Genotyping method for gene-edited hematopoietic stem cells

Abstract

The present disclosure provides a method for using reticulocytes derived from recipients of gene-edited hematopoietic stem cells (HSCs) to evaluate the results of gene editing in a transplanted population of gene-edited HSCs. Provided herein are methods and compositions relating to the use of single-cell RNA sequencing to evaluate the results of gene editing in a transplanted population of gene-edited hematopoietic stem cells. Although RBCs derived from gene-edited HSCs have had their nuclei removed and are therefore unavailable for genotyping, reticulocytes are immature RBCs that still contain some RNA that can be used to evaluate allele correction.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 382,060, filed November 2, 2022, and U.S. Provisional Application No. 63 / 383,768, filed November 15, 2022, each of which is incorporated by reference herein in its entirety for all purposes. [Background technology]

[0002] Field Provided herein are methods and compositions related to assessing gene editing outcomes in genetically modified stem cells.

[0003] Therapeutic gene editing modifies the genome of individual cells at one or more target loci associated with disease. Given that each human cell has two copies of the genome, gene editing procedures using systems such as the CRISPR / Cas system can produce a variety of genomic outcomes in a target cell population, including cells with no alterations in any of the alleles at the target locus, cells with alterations in only one allele, and cells with alterations in both alleles. In these altered alleles, modifications can include the insertion or deletion of one or more nucleotides (INDELS), or the insertion of a donor polynucleotide via homology-directed repair (HDR) to address one or more mutations at the target locus. The cell population that has undergone the editing procedure can then be administered to a patient, for example, a patient whose hematopoietic stem cells have been harvested and edited ex vivo.

[0004] Sickle cell disease (SCD) is a genetic condition commonly caused by a single point mutation in codon 6 of both copies of the β-globin (HBV) gene, resulting in the E6V mutation, which results in the production of sickle (S) hemoglobin (HbS) rather than adult (A) hemoglobin (HbA). Gene-edited autologous hematopoietic stem cell-based therapies in clinical development for SCD are designed to reduce HbS production and restore HbA expression by directly correcting the underlying point mutation. Previous allogeneic hematopoietic stem cell transplantation studies, most of which use HLA-matched related donors, have demonstrated that homozygous (AA) or heterozygous (AS) hemoglobin have a competitive advantage due to ineffective erythropoiesis of SS erythroid progenitors. However, at early time points after infusion of gene-corrected HSCs, red blood cells (RBCs) expressing the corrected HbA are indistinguishable from HbA in transfused blood. Because RBCs have their nuclei removed, it is not possible to track gene editing rates at the genome level, so a method is needed that can quantify gene editing outcomes early after HSC transplantation and engraftment in patients. Summary of the Invention

[0005] This disclosure provides a method for evaluating the outcomes of gene editing in transplanted, gene-edited hematopoietic stem cell (HSC) recipients using reticulocytes. While RBCs derived from gene-edited HSCs are not available for genotyping because their nuclei have been removed, reticulocytes are immature RBCs that still contain some RNA that can be used to evaluate allele correction. The method described herein utilizes single-cell RNA sequencing (scRNAseq) to enumerate potential gene editing outcomes in peripheral reticulocytes. The altered genotypes can then be directly combined with other single-cell measurements to evaluate whether the outcomes of gene editing are associated with cell type assigned via RNA or protein surface markers, or with other aspects of cell state, such as chromatin accessibility. [Brief explanation of the drawings]

[0006] [Figure 1] Figure 1 illustrates an exemplary method for precisely correcting a disease-causing mutation in the β-globin gene to reduce HbS and restore HbA expression. The scissors represent a CRISPR / Cas nuclease that creates a double-stranded break in the HBB target locus in hematopoietic stem cells from a subject with sickle cell disease. A correction polynucleotide consisting of the glutamic acid-encoding sequence GAG, designed to replace the valine-encoding GTG mutation at codon 6, is introduced into the cells along with the nuclease, allowing the polynucleotide to integrate into the HBB locus via homology-directed repair.

[0007] [Figure 2] Figure 2 shows the maturation of red blood cells (RBCs) from hematopoietic stem cells.

[0008] [Figure 3] Figure 3 shows the enrichment and sorting of mixed AA, AS, and SS reticulocytes. Reticulocytes isolated from AA blood were distributed across all stages of maturation. The majority of AS reticulocytes were late in development. Reticulocytes from SS donor samples were the purest, with most at early stages. SS blood contained approximately 10 times more reticulocytes than AA blood. CD, cluster of differentiation antigen; FSC-A, forward scatter area; GPA, glycophorin-A; RBC, red blood cells; SSC-A, side scatter area; SSC-H, side scatter height; TO, thiazole orange; WBC, white blood cells.

