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Optimized crispr/cas9 systems and methods for gene editing in stem cells — Editas Medicine, Inc. (US20230126434A1)

Editas Medicine, Inc. · Google Patents

Google Patents · Patents · License: Open Access

patent, google patents, intellectual property, US20230126434A1, Editas Medicine, Inc., Jennifer Leah Gori, en, 2023

ABSTRACT

Abstract

The methods and compositions described herein surprisingly increase CRISPR/Cas-mediated gene editing in stem cells by transiently treating the cells with a stem cell viability enhancer prior to and/or after contacting the cells with one or more CRISPR/Cas9 components. Further, this treatment also surprisingly results in increased engraftment of the stem cells into the target tissue of a subject. The present disclosure also provides one or more modified CRISPR/Cas9 components which, when used in combination with the stem cell viability enhancer, further increases the frequency of gene editing in stem cells, increases stem cell viability, and increases stem cell engraftment.

Description

RELATED APPLICATIONS

This application is a continuation application of U.S. patent application Ser. No. 15/572,896 filed on Nov. 9, 2017; which is a 35 U.S.C. Â§ 371 national stage filing of International Application No. PCT/US2016/031366, filed on May 6, 2016, which in turn claims priority to U.S. Provisional Patent Application No. 62/159,785, filed on May 11, 2015; U.S. Provisional Patent Application No. 62/220,648, filed on Sep. 18, 2015; U.S. Provisional Patent Application No. 62/244,577, filed on Oct. 21, 2015; and U.S. Provisional Patent Application No. 62/279,020, filed on Jan. 15, 2016. The entire contents of each of the foregoing applications are expressly incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 6, 2016, is named 2016-05-06_126454-00520_ST25.txt and is 1,122,453 bytes in size.

BACKGROUND

Gene therapy is a set of strategies used to modify the expression of an individual's genes or to correct abnormal genes. Cell therapy is the administration of live cells or maturation of a specific cell population in a patient for the treatment of a disease. Gene therapy and cell therapy are overlapping fields, with the goals of targeting the cause of diseases in the nucleic acid or cellular population. For example, hematopoietic diseases can be treated by transplantation of ex vivo gene-modified stem cells (e.g., hematopoietic stem/progenitor cells and hematopoietic stem cells, also referred to herein as HSCs) into a subject.

The discovery and application of the CRISPR/Cas9 system in mammalian cells results in effective and precise editing of target genes, e.g., through the non-homologous end joining pathway (NHEJ), homology directed repair (HDR), or other DNA repair pathways. Co-delivery of a Cas9 molecule and a target-specific guide RNA (gRNA) molecule, optionally along with a donor DNA repair template molecule, facilitates gene-editing of a target sequence (e.g., a disease-related mutation) in the genome. Thus, the use of the CRISPR/Cas9 system to modify genes in stem cells is a promising strategy for treating multiple genetic disorders. However, stem cells are extremely sensitive to manipulation in vitro and ex vivo and, thus, manipulation of stem cells using CRISPR/Cas9 systems, to date, has been inefficient and has resulted in little, if any, long-term viability and engraftment of the stem cells in vivo.

In order to facilitate the use of stem cells (e.g., HSCs), methods of expanding stem cells ex vivo have been developed. For example, some small molecules have been used to expand stem cells ex vivo, e.g., to increase proliferation, i.e., increase the number of stem cells by several fold in the culture, over a prolonged exposure period (typically an exposure period of more than one week). In those instances, a minimum exposure period of at least seven days is required in order to promote expansion of the stem cells, e.g., to lead to a significant increase in the number of stem cells in the culture ex vivo. However, this long period of ex vivo culturing required to expand the stem cell populations is not optimal for clinical applications.

Moreover, clinical efficacy of cell transplantation using the expanded stem cells is often contingent upon achieving a threshold level of engraftment which, to date, has been extremely difficult to achieve after manipulation of stem cells using CRISPR/Cas9 systems. See, for example, Walasek et al., Ann. N.Y. Acad. Sci . (2012), 1266:138-150. Indeed, there have been no published reports to date showing greater than 1% long term engraftment of HSCs manipulated with any CRISPR/Cas9 system, even after expansion. (Mandal et al. (2014) Cell Stem Cell 15(5):643-52). Thus, there remains a need for additional methods and compositions that can be used to optimize gene editing or regulation in stem cells (e.g., HSCs) to preserve the viability, multipotency, and self-renewal capability of stem cells.

SUMMARY

The methods and compositions described herein increase CRISPR/Cas-mediated gene editing in stem cells, e.g., HSCs. Exposing stem cells to foreign molecules, such as a CRISPR/Cas9 system component (e.g., a gRNA molecule, Cas9 molecule, or a template nucleic acid) stresses the stem cell and likely induces an innate immune response that triggers, for example, programmed cell death or stem cell differentiation. The use of the methods and systems described herein reduces or abrogates innate immune response signaling events that ultimately may lead to programmed cell death in stem cells after exposure to foreign CRISPR/Cas9 components. Specifically, by transiently (e.g., for a period of less than 120 hours) exposing stem cells to a stem cell viability enhancer prior to and/or after contacting stem cells with one or more CRISPR/Cas9 components, an innate immune response in the stem cell to the foreign CRISPR/Cas9 components is decreased, or prevented, thereby dramatically increasing the viability of the stem cell.

