CD34 + cell pre-stimulation method for realizing clinical related gene editing frequency

Abstract

The invention relates to a CD34 + cell pre-stimulation method for realizing clinical related gene editing frequency. Specifically, the present invention relates to different elements of an integrated procedure that entails the ex vivo amplification of HSPC, preferably about 48 hours, in the presence of hematopoietic cytokines, as well as UM171 and stem cell regeneration 1, and introduces i) a system (e.g., CRISPR / Cas9 system) that produces a DNA double strand break (DSB) upstream of the transcription initiation site of the target gene, and i) a system (e.g., CRISPR / Cas9 system) that produces a DNA double strand break (DSB) upstream of the transcription initiation site of the target gene. And introducing ii) a donor matrix comprising cDNA having homologous arms on both sides (left [LHA] and right [RHA]).

Description

Technical Field

[0001] The present invention relates to different elements of an integration procedure that requires the ex vivo expansion of HSPCs in the presence of hematopoietic cytokines and UM171 and stem cell regeneration factor 1 for preferably about 48 hours, and the introduction of i) a system (e.g., a CRISPR / Cas9 system) that generates a DNA double-strand break (DSB) upstream of the transcription start site of the target gene, and the introduction of ii) a donor matrix comprising a cDNA flanked by homology arms (left side [LHA] and right side [RHA]). Background Art

[0002] Hematopoietic stem cells (HSCs) have the ability to reconstitute the entire hematopoietic system (Baum et al., Proceedings of the National Academy of Sciences USA, 89, 2804-2808 (1992)), and genome editing of HSCs may be able to cure a variety of blood genetic diseases (Mukherjee and Thrasher, Gene, 525, 174-181 (2013); Cavazzana-Calvo et al., Nature, 467, 318-322 (2010); Naldini, Nature, 526, 351-360 (2015)) and acquired blood diseases (Jenq & van den Brink, Nature Reviews Cancer, 10, 213-221 (2010)). Recent studies have shown that by combining ZFN expression with exogenous homologous recombination donors delivered by single-stranded oligonucleotides (ssODN) (Hoban et al., Blood 125, 2597-2604 (2015)), integrase-deficient lentiviral vectors (IDLV) (Genovese et al., Nature 510, 235-240 (2014)) or recombinant adeno-associated virus vector serotype 6 (rAAV6) (Wang et al., Nature Biotechnology, 33, 1256-1263 (2015)), efficient targeted integration can be performed in hematopoietic stem cells and progenitor cells (HSPCs) derived from mobilized peripheral blood, fetal liver or umbilical cord blood. However, after transplantation into immunodeficient mice, high editing frequencies in vitro did not produce high frequencies of edited cells. The present invention directly addresses the problem of providing high frequencies of edited cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0003] Figure 1. Flowchart of the gene editing production process. After purification, cells are pre-stimulated with defined media and then electroporated with Cas9 / gRNA ribonucleoprotein (RNP) using a commercially available device. Afterwards, cells are transduced with the corresponding AAV vector carrying the genome editing donor sequence and cultured again for a period of time. Cells are then cryopreserved to generate samples for quality control assays and drug product for patient infusion.

[0004] Figure 2 .Comparison of hematopoietic stem cell phenotypes of unedited and edited hematopoietic stem and progenitor cells by flow cytometry. Scatter plots were obtained within the CD34+CD38- gate of the total population. The CD34+CD38-CD90+CD45RA+ population was increased in edited cells compared to unedited cells. Two different validation experiments (VAL1 and VAL2) were performed at large scale (150 million cells and 300 million cells edited, respectively).

[0005] Figure 3 .Side-by-side comparison of different compound combinations and different virus doses. CD34+ cells obtained from mobilized peripheral blood were subjected to gene editing protocols in the presence or absence of stem cell regeneration factor 1 (+ / -SR1), AZD-7648 (+ / -AZD), and in which the AAV vector had different viral genomes per cell (MOI) (3K, 3000; 4K, 4000); R-468 is the identifier of the pre-GMP batch. All other conditions were performed as cGMP batches. The upper figure reflects the gene editing efficiency obtained by specific ddPCR on the 5th day after the end of the editing process. The lower figure represents the colony forming ability of different samples obtained by semi-solid culture. BFU-E, burst erythroid colony-forming unit; GM, granulocyte-macrophage colony-forming unit; GEMM, mixed bone marrow erythroid colony-forming unit. Results of two different experiments (named 23-Da14 and 23-Da15) are shown.

[0006] Abbreviations and Definitions

[0007] CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats

[0008] Cas9: CRISPR-associated protein 9

[0009] rAAV: recombinant adeno-associated vector

[0010] RNP: ribonucleoprotein

[0011] HDR: Homology-Directed Repair

[0012] sgRNA: single guide RNA

[0013] MOI: Multiplicity of Infection

[0014] As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0015] As used herein, the terms "a," "an," or "the" include not only aspects having one member, but also aspects having more than one member. For example, the singular form "a," "an," or "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "an agent" includes reference to one or more agents known to those skilled in the art, and so forth.

[0016] The term "gene" refers to a combination of polynucleotide elements that provide some product or function when they are operably linked in a natural or recombinant manner. The term "gene" is to be interpreted broadly and can include mRNA, cDNA, cRNA and genomic DNA forms of a gene.

