”. Retroviruses may be even further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses.
  • retrovirus and lentivirus genomes share many common features such as a 5’ LTR and a 3’ LTR. Between or within these are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome, and gag, pol and env genes encoding the packaging components - these are polypeptides required for the assembly of viral particles.
  • Lentiviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell.
  • LTRs long terminal repeats
  • the LTRs themselves are identical sequences that can be divided into three elements: U3, R and U5.
  • U3 is derived from the sequence unique to the 3’ end of the RNA.
  • R is derived from a sequence repeated at both ends of the RNA.
  • U5 is derived from the sequence unique to the 5’ end of the RNA.
  • the sizes of the three elements can vary considerably among different retroviruses.
  • gag, pol and env may be absent or not functional.
  • a retroviral vector In a typical retroviral vector, at least part of one or more protein coding regions essential for replication may be removed from the virus. This makes the viral vector replication-defective. Portions of the viral genome may also be replaced by a library encoding candidate modulating moieties operably linked to a regulatory control region and a reporter moiety in the vector genome in order to generate a vector comprising candidate modulating moieties which is capable of transducing a target host cell and/or integrating its genome into a host genome.
  • Lentivirus vectors are part of the larger group of retroviral vectors.
  • lentiviruses can be divided into primate and non-primate groups.
  • primate lentiviruses include but are not limited to human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SI ).
  • non-primate lentiviruses examples include the prototype “slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
  • VMV visna/maedi virus
  • CAEV caprine arthritis-encephalitis virus
  • EIAV equine infectious anaemia virus
  • FIV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • the lentivirus family differs from retroviruses in that lentiviruses have the capability to infect both dividing and non-dividing cells.
  • other retroviruses such as MLV, are unable to infect non-dividing or slowly dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.
  • a lentiviral vector is a vector which comprises at least one component part derivable from a lentivirus.
  • that component part is involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated.
  • the lentiviral vector may be a “primate” vector.
  • the lentiviral vector may be a “non-primate” vector (i.e. derived from a virus which does not primarily infect primates, especially humans).
  • non-primate lentiviruses may be any member of the family of lentiviridae which does not naturally infect a primate.
  • HIV-1- and HIV-2-based vectors are described below.
  • the HIV-1 vector contains cis-acting elements that are also found in simple retroviruses. It has been shown that sequences that extend into the gag open reading frame are important for packaging of HIV-1. Therefore, HIV-1 vectors often contain the relevant portion of gag in which the translational initiation codon has been mutated. In addition, most HIV-1 vectors also contain a portion of the env gene that includes the RRE. Rev binds to RRE, which permits the transport of full-length or singly spliced mRNAs from the nucleus to the cytoplasm. In the absence of Rev and/or RRE, full-length HIV-1 RNAs accumulate in the nucleus. Alternatively, a constitutive transport element from certain simple retroviruses such as Mason-Pfizer monkey virus can be used to relieve the requirement for Rev and RRE. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.
  • HIV-2-based vectors are structurally very similar to HIV-1 vectors. Similar to HIV-1-based vectors, HIV-2 vectors also require RRE for efficient transport of the full-length or singly spliced viral RNAs.
  • the viral vector used in the present invention has a minimal viral genome.
  • minimal viral genome it is to be understood that the viral vector has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in WO 1998/017815.
  • the plasmid vector used to produce the viral genome within a host cell/packaging cell will have sufficient lentiviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle which is capable of infecting a target cell, but is incapable of independent replication to produce infectious viral particles within the final target cell.
  • the vector lacks a functional gag-pol and/or env gene and/or other genes essential for replication.
  • the plasmid vector used to produce the viral genome within a host cell/packaging cell will also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a host cell/packaging cell.
  • transcriptional regulatory control sequences may be the natural sequences associated with the transcribed viral sequence (i.e. the 5’ U3 region), or they may be a heterologous promoter, such as another viral promoter (e.g. the CMV promoter).
  • the vectors may be self-inactivating (SIN) vectors in which the viral enhancer and promoter sequences have been deleted.
  • SIN vectors can be generated and transduce non-dividing cells in vivo with an efficacy similar to that of wild-type vectors.
  • the transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus should prevent mobilisation by replication- competent virus. This should also enable the regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTR.
  • LTR long terminal repeat
  • the vectors may be integration-defective.
  • Integration defective lentiviral vectors can be produced, for example, either by packaging the vector with catalytically inactive integrase (such as an HIV integrase bearing the D64V mutation in the catalytic site) or by modifying or deleting essential att sequences from the vector LTR, or by a combination of the above.
  • the vector of the present invention may be an adenoviral vector.
  • the vector of the present invention may be an adenoviral vector particle.
  • the adenovirus is a double-stranded, linear DNA virus that does not go through an RNA intermediate.
  • adenovirus There are over 50 different human serotypes of adenovirus divided into 6 subgroups based on the genetic sequence homology.
  • the natural targets of adenovirus are the respiratory and gastrointestinal epithelia, generally giving rise to only mild symptoms.
  • Serotypes 2 and 5 (with 95% sequence homology) are most commonly used in adenoviral vector systems and are normally associated with upper respiratory tract infections in the young.
  • Adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes.
  • the large (36 kb) genome can accommodate up to 8 kb of foreign insert DNA and is able to replicate efficiently in complementing cell lines to produce very high titres of up to 10 12 .
  • Adenovirus is thus one of the best systems to study the expression of genes in primary non- replicative cells.
  • Adenoviral vectors enter cells by receptor mediated endocytosis. Once inside the cell, adenovirus vectors rarely integrate into the host chromosome. Instead, they function episomally (independently from the host genome) as a linear genome in the host nucleus. Hence the use of recombinant adenovirus alleviates the problems associated with random integration into the host genome.
  • Herpes simplex viral vector Herpes simplex viral vector
  • the vector of the present invention may be a herpes simplex viral vector.
  • the vector of the present invention may be a herpes simplex viral vector particle.
  • Herpes simplex virus is a neurotropic DNA virus with favorable properties as a gene delivery vector.
  • HSV is highly infectious, so HSV vectors are efficient vehicles for the delivery of exogenous genetic material to cells.
  • Viral replication is readily disrupted by null mutations in immediate early genes that in vitro can be complemented in trans, enabling straightforward production of high-titre pure preparations of non-pathogenic vector.
  • the genome is large (152 Kb) and many of the viral genes are dispensable for replication in vitro, allowing their replacement with large or multiple transgenes.
  • Latent infection with wild-type virus results in episomal viral persistence in sensory neuronal nuclei for the duration of the host lifetime.
  • the vectors are non-pathogenic, unable to reactivate and persist long-term.
  • HSV vectors transduce a broad range of tissues because of the wide expression pattern of the cellular receptors recognized by the virus. Increasing understanding of the processes involved in cellular entry has allowed targeting the tropism of HSV vectors.
  • the vector of the present invention may be a vaccinia viral vector.
  • the vector of the present invention may be a vaccinia viral vector particle.
  • Vaccinia virus is large enveloped virus that has an approximately 190 kb linear, doublestranded DNA genome. Vaccinia virus can accommodate up to approximately 25 kb of foreign DNA, which also makes it useful for the delivery of large genes.
  • a number of attenuated vaccinia virus strains are known in the art that are suitable for gene therapy applications, for example the MVA and NYVAC strains.
  • the vector of the present invention may be used to deliver a polynucleotide into a cell. Subsequently, a nucleotide sequence insert can be introduced into the cell’s genome at a site of a double strand break (DSB) by homology-directed repair (HDR).
  • the site of the doublestrand break (DSB) can be introduced specifically by any suitable technique, for example by using an RNA-guided gene editing system.
  • RNA-guided gene editing system can be used to introduce a DSB and typically comprises a guide RNA and a RNA-guided nuclease.
  • a CRISPR/Cas9 system is an example of a commonly used RNA-guided gene editing system, but other RNA-guided gene editing systems may also be used.
  • a “guide RNA” confers target sequence specificity to a RNA-guided nuclease.
  • Guide RNAs are non-coding short RNA sequences which bind to the complementary target DNA sequences. For example, in the CRISPR/Cas9 system, guide RNA first binds to the Cas9 enzyme and the gRNA sequence guides the resulting complex via base-pairing to a specific location on the DNA, where Cas9 performs its nuclease activity by cutting the target DNA strand.
  • guide RNA encompasses any suitable gRNA that can be used with any RNA- guided nuclease, and not only those gRNAs that are compatible with a particular nuclease such as Cas9.
  • the guide RNA may comprise a trans-activating CRISPR RNA (tracrRNA) that provides the stem loop structure and a target-specific CRISPR RNA (crRNA) designed to cleave the gene target site of interest.
  • tracrRNA and crRNA may be annealed, for example by heating them at 95°C for 5 minutes and letting them slowly cool down to room temperature for 10 minutes.
  • the guide RNA may be a single guide RNA (sgRNA) that consists of both the crRNA and tracrRNA as a single construct.
  • the guide RNA may comprise of a 3’-end, which forms a scaffold for nuclease binding, and a 5'-end which is programmable to target different DNA sites.
  • the targeting specificity of CRISPR-Cas9 may be determined by the 15-25 bp sequence at the 5' end of the guide RNA.
  • the desired target sequence typically precedes a protospacer adjacent motif (PAM) which is a short DNA sequence usually 2-6 bp in length that follows the DNA region targeted for cleavage by the CRISPR system, such as CRISPR-Cas9.
  • PAM protospacer adjacent motif
  • the PAM is required for a Cas nuclease to cut and is typically found 3-4 bp downstream from the cut site.