Additionally, the transient treatment of stem cells with stem cell viability enhancers (e.g., small molecules) that improve viability, prevent intracellular innate immune response, or both, before and/or after delivery of a CRISPR/Cas9 component also results in increased multipotency and self-renewal capability of the stem cell. Furthermore, the transient treatment of stem cells with stem cell viability enhancers (e.g., small molecules) that improve viability, prevent intracellular innate immune response, or both, before and/or after delivery of a CRISPR/Cas9 component also results in increased engraftment of the stem cell in a target tissue upon introduction of the modified stem cell into a subject. The unforeseen benefits of transiently exposing the stem cells to the reagents disclosed herein is surprising, given that they were typically used as stem cell expansion agents, e.g., to increase proliferation and the number of stem cells by several fold, and in view of the fact that the benefits for expansion of the stem cells required contact with the agents for a prolonged period of time (e.g., exposure for more than a week in culture). Thus, the methods and compositions described herein optimize the editing of a target nucleic acid sequence in a viable stem cell, and are particularly advantageous to advance the field of stem cell therapy in a multitude of clinical applications.

In one aspect, disclosed herein is a method of making a modified cell, e.g., stem cell, for transplantation, comprising (a) contacting a cell, e.g., stem cell, with a stem cell viability enhancer for a period of fewer than 120 hours, followed by (b) contacting the cell with a gRNA molecule and a Cas9 molecule in the absence of the stem cell viability enhancer.

In another aspect, disclosed herein is a method of modifying a target nucleic acid in a cell, e.g., stem cell, the method comprising contacting the cell with a stem cell viability enhancer; a modified gRNA molecule; and a Cas9 molecule. In one embodiment, the contacting step comprises (a) contacting the cell with the stem cell viability enhancer for a period of fewer than 120 hours, followed by (b) contacting the cell with the gRNA molecule and the Cas9 molecule in the absence of the stem cell viability enhancer.

In another aspect, disclosed herein is a method of transplanting a modified cell, e.g., stem cell, into a subject, the method comprising contacting a cell, e.g., stem cell, with a stem cell viability enhancer; a gRNA molecule; and a Cas9 molecule; thereby making a modified cell, e.g., modified stem cell, and transferring the modified cell, e.g., modified stem cell, to the subject. In one embodiment, the contacting step comprises (a) contacting the cell, e.g., stem cell, with the stem cell viability enhancer for a period of fewer than 120 hours, followed by (b) contacting the cell, e.g., stem cell, with the gRNA molecule and the Cas9 molecule in the absence of the stem cell viability enhancer.

In one embodiment, the step of contacting the cell, e.g., stem cell, with the gRNA molecule and the Cas9 molecule is performed using electroporation.

In one embodiment, the method further comprises cold-shocking the cell, e.g., stem cell, before electroporation. In one embodiment, the method further comprises cold-shocking the cell, e.g., stem cell, after electroporation. In one embodiment, the cell, e.g., stem cell, is cold-shocked at a temperature of about 30Â° C. to about 32Â° C.

In another embodiment, the period of fewer than 120 hours is about 96 hours. In one embodiment, the period of fewer than 120 hours is about 72 hours. In another embodiment, the period of fewer than 120 hours is about 48 hours. In another embodiment, the period of fewer than 120 hours is about 36 hours. In another embodiment, the period of fewer than 120 hours is about 24 hours. In another embodiment, the period of fewer than 120 hours is about 12 hours. In another embodiment, the period of fewer than 120 hours is about 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 hours. In another embodiment, the period of fewer than 120 hours is about 24 to about 48 hours. In another embodiment, the period of fewer than 120 hours is about 1 to about 120 hours. In another embodiment, the period of fewer than 120 hours is about 48 to about 120 hours. In another embodiment, the period of fewer than 120 hours is about 24 to about 96 hours. In another embodiment, the period of fewer than 120 hours is about 24 to about 72 hours. In another embodiment, the period of fewer than 120 hours is about 36 to about 48 hours. In another embodiment, the period of fewer than 120 hours is about 24 to about 36 hours. In another embodiment, the period of fewer than 120 hours is about 12 to about 24 hours. In another embodiment, the period of fewer than 120 hours is about 12 to about 36 hours. In one embodiment, the period of fewer than 120 hours is a period not long enough to promote expansion of the stem cell.

In one embodiment, the method further comprises (c) contacting the cell, e.g., stem cell, with the stem cell viability enhancer for a period of fewer than 96 hours after step (b). In one embodiment, the method further comprises (c) contacting the cell, e.g., stem cell, with the stem cell viability enhancer for a period of fewer than 84 hours after step (b).

In one embodiment, the method further comprises (c) contacting the cell, e.g., stem cell, with the stem cell viability enhancer for a period of fewer than 72 hours after step (b). In one embodiment, the period of fewer than 72 hours after step (b) is a period not long enough to result in expansion of the cell.

In one embodiment, the cell is a population of cells, and the number of cells in the population of cells does not increase more than 10-fold during the period of fewer than 72 hours after step (b). In one embodiment, the cell is a population of cells, and the number of cells in the population of cells does not increase more than 5-fold during the period of fewer than 72 hours after step (b). In another embodiment, the cell is a population of cells, and the number of cells in the population of cells does not increase more than 4-fold during the period of fewer than 72 hours after step (b). In another embodiment, the cell is a population of cells, and the number of cells in the population of cells does not increase more than 3-fold during the period of fewer than 72 hours after step (b). In another embodiment, the cell is a population of cells, and the number of cells in the population of cells does not increase more than 2-fold during the period of fewer than 72 hours after step (b).

In one embodiment, the period of fewer than 72 hours after step (b) is a period of about 24 hours to about 48 hours after step (b). In one embodiment, the period of fewer than 72 hours after step (b) is a period of about 12 hours, 24 hours, 36 hours, 48 hours, or 60 hours after step (b).