[0017] The term "homologous directed repair" or "HDR" refers to a mechanism in cells that uses a homologous template to direct repair to precisely repair double-stranded DNA breaks. The most common form of HDR is homologous recombination (HR).

[0018] The term "homologous recombination" or "HR" refers to the genetic process of exchanging nucleotide sequences between two similar DNA molecules. Cells use homologous recombination (HR) to accurately repair harmful breaks that occur in both strands of DNA (called double-strand breaks or other breaks that produce overhanging sequences).

[0019] The term "single guide RNA" or "sgRNA" refers to a DNA-targeting RNA that comprises a guide sequence that targets a Cas nuclease to a target genomic DNA and a scaffold sequence (e.g., tracrRNA) that interacts with the Cas nuclease and, optionally, a donor repair template.

[0020] The term "Cas polypeptide" or "Cas nuclease" refers to a clustered regularly interspaced short palindromic repeat-related polypeptide or nuclease that cuts DNA to produce blunt ends at double-strand breaks at sites specified by a 20-nucleotide guide sequence contained within the crRNA transcript. Cas nucleases require both crRNA and tracrRNA for site-specific DNA recognition and cleavage. The crRNA binds to the tracrRNA through a partially complementary region to guide the Cas nuclease to a region in the target DNA that is homologous to the crRNA, called the "pre-spacer sequence."

[0021] The term "ribonucleoprotein complex" or "RNP complex" refers to a complex comprising an sgRNA and a Cas polypeptide.

[0022] The term "homologous donor AAV vector" or "donor AAV vector" refers to an AAV particle that can express a recombinant donor template for CRISPR-based gene editing via homology-directed repair in a host cell (e.g., a primary cell).

[0023] The term "recombination donor template" refers to a nucleic acid strand, such as a DNA strand, that serves as an acceptor strand during homologous recombination strand invasion initiated by damaged DNA (caused by double-strand breaks in some cases). The donor polynucleotide serves as a template material to guide the repair of damaged DNA regions.

[0024] The term "primary cell" refers to a cell that is directly isolated from a multicellular organism. Compared to continuous (tumor or artificial immortalization) cell lines, primary cells have typically undergone very few population doublings and are therefore more representative of the main functional components of the tissue from which they are derived. In some cases, primary cells are cells that have been separated and then used immediately. In other cases, primary cells cannot divide indefinitely and therefore cannot be cultured for a long time in vitro.

[0025] The term "genetically modified primary cell" or "genome-edited primary cell" refers to a primary cell into which, under certain circumstances, a heterologous nucleic acid has been introduced into its endogenous genomic DNA.

[0026] The term "primary blood cell" refers to a primary cell obtained from blood or its progeny. The primary blood cell can be a stem cell or a progenitor cell obtained from blood. For example, the primary blood cell can be a hematopoietic stem cell or a hematopoietic progenitor cell.

[0027] The term "pharmaceutical composition" refers to a physiologically acceptable and pharmacologically acceptable composition. In some cases, the composition includes agents for buffering and preservation during storage, and may include buffers and carriers for proper delivery, depending on the route of administration.

[0028] The term "pharmaceutically acceptable carrier" refers to a substance that facilitates the administration of an agent (e.g., Cas nuclease, modified single guide RNA, genetically modified primary cells, etc.) to a cell, an organism, or an object. "Pharmaceutically acceptable carrier" refers to a carrier or excipient that can be included in a composition or formulation and does not cause significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, and pigments, etc. Those skilled in the art will recognize that other pharmaceutical carriers can be used in the present invention.

[0029] The term "administering" or "administration" refers to the process of delivering the agents, compositions, dosage forms and / or combinations disclosed herein to a subject for therapeutic or preventive purposes. Compositions, dosage forms and / or combinations disclosed herein are administered according to good medical practice, taking into account the subject's clinical condition, the site and method of administration, dosage, subject age, sex, weight and other factors known to the physician. For example, the term "administering" or "administration" includes providing, giving, administering and / or prescribing the agents, compositions, dosage forms and / or combinations disclosed herein by a clinician or other clinical professional.

[0030] The term "treatment" refers to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to any treatment-related improvement or effect on one or more diseases, conditions, or symptoms in treatment. For prophylactic benefit, the composition can be administered to a subject at risk for developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even though the disease, condition, or symptom may not yet be manifested.

[0031] The terms "culture," "culturing," "grow," "growing," "maintain," "maintaining," "expand," "expanding," and the like are used interchangeably when referring to cell culture itself or the process of culturing, and mean that cells (e.g., primary cells) are maintained under controlled conditions (e.g., under conditions suitable for survival) outside of their normal environment. Cultured cells can survive, and culturing can cause cells to grow, stagnate, differentiate, or divide. The terms do not mean that all cells in the culture can survive, grow, or divide, as some cells may die or age naturally. Cells are typically cultured in a culture medium, and the culture medium can be changed during the culture process.

[0032] The terms "subject", "patient" and "individual" are used interchangeably herein to include humans or animals. For example, an animal subject can be a mammal, a primate (e.g., a monkey), a livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance.

[0033] The term "StemSpan AOF" is to be understood as the cGMP cell culture medium used for the culture of CD34+ cells during the pre-stimulation step or elsewhere during the genetic manipulation protocol.