[0009] [Figure 4] Figure 4 shows the single-cell RNAseq workflow to capture the 5' end of the HBB transcript and ensure high-resolution coverage of E6V mutations located near the transcription start site in exon 1. HBB, β-globin; TSO, template switch oligo; UMI, unique molecular identifier.

[0010] [Figure 5]FIG. 5 shows an exemplary single-cell RNA sequencing and genotyping workflow of the present disclosure.

[0011] [Figure 6] FIG. 6 shows bulk cDNA sequencing of reticulocytes and blood mixtures.

[0012] [Figure 7] FIG. 7 shows the results of variant calling (either wild type (AA) or sickle cell (SS)) in a mixed reticulocyte population using the methods described herein.

[0013] [Figure 8] FIG. 8 shows variant allele calling in a 1:1:1 mixture of AA, AS and SS reticulocytes.

[0014] [Figure 9] FIG. 9 shows the mRNA expression profiles and t-SNE plots of AA, AS, and SS reticulocytes.

[0015] [Figure 10] FIG. 10 shows the results showing that SS reticulocytes have higher expression of HBB and HGD2 than AA and AS reticulocytes.

[0016] [Figure 11] Figure 11 shows results demonstrating that scRNAseq data from AA, AS, and SS reticulocytes can be used to investigate gene expression differences associated with genotype. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Invention Single-cell RNA sequencing of reticulocytes from subjects receiving gene-edited HSCs

[0018] In one aspect, provided herein is a method that can be used to determine the editing status of the hemoglobin (HBB) gene in reticulocytes from patients transplanted with gene-edited HSCs designed to correct E6V mutations (Figure 1). The HBB gene is expressed exclusively in the erythroid lineage, which ultimately develops into mature red blood cells that contain no genomic DNA and few gene transcripts. Reticulocytes are immature red blood cells that still contain some gene transcripts (Figure 2). By applying the disclosed single-cell RNA sequencing and genotyping approach to these cells, it is possible to assess the editing status of the HBB gene in particular and how this status may affect red blood cell maturation, as well as the functional impact on the expression of other hemoglobin genes.

[0019] The method provided herein utilizes 10X single-cell RNA sequencing. See Technical Note, CG000425, ChromiumNextGEM_SingleCell5'_HT_v2_Reagent, Workflow & Data Overview_RevA, 10xGenomics (August 9, 2021). The 10X method is primarily intended to count mRNA transcripts to determine gene expression levels and utilizes short sequencing reads to do so. The method provided herein utilizes a modified method that allows for longer sequencing reads and can provide sequencing coverage of the gene edits of interest (Figure 3). The resulting gene-specific sequencing reads are then analyzed based on barcodes linking them to individual cells and analyzed in parallel using Crispresso2, a software tool intended to analyze editing results based on amplicon sequencing data. See https: / / github.com / pinellolab / CRISPResso2. The Crispresso2 results are further processed to determine the editing outcome and zygosity of each barcoded single cell. The single-cell editing outcome and barcode are paired and uploaded to the 10X analysis software, Loupe. The 10X software can then be used to examine associations between single-cell editing outcomes and phenotypic differences, such as transcriptional changes that may result from specific genotypic modifications.

[0020] The methods provided herein can be useful for assessing the editing status of any target gene of interest in cells, e.g., genetically modified HSCs. Gene editing, transplantation and engraftment of HSCs

[0021] In certain embodiments, the method comprises administering to the patient a therapeutically effective amount of genetically modified hematopoietic stem cells. In certain embodiments of the methods provided herein, the patient is administered a therapeutically effective amount of genetically modified hematopoietic stem and progenitor cells. In certain embodiments, the genetically modified cells administered are donor bone marrow cells, umbilical cord blood cells, hematopoietic stem and progenitor cells (HSPCs), peripheral blood CD34 cells, or the like. + Cells, peripheral blood CD34 + and CD90 + The composition may include a cell, a mammalian ...

[0022] Genetically modified hematopoietic stem cells can be derived from any hematopoietic stem cells that a person skilled in the art deems useful. In certain embodiments, once engrafted, the genetically modified hematopoietic stem cells can reconstitute hematopoiesis in a patient. Human hematopoiesis is defined by a hierarchy based on cell surface marker expression, initiated by hematopoietic stem cells that undergo both self-renewal and differentiation into multipotent progenitor cells, which in turn give rise to lineage-restricted progenitor cells and ultimately to terminally differentiated blood cells (Baum et al., PNAS 89, 2804-2808 (1992); Majeti et al., Cell Stem Cell 1, 635-645 (2007); Doulatov et al., Cell Stem Cell 10, 120-136 (2012)). CD34 - Expression defines a heterogeneous HSPC population and includes multipotent progenitor cells (CD34 + / CD38 - / CD45RA - ), long-term repopulating cells in xenografted mice (CD34 + / CD38 - / CD90 + ), and a population highly enriched in hematopoietic stem cells (CD34 + / CD38 - / CD90 + / CD45RA - ) can be further classified as