  • Cas9 mediates a double strand break about 3-nt upstream of PAM.
  • Numerous tools exist for designing guide RNAs e.g.
  • COSMID is a webbased tool for identifying and validating guide RNAs (Cradick TJ, et al. Mol Ther - Nucleic Acids. 2014;3(12):e214).
  • the present invention provides a guide RNA comprising or consisting of a nucleotide sequence that has at least 90% identity or at least 95% identity to any of SEQ ID NOs: 41-52, optionally wherein the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity or at least 95% identity to SEQ ID NO: 41.
  • the guide RNA comprises or consists of the nucleotide sequence of any of SEQ ID NOs: 41-52, optionally wherein the guide RNA comprises or consists of the nucleotide sequence of SEQ ID NO: 41.
  • sequences for guides 9, 3 and 7 may be extended as shown below, for example when used as crRNA:
  • the present invention provides a guide RNA comprising or consisting of a nucleotide sequence that has at least 90% identity or at least 95% identity to any of SEQ ID NOs: 53-55, optionally wherein the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity or at least 95% identity to SEQ ID NO: 53.
  • the guide RNA comprises or consists of the nucleotide sequence of any of SEQ ID NOs: 53-55, optionally wherein the guide RNA comprises or consists of the nucleotide sequence of SEQ ID NO: 53.
  • the guide RNA is chemically modified.
  • the chemical modification may enhance the stability of the guide RNA.
  • from one to five (e.g. three) of the terminal nucleotides at 5’ end and/or 3’ end of the guide RNA may be chemically modified to enhance stability.
  • any chemical modification which enhances the stability of the guide RNA may be used.
  • the chemical modification may be modification with 2'-O-methyl 3'-phosphorothioate, as described in Hendel A, et al. Nat Biotechnol. 2015;33(9):985-9.
  • RNA-quided nuclease is an enzyme that can cleave the phosphodiester bond present within a polynucleotide chain.
  • the nuclease is an endonuclease. Endonucleases are capable of breaking the bond from the middle of a chain.
  • RNA-guided nuclease is a nuclease which can be directed to a specific site by a guide RNA.
  • the present invention can be implemented using any suitable RNA-guided nuclease, for example any RNA-guided nuclease described in Murugan, K., et al., 2017. Molecular cell, 68(1), pp.15-25.
  • RNA-guided nucleases include, but are not limited to, Type II CRISPR nucleases such as Cas9, and Type V CRISPR nucleases such as Cas12a and Cas12b, as well as other nucleases derived therefrom.
  • RNA-guided nucleases can be defined, in broad terms, by their PAM specificity and cleavage activity.
  • the RNA-guided nuclease is a Type II CRISPR nuclease, for example a Cas9 nuclease.
  • Cas9 is a dual RNA-guided endonuclease enzyme associated with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) adaptive immune system.
  • Cas9 nucleases include the well-characterized ortholog from Streptococcus pyogenes (SpCas9). SpCas9 and other orthologs (including SaCas9, FnCa9, and AnaCas9) have been reviewed by Jiang, F. and Doudna, J. A., 2017. Annual review of biophysics, 46, pp.505-529.
  • the RNA-guided nuclease may be in a complex with the guide RNA, i.e. the guide RNA and the RNA-guided nuclease may together form a ribonucleoprotein (RNP).
  • RNP ribonucleoprotein
  • the RNP is a Cas9 RNP.
  • a RNP may be formed by any method known in the art, for example by incubating a RNA-guided nuclease with a guide RNA for 5-30 minutes at room temperature. Delivering Cas9 as a preassembled RNP can protect the guide RNA from intracellular degradation thus improving stability and activity of the RNA-guided nuclease (Kim S, et al. Genome Res. 2014;24(6):1012-9).
  • Kit composition, gene-editing system
  • the present invention provides a kit, composition, or gene-editing system comprising the polynucleotide of the invention, the vector of the invention, and/or the guide RNA of the invention.
  • a “gene-editing system” is a system which comprises all components necessary to edit a genome using the polynucleotide of the invention.
  • the kit, composition, or gene-editing system comprises a polynucleotide and/or vector of the invention and a guide RNA.
  • the guide RNA may correspond to the same DSB site targeted by the homology arms.
  • the kit, composition, or gene-editing system comprises: (i) a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region is homologous to a region upstream of chr 11 : 36569295 and the second homology region is homologous to a region downstream of chr 11 : 36569298, and/or a vector comprising said polynucleotide; and a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to S
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region is homologous to a region upstream of chr 11 : 36573790 and the second homology region is homologous to a region downstream of chr 11 : 36573793 and/or a vector comprising said polynucleotide; and a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 42;
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region is homologous to a region upstream of chr 11 : 36573641 and the second homology region is homologous to a region downstream of chr 11 : 36573644 and/or a vector comprising said polynucleotide; and a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 43;
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region is homologous to a region upstream of chr 11 : 36573351 and the second homology region is homologous to a region downstream of chr 11 : 36573354 and/or a vector comprising said polynucleotide; and a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 44 or 54 (preferably SEQ ID NO: 44);
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region is homologous to a region upstream of chr 11 : 36569080 and the second homology region is homologous to a region downstream of chr 11 : 36569083 and/or a vector comprising said polynucleotide; and a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 45;
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region is homologous to a region upstream of chr 11 : 36572472 and the second homology region is homologous to a region downstream of chr 11 : 36572475 and/or a vector comprising said polynucleotide; and a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 46;
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region is homologous to a region upstream of chr 11 : 36571458 and the second homology region is homologous to a region downstream of chr 11 : 36571461 and/or a vector comprising said polynucleotide; and a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 47;
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region is homologous to a region upstream of chr 11 : 36571366 and the second homology region is homologous to a region downstream of chr 11 : 36571369 and/or a vector comprising said polynucleotide; and a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 48 or 55 (preferably SEQ ID NO: 48);
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region is homologous to a region upstream of chr 11 : 36572859 and the second homology region is homologous to a region downstream of chr 11 : 36572862 and/or a vector comprising said polynucleotide; and a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 49;
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region is homologous to a region upstream of chr 11 : 36571457 and the second homology region is homologous to a region downstream of chr 11 : 36571460 and/or a vector comprising said polynucleotide; and a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 50;
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region is homologous to a region upstream of chr 11 : 36569351 and the second homology region is homologous to a region downstream of chr 11 : 36569354 and/or a vector comprising said polynucleotide; and a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 51 ; or
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region is homologous to a region upstream of chr 11 : 36572375 and the second homology region is homologous to a region downstream of chr 11 : 36572378 and/or a vector comprising said polynucleotide; and a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 52.
  • the kit, composition, or gene-editing system comprises:
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region is homologous to a region upstream of chr 11 : 36569295 and the second homology region is homologous to a region downstream of chr 11 : 36569298, and/or a vector comprising said polynucleotide; and
  • a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 41 or 53 (preferably SEQ ID NO: 41).
  • the kit, composition, or gene-editing system comprises:
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region is homologous to a region comprising chr 11 : 36569245-36569294 and the second homology region is homologous to a region comprising chr 11 : 36569299-36569348, and/or a vector comprising said polynucleotide; and
  • a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 41 or 53 (preferably SEQ ID NO: 41).
  • the kit, composition, or gene-editing system comprises:
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 7 and the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 19, and/or a vector comprising said polynucleotide; and
  • a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 41 or 53 (preferably SEQ ID NO: 41).
  • the kit, composition, or gene-editing system comprises:
  • a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
  • the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
  • a guide RNA which comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 41 or 53 (preferably SEQ ID NO: 41).
  • the kit, composition, or gene-editing system may further comprise an RNA-guided nuclease.
  • the RNA-guided nuclease corresponds to the guide RNA used.
  • the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to any one of SEQ ID NOs: 41-52
  • the RNA-guided nuclease is suitably a Cas9 endonuclease.
  • the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to any one of SEQ ID NOs: 53-55
  • the RNA-guided nuclease is suitably a Cas9 endonuclease.
  • RNA-guided nuclease may be in a complex with the guide RNA, i.e. the guide RNA and the RNA-guided nuclease together form a ribonucleoprotein (RNP).
  • RNP ribonucleoprotein
  • the present invention provides a cell which has been edited using the polynucleotide, vector, kit, composition, or gene-editing system of the present invention.
  • the present invention provides a cell comprising the polynucleotide, vector and/or genome of the present invention.
  • the cell is an isolated cell.
  • the cell is a mammalian cell, for example a human cell.
  • the cell is a hematopoietic stem cell (HSC), a hematopoietic progenitor cell (HPC), or a lymphoid progenitor cell (LPC).
  • HSC hematopoietic stem cell
  • HPC hematopoietic progenitor cell
  • LPC lymphoid progenitor cell
  • the cell is a HSC or a HPC, optionally the cell is a HSC.
  • hematopoietic stem cells are stem cells that have no differentiation potential to cells other than hematopoietic cells
  • hematopoietic progenitor cells are progenitor cells that have no differentiation potential to cells other than hematopoietic cells
  • lymphoid progenitor cells progenitor cells that have no differentiation potential to cells other than lymphocytes.
  • the cell can be obtained from any source.
  • the cell may be autologous or allogeneic.
  • the cell may be obtained or obtainable from any biological sample, such as peripheral blood or cord blood.
  • Peripheral blood may be treated with mobilising agent, i.e. may be mobilised peripheral blood.
  • the cell may be a universal cell.
  • the cell may be isolated or isolatable using commercially available antibodies that bind to cell surface antigens, e.g. CD34, using methods known to those of skill in the art.
