In vivo hematopoietic stem cell gene editing

Abstract

The present invention relates to methods for treating diseases by the in vivo gene editing of hematopoietic stem and progenitor cells (HSPCs), and in particular to the use of a CXCR4 antagonist to increase the efficiency of HSPC gene editing in vivo when administered prior to or in conjunction with administration of a gene editing system to a subject in need thereof.

Description

In Vivo Gene EditingFIELD

[0001] The present invention relates to methods for treating diseases by the in vivo gene editing of hematopoietic stem and progenitor cells (HSPCs), and in particular to the use of a CXCR4 antagonist to increase the efficiency of HSPC gene editing in vivo when administered prior to or in conjunction with administration of a gene editing system to a subject in need thereof. The disclosure also provides compounds and methods for increasing the efficiency of HSPC gene editing in vivo by administration of cycling agent(s) to alter the cellular state of HSPCs to a more active metabolic state or proliferative state.BACKGROUND

[0002] Hereditary blood disorders such as including hemoglobinopathies, primary immunodeficiencies (PIDs) and congenital cytopenias are caused by genetic aberrations in hematopoietic stem cells (HSCs) that affect production or function of one or more hematopoietic lineages. There is a possibility to treat these diseases by replacement of mutant HSCs with genetically normal HSCs, for example, by allogenic HSC transplantation. However, application of allogenic HSC transplantation is restricted by the need to identify HLA-compatible donors.

[0003] Due to the problems associated with allogenic therapies, a number of autologous HSC gene therapies have been developed. These therapies have generally utilized viral vectors to deliver a desired copy of a gene to HSCs harvested from a patient and then transplanting the cells back into the patient. There are several problems associated with viral delivery of genes including limited packaging capacity, inability to treat toxic gain of function mutations, and unpredictability of the gene insertion site which can causes problems such as insertional mutagenesis, oncogene transactivation and aberrant expression of the transgene or neighboring genes. Moreover, these methods, like allogenic transplantation therapies, require a myeloablative conditioning regimen such as chemotherapy or radiation to eliminate the subjects own HSCs following harvest of the HSCs used for the gene therapy. In addition to being expensive and requiring a long hospital stay, conditioning procedures can be toxic and cause side effects such as infertility and secondary malignancies due to accumulated DNA damage.

[0004] Gene editing via systems such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromicrepeats (CRISPR) / CRISPR-associated protein (Cas) have emerged as powerful systems for editing mutant genes. Recently, the FDA approved the treatment Casgevy® for the treatment of sickle cell disease in patients 12 years of age and older with recurrent vaso-occlusive crises. In this treatment, CRISPR / Cas9 is directed to cut DNA in targeted areas, enabling the ability to accurately edit (remove, add, or replace) DNA where it was cut. The modified blood stem cells are transplanted back into the patient where they engraft (attach and multiply) within the bone marrow and increase the production of fetal hemoglobin (HbF), a type of hemoglobin that facilitates oxygen delivery. However, like some of the earlier gene therapies, this treatment also requires myeloablative conditioning.

[0005] What is needed in the art are HSC gene editing therapies for diseases including hemoglobinopathies, primary immunodeficiencies (PIDs) and congenital cytopenias that do not require myeloablative conditions and most preferably which could be done on an outpatient basis or with a very short hospital stay.SUMMARY

[0006] The present disclosure relates to methods and compositions for treating diseases by the in vivo gene editing of hematopoietic stem and progenitor cells (HSPCs), including hematopoietic stem cells (HSC) and progenitor cells, involving the administration of a cycling agent to increase the efficiency of HSPC gene editing in vivo or ex vivo. For example, in one aspect, the disclosure provides for administration of one, two or more cycling agents prior to or in conjunction with a gene editing system, to increase efficiency of HSPC gene editing in vivo.

[0007] In some aspects, the present disclosure provides a method, kit or the use of a CXCR4 antagonist, such as plerixafor, that is administered in the absence of administering G- CSF or in the absence of administrating other mobilizing agents that have primarily mobilizing activity.

[0008] In any of the embodiments of the in vivo editing or treatment methods described herein, the subject being treated may be a subject that does not undergo myeloablative conditioning, and / or a subject that does not undergo HSC harvest.

[0009] In some aspects, the present disclosure provides an in vivo method of editing the genome of one or more hematopoietic stem and progenitor cells (HSPCs, including HSCs) in a subject in need thereof comprising: administering a cycling agent; and administering a gene editing system to the subject so that the genome(s) of one or more HSPCs in the subject are edited. In some embodiments, the cycling agent is administered in an amount effective toincrease the gene editing efficiency of a gene editing system. In some embodiments, the cycling agent is administered in an amount effective to increase the percentage of HSPCs, including HSCs, that are in a more active metabolic state or proliferative state. In some embodiments, the administration of the cycling agent occurs within about six hours prior to administering the gene editing system. In alternate embodiments, the administration of the cycling agent occurs within about an hour before administering the gene editing system.

[0010] In some aspects, the present disclosure provides a cycling agent for use in increasing the efficiency of in vivo gene editing of one or more hematopoietic stem and progenitor cells (HSPCs, including HSCs) in a subject in need thereof.

[0011] In some aspects, the present disclosure provides an ex vivo method of editing the genome of one or more hematopoietic stem and progenitor cells (HSPCs, including HSCs) from a subject comprising: obtaining HSPCs, including HSCs, from a subject; contacting the HSPCs, including HSCs, with a cycling agent; and introducing a gene editing system into the HSPCs, including HSCs, so that the genome(s) of the HSPCs, including HSCs, are edited. In some embodiments, the cycling agent is administered in an amount effective to increase the gene editing efficiency of a gene editing system. In some embodiments, the cycling agent is administered in an amount effective to increase the percentage of HSPCs, including HSCs, that are in a more active metabolic state or proliferative state.

[0012] In some aspects, the present disclosure provides a cycling agent for use in increasing the efficiency of ex vivo gene editing of one or more hematopoietic stem and progenitor cells (HSPCs, including HSCs) from a subject in need thereof. In some embodiments, the HSPCs, including HSCs, are infused back into the subject. In some embodiments, the patient is not subjected to a myeloablative conditioning treatment.

[0013] In any of the embodiments described herein, the subject in need of the gene editing may be a subject that has or is at risk of having an HSPC-treatable disease. In some embodiments, the HSPC-treatable disease, including HSC-treatable disease, or condition is selected from the group consisting of a hemoglobinopathy, immunodeficiency, hematological malignancies, cancer, anemias, autoimmune diseases, and cytopenias. In some embodiments, the subject has or is at risk of having a disease or condition selected from the group consisting of Sickle cell disease, Thalassemia, Pyruvate kinase deficiency, metabolic disorders such as hypophosphatasia, Hemophilia A, Hemophilia B, Factor X deficiency, Wiskott-Aldrich syndrome (WAS), Diamond-Blackfan anemia, primary immunodeficiency disorder (PIDs) including Severe Combined Immunodeficiency (SCID), chronic granulomatous disease (CGD), etc., X-linked chronic granulomatous disease, Kostmann’ssyndrome, X-linked adrenoleukodystrophy, Metachromatic leukodystrophy, CNS diseases such as Friedreich’s ataxia and Alzheimer’s diseases, lysosomal storage diseases (LSD), such as Gaucher disease, Fabry’s disease, Pompe disease, Mucopolysaccharidosis Type I (MPSOI) and MPS-II; autoimmune conditions such as lupus (SLE / LN), scleroderma, myositis, myasthenia gravis, multiple sclerosis, Crohn’s disease, and stiff-persons-syndrome; Hunter syndrome, Mucopolysaccharidosis type 1, Osteopetrosis, X-linked agammaglobulinemia, X- linked hyper IgM syndrome, Immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, Early-onset inflammatory disease, Hemophagocytic lymphohistiocytosis, VEXAS (vacuoles, El enzyme, X-linked, autoinflammatory, somatic) syndrome, and erythropoietic protoporphyria. In some embodiments, the SCID is selected from the group consisting of Adenosine deaminase-deficient SCID, Artemis SCID, X-linked SCID, and Recombination activating gene SCID.

[0014] In any of the embodiments described herein, the gene editing system may be selected from the group consisting of a zinc finger nuclease (ZFN) gene editing system, a transcription activator-like effector nuclease (TALEN) gene editing system, a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease gene editing system, a base or prime editing system, a reverse transcriptase (RT) editing system, a non-LTR retrotransposon editing system, and a retron gene editing system. In some embodiments, the gene editing system is a CRISPR / Cas nuclease gene editing system. In some embodiments, the gene editing system is a reverse transcriptase (RT) editing system. In some embodiments, the gene editing system is a base editing system. In some embodiments, the gene editing system is a non-LTR retrotransposon system. In some embodiment, the gene editing system is a retron gene editing system. In some embodiments, the gene editing system comprises a Cas nuclease selected from the group consisting of Casl , CasI B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Casl 00, Csyl , Csy2, Csy3, Csel , Cse2, Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl , Cmr3, Cmr4, Cmr5, Cmr6, Csbl , Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl , Csxl5, Csfl , Csf2, Csf3, Csf4, Cpfl, or SluCas9 endonuclease; a homolog thereof, a recombination of the naturally occurring molecule thereof, codon-optimized thereof, or modified versions thereof, and combinations thereof. In some embodiments, the Cas nuclease is encoded by a nucleic acid molecule.

[0015] In some embodiments, the gene editing system comprises a nucleic acid molecule encoding the Cas nuclease. In some aspects, an mRNA or DNA encodes the Cas nuclease and one or two or more guide RNAs or nucleic acid(s) encoding the guide RNA. In someembodiments, the gene editing system comprises an mRNA encoding the Cas nuclease and one or two or more guide RNAs. In some embodiments, the gene editing system comprises an mRNA encoding the Cas nuclease and a DNA encoding one or two or more guide RNAs. In some aspects, the DNA encoding one or two or more guide RNAs is in an expression vector comprising one or more regulatory sequences. In some embodiments, the gene editing system comprises a DNA encoding the Cas nuclease. In some aspects, the DNA encoding the Cas nuclease is in an expression vector comprising one or more regulatory sequences, and a DNA encoding one or two or more guide RNAs. In some aspects, the DNA encoding one or two or more guide RNAs is in an expression vector comprising one or more regulatory sequences. In some embodiments, the gene editing system comprises a Cas nuclease polypeptide and one or two or more guide RNAs. In some aspects, the Cas nuclease polypeptide and one or two or more guide RNAs are pre-complexed.

[0016] In some embodiments, the gene editing system effects one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within a target gene in the genomes of one or more HSPCs in the subject. In some embodiments, the gene editing system introduces an indel mutation into a target gene in the genomes of the one or more HSPCs, including HSCs, of the subject.

[0017] In some embodiments, the gene editing system further comprises a donor template. In some embodiments, the donor template is a DNA molecule.

[0018] In some embodiments, the gene editing system is administered via a lipid nanoparticle (LNP). In some embodiments, the components of the gene editing system are delivered in the same LNP or in separate LNPs. \

[0019] In one aspect of the invention, the disclosure provides a method or use of a cycling agent for increasing gene editing efficiency of HSPCs, included HSCs, comprising a repeated administration of a cycling agent and a gene editing system, wherein the repeated administration is repeated one, two, three, four or more times, or up to five times. In some embodiments, a combination of two more cycling agents is administered.

[0020] In another aspect of the invention, the disclosure provides an in vivo method of editing the genome of one or more hematopoietic stem and progenitor cells (HSPCs, including HSCs) in a subject in need thereof comprising: (a) a first step of administering a cycling agent prior to or in conjunction with administering a gene editing system to the subject so that the genome(s) of HSPCs, including HSCs, in the subject are edited, and (b) a second step of administering a cycling agent prior to or in conjunction with administering a gene editing system to the subject so that the genome(s) of HSPCs, including HSCs, in thesubject are edited. In some embodiments, the cycling agent is administered in an amount effective to increase the gene editing efficiency of a gene editing system. In some embodiments, the cycling agent is administered in an amount effective to increase the percentage of HSPCs, including HSCs that are in a more active metabolic state of proliferative state.

[0021] In some embodiments, the second step is performed about 3 months after the first step. In some embodiments, the second step is performed about 3 to 6 months after the first step. In some embodiments, the second step is performed about 3 to 9 months after the first step, or about 3 months to 1 year after the first step, or about 6 months to about 1 year after the first step.

[0022] In a related aspect of the invention, the disclosure provides, an in vivo method of editing the genome of HSPCs, including HSCs, comprising administering to a subject in need thereof a second dose of a cycling agent and a second dose of a gene editing system, wherein the subject has previously received a first dose of a cycling agent and a first dose of a gene editing system, and wherein the first dose and second dose of cycling agent are of the same compound or different compounds.

[0023] In any embodiments of the methods described herein, the percentage of HSPCs, including HSCs, in the bone marrow that maintain editing five months after the first administration of the gene editing system is increased relative to the percentage without administration of the cycling agent. In some embodiments, the percentage of HSPCs, including HSCs, in the bone marrow that maintain editing five months after the first administration of the gene editing system is at least 10%. In some embodiments, the percentage of HSPCs, including HSCs, in the bone marrow that maintain editing five months after the first administration of the gene editing system is at least 15%, at least 20%, at least 25%, at least between 10-50%, at least between 15-40%, at least between 20-30%.

[0024] In some embodiments, one or more of the cell lineages of the HSPCs, including HSCs, is selected from myeloid cells, erythroid cells, B cells and T cells and / or combinations thereof and exhibit editing at least five months after the first administration of the gene editing system.

[0025] In some embodiments, the cell lineage is myeloid cells and the percent of editing is greater than 10%. In some embodiments, the cell lineage is erythroid cells and the percent of editing is greater than 10%. In some embodiments, the cell lineage is B cells and the percent of editing is greater than 10%. In some embodiments, the cell lineage is T cells and the percent of editing is greater than 7%.

[0026] In another aspect of the invention, the disclosure provides, an in vivo method of editing the genome of one or more hematopoietic stem and progenitor cells (HSPCs, including HSCs) in a subject in need thereof comprising: (a) administering two or more cycling agents, in amounts effective to increase the gene editing efficiency of a gene editing system, and (b) administering a gene editing system to the subject so that the genome(s) of HSPCs, including HSCs, in the subject are edited. In some embodiments, the administration of step (a) is before the administration of step (b). In some embodiments, the administration of step (a) and (b) are done at the same time, or within 30 to 60 minutes of each other. In some embodiments, the two or more cycling agents are administered as a single composition.

[0027] In some embodiments, the cycling agent is a CXCR4 antagonist, antimetabolite, alkylating agent, or cytotoxic agent or chemotherapeutic agent, optionally plerixafor, 5- fluorouracil, and cyclophosphamide. In some embodiments, the cycling agent is radiation.

[0028] In some embodiments, the gene editing system is administered in an LNP formulation. In some embodiments, the editing efficiency of the gene editing system is higher after the administration of the two or more of cycling agents than that of the editing efficiency of the gene editing system after the administration of one of the cycling agents alone. In some embodiments, the effect of the two or more of the cycling agents is synergistic. In some embodiments, the effect of the two or more of the cycling agents is not additive.

[0029] In any of the embodiments, including kits, compositions or methods described herein, the HSPC may be an HSC. In any of the embodiments described herein, the HSPC may be a progenitor cell.

[0030] In some embodiments, including kits, compositions or methods described herein, the gene editing system comprises a nucleic acid molecule encoding the Cas nuclease. In some aspects, an mRNA or DNA encodes the Cas nuclease and one or two or more guide RNAs or nucleic acid(s) encoding the guide RNA. In some embodiments, the gene editing system comprises an mRNA encoding the Cas nuclease and one or two or more guide RNAs. In some embodiments, the gene editing system comprises an mRNA encoding the Cas nuclease and a DNA encoding one or two or more guide RNAs. In some aspects, the DNA encoding one or two or more guide RNAs is in an expression vector comprising one or more regulatory sequences. In some embodiments, the gene editing system comprises a DNA encoding the Cas nuclease and a DNA encoding one or two or more guide RNAs. In some aspects, the DNA encoding the Cas nuclease is in an expression vector comprising one or more regulatory sequences. In some aspects, the DNA encoding one or two or more guideRNAs is in an expression vector comprising one or more regulatory sequences. In some embodiments, the gene editing system comprises a Cas nuclease polypeptide and one or two or more guide RNAs. In some aspects, the Cas nuclease polypeptide and one or two or more guide RNAs are pre-complexed.

[0031] In some embodiments including kits, compositions or methods described herein, the mRNA encoding the Cas nuclease of the gene editing system is chemically modified. In some aspects, the mRNA encoding the Cas nuclease is modified with a polyA tail, a 5' cap analog, modified 5' or 3' untranslated regions (UTRs), or modified bases, optionally Pseudo- U, N6-Methyl-A, 2-Thio-U and / or 5-Methyl-C modifications.

[0032] In some embodiments including kits, compositions or methods described herein, the guide RNA of the gene editing system is chemically modified. In some aspects, the guide RNA is modified with a modified backbone, one or more substituted sugar moieties, and / or nucleobase modifications or substitutions, optionally a combination of (a) PS backbone modifications and (b) 2’ substitutions, optionally 2'-O-Methyl, 2'-Fluoro, or 2'-Hydro.

[0033] In some embodiments including kits, compositions or methods described herein, the gene editing system is administered via a lipid nanoparticle (LNP). In some aspects, the components of the gene editing system are delivered in the same LNP or in separate LNPs.

[0034] In some embodiments including kits, compositions or methods described herein, the cycling agent is a CXCR4 antagonist, Thrombopoietin Receptor Agonist, CXCR2 agonist, VLA-4 antagonist, antimetabolite, alkylating agent, cytotoxic agent or other chemotherapeutic agent. In some embodiments, the cycling agent is a CXCR4 antagonist. In some aspects, the CXCR4 antagonist is AMD-3100 (Plerixafor), AMD-11070 (Mavorixafor), Motixafortide, AMD-3465, BMS-936564 / MDX-1338, LY2510924, N, N-dipropyl-N-[4- ({[(lH-imidazol-2-yl)methyl)benzyl][(l-methyl-lH-imidazol-2-yl) methyl]amino]methyl)benzyl]-N-methylbutane-l, 4-diamine tri(2R, 3R)-tartrate (KRH- 3955), and ([5-(4-methyl-l-piperazinyl)-2-({methyl[(8S)-5,6,7,8-tetrahydro-8- quinolinyl]amino}methyl)imidazo[l,2-a]pyri din-3 -yl]methanol) (GSK812397), an antibody or fragment thereof that binds CXCR4, or ALB408-423.

[0035] In some embodiments including kits, compositions or methods described herein, the cycling agent is an antimetabolite, alkylating agent, cytotoxic agent or other chemotherapeutic agent, optionally camptothecin, cisplatin, carboplatin, oxaliplatin, bleomycin, mitomycin C, calicheamicins, maytansinoids, geldanamycin, doxorubicin, idarubicin, daunorubicin, epirubicin, busulfan, carmustine (BCNU), lomustine (CCNU), semustine, thalidomide, lenalidomide, methotrexate, azathioprine, 6-mercaptopurine,fludarabine, 5 -azacytidine, pentostatin (2'-deoxycoformycin), cytarabine (cytosine arabinoside), gemcitabine, 5 -fluorouracil, hydroxyurea, elesclomol, etoposide, teniposide, amsacrine, topotecan, irinotecan, chlorambucil, cyclophosphamide, ifosfamide, melphalan, bortezomib, vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, amphotericin B, rifampicin, pentamidine, cyclosporine A, tacrolimus (FK506), sirolimus (rapamycin), everolimus, temsirolimus, zotarolimus, biolimus, oxaliplatin or mitomycin, 5 -fluorouracil (5- FU), hydroxyurea, gemcitabine, mercaptopurine, thioguanine, cladribine, fludarabine phosphate, fluorouracil (5-FU), floxuridine, cytarabine, pentostatin, methotrexate, azathioprine, acyclovir, adenine p-l-D-arabinoside, am ethopterin, aminopterin, 2- aminopurine, aphidicolin, 8 azaguanine, azaserine, 6-azauracil, 2'-azido-2'-deoxynucleosides, 5-bromodeoxy cytidine, cytosine P-l-D-arabinoside, diazooxynorleucine, dideoxynucleosides, 5-fluorodeoxycytidine, 5 fluorodeoxyuridine, hydroxyurea, cyclophosphamide, alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as a benzodizepa, carboquone, meturedepa, and uredepa; ehylenimines and methylmelamines, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylol melamine; nitrogen mustards such as chlorambucil, chlomaphazine, cyclophosphamide, estramustine, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, and uracil mustard; and nitroso ureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, or ranimustine. In some aspects, the cycling agent is plerixafor, 5-fluorouracil, and / or cyclophosphamide.

[0036] In some embodiment including kits, compositions or methods described herein, the cycling agent is plerixafor administered at a dose ranging from about 0.06 mg / kg to about 0.72 mg / kg.

[0037] In some embodiments including kits, compositions or methods described herein, the cycling agent is 5-fluorouracil administered parenterally at a dose ranging from about 200 to about 1200 mg / m2.

[0038] In some embodiments including kits, compositions or methods described herein, the cycling agent is cyclophosphamide administered orally or parenterally at a dose ranging from about 1 mg / kg to about 10 mg / kg.

[0039] In some embodiments including kits, compositions or methods described herein, the gene editing system is a CRISPR / Cas nuclease gene editing system administered via an LNP at a dose of about 0.1 mg / kg to about 10 mg / kg.

[0040] In some illustrative embodiments, the present invention relates to methods fortreating diseases by the in vivo gene editing of hematopoietic stem and progenitor cells (HSPCs), and in particular to the use of a CXCR4 antagonist to increase the efficiency of HSPC gene editing in vivo when administered prior to or in conjunction with administration of a gene editing system to a subject in need thereof.

[0041] In one aspect, the present disclosure provides in vivo methods of editing the genome of one or more hematopoietic stem and progenitor cells (HSPCs, including HSCs) in a subject in need thereof comprising: administering a CXCR4 antagonist to the subject; and administering a gene editing system to the subject so that the genome(s) of HSPCs, including HSCs, in the subject are edited.

[0042] In another aspect, the present disclosure provides a compound comprising a CXCR4 antagonist for use in increasing the efficiency of in vivo gene editing of one or more hematopoietic stem and progenitor cells (HSPCs, including HSCs) in a subject in need thereof.

[0043] In another aspect, the present disclosure provides ex vivo methods of editing the genome of one or more hematopoietic stem and progenitor cells (HSPCs, including HSCs) from a subject comprising: obtaining HSPCs, including HSCs, from a subject; contacting the HSPCs, including HSCs, with a CXCR4 antagonist; and introducing a gene editing system into the HSPCs, including HSCs, so that the genome(s) of HSPCs, including HSCs, are edited.

[0044] In another aspect, the present disclosure provides ex vivo methods of editing the genome of one or more hematopoietic stem and progenitor cells (HSPCs, including HSCs) from a subject comprising: obtaining HSPCs from a subject; treating the subject with a CXCR4 antagonist before or after obtaining the HSPCs; and introducing a gene editing system into the HSPCs so that the genome(s) of HSPCs are edited.

[0045] In another aspect, the present disclosure provides a compound comprising a CXCR4 antagonist for use in increasing the efficiency of ex vivo gene editing of one or more hematopoietic stem and progenitor cells (HSPCs, including HSCs) from a subject in need thereof.

[0046] In some embodiments, where the HSPCs, including HSCs, are edited ex vivo, the HSPCs, including HSCs, are infused back into the subject. In some preferred embodiments, the patient is not subjected to a conditioning treatment.

[0047] In some embodiments, the CRCX4 antagonist is plerixafor or a functional derivative thereof.