[0023] In certain embodiments, the genetically modified hematopoietic stem cells are any subtype or colony-forming unit. In certain embodiments, the genetically modified hematopoietic stem cells are granulocyte-erythroid-monocyte-megakaryocyte colony-forming units. In certain embodiments, the genetically modified hematopoietic stem cells are erythroid colony-forming units. In certain embodiments, the genetically modified hematopoietic stem cells are granulocyte-macrophage colony-forming units. In certain embodiments, the genetically modified hematopoietic stem cells are megakaryocyte colony-forming units. In certain embodiments, the genetically modified hematopoietic stem cells are basophil colony-forming units. In certain embodiments, the genetically modified hematopoietic stem cells are eosinophil colony-forming units.

[0024] The genetically modified hematopoietic stem cells can be derived from any source deemed useful by one of skill in the art. In certain embodiments, the hematopoietic stem cells are derived from a donor. In certain embodiments, the donor is a patient. In certain embodiments, the donor is another subject of the same species, e.g., another human. In certain embodiments, the genetically modified hematopoietic stem cells are autologous. In certain embodiments, the genetically modified hematopoietic stem cells are allogeneic. In certain embodiments, the genetically modified hematopoietic stem cells are syngeneic.

[0025] The donor's hematopoietic stem cells to be genetically modified can be harvested by any technique deemed useful by one of skill in the art. In certain embodiments, the donor subject is administered a hematopoietic stem cell mobilizing agent (e.g., plerixafor (Mozobil®), G-CSF, GM-CSF) prior to harvest. In certain embodiments, hematopoietic stem cells are harvested from peripheral blood. In certain embodiments, hematopoietic stem cells are harvested from umbilical cord blood. In certain embodiments, hematopoietic stem cells are harvested from bone marrow. In certain embodiments, the population of donor cells can be obtained from a product collected from a subject, such as a patient or subject in need of autologous HSCT. The product can be an apheresis product containing a heterogeneous mixture of cells collected from the subject. The heterogeneous mixture of cells can include primary cells, and primary CD34 cells. +cells, and / or human stem progenitor cells (HSPCs). + The cells and / or HSPCs can be isolated or separated from other cells to obtain a population of stem cells. + After HSPC isolation, the resulting stem cell population is substantially non-CD34 + It is free of cells and ready for further genetic manipulation.

[0026] In certain embodiments, the collected hematopoietic stem cells are separated from the population of primary cells using flow cytometry. In some instances, the flow cytometry comprises fluorescence-activated cell sorting (FACS). In certain other embodiments, the collected hematopoietic stem cells are separated from the population of primary cells using magnetic bead separation. In some instances, the magnetic bead separation comprises magnetic-activated cell sorting (MACS). In certain other embodiments, the collected hematopoietic stem cells are separated using a device configured for hematopoietic stem cell enrichment, for example, the Miltenyi Biotec CliniMACS Cell Manufacturing Platform.

[0027] Methods for culturing or expanding primary hematopoietic stem cells are known in the art, including those described in International Patent Application No. PCT / US2022 / 72014, the entire contents of which are incorporated herein by reference. Methods for culturing primary cells and their progeny are known, and suitable culture media, additives, growth factors, etc. are known and commercially available. Typically, human primary cells are maintained and expanded under serum-free conditions. Alternative media, additives, growth factors, and / or alternative concentrations can be readily determined by one of skill in the art and are fully described in the literature. In certain embodiments, isolated or purified genetically modified cells can be expanded in vitro according to standard methods known to those of skill in the art.

[0028] Genetically modified hematopoietic stem cells

[0029] In certain embodiments, hematopoietic stem cells are genetically modified to contain a therapeutic xenogeneic donor polynucleotide sequence. The donor polynucleotide sequences described herein may be incorporated into various gene therapy constructs, for example, to deliver a nucleic acid encoding a protein to a subject in need thereof. A vector construct refers to a polynucleotide molecule that contains all or part of a viral genome and an exogenous polynucleotide sequence. In some cases, gene transfer can be mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV). Other vectors useful for gene therapy are known in the art. For example, constructs of the present disclosure may include alphaviruses, herpesviruses, retroviruses, lentiviruses, or vaccinia viruses. The exogenous sequence generally encodes a recombinant molecule that is expressed in cells, e.g., cells for use in cell therapy. The processing steps of these methods may also or alternatively include all or part of cell washing, dilution, selection, isolation, separation, culturing, stimulation, packaging, and / or formulation. These methods generally allow for the processing, eg, selection or separation and / or transduction, of cells on a large scale (eg, in compositions of greater than or about 50 mL volume).