  • the antibodies may be conjugated to magnetic beads and immunological procedures utilized to recover the desired cell type.
  • the cell is identified by the presence or absence of one or more antigenic markers. Suitable antigenic markers include CD34, CD133, CD90, CD45, CD4, CD19, CD13, CD3, CD56, CD14, CD61/41 , CD135, CD45RA, CD33, CD66b, CD38, CD45, CD10, CD11c, CD19, CD7, and CD71.
  • the cell is identified by the presence of the antigenic marker CD34 (CD34+), i.e. the cell is a CD34+ cell.
  • the cell may be a cord blood CD34+ cell or a (mobilised) peripheral blood CD34+ cell.
  • the cell may be a CD34+ HSC, a CD34+ HPC, or a CD34+ LPC, optionally the cell is a CD34+ HSC.
  • the cell is identified by the presence of CD34 and the presence or absence or one or more further antigenic markers.
  • the further antigenic markers may be selected from one or more of CD133, CD90, CD3, CD56, CD14, CD61/41 , CD135, CD45RA, CD33, CD66b, CD38, CD45, CD10, CD11c, CD19, CD7, and CD71.
  • the cell may be a CD34+CD133+CD90+ cell, a CD34+CD133+CD90- cell, or a CD34+CD133-CD90- cell.
  • the cell is a NALM6 cell, a K562 cell, or other human cell (e.g. a Molt4 cell, a LI937 cell, etc.).
  • the cell is a T cell.
  • the present invention provides a population or cells comprising the cell of the present invention.
  • at least 1 %, at least 2%, at least 5%, at least 10%, or at least 20% of the cells in the population of cells are cells of the present invention.
  • the population of cells comprises at least 10x10 5 , at least 50x10 5 , or at least 100x10 5 cells of the present invention.
  • the present invention provides a population of cells which have been edited using the polynucleotide, vector, kit, composition, or gene-editing system of the present invention.
  • at least 1 %, at least 2%, at least 5%, at least 10%, or at least 20% of the cells in the population of cells are cells which have been edited using the polynucleotide, vector, kit, composition, or gene-editing system of the present invention.
  • the population of cells comprises at least 10x10 5 , at least 50x10 5 , or at least 100x10 5 cells which have been edited using the polynucleotide, vector, kit, composition, or gene-editing system of the present invention.
  • the present invention provides a population of cells comprising the polynucleotide, vector and/or genome of the present invention.
  • at least 1 %, at least 2%, at least 5%, at least 10%, or at least 20% of the cells in the population of cells are cells comprising the polynucleotide, vector and/or genome of the present invention.
  • the population of cells comprises at least 10x10 5 , at least 50x10 5 , or at least 100x10 5 cells comprising the polynucleotide, vector and/or genome of the present invention.
  • the population of cells are mammalian cells, for example human cells.
  • the population of cells may be autologous or allogeneic.
  • the population of cells are obtained or obtainable from (mobilised) peripheral blood or cord blood.
  • the population of cells may be universal cells.
  • At least 50%, at least 60%, at least 70%, or at least 80% of the population of cells are HSCs, HPCs, and/or LPCs.
  • at least 50%, at least 60%, at least 70%, or at least 80% of the population of cells are CD34+ cells.
  • At least 1%, at least 2%, at least 5%, at least 10%, or at least 20% of the population of cells are CD34+ cells comprising the polynucleotide, vector and/or genome of the present invention.
  • at least 20% of the population of cells are CD34+ cells comprising the genome of the present invention.
  • the population of cells comprises at least 10x10 5 , at least 50x10 5 , or at least 100x10 5 CD34+ cells comprising the polynucleotide, vector and/or genome of the present invention.
  • the population of cells comprises at least 100x10 5 CD34+ cells comprising the genome of the present invention.
  • the present invention provides a method of gene editing a cell or a population of cells using polynucleotides, vectors, guide RNAs, kits, compositions and/or gene-editing system of the present invention.
  • the present invention also provide a population of gene- edited cells obtained or obtainable by said methods.
  • the present invention provides use of a polynucleotide, vector, guide RNA, kit, composition, and/or gene-editing system of the present invention for gene editing a cell or a population of cells.
  • the method of gene editing a cell or a population of cells comprises:
  • the method of gene editing a cell or a population of cells comprises:
  • RNA-guided nuclease a guide RNA, and/or a polynucleotide or vector of the present invention to the cell or population of cells to obtain a gene-edited cell or a population of gene-edited cells.
  • the gene-edited cell or population of gene-edited cells may be as defined herein.
  • the present invention also provides a gene-edited cell or population of gene-edited cells obtained or obtainable by said method.
  • Step (a) providing a cell or a population of cells
  • the population of cells may be obtained or obtainable from any suitable source.
  • the population of cells are obtained or obtainable from (mobilised) peripheral blood or cord blood.
  • the population of cells may be obtained or obtainable from a subject, e.g. a subject to be treated.
  • the population of cells may be isolated and/or enriched from a biological sample by any method known in the art, for example by FACS and/or magnetic bead sorting.
  • the population of cells are mammalian cells, for example human cells.
  • the population of cells may be, for example, autologous or allogeneic.
  • the population of cells may be, for example, universal cells.
  • the population of cells comprises about 1 x 10 5 cells per well to about 10 x 10 5 cells per well, e.g. about 2 x 10 5 cells per well, or about 5 x 10 5 cells per well.
  • the population of cells may comprise HSCs, HPCs, and/or LPCs.
  • at least 50%, at least 60%, at least 70%, or at least 80% of the population of cells are HSCs, HPCs, and/or LPCs.
  • the population of cells consists essentially of HSCs, HPCs, and/or LPCs, or consists of HSCs, HPCs, and/or LPCs.
  • the population of cells may comprise CD34+ cells, e.g. CD34+ HSCs, HPCs, and/or LPCs.
  • CD34+ cells e.g. CD34+ HSCs, HPCs, and/or LPCs.
  • at least 50%, at least 60%, at least 70%, or at least 80% of the population of cells are CD34+ cells, e.g. CD34+ HSCs, HPCs, and/or LPCs.
  • the population of cells consists essentially of CD34+ cells, e.g. CD34+ HSCs, HPCs, and/or LPCs, or consists of CD34+ cells, e.g. CD34+ HSCs, HPCs, and/or LPCs.
  • the population of cells may comprise CD34+CD133+CD90+ cells, CD34+CD133+CD90- cells, and/or CD34+CD133-CD90-.
  • at least 50%, at least 60%, at least 70%, or at least 80% of the population of cells are CD34+CD133+CD90+ cells, CD34+CD133+CD90- cells, and/or CD34+CD133-CD90- cells.
  • the population of cells consists essentially of CD34+CD133+CD90+ cells, CD34+CD133+CD90- cells, and/or CD34+CD133-CD90- cells, or consists of CD34+CD133+CD90+ cells, CD34+CD133+CD90- cells, and/or CD34+CD133-CD90- cells.
  • the cell or population of cells may be cultured prior to step (b).
  • the pre-culturing step may comprise a pre-activation step and/or a pre-expansion step, optionally the pre-culturing step is a pre-activation step.
  • a “pre-culturing step” refers to a culturing step which occurs prior to genetic modification of the cells.
  • a “pre-activating step” refers to an activation step or stimulation step which occurs prior to genetic modification of the cells.
  • a “preexpansion step” refers to an expansion step which occurs prior to genetic modification of the cells.
  • the method may comprise:
  • pre-culturing e.g. pre-activating and/or pre-expanding
  • the population of cells to obtain a pre-cultured (e.g. pre-activated and/or pre-expanded) population of cells
  • RNA-guided nuclease e.g. pre-activated and/or pre-expanded population of cells to obtain a population of gene-edited cells.
  • the pre-culturing step e.g. pre-activation step and/or pre-expansion step
  • the population of cells may be seeded at a concentration of about 1 x 10 5 cells/ml to about 10 x 10 5 cells/ml, e.g. about 2 x 10 5 cells/ml, or about 5 x 10 5 cells/ml.
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) is at least 1 day, at least 2 days, or at least 3 days.
  • the population of cells are pre-cultured (e.g. pre-activated and/or pre-expanded) for about 3 days.
  • the population of cells are precultured in a 5% CO2 humidified atmosphere at 37°C.
  • Any suitable culture medium may be used.
  • commercially available medium such as StemSpan medium may be used, which contains bovine serum albumin, insulin, transferrin, and supplements in Iscove's MDM.
  • the culture medium may be supplemented with one or more antibiotic (e.g. penicillin, streptomycin).
  • the pre-culturing step may be carried out in the presence in of one or more cytokines and/or growth factors.
  • cytokines are any cell signalling substance and includes chemokines, interferons, interleukins, lymphokines, and tumour necrosis factors.
  • growth factor is any substance capable of stimulating cell proliferation, wound healing, or cellular differentiation. The terms “cytokine” and “growth factor” may overlap.
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) may be carried out in the presence of one or more early-acting cytokine, one or more transduction enhancer, and/or one or more expansion enhancer.
  • an “early-acting cytokine” is a cytokine which stimulates HSCs, HPCS, and/or LPCs or CD34+ cells.
  • Early-acting cytokines include thrombopoietin (TPO), stem cell factor (SCF), Flt3-ligand (FLT3-L), interleukin (IL)-3, and IL-6.
  • the preculturing step e.g. pre-activation step and/or pre-expansion step
  • Any suitable concentration of early-acting cytokine may be used. For example, 1-1000 ng/ml, or 10-1000 ng/ml, or 10-500 ng/ml.