[0048] In some embodiments, the subject has or is at risk of having a disease or conditionselected from the group consisting of a hemoglobinopathy, a primary immunodeficiency and a congenital cytopenia.

[0049] In some embodiments, the subject has or is at risk of having a disease or condition selected from the group consisting of Sickle cell disease, Thalassemia, Pyruvate kinase deficiency, Hemophilia A, Hemophilia B, Factor X deficiency, Wiskott-Aldrich syndrome, Diamond-Blackfan anemia, Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease, Kostmann’s syndrome, X-linked adrenoleukodystrophy, Metachromatic leukodystrophy, Friedreich’s ataxia, Gaucher disease, Hunter syndrome, Mucopolysaccharidosis type 1, Osteopetrosis, X-linked agammaglobulinemia, X-linked hyper IgM syndrome, Immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, Early-onset inflammatory disease, Hemophagocytic lymphohistiocytosis, VEXAS (vacuoles, El enzyme, X-linked, autoinflammatory, somatic) syndrome, and erythropoietic protoporphyria. In some embodiments, SCID is selected from the group consisting of Adenosine deaminase-deficient SCID, Artemis SCID, X-linked SCID, and Recombination activating gene SCID.

[0050] In some embodiments, the gene editing system is selected from the group consisting of a zinc finger nuclease (ZFN) gene editing system, a transcription activator-like effector nuclease (TALEN) gene editing system, a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease gene editing system, a base or prime editing system, a reverse transcriptase (RT) editing system, a non-LTR retrotransposon editing system, and a retron gene editing system.

[0051] In some instances, the editing system is a base editing system or an RT editing system. In some instances, the editing system is a non-LTR retrotransposon editing system. In some instances, the editing system is a retron gene editing system.

[0052] In some embodiments, the gene editing system is a CRISPR / Cas nuclease gene editing system. In some embodiments, the CRISPR / Cas nuclease gene editing system comprises a gRNA that targets a sequence in the genome of the one or more HSCs. In some embodiments, the gRNA is a single-molecule guide RNA (sgRNA). In some embodiments, the CRISPR / Cas nuclease gene editing system comprises a Cas nuclease selected from the group consisting of Casl , CasI B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Casl OO, Csyl , Csy2, Csy3, Csel , Cse2, Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl , Cmr3, Cmr4, Cmr5, Cmr6, Csbl , Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl , Csxl5, Csfl , Csf2, Csf3, Csf4, Cpfl, or SluCas9 endonuclease; a homolog thereof, a recombination of the naturallyoccurring molecule thereof, codon-optimized thereof, or modified versions thereof, and combinations thereof. In some embodiments, the Cas nuclease is encoded by a nucleic acid molecule.

[0053] In some embodiments, the gene editing system effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within a target gene in the genomes of the one or more the subject HSCs.

[0054] In some embodiments, the gene editing system introduces an indel mutation into a target gene in the genomes of the one or more subject HSPCs.

[0055] In some embodiments, the CRISPR / Cas nuclease gene editing system further comprises a donor template. In some embodiments, the donor template is a DNA molecule.

[0056] In some embodiments, the gene editing system is administered via a lipid nanoparticle (LNP) or adeno-associated virus (AAV) vector or combination thereof. In some preferred embodiments, the gene editing system is administered via a lipid nanoparticle (LNP).

[0057] In some embodiments, the editing step further comprises introducing into the subject HSCs one or more deoxyribonucleic acid (DNA) endonucleases to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the BCL11A gene or other DNA sequence that encodes a regulatory element of the BCL11A gene that results in a permanent deletion, modulation, or inactivation of a transcriptional control sequence of the BCL11A gene.

[0058] In some embodiments, the method or use further comprises introducing into the subject HSCs one guide ribonucleic acid (gRNA) and a polynucleotide donor template comprising a wild-type gene, such as BCL11A gene or cDNA comprising a modified transcriptional control sequence and one or more DNA endonucleases that effect one singlestrand break (SSB) or double-strand break (DSB), at a locus within or near the BCL11A gene or other DNA sequence that encodes a regulatory element of the BCL11A gene, that facilitates insertion of a new sequence from the polynucleotide donor template into the chromosomal DNA at the locus that results in a permanent insertion, modulation, or inactivation of the transcriptional control sequence of the chromosomal DNA proximal to the locus, and wherein the gRNA comprises a spacer sequence that is complementary to a segment of the locus. In some embodiments, the method or use further comprises introducing into the subject HSCs one guide ribonucleic acid (gRNA) and a polynucleotide donor template comprising a wild-type gene, such as a CCR5 gene or cDNA comprising a modified transcriptional control sequence and one or more DNA endonucleases that effect one single-strand break (SSB) or double-strand break (DSB), at a locus within or near the BCL11A gene or other DNA sequence that encodes a regulatory element of the CCR5 gene, that facilitates insertion of a new sequence from the polynucleotide donor template into the chromosomal DNA at the locus that results in a permanent insertion, modulation, or inactivation of the transcriptional control sequence of the chromosomal DNA proximal to the locus, and wherein the gRNA comprises a spacer sequence that is complementary to a segment of the locus. In some embodiments, the method or use further comprises introducing into the subject HSCs one guide ribonucleic acid (gRNA) and a polynucleotide donor template comprising a wildtype gene of interest or cDNA comprising a modified transcriptional control sequence and one or more DNA endonucleases that effect one single-strand break (SSB) or double-strand break (DSB), at a locus within or near the gene of interest or other DNA sequence that encodes a regulatory element of the gene of interest, that facilitates insertion of a new sequence from the polynucleotide donor template into the chromosomal DNA at the locus that results in a permanent insertion, modulation, or inactivation of the transcriptional control sequence of the chromosomal DNA proximal to the locus, and wherein the gRNA comprises a spacer sequence that is complementary to a segment of the locus.

[0059] In some embodiments, the method or use further comprises introducing into the cell one or more guide ribonucleic acid (gRNAs) and a polynucleotide donor template comprising a wild-type BCL11A gene or cDNA comprising a modified transcriptional control sequence and one or more DNA endonucleases that effect or create a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs), the first break at a 5' locus and the second break at a 3' locus, within or near the BCL11A gene or other DNA sequence that encodes a regulatory element of the BCL11A gene, that facilitates insertion of a new sequence from the polynucleotide donor template into the chromosomal DNA between the 5' locus and the 3' locus that results in a permanent insertion, modulation, or inactivation of the transcriptional control sequence of the chromosomal DNA between the 5' locus and the 3' locus. In some embodiments, the method or use further comprises introducing into the cell one or more guide ribonucleic acid (gRNAs) and a polynucleotide donor template comprising a wild-type CCR5 gene or cDNA comprising a modified transcriptional control sequence and one or more DNA endonucleases that effect or create a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs), the first break at a 5' locus and the second break at a 3' locus, within or near the CCR5 gene or other DNA sequence that encodes a regulatory element of the CCR5 gene, that facilitates insertion of a new sequence from the polynucleotide donor template into the chromosomal DNA between the 5' locus and the 3' locus that results in apermanent insertion, modulation, or inactivation of the transcriptional control sequence of the chromosomal DNA between the 5' locus and the 3' locus. In some embodiments, the method or use further comprises introducing into the cell one or more guide ribonucleic acid (gRNAs) and a polynucleotide donor template comprising a wild-type gene of interest or cDNA comprising a modified transcriptional control sequence and one or more DNA endonucleases that effect or create a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs), the first break at a 5' locus and the second break at a 3' locus, within or near the gene of interest or other DNA sequence that encodes a regulatory element of the gene of interest, that facilitates insertion of a new sequence from the polynucleotide donor template into the chromosomal DNA between the 5' locus and the 3' locus that results in a permanent insertion, modulation, or inactivation of the transcriptional control sequence of the chromosomal DNA between the 5' locus and the 3' locus.

[0060] In some embodiments, the method or use further comprises introducing into the cell one or more guide ribonucleic acid (gRNAs and one or more DNA endonucleases that effect or create a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs), a first SSB or DSB at a 5' locus and a second SSB or DSB at a 3' locus, within or near the BCL11A gene or other DNA sequence that encodes a regulatory element of the BCL11A gene that causes a deletion of the chromosomal DNA between the 5' locus and the 3' locus that results in a permanent deletion, modulation, or inactivation of the transcriptional control sequence of the chromosomal DNA between the 5' locus and the 3' locus. In some embodiments, the method or use further comprises introducing into the cell one or more guide ribonucleic acid (gRNAs and one or more DNA endonucleases that effect or create a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs), a first SSB or DSB at a 5' locus and a second SSB or DSB at a 3' locus, within or near the CCR5 gene or other DNA sequence that encodes a regulatory element of the CCR5 gene that causes a deletion of the chromosomal DNA between the 5' locus and the 3' locus that results in a permanent deletion, modulation, or inactivation of the transcriptional control sequence of the chromosomal DNA between the 5' locus and the 3' locus. In some embodiments, the method or use further comprises introducing into the cell one or more guide ribonucleic acid (gRNAs and one or more DNA endonucleases that effect or create a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs), a first SSB or DSB at a 5' locus and a second SSB or DSB at a 3' locus, within or near the gene of interest or other DNA sequence that encodes a regulatory element of the gene of interest that causes a deletion of the chromosomal DNA between the 5' locus and the 3' locus that results in a permanent deletion, modulation, or inactivation of thetranscriptional control sequence of the chromosomal DNA between the 5' locus and the 3' locus.

[0061] In some embodiments, the method or use further comprises repeating one or more times one or both steps of administering a CXCR4 antagonist to the subject or administering a gene editing system to the subject so that the genome(s) of HSPCs, including HSCs, in the subject are edited.

[0062] In still other aspects, the disclosure provides a kit comprising: a container containing a CXCR4 agonist; and a container containing a formulation comprising one or more components of a gene editing system sufficient for editing a gene or nucleic acid sequence associated with a disease or condition selected from the group consisting of a hemoglobinopathy, a primary immunodeficiency and a congenital cytopenia.

[0063] In some embodiments, the CRCX4 antagonist is plerixafor or a functional derivative thereof.

[0064] In some embodiments, the subject has or is at risk of having a disease or condition selected from the group consisting of Sickle cell disease, Thalassemia, Pyruvate kinase deficiency, metabolic disorders such as hypophosphatasia, Hemophilia A, Hemophilia B, Factor X deficiency, Wiskott-Aldrich syndrome (WAS), Diamond-Blackfan anemia, primary immunodeficiency disorders (PIDs) including Severe Combined Immunodeficiency (SCID), chronic granulomatous disease (CGD), etc., X-linked chronic granulomatous disease, Kostmann’s syndrome, X-linked adrenoleukodystrophy, Metachromatic leukodystrophy, CNS diseases such as Friedreich’s ataxia and Alzheimer’s disease, lysosomal storage diseases (LSD), such as Gaucher disease, Fabry’s disease, Pompe disease, Mucopolysaccharidosis Type I (MPS-I) and MPS-II; autoimmune conditions such as lupus (SLE / LN), scleroderma, myositis, myasthenia gravis, multiple sclerosis, Crohn’s disease, and stiff-person-syndrome, Hunter syndrome, Mucopolysaccharidosis type 1, Osteopetrosis, X-linked agammaglobulinemia, X-linked hyper IgM syndrome, Immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, Early-onset inflammatory disease, Hemophagocytic lymphohistiocytosis, VEXAS (vacuoles, El enzyme, X-linked, autoinflammatory, somatic) syndrome, and erythropoietic protoporphyria. In some embodiments, the SCID is selected from the group consisting of Adenosine deaminase- deficient SCID, Artemis SCID, X-linked SCID, and Recombination activating gene SCID.

[0065] In some embodiments, the gene editing system is selected from the group consisting of a zinc finger nuclease (ZFN) gene editing system, a transcription activator-like effector nuclease (TALEN) gene editing system, a clustered regularly interspaced shortpalindromic repeats (CRISPR) / Cas nuclease gene editing system, a base or prime editing system, a reverse transcriptase (RT) editing system, a non-LTR retrotransposon editing system, and a retron gene editing system.

[0066] In some embodiments, the gene editing system is a CRISPR / Cas nuclease gene editing system. In some embodiments, CRISPR / Cas nuclease gene editing system comprises a gRNA that targets a sequence in the genome of the one or more HSCs. In some embodiments, gRNA is a single-molecule guide RNA (sgRNA). In some embodiments, the CRISPR / Cas nuclease gene editing system comprises a Cas nuclease selected from the group consisting of Casl , CasI B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Casl 00, Csyl , Csy2, Csy3, Csel , Cse2, Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl , Cmr3, Cmr4, Cmr5, Cmr6, Csbl , Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl , Csxl5, Csfl , Csf2, Csf3, Csf4, Cpfl, or SluCas9 endonuclease; a homolog thereof, a recombination of the naturally occurring molecule thereof, codon-optimized thereof, or modified versions thereof, and combinations thereof. In some embodiments, the Cas nuclease is encoded by a nucleic acid molecule. In some embodiments, the CRISPR / Cas nuclease gene editing system further comprises a donor template. In some embodiments, the donor template is a DNA molecule.

[0067] In some embodiments, the one or more components of the gene editing system are formulated in a LNP. Guide RNA polynucleotides (RNA or DNA) and / or endonuclease polynucleotide(s) (RNA or DNA) can be delivered by viral or non-viral delivery vehicles known in the art, such as lipid nanoparticles. In further alternative aspects, the DNA endonuclease can be delivered as one or more polypeptides, either alone or pre-complexed with one or more guide RNAs, or one or more crRNA together with a tracrRNA. Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding an endonuclease, can be delivered to a cell or a patient by a lipid nanoparticle (LNP).

[0068] In some embodiments, the one or more components of the gene editing system edit BCLUA gene or control sequence of the BCL11 A gene. In some embodiments, the one or more components of the gene editing system edit a CCR5 gene or control sequence of the CCR5 gene.

[0069] In some the aspects, the foregoing kits are provided for use in treating a with a disease or condition selected from the group consisting of a hemoglobinopathy, a primary immunodeficiency and a congenital cytopenia. In some embodiments, the subject has or is at risk of having a disease or condition selected from the group consisting of Sickle cell disease, Thalassemia, Pyruvate kinase deficiency, Hemophilia A, Hemophilia B, Factor X deficiency,Wiskott-Aldrich syndrome, Diamond-Blackfan anemia, Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease, Kostmann’s syndrome, X-linked adrenoleukodystrophy, Metachromatic leukodystrophy, Friedrich’s ataxia, Gaucher disease, Hunter syndrome, Mucopolysaccharidosis type 1, Osteopetrosis, X-linked agammaglobulinemia, X-linked hyper IgM syndrome, Immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, Early-onset inflammatory disease, Hemophagocytic lymphohistiocytosis, VEXAS (vacuoles, El enzyme, X-linked, autoinflammatory, somatic) syndrome, and erythropoietic protoporphyria. In some embodiments, the SCID is selected from the group consisting of Adenosine deaminase- deficient SCID, Artemis SCID, X-linked SCID, and Recombination activating gene SCID.BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG. 1 A-1B. Graphical data from experiments showing the effect of plerixafor and G-CSF on HSC mobilization to peripheral blood.

[0071] FIG. 2A-2B. Graphs from experiments showing 1 week post LNP administration enhanced editing of mBCLl la in Lin-Sca+c-Kit+ cells that are enriched for HSPCs with or without plerixafor administration.

[0072] FIG. 3 A-3D. Graphs from experiments showing that plerixafor pre-treatment leads to increased editing of mBCLl la over time in peripheral blood (n=3 mice) at 1 week, 1 month, 2 months, 3 months, 4 months, and 5 months after LNP administration. FIG. 3C shows editing efficiency in the bone marrow. FIG. 3 A, 3B & 3D show editing efficiency in peripheral blood cells.

[0073] FIG. 4A-4C. Graph showing that plerixafor administration reproducibly increases LNP -mediated gene editing of phenotypic HSCs in total bone marrow cells (FIG. 4A), LK cells (FIG. 4B and HSCs (FIG. 4C) at the 5-month time-point.

[0074] FIG. 5 A-5D. Graph showing that plerixafor administration reproducibly increases LNP -mediated gene editing of phenotypic HSCs in the peripheral blood. Figs. 5A-5D respectively show editing of myeloid cells (CD1 lb+GR1+), erythroid cells (TERI 19+), B cells (B220+), and T cells (CD3+) when they were separately evaluated at the 5-month timepoint.

[0075] FIG. 6A-B. Graph showing increase in indel frequencies at different time-points in cells in mice pre-treated with plerixafor alone.

[0076] FIG. 7. Graph showing effect of addition of plerixafor in the media on editing efficiency in human CD34+ HSPCs in vitro.

[0077] FIG. 8A-8D. Graph showing percent editing in hNSG mice with or without plerixafor pre-treatment 12 days after LNP administration in total bone marrow cells (FIG. 8 A), CD34+ cells (FIG. 8B), and sorted CD34+CD38- cell (FIG. 8C) populations. The CD34+ / CD38- immunophenotype is used to identify HSCs. FIG. 8D. Graph showing editing efficiency in bone marrow HSC cells 2 months after LNP administration.

[0078] FIG. 9A-9C. Graphs showing increased editing efficiencies after plerixafor pretreatment in HSCs (Lin“Sca+cKit+CD48“ CD150+) (FIG. 9 A), progenitor LK cells (Lin“Sca“ cKit+) (FIG. 9B), and total BM cells (FIG. 9C).

[0079] FIG. 10. Graph showing increased indel rates in peripheral blood over 3 months in mice pre-treated with plerixafor before LNP administration. Plots show the average rates in 5 mice.

[0080] FIG. 11 A-l 1C. Graphs showing that plerixafor pretreatment and redosing with plerixafor and LNP leads to increased editing of mBCLl la over time in total bone marrow cells (FIG. 11 A), HSCs (FIG. 1 IB), and LK cells (FIG. 11C).

[0081] FIG. 12A-12G. Graphs showing that plerixafor pretreatment and redosing with plerixafor and LNP leads to increased editing of mBCLl la over time in peripheral blood (n=3 mice), HSCs, LKs and total bone marrow. (FIG. 12 A) Graph superimposing all groups (PBS control group, LNP group, PLX+LNP group) with data to 8 -months. (FIG. 12B) Graph of PBS control group re-dosed at 3.5 months with plerixafor and LNP with data to 10 months. (FIG. 12C) Graph of LNP only group re-dosed at 3.5 months with plerixafor and LNP with data to 10 months. (FIG. 12D) Graph of Plerixafor and LNP treatment group re-dosed at 3.5 months with plerixafor and LNP with data to 10 months. (FIGs. 12E-12G) Graph of mBCLl la editing in HSCs (FIG. 12E), LKs (FIG.12F) and total bone marrow (FIG. 12G) for all groups (PBS control, LNP group and PLX+LNP group) at 2-weeks, 5-months and 6- months.

[0082] FIG. 13A-13D. Graphs showing that redosing with plerixafor and LNP leads to increased editing of mBCLl la in lineages (T cells (FIG. 13 A), B cells (FIG. 13B), myeloid cells (FIG. 13C) and erythroid cells (FIG. 13D)) in bone marrow at 2 weeks and 6 months post re-dose.

[0083] FIG. 14A-14D. Graphs showing long-term engraftment of edited CD45.2 HSCs in recipient CD45.1 mice. (FIG. 14A and FIG. 14B) Graphs of the percentage of donor CD45.2 cells over time show the donor CD45.2 cells become dominant in animals treated with sublethal irradiation and lethal irradiation, respectively. (FIG. 14C and FIG. 14D) Graphs ofthe editing of mBCLl la over time in peripheral blood of animals treated with sublethal irradiation and lethal irradiation, respectively.

[0084] FIG. 15A-15D. Graphs showing effect of the pretreatment with PIPC, 5-FU and cyclophosphamide on editing of mBCLl la in sorted HSCs (FIG. 15 A), LKs (FIG. 15B) and total bone marrow (FIG. 15C) at 10-days and 6-months post-administration. FIG. 15D is a graph of mBCLl la editing in peripheral blood over a 6-month period; highest editing is seen in mice administered 5-FU or cyclophosphamide.

[0085] FIG. 16. A diagram illustrating a study design that administers various combinations of cycling agents (5FU, cyclophosphamide) with plerixafor or various combinations of mobilizing agents (Gro-b, BIO5192, mavorixafor) with plerixafor.

[0086] FIG. 17A-17K. Graphs showing synergistic increase in editing efficiency of the pretreatment with a combination of cycling agents, either 5-FU or cyclophosphamide, and pleriaxfor, on editing of mBCLl la in sorted HSCs at 2 weeks and 1.5 months (FIGs. 17A & 17B, respectively), in bone marrow at 2 weeks and 1.5 months (FIGs. 17C & 17D, respectively), in peripheral blood at 10-days, 1-month, 1.5 months, 3- months and 4-months (FIG. 17E), in peripheral blood at 2 weeks (FIG. 17F), in LKs at 2 weeks and 1.5 months, (FIG.17G & 17H), in erythroid cells at 2 weeks (FIG.171), myeloid cells at 2 weeks (FIG.17J) and B cells at 2 weeks (FIG. 17K) post LNP administration. More than 50% editing of HSCs, total bone marrow and LK cells were observed 2 weeks and 1.5 months post LNP dosing, with 5-FU and PLX combination.

[0087] FIG. 18A-18H. Graphs showing effect of the plerixafor and mobilizing agents, Gro-P, BIO5192 and their combinations on stem cell mobilization to peripheral blood and editing efficiency. (FIG. 18A) Graph showing the effect on mobilization of cells, expressed in the CFU count, by the mobilizing agents, Gro-beta, BIO5192, as well as plerixafor. Plerixafor was administered 1 hour, Gro-P was administered 15 minutes and BIO5192 was administered 30 minutes prior to blood collection. (FIG. 18B-18G) Effect on editing efficiency of mBCLl la at 2 weeks post LNP administration, in peripheral blood (FIG. 18B), sorted HSCs (FIG. 18C), LKs cells (FIG.18D), erythroid cells (FIG.18E), myeloid cells (FIG.18F) and B cells (FIG.18G) with pre-treatment with a combination of the mobilizing agent and plerixafor. (FIG. 18H) Shows effect on editing efficiency of mBCLl la in peripheral blood at 10-days, 1-month, 1.5 months, 3- months and 4-month post LNP administration. In FIG.18B the editing rate for LNP ranged from 1.4-5.5 with a sacrificed mouse editing rate of 1.8. The editing rate for PLX+LNP ranged from 2.4-10.3 with a sacrificed mouse editing rate of 2.4. The editing rate for GroB+PLX+LNP ranged from 3.4-8.2 with a sacrificed mouse editingrate of 3.4. The editing rate for BIO5129+PLX+LNP ranged from 4.5-9.7 with a sacrificed mouse editing rate of 6.7. The editing rate for Mavorixafor+PLX+LNP ranged from 1-8.5 with a sacrificed mouse editing rate of 2.6.

[0088] FIG. 19A-19C. Graphs showing percent editing of CCR5 in hu-NSG mice pretreated with plerixafor + LNP. FIG. 19A shows the percent of CCR5 gene editing at 2 weeks after LNP administration in bone marrow cells, CD34+ cells, CD34+ / CD38- / CD90- cells, and CD34+ / CD38- / CD90+ cells. FIG. 19B shows the percent of CCR5 gene editing at 6 weeks in peripheral blood. FIG. 19C shows the percent of CCR5 gene editing at 8 weeks after LNP administration in bone marrow cells, CD34+ cells, CD34+ / CD38- / CD90- cells, and CD34+ / CD38- / CD90+ cells.DETAILED DESCRIPTION

[0089] The present disclosure relates to methods and compositions for treating diseases by the in vivo gene editing of hematopoietic stem and progenitor cells (HSPCs), including hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPC), involving the administration of a cycling agent to increase the efficiency of HSPC gene editing in vivo or ex vivo. All references herein to HSPCs are specifically intended to reference all subsets of such cells, including HSCs and progenitor cells described herein.