[0030] In certain embodiments, hematopoietic stem cells are genetically modified using site-specific nuclease-based gene editing techniques to knock out target genomic sequences or knock in exogenous sequences, and to introduce exogenous sequences into cells by viral transduction using recombinant viral vectors. In some such embodiments, hematopoietic stem cells are collected by apheresis, concentrated from the apheresis product, and then cryopreserved before performing any gene editing method (e.g., gene knockout, gene knock-in, gene correction). Cryopreservation may be performed after stem cell mobilization and collection (e.g., apheresis) and hematopoietic stem cell selection. After cryopreservation, whether a threshold number of hematopoietic stem cells has been collected from the donor can be assessed to proceed to subsequent gene editing steps. If the threshold number of cells is not reached in a single round of mobilization, collection, selection, and cryopreservation, subsequent rounds may be performed until the threshold number of cells is reached. The threshold number of hematopoietic stem cells to be collected may vary depending on several factors. Some factors include, but are not limited to, the gene editing procedure being performed (e.g., gene knockout, gene knockin, gene correction), the target gene being edited, the mechanism by which the target gene is modified (e.g., homology-dependent repair (HDR)), the efficiency of the editing procedure (e.g., HDR efficiency), and the therapeutic threshold for treating a particular disease. In one embodiment, the threshold number of hematopoietic stem cells to be collected from a donor prior to gene editing is about 1 x 10 4 From 1x10 5 , 1x10 5 From 1x10 6 , 1x10 6 From 1x10 7 cells / kg or more. In one embodiment, at least about 1 x 10 5 From 1x10 7 Cells / kg are collected prior to gene editing. In one embodiment, at least about 1 x 10 4 , 2x10 4 , 3x10 4 , 4x10 4 , 5x10 4 , 6x10 4 , 7x10 4, 8x10 4 , 9x10 4 , 1x10 5 , 2x10 5 , 3x10 5 , 4x10 5 , 5x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , or about 1x10 8 Hematopoietic stem cells / kg are collected before proceeding with gene editing of the collected cells. Once a threshold number of hematopoietic stem cells have been mobilized, collected, sorted, and cryopreserved, the cells can then proceed to thawing, culture, and gene editing.

[0031] In some embodiments, gene editing utilizes a nuclease that can be introduced into cells and cause double-strand breaks near or within the genomic target site, which can be useful for increasing the frequency of homologous recombination and HDR at or near the break site. In a more preferred embodiment, the recognition sequence of the nuclease is present only at the target site in the host cell genome, thereby minimizing any off-target genome binding and cleavage by the nuclease. Gene editing nucleases useful in the methods provided herein include TAL effector DNA binding domain-nuclease fusion proteins (TALENs), site-specific recombinases (e.g., serine recombinases or tyrosine recombinases, integrases (FLP, Cre, lambda integrase) or resolvases, transposases, These include, but are not limited to, zinc finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, and Csy1. , Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.

[0032] In one embodiment, genetically modified CD34 + Stem cells express a CRISPR-associated Cas nuclease (e.g., Cas9), a guide RNA polynucleotide, and a donor polynucleotide sequence in primary CD34 +The vector is generated by introducing the vector into a stem cell. By introducing these components into the cell, a double-stranded break can be introduced at a specific site, directed by a guide polynucleotide sequence and a CRISPR-associated Cas9 nuclease. A donor polynucleotide containing a sequence of interest can then be introduced into the cell, allowing the sequence of interest to be inserted into the cell through homologous-directed recombination. The transfer of the donor polynucleotide sequence can be achieved by transduction. Viral transfer, e.g., transduction, generally involves at least initiating transduction by centrifugally culturing an input composition containing cells to be transduced and viral vector particles containing the vector under conditions such that the cells are transduced or transduction is initiated in at least some of the cells of the input composition, whereby the method produces an output composition containing transduced cells.

[0033] Methods for introducing polypeptides, nucleic acids, and viral vectors (e.g., viral particles) into primary cells, target cells, or host cells are known in the art. Any known method can be used to introduce polypeptides or nucleic acids (e.g., nucleotide sequences encoding DNA nucleases or modified sgRNAs) into primary cells, such as human primary cells. Non-limiting examples of suitable methods include electroporation (e.g., nucleofection), virus or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

[0034] In some embodiments, the Cas nuclease can be in the form of a protein. In some embodiments, the Cas nuclease can be in the form of a plasmid, which allows cells harboring this expression construct to subsequently express the Cas nuclease. In other embodiments, the Cas nuclease is pre-complexed with a guide RNA and introduced into cells as a ribonucleoprotein (RNP). In some embodiments, the Cas nuclease and guide polynucleotide sequence are introduced into CD34+ cells via electroporation.