In one embodiment, the cell, e.g., stem cell, is transferred into a human subject within 96 hours of the end of the contacting step or steps. In one embodiment, the stem cell is transferred into a human subject within 72 hours of the end of the contacting step or steps. In one embodiment, the stem cell is transferred into a human subject within 60 hours of the end of the contacting step or steps. In one embodiment, the stem cell is transferred into a human subject within 48 hours of the end of the contacting step or steps. In one embodiment, the stem cell is transferred into a human subject within 36 hours of the end of the contacting step or steps. In one embodiment, the stem cell is transferred into a human subject within 24 hours of the end of the contacting step or steps.

In one embodiment, the cell, e.g., stem cell, is cryopreserved within 96 hours of the end of the contacting step or steps. In one embodiment, the stem cell is cryopreserved within 72 hours of the end of the contacting step or steps. In one embodiment, the stem cell is cryopreserved within 60 hours of the end of the contacting step or steps. In one embodiment, the stem cell is cryopreserved within 48 hours of the end of the contacting step or steps. In one embodiment, the stem cell is cryopreserved within 36 hours of the end of the contacting step or steps. In one embodiment, the stem cell is cryopreserved within 24 hours of the end of the contacting step or steps.

In one embodiment, the stem cell viability enhancer inhibits differentiation of the stem cell. In another embodiment, the stem cell viability enhancer inhibits programmed cell death of the stem cell. In another embodiment, the stem cell viability enhancer inhibits senescence of the stem cell. In another embodiment, the stem cell viability enhancer inhibits an innate immune response of the stem cell. In one embodiment, the stem cell viability enhancer inhibits programmed cell death by inhibiting autophagy or apoptosis of the stem cell.

In one embodiment, the stem cell engrafts into a target tissue of the subject.

In one embodiment, the stem cell comprises a population of stem cells, and wherein at least 2% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cell comprises a population of stem cells, and wherein at least 3% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cell comprises a population of stem cells, and wherein at least 4% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cell comprises a population of stem cells, and wherein at least 5% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cell comprises a population of stem cells, and wherein at least 6%, 7%, 8%, 9%, or 10% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cell comprises a population of stem cells, and wherein at least 15% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cell comprises a population of stem cells, and wherein at least 20% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cell comprises a population of stem cells, and wherein at least 25% of the stem cells engraft into the target tissue of the subject. In one embodiment, the stem cell comprises a population of stem cells, and wherein at least 30%, 35%, 40%, 45%, or 50% of the stem cells engraft into the target tissue of the subject.

In one embodiment, the cell comprises a population of cells, and wherein at least 1% of the cells are capable of engrafting into bone marrow of the subject. In another embodiment, at least 5% of the cells are capable of engrafting into bone marrow of the subject. In another embodiment, at least 10% of the cells are capable of engrafting into bone marrow of the subject. In another embodiment,

RELATED APPLICATIONS

SEQUENCE LISTING

BACKGROUND

SUMMARY

In one embodiment, the step of contacting the cell, e.g., stem cell, with the gRNA molecule and the Cas9 molecule is performed using electroporation.

In one embodiment, the stem cell engrafts into a target tissue of the subject.

In one embodiment, the cell comprises a population of cells, and at least 1% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 2.5% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 5% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 10% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 20% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 25% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 30% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 35% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 40% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 45% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 50% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 55% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 60% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 65% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 70% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 75% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 80% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 85% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 80% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 85% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 90% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 95% of the cells reconstitute the peripheral blood of the subject. In another embodiment, at least 100% of the cells reconstitute the peripheral blood of the subject. In one embodiment, the stem cell remains engrafted in the target tissue of the subject for at least 16 weeks. In another embodiment, the stem cell remains engrafted in the target tissue of the subject for at least 20 weeks. In another embodiment, the stem cell remains engrafted in the target tissue of the subject for at least 24 weeks. In another embodiment, the stem cell remains engrafted in the target tissue of the subject for at least 6 months. In another embodiment, the stem cell remains engrafted in the target tissue of the subject for at least 9 months. In another embodiment, the stem cell remains engrafted in the target tissue of the subject for at least 1 year. In another embodiment, the cell is capable of engrafting into the target tissue of the subject for the remainder of the life of the subject.

In one embodiment, the target tissue is peripheral blood, bone marrow, or spleen. In one embodiment, the cell is a stem cell. In one embodiment, the stem cell is a hematopoietic stem/progenitor cell (HSC). As used herein, the term HSC refers to both hematopoietic stem cells and hematopoietic stem progenitor cells. In one embodiment, the stem cell is selected from the group consisting of a circulating blood cell, a mobilized blood cell, a bone marrow cell, a myeloid progenitor cell, a lymphoid progenitor cell, a multipotent progenitor cell, a lineage restricted progenitor cell, an endothelial cell, or a mesenchymal stromal cell. In another embodiment, the HSC is from a non-cord blood source, an umbilical cord source, or a cord blood source. In one embodiment, the HSC is a CD34+ cell.

In one embodiment, the method further comprises isolating the cell, e.g., stem cell, from the subject before the contacting step or steps.

In one embodiment, the method further comprises culturing the cell, e.g., stem cell, in a medium comprising one or more cytokines after step (b). In one embodiment, the medium comprises the one or more cytokines and the stem cell viability enhancer. In one embodiment, the one or more cytokines is selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), Flt-3 ligand (FL), interleukin-6 (IL-6), and interleukin-11 (IL-11).