[0090] For example, in one aspect, the disclosure provides for administration of one, two or more cycling agents prior to or in conjunction with a gene editing system. In some illustrative embodiments, the present invention relates to methods for treating diseases by the in vivo gene editing of hematopoietic stem and progenitor cells (HSPCs), and in particular to the use of a CXCR4 antagonist to increase the efficiency of HSPC gene editing in vivo when administered prior to or in conjunction with administration of a gene editing system to a subject in need thereof. In a related aspect, the present invention relates to methods for treating diseases by the in vivo gene editing of hematopoietic stem and progenitor cells (HSPCs), and in particular administering a cycling agent to alter the cellular state of HSPCs to a more active metabolic state or a proliferative state (e.g. inducing HSPCs to undergo transition from a GO or quiescent state to a more active cell cycle state, e.g. Gl) prior to or in in conjunction with administering a gene editing system to a subject in need thereof. As shown in the examples, the efficiency of HSPC gene editing in vivo and in vitro is increased by inducing HSPCs to transition out of a GO state to a more active metabolic state or proliferative state, and then administering a gene editing system to a subject in need thereof.Gene therapy, including therapy by gene editing, of HSPCs has the ability to treat many different diseases such as hemoglobinopathies, immunodeficiency, primary immunodeficiencies (PIDs), hematological malignancies, cancers, non-malignant hematological disorders, anemias, autoimmune diseases, cytopenias, and congenital cytopenias. However, current methods require myeloablative conditioning and associated long and expensive hospital stays. This makes the therapies inaccessible to many patients. To date, in vivo gene editing of long-term hematopoietic stem cells through non-viral means has not been sufficiently achieved. The present invention addresses this problem by providing methods and kits for the in vivo editing of HSPCs in a subject in need thereof. As shown in the examples, the efficiency of gene editing in vivo can be greatly improved by perturbation of chemokine pathways through the use of a CXCR4 antagonist, preferably plerixafor or a derivative thereof. In some embodiments, the plerixafor is administered in the absence of G- CSF (granulocyte-colony stimulating factor). Described herein are compositions and methods for altering the cellular state of HSPCs to a more active metabolic state or a proliferative state (for example, inducing HSPCs to transition out of a GO state) prior to or in conjunction with administration of a gene editing system. It is not obvious why perturbing chemokine pathways alone may increase LNP mediated gene editing in vivo in HSC. G-CSF, which has multiple mechanisms of action, along with plerixafor, can lead to greater HSPC mobilization out of the bone marrow than plerixafor alone (See FIG. 1) and has been described to enhance viral-vector mediated gene editing in vivo, as some viral vectors do not efficiently transduce cells in the bone marrow. Therefore, it is surprising that as shown in Examples that LNPs can deliver mRNA to bone marrow HSPC and that plerixafor can enhance gene editing when administered before LNP injection. Further, without being limited to any mechanism of action, it is surprising that G-CSF administration along with plerixafor reduced the enhancing effects of plerixafor suggesting that HSPC mobilization is likely not the mechanism of the effect and that other plerixafor / CXCR4 chemokine responsive pathways may help with LNP mediated gene editing in HSPC. Indeed, the data indicates the mechanism of action is not increased HSPC mobilization but rather altering the cellular state of HSPCs to a more active metabolic state or a proliferative state (for example, a transition out of a GO state). This surprising result is supported by experiments demonstrating that administration of diverse cycling agents that drive cell cycle transition by different mechanisms, such as 5-fluorouracil, cyclophosphamide and CXCR4 antagonists, can increase HSPC editing efficiency. Thus, described herein are compositions and methods for treating diseases and / or increasing HSPC editing efficiency by altering the cellular state of HSPCs to a more active metabolic state or aproliferative state (for example, inducing HSPCs to transition out of a GO state) prior to or in conjunction with administration of a gene editing system.

[0091] Accordingly, in some preferred embodiments, the present invention provides therapeutic methods comprising administering a CXCR4 antagonist, e.g., plerixafor or a derivative thereof, to a subject in need thereof prior to or in conjunction with administration of a gene editing system that effects editing of a target in a HSPC. In some other embodiments, the present invention provides therapeutic methods comprising increasing the metabolic and cellular activity of HSPCs resulting in a change from a quiescent to a proliferative state (i.e. inducing HSPCs to undergo a GO to G1 transition) in a subject in need thereof prior to or in conjunction with administration of a gene editing system that effects editing of a target in a HSPC. Metabolic and cellular activity of HSPCs resulting in a change from a quiescent to a proliferative state and inducing a GO to G1 transition may be accomplished with cycling agents known in the art. Thus, such methods may comprise administering one or more cycling agents, e.g., in an amount effective to increase the metabolic and cellular activity of the HSPCs resulting in a change from a quiescent to a proliferative state or induce HSPCs to undergo a GO to G1 transition, prior to or in conjunction with administration of a gene editing system that effects editing of a target in HSPCs. Such methods may also comprise a second, third, fourth, or fifth (re-dosing) step of administering one or more cycling agents in conjunction with a second, third, fourth, or fifth administration of a gene editing system that effects editing of a target in a HSPC. In some preferred embodiments, the subject either suffers from or is at risk of a hemoglobinopathy, a primary immunodeficiency (PIDs), or a congenital cytopenia.

[0092] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.

[0093] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0094] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.10095] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0096] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.1. CXCR4 antagonists and other Cycling Agents

[0097] A “cycling agent” as used herein is an agent that promotes altering the cellular state of HSPCs to a more active metabolic state or proliferative state. For example, a cycling agent can promote the transition of an HSPC from a GO or quiescent state to a more active cell cycle state, e.g. Gl.

[0098] A “mobilizing agent” as used herein is an agent that causes HSCs to mobilize from the bone marrow into peripheral blood. Typically, mobilizing agents are used to increase the numbers of HSCs in peripheral blood so that they can be harvested by apheresis.

[0099] Some cycling agents may also have some mobilizing activity, but they are still cycling agents if they increase metabolic, cellular or proliferative activity and thus induce a detectable percentage of cells to transition from a GO or quiescent state into a more active metabolic or proliferative state. The transition in cell state can be detected, for example, by measuring the proliferation and differentiation ability of individual cells within a sample. In some embodiments proliferation and differentiation ability is measured by a colony-forming unit (CFU) assay. In some embodiments, the CFUs increase from 1-5 CFU / mL to >10 CFU / mL as measured by CFU assay. In some embodiments, the CFUs increase 2.5x to lOx as measured by CFU assay. In some embodiments proliferation and differentiation ability is observed by measuring Ki -67 upregulation as a marker of increased cell division.

[0100] The compositions and methods of this disclosure are not limited to any particular cycling agent or any particular mode of administration, or any particular gene editing system.In some embodiments, HSPCs are contacted ex vivo with a cycling agent and a gene editing system. In some preferred embodiments, one, two or more cycling agents is administered to a subject in need thereof prior to or in conjunction with a gene editing system. In some embodiments, one cycling agent is administered, and in some embodiments, two cycling agents are administered, or in some embodiments, three cycling agents are administered.

[0101] The present invention is not limited to the use of any particular CXCR4 antagonist or other cycling agent(s). Indeed, the use of a variety of CXCR4 antagonists or cycling agents are contemplated, including, but not limited to, cytotoxic agents, chemotherapeutic agents, radiation, antimetabolites such as 5 -fluorouracil (5-FU), alkylating agents such as cyclophosphamide, Thrombopoietin Receptor Agonists (TPO-RAs) such as eltrombopag, and / or agents that perturb CXCR4 signaling (e.g. antagonist or reduce CXCR4 signaling) by affecting key CXCR4 signaling associated molecules.In some embodiments, the cycling agent is administered with a mobilizing agent. In other preferred embodiments, the cycling agent is administered without a mobilizing agent whose primary activity is mobilizing (e.g. G-CSF or GM-CSF). Mobilizing agents include, but are not limited to Plerixafor (AMD3100), cyclophosphamide, benzylamines, cy clams, heterocyclic compounds.

[0102] The use of a variety of CXCR4 antagonists is contemplated, including but not limited to AMD-3100 (Plerixafor), AMD-11070 (Mavorixafor), Motixafortide, AMD-3465, BMS-936564 / MDX-1338, LY2510924, N, N-dipropyl-N-[4-({[(lH-imidazol-2- yl)methyl)benzyl][(l-methyl-lH-imidazol-2-yl) methyl]amino]methyl)benzyl]-N- methylbutane-1, 4-diamine tri(2R, 3R)-tartrate (KRH-3955), and ([5-(4-methyl-l- piperazinyl)-2-({methyl[(8S)-5,6,7,8-tetrahydro-8-quinolinyl]amino}methyl)imidazo[l,2- a]pyri din-3 -yl]methanol) (GSK812397) as well as antigen binding molecules such as antibodies and antigen binding fragments thereof that bind to CXCR4 and inhibitory polypeptides such as ALB408-423. Suitable CXCR4 antagonists are described in the following publications and patents, each of which is incorporated by reference herein in its entirety: U.S. Pat. Nos. 5,021,409; 6,001,826; 5,583,131; RE42,152; 5,698,546; 5,817,807; 6,756,391; 7,022,717; 7,160,872; 7,414,065; 7,7094,86; 6,506,770; 6,667,320; 6,872,714, 5,612,478; 5,756,728; 5,801,281; 5,606,053; 6,489,472; 9,045,563; 9,708,405; 10,709,763; 10,988,465; and 12,115,156; in U.S. Patent Application Publication Nos. 2018 / 0228894 and 2007 / 0060591; and in PCT Publication Nos. WO 92 / 016494; WO 93 / 012096; WO 95 / 018808; WO 00 / 002870, WO 01 / 044229, and WO 02 / 26721.

[0103] In some particularly preferred embodiments, the CXCR4 antagonist is plerixafor, which has the following structure:

[0104] In some embodiments, the CXCR4 antagonist is mavorixafor, which has the following structure:

[0105] The use of a variety of cytotoxic agents as cycling agents is contemplated, including but not limited to camptothecin, cisplatin, carboplatin, oxaliplatin, bleomycin, mitomycin C, calicheamicins, maytansinoids, geldanamycin, doxorubicin, idarubicin, daunorubicin, epirubicin, busulfan, carmustine (BCNU), lomustine (CCNU), semustine, thalidomide, lenalidomide, methotrexate, azathioprine, 6-mercaptopurine, fludarabine, 5- azacytidine, pentostatin (2'-deoxycoformycin), cytarabine (cytosine arabinoside), gemcitabine, 5-fluorouracil, hydroxyurea, elesclomol, etoposide, teniposide, amsacrine, topotecan, irinotecan, chlorambucil, cyclophosphamide, ifosfamide, melphalan, bortezomib, vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, amphotericin B, rifampicin, pentamidine, cyclosporine A, tacrolimus (FK506), sirolimus (rapamycin), everolimus, temsirolimus, zotarolimus, biolimus, oxaliplatin or mitomycin, also including chemotherapeutic agents and / or radiation.

[0106] The use of a variety of antimetabolites as cycling agents is contemplated, including but not limited to 5-fluorouracil (5-FU), hydroxyurea, gemcitabine, mercaptopurine, thioguanine, cladribine, fludarabine phosphate, fluorouracil (5-FU), floxuridine, cytarabine, pentostatin, methotrexate, azathioprine, acyclovir, adenine p-l-D-arabinoside, amethopterin, aminopterin, 2-aminopurine, aphidicolin, 8 azaguanine, azaserine, 6-azauracil, 2'-azido-2'- deoxynucleosides, 5-bromodeoxycytidine, cytosine P-l-D-arabinoside, diazooxynorleucine, dideoxynucleosides, 5-fluorodeoxycytidine, 5 fluorodeoxyuridine, and hydroxyurea.

[0107] The use of a variety of alkylating agents as cycling agents is contemplated, including but not limited to cyclophosphamide, alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as a benzodizepa, carboquone, meturedepa, and uredepa; ethylenimines and methylmelamines, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylol melamine; nitrogen mustards such as chlorambucil, chlomaphazine, cyclophosphamide, estramustine, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, and uracil mustard; and nitroso ureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine.

[0108] The use of a variety of Thrombopoietin Receptor Agonists (TPO-RAs) as cycling agents is contemplated, including but not limited to eltrombopag or romiplostim.

[0109] Cycling agents also include agents that perturb CXCR4 signaling by affecting key CXCR4-associated molecules, including CD164, VLA-4, VLA-5, CXCL-12 / SDF-1, LPA-1, and / or lysosphosphatidic acid, by agonism or antagonism. Such agents may comprise antibodies, small molecules, siRNA, antisense oligonucleotides, miRNA, or any RNA interfering agents that comprise nucleobase sequences that hybridize to (and thereby specifically target) the target genes or mRNA. In some embodiments, such cycling agents are antagonists.

[0110] In contrast, mobilizing agents include CXCR2 agonists and VLA-4 antagonists, G- CSF and GM-CSF. CXCR2 agonists include, but are not limited to, Gro-p.VLA-4 antagonists include, but are not limited to, BIO5192, GW559090, and BIO1211 as well as antigen binding molecules such as antibodies and antigen binding fragments thereof that bind to VLA-4.

[0111] Also contemplated herein are combination of agents. In some embodiments, a cycling agent is administered in combination with a second and different cycling agent. In some embodiments, a cycling agent is administered in combination with a mobilizing agent. Preferably, the one or more cycling agents are administered without (i.e., excluding administration of) a mobilizing agent whose primary activity is mobilizing. Preferably, the cycling agent(s) are administered without (i.e., excluding administration of) G-CSF or GM- CSF. In some embodiments, a cycling agent (e.g., a CXCR4 antagonist such as plerixafor) is administered in combination with a cytotoxic agent. For example, a cycling agent such as plerixafor is administered in combination with an antimetabolite. As another example, a cycling agent such as plerixafor is administered in combination with an alkylating agent. Insome embodiments, a CXCR4 antagonist is administered in combination with a second and different CXCR4 antagonist, e.g., plerixafor and mavorixafor.

[0112] In some embodiments, a CXCR4 antagonist is administered with a cytotoxic agent. In some embodiments, a CXCR4 antagonist is administered with an antimetabolite, for example, plerixafor and 5-FU or hydroxyurea. In some embodiments, the CXCR4 antagonist is administered with an alkylating agent, for example, plerixafor and cyclophosphamide.2. Gene editing systems

[0113] The present invention is not limited to the use of any particular gene editing system. A number of gene editing systems may be utilized including, but not limited to, zinc finger nuclease (ZFN) gene editing systems, transcription activator-like effector nuclease (TALEN) gene editing systems, and clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease gene editing systems.

[0114] Genome editing generally refers to the process of modifying the nucleotide sequence of a genome, preferably in a precise or pre-determined manner. Examples of methods of genome editing described herein include methods of using site-directed nucleases to cut deoxyribonucleic acid (DNA) at precise target locations in the genome, thereby creating single-strand or double-strand DNA breaks at particular locations within the genome. Such breaks can be and regularly are repaired by natural, endogenous cellular processes, such as homology-directed repair (HDR) and NHEJ, as reviewed in Cox et al., Nature Medicine 21(2), 121-31 (2015). These two main DNA repair processes consist of a family of alternative pathways. NHEJ directly joins the DNA ends resulting from a double-strand break, sometimes with the loss or addition of nucleotide sequence, which may disrupt or enhance gene expression. HDR utilizes a homologous sequence, or donor sequence, as a template for inserting a defined DNA sequence at the break point. The homologous sequence can be in the endogenous genome, such as a sister chromatid. Alternatively, the donor can be an exogenous nucleic acid, such as a plasmid, a single-strand oligonucleotide, a double-stranded oligonucleotide, a duplex oligonucleotide or a virus, that has regions of high homology with the nuclease-cleaved locus, but which can also contain additional sequence or sequence changes including deletions that can be incorporated into the cleaved target locus. A third repair mechanism can be microhomology -mediated end joining (MMEJ), also referred to as “Alternative NHEJ”, in which the genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ can make use of homologous sequences of a few basepairs flanking the DNA break site to drive a more favored DNA end joiningrepair outcome, and recent reports have further elucidated the molecular mechanism of this process; see, e.g., Cho and Greenberg, Nature 518, 174-76 (2015); Kent et al., Nature Structural and Molecular Biology, Adv. Online doi: 10.1038 / nsmb.2961(2015); Mateos- Gomez et al., Nature 518, 254-57 (2015); Ceccaldi et al., Nature 528, 258-62 (2015). In some instances it may be possible to predict likely repair outcomes based on analysis of potential microhomologies at the site of the DNA break.

[0115] Each of these genome editing mechanisms can be used to create desired genomic alterations. A step in the genome editing process can be to create one or two DNA breaks, the latter as double-strand breaks or as two single-stranded breaks, in the target locus as near the site of intended mutation. This can be achieved via the use of site-directed polypeptides, as described and illustrated herein.

[0116] Site-directed polypeptides, such as a DNA endonuclease, can introduce doublestrand breaks or single-strand breaks in nucleic acids, e.g., genomic DNA. The double-strand break can stimulate a cell's endogenous DNA-repair pathways (e.g., homology-dependent repair or non-homologous end joining or alternative non-homologous end joining (A-NHEJ) or microhomology-mediated end joining). NHEJ can repair cleaved target nucleic acid without the need for a homologous template. This can sometimes result in small deletions or insertions (indels) in the target nucleic acid at the site of cleavage, and can lead to disruption or alteration of gene expression. HDR can occur when a homologous repair template, or donor, is available. The homologous donor template can comprise sequences that can be homologous to sequences flanking the target nucleic acid cleavage site. The sister chromatid can be used by the cell as the repair template. However, for the purposes of genome editing, the repair template can be supplied as an exogenous nucleic acid, such as a plasmid, duplex oligonucleotide, single-strand oligonucleotide, double-stranded oligonucleotide, or viral nucleic acid. With exogenous donor templates, an additional nucleic acid sequence (such as a transgene) or modification (such as a single or multiple base change or a deletion) can be introduced between the flanking regions of homology so that the additional or altered nucleic acid sequence also becomes incorporated into the target locus. MMEJ can result in a genetic outcome that is similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ can make use of homologous sequences of a few basepairs flanking the cleavage site to drive a favored end-joining DNA repair outcome. In some instances it may be possible to predict likely repair outcomes based on analysis of potential microhomologies in the nuclease target regions.

[0117] Thus, in some cases, homologous recombination can be used to insert an exogenous polynucleotide sequence into the target nucleic acid cleavage site. An exogenous polynucleotide sequence is termed a donor polynucleotide (or donor or donor sequence or polynucleotide donor template) herein. The donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide can be inserted into the target nucleic acid cleavage site. The donor polynucleotide can be an exogenous polynucleotide sequence, i.e., a sequence that does not naturally occur at the target nucleic acid cleavage site.

[0118] The modifications of the target DNA due to NHEJ and / or HDR can lead to, for example, mutations, deletions, alterations, integrations, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocations and / or gene mutation. The processes of deleting genomic DNA and integrating non-native nucleic acid into genomic DNA are examples of genome editing.CRISPR Endonuclease System

[0119] A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic locus can be found in the genomes of many prokaryotes (e.g., bacteria and archaea). In prokaryotes, the CRISPR locus encodes products that function as a type of immune system to help defend the prokaryotes against foreign invaders, such as virus and phage. There are three stages of CRISPR locus function: integration of new sequences into the CRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreign invader nucleic acid. Five types of CRISPR systems (e.g., Type I, Type II, Type III, Type U, and Type V) have been identified.

[0120] A CRISPR locus includes a number of short repeating sequences referred to as “repeats.” When expressed, the repeats can form secondary structures (e.g., hairpins) and / or comprise unstructured single-stranded sequences. The repeats usually occur in clusters and frequently diverge between species. The repeats are regularly interspaced with unique intervening sequences referred to as “spacers,” resulting in a repeat-spacer-repeat locus architecture. The spacers are identical to or have high homology with known foreign invader sequences. A spacer-repeat unit encodes a crisprRNA (crRNA), which is processed into a mature form of the spacer-repeat unit. A crRNA comprises a “seed” or spacer sequence that is involved in targeting a target nucleic acid (in the naturally occurring form in prokaryotes, the spacer sequence targets the foreign invader nucleic acid). A spacer sequence is located at the 5' or 3' end of the crRNA.

[0121] A CRISPR locus also comprises polynucleotide sequences encoding CRISPR Associated (Cas) genes. Cas genes encode endonucleases involved in the biogenesis and the interference stages of crRNA function in prokaryotes. Some Cas genes comprise homologous secondary and / or tertiary structures.Type II CRISPR Systems

[0122] crRNA biogenesis in a Type II CRISPR system in nature requires a trans-activating CRISPR RNA (tracrRNA). The tracrRNA can be modified by endogenous RNaselll, and then hybridizes to a crRNA repeat in the pre-crRNA array. Endogenous RNaselll can be recruited to cleave the pre-crRNA. Cleaved crRNAs can be subjected to exoribonuclease trimming to produce the mature crRNA form (e.g., 5' trimming). The tracrRNA can remain hybridized to the crRNA, and the tracrRNA and the crRNA associate with a site-directed polypeptide (e.g., Cas9). The crRNA of the crRNA-tracrRNA-Cas9 complex can guide the complex to a target nucleic acid to which the crRNA can hybridize. Hybridization of the crRNA to the target nucleic acid can activate Cas9 for targeted nucleic acid cleavage. The target nucleic acid in a Type II CRISPR system is referred to as a protospacer adjacent motif (PAM). In nature, the PAM is essential to facilitate binding of a site-directed polypeptide (e.g., Cas9) to the target nucleic acid. Type II systems (also referred to as Nmeni or CASS4) are further subdivided into Type II-A (CASS4) and II-B (CASS4a). Jinek et al., Science, 337(6096):816-821 (2012) showed that the CRISPR / Cas9 system is useful for RNA- programmable genome editing, and international patent application publication number WO2013 / 176772 provides numerous examples and applications of the CRISPR / Cas endonuclease system for site-specific gene editing.Type V CRISPR Systems

[0123] Type V CRISPR systems have several important differences from Type II systems. For example, Cpfl is a single RNA-guided endonuclease that, in contrast to Type II systems, lacks tracrRNA. In fact, Cpfl -associated CRISPR arrays can be processed into mature crRNAs without the requirement of an additional trans-activating tracrRNA. The Type V CRISPR array can be processed into short mature crRNAs of 42-44 nucleotides in length, with each mature crRNA beginning with 19 nucleotides of direct repeat followed by 23-25 nucleotides of spacer sequence. In contrast, mature crRNAs in Type II systems can start with 20-24 nucleotides of spacer sequence followed by about 22 nucleotides of direct repeat. Also, Cpfl can utilize a T-rich protospacer-adjacent motif such that Cpfl -crRNA complexesefficiently cleave target DNA preceded by a short T-rich PAM, which is in contrast to the G- rich PAM following the target DNA for Type II systems. Thus, Type V systems cleave at a point that is distant from the PAM, while Type II systems cleave at a point that is adjacent to the PAM. In addition, in contrast to Type II systems, Cpfl cleaves DNA via a staggered DNA double-stranded break with a 4 or 5 nucleotide 5' overhang. Type II systems cleave via a blunt double-stranded break. Similar to Type II systems, Cpfl contains a predicted RuvC- like endonuclease domain, but lacks a second HNH endonuclease domain, which is in contrast to Type II systems.Cas Genes / Polypeptides and Protospacer Adjacent Motifs

[0124] Exemplary CRISPR / Cas polypeptides include the Cas9 polypeptides in FIG. 1 of Fonfara et al., Nucleic Acids Research, 42: 2577-2590 (2014). The CRISPR / Cas gene naming system has undergone extensive rewriting since the Cas genes were discovered. FIG. 5 of Fonfara, supra, provides PAM sequences for the Cas9 polypeptides from various species.Site-Directed Polypeptides

[0125] A site-directed polypeptide is a nuclease used in genome editing to cleave DNA. The site-directed nuclease or polypeptide can be administered to a cell or a patient as either: one or more polypeptides, or one or more mRNAs encoding the polypeptide.