[0035] Introduction of the donor polynucleotide can be achieved through viral transduction using a delivery vector such as an adeno-associated virus (AAV). Any serotype or pseudotype of AAV can be used. Certain AAV vectors are derived from single-stranded (ss) DNA parvoviruses that are nonpathogenic to mammals. Briefly, the rep and cap viral genes, which comprise 96% of a typical wild-type AAV genome, can be removed during the generation of certain AAV vectors, leaving adjacent inverted terminal repeats (ITRs) that can be used to initiate viral DNA replication, packaging, and integration. Wild-type AAV integrates into the human host cell genome with preferential site specificity at chromosome 19q13.3. Alternatively, AAV can be maintained episomally. At least 12 human AAV serotypes (AAV serotype 1 (AAV-1) to AAV-12) and over 100 nonhuman primate serotypes have been discovered to date. Any of these serotypes, and combinations thereof, can be used within the scope of the present disclosure. The serotype of the viral vector can be selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In one embodiment, the serotype is AAV6.

[0036] In some embodiments, viral transduction occurs within 30 minutes of electroporation. In some embodiments, viral transduction occurs simultaneously with electroporation. In some embodiments, viral transduction occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 minutes of electroporation.

[0037] In another embodiment, hematopoietic stem cells are genetically modified using gene editing techniques that utilize base editing. Base editors are genome editing techniques based on CRISPR-Cas9 that can introduce point mutations into DNA without generating DSBs. Two major classes of base editors have been developed: cytidine base editors (CBEs) that can convert C to T, and adenine base editors (ABEs) that can convert A to G (see Rees et al. (2018) Nat Rev Genet 19:770-788).

[0038] In another embodiment, hematopoietic stem cells are genetically modified using gene editing techniques that utilize a prime editor. The prime editor (PE) consists of nCas9 fused to a reverse transcriptase and is used in combination with a prime editing RNA (pegRNA, a guide RNA containing a template region for reverse transcription). Prime editing can introduce insertions, deletions (indels), and 12-base pair transversions. Prime editing relies on the ability of reverse transcriptase (RT) fused to a Cas nickase mutant to convert the RNA sequence provided by the prime editing guide RNA (pegRNA) into DNA at nick sites generated by the Cas protein. The DNA flap generated in this process can then be incorporated into the target DNA sequence or not. See Anzalone et al. (2019) Nature 576:149-157. Non-limiting examples of prime editing systems include PE1, PEI-M1, PE1-M2, PE1-M3, PE1-M6, PE1-M15, PE1-M3inv, PE2, PE3, PE3b.

[0039] Dosing and administration of genetically modified HSCs

[0040] In certain embodiments, the method comprises administering to an individual in need of treatment a composition comprising an effective amount of genetically modified hematopoietic stem cells. A therapeutically effective amount of hematopoietic stem cells can range from about 1 million to about 200 billion cells. For example, 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the above values), such as about 10 million cells to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, In some embodiments, the cell density may be greater than about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the above values), and in some cases, about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value within these ranges. 6 From 2x10 8 The method includes administering viable hematopoietic stem cells between 100 and 150 mg / kg.

[0041] In certain embodiments, pharmaceutical compositions comprising hematopoietic stem cells genetically modified according to the present disclosure contain, for example, about 1 x 10 4 From 1x10 5 , 1x10 5 From 1x10 6 , 1x10 6 From 1x10 7The modified host cell compositions can be administered at a dosage level sufficient to deliver 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more than 10 years of therapeutic benefit. In some embodiments, only a single dose is required to provide treatment or prevention of a disease or disorder described herein. In other embodiments, a subject in need thereof can receive more than one dose, e.g., two, three, or more than three doses, of the pharmaceutical hematopoietic stem cell compositions described herein to provide treatment or prevention of a disease or disorder. The hematopoietic stem cells can be used in combination, sequentially or simultaneously, with one or more other therapeutic, prophylactic, research, or diagnostic agents or medical procedures. Generally, each agent is administered at a dose and / or time schedule determined for that agent.

[0042] The infusion group and its compositions can be administered to individuals in need thereof using standard administration techniques, formulations, and / or devices. Formulations and administration devices, such as syringes and vials, are provided, along with devices for storing and administering the compositions. Formulations or pharmaceutical compositions containing exogenous hematopoietic stem cells include those for intravenous, intraperitoneal, subcutaneous, intramuscular, or intrapulmonary administration. The exogenous hematopoietic stem cell composition can be provided as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which may be buffered to a selected pH in some embodiments. Viscous compositions can be formulated within an appropriate viscosity range to provide extended contact time with specific tissues. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating the hematopoietic stem cells into a solvent such as sterile water, saline, glucose, dextrose, or the like mixed with a suitable carrier, diluent, or excipient.