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) is carried out in the presence of SCF.
  • concentration of SCF may be about 10-1000 ng/ml, about 50-500 ng/ml, or about 100-300 ng/ml.
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) is carried out in the presence of FLT3-L.
  • the concentration of FLT3-L may be about 10- 1000 ng/ml, about 50-500 ng/ml, or about 100-300 ng/ml.
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) is carried out in the presence of TPO.
  • concentration of TPO may be about 5-500 ng/ml, about 10-200 ng/ml, or about 20-100 ng/ml.
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) is carried out in the presence of IL-3.
  • concentration of IL-3 may be about 10-200 ng/ml, about 20-100 ng/ml, or about 60 ng/ml.
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) is carried out in the presence of IL-6.
  • concentration of IL-6 may be about 5-100 ng/ml, about 10-50 ng/ml, or about 20 ng/ml.
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) is carried out in the presence of SCF (e.g. in a concentration of about 100 ng/ml), FLT3- L (e.g. in a concentration of about 100 ng/ml), TPO (e.g. in a concentration of about 20 ng/ml) and IL-6 (e.g. in a concentration of about 20 ng/ml), in particular when the population of cells are cord-blood CD34+ cells.
  • SCF e.g. in a concentration of about 100 ng/ml
  • FLT3- L e.g. in a concentration of about 100 ng/ml
  • TPO e.g. in a concentration of about 20 ng/ml
  • IL-6 e.g. in a concentration of about 20 ng/ml
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) is carried out in the presence of SCF (e.g. in a concentration of about 300 ng/ml), FLT3- L (e.g. in a concentration of about 300 ng/ml), TPO (e.g. in a concentration of about 100 ng/ml) and IL-3 (e.g. in a concentration of about 60 ng/ml), in particular when the population of cells are (mobilised) peripheral blood CD34+ cells.
  • SCF e.g. in a concentration of about 300 ng/ml
  • FLT3- L e.g. in a concentration of about 300 ng/ml
  • TPO e.g. in a concentration of about 100 ng/ml
  • IL-3 e.g. in a concentration of about 60 ng/ml
  • transduction enhancer is a substance that is capable of improving viral transduction of HSCs, HPCS, and/or LPCs or CD34+ cells.
  • Suitable transduction enhancers include LentiBOOST, prostaglandin E2 (PGE2), protamine sulfate (PS), Vectofusin-1 , ViraDuctin, RetroNectin, staurosporine (Stauro), 7-hydroxy-stauro, human serum albumin, polyvinyl alcohol, and cyclosporin H (CsH).
  • the pre-culturing step e.g.
  • pre-activation step and/or pre-expansion step is carried out in the presence of at least one transduction enhancer.
  • Any suitable concentration of transduction enhancer may be used, for example as described in Schott, J.W., et al., 2019. Molecular Therapy-Methods & Clinical Development, 14, pp.134-147 or Yang, H., et al., 2020. Molecular Therapy-Nucleic Acids, 20, pp. 451-458.
  • the pre-culturing step e.g. pre-activation step and/or pre-expansion step
  • the PGE2 is 16,16-dimethyl prostaglandin E2 (dmPGE2).
  • the concentration of PGE2 may be about 1-100 pM, about 5-20 pM, or about 10 pM.
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) is carried out in the presence of CsH.
  • concentration of CsH may be about 1-50 pM, 5-50 pM, about 10-50 pM, or about 10 pM.
  • an “expansion enhancer” is a substance that is capable of improving expansion of HSCs, HPCS, and/or LPCs or CD34+ cells.
  • Suitable expansion enhancers include LIM171 , LIM729, StemRegeninl (SR1), diethylaminobenzaldehyde (DEAB), LG1506, BIO (GSK3P inhibitor), NR-101 , trichostatin A (TSA), garcinol (GAR), valproic acid (VPA), copper chelator, tetraethylenepentamine, and nicotinamide.
  • the preculturing step e.g.
  • pre-activation step and/or pre-expansion step is carried out in the presence of at least one expansion enhancer.
  • Any suitable concentration of expansion enhancer may be used, for example as described in Huang, X., et al., 2019. F1000Research, 8, 1833.
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) is carried out in the presence of LIM171 or LIM729.
  • concentration of LIM171 may be about 10-200 nM, about 20-100 nM, or about 50 nM.
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) is carried out in the presence of SR1.
  • concentration of SR1 may be about 0.1-10 pM, about 0.5-5 pM, or about 1 pM.
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) is carried out in the presence of UM 171 (e.g. in a concentration of about 50 nM) or UM729 and SR1 (e.g. in a concentration of about 1 pM).
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) is carried out in the presence of SCF (e.g. in a concentration of about 100 ng/ml), FLT3- L (e.g. in a concentration of about 100 ng/ml), TPO (e.g. in a concentration of about 20 ng/ml), IL-6 (e.g. in a concentration of about 20 ng/ml), PGE2 (e.g. in a concentration of about 10 pM), UM 171 (e.g. in a concentration of about 50 nM), and SR1 (e.g.
  • SCF e.g. in a concentration of about 100 ng/ml
  • FLT3- L e.g. in a concentration of about 100 ng/ml
  • TPO e.g. in a concentration of about 20 ng/ml
  • IL-6 e.g. in a concentration of about 20 ng/ml
  • the pre-culturing step (e.g. pre-activation step and/or pre-expansion step) is carried out in the presence of SCF (e.g. in a concentration of about 300 ng/ml), FLT3- L (e.g. in a concentration of about 300 ng/ml), TPO (e.g. in a concentration of about 100 ng/ml), IL-3 (e.g. in a concentration of about 60 ng/ml), PGE2 (e.g. in a concentration of about 10 pM), UM 171 (e.g. in a concentration of about 50 nM), and SR1 (e.g. in a concentration of about 1 pM), in particular when the population of cells are (mobilised) peripheral blood CD34+ cells.
  • SCF e.g. in a concentration of about 300 ng/ml
  • FLT3- L e.g. in a concentration of about 300 ng/ml
  • TPO e.g. in a concentration
  • Step (b) obtaining a gene-edited cell or a population of gene-edited cells
  • a kit, composition, and/or gene-editing system comprising an RNA-guided nuclease, a guide RNA, and/or a polynucleotide or vector of the present invention may, for example, be used to obtain the gene-edited cell or a population of gene-edited cells.
  • RNA-guided nuclease, guide RNA, and/or polynucleotide or vector may be any suitable combination described herein.
  • the guide RNA may correspond to the same DSB site targeted by the homology arms.
  • the RNA-guided nuclease may correspond to the guide RNA used. For example:
  • RNA-guided nuclease may be a Cas9 endonuclease
  • the guide RNA may be a guide RNA comprising or consisting of a nucleotide seguence that has at least 90% identity or at least 95% identity to any of SEQ ID NOs: 41-52 or 53-55, optionally wherein the guide RNA comprises or consists of a nucleotide seguence that has at least 90% identity or at least 95% identity to SEQ ID NO: 41 or 53 (preferably SEQ ID NO: 41); and
  • the polynucleotide may be a polynucleotide comprising from 5’ to 3’: a first homology region, a splice acceptor seguence, a nucleotide seguence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region comprises or consists of a nucleotide seguence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to any of SEQ ID NOs: 7-18 and/or the second homology region comprises or consists of a nucleotide seguence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to any of SEQ ID NOs: 19-30; or the vector may be a vector comprising said polynucleotide.
  • the RNA-guided nuclease may be a Cas9 endonuclease;
  • the guide RNA comprises or consists of a nucleotide sequence that has at least 90% identity, at least 95% identity or 100% identity to SEQ ID NO: 41 or 53 (preferably SEQ ID NO: 41);
  • the polynucleotide comprises from 5’ to 3’: a first homology region, a splice acceptor sequence, a nucleotide sequence encoding a RAG1 polypeptide, and a second homology region, wherein the first homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
  • the second homology region comprises or consists of a nucleotide sequence that has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO:
  • the vector comprises said polynucleotide.
  • RNA-guided nuclease Delivery of a RNA-guided nuclease, guide RNA, and/or polynucleotide or vector
  • RNA-guided nuclease, guide RNA, and/or polynucleotide or vector may be delivered to the cell by any suitable technique.
  • the RNA-guided nuclease may be delivered directly using electroporation, microinjection, bead loading or the like, or indirectly via transfection and/or transduction.
  • the guide RNA, and/or polynucleotide or vector may be introduced by transfection and/or transduction.
  • transfection is a process using a non-viral vector to deliver a polypeptide and/or polynucleotide to a target cell.
  • Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs) and combinations thereof.
  • transduction is a process using a viral vector to deliver a polynucleotide to a target cell.
  • Typical transduction methods include infection with recombinant viral vectors, such as adeno-associated viral, retroviral, lentiviral, adenoviral, baculoviral and herpes simplex viral vectors.
  • the RNA-guided nuclease and the guide RNA may be delivered by any suitable method, for instance any method described in Wilbie, D., et al., 2019. Accounts of chemical research, 52(6), pp.1555-1564.
  • the RNA-guided nuclease and the guide RNA are delivered together preassembled as in the form of a RNP complex.
  • the RNP complex may be delivered by electroporation. Any suitable dose of the RNA-guided nuclease and/or the guide RNA may be used.
  • the guide RNA may be delivered at a dose of about 10-100 pmol/well, optionally about 50 pmol/well.
  • the RNP may be delivered at a dose of about 1-10 pM, optionally 1-2.5 pM.