[0126] In the context of a CRISPR / Cas or CRISPR / Cpfl system, the site-directed polypeptide can bind to a guide RNA that, in turn, specifies the site in the target DNA to which the polypeptide is directed. In the CRISPR / Cas or CRISPR / Cpfl systems disclosed herein, the site-directed polypeptide can be an endonuclease, such as a DNA endonuclease. Suitable DNA nucleases include, but are not limited to, Casl , Casl B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Casl OO, Csyl , Csy2, Csy3, Csel , Cse2, Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl , Cmr3, Cmr4, Cmr5, Cmr6, Csbl , Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl , Csxl5, Csfl , Csf2, Csf3, Csf4, Cpfl, and SluCas9.

[0127] A site-directed polypeptide can comprise a plurality of nucleic acid-cleaving (i.e., nuclease) domains. Two or more nucleic acid-cleaving domains can be linked together via a linker. For example, the linker can comprise a flexible linker. Linkers can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or more amino acids in length.

[0128] Naturally-occurring wild-type Cas9 enzymes comprise two nuclease domains, a HNH nuclease domain and a RuvC domain. Herein, the “Cas9” refers to both naturally- occurring and recombinant Cas9s. Cas9 enzymes contemplated herein can comprise a HNH or HNH-like nuclease domain, and / or a RuvC or RuvC-like nuclease domain.

[0129] HNH or HNH-like domains comprise a McrA-like fold. HNH or HNH-like domains comprises two antiparallel P-strands and an a-helix. HNH or HNH-like domains comprises a metal binding site (e.g., a divalent cation binding site). HNH or HNH-like domains can cleave one strand of a target nucleic acid (e.g., the complementary strand of the crRNA targeted strand).

[0130] RuvC or RuvC-like domains comprise an RNaseH or RnaseH-like fold. RuvC / RnaseH domains are involved in a diverse set of nucleic acid-based functions including acting on both RNA and DNA. The RnaseH domain comprises 5P-strands surrounded by a plurality of a-helices. RuvC / RnaseH or RuvC / RnaseH-like domains comprise a metal binding site (e.g., a divalent cation binding site). RuvC / RnaseH or RuvC / RnaseH-like domains can cleave one strand of a target nucleic acid (e.g., the non-complementary strand of a doublestranded target DNA).

[0131] Site-directed polypeptides can introduce double-strand breaks or single-strand breaks in nucleic acids, e.g., genomic DNA. The double-strand break can stimulate a cell's endogenous DNA-repair pathways (e.g., homology-dependent repair (HDR) or NHEJ or alternative non-homologous end joining (A-NHEJ) or microhomology-mediated end joining (MMEJ)). NHEJ can repair cleaved target nucleic acid without the need for a homologous template. This can sometimes result in small deletions or insertions (indels) in the target nucleic acid at the site of cleavage, and can lead to disruption or alteration of gene expression. HDR can occur when a homologous repair template, or donor, is available. The homologous donor template can comprise sequences that are homologous to sequences flanking the target nucleic acid cleavage site. The sister chromatid can be used by the cell as the repair template. However, for the purposes of genome editing, the repair template can be supplied as an exogenous nucleic acid, such as a plasmid, duplex oligonucleotide, singlestrand oligonucleotide or viral nucleic acid. With exogenous donor templates, an additional nucleic acid sequence (such as a transgene) or modification (such as a single or multiple base change or a deletion) can be introduced between the flanking regions of homology so that the additional or altered nucleic acid sequence also becomes incorporated into the target locus. MMEJ can result in a genetic outcome that is similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ can make use of homologous sequences of afew basepairs flanking the cleavage site to drive a favored end-joining DNA repair outcome. In some instances it may be possible to predict likely repair outcomes based on analysis of potential microhomologies in the nuclease target regions.

[0132] Thus, in some cases, homologous recombination can be used to insert an exogenous polynucleotide sequence into the target nucleic acid cleavage site. An exogenous polynucleotide sequence is termed a donor polynucleotide (or donor or donor sequence) herein. The donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide can be inserted into the target nucleic acid cleavage site. The donor polynucleotide can be an exogenous polynucleotide sequence, i.e., a sequence that does not naturally occur at the target nucleic acid cleavage site.

[0133] The modifications of the target DNA due to NHEJ and / or HDR can lead to, for example, mutations, deletions, alterations, integrations, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocations and / or gene mutation. The processes of deleting genomic DNA and integrating non-native nucleic acid into genomic DNA are examples of genome editing.

[0134] The site-directed polypeptide can comprise an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity to a wild-type exemplary site-directed polypeptide [e.g., Cas9 from S. pyogenes, US2014 / 0068797 Sequence ID No. 8 or Sapranauskas et al., Nucleic Acids Res, 39(21): 9275-9282 (2011), both of which are incorporated herein by reference in their entirety], and various other site-directed polypeptides. The site-directed polypeptide can comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids. The site- directed polypeptide can comprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids. The site-directed polypeptide can comprise at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a HNH nuclease domain of the site-directed polypeptide. The site-directed polypeptide can comprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a HNH nuclease domain of the site-directed polypeptide. The site-directed polypeptide can comprise at least: 70, 75, 80, 85, 90, 95, 97,99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a RuvC nuclease domain of the site-directed polypeptide. The site-directed polypeptide can comprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a RuvC nuclease domain of the site-directed polypeptide.

[0135] The site-directed polypeptide can comprise a modified form of a wild-type exemplary site-directed polypeptide. The modified form of the wild-type exemplary site- directed polypeptide can comprise a mutation that reduces the nucleic acid-cleaving activity of the site-directed polypeptide. The modified form of the wild-type exemplary site-directed polypeptide can have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity of the wild-type exemplary site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra). The modified form of the site-directed polypeptide can have no substantial nucleic acid-cleaving activity. When a site-directed polypeptide is a modified form that has no substantial nucleic acid-cleaving activity, it is referred to herein as “enzymatically inactive.”

[0136] The modified form of the site-directed polypeptide can comprise a mutation such that it can induce a single-strand break (SSB) on a target nucleic acid (e.g., by cutting only one of the sugar-phosphate backbones of a double-strand target nucleic acid). The mutation can result in less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity in one or more of the plurality of nucleic acid-cleaving domains of the wild-type site directed polypeptide (e.g., Cas9 from S. pyogenes, supra). The mutation can result in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the complementary strand of the target nucleic acid, but reducing its ability to cleave the non-complementary strand of the target nucleic acid. The mutation can result in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the non-complementary strand of the target nucleic acid, but reducing its ability to cleave the complementary strand of the target nucleic acid. For example, residues in the wild-type exemplary S. pyogenes Cas9 polypeptide, such as AsplO, His840, Asn854 and Asn856, are mutated to inactivate one or more of the plurality of nucleic acid-cleaving domains (e.g., nuclease domains). The residues to be mutated can correspond to residues AsplO, His840, Asn854 and Asn856 in the wild-type exemplary S. pyogenes Cas9 polypeptide (e.g., asdetermined by sequence and / or structural alignment). Non-limiting examples of mutations include D10A, H840A, N854A or N856A. One skilled in the art will recognize that mutations other than alanine substitutions can be suitable.

[0137] A D10A mutation can be combined with one or more of H840A, N854A, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity. A H840A mutation can be combined with one or more of D10A, N854A, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity. A N854A mutation can be combined with one or more of H840A, D10A, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity. A N856A mutation can be combined with one or more of H840A, N854A, or D10A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity. Site-directed polypeptides that comprise one substantially inactive nuclease domain are referred to as “nickases”.

[0138] Nickase variants of RNA-guided endonucleases, for example Cas9, can be used to increase the specificity of CRISPR-mediated genome editing. Wild type Cas9 is typically guided by a single guide RNA designed to hybridize with a specified ~20 nucleotide sequence in the target sequence (such as an endogenous genomic locus). However, several mismatches can be tolerated between the guide RNA and the target locus, effectively reducing the length of required homology in the target site to, for example, as little as 13 nt of homology, and thereby resulting in elevated potential for binding and double-strand nucleic acid cleavage by the CRISPR / Cas9 complex elsewhere in the target genome — also known as off-target cleavage. Because nickase variants of Cas9 each only cut one strand, in order to create a double-strand break it is necessary for a pair of nickases to bind in close proximity and on opposite strands of the target nucleic acid, thereby creating a pair of nicks, which is the equivalent of a double-strand break. This requires that two separate guide RNAs — one for each nickase — must bind in close proximity and on opposite strands of the target nucleic acid. This requirement essentially doubles the minimum length of homology needed for the double-strand break to occur, thereby reducing the likelihood that a double-strand cleavage event will occur elsewhere in the genome, where the two guide RNA sites — if they exist — are unlikely to be sufficiently close to each other to enable the double-strand break to form. As described in the art, nickases can also be used to promote HDR versus NHEJ. HDR can be used to introduce selected changes into target sites in the genome through the use of specific donor sequences that effectively mediate the desired changes.

[0139] Mutations contemplated can include substitutions, additions, and deletions, or any combination thereof. The mutation converts the mutated amino acid to alanine. The mutation converts the mutated amino acid to another amino acid (e.g., glycine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagines, glutamine, histidine, lysine, or arginine). The mutation converts the mutated amino acid to a non -natural amino acid (e.g., selenomethionine). The mutation converts the mutated amino acid to amino acid mimics (e.g., phosphomimics). The mutation can be a conservative mutation. For example, the mutation converts the mutated amino acid to amino acids that resemble the size, shape, charge, polarity, conformation, and / or rotamers of the mutated amino acids (e.g., cysteine / serine mutation, lysine / asparagine mutation, histidine / phenylalanine mutation). The mutation can cause a shift in reading frame and / or the creation of a premature stop codon. Mutations can cause changes to regulatory regions of genes or loci that affect expression of one or more genes.

[0140] The site-directed polypeptide (e.g., variant, mutated, enzymatically inactive and / or conditionally enzymatically inactive site-directed polypeptide) can target nucleic acid. The site-directed polypeptide (e.g., variant, mutated, enzymatically inactive and / or conditionally enzymatically inactive endoribonuclease) can target DNA. The site-directed polypeptide (e.g., variant, mutated, enzymatically inactive and / or conditionally enzymatically inactive endoribonuclease) can target RNA.

[0141] The site-directed polypeptide can comprise one or more non-native sequences (e.g., the site-directed polypeptide is a fusion protein).

[0142] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), a nucleic acid binding domain, and two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain).

[0143] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain).

[0144] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), and two nucleic acid cleaving domains, wherein one or both of the nucleic acid cleaving domains comprise at least 50% amino acid identity to a nuclease domain from Cas9 from a bacterium (e.g., S. pyogenes).

[0145] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain), and non-native sequence (for example, a nuclear localization signal) or a linker linking the site-directed polypeptide to a non-native sequence.

[0146] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain), wherein the site-directed polypeptide comprises a mutation in one or both of the nucleic acid cleaving domains that reduces the cleaving activity of the nuclease domains by at least 50%.

[0147] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to a Cas9 from a bacterium (e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain), wherein one of the nuclease domains comprises mutation of aspartic acid 10, and / or wherein one of the nuclease domains can comprise a mutation of histidine 840, and wherein the mutation reduces the cleaving activity of the nuclease domain(s) by at least 50%.

[0148] The one or more site-directed polypeptides, e.g. DNA endonucleases, can comprise two nickases that together effect one double-strand break at a specific locus in the genome, or four nickases that together effect or cause two double-strand breaks at specific loci in the genome. Alternatively, one site-directed polypeptide, e.g. DNA endonuclease, can effect or cause one double-strand break at a specific locus in the genome.

[0149] The site-directed polypeptide can be flanked at the N-terminus, the C-terminus, or both the N-terminus and C-terminus by one or more nuclear localization signals (NLSs). For example, a Cas9 endonuclease can be flanked by two NLSs, one NLS located at the N- terminus and the second NLS located at the C-terminus. The NLS can be any NLS known in the art, such as a SV40 NLS.Additional exemplary editing

[0150] In some embodiments, a gene can be edited using additional exemplary editing. In some embodiments, additional editing can be used to introduce a loss-of-function mutation (e.g., premature stop codons, destabilizing mutations, altering splicing, etc.). In other embodiments, the editing can be used to correct a mutation (e.g., a disease-causing mutation). The editing systems generally include an editor comprising a polynucleotide programmable nucleotide binding domain (e.g., a nickase Cas9) and a DNA-polymerase domain (e.g., areverse transcriptase (RT), such as a Moloney murine leukemia virus reverse transcriptase (M-MLV RT)). The guide nucleic acid can contain an editing template. The gRNA can also include a primer-binding site (PBS). The PBS may be designed to hybridize with the displaced strand on the 5’ side of the introduced cut generated by the nickase. The PBS may be complementary to a portion of the protospacer sequence. The editing template sequence includes the edit to be installed and is typically located between the tracr region (e.g., scaffold or core region) and the PBS. The length of the edit to be installed may vary, e.g., from deletions of 10 or fewer nucleotides to insertions of more than 80 nucleotides. In some embodiments, the edit comprises a substitution of 1 or more nucleotides.

[0151] In some embodiments, the target sequence is bound by the nickase Cas9 (e.g., Cas9-H840A domain) via the spacer region of the guide RNA (gRNA). The hybridization of the spacer sequence to the complementary target sequences can result in displacement of the other strand (e.g., the PAM-strand or the edit strand). The Cas9-H840A domain can cut the displaced strand, and the displaced strand then may pair with the PBS. The RT may recognize the RNA-DNA duplex formed by the displaced strand and PBS and extend the DNA of the displaced strand in the 3’ direction, using the editing template (e.g., RT template) of the gRNA as a template. This can create a “flap” of single- stranded DNA on the displaced strand including the desired edit. The editor may then dissociate from the DNA, leaving two redundant “flaps” on the displaced strand, wherein one flap is the original sequence, and one flap is the edited sequence. Through a process called “flap equilibration”, one of the sequences will bind the target sequence, and the other will remain attached to the displaced strand as a single-stranded flap. If the flap with the edited sequence is bound by the target sequence, the complex may be called a “DNA heteroduplex”, in view of the mismatch caused by the edit. Cellular DNA repair machinery may then act on the DNA heteroduplex, incorporating the edit.

[0152] In some embodiments, where an editor comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can include a D10A mutation and a histidine at position 840. In another example, a Cas9-derived nickase domain comprises an H840A mutation, while the amino acid residue at position 10 remains a D. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase, referred to as an “nCas9” protein. The Cas9 nickase may be a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g., a duplexed DNA molecule). Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the field and are within the scope of thisdisclosure. In some embodiments, an editor comprises an RNA-dependent DNA polymerase domain, such as a reverse transcriptase (RT). In some embodiments, an editor comprises a virus RT, such as a retrovirus RT (e.g., Moloney murine leukemia virus (M-MLV or MLVRT)). In some embodiments, an editor may comprise a fusion of an S. pyogenes Cas9 polypeptide and a Moloney murine leukemia virus (M-MLV) reverse transcriptase polypeptide.

[0153] The gRNA can refer to a guide polynucleotide that comprises one or more intended nucleotide edits for incorporation into the target DNA. In some embodiments, the gRNA associates with and directs an editor to incorporate the one or more intended nucleotide edits into the target gene via editing. “Nucleotide edit” or “intended nucleotide edit” shall be given their ordinary meaning and shall also refer to a specified deletion of one or more nucleotides at one specific position, insertion of one or more nucleotides at one specific position, substitution of a single nucleotide, or other alterations at one specific position to be incorporated into the sequence of the target gene. Intended nucleotide edit may refer to the edit on the editing template as compared to the sequence on the target strand of the target gene or may refer to the edit encoded by the editing template on the newly synthesized single stranded DNA. In some embodiments, a gRNA comprises a spacer sequence that is complementary or substantially complementary to a sequence on a target strand of the target gene. In some embodiments, the gRNA comprises a gRNA core that associates with a DNA binding domain, e.g., a CRISPR-Cas protein domain, of an editor. In some embodiments, the gRNA further comprises an extended nucleotide sequence comprising one or more intended nucleotide edits compared to the endogenous sequence of the target gene, wherein the extended nucleotide sequence may be referred to as an extension arm.

[0154] The extension arm can comprise a primer binding site sequence (PBS) that can initiate target-primed DNA synthesis. In some embodiments, the PBS is complementary or substantially complementary to a free 3’ end on the edit strand of the target gene at a nick site generated by the editor. In some embodiments, the extension arm further comprises an editing template that comprises one or more intended nucleotide edits to be incorporated in the target gene by editing. In some embodiments, the editing template is a template for an RNA-dependent DNA polymerase domain or polypeptide of the editor, for example, a reverse transcriptase domain. In some embodiments, the editing template comprises partial complementarity to an editing target sequence in the target gene. In some embodiments, the editing template comprises substantial or partial complementarity to the editing targetsequence except at the position(s) of the intended nucleotide edit(s) to be incorporated into the target gene.10155] Some editors include a Cas9 variant comprising an H840A mutation (i.e., a Cas9 nickase) and an M-MLV RT wild type, as well as an N-terminal NLS sequence (19 amino acids) and an amino acid linker (32 amino acids) that joins the C-terminus of the Cas9 nickase domain to the N-terminus of the RT domain. The fusion protein can have the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(wt)]. The editor proteins can, in some instances, include a Cas9 variant comprising an H840A mutation (i.e., a Cas9 nickase) and an M-MLV RT comprising mutations D200N, T330P, L603W, T306K, and W313F, as well as an N-terminal NLS sequence (19 amino acids) and an amino acid linker (33 amino acids) that joins the C-terminus of the Cas9 nickase domain to the N-terminus of the RT domain.

[0156] In some embodiments, the editing system or composition further comprises a nick guide polynucleotide, such as a nick guide RNA (ngRNA). Without wishing to be bound by any particular theory, the non-edit strand of a double stranded target DNA in the target gene may be nicked by a CRISPR-Cas nickase directed by an ngRNA. In some embodiments, the nick on the non-edit strand directs endogenous DNA repair machinery to use the edit strand as a template for repair of the non-edit strand, which may increase efficiency of editing. Some editor systems have an editor plus a second-strand nicking guide RNA that complexes with the editor and introduces a nick in the non-edited DNA strand in order to induce preferential replacement of the edited strand. In some editors, the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing a gRNA with a spacer sequence that matches only the edited strand, but not the original allele. Using this strategy, mismatches between the protospacer and the unedited allele should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place. Some additional editors comprise a fusion protein comprising Cas9(R221K N39K H840A) and a variant MMLV RT pentamutant (D200N T306K W313F T330P L603W) having the following structure: [bipartite NLS]-[Cas9(R221K)(N394K)(H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)]-[bipartite NLS]-[NLS] + a desired gRNA.

[0157] Some of the methods and compositions related to the editing disclosed herein are also described in W02023015309, W02022150790, W02022067130, WO2020191233, WO2020191234, WO2020191239, W02020191241, WO2020191242, WO2020191243, WO2020191245, WO2020191246, WO2020191248, WO2020191249, W02020191153, andW02020191171, the contents of which are incorporated herein by reference in their entireties.10158] Some of the methods, compositions, and kits related to RT editing are described in PCT Application No. PCT / IB2025 / 052079, entitled, “RT EDITING COMPOSITIONS AND METHODS,” filed February 26, 2025, the content of which is incorporated herein by reference in its entirety.Genome-Targeting Nucleic Acid

[0159] The present disclosure provides a genome-targeting nucleic acid that can direct the activities of an associated polypeptide (e.g., a site-directed polypeptide) to a specific target sequence within a target nucleic acid. The genome-targeting nucleic acid can be an RNA. A genome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein. A guide RNA can comprise at least a spacer sequence that hybridizes to a target nucleic acid sequence of interest, and a CRISPR repeat sequence. In Type II systems, the gRNA also comprises a second RNA called the tracrRNA sequence. In the Type II guide RNA (gRNA), the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. In the Type V guide RNA (gRNA), the crRNA forms a duplex. In both systems, the duplex can bind a site-directed polypeptide, such that the guide RNA and site-direct polypeptide form a complex. The genome-targeting nucleic acid can provide target specificity to the complex by virtue of its association with the site-directed polypeptide. The genome-targeting nucleic acid thus can direct the activity of the site-directed polypeptide.

[0160] The genome-targeting nucleic acid can be a double-molecule guide RNA. The genome-targeting nucleic acid can be a single-molecule guide RNA.

[0161] A double-molecule guide RNA can comprise two strands of RNA. The first strand comprises in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence and a minimum CRISPR repeat sequence. The second strand can comprise a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3' tracrRNA sequence and an optional tracrRNA extension sequence.

[0162] A single-molecule guide RNA (sgRNA) in a Type II system can comprise, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3' tracrRNA sequence and an optional tracrRNA extension sequence. The optional tracrRNA extension can comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker can link the minimum CRISPR repeat and theminimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can comprise one or more hairpins.10163] A single-molecule guide RNA (sgRNA) in a Type V system can comprise, in the 5' to 3' direction, a minimum CRISPR repeat sequence and a spacer sequence.

[0164] The sgRNA can comprise a 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence. The sgRNA can comprise a less than a 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence. The sgRNA can comprise a more than 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence. The sgRNA can comprise a variable length spacer sequence with 17-30 nucleotides at the 5' end of the sgRNA sequence (see, e.g., Table 1 of U.S Pat. Publ. 2022 / 0211874, which is incorporated herein by reference in its entirety).

[0165] The sgRNA can comprise no uracil at the 3 'end of the sgRNA sequence. The sgRNA can comprise one or more uracil at the 3 'end of the sgRNA sequence. For example, the sgRNA can comprise 1 uracil (U) at the 3' end of the sgRNA sequence. The sgRNA can comprise 2 uracil (UU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 3 uracil (UUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 4 uracil (UUUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 5 uracil (UUUUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 6 uracil (UUUUUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 7 uracil (UUUUUUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 8 uracil (UUUUUUUU) at the 3' end of the sgRNA sequence.

[0166] The sgRNA can be unmodified or modified. For example, modified sgRNAs can comprise one or more 2'-O-methyl phosphorothioate nucleotides.

[0167] By way of illustration, guide RNAs used in the CRISPR / Cas / Cpfl system, or other smaller RNAs can be readily synthesized by chemical means, as illustrated below and described in the art. While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high performance liquid chromatography (H PLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides. One approach used for generating RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding a Cas9 or Cpfl endonuclease, are more readily generated enzymatically. Various types of RNA modifications can be introduced during or after chemical synthesis and / or enzymaticgeneration of RNAs, e.g., modifications that enhance stability, reduce the likelihood or degree of innate immune response, and / or enhance other attributes, as described in the art.Spacer Extension Sequence

[0168] In some examples of genome-targeting nucleic acids, a spacer extension sequence can modify activity, provide stability and / or provide a location for modifications of a genome-targeting nucleic acid. A spacer extension sequence can modify on- or off-target activity or specificity. In some examples, a spacer extension sequence can be provided. The spacer extension sequence can have a length of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, or 7000 or more nucleotides. The spacer extension sequence can have a length of less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, 7000 or more nucleotides. The spacer extension sequence can be less than 10 nucleotides in length. The spacer extension sequence can be between 10-30 nucleotides in length. The spacer extension sequence can be between 30-70 nucleotides in length.