In one embodiment, the method further comprises culturing the cell, e.g., stem cell, in a medium after step (b), wherein the medium comprises one or more of a basic fibroblast growth factor (bFGF), a vascular endothelial growth factor (VEGF), a Notch signaling modulator, a TGF-Î² signaling modulator, insulin-like growth factor-binding protein 1 (IGFBP1), insulin-like growth factor binding protein 2 (IGFBP2), insulin- like growth factor 1, insulin-like growth factor 2 (IGF2), insulin-like growth factor 3 (IGF3), an angiopoietin (ANG1), an angiopoietin-like protein (ANGPTL4), a SDF1/CXCR4 axis modulator, a Wnt signaling modulator, or combinations thereof.

In one embodiment, the cell, e.g., stem cell, is cultured in the medium for at least 1, 2, 3, 4, 5, 6, or 7 days.

In one embodiment, the stem cell viability enhancer is an aryl hydrocarbon receptor (AhR) antagonist or an innate immune response antagonist. In one embodiment, the AhR antagonist is selected from the group consisting of StemRegenin-1 (SR1), LGC0006, alpha-napthoflavone, and CH-223191. In one embodiment, the AhR antagonist is SR1. In one embodiment, the innate immune response antagonist is selected from the group consisting of cyclosporin A, dexamethasone, reservatrol, a MyD88 inhibitory peptide, an RNAi agent targeting Myd88, a B18R recombinant protein, a glucocorticoid, OxPAPC, a TLR antagonist, rapamycin, BX795, and a RLR shRNA. In one embodiment, the stem cell viability enhancer is selected from the group consisting of MG132, SB431542, UM171, UM729, and 16, 16-dimethyl prostaglandin E2 (dmPGE2).

In one embodiment, the Cas9 molecule is an enzymatically active Cas9 (eaCas9). In one embodiment, the Cas9 molecule is selected from the group consisting of wild-type Cas9, a nickase Cas9, a dead Cas9 (dCas9), a split Cas9, and an inducible Cas9. In one embodiment, the Cas9 molecule comprises N-terminal RuvC-like domain cleavage activity, but has no HNH-like domain cleavage activity. In one embodiment, the Cas9 molecule comprises an amino acid mutation at an amino acid position corresponding to amino acid position N863 of Streptococcus pyogenes Cas9. In one embodiment, the Cas9 molecule comprises HNH-like domain cleavage activity but has no N-terminal RuvC-like domain cleavage activity. In one embodiment, the Cas9 molecule comprises an amino acid mutation at an amino acid position corresponding to amino acid position D10 of Streptococcus pyogenes Cas9.

In one embodiment, the Cas9 molecule is a Cas9 polypeptide. In one embodiment, the gRNA molecule and the Cas9 polypeptide are associated in a pre-formed ribonucleotide complex. In one embodiment, the Cas9 molecule is a nucleic acid encoding a Cas9 polypeptide.

In one embodiment, the gRNA molecule comprises a 5â²-end cap structure. In one embodiment, the gRNA molecule comprises a 3â²-end poly-A tail.

In one embodiment, the method further comprises contacting the cell with a template nucleic acid. In one embodiment, the cell is contacted with the template nucleic acid during the same contacting step as the gRNA molecule and the Cas9 molecule. In one embodiment, the template nucleic acid is a single stranded oligodeoxynucleotide (ssODN). In one embodiment, the ssODN comprises a 5â² phosphorothionate modification, a 3â² phosphorothionate modification, or a combination thereof.

In one embodiment, the method further comprises contacting the stem cell with a transgene, wherein the contacting occurs under conditions that allow the transgene to integrate into the genome of the stem cell. In one embodiment, the transgene is a gene is a chemotherapy selection marker, a cell surface antigen, or a suicide gene.

In one embodiment, the transgene integrates into a safe harbor locus. In one embodiment, the safe harbor locus is the AAVS1 safe harbor locus.

In one embodiment, the transgene is a chemotherapy selection marker, a cell surface antigen, or a suicide gene. In one embodiment, the chemotherapy selection marker is a gene encoding the P140K variant of methylguanine methyltransferase (P140K). In another embodiment, the cell surface antigen is a gene encoding a truncated CD19 (tCD19) or a gene encoding a truncated CD20 (tCD20). In another embodiment, the suicide gene is a gene encoding tCD20 or an inducible Caspase-9 transgene (iCaspase-9).

In one embodiment, the transgene is a gene encoding P140K, a gene encoding tCD19, a gene encoding tCD20, or a gene encoding iCaspase-9.

In another embodiment, the cell is contacted with a plurality of transgenes. In one embodiment, the plurality of transgenes are integrated into the genome of the cell. In another embodiment, the plurality of transgenes are integrated into a safe harbor locus in the genome of the cell. In one embodiment, the safe harbor locus is the AAVS1 safe harbor locus.

In one embodiment, the plurality of transgenes comprise two, three, or all of: a gene encoding the P140K variant of methylguanine methyltransferase, a gene encoding tCD19, a gene encoding tCD20, or an iCaspase-9. In another embodiment, the plurality of transgenes comprise or consist of a gene encoding the P140K variant of methylguanine methyltransferase and a gene encoding tCD20.

In one embodiment, the method further comprises contacting the cell with an enzymatically inactive (eiCas9) molecule. In one embodiment, the eiCas9 is fused to a transcriptional repressor or a transcriptional activator.

In one embodiment, the gRNA molecule comprises a targeting domain which is complementary to a target domain in a target gene. In one embodiment, the target gene is described in Table 4.

In one aspect, disclosed herein is a cell altered by any method disclosed herein.

In another aspect, disclosed herein is a pharmaceutical composition comprising a cell disclosed herein.

In one aspect, disclosed herein is a method of treating or preventing a disease in a subject comprising administering to the subject a modified cell or a cell altered by a method disclosed herein.