[0034] The term "StemSpan SFEMII" is to be understood as a cell culture medium used for the culture of CD34+ cells during the pre-stimulation step or at other times during the genetic manipulation protocol.

[0035] The term "UM171" should be understood as a small molecule used for ex vivo expansion of human hematopoietic stem cells. CAS No. 1448724-09-1. Chemical or molecular structure:

[0036]

[0037] The term "Stemregenin 1" should be understood as an anti-differentiation / apoptosis small molecule for in vitro cell culture of human hematopoietic stem cells. CAS No. 1227633-49-9. Chemical or molecular structure:

[0038]

[0039] The term "SCF" is to be understood as "stem cell factor" and is used in the in vitro cell culture of human hematopoietic stem cells and is associated with the survival, motility and possible self-renewal of hematopoietic stem cells in culture and in the hematopoietic stem cell niche.

[0040] The term "TPO" is to be understood as "thrombopoietin" which is used to regulate hematopoietic stem cells.

[0041] The term "Flt3-ligand" is to be understood as "Fms-related tyrosine kinase 3 ligand", which is involved in the ex vivo expansion of hematopoietic stem cells.

[0042] The term "IL-6" is to be understood as interleukin-6, which is associated with the production of hematopoietic stem cells.

[0043] The term "AZD" is understood to mean a DNA-PK inhibitor that, when added during stem cell transduction, can enhance HDR-mediated correction. CAS No. 2230820-11-6. Chemical or Molecular Formula:

[0044]

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which this technology belongs. Although exemplary methods, devices and materials are described herein, any methods and materials similar or equivalent to those explicitly described herein can be used in the practice or testing of this technology. For example, the reagents described herein are merely exemplary and their equivalents are known in the art. Unless otherwise indicated, the implementation of this technology can adopt conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the technical scope of this area. See, for example, Sambrook and Russell, eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed.; Ausubel et al., eds. (2007) Current Protocols in Molecular Biology; Enzymology Methods Series (Academic Press, Inc., NY); MacPherson et al. (1991) PCR I: A Practical Approach (IRL Press of Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane, eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th ed.; Miller and Calos, eds. (1987) Gene Transfer Vectors for Mammalian Cells. Cells) (Cold Spring Harbor Laboratory); and Makrides, eds. (2003) Gene Transfer and Expression in Mammalian Cells (Cold Spring Harbor Laboratory). DETAILED DESCRIPTION

[0046] The present invention relates to different elements of an integration procedure, which requires that HSPCs be expanded in vitro in the presence of hematopoietic cytokines and UM171 and stem cell regeneration factor 1, preferably for about 48 hours, and that i) a system (e.g., CRISPR / Cas9 system) that produces a DNA double-strand break (DSB) upstream of the transcription start site of the target gene is introduced, and ii) a donor matrix of cDNA comprising homology arms (left side [LHA] and right side [RHA]) on both sides is introduced. LHA and RHA are identical to the genomic sequence at the insertion site of the exogenous sequence. CRISPR / Cas9 should be introduced by electroporation to facilitate the entry of DNA nucleases into the nucleus, and the donor matrix should be introduced by an adeno-associated virus vector (preferably serotype 6). In order to assemble ribonucleoproteins (RNPs), Cas9 proteins are combined with sgRNAs. In order to transfect RNPs into cells, an electroporation device is used. The cells are pre-stimulated and then resuspended in an electroporation solution. The RNP complex is added to the cell suspension and the cells are then electroporated. After the electric pulse, HSPCs were incubated at 37 °C for 10 min. Then, pre-warmed culture medium was added and cells were transferred to the culture system. Nucleofected cells were immediately transduced with different concentrations of the corresponding AAV.

[0047] Thus, cells (primary cells, such as hematopoietic stem cells (HSCs)) are obtained from the patient, manipulated in vitro, and once the product has been characterized and shown to meet the acceptance criteria for drug release, these cells are infused into the patient who has previously been chemotherapeutically conditioned to allow the infused corrected cells to engraft.

[0048] The present invention is based on the unexpected discovery that the method of the present invention for stimulating the ex vivo expansion of primary cells (e.g., hematopoietic stem / progenitor cells (HSPCs)) (hereinafter referred to as the "stimulation method of the present invention"), when used as a pre-stimulation step in a method for inducing stable genetic modification of a target nucleic acid via homologous recombination in a cell population comprising primary cells, directly has a considerable impact on the HDR efficacy. In fact, among other beneficial effects, the established pre-stimulation time and the type of culture medium used favor the entry of hematopoietic stem cells into the cell cycle, thereby increasing the HDR frequency. Therefore, the present invention directly addresses the problem of providing a high frequency of edited cells.

[0049] Therefore, in some aspects, the present invention provides a method for stimulating ex vivo expansion of primary cells (e.g., hematopoietic stem / progenitor cells (HSPCs)) (hereinafter referred to as "the stimulation method of the present invention"), which comprises culturing primary cells in a suitable basal medium (e.g., StemSpan AOF or StemSpan SFEMII) supplemented with UM171 and stem cell regeneration factor 1 and cytokines and optional other compounds, or any other cell culture medium for CD34+ cell culture. Preferably, in a specific embodiment, the cytokines and other compounds are selected from any one of the following lists: SCF, TPO, Flt3-ligand, IL3 and IL-6. More preferably, UM171 is added to the culture medium at a final concentration of ±30%, ±20%, ±10%, ±5% or ±1% of 35nM, and stem cell regeneration factor 1 is added to the culture medium at a final concentration of ±30%, ±20%, ±10%, ±5% or ±1% of 0.75mM.