[0169] The spacer extension sequence can comprise another moiety (e.g., a stability control sequence, an endoribonuclease binding sequence, a ribozyme). The moiety can decrease or increase the stability of a nucleic acid targeting nucleic acid. The moiety can be a transcriptional terminator segment (i.e., a transcription termination sequence). The moiety can function in a eukaryotic cell. The moiety can function in a prokaryotic cell. The moiety can function in both eukaryotic and prokaryotic cells. Non-limiting examples of suitable moieties include: a 5' cap (e.g., a 7-methylguanylate cap (m7 G)), a riboswitch sequence (e.g., to allow for regulated stability and / or regulated accessibility by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, and the like), a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.), and / or a modification or sequence that provides a binding site for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional controls, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and the like).Spacer Sequence

[0170] The spacer sequence hybridizes to a sequence in a target nucleic acid of interest. The spacer of a genome-targeting nucleic acid can interact with a target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing). The nucleotide sequence of the spacer can vary depending on the sequence of the target nucleic acid of interest.

[0171] In a CRISPR / Cas system herein, the spacer sequence can be designed to hybridize to a target nucleic acid that is located 5' of a PAM of the Cas9 enzyme used in the system. The spacer may perfectly match the target sequence or may have mismatches. Each Cas9 enzyme has a particular PAM sequence that it recognizes in a target DNA. For example, S. pyogenes recognizes in a target nucleic acid a PAM that comprises the sequence 5'-NRG-3', where R comprises either A or G, where N is any nucleotide and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence.

[0172] The target nucleic acid sequence can comprise 20 nucleotides. The target nucleic acid can comprise less than 20 nucleotides. The target nucleic acid can comprise more than 20 nucleotides. The target nucleic acid can comprise at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. The target nucleic acid can comprise at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. The target nucleic acid sequence can comprise 20 bases immediately 5' of the first nucleotide of the PAM. For example, in a sequence comprising 5'-NNNNNNNNNNNNNNNNNNNNNRG-3' (SEQ ID NO: 1), the target nucleic acid can comprise the sequence that corresponds to the Ns, wherein N is any nucleotide, and the underlined NRG sequence is the S. pyogenes PAM.

[0173] The spacer sequence that hybridizes to the target nucleic acid can have a length of at least about 6 nucleotides (nt). The spacer sequence can be at least about 6 nt, at least about 10 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt or at least about 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50 nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, from about 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt to about 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about 45 nt, from about 10 nt to about 40 nt, from about 10 nt to about 35 nt, from about 10 nt to about 30 nt, from about 10 nt to about 25 nt, from about 10 nt to about 20 nt, from about 10 nt to about 19 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt toabout 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt. In some examples, the spacer sequence can comprise 20 nucleotides. In some examples, the spacer can comprise 19 nucleotides.

[0174] In some examples, the percent complementarity between the spacer sequence and the target nucleic acid is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. In some examples, the percent complementarity between the spacer sequence and the target nucleic acid is at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, at most about 97%, at most about 98%, at most about 99%, or 100%. In some examples, the percent complementarity between the spacer sequence and the target nucleic acid is 100% over the six contiguous 5 '-most nucleotides of the target sequence of the complementary strand of the target nucleic acid. The percent complementarity between the spacer sequence and the target nucleic acid can be at least 60% over about 20 contiguous nucleotides. The length of the spacer sequence and the target nucleic acid can differ by 1 to 6 nucleotides, which may be thought of as a bulge or bulges.

[0175] The spacer sequence can be designed or chosen using a computer program. The computer program can use variables, such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, % GC, frequency of genomic occurrence (e.g., of sequences that are identical or are similar but vary in one or more spots as a result of mismatch, insertion or deletion), methylation status, presence of SNPs, and the like.Minimum CRISPR Repeat Sequence

[0176] A minimum CRISPR repeat sequence can be a sequence with at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference CRISPR repeat sequence (e.g., crRNA from S. pyogenes).

[0177] A minimum CRISPR repeat sequence can comprise nucleotides that can hybridize to a minimum tracrRNA sequence in a cell. The minimum CRISPR repeat sequence and a minimum tracrRNA sequence can form a duplex, i.e. a base-paired double-stranded structure. Together, the minimum CRISPR repeat sequence and the minimum tracrRNA sequence canbind to the site-directed polypeptide. At least a part of the minimum CRISPR repeat sequence can hybridize to the minimum tracrRNA sequence. At least a part of the minimum CRISPR repeat sequence can comprise at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementary to the minimum tracrRNA sequence. At least a part of the minimum CRISPR repeat sequence can comprise at most about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementary to the minimum tracrRNA sequence.

[0178] The minimum CRISPR repeat sequence can have a length from about 7 nucleotides to about 100 nucleotides. For example, the length of the minimum CRISPR repeat sequence is from about 7 nucleotides (nt) to about 50 nt, from about 7 nt to about 40 nt, from about 7 nt to about 30 nt, from about 7 nt to about 25 nt, from about 7 nt to about 20 nt, from about 7 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt to about 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt, or from about 15 nt to about 25 nt. In some examples, the minimum CRISPR repeat sequence can be approximately 9 nucleotides in length. The minimum CRISPR repeat sequence can be approximately 12 nucleotides in length.

[0179] The minimum CRISPR repeat sequence can be at least about 60% identical to a reference minimum CRISPR repeat sequence (e.g., wild-type crRNA from S. pyogenes) over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the minimum CRISPR repeat sequence can be at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical or 100% identical to a reference minimum CRISPR repeat sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.Minimum tracrRNA Sequence

[0180] A minimum tracrRNA sequence can be a sequence with at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., wild type tracrRNA from S. pyogenes).

[0181] A minimum tracrRNA sequence can comprise nucleotides that hybridize to a minimum CRISPR repeat sequence in a cell. A minimum tracrRNA sequence and a minimum CRISPR repeat sequence form a duplex, i.e. a base-paired double-stranded structure.Together, the minimum tracrRNA sequence and the minimum CRISPR repeat can bind to a site-directed polypeptide. At least a part of the minimum tracrRNA sequence can hybridize to the minimum CRISPR repeat sequence. The minimum tracrRNA sequence can be at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementary to the minimum CRISPR repeat sequence.

[0182] The minimum tracrRNA sequence can have a length from about 7 nucleotides to about 100 nucleotides. For example, the minimum tracrRNA sequence can be from about 7 nucleotides (nt) to about 50 nt, from about 7 nt to about 40 nt, from about 7 nt to about 30 nt, from about 7 nt to about 25 nt, from about 7 nt to about 20 nt, from about 7 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt to about 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt or from about 15 nt to about 25 nt long. The minimum tracrRNA sequence can be approximately 9 nucleotides in length. The minimum tracrRNA sequence can be approximately 12 nucleotides. The minimum tracrRNA can consist of tracrRNA nt 23-48 described in Jinek et al., supra.

[0183] The minimum tracrRNA sequence can be at least about 60% identical to a reference minimum tracrRNA (e.g., wild type, tracrRNA from S. pyogenes) sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the minimum tracrRNA sequence can be at least about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 98% identical, about 99% identical or 100% identical to a reference minimum tracrRNA sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.

[0184] The duplex between the minimum CRISPR RNA and the minimum tracrRNA can comprise a double helix. The duplex between the minimum CRISPR RNA and the minimum tracrRNA can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. The duplex between the minimum CRISPR RNA and the minimum tracrRNA can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides.

[0185] The duplex can comprise a mismatch (i.e., the two strands of the duplex are not 100% complementary). The duplex can comprise at least about 1, 2, 3, 4, or 5 or mismatches.The duplex can comprise at most about 1, 2, 3, 4, or 5 or mismatches. The duplex can comprise no more than 2 mismatches.Bulges

[0186] In some cases, there can be a “bulge” in the duplex between the minimum CRISPR RNA and the minimum tracrRNA. A bulge is an unpaired region of nucleotides within the duplex. A bulge can contribute to the binding of the duplex to the site-directed polypeptide. The bulge can comprise, on one side of the duplex, an unpaired 5'-XXXY-3' where X is any purine and Y comprises a nucleotide that can form a wobble pair with a nucleotide on the opposite strand, and an unpaired nucleotide region on the other side of the duplex. The number of unpaired nucleotides on the two sides of the duplex can be different.

[0187] In one example, the bulge can comprise an unpaired purine (e.g., adenine) on the minimum CRISPR repeat strand of the bulge. In some examples, the bulge can comprise an unpaired 5'-AAGY-3' of the minimum tracrRNA sequence strand of the bulge, where Y comprises a nucleotide that can form a wobble pairing with a nucleotide on the minimum CRISPR repeat strand.

[0188] A bulge on the minimum CRISPR repeat side of the duplex can comprise at least 1, 2, 3, 4, or 5 or more unpaired nucleotides. A bulge on the minimum CRISPR repeat side of the duplex can comprise at most 1, 2, 3, 4, or 5 or more unpaired nucleotides. A bulge on the minimum CRISPR repeat side of the duplex can comprise 1 unpaired nucleotide.

[0189] A bulge on the minimum tracrRNA sequence side of the duplex can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. A bulge on the minimum tracrRNA sequence side of the duplex can comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. A bulge on a second side of the duplex (e.g., the minimum tracrRNA sequence side of the duplex) can comprise 4 unpaired nucleotides.

[0190] A bulge can comprise at least one wobble pairing. In some examples, a bulge can comprise at most one wobble pairing. A bulge can comprise at least one purine nucleotide. A bulge can comprise at least 3 purine nucleotides. A bulge sequence can comprise at least 5 purine nucleotides. A bulge sequence can comprise at least one guanine nucleotide. In some examples, a bulge sequence can comprise at least one adenine nucleotide.Hairpins

[0191] In various examples, one or more hairpins can be located 3' to the minimum tracrRNA in the 3' tracrRNA sequence.

[0192] The hairpin can start at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more nucleotides 3' from the last paired nucleotide in the minimum CRISPR repeat and minimum tracrRNA sequence duplex. The hairpin can start at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides 3' of the last paired nucleotide in the minimum CRISPR repeat and minimum tracrRNA sequence duplex.

[0193] The hairpin can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more consecutive nucleotides. The hairpin can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more consecutive nucleotides.

[0194] The hairpin can comprise a CC dinucleotide (i.e., two consecutive cytosine nucleotides).

[0195] The hairpin can comprise duplexed nucleotides (e.g., nucleotides in a hairpin, hybridized together). For example, a hairpin can comprise a CC dinucleotide that is hybridized to a GG dinucleotide in a hairpin duplex of the 3' tracrRNA sequence.

[0196] One or more of the hairpins can interact with guide RNA-interacting regions of a site-directed polypeptide.3 ' tracrRNA Sequence

[0197] A 3' tracrRNA sequence can comprise a sequence with at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., a tracrRNA from S. pyogenes).

[0198] The 3' tracrRNA sequence can have a length from about 6 nucleotides to about 100 nucleotides. For example, the 3' tracrRNA sequence can have a length from about 6 nucleotides (nt) to about 50 nt, from about 6 nt to about 40 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt to about 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt, or from about 15 nt to about 25 nt. The 3' tracrRNA sequence can have a length of approximately 14 nucleotides.

[0199] The 3' tracrRNA sequence can be at least about 60% identical to a reference 3' tracrRNA sequence (e.g., wild type 3' tracrRNA sequence from S. pyogenes) over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the 3' tracrRNA sequence can be at least about 60% identical, about 65% identical, about 70% identical, about 75% identical,about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 98% identical, about 99% identical, or 100% identical, to a reference 3' tracrRNA sequence (e.g., wild type 3' tracrRNA sequence from S. pyogenes) over a stretch of at least 6, 7, or 8 contiguous nucleotides.

[0200] The 3' tracrRNA sequence can comprise more than one duplexed region (e.g., hairpin, hybridized region). The 3' tracrRNA sequence can comprise two duplexed regions.

[0201] The 3' tracrRNA sequence can comprise a stem loop structure. The stem loop structure in the 3' tracrRNA can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 or more nucleotides. The stem loop structure in the 3' tracrRNA can comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides. The stem loop structure can comprise a functional moiety. For example, the stem loop structure can comprise an aptamer, a ribozyme, a proteininteracting hairpin, a CRISPR array, an intron, or an exon. The stem loop structure can comprise at least about 1, 2, 3, 4, or 5 or more functional moieties. The stem loop structure can comprise at most about 1, 2, 3, 4, or 5 or more functional moieties.

[0202] The hairpin in the 3' tracrRNA sequence can comprise a P-domain. In some examples, the P-domain can comprise a double-stranded region in the hairpin. tracrRNA Extension Sequence

[0203] A tracrRNA extension sequence may be provided whether the tracrRNA is in the context of single-molecule guides or double-molecule guides. The tracrRNA extension sequence can have a length from about 1 nucleotide to about 400 nucleotides. The tracrRNA extension sequence can have a length of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400 nucleotides. The tracrRNA extension sequence can have a length from about 20 to about 5000 or more nucleotides. The tracrRNA extension sequence can have a length of more than 1000 nucleotides. The tracrRNA extension sequence can have a length of less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 or more nucleotides. The tracrRNA extension sequence can have a length of less than 1000 nucleotides. The tracrRNA extension sequence can comprise less than 10 nucleotides in length. The tracrRNA extension sequence can be 10-30 nucleotides in length. The tracrRNA extension sequence can be 30-70 nucleotides in length.

[0204] The tracrRNA extension sequence can comprise a functional moiety (e.g., a stability control sequence, ribozyme, endoribonuclease binding sequence). The functional moiety can comprise a transcriptional terminator segment (i.e., a transcription terminationsequence). The functional moiety can have a total length from about 10 nucleotides (nt) to about 100 nucleotides, from about 10 nt to about 20 nt, from about 20 nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90 nt to about 100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt, or from about 15 nt to about 25 nt. The functional moiety can function in a eukaryotic cell. The functional moiety can function in a prokaryotic cell. The functional moiety can function in both eukaryotic and prokaryotic cells.

[0205] Non-limiting examples of suitable tracrRNA extension functional moieties include a 3' poly-adenylated tail, a riboswitch sequence (e.g., to allow for regulated stability and / or regulated accessibility by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, and the like), a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.), and / or a modification or sequence that provides a binding site for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional controls, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and the like). The tracrRNA extension sequence can comprise a primer binding site or a molecular index (e.g., barcode sequence). The tracrRNA extension sequence can comprise one or more affinity tags.Single-Molecule Guide Linker Sequence

[0206] The linker sequence of a single-molecule guide nucleic acid can have a length from about 3 nucleotides to about 100 nucleotides. In Jinek et al., supra, for example, a simple 4 nucleotide “tetraloop” (-GAAA-) was used, Science, 337(6096):816-821 (2012). An illustrative linker has a length from about 3 nucleotides (nt) to about 90 nt, from about 3 nt to about 80 nt, from about 3 nt to about 70 nt, from about 3 nt to about 60 nt, from about 3 nt to about 50 nt, from about 3 nt to about 40 nt, from about 3 nt to about 30 nt, from about 3 nt to about 20 nt, from about 3 nt to about 10 nt. For example, the linker can have a length from about 3 nt to about 5 nt, from about 5 nt to about 10 nt, from about 10 nt to about 15 nt, from about 15 nt to about 20 nt, from about 20 nt to about 25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 35 nt, from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about80 nt, from about 80 nt to about 90 nt, or from about 90 nt to about 100 nt. The linker of a single-molecule guide nucleic acid can be between 4 and 40 nucleotides. The linker can be at least about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides. The linker can be at most about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides.

[0207] Linkers can comprise any of a variety of sequences, although in some examples the linker will not comprise sequences that have extensive regions of homology with other portions of the guide RNA, which might cause intramolecular binding that could interfere with other functional regions of the guide. In Jinek et al., supra, a simple 4 nucleotide sequence -GAAA- was used, Science, 337(6096):816-821 (2012), but numerous other sequences, including longer sequences can likewise be used.

[0208] The linker sequence can comprise a functional moiety. For example, the linker sequence can comprise one or more features, including an aptamer, a ribozyme, a proteininteracting hairpin, a protein binding site, a CRISPR array, an intron, or an exon. The linker sequence can comprise at least about 1, 2, 3, 4, or 5 or more functional moieties. In some examples, the linker sequence can comprise at most about 1, 2, 3, 4, or 5 or more functional moieties.

[0209] A step of the in vivo methods of the present disclosure can comprise editing the patient HPSC using genome engineering. Likewise, a step of the in vivo methods of the present disclosure can comprise editing HPSC in a patient having hemoglobinopathy, primary immunodeficiency or congenital cytopenia using genome engineering.

[0210] Different patients with hemoglobinopathy, primary immunodeficiency or congenital cytopenia will generally require different deletion, modulation, or inactivation strategies. Any CRISPR endonuclease may be used in the methods of the present disclosure, each CRISPR endonuclease having its own associated PAM, which may or may not be disease specific. For example, CRISPR gene editing system components for the treatment of Sickle cell disease by altering the expression of the BCL11 A gene are disclosed in U.S. Pat. Publ. 2022 / 0211874, which is incorporated by reference herein in its entirety.

[0211] For example, the transcriptional control sequence of the BCL11 A gene can be modulated or inactivated by deletions that arise due to the NHEJ pathway. NHEJ can be used to delete segments of the transcriptional control sequence of the BCL11 A gene, either directly or by altering splice donor or acceptor sites through cleavage by one gRNA targeting several locations, or several gRNAs.

[0212] Fetal hemoglobin (HbF, a2y2) is the main oxygen transport protein in a human fetus and includes alpha (a) and gamma (y) subunits. HbF expression ceases about 6 months after birth. Adult hemoglobin (HbA, a2p2) is the main oxygen transport protein in a human after ~34 weeks from birth, and includes alpha (a) and beta (P) subunits. After 34 weeks, a developmental switch results in decreased transcription of the y-globin genes and increased transcription of P-globin genes. Since many of the forms of hemoglobinopathies are a result of the failure to produce normal P-globin protein in sufficient amounts or failure to produce normal 3-globin protein entirely, increased expression of y-globin (i.e., HbF) will ameliorate P-globin disease severity.

[0213] B-cell lymphoma 11 A (BCL11 A) is a gene located on Chromosome 2 and ranges from 60,451,167-60,553,567 bp (GRCh38). BCL11A is a zinc finger transcription factor that represses fetal hemoglobin (HbF) and downregulates HbF expression starting at about 6 weeks after birth. The BCL11 A gene contains 4 exons, spanning 102.4 kb of genomic DNA. BCL11 A also is under transcription regulation, including a binding domain in intron 2 for the master transcription factor GATA-1. GATA-1 binding enhances BCL11A expression which, in turn, represses HbF expression. Intron 2 contains multiple DNase hypersensitive sites (DHS), including sites referred to as +55, +58, and +62 based on the distance in kilobases from the transcriptional start site. Various editing strategies are discussed below to delete, modulate, or inactivate the transcriptional control sequences of BCL11 A. Naturally occurring SNPs within this region have been associated with decreased BCL11 A expression and increased fetal Hb levels (Orkin et al. 2013 GWAS study). These SNPs are organized around 3 DNA Hypersensitivity sites, +55DHS, +58DHS and +62DHS. Of the 3 regions, the +58 DHS region, appears to be the key region associated with increased fetal Hb levels and also harbors a GATA1 transcriptional control region.

[0214] Non-homologous end joining (NHEJ) can be used to delete segments of the transcriptional control sequence of BCL11 A, either directly or by altering splice donor or acceptor sites through cleavage by one gRNA targeting several locations, or several gRNAs.

[0215] The transcriptional control sequence of the BCL11 A gene can also be modulated or inactivated by inserting a wild-type BCL11 A gene or cDNA comprising a modified transcriptional control sequence. For example, the donor for modulating or inactivating by homology directed repair (HDR) contains the modified transcriptional control sequence of the BCL11 A gene with small or large flanking homology arms to allow for annealing. HDR is essentially an error-free mechanism that uses a supplied homologous DNA sequence as a template during DSB repair. The rate of homology directed repair (HDR) is a function of thedistance between the transcriptional control sequence and the cut site so choosing overlapping or nearby target sites is important. Templates can include extra sequences flanked by the homologous regions or can contain a sequence that differs from the genomic sequence, thus allowing sequence editing.

[0216] In addition to deleting, modulating, or inactivating the transcriptional control sequence of the BCL11 A gene by NHEJ or HDR, a range of other options are possible. If there are small or large deletions, a cDNA can be knocked in that contains a modified transcriptional control sequence of the BCL11 A gene. A full length cDNA can be knocked into any "safe harbor"-i.e., non-deleterious insertion point that is not the BCL11 A gene itself- , with or without suitable regulatory sequences. If this construct is knocked-in near the BCL11 A regulatory elements, it should have physiological control, similar to the normal gene. Two or more (e.g., a pair) nucleases can be used to delete transcriptional control sequence regions, though a donor would usually have to be provided to modulate or inactivate the function. In this case two gRNA and one donor sequence would be supplied.

[0217] These approaches may be applied to other target genes that are involved in hemoglobinopathy, primary immunodeficiency or congenital cytopenia. Components for gene editing of these disease and conditions, for example, hemophilia A, hemophilia B, SCID, Wiskott-Aldrich syndrome and other related diseases and conditions are provided in the following U.S. Pat. Publications, all of which are incorporated herein by reference in their entirety: US20180127786, US20190247517, US20190112353 and US20210254057.

[0218] In general, it is contemplated that the gene editing systems can be utilized to modulate or inactivate transcriptional control of a target gene by NHEJ or HDR or to knock- in a desired sequence, such as a wild-type sequence or cDNA at a target site. Accordingly, some genome engineering strategies useful in the present invention involve modulating or inactivating a transcriptional control sequence of a target gene by deleting at least a portion of the transcriptional control sequence of the target gene and / or knocking-in a wild-type gene of interest (GOI) or cDNA comprising a modified transcriptional control sequence into the locus of the corresponding gene or a safe harbor locus by homology directed repair (HDR), which is also known as homologous recombination (HR). This strategy can modulate or inactivate the transcriptional control sequence of the target gene and reverse, treat, and / or mitigate the diseased state. Donor nucleotides for modulating / inactivating transcriptional control sequences often are small (<300 bp). This is advantageous, as HDR efficiencies may be inversely related to the size of the donor molecule. Also, it is expected that the donortemplates can fit into size constrained adeno-associated virus (AAV) molecules, which have been shown to be an effective means of donor template delivery.

[0219] Homology direct repair is a cellular mechanism for repairing double-stranded breaks (DSBs). The most common form is homologous recombination. There are additional pathways for HDR, including single-strand annealing and altemative-HDR. Genome engineering tools allow researchers to manipulate the cellular homologous recombination pathways to create site-specific modifications to the genome. It has been found that cells can repair a double-stranded break using a synthetic donor molecule provided in trans. Therefore, by introducing a double-stranded break near a specific mutation and providing a suitable donor, targeted changes can be made in the genome. Specific cleavage increases the rate of HDR more than 1,000 fold above the rate of 1 in 106 cells receiving a homologous donor alone. The rate of homology directed repair (HDR) at a particular nucleotide is a function of the distance to the cut site, so choosing overlapping or nearest target sites is important. Gene editing offers the advantage over gene addition, as correcting in situ leaves the rest of the genome unperturbed.