[0043] The genetically modified hematopoietic stem cells contained in the pharmaceutical composition described above can be administered by any delivery route, whether systemic or local, to produce therapeutically effective results. These routes include, but are not limited to, enteral, gastrointestinal, epidural, oral, transdermal, intracerebral, intraventricular, epicutaneous, intradermal, subcutaneous, nasal, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intrathecal, intraparenchymal, intraperitoneal, intravesical, intravitreal, intracavernous, interstitial, intraperitoneal, intralymphatic, intramedullary, intrapulmonary, intraspinal, intrasynovial, intrathecal, intraductal, parenteral, transdermal, periarticular, epidural, perineural, periodontal, rectal, soft tissue, and topical. In certain embodiments, the cells are administered intravenously. The pharmaceutical composition can be administered to a subject at any dosage and via any route effective for the prevention, treatment, or management of the diseases described herein. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its method of administration, its mechanism of action, and the like.

[0044] In some embodiments, the pharmaceutical composition comprises a modified host cell that has been genetically engineered to contain a donor sequence integrated into a target locus of the host cell. In some embodiments, the modified host cell is genetically engineered to contain an integrated functional donor sequence, such as a SNP donor that corrects one or more mutations in a target gene (e.g., HBB), or inserts a wild-type allele into part or all of a mutated allele, or replaces part or all of a mutated allele with a wild-type allele. In certain embodiments, the functional donor sequence is integrated into the translation start site of the endogenous locus of the target gene. In certain embodiments, the functional donor sequence integrated into the host cell genome is expressed under the control of the native promoter sequence of the target gene.

[0045] In some embodiments, the pharmaceutical composition comprises a plurality of modified host cells, and further comprises unmodified host cells and / or host cells that have undergone nuclease cleavage resulting in INDELS at the target locus but have not undergone integration of the donor sequence. In some embodiments, the pharmaceutical composition comprises at least 5% of the modified host cells that contain the integrated donor sequence. In some embodiments, the pharmaceutical composition comprises about 9% to 50% of the modified host cells that contain the integrated donor sequence. In certain embodiments, the pharmaceutical composition comprises at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 8 At least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, or more of the pharmaceutical compositions described herein can be formulated with one or more excipients to, for example, (1) improve stability; (2) modify biodistribution (e.g., to target cells to a particular tissue or cell type, e.g., hematopoietic stem cells); and / or (3) enhance engraftment in the recipient. Indications

[0046] The genetically modified HSCs described herein may be used as part of a treatment regimen for any disease or condition for which HSC transplantation (HSCT) is useful. HSCT may be used to treat several conditions, including congenital and acquired conditions. In some embodiments, the acquired conditions treatable with HSCT include: (1) malignant tumors, such as leukemia (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), lymphoma (e.g., Hodgkin's disease, non-Hodgkin's lymphoma), myeloma (e.g., (1) hematological malignancies such as multiple myeloma (Kahler's disease), solid tumor cancers (e.g., neuroblastoma, desmoplastic small round cell tumor, Ewing's sarcoma, choriocarcinoma); (2) hematological disorders, including, but not limited to, phagocytic disorders (e.g., chronic granulomatous disease), bone marrow dysfunction disorders (e.g., myelodysplastic syndrome, Fanconi anemia, dyskeratosis congenita), anemias (e.g., paroxysmal nocturnal hemoglobinuria, aplastic anemia, acquired pure red cell aplasia), and myeloproliferative disorders (e.g., polycythemia vera, essential thrombocytosis, myelofibrosis); (3) metabolic disorders, including, but not limited to, amyloidosis (e.g., amyloid light chain (AL) amyloidosis); (4) environmentally induced diseases, such as radiation poisoning; (5) viral diseases (e.g., HTLV-1, HIV); and (6) autoimmune diseases (e.g., multiple sclerosis).

[0047] In one embodiment, congenital conditions treatable with HSCT include: (1) lysosomal storage disorders, such as lipidosis (a disorder of lipid storage, e.g., neuronal ceroid lipofuscinosis (e.g., infantile neuronal ceroid lipofuscinosis (INCL, Santavuori disease) and Jansky-Bielschowsky disease (late infantile neuronal ceroid lipofuscinosis)); sphingolipidoses (e.g., Niemann-Pick disease and Gaucher disease); leukodystrophies (e.g., adrenoleukodystrophy, metachromatic leukodystrophy, Krabbe disease (globoid cell leukodystrophy)); mucopolysaccharidoses (e.g., Hurler syndrome (MPS IH, α-L-iduronidase deficiency), Scheie syndrome (MPS IS), Hurler-Scheie syndrome (MPS I HS), Hunter syndrome (MPS (2) immunodeficiencies, including T-cell deficiencies (e.g., ataxia-telangiectasia and DiGeorge syndrome), combined T-cell and B-cell deficiencies (e.g., severe combined immunodeficiency (SCID)), and well-defined syndromes. syndromes) (e.g., Wiskott-Aldrich syndrome), phagocytic disorders (e.g., Kostmann syndrome, Shwachman-Diamond syndrome), immune dysregulation disorders (e.g., Griscelli syndrome, type II), innate immune deficiencies (e.g., NF-κB essential modulator (NEMO) deficiency (inhibitor of kappa light polypeptide gene enhancer in B-cell gamma kinase deficiency));(3) Blood disorders include, but are not limited to, hemoglobinopathies (e.g., sickle cell disease, thalassemia (e.g., β-thalassemia)), anemias (e.g., aplastic anemias such as Diamond-Blackfan anemia and Fanconi anemia), cytopenias (e.g., amegakaryocytic thrombocytopenia), and hemophagocytic syndromes (e.g., hemophagocytic lymphohistiocytosis (HLH)).