In one aspect, disclosed herein is a stem cell gene editing system comprising a stem cell viability enhancer; a gRNA molecule, and a Cas9 molecule. The gRNA molecule may be a modified gRNA molecule. In one embodiment, the stem cell gene editing system further comprises a stem cell, wherein the stem cell comprises the gRNA molecule and the Cas9 molecule. In one embodiment, the stem cell gene editing system further comprises a stem cell, wherein the stem cell comprises the stem cell viability enhancer.

In one embodiment, the stem cell gene editing system is a kit comprising each of the components. In another embodiment, the stem cell gene editing system is a composition. In one embodiment, the composition is part of a kit. In one embodiment, the kit further comprises instructions for modifying a target nucleic acid in a stem cell.

In one embodiment, the stem cell viability enhancer is selected from the group consisting of an aryl hydrocarbon receptor (AhR) antagonist or an innate immune response antagonist. In one embodiment, the AhR antagonist is selected from the group consisting of StemRegenin-1 (SR1), AhRA, dimethoxyflavone, 6,2â²,4â²-trimethoxyflavone, LGC0006, alpha-napthoflavone, and CH-223191. In one embodiment, the AhR antagonist is SR1. In one embodiment, the innate immune response antagonist is selected from the group consisting of cyclosporin A, dexamethasone, resveratrol, a MyD88 inhibitory peptide, an RNAi agent targeting Myd88, a B18R recombinant protein, a glucocorticoid, OxPAPC, a TLR antagonist, rapamycin, BX795, and a RLR inhibitor. In one embodiment, the stem cell viability enhancer is selected from the group consisting of MG132, SB431542, UM171, UM729, and 16, 16-dimethyl prostaglandin E2 (dmPGE2).

In one embodiment, the modified gRNA molecule comprises a 5â²-end cap structure. In one embodiment, the modified gRNA molecule comprises a 3â²-end poly-adenine tail. In one embodiment, the modified gRNA molecule comprises a 5â² end cap structure and a 3â²-end poly-adenine tail.

In one embodiment, the Cas9 molecule is selected from the group consisting of wild-type Cas9, a nickase Cas9, a dead Cas9 (dCas9), a split Cas9, and an inducible Cas9. In one embodiment, the Cas9 molecule is an enzymatically active Cas9 (eaCas9).

In one embodiment, the Cas9 molecule comprises N-terminal RuvC-like domain cleavage activity, but has no HNH-like domain cleavage activity. In one embodiment, the Cas9 molecule comprises an amino acid mutation at an amino acid position corresponding to amino acid position N863 of Streptococcus pyogenes Cas9. In one embodiment, the Cas9 molecule comprises HNH-like domain cleavage activity but has no N-terminal RuvC-like domain cleavage activity. In one embodiment, the Cas9 molecule comprises an amino acid mutation at an amino acid position corresponding to amino acid position D10 of Streptococcus pyogenes Cas9.

In one embodiment, the Cas9 molecule is a Cas9 polypeptide. In one embodiment, the modified gRNA molecule and the Cas9 polypeptide are associated in a pre-formed ribonucleotide complex. In one embodiment, the Cas9 molecule is a nucleic acid encoding a Cas9 polypeptide.

In one embodiment, the stem cell gene editing system further comprises a cytokine. In one embodiment, the cytokine is selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), Flt-3 ligand (FL), interleukin-6 (IL-6), and interleukin-11 (IL-11).

In one embodiment, the composition further comprises one or more of: a basic fibroblast growth factor (bFGF), a vascular endothelial growth factor (VEGF), a Notch signaling modulator, a TGF-Î² signaling modulator, insulin-like growth factor-binding protein 1 (IGFBP1), insulin-like growth factor binding protein 2 (IGFBP2), insulin- like growth factor 1, insulin-like growth factor 2 (IGF2), insulin-like growth factor 3 (IGF3), an angiopoietin (ANG1), an angiopoietin-like protein (ANGPTL4), a SDF1/CXCR4 axis modulator, or a Wnt signaling modulator.

In one embodiment, the stem cell gene editing system further comprises a template nucleic acid. In one embodiment, the template nucleic acid is a single stranded oligodeoxynucleotide (ssODN). In one embodiment, the ssODN comprises a 5â² phosphorothionate modification, a 3â² phosphorothionate modification, or both a 5â² phosphorothionate modification and a 3â² phosphorothionate modification.

In one embodiment, the gRNA molecule comprises a targeting domain which is complementary to a target domain in a target gene. In one embodiment, the target gene is described in Table 4.

In one aspect, disclosed herein is a cell comprising a composition disclosed herein.

In another aspect, disclosed herein is a pharmaceutical composition comprising a composition disclosed herein, and a pharmaceutically acceptable carrier. In another aspect, disclosed herein is a pharmaceutical composition comprising a cell disclosed herein, and a pharmaceutically acceptable carrier.

In one embodiment, the HSC cell differentiates in vivo after transplantation. In one embodiment, the HSC cell differentiates into B cells, T cells, erythroid cells, and/or myeloid cells. In one embodiment, the HSC cell reconstitutes hematopoiesis in the subject.

In one embodiment, the cell is transplanted into the subject via intravenous infusion.

In one embodiment, the contacting step or steps occur ex vivo.

In one embodiment, the method further comprises generating the cell from an iPS cell or from an endothelial cell.

In one embodiment, the one or more cell viability enhancers has one or more of the following properties: enhances cell maintenance, enhances cell survival, enhances cell viability, or enhances cell proliferation. In another embodiment, the one or more cell viability enhancers has one or more of the following properties: inhibits differentiation, inhibits cell death via apoptosis, inhibits necrosis, inhibits autophagy, or inhibits senescence. In one embodiment, the one or more cell viability enhancers inhibits senescence associated with DNA damage response.