[0050] It is noted herein that although different stimulation media can be used for the ex vivo expansion of primary cells (e.g., hematopoietic stem / progenitor cells (HSPCs)), the addition of UM171 and StemSpan Regenerin 1 to a suitable basal medium (e.g., StemSpan AOF or StemSpan SFEMII) supplemented with cytokines and optionally other compounds, or any cell culture medium for CD34+ cell culture, significantly stimulated the ex vivo expansion of primary cells (e.g., HSPCs).

[0051] Preferably, in another specific embodiment, the stimulation method of the present invention comprises culturing primary cells in a suitable basal medium (e.g., StemSpan AOF or StemSpan SFEMII) supplemented with UM171 and StemSpan Regenerin 1 and the following cytokines and other compounds: SCF, TPO, Flt3-ligand, IL3 and IL-6. Preferably, the cytokines and other compounds are present in the culture medium at a final concentration of ±30%, ±20%, ±10%, ±5% or ±1% SCF at any value in the range of 100 μg / ml to 300 μg / ml, ±30%, ±20%, ±10%, ±5% or ±1% FLT3 at any value in the range of 100 μg / ml to 300 μg / ml, ±30%, ±20%, ±10%, ±5% or ±1% TPO at any value in the range of 100 μg / ml to 300 μg / ml, ±30%, ±20%, ±10%, ±5% or ±1% IL6 at any value in the range of 100 μg / ml to 300 μg / ml, ±30%, ±20%, ±10%, ±5% or ±1% IL3 at any value in the range of 100 μg / ml to 300 μg / ml. More preferably, wherein the cytokines and other compounds are present in the culture medium at a final concentration of ±30%, ±20%, ±10%, ±5% or ±1% of any value in the range of 100 μg / ml to 300 μg / ml for SCF, ±30%, ±20%, ±10%, ±5% or ±1% of any value in the range of 100 μg / ml to 300 μg / ml for FLT3, ±30%, ±20%, ±10%, ±5% or ±1% of any value in the range of 100 μg / ml to 300 μg / ml for TPO, ±30%, ±20%, ±10%, ±5% or ±1% of any value in the range of 100 μg / ml to 300 μg / ml for IL6, and ±30%, ±20%, ±10%, ±5% or ±1% of any value in the range of 100 μg / ml to 300 μg / ml for IL3, and the culture medium is StemSpan AOF or StemSpan SFEMII, more preferably StemSpan AOF, or any cell culture medium for CD34+ cell culture.Still more preferably, wherein the cytokines and other compounds are present in the culture medium at a final concentration of 100 μg / ml of SCF, 100 μg / ml of FLT3, 100 μg / ml of TPO, and 100 μg / ml of IL6, and the culture medium is StemSpan AOF or StemSpan SFEMII, more preferably StemSpan AOF, and UM171 at a final concentration of 35 nM is added to the culture medium, and stem cell regeneration factor 1 at a final concentration of 0.75 nM is also added to the culture medium. Still more preferably, wherein the cytokines are present in the culture medium at a final concentration of 100 μg / ml SCF, 100 μg / ml FLT3, 100 μg / ml TPO and 100 μg / ml IL6, the culture medium is StemSpan AOF or StemSpanSFEM II, more preferably StemSpan AOF, and UM171 at a final concentration of 35 nM is added to the culture medium, and stem cell regeneration factor 1 at a final concentration of 0.75 nM is also added to the culture medium.

[0052] It is worth noting that, preferably, in the stimulation method of the present invention, primary cells (preferably isolated CD34+ HSPCs) are not cryopreserved before the pre-stimulation step, because the viability of hematopoietic progenitor cells is affected by cryopreservation.

[0053] It is also worth noting that the established stimulation time is conducive to the entry of primary cells (e.g., hematopoietic stem cells) into the cell cycle, thereby increasing the HDR frequency. In particular, according to the stimulation method of the present invention, primary cells (preferably isolated CD34+HSPCs) are cultured for 24 hours or longer, preferably 48±6 hours.

[0054] In some embodiments, primary cells are selected from the group consisting of primary blood cells, primary mesenchymal cells and combinations thereof. In some embodiments, primary blood cells are selected from the group consisting of immune cells, erythrocytes, progenitor cells or stem cells and combinations thereof. In some cases, immune cells are selected from the group consisting of T cells, B cells, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, mast cells, precursor cells and combinations thereof. Progenitor cells or stem cells may be selected from the group consisting of hematopoietic progenitor cells, hematopoietic stem cells and combinations thereof. In some cases, erythrocytes are blood stem cells. In some cases, primary mesenchymal cells are selected from the group consisting of mesenchymal stem cells, mesenchymal progenitor cells, mesenchymal precursor cells, differentiated mesenchymal cells and combinations thereof. Differentiated mesenchymal cells may be selected from the group consisting of osteocytes, chondrocytes, muscle cells, adipocytes, stromal cells, fibroblasts, dermal cells and combinations thereof. Preferably, primary cells are CD34+HSPC (hematopoietic stem cells and progenitor cells).