[0048] In certain embodiments, the disease or condition is selected from the group consisting of hemoglobinopathies, viral infections, X-linked severe combined immunodeficiency, Fanconi anemia, hemophilia, neoplasms, cancer, amyotrophic lateral sclerosis, α-antitrypsin deficiency, 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 nerve cell diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, and lysosomal storage disorders. In certain embodiments, the hemoglobinopathies are selected from the group consisting of sickle cell disease, α-thalassemia, β-thalassemia, and δ-thalassemia.

[0049] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that these embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby. [Example]

[0050] Example 1: Single-cell RNA sequencing of sickle cell-reticulocytes to identify beta-globin genotypes and associated gene expression differences To demonstrate that the custom bioinformatics pipeline described herein can be used to unambiguously determine the HBB genotype of individual cells, a proof-of-concept single-cell experiment was performed using a mixture containing only wild-type (AA) and sickle cell (SS) reticulocytes.

[0051] Methods: Reticulocytes were isolated from peripheral blood of healthy donors (AA), donors with sickle cell trait (AS), and donors with sickle cell disease (SS) using Live / CD235a antibodies. + / CD45 - / TO + Based on phenotype and CD71 surface expression, cells were isolated using a combination of density-based enrichment and fluorescence-activated cell sorting (FACS) (Figure 3). Portions of each isolated population were premixed at a predetermined ratio based on cell number, requantified, and diluted to the target loading volume before applying the 10x genomics workflow for scRNAseq.

[0052] The 10x Genomics 5' Single-Cell RNA Sequencing Kit was used to ensure sufficient sequence coverage across the 5' end of the HBB transcript. The 10x scRNAseq workflow tags individual mRNA molecules with molecular barcodes (UMIs), which enable cellular barcoding and transcript quantification. Sequencing is performed using long reads rather than the protocol outlined in the standard 10x Genomics scRNAseq protocol to fully sequence the 5' end of the HBB transcript (Figure 4). A custom bioinformatics analysis pipeline (Figure 5) was developed to identify HBB variants within the scRNAseq data and identify individual reticulocytes expressing normal HBB, sickle HBB, or both.

[0053] The results obtained with these scRNAseq methods were compared with those obtained by RNA bulk sequencing of individual sorted reticulocyte samples and a mixture of blood and reticulocytes. RNA was also extracted from individual AA, AS, and SS reticulocyte samples, as well as a mixture of blood volume and sorted reticulocytes. HBB transcripts were sequenced using a targeted cDNA sequencing assay. Briefly, RNA was converted to cDNA using a standard reverse transcription reaction. The region of HBB containing the editing site was amplified by PCR, indexed, and sequenced.

[0054] result

[0055] Bulk cDNA sequencing reproducibly estimated HBB allele content, but RNA content differed between AA and SS donors. HBB allele frequency was reproducibly determined using cDNA sequencing of reticulocyte pools and whole blood samples (Figure 6). Bulk cDNA sequencing of reticulocyte and blood mixtures may overestimate the number of SS cells. cDNA sequencing of pure AA and SS reticulocytes yielded very clean and predictable allele frequency results. AS sequencing results were slightly biased toward the A allele. Reticulocyte mixtures combined equal numbers of AA, AS, and SS reticulocytes. Flow data showed that SS reticulocytes stained brighter with thiazole orange and had a higher RNA content. Higher HBB transcript levels in less mature SS reticulocytes resulted in a higher S allele frequency in reticulocyte mixtures. The effect of SS reticulocytes was even more pronounced in whole blood mixtures. The reticulocyte frequency in SS blood was 10 times higher than in AA or AS blood, so that an equal mixture of blood volumes would result in a very high S allele frequency.

[0056] Estimating AA / AS / SS allele zygosity through single-cell RNA sequencing. To measure whether the HBB genotype of individual cells can be accurately determined from scRNAseq data, we performed a single-cell experiment using a mixture containing only wild-type (AA) and sickle cell disease (SS) reticulocytes as a proof-of-concept and demonstrated that the HBB genotype of individual cells can be unambiguously determined using a custom bioinformatics pipeline. A histogram of sickle allele frequency shows that most cells have an S allele frequency of 0% or 100%. Very few reticulocytes have both the A and S alleles, indicating mixed cells after single-cell sequencing (Figure 7).