  • RNA-guided nuclease and/or the guide RNA may be delivered prior to the vector and/or simultaneously with the polynucleotide or vector of the invention.
  • the RNA-guided nuclease and/or the guide RNA are delivered prior to the polynucleotide or vector.
  • the RNA-guided nuclease and/or the guide RNA may be delivered about 1-100 minutes, about 5-30, or about 15 minutes, prior to the polynucleotide or vector.
  • the polynucleotide or vector of the invention may be delivered by any suitable method.
  • the polynucleotide may be in a viral vector or the vector may be a viral vector and delivered by transduction.
  • the vector may be delivered at a MOI of about 10 4 to 10 5 vg/cell, optionally about 10 4 vg/cell.
  • the method may further comprise a step of delivering a p53 inhibitor and/or HDR enhancer.
  • the p53 inhibitor and/or HDR enhancer may be delivered simultaneously.
  • the p53 inhibitor and/or HDR enhancer may be delivered simultaneously with or after the RNA-guided nuclease and/or the guide RNA.
  • a “p53 inhibitor” is a substance which inhibits activation of the p53 pathway.
  • the p53 pathway plays a role in regulation or progression through the cell cycle, apoptosis, and genomic stability by means of several mechanisms including: activation of DNA repair proteins, arrest of the cell cycle; and initiation of apoptosis. Inhibition of this p53 response by delivery during editing has been shown to increase hematopoietic repopulation by treated cells (Schiroli, G. et al. 2019. Cell Stem Cell 24, 551-565).
  • the p53 inhibitors is a dominantnegative p53 mutant protein, e.g. GSE56.
  • GSE56 may have the amino acid sequence:
  • the p53 dominant negative peptide is a variant of GSE56 comprising 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions or deletions, while retaining the activity of GSE56, for example in reducing or preventing p53 signalling.
  • the p53 dominant negative peptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 67.
  • an “HDR enhancer” is a substance that is capable of improving HDR efficiency in HSCs, HPCS, and/or LPCs or CD34+ cells. HDR is constrained in long-term-repopulating HSCs. Any suitable HDR enhancer may be used, for example as described in Ferrari, S., et al., 2020. Nature Biotechnology, pp.1-11. Suitably, the HDR enhancer is the adenovirus 5 E4orf6/7 protein. Adenovirus 5 E4orf6/7 proteins may be as disclosed in WO 2020/002380 (incorporated herein by reference).
  • the p53 inhibitor and the HDR enhancer may be delivered by any suitable method.
  • the p53 inhibitor and/or the HDR enhancer may be transiently expressed, for example the p53 inhibitor and/or the HDR enhancer may delivered via mRNA.
  • the p53 inhibitor and the HDR enhancer may be delivered by separate mRNAs or on a single mRNA encoding a fusion protein, optionally with a self-cleaving peptide (e.g. P2A).
  • Any suitable dose of the p53 inhibitor and/or the HDR enhancer may be used, for example mRNA be delivered at a concentration of about 10-1000 pg/ml, about 50-500 pg/ml, or about 150 pg/ml.
  • step (b) comprises:
  • (b2) optionally, delivering a p53 inhibitor and/or a HDR enhancer
  • the method may further comprise a step of culturing the population of gene-edited cells. This may be an expansion step, i.e. the method may further comprises a step of expanding the population of gene-edited cells.
  • the culturing step may be carried out using any suitable conditions.
  • the population of cells may be seeded at a concentration of about 1 x 10 5 cells/ml to about 10 x 10 5 cells/ml, e.g. about 2 x 10 5 cells/ml, or about 5 x 10 5 cells/ml.
  • the culturing step e.g. expansion step
  • the culturing step is for at least one day, or one to five days.
  • the culturing step e.g. expansion step
  • the population of cells are cultured in a 5% CO2 humidified atmosphere at 37°C.
  • Any suitable culture medium may be used.
  • commercially available medium such as StemSpan medium may be used, which contains bovine serum albumin, insulin, transferrin, and supplements in Iscove's MDM.
  • the culture medium may be supplemented with one or more antibiotic (e.g. penicillin, streptomycin).
  • antibiotic e.g. penicillin, streptomycin
  • the culturing step (e.g. expansion step) may be carried out in the presence in of one or more cytokines and/or growth factors.
  • step (b) comprises:
  • (b2) optionally, delivering a p53 inhibitor and/or a HDR enhancer
  • the present invention provides a method of treating a subject using polynucleotides, vectors, guide RNAs, kits, compositions, gene-editing systems, cells and/or populations of cells of the present invention.
  • the method of treating a subject may comprise administering a cell or population of cells of the present the invention.
  • the present invention provides a polynucleotide, vector, guide RNA, kit, composition, gene-editing system, cell and/or populations of cells of the present invention for use as a medicament.
  • the cell or population of cells of the present the invention may be used as a medicament.
  • the present invention provides use of a polynucleotide, vector, guide RNA, kit, composition, gene-editing system, cell and/or populations of cells of the present invention for the manufacture of a medicament.
  • the cell or population of cells of the present the invention may be used for the manufacture of a medicament.
  • a method of treating a subject may comprise:
  • a method of treating a subject may comprise:
  • Steps (a) and (b) may be identical to the steps described in the section above.
  • the cell of population of cells may be isolated and/or enriched from the subject to be treated, e.g. the population of cells may be an autologous population of CD34+ cells.
  • the population of cells are isolated from (mobilised) peripheral blood or cord blood of the subject to be treated and subsequently enriched (e.g. by FACS and/or magnetic bead sorting).
  • the subject may be immunocompromised and/or the disease to be treated may be an immunodeficiency, i.e. the medicament may be for treating an immunodeficiency.
  • an “immunodeficiency” is a disease in which the immune system's ability to fight infectious disease and cancer is compromised or entirely absent. A subject who has an immunodeficiency is said to be “immunocompromised”. An immunocompromised person may be particularly vulnerable to opportunistic infections, in addition to normal infections that could affect everyone.
  • the subject may have RAG deficiency, e.g. a RAG1 deficiency.
  • RAG1 deficiency may be due to a loss-of-function mutation in the RAG1 gene, optionally a loss-of-function mutation in the RAG1 exon 2.
  • the immunodeficiency may be a RAG deficient-immunodeficiency.
  • a “RAG deficient-immunodeficiency” is an immunodeficiency characterised by loss of RAG1/RAG2 activity.
  • a RAG deficient-immunodeficiency may, for example be caused by a mutation in RAG genes.
  • the RAG deficient-immunodeficiency may be a RAG1 deficiency.
  • a RAG1 deficiency may be due to a loss-of-function mutation in the RAG1 gene, optionally a loss-of-function mutation in the RAG1 exon 2.
  • RAG1 deficiency can cause a broad spectrum of phenotypes, including T- B- SCID, Omenn syndrome (OS), atypical SCID (AS) and combined immunodeficiency with granuloma/autoimmunity (CID-G/AI).
  • OS Omenn syndrome
  • AS atypical SCID
  • CID-G/AI combined immunodeficiency with granuloma/autoimmunity
  • the RAG deficient-immunodeficiency is T- B- SCID, Omenn syndrome, atypical SCID, or CID-G/AI.
  • Severe combined immunodeficiency comprises a heterogeneous group of disorders that are characterized by profound abnormalities in the development and function of T cells (and also B cells in some forms of SCID), and are associated with early-onset severe infections. This condition is inevitably fatal early in life, unless immune reconstitution is achieved, usually with HSCT. Following the introduction of newborn screening for SCID in the United States, it has become possible to establish that RAG mutations account for 19% of all cases of SCID and SCID-related conditions, and are a prominent cause of atypical SCID and Omenn syndrome in particular. (Notarangelo, L.D., et al., 2016. Nature Reviews Immunology, 16(4), pp.234-246).
  • RAG mutations were identified as the main cause of T-B- SCID with normal cellular radiosensitivity.
  • a distinct phenotype characterizes Omenn syndrome, which was first described in 1965. These patients manifest early-onset generalized erythroderma, lymphadenopathy, hepatosplenomegaly, eosinophilia and severe hypogammaglobulinaemia with increased IgE levels, which are associated with the presence of autologous, oligoclonal and activated T cells that infiltrate multiple organs.
  • a residual presence of autologous T cells was demonstrated without clinical manifestations of Omenn syndrome. This condition is referred to as 'atypical' or 'leaky' SCID.
  • y ⁇ 5 T+ SCID A distinct SCID phenotype involving the oligoclonal expansion of autologous y ⁇ 5 T cells (referred to here as y ⁇ 5 T+ SCID) has been reported in infants with RAG deficiency and disseminated cytomegalovirus (CMV) infection.
  • CMV cytomegalovirus
  • Additional phenotypes that are associated with RAG deficiency include idiopathic CD4+ T cell lymphopaenia, common variable immunodeficiency, IgA deficiency, selective deficiency of polysaccharide-specific antibody responses, hyper-IgM syndrome and sterile chronic multifocal osteomyelitis. (Notarangelo, L.D., et al., 2016. Nature Reviews Immunology, 16(4), pp.234-246).
  • RNP Cas9 ribonucleoprotein
  • DSB DNA double strand break
  • HDR homology directed repair
  • SA alternative splicing acceptor
  • NALM6 and K562 cell lines were transduced with a lentiviral vector carrying the Cas9 cassette under the control of a TET-inducible promoter and a cassette that confers resistance to puromycin. After transduction with MOI 20 the two cell lines were kept in culture with puromycin 1 .5 pg/ml for one week to select the transduced cells (Figure 1 panel B). After puromycin selection, a VON 3.65 and a VON 4.35 were verified by LTR specific ddPCR in NALM6 Cas9 and K562 Cas9 cell line respectively ( Figure 1 panel C).