[0055] Another aspect of the invention relates to a method for inducing stable genetic modification of a target nucleic acid via homologous recombination in a primary cell population, the method comprising:

[0056] a) pre-stimulating a primary cell population (e.g., CD34+ HSPC) according to the stimulation method of the present invention for 24 hours or longer, preferably 48±6 hours,

[0057] b) preferably resuspending the primary cell population in an electroporation solution;

[0058] c) introducing into the pre-stimulated primary cell population:

[0059] a. A modified single guide RNA (sgRNA) comprising a first nucleotide sequence complementary to a target nucleic acid and a second nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide;

[0060] b. Cas polypeptide, mRNA encoding Cas polypeptide, and / or recombinant nucleotide sequence encoding Cas polypeptide

[0061] An expression vector, wherein the modified sgRNA guides the Cas polypeptide to the target nucleic acid; and

[0062] c. A homologous donor adeno-associated virus (AAV) vector comprising a recombinant donor template comprising two nucleotide sequences containing two non-overlapping homologous portions of a target nucleic acid, wherein the nucleotide sequences are located at the 5' and 3' ends of the nucleotide sequence corresponding to the target nucleic acid for homologous recombination.

[0063] Specifically, after step a), the primary cell group is resuspended in an electroporation solution. The RNP complex is added to the cell suspension and the cells are electroporated. After the electric pulse, the cell group comprising primary cells (e.g., HSPC) is incubated at about 37 ° C for about 5 to 30 minutes. Then, preheated culture medium is added, and the cells are transferred to a cell culture system. Immediately transduce the nucleofected cells with the corresponding AAV transduction of different concentrations, preferably within 30 minutes after electroporation.

[0064] In some embodiments, primary cells are isolated from mammals before the modified sgRNA, Cas polypeptide, and homologous donor AAV vector are introduced into primary cells. For example, primary cells can be obtained from human subjects. In some cases, after the modified sgRNA, Cas polypeptide, and homologous donor AAV vector are introduced into primary cells, the primary cells or their progeny are returned to the mammal. In other words, the genetically modified primary cells are autologously transplanted. In other cases, the genetically modified primary cells are allogeneically transplanted. For example, primary cells that have not been stably genetically modified are isolated from the donor object, and then the genetically modified primary cells are transplanted into a recipient object different from the donor object.

[0065] Primary cells can include primary cell populations. In some cases, primary cell populations include heterologous primary cell populations. In other cases, primary cell populations include homologous primary cell populations. Preferably, primary cell populations include CD34+HSPCs.

[0066] In some embodiments, the homologous donor AAV vector is selected from wild-type AAV serotype 1 (AAV1), wild-type AAV serotype 2 (AAV2), wild-type AAV serotype 3 (AAV3), wild-type AAV serotype 4 (AAV4), wild-type AAV serotype 5 (AAV5), wild-type AAV serotype 6 (AAV6), wild-type AAV serotype 7 (AAV7), wild-type AAV serotype 8 (AAV8), wild-type AAV serotype 9 (AAV9), wild-type AAV serotype 10 (AAV10), wild-type AAV serotype 11 (AAV11), wild-type AAV serotype 12 (AAV12), variants thereof, and any shuffled chimeras thereof. In some cases, the homologous donor AAV vector has at least about 90% sequence identity with any one selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In other cases, the homologous donor AAV vector is wild-type AAV6, or an AAV6 variant having at least 95% sequence identity with wild-type AAV6 (e.g., 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with wild-type AAV6).

[0067] In some embodiments, stable genetic modification of a target nucleic acid is induced in greater than about 70% of a population of primary cells (e.g., about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of a population of primary cells). In other embodiments, in greater than about 80% of the primary cell population (e.g., about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%) of the primary cell population, a stable genetic modification of the target nucleic acid is induced. In still other embodiments, in greater than about 90% of the primary cell population (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%) of the primary cell population, a stable genetic modification of the target nucleic acid is induced.

[0068] In other embodiments, stable genetic modification of the target nucleic acid includes replacement of a genetic mutation in the target nucleic acid (e.g., to correct a disease-associated point mutation or single nucleotide polymorphism (SNP) in the target nucleic acid), or insertion of an open reading frame (ORF) containing a normal copy of the target nucleic acid (e.g., knocking in a disease-associated wild-type cDNA of the target nucleic acid).

[0069] In some embodiments, the Cas polypeptide is a Cas9 polypeptide, a variant thereof, or a fragment thereof. In some cases, the Cas polypeptide variant comprises a Cas9 polypeptide variant with high fidelity or enhanced specificity. In some embodiments, the modified sgRNA and Cas polypeptide are introduced into primary cells simultaneously. In other embodiments, the modified sgRNA and Cas polypeptide are introduced into primary cells sequentially. In some cases, the modified sgRNA is introduced first, and then the Cas polypeptide is introduced. In other cases, the Cas polypeptide is introduced first, and then the modified sgRNA is introduced.