In one embodiment, the one or more cell viability enhancers inhibits an innate immune response and/or prevents apoptosis of the cell. In another embodiment, the one or more cell viability enhancers inhibits an innate immune response and/or prevents apoptosis of the cell in response to a CRISPR/Cas9 component. In another embodiment, the one or more cell viability enhancers inhibits the interferon or toll-like receptor response to foreign nucleic acids.

In one embodiment, the method further comprises contacting the cell with a chemotherapeutic agent to increase the number of cells. In one embodiment, the cell comprises a chemotherapy selection marker. In one embodiment, the chemotherapy selection marker is the P140K variant of methylguanine methyltransferase. In one embodiment, the chemotherapeutic agent is 06BG/BCNU. In one embodiment, the contacting occurs ex vivo or in vivo.

In one embodiment, the method further comprises contacting the cell with a cell surface antigen binding agent for selection of the cell. In one embodiment, the cell comprises a cell surface antigen. In another embodiment, the cell surface antigen is tCD19 or tCD20. In one embodiment, the cell surface antigen binding agent is a tCD19- or tCD20-binding reagent. In one embodiment, the contacting occurs ex vivo or in vivo.

In one embodiment, the method further comprises contacting the cell with an agent that results in cell death. In one embodiment, the cell is an HSC comprising a suicide gene.

In one embodiment, the method further comprises contacting the cell with an anti-CD20 antibody. In one embodiment, the anti-CD20 antibody is Rituximab.

In one embodiment, the method further comprises contacting the cell with an agent that dimerizes iCaspase-9 wherein the cell comprises iCaspase-9. In one embodiment, the agent that dimerizes iCaspase-9 is a cell permeable dimerizer. In another embodiment, the cell permeable dimerizer is AP20187 or AP1903.

In one embodiment, the method further comprises contacting the cell with a template nucleic acid comprising the transgene, and one or more gRNAs comprising a targeting domain which is complementary with a target domain from a region into which the transgene is integrated.

In one embodiment, the method further comprises culturing the cell under conditions that allow expression of the transgene.

In another embodiment, the eiCas9 molecule is a fusion molecule that regulates a target gene. In one embodiment, the target gene is a target gene that transiently prevents cell death, enhances cell survival, enhances cell viability, or enhances proliferation. In another embodiment, the eiCas9 molecule is fused to a KRAB domain.

In one embodiment, expression of the transgene does not significantly reduce multipotency, cellular fitness, or both. In another embodiment, expression of the transgene does not significantly reduce viability, multipotency, cellular fitness, or both, upon acute exposure to a CRISPR/Cas9 component.

In one embodiment, the cell is cultured under hypoxic culture conditions. In one embodiment, hypoxic culture conditions comprise 10% or less, 8% or less, 5% or less, 3% or less, 1% or less, or 0.5% or less O 2 .

In one embodiment, the cell is cultured in a three dimensional culture system. In one embedment, the three dimensional culture system is a NANEXâ¢ 3D culture system.

In one embodiment, the method further comprises co-culturing the cell with an endothelial cell, a mesenchymal cell, or both. In one embodiment, the endothelial cell is a VeraVecs cell. In another embodiment, the mesenchymal cell is a mesenchymal stromal cell, or a perivascular mesenchymal cell.

In one embodiment, the method further comprises purifying the cell after the contacting step. In one embodiment, the method further comprises washing the cell between the contacting step with the cell viability enhancer and the contacting step with the gRNA molecule and the Cas9 molecule.

In one embodiment, the subject is the same subject from whom the cell is isolated. In another embodiment, the subject is a different subject from whom the cell is isolated.

In another aspect, disclosed herein is a method of treating or preventing a disease in a subject comprising administering to the subject a modified cell or a cell altered by the method disclosed herein. In one embodiment, the subject is suffering from a disease, or is at risk of developing, a disease listed in Table 4. In another embodiment, the disease is a hemoglobinopathy, an anemia, a disorder of hemostasis, a metabolic disorder, a severe immunodeficiency, a myeloid immunodeficiency, a B-lymphoid and immunoglobulin immunodeficiency, a cytopenia disorder, a metabolic, enzyme deficiency, trafficking, and storage disease, an erythroid disease, an autoimmune disease an inflammatory disease, an infectious disease, or an oncologic disease. In one embodiment, the cytopenia disorder has neurological complications. In another embodiment, the oncologic disease is a lymphoma or a leukemia.

In one embodiment, between about 1Ã10 5 and about 1Ã10 8 altered or modified cells per kg bodyweight are administered to the subject. In another embodiment, between about 1Ã10 6 and about 1Ã10 7 altered or modified cells per kg bodyweight are administered to the subject. In another embodiment, between about 1Ã10 6 , about 2Ã10 6 , or about 5Ã10 6 altered or modified cells per kg bodyweight are administered to the subject.

In one embodiment, the cell is for use in the manufacture of a medicament for treating or preventing disease. In one embodiment, the disease is a disease listed in Table 4.

In one embodiment, the cell is a hematopoietic stem/progenitor cell (HSC). In one embodiment, the HSC cell is capable of differentiating in vivo after transplantation into the subject. In one embodiment, the HSC cell is capable of differentiating into B cells, T cells, erythroid cells, and/or myeloid cells. In another embodiment, the HSC cell is capable of reconstituting hematopoiesis in the subject.

In another aspect, disclosed herein is a reaction mixture comprising: (a) a cell; (b) one or more CRISPR/Cas9 components; and (c) one, two, or all of the following: (i) a cell viability enhancer; (ii) a transgene; or (iii) an eiCas9 molecule.