  • Guide 9 was the best performing guide targeting the intron with a cutting frequency up to 72.7% in K562 Cas9 and 78.5% in NALM6 Cas9. Similar cutting frequencies were also achieved by Guide 7, that showed a cutting frequency up to 67.5% in K562 Cas9 and 70.5% in NALM6 Cas9 cell lines.
  • Guide 3 was the best performing guide targeting the exon with a cutting frequency up to 58.9% in K562 Cas9 ( Figure 2 C) and 73.5% in NALM6 Cas9 ( Figure 2 D).
  • RAG1 genomic region is composed of two exons and the whole coding sequence, which is 3.1 Kb, is encoded by the second exon, followed by a long 3’IITR region of 3.3 Kb.
  • Our correction strategy plans to deliver an AAV6 vector containing the entire coding sequence targeting the intronic region upstream of exon 2.
  • the 3’IITR region (>3 Kb) downstream of the RAG1 coding sequence was not inserted because of the limited size hosted by the AAV6 vector.
  • NALM6 Cas 9 and K562 Cas9 cell lines previously stimulated with doxycycline to induce Cas9, were transfected with guide 9 plasmid DNA (100ng/well) and of various linearized DNA donors (1600ng/well). Stable integration of the donor DNA was verified by flow cytometry as GFP expression.
  • the PGK_GFP positive control was stably integrated in both cell lines.
  • ten days after transfection 14% K562 Cas9 and 1.8% of NALM6 Cas9 were GFP positive (Figure 3 D).
  • NALM6 cell line is particularly tricky to edit and we expected a lower efficiency as compared K562.
  • Similar frequencies of GFP+ cells were observed in NALM6 Cas9 transfected with the different SA_GFP donors, while almost no GFP + cells were detectable in the K562 cell lines transfected with the SA_GFP donors.
  • This observation confirms that the endogenous RAG1 promoter efficiently induces the expression of the SA_GFP cassette in the NALM6 Cas9 cell line.
  • the absence of GFP + cells in K562 Cas9 cell line which lacks RAG1 expression, further confirms that the GFP expression observed in NALM6 is specifically dependent on RAG1 promoter activity.
  • hCB-CD34 human CD34 + cells from cord blood
  • hCB-CD34 cells were thawed at day 0 and prestimulated for three days seeding 1x10 6 cells/ml in StemSpan enriched with cytokines (hTPO 20ng/ml, hlL6 20ng/ml, hSCF 100ng/ml, hFlt3-L 100ng/ml, SR1 1 uM, UM171 50nM).
  • guides 3 and 9 were delivered by electroporation as in vitro preassembled RNPs and two doses were considered 25 and 50 pmol/well.
  • chemical modification consisting in 2'-O-methyl 3'phosphorothioate were added at the last three terminal nucleotides at 5’ and 3’ ends of the guide RNAs.
  • AAV6 vectors were added to the medium using three (10 4 , 5x10 4 , 10 5 ) MOI doses ( Figure 5 A).
  • two AAV6 donors one for each guide
  • the toxicity of the procedure was assessed 24 hours after the treatment, by staining the cells with 7AAD and Annexin V and measuring the fraction of necrotic and apoptotic cells by flow cytometry.
  • Four days after electroporation we performed multiparametric flow cytometry analysis to evaluate the composition of various cellular subpopulations composing the bulk treated cell culture and measure the percentage of GFP+ cells within these subpopulations. For this analysis, we took advantage of surface markers that allow identifying the primitive (CD34 + CD133 + CD90 + ), early (CD34 + CD133 + CD90‘) and more committed (CD34 + CD133‘ CD90') progenitors (Figure 5 B). Moreover, genomic DNA was extracted to determine the activity of the nucleases by T7 nuclease assay.
  • Guide 9 retained an activity comparable to that verified in NALM6 and K562 cell lines, 73.9% cutting frequency was observed with 25pmol/well and 80.1% with 50pmol/well.
  • Guide 3 displayed a lower activity in hCB-CD34 with a cutting frequency of 16.9% and 19.3% with 25 and 50pmol/well respectively (Figure 5 C).
  • targeted integration with guide 3 was less efficient and at the dose of 25pmol/well, with the highest MOI (10 5 ), levels of integration were 18.3% in the bulk CD34 + and 1.25% in the most primitive subpopulation (Figure 5 D, E).
  • edited CD34 + cells were transplanted into sublethally irradiated NOD-scid IL2Rg nu " mice (NSG) mice.
  • NSG sublethally irradiated NOD-scid IL2Rg nu " mice
  • hCB-CD34 + cells were electroporated with 50pmol/well of guide 9 RNP and 15 minutes later transduced with AAV6 at MOI 10 4 Vg/cell.
  • two distinct AAV6 vectors were used.
  • the first AAV6 vector carrying the PGK_GFP_BGH was used as a positive control to easily follow engraftment of edited cells.
  • the second donor carrying a SA_GFP_BGH was used to assess the in vivo expression of GFP gene under the control of RAG1 endogenous promoter.
  • the day following the editing procedure treated hCB-CD34 + 350,000 cells/mouse were injected in 4-5 NSG mice per group, 6 hours after sublethal total body irradiation (120 rad).
  • few cells were maintained in culture for 4 more days.
  • Using both the AAV6 vectors we measured -80% of targeted integration by ddPCR (Figure 7 A), thus recapitulating the results obtained in the previous experiments.
  • Flow cytometric analysis of the peripheral blood obtained from transplanted mice was performed 6, 9, 13 weeks after transplantation and at sacrifice at 17 weeks.
  • mice showed no major skew in the subpopulation composition and a normal presence of B, T and myeloid cells in both the groups confirming that the editing procedure does not affect multi-lineage differentiation (Figure 7 D, F, H).
  • Myeloid and circulating T cells were GFP negative, as expected, because these two cell populations do not express RAG1 (Figure 7 G, I). Conversely, relevant percentage (-18%) of GFP + cells was observed among circulating B cells ( Figure 7 E) likely due to their immature phenotype as the majority of B cells expressed CD24 and CD38.
  • the corrective donor included the two homology arms at the 3’ and 5’ extremities, a splice acceptor followed by the Kozak sequence, the RAG1 coding sequence and the BGH PolyA for a total length of 4.1 Kb ( Figure 8 A).
  • RAG1 coding sequence was codon optimized replacing more “rare” codons with more frequent ones without changing the amino acid sequence, thus enhancing protein translation.
  • MPB mobilized peripheral blood
  • MPB-CD34* cells from normal donors (commercially purchased by AllCells California, US) were thawed and prestimulated for three days.
  • SCF Stem cell factor
  • Flt-3L Flt3 ligand
  • TPO Thrombopoietin
  • IL-3L Interleukin 3
  • IL-3L Interleukin 3
  • dmPGE2 16,16-dimethyl prostaglandin E2
  • Cas9 was electroporated as in vitro preassembled RNP at two doses (25pmol/well and 50pmol/well). Since our previous observation suggested that high AAV6 vector MOI could impair cell fitness, we considered two low MOI (10 4 and 2*10 4 ).
  • the editing protocol did not affect cell phenotype based on the expression of CD133 and CD90 (data not shown) and high on-target integration frequency was observed in all CD34 subpopulation.
  • a targeting frequency of 45.3% was observed using 50pmol/well Cas9 and 10 4 MOI of AAV6 vector ( Figure 8 C) also showing lower impact on cell growth as compared to the higher MOI ( Figure 8 D).
  • No differences were noticed between hCD34 + cells from MPB or CB both in terms of efficiency and toxicity.
  • edited hMPB-CD34 + cells were transplanted into sub-lethally irradiated NSG mice. Following the same protocol used in the previous experiment, after 3 days of stimulation, hMPB-CD34 + cells were electroporated with 50pmol/well of guide 9 RNP and 15 minutes later transduced with corrective AAV6 at MOI 10 4 Vg/cell. To dampen the previously reported editing-induced p53 response, which decreases hematopoietic reconstitution by edited HSPCs, we added to the electroporation mixture an mRNA encoding for the dominant-negative p53 inhibitor GSE56 (Schiroli G, et al. Cell Stem Cell. 2019;24(4):551-565.e8).
  • GSE56 dominant-negative p53 inhibitor
  • NIHPID0021 is an adult patient with CID-G/AI due to missense RAG1 mutations (C1228T; G1520A) allowing residual development of B and T cells.
  • the very low B cell counts in the periphery was also due to the treatment with anti-CD20 mAb to control severe autoimmune manifestations.
  • NSG mice transplanted with treated HD cells showed no major skewing in the subpopulation composition and a comparable frequency of B, T and myeloid cells was observed in mice receiving treated or untreated cells, confirming that multilineage differentiation was not impaired (Figure 9 E).
  • Untreated patient cells showed a partial skew in B- and T- cell compartment, when compared to the HD, in line with the immune phenotype of patients carrying hypomorphic mutations (Delmonte OM, et al. Blood. 2020; 135(9):610-9).
  • mice were sacrificed 17 weeks after the transplant to analyze the engraftment of edited cells in bone marrow, thymus and spleen.
  • frequencies of human CD45+ cells were higher than those retrieved mice peripheral blood (Figure 8G, H left panels and 8C) .
  • NSG mice transplanted with edited MPB CD34 cells from HD showed 13.9% of hCD45 + in the bone marrow, whilst 23.4% in untreated group (Figure 9 G, left panel).