[0070] In some embodiments, the sgRNA and Cas polypeptide can be incubated together before being introduced into primary cells to form a ribonucleoprotein (RNP) complex. For example, the modified sgRNA and Cas polypeptide can be mixed together in a container to form an RNP complex, and then the RNP complex is introduced into the primary cell. In other embodiments, the Cas polypeptide described herein can be an mRNA encoding a Cas polypeptide, and the Cas mRNA is introduced into the primary cell as a "full RNA" CRISPR system together with the modified sgRNA. In some cases, the modified sgRNA and Cas mRNA are introduced into the primary cell at the same time. In other cases, the modified sgRNA and Cas mRNA are introduced into the primary cell in sequence. In some cases, the modified sgRNA is first introduced, and then the Cas mRNA is introduced. In other cases, the Cas mRNA is first introduced, and then the modified sgRNA is introduced.

[0071] In some embodiments, the RNP complex and the homologous donor AAV vector are introduced into primary cells simultaneously. In other embodiments, the RNP complex and the homologous donor AAV vector are introduced into primary cells sequentially. In some cases, the RNP complex is introduced into primary cells before the homologous donor AAV vector. In other cases, the homologous donor AAV vector is introduced into primary cells before the RNP complex. For example, the RNP complex can be introduced into primary cells about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 90, 120, 150, 180, 210 or 240 minutes or longer before the homologous donor AAV vector, or vice versa. In a specific embodiment, the RNP complex is introduced into the primary cells about 15 minutes (e.g., about 10 minutes to about 20 minutes) prior to the cognate donor AAV vector.

[0072] In some embodiments, the "all RNA" CRISPR system and the homologous donor AAV vector are introduced into primary cells simultaneously. In other embodiments, the "all RNA" CRISPR system and the homologous donor AAV vector are introduced into primary cells sequentially. In some cases, the "all RNA" CRISPR system is introduced into primary cells before the homologous donor AAV vector. In other cases, the homologous donor AAV vector is introduced into primary cells before the "all RNA" CRISPR system. For example, the "all RNA" CRISPR system can be introduced into primary cells about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 90, 120, 150, 180, 210 or 240 minutes or more before the homologous donor AAV vector, or vice versa. In a specific embodiment, the "all RNA" CRISPR system is introduced into primary cells about 15 minutes (e.g., about 10 minutes to about 20 minutes) prior to the cognate donor AAV vector.

[0073] In some embodiments, the recombinant donor template further contains a nucleotide sequence encoding a marker selected from the group consisting of a selectable marker, a detectable marker, a cell surface marker, or a combination thereof.

[0074] In some embodiments, any of the methods described herein may also include using a marker to purify a stable genetically modified primary cell with a target nucleic acid. In some cases, the composition separated by the purification step comprises at least about 80% of a stable genetically modified primary cell with a target nucleic acid, such as about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more of a stable genetically modified primary cell with a target nucleic acid.

[0075] In some embodiments, the step of introducing the sgRNA and Cas polypeptide into the primary cells comprises electroporating the modified sgRNA and Cas polypeptide into the primary cells. In some embodiments, the step of introducing the homologous donor AAV vector into the primary cells comprises transducing the primary cells. In some embodiments, the cells are contacted with the compound AZD prior to simultaneously contacting the cells with the recombinant donor template.

[0076] In other aspects, provided herein are genetically modified primary cells produced by any of the methods described herein. In some embodiments, genetically modified primary cells are selected from the group consisting of genetically modified primary blood cells, genetically modified primary mesenchymal cells, and combinations thereof. In some embodiments, genetically modified primary blood cells are selected from the group consisting of genetically modified immune cells, genetically modified erythrocytes, its genetically modified progenitor cells or stem cells, and combinations thereof. In some cases, genetically modified immune cells are selected from the group consisting of genetically modified T cells, genetically modified B cells, genetically modified dendritic cells, genetically modified natural killer cells, genetically modified macrophages, genetically modified neutrophils, genetically modified eosinophils, genetically modified basophils, genetically modified mast cells, its precursors, and combinations thereof. Genetically modified progenitor cells or stem cells can be selected from the group consisting of genetically modified hematopoietic progenitor cells, genetically modified hematopoietic stem cells, and combinations thereof. In some cases, genetically modified erythrocytes are genetically modified blood stem cells. In some cases, the genetically modified primary mesenchymal cells are selected from the group consisting of genetically modified mesenchymal stem cells, genetically modified mesenchymal progenitor cells, genetically modified mesenchymal precursor cells, genetically modified differentiated mesenchymal cells, and combinations thereof. The genetically modified differentiated mesenchymal cells can be selected from the group consisting of genetically modified osteocytes, genetically modified chondrocytes, genetically modified muscle cells, genetically modified adipocytes, genetically modified stromal cells, genetically modified fibroblasts, genetically modified dermal cells, and combinations thereof.

[0077] On the other hand, the genetically modified cell population of the present invention produced by stimulating the ex vivo expansion of primary cells (e.g., hematopoietic stem / progenitor cells (HSPCs)), when used as a pre-stimulation step in a method for inducing stable genetic modification of a target nucleic acid via homologous recombination in a cell population comprising primary cells, has the characteristic of comprising a double-positive CD45RA+CD90+ population, which appears in edited cells but is absent in unoperated cells.