[0220] Supplied donors for editing by HDR vary markedly but can contain the intended sequence with small or large flanking homology arms to allow annealing to the genomic DNA. The homology regions flanking the introduced genetic changes can be 30 bp or smaller, or as large as a multi-kilobase cassette that can contain promoters, cDNAs, etc. Both single-stranded and double-stranded oligonucleotide donors have been used. These oligonucleotides range in size from less than 100 nt to over many kb, though longer ssDNA can also be generated and used. Double-stranded donors can be used, including PCR amplicons, plasmids, and mini-circles. In general, it has been found that an AAV vector can be a very effective means of delivery of a donor template, though the packaging limits for individual donors is <5 kb. Active transcription of the donor increased HDR three-fold, indicating the inclusion of promoter may increase conversion. Conversely, CpG methylation of the donor decreased gene expression and HDR.

[0221] In addition to wildtype endonucleases, such as Cas9, nickase variants exist that have one or the other nuclease domain inactivated resulting in cutting of only one DNA strand. HDR can be directed from individual Cas nickases or using pairs of nickases that flank the target area. Donors can be single-stranded, nicked, or dsDNA.

[0222] The repair pathway choice can be guided by a number of culture conditions, such as those that influence cell cycling, or by targeting of DNA repair and associated proteins.For example, to increase HDR, key NHEJ molecules can be suppressed, such as KU70, KU80 or DNA ligase IV.

[0223] Without a donor present, the ends from a DNA break or ends from different breaks can be joined using the several nonhomologous repair pathways in which the DNA ends are joined with little or no base-pairing at the junction. In addition to canonical NHEJ, there are similar repair mechanisms, such as alt-NHEJ. If there are two breaks, the intervening segment can be deleted or inverted. NHEJ repair pathways can lead to insertions, deletions or mutations at the joints.[0224| In addition to genome editing by NHEJ or HDR, site-specific gene insertions have been conducted that use both the NHEJ pathway and HR. A combination approach may be applicable in certain settings, possibly including intron / exon borders. NHEJ may prove effective for ligation in the intron, while the error-free HDR may be better suited in the coding region.[0225J As a further alternative, a wild-type GOI or cDNA comprising a modified transcriptional control sequence can be knocked-in to the locus of the corresponding gene or knocked-in to a safe harbor site, such as AAVS1. In some examples, the methods can provide one gRNA or a pair of gRNAs that can be used to facilitate incorporation of a new sequence from a polynucleotide donor template to knock-in a part of or the entire wild-type GOI or cDNA comprising a modified transcriptional control sequence.

[0226] The methods can provide gRNA pairs that make a deletion by cutting the gene twice, one gRNA cutting at the 5' end of one or more mutations and the other gRNA cutting at the 3' end of one or more mutations that facilitates insertion of a new sequence from a polynucleotide donor template to replace the transcriptional control sequence of the target gene. The cutting can be accomplished by a pair of DNA endonucleases that each makes a DSB in the genome, or by multiple nickases that together make a DSB in the genome.

[0227] Alternatively, the methods can provide one gRNA to make one double-strand cut around a transcriptional control sequence of the target gene gene that facilitates insertion of a new sequence from a polynucleotide donor template to replace the transcriptional control sequence of the target gene with a wild-type sequence or cDNA comprising a modified transcriptional control sequence. The double-strand cut can be made by a single DNA endonuclease or multiple nickases that together make a DSB in the genome.

[0228] Illustrative modifications within or near the target gene or other DNA sequence that encodes a regulatory element of the target gene include replacements within or near (proximal) the transcriptional control sequence of the target gene referred to above, such aswithin the region of less than 3 kb, less than 2 kb, less than 1 kb, less than 0.5 kb upstream or downstream of the transcriptional control sequence.

[0229] Such variants can include replacements that are larger in the 5' and / or 3' direction than the specific replacement in question, or smaller in either direction. Accordingly, by “near” or “proximal” with respect to specific replacements, it is intended that the SSB or DSB locus associated with a desired replacement boundary (also referred to herein as an endpoint) can be within a region that is less than about 3 kb from the reference locus noted. The SSB or DSB locus can be more proximal and within 2 kb, within 1 kb, within 0.5 kb, or within 0.1 kb. In the case of small replacement, the desired endpoint can be at or “adjacent to” the reference locus, by which it is intended that the endpoint can be within 100 bp, within 50 bp, within 25 bp, or less than about 10 bp to 5 bp from the reference locus.

[0230] Examples comprising larger or smaller replacements can be expected to provide the same benefit, as long as the transcriptional control activity is modulated or inactivated. It is thus expected that many variations of the replacements described and illustrated herein can be effective for ameliorating hemoglobinopathies.

[0231] Another genome engineering strategy involves exon or intron deletion. Targeted deletion of specific exons or introns can be an attractive strategy for treating a large subset of patients with a single therapeutic cocktail. Deletions can either be single exon or intron deletions or multi-exon or intron deletions. While multi -exon deletions can reach a larger number of patients, for larger deletions the efficiency of deletion greatly decreases with increased size. Therefore, deletions range can be from 40 to 10,000 base pairs (bp) in size. For example, deletions can range from 40-100; 100-300; 300-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; or 5,000-10,000 base pairs in size. It may be desirable to delete an intron if the intron contains a regulatory element, such as a transcriptional control element (e.g., a transcription factor binding site).

[0232] In order to ensure that the pre-mRNA is properly processed following deletion, the surrounding splicing signals can be deleted. Splicing donor and acceptors are generally within 100 base pairs of the neighboring intron. Therefore, in some examples, methods can provide all gRNAs that cut approximately + / - 100-3100 bp with respect to each exon / intron junction of interest.

[0233] For any of the genome editing strategies, gene editing can be confirmed by sequencing or PCR analysis.Target Sequence Selection

[0234] Shifts in the location of the 5' boundary and / or the 3' boundary relative to particular reference loci can be used to facilitate or enhance particular applications of gene editing, which depend in part on the endonuclease system selected for the editing, as further described and illustrated herein.

[0235] In a first nonlimiting example of such target sequence selection, many endonuclease systems have rules or criteria that can guide the initial selection of potential target sites for cleavage, such as the requirement of a PAM sequence motif in a particular position adjacent to the DNA cleavage sites in the case of CRISPR Type II or Type V endonucleases.

[0236] In another nonlimiting example of target sequence selection or optimization, the frequency of off-target activity for a particular combination of target sequence and gene editing endonuclease (i.e. the frequency of DSBs occurring at sites other than the selected target sequence) can be assessed relative to the frequency of on-target activity. In some cases, cells that have been correctly edited at the desired locus can have a selective advantage relative to other cells. Illustrative, but nonlimiting, examples of a selective advantage include the acquisition of attributes such as enhanced rates of replication, persistence, resistance to certain conditions, enhanced rates of successful engraftment or persistence in vivo following introduction into a patient, and other attributes associated with the maintenance or increased numbers or viability of such cells. In other cases, cells that have been correctly edited at the desired locus can be positively selected for by one or more screening methods used to identify, sort or otherwise select for cells that have been correctly edited. Both selective advantage and directed selection methods can take advantage of the phenotype associated with the correction. In some cases, cells can be edited two or more times in order to create a second modification that creates a new phenotype that is used to select or purify the intended population of cells. Such a second modification could be created by adding a second gRNA for a selectable or screenable marker. In some cases, cells can be correctly edited at the desired locus using a DNA fragment that contains the cDNA and also a selectable marker.

[0237] Whether any selective advantage is applicable or any directed selection is to be applied in a particular case, target sequence selection can also be guided by consideration of off-target frequencies in order to enhance the effectiveness of the application and / or reduce the potential for undesired alterations at sites other than the desired target. As described further and illustrated herein and in the art, the occurrence of off-target activity can be influenced by a number of factors including similarities and dissimilarities between the targetsite and various off-target sites, as well as the particular endonuclease used. Bioinformatics tools are available that assist in the prediction of off-target activity, and frequently such tools can also be used to identify the most likely sites of off-target activity, which can then be assessed in experimental settings to evaluate relative frequencies of off-target to on-target activity, thereby allowing the selection of sequences that have higher relative on-target activities. Illustrative examples of such techniques are provided herein, and others are known in the art.

[0238] Another aspect of target sequence selection relates to homologous recombination events. Sequences sharing regions of homology can serve as focal points for homologous recombination events that result in deletion of intervening sequences. Such recombination events occur during the normal course of replication of chromosomes and other DNA sequences, and also at other times when DNA sequences are being synthesized, such as in the case of repairs of double-strand breaks (DSBs), which occur on a regular basis during the normal cell replication cycle but can also be enhanced by the occurrence of various events (such as UV light and other inducers of DNA breakage) or the presence of certain agents (such as various chemical inducers). Many such inducers cause DSBs to occur indiscriminately in the genome, and DSBs can be regularly induced and repaired in normal cells. During repair, the original sequence can be reconstructed with complete fidelity, however, in some cases, small insertions or deletions (referred to as “indels”) are introduced at the DSB site.

[0239] DSBs can also be specifically induced at particular locations, as in the case of the endonucleases systems described herein, which can be used to cause directed or preferential gene modification events at selected chromosomal locations. The tendency for homologous sequences to be subject to recombination in the context of DNA repair (as well as replication) can be taken advantage of in a number of circumstances, and is the basis for one application of gene editing systems, such as CRISPR, in which homology directed repair is used to insert a sequence of interest, provided through use of a “donor” polynucleotide, into a desired chromosomal location.

[0240] Regions of homology between particular sequences, which can be small regions of “microhomology” that can comprise as few as ten basepairs or less, can also be used to bring about desired deletions. For example, a single DSB can be introduced at a site that exhibits microhomology with a nearby sequence. During the normal course of repair of such DSB, a result that occurs with high frequency is the deletion of the intervening sequence as a result of recombination being facilitated by the DSB and concomitant cellular repair process.

[0241] In some circumstances, however, selecting target sequences within regions of homology can also give rise to much larger deletions, including gene fusions (when the deletions are in coding regions), which may or may not be desired given the particular circumstances.Nucleic Acid Modifications

[0242] In some cases, polynucleotides introduced into cells can comprise one or more modifications that can be used individually or in combination, for example, to enhance activity, stability or specificity, alter delivery, reduce innate immune responses in host cells, or for other enhancements, as further described herein and known in the art.

[0243] In certain examples, modified polynucleotides can be used in the CRISPR / Cas9 / Cpfl system, in which case the guide RNAs (either single-molecule guides or double-molecule guides) and / or a DNA or an RNA encoding a Cas or Cpfl endonuclease introduced into a cell can be modified, as described and illustrated below. Such modified polynucleotides can be used in the CRISPR / Cas9 / Cpfl system to edit any one or more genomic loci.

[0244] Using the CRISPR / Cas9 / Cpfl system for purposes of nonlimiting illustrations of such uses, modifications of guide RNAs can be used to enhance the formation or stability of the CRISPR / Cas9 / Cpfl genome editing complex comprising guide RNAs, which can be single-molecule guides or double-molecule, and a Cas or Cpfl endonuclease. Modifications of guide RNAs can also or alternatively be used to enhance the initiation, stability or kinetics of interactions between the genome editing complex with the target sequence in the genome, which can be used, for example, to enhance on-target activity. Modifications of guide RNAs can also or alternatively be used to enhance specificity, e.g., the relative rates of genome editing at the on-target site as compared to effects at other (off-target) sites.

[0245] Modifications can also or alternatively be used to increase the stability of a guide RNA, e.g., by increasing its resistance to degradation by ribonucleases (RNases) present in a cell, thereby causing its half-life in the cell to be increased. Modifications enhancing guide RNA half-life can be particularly useful in aspects in which a Cas or Cpfl endonuclease is introduced into the cell to be edited via an RNA that needs to be translated in order to generate endonuclease, because increasing the half-life of guide RNAs introduced at the same time as the RNA encoding the endonuclease can be used to increase the time that the guide RNAs and the encoded Cas or Cpfl endonuclease co-exist in the cell.

[0246] Modifications can also or alternatively be used to decrease the likelihood or degree to which RNAs introduced into cells elicit innate immune responses. Such responses, which have been well characterized in the context of RNA interference (RNAi), including smallinterfering RNAs (siRNAs), as described below and in the art, tend to be associated with reduced half-life of the RNA and / or the elicitation of cytokines or other factors associated with immune responses.

[0247] One or more types of modifications can also be made to RNAs encoding an endonuclease that are introduced into a cell, including, without limitation, modifications that enhance the stability of the RNA (such as by increasing its degradation by RNAses present in the cell), modifications that enhance translation of the resulting product (i.e. the endonuclease), and / or modifications that decrease the likelihood or degree to which the RNAs introduced into cells elicit innate immune responses.

[0248] Combinations of modifications, such as the foregoing and others, can likewise be used. In the case of CRISPR / Cas9 / Cpfl, for example, one or more types of modifications can be made to guide RNAs (including those exemplified above), and / or one or more types of modifications can be made to RNAs encoding Cas endonuclease (including those exemplified above).

[0249] By way of illustration, guide RNAs used in the CRISPR / Cas9 / Cpfl system, or other smaller RNAs can be readily synthesized by chemical means, enabling a number of modifications to be readily incorporated, as illustrated below and described in the art. While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides. One approach that can be used for generating chemically-modified RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding a Cas9 endonuclease, are more readily generated enzymatically. While fewer types of modifications are available for use in enzymatically produced RNAs, there are still modifications that can be used to, e.g., enhance stability, reduce the likelihood or degree of innate immune response, and / or enhance other attributes, as described further below and in the art; and new types of modifications are regularly being developed.

[0250] By way of illustration of various types of modifications, especially those used frequently with smaller chemically synthesized RNAs, modifications can comprise one or more nucleotides modified at the 2' position of the sugar, in some aspects a 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro-modified nucleotide. In some examples, RNA modifications can comprise 2'-fluoro, 2'-amino or 2' O-methyl modifications on the ribose of pyrimidines, abasic residues, or an inverted base at the 3' end of the RNA. Such modifications can be routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2'-deoxyoligonucleotides against a given target.

[0251] A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligonucleotide; these modified oligos survive intact for a longer time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Some oligonucleotides are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2 — NH — O — CH2, CH, ~N(CH3)~O~CH2 (known as a methylene(methylimino) or MMI backbone), CH2— O— N(CH3)— CH2, CH2— N(CH3)— N(CH3)— CH2 and O— N(CH3)— CH2— CH2 backbones, wherein the native phosphodiester backbone is represented as O — P — O — CH); amide backbones [see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)]; morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3 'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 '-5 ' to 5 '-3 ' or 2'-5 ' to 5 '-2'; see U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0252] Morpholino-based oligomeric compounds are described in Braasch and David Corey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Volume 30, Issue 3, (2001); Heasman, Dev. Biol., 243: 209-214 (2002); Nasevicius et al., Nat. Genet., 26:216-220 (2000); Lacerra et al., Proc. Natl. Acad. Sci., 97: 9591-9596 (2000); and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.

[0253] Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 122: 8595-8602 (2000).

[0254] Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

[0255] One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3OCH3, OCH3O(CH2)n CH3, O(CH2)n NH2, or O(CH2)n CH3, where n is from 1 to about 10; Cl to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. In some aspects, a modification includes 2 '-methoxy ethoxy (2'-0 — CH2CH2OCH3, also known as 2'-O-(2- methoxy ethyl)) (Martin et al, Helv. Chim. Acta, 1995, 78, 486). Other modifications include 2'-methoxy (2'-0 — CH3), 2'-propoxy (2'-OCH2CH2CH3) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminalnucleotide. Oligonucleotides can also have sugar mimetics, such as cyclobutyls in place of the pentofuranosyl group.10256] In some examples, both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units can be replaced with novel groups. The base units can be maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar- backbone of an oligonucleotide can be replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases can be retained and bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds comprise, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNA compounds can be found in Nielsen et al, Science, 254: 1497-1500 (1991).

[0257] Guide RNAs can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5- Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5 -bromouracil, 5- hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine, and 2,6- diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, pp 75-77 (1980); Gebeyehu et al., Nucl. Acids Res. 15:4513 (1997). A “universal” base known in the art, e.g., inosine, can also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are aspects of base substitutions.

[0258] Modified nucleobases can comprise other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8- hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7- methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3- deazaguanine and 3 -deazaadenine.

[0259] Further, nucleobases can comprise those disclosed in U.S. Pat. No. 3,687,808, those disclosed in ‘The Concise Encyclopedia of Polymer Science And Engineering’, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandle Chemie, International Edition’, 1991, 30, page 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications’, pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Research and Applications’, CRC Press, Boca Raton, 1993, pp. 276-278) and are aspects of base substitutions, even more particularly when combined with 2 '-O-m ethoxy ethyl sugar modifications. Modified nucleobases are described in U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617;5,681,941; 5,750,692; 5,763,588; 5,830,653; 6,005,096; and US Patent Application Publication 2003 / 0158403.

[0260] Thus, the term “modified” refers to a non-natural sugar, phosphate, or base that is incorporated into a guide RNA, an endonuclease, or transcriptional control sequence of BCL11 A or both a guide RNA and an endonuclease. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single oligonucleotide, or even in a single nucleoside within an oligonucleotide.

[0261] The guide RNAs and / or mRNA (or DNA) encoding an endonuclease can be chemically linked to one or more moi eties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties comprise, but are not limited to, lipid moieties such as a cholesterol moiety [Letsinger et al., Proc. Natl. Acad. Sci.USA, 86: 6553-6556 (1989)]; cholic acid [Manoharan et al., Bioorg. Med. Chem. Let., 4: 1053-1060 (1994)]; a thioether, e.g., hexyl-S-tritylthiol [Manoharan et al, Ann. N. Y. Acad. Sci., 660: 306-309 (1992) and Manoharan et al., Bioorg. Med. Chem. Let., 3: 2765-2770 (1993)]; a thiocholesterol [Oberhauser et al., Nucl. Acids Res., 20: 533-538 (1992)]; an aliphatic chain, e.g., dodecandiol or undecyl residues [Kabanov et al., FEBS Lett., 259: 327- 330 (1990) and Svinarchuk et al., Biochimie, 75: 49-54 (1993)]; a phospholipid, e.g., dihexadecyl -rac-glycerol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H- phosphonate [Manoharan et al., Tetrahedron Lett., 36: 3651-3654 (1995) and Shea et al., Nucl. Acids Res., 18: 3777-3783 (1990)]; a polyamine or a polyethylene glycol chain [Mancharan et al., Nucleosides & Nucleotides, 14: 969-973 (1995)]; adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36: 3651-3654 (1995)]; a palmityl moiety [(Mishra et al., Biochim. Biophys. Acta, 1264: 229-237 (1995)]; or an octadecyl amine or hexylaminocarbonyl -t oxy cholesterol moiety [Crooke et al., J. Pharmacol. Exp. Ther., 277: 923-937 (1996)]. See also U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941.

[0262] Sugars and other moieties can be used to target proteins and complexes comprising nucleotides, such as cationic polysomes and liposomes, to particular sites. For example, hepatic cell directed transfer can be mediated via asialoglycoprotein receptors (ASGPRs); see, e.g., Hu, et al., Protein Pept Lett. 21(10): 1025-30 (2014).

[0263] Other systems known in the art and regularly developed can be used to target biomolecules of use in the present case and / or complexes thereof to particular target cells of interest.

[0264] These targeting moieties or conjugates can include conjugate groups covalently bound to functional groups, such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this disclosure, include groups that improve uptake, enhance resistance to degradation, and / or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT / US92 / 09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium I,2-di- O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecyl amine or hexylamino-carbonyl-oxy cholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

[0265] Longer polynucleotides that are less amenable to chemical synthesis and are typically produced by enzymatic synthesis can also be modified by various means. Such modifications can include, for example, the introduction of certain nucleotide analogs, the incorporation of particular sequences or other moieties at the 5' or 3' ends of molecules, and other modifications. By way of illustration, the mRNA encoding Cas9 is approximately 4 kb in length and can be synthesized by in vitro transcription. Modifications to the mRNA can be applied to, e.g., increase its translation or stability (such as by increasing its resistance to degradation with a cell), or to reduce the tendency of the RNA to elicit an innate immune response that is often observed in cells following introduction of exogenous RNAs, particularly longer RNAs such as that encoding Cas9.

[0266] Numerous such modifications have been described in the art, such as polyA tails, 5' cap analogs (e.g., Anti Reverse Cap Analog (ARC A) or m7G(5')ppp(5')G (mCAP)), modified 5' or 3' untranslated regions (UTRs), use of modified bases (such as Pseudo-UTP,2-Thio-UTP, 5-Methylcytidine-5 '-Triphosphate (5-Methyl-CTP) or N6-Methyl-ATP), or treatment with phosphatase to remove 5' terminal phosphates. These and other modifications are known in the art, and new modifications of RNAs are regularly being developed.

[0267] There are numerous commercial suppliers of modified RNAs, including for example, TriLink Biotech, AxoLabs, Bio-Synthesis Inc., Dharmacon and many others. As described by TriLink, for example, 5-Methyl-CTP can be used to impart desirable characteristics, such as increased nuclease stability, increased translation or reduced interaction of innate immune receptors with in vitro transcribed RNA. 5-Methylcytidine-5'- Triphosphate (5-Methyl-CTP), N6-Methyl-ATP, as well as Pseudo-UTP and 2-Thio-UTP, have also been shown to reduce innate immune stimulation in culture and in vivo while enhancing translation, as illustrated in publications by Kormann et al. and Warren et al. referred to below.

[0268] It has been shown that chemically modified mRNA delivered in vivo can be used to achieve improved therapeutic effects; see, e.g., Kormann et al., Nature Biotechnology 29,154-157 (2011). Such modifications can be used, for example, to increase the stability of the RNA molecule and / or reduce its immunogenicity. Using chemical modifications such as Pseudo-U, N6-Methyl-A, 2-Thio-U and 5-Methyl-C, it was found that substituting just one quarter of the uridine and cytidine residues with 2-Thio-U and 5-Methyl-C respectively resulted in a significant decrease in toll-like receptor (TLR) mediated recognition of the mRNA in mice. By reducing the activation of the innate immune system, these modifications can be used to effectively increase the stability and longevity of the mRNA in vivo; see, e.g., Kormann et al., supra.

[0269] It has also been shown that repeated administration of synthetic messenger RNAs incorporating modifications designed to bypass innate anti-viral responses can reprogram differentiated human cells to pluripotency. See, e.g., Warren, et al., Cell Stem Cell, 7(5):618- 30 (2010). Such modified mRNAs that act as primary reprogramming proteins can be an efficient means of reprogramming multiple human cell types. Such cells are referred to as induced pluripotency stem cells (iPSCs), and it was found that enzymatically synthesized RNA incorporating 5-Methyl-CTP, Pseudo-UTP and an Anti Reverse Cap Analog (ARCA) could be used to effectively evade the cell's antiviral response; see, e.g., Warren et al., supra.

[0270] Other modifications of polynucleotides described in the art include, for example, the use of polyA tails, the addition of 5' cap analogs (such as m7G(5')ppp(5')G (mCAP)), modifications of 5' or 3' untranslated regions (UTRs), or treatment with phosphatase to remove 5' terminal phosphates — and new approaches are regularly being developed.

[0271] A number of compositions and techniques applicable to the generation of modified RNAs for use herein have been developed in connection with the modification of RNA interference (RNAi), including small-interfering RNAs (siRNAs). siRNAs present particular challenges in vivo because their effects on gene silencing via mRNA interference are generally transient, which can require repeat administration. In addition, siRNAs are doublestranded RNAs (dsRNA) and mammalian cells have immune responses that have evolved to detect and neutralize dsRNA, which is often a by-product of viral infection. Thus, there are mammalian enzymes such as PKR (dsRNA-responsive kinase), and potentially retinoic acidinducible gene I (RIG-I), that can mediate cellular responses to dsRNA, as well as Toll-like receptors (such as TLR3, TLR7 and TLR8) that can trigger the induction of cytokines in response to such molecules; see, e.g., the reviews by Angart et al., Pharmaceuticals (Basel) 6(4): 440-468 (2013); Kanasty et al., Molecular Therapy 20(3): 513-524 (2012); Burnett et al., Biotechnol J. 6(9): 1130-46 (2011); Judge and MacLachlan, Hum Gene Ther 19(2): 111-24 (2008); and references cited therein.