[0057] This approach was then applied to a mixture of AA, AS, and SS reticulocytes. The 10x Genomics approach was performed using different loading concentrations to examine its impact on cell yield and genotyping. The percentage of cells recovered after single-cell sequencing was lower than expected based on other experiments with reticulocytes. Reticulocytes express fewer genes than most common cells evaluated by single-cell sequencing, but based on 10x Genomics QC metrics such as the percentage of reads assigned to the cell, this appears to have little impact on cell identification. Overall, even with the lowest input, we identified and genotyped well over 1,000 single cells. Despite the low recovery rate, the results of these experiments demonstrate that all three HBB genotypes can be reproducibly determined using the scRNAseq5' protocol (Figure 8), sufficient to assess the potential impact of zygosity editing and editing on the genetic profile of cells.

[0058] scRNA-seq can distinguish SS reticulocytes from AA and AS reticulocytes. Genotyping overlays on single-cell clustering results (t-SNE plots) reveal that SS reticulocytes form a distinct population from AA and AS reticulocytes. Furthermore, differential gene expression analysis of the three genotypes highlights the similarities between AA and AS reticulocytes and their striking differences from the SS reticulocyte population (Figure 9). Compared to AA and AS reticulocytes, SS reticulocytes exhibited higher HBB and HBG2 (fetal hemoglobin) levels, resulting in higher overall transcript (UMI) counts (Figure 10). Interestingly, both the total transcript count and HBB transcript counts were consistent with the results of thiazole orange staining, suggesting that the total UMI count can be used to classify reticulocyte age / maturity. Although the total expression level in SS reticulocytes was high, the ratio of HBB expression to HBA1 / HBA2 expression was lower than in AA and AS reticulocytes (FIG. 11).

[0059] Conclusion: These data demonstrate the utility of scRNAseq for assessing differential HBB editing outcomes in erythroid progenitor cells from patients treated with gene-edited autologous hematopoietic stem cell-based therapy. The workflow and bioinformatic pipeline described herein enable genotyping and classification of editing outcomes from single-cell RNA sequencing data. The results of this example demonstrate that this approach can determine the HBB genotype of individual reticulocytes and is suitable for determining HBB editing outcomes in erythroid progenitor cells. Furthermore, this analysis allows correlation of genotype with gene expression profile, as demonstrated by the distinct expression profiles of HbAA and HbAS cells compared with HbSS cells. In principle, the approach described herein can be applied to any region of an expression-edited gene of interest via single-cell targeted RNA sequencing.

[0060] All publications, patents, and applications cited herein are incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. While the claimed subject matter has been described in terms of various embodiments, those skilled in the art will understand that various modifications, substitutions, omissions, and changes can be made therein without departing from the spirit thereof. Accordingly, it is intended that the scope of the subject matter be limited only by the following claims, including equivalents thereof.

Claims

1. 1. A method for assessing the outcome of gene editing in a population of hematopoietic stem cells, comprising: a. isolating a population of reticulocytes from a subject receiving a population of gene-edited hematopoietic stem cells; and b. determining the genotype of said population of reticulocytes by quantifying RNA transcripts of single cells of said population of reticulocytes; A method comprising:

2. 10. The method of claim 1, wherein said quantifying RNA transcripts comprises single-cell RNA sequencing.

3. 3. The method of claim 1 or 2, wherein the gene-edited hematopoietic stem cells are autologous hematopoietic stem cells.

4. The gene-edited hematopoietic stem cells are CD34 ＋ The method of any one of claims 1 to 3, comprising hematopoietic stem progenitor cells (HSPCs).

5. CD34 ＋ HSPCs express CD34 ＋ / CD38 － / CD90 ＋ 5. The method of claim 4, comprising HSPCs.

6. CD34 ＋ HSPCs express CD34 ＋ / CD38 － / CD90 ＋ / CD45RA － 5. The method of claim 4, comprising HSPCs.

7. gene editing the population of hematopoietic stem cells, contacting said population of hematopoietic stem cells with a CRISPR-associated Cas nuclease and a guide polynucleotide sequence that hybridizes to a target sequence within the genome of said hematopoietic stem cells; The method according to any one of claims 1 to 6.

8. 8. The method of claim 7, further comprising contacting said population of hematopoietic stem cells with an AAV vector comprising a donor polynucleotide sequence.

9. 9. The method of any one of claims 1 to 8, wherein the gene editing is aimed at correcting a genetic mutation, replacing a mutant allele with a wild-type allele, or inserting a nucleic acid sequence encoding a therapeutic protein.

10. The method of any one of claims 1 to 9, wherein the subject is suffering from a disease.

11. 11. The method of claim 10, wherein the disease is a hemoglobinopathy.

12. 12. The method of claim 11, wherein the hemoglobinopathy is selected from the group consisting of sickle cell disease, alpha thalassemia, beta thalassemia, and delta thalassemia.

13. The method of any one of claims 1 to 12, wherein the subject is a human.

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