  • Similar engraftment levels were achieved in mice receiving edited RAG1 patient cells (10.2%), but lower proportion of hCD45 + cells was found in mice receiving untreated RAG1 patient cells (6.9%) (Figure 9 G, left panel).
  • hCD45 + cells engraftment was even higher in the spleen for both edited and untreated cells of HD and patient.
  • the frequency of hCD45 + cells was 37.4% and 43.3% in mice with edited or untreated cells, respectively ( Figure 9 H, left panel), indicating the absence of differences between edited and not edited cells.
  • the frequency of hCD45 + cells was 24% and 23.7% in mice with edited or untreated cells derived from the RAG1-patient, respectively ( Figure 9 H, left panel).
  • HDR targeting efficiency assessed by ddPCR on DNA samples extracted from bone marrow and spleen showed a range from 1.1% to 19.6% in edited cells from the bone marrow, while 2.1 % to 8.5% in the case of patient cells (Figure 9 G, right panel).
  • the spleen showed the highest targeting frequency, with a range between 6.1 % and 22.2% for mice with edited HD cells, and between 11.9% and 14.8% for mice with edited patient cells (Figure 9 H, right panel).
  • DNA double strand breaks are per se dangerous lesions that can result in pathological genome rearrangements or chromosomal translocations.
  • An important mechanism that ensures the fidelity of V(D)J recombination resides in the fine control of RAG1 expression that is restricted to specific target cells at specific developmental stages.
  • RAG1 expression regulation is also indispensable for the selection of functional, non-self-reactive lymphocyte through complex mechanisms of “allelic exclusion” or BCR and TCR receptor editing (Ten Boekel E, et al. Immunity. 1998;8(2): 199-207).
  • Cas9 was electroporated as in vitro preassembled RNP in order to ensure a robust and short-term persistence in cells as prolonged persistence of Cas9 protein in primary cells could lead to off-target cleavage, potentially affecting cell homeostasis and functionality (Kim S, et al. Genome Res. 2014;24(6):1012-9). Delivering Cas9 as preassembled RNP is well tolerated and partially protect the gRNA from intracellular degradation thus improving stability and activity of the nuclease (Hendel A, et al. Nat Biotechnol. 2015; 33(9): 985-9).
  • HSPC were prestimulated to favour the transit through S/G2 phases when HDR preferably occurs (Genovese P, et al. Nature. 2014;510(7504):235- 40; and Kass EM, Jasin M. Vol. 584, FEBS Letters. 2010. p. 3703-8) resulting in a moderate cell expansion while preserving original sternness phenotype considering expression of CD34, CD133 and CD90 markers.
  • the newly designed donor AAV6 vector (including a SA sequence followed by the Kozak sequence, the RAG1 codon optimized followed by BGH_PolyA) was tested also in hMPB- CD34 + cells. We observed the same efficiency obtained with the previous donors, confirming that our protocol is reproducible using several donors and several HSPC sources. Moreover, the multiparametric analysis of HSPC composition in untreated and edited HD cells showed a redistribution of HSPC subtypes in cultured cells as compared to cells analyzed before the expansion phase ( Figure 10 A).
  • HSC hematopoietic stem cells
  • MLP multipotent progenitors
  • CMP common myeloid progenitors
  • ddPCR analysis showed more than 80% HDR in total CD34 + cells and 45% of targeting frequency was observed in the most primitive (CD133 + CD90 + ) subpopulation subset.
  • In vivo experiments in NSG mice transplanted with treated hMPB-CD34 + cells showed good level of engraftment and multilineage differentiation capability as those treated with unedited cells.
  • LVs were produced by transient transfection of 293T cells. 24 hours before transfection 9x10 6 cells were plated in a 15 cm dish, 2 hours before transfection Iscove's Modified Dulbecco's (IM DM) medium was changed. The required transfer vector (34 pg) was mixed with 9 pg of VSV-G envelope encoding plasmid, 12.5 pg pMDLg/pRRE, 6.25 pg of REV plasmid and 15 pg of pADVANTAGE per 15 cm dish. This mixture was added to 293T cells by calcium phosphate precipitation. After 12-14 hours the medium was replaced with fresh complete IMDM supplemented with 1mM of sodium butyrate.
  • IM DM Iscove's Modified Dulbecco's
  • NALM6 Cas9 cell line was generated by transducing NALM6 cells with a lentiviral vector expressing Cas9 protein under the control of a TET-inducible promoter and with a vector that constitutively expresses the TET transactivator (Clackson T. Vol. 7, Gene Therapy. 2000. p. 120-5). When doxycycline is administered to the culture media, the TET transactivator can bind the promoter of the Cas9 and induce its expression in the cells. K562 Cas9 cell line was generated with the same vector. Doxycycline was administered 24h before electroporation of the nuclease. Cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS, glutamine and penicillin/streptomycin antibiotics (complete medium). gRNA and RNP assembly
  • Cas9 protein and custom RNA guides were purchased from Integrated DNA Technologies (IDT) and assembled following the manufacturer protocol. To enhance cellular stability, chemically modified guide RNAs were used. Briefly crRNA and trRNA were annealed heating them at 95°C for 5 minutes and letting them slowly cool down at RT for 10 minutes. Cas9 protein was then incubated for 15 minutes at room temperature with the annealed guide RNA fragments, to assemble the ribonucleoprotein (RNP).
  • IDT Integrated DNA Technologies
  • T7E1 assay was used to measure indels induced by NHEJ. Briefly, gDNA of gene edited cells was extracted and amplified by PCR with primers flanking the Cas9 RNP target site. The PCR product was denatured, slowly re- annealed and digested with T7 endonuclease (New England BioLabs) for 1 h, 37°. T7 nuclease only cut DNA at sites where there is a mismatch between the DNA strands, thus between re-annealed wild type and mutant alleles. Fragments were separated on LabChip GXII Touch High Resolution DNA Chip (PerkinElmer®) and analysed by the provided software.
  • T7E1 T7 endonuclease
  • dsODN integration sites in genomic DNA were precisely mapped at the nucleotide level using unbiased amplification and next-generation sequencing (Tsai SQ, et al. Nat Biotechnol. 2015;33(2): 187-97).
  • Library construction and GUIDE-Seq sequencing were performed by Creative Biogen Biotechnology (NY, USA) using Unique Molecular Identifier (UMI) for tracking PCR duplicates.
  • UMI Unique Molecular Identifier
  • Quality checking and trimming were performed on the sequencing reads, using FastQC and Trim_galore, respectively.
  • High quality reads were aligned against the human reference genome (GRCh38), using Bowtie2 (Langmead B, Salzberg SL. Nat Methods.
  • GUIDE-Seq data analysis was performed employing the R/Bioconductor package GUIDE-seq (Zhu LJ, et al. BMC Genomics. 2017;18(1)), and using UMI to deduplicate reads.
  • mice analysis single-cell suspensions were obtained from bone marrow, spleen, thymus and peripheral blood and stained with the following anti-human antibodies: CD45 (clone REA757), CD3(clone REA613) (Miltenyi biotech), CD19 (clone SJ25C1), CD13 (clone WM15) (BD Biosciences).
  • Human and murine Fc blocking was performed before each staining using human F-Block and murine CD16/CD32 from BD Pharmingen.
  • Live/Dead Fixable Yellow (Thermo Fisher Scientific, Waltham, MA) was added to the antibody mix to exclude dead cells. Samples were acquired on a FACSCanto II (BD) and analyzed with FlowJo software (TreeStar, Ashland, Ore).
  • AAV vectors were produced by transient triple transfection of HEK293 cells by calcium phosphate. The following day, the medium was changed with serum-free DMEM and cells were harvested 72 hours after transfection. Cells were lysed by three rounds of freeze-thaw to release the viral particles and the lysate was incubated with DNAsel and RNAse I to eliminate nucleic acids. AAV vector was then purified by two sequential rounds of Cesium Cloride (CsCI2) gradient. For each viral preparation, physical titres (genome copies/mL) were determined by PCR quantification using TaqMan.
  • CsCI2 Cesium Cloride
  • 2x10 5 / 5x10 5 cells per well were electroporated (Lonza, SF Cell line 4D Nucleofector X Kit, program FF120 for K562 or program DC100 for NALM6) with either plasmids or RNPs. Fifteen minutes after electroporation, cells were infected with AAV6 at different MOI: 10 4 ; 5x10 4 ; 10 5 Vector Genome/cell, Vg/cell.
  • CB CD34+ cells Human cord blood CD34+ cells (CB CD34+ cells) were obtained from Lonza (PoieticsTM cat# 2C 101). CB CD34+ cells/ml were stimulated in StemSpan medium supplemented with penicillin/streptomycin antibiotics and early-acting cytokines: Stem cell factor (SCF) 100 ng/ml, Flt3 ligand (Flts-L) 100 ng/ml, Thrombopoietin (TPO) 20 ng/ml, Interleukin 6 (IL- 6) 20 ng/ml, StemRegeninl (SR1) (1 uM) and 16,16-dimethyl prostaglandin E2 (dmPGE2) (10uM), UM171 50nM.
  • SCF Stem cell factor
  • Flt3 ligand Flt3 ligand
  • TPO Thrombopoietin
  • TPO Thrombopoietin
  • CB CD34+ cells Patient mobilized peripheral blood CD34+ cells (CB CD34+ cells) were kindly provided by Dr. Luigi Notarangelo (Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States).