[0272] A large variety of modifications have been developed and applied to enhance RNA stability, reduce innate immune responses, and / or achieve other benefits that can be useful in connection with the introduction of polynucleotides into human cells, as described herein; see, e.g., the reviews by Whitehead K A et al., Annual Review of Chemical and Biomolecular Engineering, 2: 77-96 (2011); Gaglione and Messere, Mini Rev Med Chem, 10(7):578-95 (2010); Chemolovskaya et al, Curr Opin Mol Ther., 12(2): 158-67 (2010); Deleavey et al., Curr Protoc Nucleic Acid Chem Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19 (2008); Fucini et al., Nucleic Acid Ther 22(3): 205-210 (2012); Bremsen et al., Front Genet 3: 154 (2012).

[0273] As noted above, there are a number of commercial suppliers of modified RNAs, many of which have specialized in modifications designed to improve the effectiveness of siRNAs. A variety of approaches are offered based on various findings reported in the literature. For example, Dharmacon notes that replacement of a non -bridging oxygen with sulfur (phosphorothioate, PS) has been extensively used to improve nuclease resistance of siRNAs, as reported by Kole, Nature Reviews Drug Discovery 11 : 125-140 (2012). Modifications of the 2'-position of the ribose have been reported to improve nuclease resistance of the intemucleotide phosphate bond while increasing duplex stability (Tm), which has also been shown to provide protection from immune activation. A combination of moderate PS backbone modifications with small, well-tolerated 2'-substitutions (2'-O-Methyl, 2'-Fluoro, 2'-Hydro) have been associated with highly stable siRNAs for applications in vivo,as reported by Soutschek et al. Nature 432: 173-178 (2004); and 2'-O-Methyl modifications have been reported to be effective in improving stability as reported by Volkov, Oligonucleotides 19: 191-202 (2009). With respect to decreasing the induction of innate immune responses, modifying specific sequences with 2'-O-Methyl, 2'-Fluoro, 2'-Hydro have been reported to reduce TLR7 / TLR8 interaction while generally preserving silencing activity; see, e.g., Judge et al., Mol. Ther. 13:494-505 (2006); and Cekaite et al., J. Mol. Biol. 365:90- 108 (2007). Additional modifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine, 5-methyluracil, and N6-methyladenosine have also been shown to minimize the immune effects mediated by TLR3, TLR7, and TLR8; see, e.g., Kariko, K. et al., Immunity 23: 165- 175 (2005).

[0274] As is also known in the art, and commercially available, a number of conjugates can be applied to polynucleotides, such as RNAs, for use herein that can enhance their delivery and / or uptake by cells, including for example, cholesterol, tocopherol and folic acid, lipids, peptides, polymers, linkers and aptamers; see, e.g., the review by Winkler, Ther. Deliv. 4:791-809 (2013), and references cited therein.Codon-Optimization

[0275] A polynucleotide encoding a site-directed polypeptide can be codon-optimized according to methods standard in the art for expression in the cell containing the target DNA of interest. For example, if the intended target nucleic acid is in a human cell, a human codon-optimized polynucleotide encoding Cas9 is contemplated for use for producing the Cas9 polypeptide.Complexes of a Genome-Targeting Nucleic Acid and a Site-Directed Polypeptide

[0276] A genome-targeting nucleic acid interacts with a site-directed polypeptide (e.g., a nucleic acid-guided nuclease such as Cas9), thereby forming a complex. The genometargeting nucleic acid guides the site-directed polypeptide to a target nucleic acid.RNPs

[0277] The site-directed polypeptide and genome-targeting nucleic acid can each be administered separately to a cell or a patient. On the other hand, the site-directed polypeptide can be pre-complexed with one or more genome-targeting nucleic acids (guide RNA, sgRNA, or crRNA together with a tracrRNA). The pre-complexed material can then be administered to a cell or a patient. Such pre-complexed material is known as a ribonucleoprotein particle(RNP). The site-directed polypeptide in the RNP can be, for example, a Cas9 endonuclease or a Cpfl endonuclease. The site-directed polypeptide can be flanked at the N-terminus, the C- terminus, or both the N-terminus and C-terminus by one or more nuclear localization signals (NLSs). For example, a Cas9 endonuclease can be flanked by two NLSs, one NLS located at the N-terminus and the second NLS located at the C-terminus. The NLS can be any NLS known in the art, such as a SV40 NLS. The weight ratio of genome-targeting nucleic acid to site-directed polypeptide in the RNP can be 1 : 1. For example, the weight ratio of sgRNA to Cas9 endonuclease in the RNP can be 1 : 1.Nucleic Acids Encoding System Components

[0278] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a genome-targeting nucleic acid of the disclosure, a site-directed polypeptide of the disclosure, and / or any nucleic acid or proteinaceous molecule necessary to carry out the aspects of the methods of the disclosure.

[0279] The nucleic acid encoding a genome-targeting nucleic acid of the disclosure, a site- directed polypeptide of the disclosure, and / or any nucleic acid or proteinaceous molecule necessary to carry out the aspects of the methods of the disclosure can comprise a vector (e.g., a recombinant expression vector).

[0280] The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double-stranded DNA loop into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector, wherein additional nucleic acid segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0281] In some examples, vectors can be capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors”, or more simply “expression vectors”, which serve equivalent functions.

[0282] The term “operably linked” means that the nucleotide sequence of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence. The term “regulatory sequence” is intended to include, for example, promoters, enhancersand other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the target cell, the level of expression desired, and the like.

[0283] Expression vectors contemplated include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus) and other recombinant vectors. Other vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pXTl, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Additional vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pCTx-1, pCTx-2, and pCTx-3, which are described in FIGS. 1 A to 1C. Other vectors can be used so long as they are compatible with the host cell.

[0284] In some examples, a vector can comprise one or more transcription and / or translation control elements. Depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. can be used in the expression vector. The vector can be a self-inactivating vector that either inactivates the viral sequences or the components of the CRISPR machinery or other elements.

[0285] Non-limiting examples of suitable eukaryotic promoters (i.e., promoters functional in a eukaryotic cell) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, human elongation factor-1 promoter (EFl), a hybrid construct comprising the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter (CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase- 1 locus promoter (PGK), and mouse metallothionein-I.

[0286] For expressing small RNAs, including guide RNAs used in connection with Cas endonuclease, various promoters such as RNA polymerase III promoters, including forexample U6 and Hl, can be advantageous. Descriptions of and parameters for enhancing the use of such promoters are known in art, and additional information and approaches are regularly being described; see, e.g., Ma, H. et al., Molecular Therapy — Nucleic Acids 3, el 61 (2014) doi: 10.1038 / mtna.2014.12.

[0287] The expression vector can also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector can also comprise appropriate sequences for amplifying expression. The expression vector can also include nucleotide sequences encoding non-native tags (e.g., histidine tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the site-directed polypeptide, thus resulting in a fusion protein.

[0288] A promoter can be an inducible promoter (e.g., a heat shock promoter, tetracycline- regulated promoter, steroid-regulated promoter, metal -regulated promoter, estrogen receptor- regulated promoter, etc.). The promoter can be a constitutive promoter (e.g., CMV promoter, UBC promoter). In some cases, the promoter can be a spatially restricted and / or temporally restricted promoter (e.g., a tissue specific promoter, a cell type specific promoter, etc.).

[0289] The nucleic acid encoding a genome-targeting nucleic acid of the disclosure and / or a site-directed polypeptide can be packaged into or on the surface of delivery vehicles for delivery to cells. Delivery vehicles contemplated include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, and micelles. As described in the art, a variety of targeting moieties can be used to enhance the preferential interaction of such vehicles with desired cell types or locations.Delivery

[0290] Guide RNA polynucleotides (RNA or DNA) and / or endonuclease polynucleotide(s) (RNA or DNA) can be delivered by viral or non-viral delivery vehicles known in the art, such as lipid nanoparticles. In further alternative aspects, the DNA endonuclease can be delivered as one or more polypeptides, either alone or pre-complexed with one or more guide RNAs, or one or more crRNA together with a tracrRNA.

[0291] Polynucleotides can be delivered by non-viral delivery vehicles including, but not limited to, lipid nanoparticles and liposomes. Some exemplary non-viral delivery vehicles are described in Peer and Lieberman, Gene Therapy, 18: 1127-1133 (2011) (which focuses on non-viral delivery vehicles for siRNA that are also useful for delivery of other polynucleotides).

[0292] Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding an endonuclease, can be delivered to a cell or a patient by a lipid nanoparticle (LNP).

[0293] A LNP refers to any particle having a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, a nanoparticle can range in size from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25- 60 nm.

[0294] LNPs can be made from cationic, anionic, or neutral lipids. Neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in LNPs as ‘helper lipids’ to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include low efficacy owing to poor stability and rapid clearance, as well as the generation of inflammatory or anti-inflammatory responses.

[0295] LNPs can also be comprised of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0296] Ionizable lipids include synthetic lipids comprising: (i) cationic or ionizable head groups, (ii) linker groups, and (iii) hydrophobic tails. Ionizable cationic lipids include monoamino or polyamino lipids. Example monoamino acid lipids include but are not limited to DLin-MC3-DMA (MC3), SM-102, and ALC-0315 which are FDA-approved ionizable cationic lipids for RNA delivery. The lipid tail structure can include a different number of tails, linear or branching structures, and unsaturated or biodegradable bonds. Polyamino- ionizable cationic lipids include 3060 ilO, cKK-E12, C12-200, 5A2-SC8, TT3, FTT5. (Albertsen et al., Adv Drug Deliv Rev. 188: 114416 (2022); Hou et al., Nat Rev Mater, 6: 1078-94 (2021); Tenchov et al., Bioconjugate Chem. 34(6): 941-60 (2023).

[0297] Any lipid or combination of lipids that are known in the art can be used to produce a LNP. Examples of lipids used to produce LNPs are: DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE- polyethylene glycol (PEG). Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2- DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are: DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are: PEG-DMG, PEG-CerC14, and PEG-CerC20. In some embodiments, the lipid to produce a LNP is an ionizable lipid.

[0298] The lipids can be combined in any number of molar ratios to produce a LNP. In addition, the polynucleotide(s) can be combined with lipid(s) in a wide range of molar ratios to produce a LNP.

[0299] As stated previously, the site-directed polypeptide and genome-targeting nucleic acid can each be administered separately to a cell or a patient. On the other hand, the site- directed polypeptide can be pre-complexed with one or more guide RNAs, or one or morecrRNA together with a tracrRNA. The pre-complexed material can then be administered to a cell or a patient. Such pre-complexed material is known as a ribonucleoprotein particle (RNP).

[0300] RNA is capable of forming specific interactions with RNA or DNA. While this property is exploited in many biological processes, it also comes with the risk of promiscuous interactions in a nucleic acid-rich cellular environment. One solution to this problem is the formation of ribonucleoprotein particles (RNPs), in which the RNA is pre-complexed with an endonuclease. Another benefit of the RNP is protection of the RNA from degradation.

[0301] The endonuclease in the RNP can be modified or unmodified. Likewise, the gRNA, crRNA, tracrRNA, or sgRNA can be modified or unmodified. Numerous modifications are known in the art and can be used.

[0302] The endonuclease and sgRNA can be generally combined in a 1 : 1 molar ratio. Alternatively, the endonuclease, crRNA and tracrRNA can be generally combined in a 1 : 1 : 1 molar ratio. However, a wide range of molar ratios can be used to produce a RNP.

[0303] A recombinant adeno-associated virus (AAV) vector can be used for delivery. Techniques to produce rAAV particles, in which an AAV genome to be packaged that includes the polynucleotide to be delivered, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV typically requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived, and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13 and AAV rh.74. Production of pseudotyped rAAV is disclosed in, for example, international patent application publication number WO 01 / 83692.

[0304] A method of generating a packaging cell involves creating a cell line that stably expresses all of the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) or by direct,blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line can then be infected with a helper virus, such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus, rather than plasmids, to introduce rAAV genomes and / or rep and cap genes into packaging cells.

[0305] General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial, and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81 :6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62: 1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat. No. 5,173,414; WO 95 / 13365 and corresponding U.S. Pat. No.5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. (1995) Vaccine 13: 1244- 1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3: 1124-1132; U.S. Pat. Nos. 5,786,211; 5,871,982; and 6,258,595. AAV vector serotypes can be matched to target cell types.

[0306] In addition to adeno-associated viral vectors, other viral vectors can be used. Such viral vectors include, but are not limited to, lentivirus, alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, Epstein Barr virus, poxvirus, vaccinia virus, and herpes simplex virus.Other gene editing systems

[0307] Gene editing can be conducted using nucleases engineered to target specific sequences. To date there are four major types of nucleases: meganucleases and their derivatives, zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and CRISPR-Cas9 nuclease systems. The nuclease platforms vary in difficulty of design, targeting density and mode of action, particularly as the specificity of ZFNs and TALENs is through protein-DNA interactions, while RNA-DNA interactions primarily guide Cas9. Cas9 cleavage also requires an adjacent motif, the PAM, which differs between different CRISPR systems. Cas9 from Streptococcus pyogenes cleaves using a NGG PAM, CRISPR from Neisseria meningitidis can cleave at sites with PAMs including NNNNGATT,NNNNNGTTT and NNNNGCTT. A number of other Cas9 orthologs target protospacer adjacent to alternative PAMs. Other gene editing system useful in the present invention include, but are not limited to, a base or prime editing system, a reverse transcriptase (RT) editing system, a non-LTR retrotransposon editing system, and a retron gene editing system.

[0308] CRISPR endonucleases, such as Cas9, can be used in the methods of the present disclosure. However, the teachings described herein, such as therapeutic target sites, can be applied to other forms of endonucleases, such as ZFNs, TALENs, HEs, or MegaTALs, or using combinations of nucleases. However, in order to apply the teachings of the present disclosure to such endonucleases, one would need to, among other things, engineer proteins directed to the specific target sites.

[0309] Additional binding domains can be fused to the Cas9 protein to increase specificity. The target sites of these constructs would map to the identified gRNA specified site, but would require additional binding motifs, such as for a zinc finger domain. In the case of Mega-TAL, a meganuclease can be fused to a TALE DNA-binding domain. The meganuclease domain can increase specificity and provide the cleavage. Similarly, inactivated or dead Cas9 (dCas9) can be fused to a cleavage domain and require the sgRNA / Cas9 target site and adjacent binding site for the fused DNA-binding domain. This likely would require some protein engineering of the dCas9, in addition to the catalytic inactivation, to decrease binding without the additional binding site.Zinc Finger Nucleases

[0310] Zinc finger nucleases (ZFNs) are modular proteins comprised of an engineered zinc finger DNA binding domain linked to the catalytic domain of the type II endonuclease Fokl. Because FokI functions only as a dimer, a pair of ZFNs must be engineered to bind to cognate target “half-site” sequences on opposite DNA strands and with precise spacing between them to enable the catalytically active Fokl dimer to form. Upon dimerization of the Fokl domain, which itself has no sequence specificity per se, a DNA double-strand break is generated between the ZFN half-sites as the initiating step in genome editing.

[0311] The DNA binding domain of each ZFN is typically comprised of 3-6 zinc fingers of the abundant Cys2-His2 architecture, with each finger primarily recognizing a triplet of nucleotides on one strand of the target DNA sequence, although cross-strand interaction with a fourth nucleotide also can be important. Alteration of the amino acids of a finger in positions that make key contacts with the DNA alters the sequence specificity of a given finger. Thus, a four-finger zinc finger protein will selectively recognize a 12 bp targetsequence, where the target sequence is a composite of the triplet preferences contributed by each finger, although triplet preference can be influenced to varying degrees by neighboring fingers. An important aspect of ZFNs is that they can be readily re-targeted to almost any genomic address simply by modifying individual fingers, although considerable expertise is required to do this well. In most applications of ZFNs, proteins of 4-6 fingers are used, recognizing 12-18 bp respectively. Hence, a pair of ZFNs will typically recognize a combined target sequence of 24-36 bp, not including the typical 5-7 bp spacer between halfsites. The binding sites can be separated further with larger spacers, including 15-17 bp. A target sequence of this length is likely to be unique in the human genome, assuming repetitive sequences or gene homologs are excluded during the design process. Nevertheless, the ZFN protein-DNA interactions are not absolute in their specificity so off-target binding and cleavage events do occur, either as a heterodimer between the two ZFNs, or as a homodimer of one or the other of the ZFNs. The latter possibility has been effectively eliminated by engineering the dimerization interface of the FokI domain to create “plus” and “minus” variants, also known as obligate heterodimer variants, which can only dimerize with each other, and not with themselves. Forcing the obligate heterodimer prevents formation of the homodimer. This has greatly enhanced specificity of ZFNs, as well as any other nuclease that adopts these FokI variants.

[0312] A variety of ZFN-based systems have been described in the art, modifications thereof are regularly reported, and numerous references describe rules and parameters that are used to guide the design of ZFNs; see, e.g., Segal et al., Proc Natl Acad Sci USA 96(6):2758- 63 (1999); Dreier B et al., J Mol Biol. 303(4):489-502 (2000); Liu Q et al., J Biol Chem. 277(6):3850-6 (2002); Dreier et al., J Biol Chem 280(42):35588-97 (2005); and Dreier et al., J Biol Chem. 276(31):29466-78 (2001).Transcription Activator-Like Effector Nucleases (TALENs)

[0313] TALENs represent another format of modular nucleases whereby, as with ZFNs, an engineered DNA binding domain is linked to the FokI nuclease domain, and a pair of TALENs operate in tandem to achieve targeted DNA cleavage. The major difference from ZFNs is the nature of the DNA binding domain and the associated target DNA sequence recognition properties. The TALEN DNA binding domain derives from TALE proteins, which were originally described in the plant bacterial pathogen Xanthomonas sp. TALEs are comprised of tandem arrays of 33-35 amino acid repeats, with each repeat recognizing a single basepair in the target DNA sequence that is typically up to 20 bp in length, giving atotal target sequence length of up to 40 bp. Nucleotide specificity of each repeat is determined by the repeat variable diresidue (RVD), which includes just two amino acids at positions 12 and 13. The bases guanine, adenine, cytosine and thymine are predominantly recognized by the four RVDs: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly, respectively. This constitutes a much simpler recognition code than for zinc fingers, and thus represents an advantage over the latter for nuclease design. Nevertheless, as with ZFNs, the protein-DNA interactions of TALENs are not absolute in their specificity, and TALENs have also benefitted from the use of obligate heterodimer variants of the FokI domain to reduce off- target activity.

[0314] Additional variants of the FokI domain have been created that are deactivated in their catalytic function. If one half of either a TALEN or a ZFN pair contains an inactive FokI domain, then only single-strand DNA cleavage (nicking) will occur at the target site, rather than a DSB. The outcome is comparable to the use of CRISPR / Cas9 / Cpfl “nickase” mutants in which one of the Cas9 cleavage domains has been deactivated. DNA nicks can be used to drive genome editing by HDR, but at lower efficiency than with a DSB. The main benefit is that off-target nicks are quickly and accurately repaired, unlike the DSB, which is prone to NHEJ-mediated mis-repair.

[0315] A variety of TALEN-based systems have been described in the art, and modifications thereof are regularly reported; see, e.g., Boch, Science 326(5959): 1509-12 (2009); Mak et al., Science 335(6069):716-9 (2012); and Moscou et al., Science 326(5959): 1501 (2009). The use of TALENs based on the “Golden Gate” platform, or cloning scheme, has been described by multiple groups; see, e.g., Cermak et al., Nucleic Acids Res. 39(12):e82 (2011); Li et al., Nucleic Acids Res. 39(14): 6315-25(2011); Weber et al., PLoS One. 6(2):el6765 (2011); Wang et al., J Genet Genomics 41(6):339-47, Epub 2014 May 17 (2014); and Cermak T et al., Methods Mol Biol. 1239: 133-59 (2015).Homing Endonucleases

[0316] Homing endonucleases (HEs) are sequence-specific endonucleases that have long recognition sequences (14-44 base pairs) and cleave DNA with high specificity — often at sites unique in the genome. There are at least six known families of HEs as classified by their structure, including LAGLID ADG (SEQ ID NO. 71,949), GIY-YIG, His-Cis box, H-N-H, PD-(DZE)xK, and Vsr-like that are derived from a broad range of hosts, including eukarya, protists, bacteria, archaea, cyanobacteria and phage. As with ZFNs and TALENs, HEs can be used to create a DSB at a target locus as the initial step in genome editing. In addition, somenatural and engineered HEs cut only a single strand of DNA, thereby functioning as sitespecific nickases. The large target sequence of HEs and the specificity that they offer have made them attractive candidates to create site-specific DSBs.

[0317] A variety of HE-based systems have been described in the art, and modifications thereof are regularly reported; see, e.g., the reviews by Steentoft et al., Glycobiology 24(8):663-80 (2014); Belfort and Bonocora, Methods Mol Biol. 1123 : 1-26 (2014); Hafez and Hausner, Genome 55(8):553-69 (2012); and references cited therein.Mega TAL / Tev-m TALEN / Mega Tev

[0318] As further examples of hybrid nucleases, the MegaTAL platform and Tev- mTALEN platform use a fusion of TALE DNA binding domains and catalytically active HEs, taking advantage of both the tunable DNA binding and specificity of the TALE, as well as the cleavage sequence specificity of the HE; see, e.g., Boissel et al., NAR 42: 2591-2601 (2014); Kleinstiver et al., G3 4: 1155-65 (2014); and Boissel and Scharenberg, Methods Mol. Biol. 1239: 171-96 (2015).

[0319] In a further variation, the MegaTev architecture is the fusion of a meganuclease (Mega) with the nuclease domain derived from the GIY-YIG homing endonuclease LTevl (Tev). The two active sites are positioned ~30 bp apart on a DNA substrate and generate two DSBs with non-compatible cohesive ends; see, e.g., Wolfs et al., NAR 42, 8816-29 (2014). It is anticipated that other combinations of existing nuclease-based approaches will evolve and be useful in achieving the targeted genome modifications described herein. dCas9-FokI or dCpfl-Fokl and Other Nucleases

[0320] Combining the structural and functional properties of the nuclease platforms described above offers a further approach to genome editing that can potentially overcome some of the inherent deficiencies. As an example, the CRISPR genome editing system typically uses a single Cas9 endonuclease to create a DSB. The specificity of targeting is driven by a 20 or 24 nucleotide sequence in the guide RNA that undergoes Watson-Crick base-pairing with the target DNA (plus an additional 2 bases in the adjacent NAG or NGG PAM sequence in the case of Cas9 from S. pyogenes). Such a sequence is long enough to be unique in the human genome, however, the specificity of the RNA / DNA interaction is not absolute, with significant promiscuity sometimes tolerated, particularly in the 5' half of the target sequence, effectively reducing the number of bases that drive specificity. One solution to this has been to completely deactivate the Cas9 or Cpfl catalytic function — retaining onlythe RNA-guided DNA binding function — and instead fusing a FokI domain to the deactivated Cas9; see, e.g., Tsai et al., Nature Biotech 32:569-76 (2014); and Guilinger et al., Nature Biotech. 32: 577-82 (2014). Because FokI must dimerize to become catalytically active, two guide RNAs are required to tether two FokI fusions in close proximity to form the dimer and cleave DNA. This essentially doubles the number of bases in the combined target sites, thereby increasing the stringency of targeting by CRISPR-based systems.