[0078] In yet other aspects, provided herein is a pharmaceutical composition comprising any genetically modified primary cell described herein and a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical composition comprises one type of genetically modified primary cell. In other embodiments, the pharmaceutical composition comprises two or more different types of genetically modified primary cells, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different types of genetically modified primary cells. DETAILED DESCRIPTION OF THE INVENTION

[0080] The present invention discloses an improved method for genetically modifying CD34+ cells by electroporation and transduction of a sgRNA / Cas9 / AAV vector system. This improved method provides a detailed protocol for pre-stimulation, electroporation, and transduction of CD34+ cells with AAV to achieve clinically relevant gene editing frequencies. Precision gene editing has been used in the clinical treatment of a variety of diseases, and more applications are under development. The programmable nuclease Cas9, under the guidance of a single guide RNA (sgRNA), can introduce double-strand breaks (DSBs) at target sites in genomic DNA, which is the initial step for gene editing using this technology. In the present invention, repairing DSBs by homology-directed repair (HDR) is a key step in providing successful results for gene editing strategies.

[0081] Specifically, in the present invention, the CRISPR-Cas9 system and the donor recombinant adeno-associated vector (rAAV) delivery are combined to construct an efficient, safe and clinically applicable system to knock in therapeutic sequences at the translation start sites of some genes in human hematopoietic progenitor cells. At present, there are some methods to introduce the CRISPR-Cas9 system in the form of ribonucleoprotein (RNP) into cells, of which electroporation is the preferred method. Electroporation induces small holes in the cell membrane, allowing some small molecules (such as RNP) to be internalized. Similarly, rAAV6 is a good tool for homology-directed repair (HDR) donor template delivery.

[0082] The method of the present invention can be carried out by using Figure 1It is worth noting that the method of the present invention is firstly to isolate CD34+ cells, preferably from mobilized peripheral blood, more preferably by using Sepax C-Pro (CD34 marker) and CliniMACS Plus (CD34 enrichment process) (PR-SOP-03, CD34 selection in CliniMACS plus). The method is further described in detail below.

[0083] CD34 enrichment and pre-stimulation

[0084] CD34+ HSPCs, preferably isolated from mobilized peripheral blood, are cultured in a suitable basal medium (e.g., StemSpan AOF or StemSpan SFEM II (Stemcell Technologies, Vancouver, Canada)) supplemented with SCF (100 ng / ml), TPO (100 ng / ml), Flt3-ligand (100 ng / ml), IL-6 (100 ng / ml) and stem cell regeneration factor 1 (0.75 mM). In addition, UM171 (Stemcell Technologies) is added to the culture medium at a final concentration of 35 nM. The cells are incubated at 37°C, 5% CO 2 and 5% O 2 Culture for 48±6 hours.

[0085] It is noteworthy that it is preferred that the CD34+HSPC separated is not frozen before the pre-stimulation step, because the viability of hematopoietic progenitor cells is affected by freezing. In addition, although different pre-stimulation culture media can be used, it is noteworthy that the addition of UM171 and stem cell regeneration factor 1 significantly stimulated HSPC ex vivo expansion. In fact, the introduction of the scheme of using UM171 and SR-1 to cultivate HSPC has successfully achieved the in vitro expansion of reproliferation cells. In addition, SCF (100ng / ml), TPO (100ng / ml), Flt3-ligand (100ng / ml), IL-6 (100ng / ml) and stem cell regeneration factor 1 (0.75mM) are added to suitable basal culture medium (for example, StemSpan AOF or StemSpan SFEM II (Stemcell Technologies, Vancouver, Canada), and further supplemented with a final concentration of 35nM UM171 is the most striking strategy tested herein, because the strategy represents the best compromise between high editing efficiency and retention of reproliferation bone marrow cells.

[0086] It was further pointed out that the established pre-stimulation time is beneficial for hematopoietic stem cells to enter the cell cycle, thereby increasing the frequency of HDR.

[0087] Electroporation and transduction

[0088] Immediately before electroporation, prepare Cas9 RNP by incubating protein with sgRNA at a 1:3 molar ratio at 25 °C for at least 10 min.

[0089] Then, CD34+ HSPCs were electroporated after the pre-stimulation step. CD34+ HSPCs were electroporated using the Maxcyte electroporation flow system under the following conditions: 200-100×10 6 cells / ml, 300-350 μg / ml Cas9 protein and sgRNA were complexed at a 1:3 molar ratio. After electroporation, the cells were incubated at 37°C for 10 minutes, and then the AAV donor vector was added (usually at an MOI of 4000 unless otherwise stated) and incubated at 37°C for 24 ± 6 hours. Figure 3 As shown, further exposure of cells to the compound AZD before or during transduction significantly increased editing efficiency while sparing myeloid cells.

[0090] Determination of targeted integration

[0091] The targeted integration rate was determined by ddPCR in liquid cultures at least 3 days after electroporation. Targeted integration of the coRPK expression cassette was determined by ddPCR using input-output specific primers to detect correct integration.

[0092] According to the results obtained, the use of CD34 enrichment and pre-stimulation medium in the present invention significantly stimulates the ex vivo expansion of HSPCs and therefore has a direct and considerable impact on the HDR efficacy. In fact, the gene-edited cell population obtained after the electroporation and transduction steps has the characteristic of containing a double-positive CD34+CD38-CD45RA+CD90+ population, which appears in edited cells and is absent in unoperated cells. Figure 2 .