In one embodiment, the one or more CRISPR/Cas9 components comprise a Cas9 molecule, a gRNA molecule, or both. In another embodiment, the reaction mixture further comprises a donor template nucleic acid. In another embodiment, the eiCas9 molecule is fused to a transcriptional repressor or transcriptional activator.

In another aspect, disclosed herein is a kit comprising: (a) one, two, or all of the following: (i) a cell viability enhancer; (ii) a transgene; or (iii) an eiCas9 molecule, and (b) instructions for altering a cell or making a modified cell.

In one embodiment, the eiCas9 molecule is fused to a transcriptional repressor or transcriptional activator. In another embodiment, the cell is an HSC.

In another aspect, disclosed herein is the use of a cell in the manufacture of a medicament for treating or preventing a disease. In one embodiment, the disease is a disease listed in Table 4.

In one aspect, disclosed herein is the use of a cell described herein in the manufacture of a medicament for treating or preventing a disease, e.g., a disease described herein, e.g., a disease listed in Table 4.

In another aspect, disclosed herein is a cell described herein for treating or preventing a disease, e.g., a disease described herein, e.g., a disease listed in Table 4.

The compositions, reaction mixtures and kits, as disclosed herein, can also include a governing gRNA molecule, e.g., a governing gRNA molecule disclosed herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Headings, including numeric and alphabetical headings and subheadings, are for organization and presentation and are not intended to be limiting.

Other features and advantages of embodiments of the present disclosure will be apparent from the detailed description, drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the detection of indels at the CCR5 locus after delivery of S. aureus gRNA and S. aureus Cas9.

FIG. 2 depicts the flow cytometry analysis of genome edited HSCs to determine co-expression of stem cell phenotypic markers CD34 and CD90 and for viability (7-AAD â AnnexinV â cells).

FIG. 3 A depicts the fold-change in the number (e.g., maintenance of survival) of Nucleofectedâ¢ CD34 + cells 96 hours after delivery of the indicated Cas9 variant paired with CXCR4 gRNA or GFP-expressing plasmid alone (pmax GFP). Treatment +/â40 nM UM171 is indicated by minus sign (â, no UM171) or plus sign (+, with 40 nM UM171).

FIG. 3 B depicts the percentage of indels as detected by T7E1 assays in CD34 + HSC after the indicated Nucleofectionsâ¢. Treatment +40 nM UM171 is indicated by minus sign (â, no UM171) or plus sign (+, with 40 nM UM171).

FIG. 4 A depicts the fold-change in the number (e.g., maintenance of survival and proliferation potential) of Nucleofectedâ¢ CD34 + cells 96 hours after co-delivery of Cas9 paired with CXCR4 gRNA (CXCR4-231) and CCR5 gRNA (CCR5-U43) plasmids.

FIG. 4 B depicts the percentage of indels detected by T7E1 assays in CD34 + HSCs at CCR5 and CXCR4 genomic loci.

FIG. 5 A depicts the kinetics of the fold-change in the number of CD34 + cells after electroporation with the indicated uncapped/untailed gRNAs or capped/tailed gRNAs with paired Cas9 mRNA (either S. pyogenes (Sp) or S. aureus Sa Cas9).

FIG. 5 B depicts the fold change in total live CD34 + cells 72 hours after electroporation with the indicated uncapped/untailed gRNAs or capped/tailed gRNAs with paired Cas9 mRNA (either S. pyogenes (Sp) or S. aureus Sa Cas9).

FIG. 5 C depicts representative flow cytometry data showing maintenance of viable (propidium iodide negative) human CD34+ cells after electroporation with capped and tailed AAVS1 gRNA and Cas9 mRNA.

FIG. 6 A depicts the percentage of insertions/deletions (indels) detected in CD34 + cells and their hematopoietic colony forming cell (CFC) progeny at the targeted AAVS1 locus after delivery of Cas9 mRNA with capped and tailed AAVS1 gRNA compared to uncapped and untailed AAVS1 gRNA.

FIG. 6 B depicts the maintenance of hematopoietic colony forming potential (CFCs) in CD34+ cells after editing with capped/tailed AAVS1 gRNA. Note loss of CFC potential for cells electroporated with uncapped/untailed AAVS1 gRNA. E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythrocyte-macrophage-monocyte.

FIG. 6 C depicts efficient targeted locus editing (% indels) in the K562 erythroleukemia cell line, a human erythroleukemia cell line has similar properties to HSCs, after delivery of capped and tailed HBB gRNA with S. pyogenes Cas9 mRNA or ribonucleoprotein (RNP).

FIG. 6 D depicts Cas9-mediated/capped and tailed gRNA mediated editing (% indels) at the indicated target genetic loci (AAVS1, HBB, CXCR4) in human cord blood CD34 + cells. Right: CFC potential of cord blood CD34+ cells after electroporation with Cas9 mRNA and capped and tailed HBB-8 gRNA, also called HBB_Sp8 herein, (SEQ ID NO:388) (unelectroporated control or cells electroporated with 2 or 10 Î¼g HBB gRNAs). Cells were electroporated with Cas9 mRNA and 2 or 10 Î¼g of gRNA.

FIG. 6 E depicts CFC assays for cells electroporated with 2 Î¼g or 10 Î¼g of capped/tailed HBB gRNA. CFCs: colony forming cells, E: erythroid, G: granulocyte, M: macrophage, GM: granulocyte-macrophage, GEMM: granulocyte-erythrocyte-macrophage-monocyte.

FIG. 6 F depicts a representative gel image showing cleavage at the indicated loci (T7E1 analysis) in cord blood CD34 + cells at 72 hours after delivery of capped and tailed AAVS1, HBB, or CXCR4 gRNA and S. pyogenes Cas9 mRNA. The example gel corresponds to the summary data shown in FIG. 6 D .