  • MPB CD34+ cells/ml were stimulated in StemSpan medium supplemented with penicillin/streptomycin antibiotics and early-acting cytokines: Stem cell factor (SCF) 300 ng/ml, Flt3 ligand (Flts-L) 300 ng/ml, Thrombopoietin (TPO) 100 ng/ml, Interleukin 3 (IL- 3) 60 ng/ml, StemRegeninl (SR1) (1 uM) and 16,16-dimethyl prostaglandin E2 (dmPGE2) (10uM), UM171 50nM.
  • SCF Stem cell factor
  • Flt3 ligand Flt3 ligand
  • TPO Thrombopoietin
  • TPO Thrombopoietin
  • IL- 3 Interleukin 3
  • SR1 StemRegeninl
  • dmPGE2 16,16-dimethyl prostaglandin E2
  • CD34+ cells per condition were electroporated (Lonza, P3 Primary Cell 4DNucleofector X Kit, CD34+ program) with RNPs, GSE56 mRNA (p53 inhibitor) was added at a dose of 150pg/ml when cells were aimed at being transplanted. 15 minutes after electroporation, CD34+ cells were infected with AAV6 at different MOI: 10 4 ; 5x10 4 ; 10 5 Vg/cell.
  • ddPCR Digital PCR
  • gDNA was quantified using Nanodrop, and diluted in H2O to reach 5-10 ng per reaction (1-2ng/ul). It is possible to increase the gDNA quantity per reaction but it is important to remain below the saturation limit of the system.
  • ddPCR master mix was prepared by adding 11 ul ddPCR Supermix for Probes (no dUTP; BioRad), 1.1 ul primer mix Primer forward + Primer reverse (final concentration 0,9uM) + Probe (final concentration 0,25 uM), 1 .1 ul normalizer primer mix, 4.9 ul H2O per reaction.
  • Primers and Probes used for ddPCR assay are the following:
  • NOD-scid IL2Rgnull mice (NSG; Charles River) were purchased from Charles River Laboratories Inc. (Calco, Italy) and were maintained in specific pathogen-free (SPF) conditions. Mice were transplanted at 8-10 weeks approximately 6 hours after sublethal total body irradiation (120 rad), via intravenous injection of treated HSCPs in phosphate-buffered saline. Gentamicin sulfate (Italfarmaco, Milan, Italy) was administered in drinking water (8mg/mL) for the first 2 weeks after transplantation to prevent infections. Mice were followed until the sacrifice and then euthanized for ex vivo analyses.
  • NALM6 cells were transfected with guide 9 and Cas9 as an RNP (25 pmol) and donors as linearized DNA fragments (1600 ng), and then kept in culture with RPMI and 10% FBS. To synchronize cell cycles at G0/G1 phase when the RAG1 gene is mainly expressed, cells were serum starved 16 days after the transfection (Figure 11 B).
  • ATO artificial thymic organoid
  • CD34+ cells obtained from healthy donor (HD) mobilized peripheral blood (MPB) or bone marrow (BM).
  • HD healthy donor
  • MPB mobilized peripheral blood
  • BM bone marrow
  • Ad5-E4orf6/7 is an adenoviral protein known as a helper in Ad-AAV co-infection, which interacts with several components involved in survival and cell cycle.
  • 5x10 5 cells per well were electroporated (Lonza, SF Cell line 4D Nucleofector X Kit, program FF120 for K562 or program DS100 for NALM6) with either plasmids or RNPs.
  • Donor DNA was delivered by electroporation as fragment plasmid spanning the region between the left and right homology arms at a dose of 1600 ng.
  • Human MPB or BM CD34+ cells were obtained from Lonza and stimulated in StemSpan medium supplemented with penicillin/streptomycin antibiotics and early-acting cytokines: Stem cell factor (SCF) 300 ng/ml, Flt3 ligand (Flt3-L) 300 ng/ml, Thrombopoietin (TPO) 100 ng/ml, StemRegeninl (SR1) (1 pM) and 16,16-dimethyl prostaglandin E2 (dmPGE2) (10pM), UM171 35nM.
  • SCF Stem cell factor
  • Flt3 ligand Flt3 ligand
  • TPO Thrombopoietin
  • TPO Thrombopoietin
  • SR1 StemRegeninl
  • dmPGE2 16,16-dimethyl prostaglandin E2
  • CD34+ cells per condition were electroporated (Lonza, P3 Primary Cell 4DNucleofector X Kit, CD34+ program) with RNPs, GSE56 mRNA (3 ug/test), Ad5-E4orf6/7 (1.5ug/test) or GSE56+Ad5-E4orf6/7 as fusion protein with P2A self cleaving peptide (5ug/test).
  • CD34+ cells were infected with AAV6 at 10 4 Vg/cell and kept in culture with StemSpan medium supplemented with penicillin/streptomycin antibiotics and early-acting cytokines: Stem cell factor (SCF) 300 ng/ml, Flt3 ligand (Flt3-L) 300 ng/ml, Thrombopoietin (TPO) 100 ng/ml , StemRegeninl (SR1) (1 pM) and UM171 35 nM.
  • SCF Stem cell factor
  • Flt3 ligand Flt3 ligand
  • TPO Thrombopoietin
  • SR1 StemRegeninl
  • Flow cytometry analysis FACS and sorting
  • unstained and single-stained cells or compensation beads were used as negative and positive controls.
  • CD34+ cells were stained with phycoerythrin cyanine 7 (PECy7) CD34 (Clone: AC136, Miltenyi Biotec), phycoerythrin (PE) CD133 (Miltenyi Biotec) allophycocyanin (APC) CD90 (BD Biosciences).
  • PECy7 phycoerythrin cyanine 7
  • CD34 CD34 (Clone: AC136, Miltenyi Biotec), phycoerythrin (PE) CD133 (Miltenyi Biotec) allophycocyanin (APC) CD90 (BD Biosciences).
  • MoFlo XDP Cell Sorter Beckman Coulter
  • T cell differentiation was analyzed after cell harvesting from ATOs by flow cytometry using the following mAb: TCRab APC (cl. IP26, eBioscience), CD4 Alexa Fluor 700 (cl. OKT4, eBioscience), CD19 PerCP-Cy5.5 (cl. HIB19, Biolegend), CD56 FITC (cl. MEM-188, Biolegend), CD8a PE/Dazzle (cl. RPA-T8, Biolegend), CD45 V500 (cl. HI30, BD Biosciences), CD3 BV421 (cl. UCHT1 , BD Biosciences), CD8b PE (cl.
  • CFLI-C assay was performed 24 h after editing procedure by plating 600 cells in methylcellulose-based medium (MethoCult H4434, StemCell Technologies) supplemented with 100 lll/ml penicillin and 100 pg/ml streptomycin. Three technical replicates were performed for each condition. Two weeks after plating, colonies were counted and identified according to morphological criteria.
  • ATOs were generated as described in Seet et al (Seet et al. (2017) Nat Methods). Briefly, one day after the editing procedure 5000-10000 CD34 + from BM or MPB samples (commercially available, Lonza) were combined with 150000 MS5-hDLL4 cells per ATO. We normalized the number of “true” live CD34+ cells according to the flow cytometry analysis excluding dead and CD34- cells.
  • Each ATO (5 l) was then plated in a 0.4 pM Millicell Transwell insert, placed on a well of a 6-well plate containing 1 ml complete RB27 medium supplemented with rhlL-7 (5 ng/ml), rhFlt3-L (5 ng/ml) and 30 pM l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate.
  • Each insert contained a maximum of two ATOs.
  • Medium was changed every 3-4 days. From weeks 4 to 9, ATOs were collected by adding MACS buffer (PBS with 7.5% BSA and 0.5 M EDTA) to each well and pipetting to dissociate the ATOs.
  • ddPCR Digital PCR
  • Primers and Probes used for the ddPCR assay are the following:
  • PGK_GFP cassette PROBE FAM CTGCTGCACCCTGGCCTCCTGAACTAA
  • Two further donor constructs were designed and generated: i) a SA_coRAG1 CDS_BGHpA donor carrying the bovine growth hormone (BGH) PolyA downstream the SA_ coRAGICDS allowing the transcription termination of the corrective RAG1 CDS ( Figure 14A); ii) a SA_coRAG1 CDS_SD containing a splice donor (SD) sequence to obtain a fusion transcript including the corrected codon optimized sequence and endogenous RAG1 followed by the 3’ UTR sequence ( Figure 14B).
  • BGH bovine growth hormone
  • SD splice donor
  • NALM6.Rag1 KO cells were transfected with guide 9 and Cas9 as RNP (50pmol) and transduced with SA_coRAG1 CDS_BGHpA or SA_coRAG1 CDS_SD AAV6 donor at two doses (10 4 and 5x10 4 ) ( Figure 15A).
  • SA_coRAG1 CDS_BGHpA or SA_coRAG1 CDS_SD AAV6 donor at two doses (10 4 and 5x10 4 ) (Figure 15A).
  • we obtained low proportion of edited alleles in bulk edited NALM6.Rag1 KO cells due to the low permissiveness of NALM6 cells to HDR-mediated editing.
  • HSPC hematopoietic stem and progenitor cells

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023062030A1 (en) * 2021-10-12 2023-04-20 Ospedale San Raffaele S.R.L. Polynucleotides useful for correcting mutations in the rag1 gene
WO2023209225A1 (en) * 2022-04-29 2023-11-02 Ospedale San Raffaele S.R.L. Gene therapy
WO2025073293A1 (en) * 2023-10-02 2025-04-10 Consorcio Centro De Investigación Biomédica En Red Gene editing methods for cd34+ cells to achieve clinically relevant efficacies

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