Claims

1. An in vitro method for stimulating the ex vivo expansion of primary cells, the method comprising culturing the primary cells in a suitable basal medium supplemented with UM171 and stem cell regeneration factor 1, preferably at 37°C, 5% CO2 and 5% O2 for 48±6 hours.

2. The in vitro method according to claim 1, wherein the suitable basal medium is further supplemented with a compound selected from any one of the list consisting of SCF, TPO, Flt3-ligand, IL3 and IL-6.

3. The in vitro method according to any one of claims 1 or 2, wherein UM171 is added to the culture medium at a final concentration of 35 nM at ±30%, ±20%, ±10%, ±5% or ±1%, and stem cell regeneration factor 1 is added to the culture medium at a final concentration of 0.75 nM at ±30%, ±20%, ±10%, ±5% or ±1%.

4. The in vitro method according to claim 3, wherein the suitable basal medium is further supplemented with at least SCF, TPO, Flt3-ligand and IL-6.

5. An in vitro method according to claim 4, wherein the suitable basal medium is further supplemented with SCF at a final concentration of 100 μg / ml at ±30%, ±20%, ±10%, ±5% or ±1%, FLT3 at 100 μg / ml at ±30%, ±20%, ±10%, ±5% or ±1%, TPO at 100 μg / ml at ±30%, ±20%, ±10%, ±5% or ±1%, and IL6 at 100 μg / ml at ±30%, ±20%, ±10%, ±5% or ±1%.

6. The in vitro method according to claim 5, wherein the culture medium is StemSpan AOF or StemSpan SFEMII, more preferably StemSpan AOF.

7. An in vitro method according to claim 1, wherein the suitable basal culture medium is supplemented with SCF at a final concentration of 100 μg / ml, FLT3 at 100 μg / ml, TPO at 100 μg / ml and IL6 at 100 μg / ml, the culture medium is StemSpan AOF, and UM171 at a final concentration of 35 nM is added to the culture medium and stem cell regeneration factor 1 at a final concentration of 0.75 nM is added to the culture medium.

8. The in vitro method according to any one of claims 1 to 7, wherein the primary cells are not frozen prior to stimulating their ex vivo expansion.

9. A method for inducing stable genetic modification of a target nucleic acid via homologous recombination in a primary cell population, the method comprising: a. pre-stimulating a primary cell population according to the method according to any one of claims 1 to 8, b. Introducing into the pre-stimulated primary cell population: i. a modified single guide RNA (sgRNA) comprising a first nucleotide sequence complementary to the target nucleic acid and a second nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide; ii. a Cas polypeptide, an mRNA encoding a Cas polypeptide, and / or a recombinant expression vector comprising a nucleotide sequence encoding a Cas polypeptide, wherein the modified sgRNA guides the Cas polypeptide to the target nucleic acid; and iii. A homologous donor adeno-associated virus (AAV) vector comprising a recombinant donor template, wherein the recombinant donor template comprises two nucleotide sequences containing two non-overlapping homologous portions of the target nucleic acid, wherein the nucleotide sequences are located at the 5' and 3' ends of the nucleotide sequence corresponding to the target nucleic acid for homologous recombination.

10. The method according to any one of claims 1 to 9, wherein the primary cells are a cell population comprising CD34+ HSPCs, preferably from mobilized peripheral blood.

11. The method of any one of claims 1 to 10, wherein the homologous donor AAV vector is selected from wild-type AAV serotype 1 (AAV1), wild-type AAV serotype 2 (AAV2), wild-type AAV serotype 3 (AAV3), wild-type AAV serotype 4 (AAV4), wild-type AAV serotype 5 (AAV5), wild-type AAV serotype 6 (AAV6), wild-type AAV serotype 7 (AAV7), wild-type AAV serotype 8 (AAV8), wild-type AAV serotype 9 (AAV9), wild-type AAV serotype 10 (AAV10), wild-type AAV serotype 11 (AAV11), wild-type AAV serotype 12 (AAV12), variants thereof, and any shuffled chimeras thereof.

12. The method according to any one of claims 9 to 11, wherein the modified sgRNA and the Cas polypeptide are incubated together to form a ribonucleoprotein (RNP) complex before being introduced into the primary cell.

13. The method according to any one of claims 9 to 12, wherein the cells are exposed to or contacted with compound AZD prior to or simultaneously with transduction with AAV.

14. A genetically modified primary cell produced by the method of any one of claims 1 to 13.

15. A composition, preferably a pharmaceutical composition, comprising the genetically modified primary cells of claim 14, which is used in a method for preventing or treating a disease in a subject in need thereof, the method comprising administering the genetically modified primary cells of claim 13 to the subject to prevent the disease or improve one or more symptoms of the disease.

16. The composition for use according to claim 15, wherein the disease is selected from the group consisting of pyruvate kinase deficiency, hemoglobinopathies, viral infections, X-linked severe combined immunodeficiency, Fanconi anemia, hemophilia, neoplasia, cancer, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, cystic fibrosis, blood diseases and disorders, inflammation, immune system diseases or disorders, metabolic diseases, liver diseases and disorders, kidney diseases and disorders, muscle diseases and disorders, bone or cartilage diseases and disorders, nervous system and neuronal diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, and lysosomal storage diseases.

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