FIG. 6 G Cell viability in CB CD34 + cells 48 hours after delivery of Cas9 mRNA and indicated gRNAs as determined by co-staining with 7-AAD and Annexin V and flow cytometry analysis.

FIGS. 7 A and 7 B depict an analysis of stability of D10A nickase RNP in vitro and ex vivo in human adult CD34+ HSCs. FIG. 7 A depicts Differential Scanning Fluorimetry Shift Assay after complexing D10A protein with the indicated HBB gRNAs added at 1:1 molar ratio gRNA:RNP. FIG. 7 B depicts detection of Cas9 protein in cell lysates 72 hours after human adult CD34+ HSCs were electroporated with D10A nickase RNP or D10A mRNA with gRNAs HBB-

CLAIMS

Claims ( 23 )

1 . A method of generating a modified cell for transplantation, comprising:

(a) contacting a cell with a stem cell viability enhancer for a period not long enough to promote expansion of the cell, wherein the cell does not expand, followed by (b) contacting the cell with a gRNA molecule and a Cas molecule in the absence of the stem cell viability enhancer, thereby generating a modified cell for transplantation.

2 . The method of claim 1 , wherein the step of contacting the cell with the gRNA molecule and the Cas molecule is performed using electroporation.

3 . The method of claim 2 , further comprising cold-shocking the cell before electroporation and/or after electroporation.

4 . The method of claim 1 , wherein the cell is contacted with the stem cell viability enhancer for a period of about 72 hours.

5 . The method of claim 1 , wherein the cell is contacted with the stem cell viability enhancer for a period of about 24-48 hours.

6 . The method of claim 1 , wherein the cell is contacted with the stem cell viability enhancer for a period of fewer than 120 hours.

7 . The method of claim 1 , further comprising

8 . The method of claim 1 , wherein the stem cell viability enhancer inhibits differentiation, inhibits programmed cell death, inhibits senescence, or inhibits an innate immune response of the cell.

9 . The method of claim 8 , wherein the stem cell viability enhancer inhibits programmed cell death by inhibiting autophagy or apoptosis.

10 . The method of claim 1 , further comprising transferring the modified cell to a subject, wherein the cell engrafts into a target tissue of the subject.

11 . The method of any one of claim 10 , wherein the target tissue is peripheral blood, bone marrow, or spleen.

12 . The method of claim 1 , wherein the cell is a stem cell.

13 . The method of claim 1 , wherein the cell is selected from the group consisting of a circulating blood cell, a mobilized blood cell, a bone marrow cell, a myeloid progenitor cell, a lymphoid progenitor cell, a multipotent progenitor cell, a lineage restricted progenitor cell, an endothelial cell, or a mesenchymal stromal cell.

14 . The method of claim 1 , further comprising culturing the cell in a medium after step (b), wherein the medium comprises one or more of a cytokines, a basic fibroblast growth factor (bFGF), a vascular endothelial growth factor (VEGF), a Notch signaling modulator, a TGF-Î² signaling modulator, insulin-like growth factor-binding protein 1 (IGFBP1), insulin-like growth factor binding protein 2 (IGFBP2), insulin-like growth factor 1, insulin-like growth factor 2 (IGF2), insulin-like growth factor 3 (IGF3), an angiopoietin (ANG1), an angiopoietin-like protein (ANGPTL4), a SDF1/CXCR4 axis modulator, a Wnt signaling modulator, or combinations thereof.

15 . The method of claim 14 , wherein the medium comprises one or more cytokines selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), Flt-3 ligand (FL), interleukin-6 (IL-6), and interleukin-11 (IL-11).

16 . The method of claim 1 , wherein the stem cell viability enhancer is an aryl hydrocarbon receptor (AhR) antagonist or an innate immune response antagonist.

17 . The method of claim 16 , wherein the AhR antagonist is selected from the group consisting of StemRegenin-1 (SR1), LGC0006, alpha-napthoflavone, and CH-223191.

18 . The method of claim 16 , wherein the innate immune response antagonist is selected from the group consisting of cyclosporin A, dexamethasone, reservatrol, a MyD88 inhibitory peptide, an RNAi agent targeting Myd88, a B18R recombinant protein, a glucocorticoid, OxPAPC, a TLR antagonist, rapamycin, BX795, and a RLR shRNA.

19 . The method of claim 1 , wherein the stem cell viability enhancer is selected from the group consisting of MG132, SB431542, UM171, UM729, and 16, 16-dimethyl prostaglandin E2 (dmPGE2).

20 . The method of claim 1 , wherein the Cas molecule is a Cas9 polypeptide.

21 . The method of claim 20 , wherein the gRNA molecule and the Cas9 polypeptide are associated in a pre-formed ribonucleotide complex.

22 . A method of making a hematopoietic stem/progenitor cell (HSC) for transplantation, comprising:

(a) contacting a HSC with a stem cell viability enhancer for a period of fewer than 72 hours, followed by (b) electroporating the HSC with a gRNA molecule and a Cas polypeptide in the absence of the stem cell viability enhancer, wherein the gRNA molecule and the Cas polypeptide are associated in a pre-formed ribonucleotide complex.

23 . A method of modifying a target nucleic acid in a cell, the method comprising contacting the cell with a stem cell viability enhancer, a gRNA molecule, and a Cas molecule,

wherein the contacting step comprises: (a) contacting the cell with the stem cell viability enhancer for a period not long enough to promote expansion of the cell, wherein the cell does not expand, followed by (b) contacting the cell with the gRNA molecule and the Cas molecule in the absence of the stem cell viability enhancer, thereby modifying a target nucleic acid in a cell.

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