[0321] As further example, fusion of the TALE DNA binding domain to a catalytically active HE, such as I-Tevl, takes advantage of both the tunable DNA binding and specificity of the TALE, as well as the cleavage sequence specificity of I-Tevl, with the expectation that off-target cleavage can be further reduced.3. Methods of treatment

[0322] The methods of the present invention can be applied to treat any disease treatable with HSPC editing (an HSPC-treatable disease). A subject in need of treatment according to the methods of the invention thus is a subject with an HSPC-treatable disease.

[0323] According to the present disclosure, one or more cycling agents are administered to a subject in need thereof, prior to or in conjunction with a gene editing system that is capable of in vivo editing of a target gene or nucleic acid sequence in an HSPC. Editing of any HSPC is contemplated, including an HSC, or including a multipotent progenitor cell type 1, type 2, type 3 or type 4.Timing

[0324] In preferred embodiments of the present invention, a CXCR4 antagonist (e.g., plerixafor) or a cycling agent is administered to a subject in need thereof prior to or in conjunction with a gene editing system formulation that effects in vivo editing of a target gene or nucleic acid sequence in a HSPC. In other embodiments, the CXCR4 antagonist or cycling agent is contacted with HSPCs ex vivo.

[0325] The timing of administration may depend on the cycling agent. For example, antimetabolites may be administered 0 to 7 days prior to administration of the gene editing system. Alkylating agents may be administered 0 to 7 days prior to administration of the gene editing system. In preferred embodiments, the cycling agent is administered prior to or in conjunction with the gene editing system.

[0326] In some embodiments, e.g., when the cycling agent is a CXCR4 antagonist (e.g. plerixafor), the CXCR4 antagonist is administered less than 11 hours prior to administrationof the gene editing system. In some embodiments, e.g., when the cycling agent is a CXCR4 antagonist (e.g. plerixafor), the CXCR4 antagonist is administered about 0 to 10 hours prior to the gene editing system, e.g. about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour prior to administering the gene editing system. In some embodiments, e.g., when the cycling agent is a CXCR4 antagonist (e.g. plerixafor), the CXCR4 antagonist is administered less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, or less than 2 hours prior to administration of the gene editing system, or about 1-8 hours, about 1-7 hours, about 1-6 hours, about 1-5 hours, about 1-4 hours, about 1-3 hours or about 1-2 hours, or about 30 minutes to about 2 hours prior to administration of the gene editing system. In some embodiments, the cycling agent is administered about 1 hour prior to administration of the gene editing system. In some embodiments, administering “in conjunction with” can include administration to a subject in need thereof or to an HSPC the cycling agent about 30-60 minutes before, or within an hour before or after the administration of the gene editing system. In some embodiments, the cycling agent is administered after administration of the gene editing system.Re-dosing

[0327] In some embodiments, a first dose of a cycling agent (or a combination of two or more cycling agents) and a first dose of a gene editing system are administered to a subject. In further embodiments, a second dose of a cycling agent (or a combination of two or more cycling agents) and a second dose of a gene editing system are administered to a subject. The cycling agent(s) used in the second dose may be the same as the cycling agent(s) used in the first dose, or they may be different. In some embodiments, the second dose of the cycling agent(s) and gene editing system is administered about 3, 4, 5, 6, 7, 8 or 9 months after the first dose of the cycling agent(s) and gene editing system. In some embodiments, the second dose of the cycling agent(s) and gene editing system is administered about 3 to 6 months after the first dose of the cycling agent(s) and gene editing system, or about 3 to 9 months after the first dose of the cycling agent(s) and gene editing system. In some embodiments, the subject is administered a third dose, fourth dose, and / or fifth dose of a cycling agent (or a combination of two or more cycling agents) and a gene editing system. The cycling agent(s) used in the third or subsequent dose may be the same as the cycling agent(s) used in the first dose, or they may be different. In further embodiments, each subsequent dose is administeredabout 3, 4, 5, 6, 7, 8 or 9 months after the preceding dose or about 3 to 6 months after the preceding dose, or about 3 to 9 months after the preceding dose.

[0328] In some embodiments, subjects being administered a first dose of a cycling agent (or a combination of two or more cycling agents) and a gene editing system are monitored for percentage of edited HSCs or HSPCs, and the percentage of edited cells is evaluated periodically, e.g. monthly, or every two months, or every three months. According to such embodiments, subjects may be administered a second or subsequent dose of cycling agent(s) and gene editing system if the percentage of edited HSC falls below 20%, 25%, 30%, 35%, 40%, 50%, 55% or 60%.Amounts

[0329] The cycling agent is administered to the subject in an amount effective to increase gene editing efficiency of the gene editing system. In some embodiments, the cycling agent is administered to the subject in an amount effective to increase the percentage of HSPCs in a more active metabolic state or proliferative state.

[0330] In some embodiments, the cycling agent is administered to the subject at a conventional dose. In some embodiments, the cycling agent is administered at a dose range that is about 25% to about 400% of the conventional dose (i.e. about 4-fold less to about 4- fold higher than the conventional dose), or about 50% to about 200% (i.e. about 2-fold less to about 2-fold higher than the conventional dose). In some embodiments, the dose range is about 25% to about 300%, or about 25% to about 200%, or about 50% to about 400%, or about 50% to about 300% or about 50% to about 200% of the conventional dose. As one illustrative example, when the cycling agent is plerixafor, 0 to 10 hours prior to administration of the gene editing system, the subject is administered a conventional dose of 0.24 mg / kg plerixafor (human dosing), or at a dose ranging from about 0.06 mg / kg to about 0.96 mg / kg, or about 0.06 mg / kg to about 0.72 mg / kg, or about 0.06 mg / kg to about 0.48 mg / kg, or about 0.12 mg / kg to about 0.96 mg / kg, or about 0.12 mg / kg to about 0.72 mg / kg, or about 0.12 mg / kg to about 0.48 mg / kg, or about 0.12 mg / kg to about 0.32 mg / kg. In some embodiments, the amount of cycling agent administered may be less than the conventional dose, e.g., less than 0.24 mg / kg plerixafor. In some embodiments, the amount of cycling agent administered may be higher than the conventional dose, e.g. more than 0.24 mg / kg plerixafor.

[0331] As another example, when the cycling agent is an antimetabolite or alkylating agent or other cytotoxic agent, the amount administered as a one-time administration of aconventional cytotoxic dose. In some embodiments, the antimetabolite or alkylating agent is administered 0 to 7 days prior to the gene editing system, at a dose range that is about 25% to about 400% of the conventional dose (i.e. about 4-fold less to about 4-fold higher than the conventional dose), or about 50% to about 200% (i.e. about 2-fold less to about 2-fold higher than the conventional dose). For example, depending on the type of cancer, the dose of 5-FU ranges from about 200 to about 1200 mg / m2parenterally in one day, or up to about 1500 mg / m2parenterally in one day. In some embodiments, the cycling agent is 5 -fluorouracil administered parenterally at a dose ranging from about about 200 to about 1200 mg / m2. As another example, depending on the type of cancer, the dose of cyclophosphamide ranges from 1-5 mg / kg (or 50 to 100 mg / m2) orally in one day, or 1-2.5 mg / kg or higher parenterally in one day. For autoimmune diseases, the dose of cyclophosphamide may be about 10 mg / kg, 500-1000 mg / m2or 1-2 mg / kg (low dose) orally or parenterally. In some embodiments, the cycling agent is cyclophosphamide administered orally or parenterally at a dose ranging from about 1 mg / kg to about 10 mg / kg.

[0332] In some embodiments, the gene editing system is administered to the subject at a conventional dose. In some embodiments, the amount of gene editing system administered is less than the conventional dose. In some embodiments, the amount of gene editing system administered is less than the conventional dose. In some embodiments, the gene editing system is a CRISPR / Cas system administered via LNP delivery at a dose of about 0.1 mg / kg to about 10 mg / kg.Mode of administration / delivery

[0333] The gene editing system (or one or more components of the gene editing system) is administered to the subject or HSPC directly, or is indirectly delivered via expression in cells or in vivo. In such cases of delivery via expression, a polynucleotide (e.g. DNA or RNA) encoding one or more of the components of the gene editing system is administered to the subject or HSPC. In some embodiments, a gene editing system comprises one or more of: a genome-targeting nucleic acid, a polynucleotide encoding a genome-targeting nucleic acid, a site-directed polypeptide such as Cas9 or Cpfl, a polynucleotide encoding a site-directed polypeptide such as Cpfl, and / or any nucleic acid or proteinaceous molecule necessary to carry out the aspects of the methods described herein, such as a guide RNA, or any combination thereof.

[0334] Thus, in some embodiments, the gene editing system (or one or more components of the gene editing system) is administered to the subject or HSPC via expression from DNAor vectors encoding the gene editing system (or encoding one or more components of the gene editing system).

[0335] In other embodiments, the gene editing system (or one or more components of the gene editing system) is administered to the subject or HSPC via expression from RNA or mRNA encoding the gene editing system (or encoding one or more components of the gene editing system). When the gene editing system (or one or more components of the gene editing system) is encoded by mRNA, the mRNA can be delivered via a nanoparticle formulation, such as a lipid nanoparticle (LNP). For example, when the gene editing system is CRISPR-Cas9 / Cpfl, the Cas9 or Cpfl can be expressed in vivo from mRNA encoding Cas9. Such mRNA, optionally with guide RNA, can be formulated in LNP for delivery. The mRNA can be modified to increase its stability or expression level. The guide RNA can also be modified to increase its stability or activity. Illustrative examples of LNPs are described herein, as well as illustrative examples of nucleic acid modifications to guide RNA or mRNA, and any of these or any combination of these can be used in the treatment methods disclosed herein.Diseases

[0336] As used herein, the phrase "HSPC-treatable disease” broadly refers to any disease, disorder, or condition that may be treated or cured by editing HSPCs, either in vivo or ex vivo. HSPCs are long-lived and multipotent, so gene correction in HSPCs leads to persistent gene correction among the different lineages such as erythroid (erythrocytes or RBCs), myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, megakaryocytes / platelets, and dendritic cells), and lymphoid (T-cells, B-cells, NK-cells). Various reviews have identified specific cellular targets, relevant genes, and the corresponding diseases for gene therapy. They include the following: HSCs: Fanconi Anemia (FANC A-F). Platelets: Hemophilia A (Factor VIII (F8)); Hemophilia B (Factor IX (F9)); Factor X deficiency (Factor X (F 10)); Wiskott-Aldrich Syndrome (Wiskott Aldrich Syndrome Protein (WASP)). Neutrophils: X-linked Chronic Granulomatous Disease (Cytochrome B-245 Beta Chain (CYBB)); Kostmann’s Syndrome (Elastase Neutrophil Expressed (ELANE)). Erythrocytes: Alpha-Thalassemia (Hemoglobin Subunit Alpha (HBA)); Beta- Thalassemia and Sickle Cell Disease (Hemoglobin Subunit Beta (HBB)); Pyruvate Kinase Deficiency (Pyruvate Kinase, Liver and RBC (PKLR)); Diamond-Blackfan Anemia (Ribosomal Protein S19 (RPS19)). Monocytes: X-linked Adrenoleukodystrophy (ATP Binding Cassette Subfamily D Member 1 (ABCD1)); Metachromatic Leukodystrophy(Aryl sulfatase A (ARSA)); Gaucher disease (Glucosylceramidase Beta (GBA)); Hunter Syndrome (Iduronate 2-Sulfatase (IDS)); Mucopolysaccharidosis type I (Iduronidase, Alpha- L (IDUA)); Osteopetrosis (T-Cell Immune Regulator 1 (TCIRG1)). B Cells: Adenosine deaminase (ADA)-deficient Severe Combined Immunodeficiency (Adenosine Deaminase (ADA)); X-linked severe combined immunodeficiency (Interleukin 2 Receptor Subunit Gamma (IL2RG)); Wiskott-Aldrich Syndrome (Wiskott Aldrich Syndrome Protein (WASP)); X-linked agammaglobulinemia (Bruton’s Tyrosine Kinase (BTK)). T Cells: Adenosine Deaminase (ADA)-deficient Severe Combined Immunodeficiency (ADA); X-linked severe combined immunodeficiency (IL2RG); Wiskott-Aldrich Syndrome Protein (WASP); X- linked Hyper IgM syndrome (CD40 Ligand (CD40LG)); IPEX Syndrome (Forkhead Box P3 (FOXP3)); Early Onset Inflammatory Disease (Interleukin 4, 10, 13 (IL-4, 10, 13));Hemophagocytic Lymphohistiocytosis (Perforin 1 (PRF1)); Cancer (Artificial T cell receptors (TCR), Cancer; Chimeric Antigen Receptor (CAR)); Human immunodeficiency virus (C-C Motif Chemokine Receptor 5 (CCR5)). See Morgan, Richard A. et al., “Hematopoietic Stem Cell Gene Therapy: Progress and Lessons Learned,” Cell Stem Cell, 21(5): 574 - 590 (2017).

[0337] Examples of HSPC-treatable disease include blood disorders, such as sickle cell anemia, thalassemia, thalassemia major, alpha-thalassemia, beta-thalassemia, Fanconi anemia, aplastic anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, Schwachman-Diamond syndrome. Pre-clinical or clinical applications of gene therapy in these diseases have been reported. Morgan et al., supra.

[0338] Other examples of HSPC-treatable disease include autoimmune disorders, such as multiple sclerosis, systemic lupus erythematosus (SLE), systemic sclerosis, scleroderma, juvenile rheumatoid arthritis, ulcerative colitis, Crohn’s disease, type I (juvenile) diabetes.

[0339] Additional examples of HSPC-treatable disease include cancer or hematologic malignancies, for example, a hematologic cancer, leukemia, lymphoma, myeloma, acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, non-Hodgkin’s lymphoma, myelodysplastic syndrome. Examples of other cancers include glioblastoma, breast cancer, colorectal cancer, renal cell carcinoma, liver cancer, hepatocellular carcinoma, lung cancer, non-small cell and small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer, gastic cancer, gallbladder cancer and cholangiocarcinoma, melanoma,testis cancer, urinary or bladder cancer, head- and neck squamous cell carcinoma, or uterine cancer.10340] Further examples of HSPC-treatable disease include metabolic disorders or inherited genetic disorders such as glycogen storage diseases, mucopolysaccharidoses, Gaucher Disease, Hurler Disease, sphingolipidoses, metachromatic leukodystrophy, globoid cell leukodystrophy, cerebral adrenoleukodystrophy, severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyper immunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta. Numerous animal studies have demonstrated that HSPC-based gene therapy is able to treat non- hematological genetic disorders through secretion of curative or deficient proteins from HSPC-derived cells, e.g. arylsulfatase A in MLD, a-L-iduronidase in mucopolysaccharidosis type I (MPS-I), alpha-glucosidase enzyme in Pompe’s disease, glucocerebrosidase in Gaucher disease, a-galactosidase in Fabry disease, lysosomal acid lipase in Wolman disease, frataxin in Friedreich’s ataxia (FRDA) and other diseases such as cystinosis, Dent disease and X-ALD. See review Buffa et al., “Hematopoietic stem and progenitors cells gene editing: Beyond blood disorders,” Front. Genome Ed., 4:997142 (2023). Pre-clinical or clinical applications of gene therapy in these storage or metabolic disorders have been reported. Morgan et al., supra.

[0341] Yet further examples of HSPC-treatable disease include immunodeficiencies.

[0342] In some preferred embodiments, the subject either suffers from or is at risk of a hemoglobinopathy, a primary immunodeficiency (PIDs), or a congenital cytopenia. In some preferred embodiments, the subject has or is at risk of having a disease or condition selected from the group consisting of Sickle cell disease, Thalassemia, Pyruvate kinase deficiency, Hemophilia A, Hemophilia B, Factor X deficiency, Wiskott-Aldrich syndrome, Diamond- Blackfan anemia, Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease, Kostmann’s syndrome, X-linked adrenoleukodystrophy, Metachromatic leukodystrophy, Friedrich’s ataxia, Gaucher disease, Hunter syndrome, Mucopolysaccharidosis type 1, Osteopetrosis, X-linked agammaglobulinemia, X-linked hyper IgM syndrome, Immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, Early-onset inflammatory disease, Hemophagocytic, and lymphohistiocytosis. In some preferred embodiments, the SCID is selected from the group consisting of Adenosine deaminase-deficient SCID, Artemis SCID, X-linked SCID, and Recombination activating gene SCID.

[0343] Other examples of HSPC-treatable disease include diseases such as blood disorders, autoimmune disorders, or cancers that were conventionally treated by transplanting a population of HSPCs to a patient or by viral gene therapy; such conditions can be treated more advantageously by in vivo gene editing as described herein. Approved HSC gene therapy products include products for treating ADA-SCID, beta-thalassemia, sickle cell disease (SCD), metachromatic leukodystrophy (MLD), and cerebral adrenoleukodystrophy (CALD). HSC gene therapy clinical trials include products for treating TAG1-SCID, Artemis-SCID, X-SCID, ADA-SCID, MLD, ALD, beta-thalassemia, sickle cell disease, Hurler syndrome, Hemophilia A, Wiskott-Aldrich syndrome, leukocyte adhesion deficiency I, and glioblastoma multiforme. See review by Giommetti and Papanikolaou, “Advancements in Hemtopoietic Stem Cell Gene Therapy: A Journey of Progress for Viral Transduction,” Cells 13(12): 1039 (2024).

[0344] In preferred embodiments, the CXCR4 antagonist and gene editing systems are provided in an effective amount to treat or modulate the disease or condition.Hematopoietic Stem and Progenitor Cells

[0345] HSPC, the progenitors of all blood and immune cells, are comprised of five subgroups of hematopoietic cells: hematopoietic stem cells (HSCs), which have long-term self-renewal capacity, and four types of multipotent progenitors (MPPs 1-4), which are defined by lower self-renewal capacity and myeloid or lymphoid differentiation biases. Progenitors include myeloid progenitors, lymphoid progenitors, erythroid progenitors, T cell progenitors, and B cell progenitors.

[0346] HSPC are capable of both proliferation and giving rise to more progenitor cells, these in turn having the ability to generate a large number of mother cells that can in turn give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. The term “stem cell” refers then, to a cell with the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retains the capacity, under certain circumstances, to proliferate without substantially differentiating. In one aspect, the term progenitor or stem cell refers to a generalized mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. Cellular differentiation is acomplex process typically occurring through many cell divisions. A differentiated cell may derive from a multipotent cell that itself is derived from a multipotent cell, and so on. While each of these multipotent cells may be considered stem cells, the range of cell types that each can give rise to may vary considerably. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity may be natural or may be induced artificially upon treatment with various factors. In many biological instances, stem cells can also be “multipotenf ’ because they can produce progeny of more than one distinct cell type, but this is not required for “stem-ness.”

[0347] Self-renewal can be another important aspect of the stem cell. In theory, selfrenewal can occur by either of two major mechanisms. Stem cells can divide asymmetrically, with one daughter retaining the stem state and the other daughter expressing some distinct other specific function and phenotype. Alternatively, some of the stem cells in a population can divide symmetrically into two stems, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise to differentiated progeny only. Generally, “progenitor cells” have a cellular phenotype that is more primitive (i.e., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell). Often, progenitor cells also have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.

[0348] Thus, HSPC are cells of a stem cell lineage that give rise to all the blood cell types, including erythroid (erythrocytes or red blood cells (RBCs)), myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, megakaryocytes / platelets, and dendritic cells), and lymphoid (T-cells, B-cells, NK-cells).

[0349] A “cell of the erythroid lineage” indicates that the cell being contacted is a cell that undergoes erythropoiesis, such that upon final differentiation it forms an erythrocyte or red blood cell. Such cells originate from bone marrow hematopoietic progenitor cells. Upon exposure to specific growth factors and other components of the hematopoietic microenvironment, hematopoietic progenitor cells can mature through a series of intermediate differentiation cellular types, all intermediates of the erythroid lineage, into RBCs. Thus, cells of the “erythroid lineage” comprise hematopoietic progenitor cells, rubriblasts, prorubricytes, erythroblasts, metarubricytes, reticulocytes, and erythrocytes.

[0350] The hematopoietic progenitor cell can express at least one of the following cell surface markers characteristic of hematopoietic progenitor cells: CD34+, CD59+,Thyl / CD90+, CD381o / -, and C-kit / CDI 17+. In some examples provided herein, the hematopoietic progenitors can be CD34+.10351] The HSPC can be a peripheral blood stem cell obtained from the patient after the patient has been treated with one or more factors such as granulocyte colony stimulating factor (optionally in combination with Plerixafor). CD34+ cells can be enriched using CliniMACS® Cell Selection System (Miltenyi Biotec). CD34+ cells can be stimulated in serum-free medium (e.g., CellGrow SCGM media, CellGenix) with cytokines (e.g., SCF, rhTPO, rhFLT3) before genome editing. Addition of SRI and dmPGE2 and / or other factors is contemplated to improve long-term engraftment.

[0352] The HSPC of the erythroid lineage can have a cell surface marker characteristic of the erythroid lineage: such as CD71 and Teri 19.

[0353] Hematopoietic stem cells (HSCs) can be an important target for gene therapy as they provide a prolonged source of the corrected cells. HSCs give rise to both the myeloid and lymphoid lineages of blood cells. Mature blood cells have a finite life-span and must be continuously replaced throughout life. Blood cells are continually produced by the proliferation and differentiation of a population of pluripotent HSCs that can be replenished by self-renewal. Bone marrow (BM) is the major site of hematopoiesis in humans and a good source for hematopoietic stem and progenitor cells (HSPCs). HSPCs can be found in small numbers in the peripheral blood (PB). In some indications or treatments their numbers increase.

[0354] The terms “individual”, “subject,” “host” and “patient” are used interchangeably herein and refer to any subject for whom diagnosis, treatment or therapy is desired. In some aspects, the subject is a mammal. In some aspects, the subject is a human being.

[0355] The terms “administering,” “introducing” and “contacting” are used interchangeably in the context of the placement of the CXCR4 antagonist and / or gene editing system formulation into a subject, by a method or route such that a desired effect(s) is produced.

[0356] The term “effective amount” refers to the amount of the cycling agent (e.g. CXCR4 antagonist) and / or gene editing system formulation needed to prevent or alleviate at least one or more signs or symptoms of a HSPC-treatable disease, including but not limited to hemoglobinopathy, primary immunodeficiency or congenital cytopenia, or any of the diseases described herein, and relates to a sufficient amount of a composition to provide the desired effect, e.g., to treat a subject having hemoglobinopathy. The term “therapeutically effective amount” therefore refers to an amount of the cycling agent (for example, a CXCR4antagonist) and / or gene editing system formulation that is sufficient to promote a particular effect when administered to a typical subject, such as one who has or is at risk for hemoglobinopathy. An effective amount would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using routine experimentation.

[0357] When provided prophylactically, the agents described herein, (i.e., the cycling agent(s) or CXCR4 antagonist and / or gene editing system formulation) can be administered to a subject in advance of any symptom of a hemoglobinopathy, e.g., prior to the development of fatigue, shortness of breath, jaundice, slow growth late puberty, joint, bone and chest pain, enlarged spleen and liver. Accordingly, the prophylactic administration of a hematopoietic progenitor cell population serves to prevent a hemoglobinopathy, such as B- thalassemia or Sickle Cell Disease.

[0358] When provided therapeutically, the agents described herein, (i.e., the cycling agent(s) or CXCR4 antagonist and / or gene editing system formulation) are provided at (or after) the onset of a symptom or indication of hemoglobinopathy, e.g., upon the onset of disease.4. Kits

[0359] The present disclosure provides kits for carrying out the methods described herein. A kit can include a cycling agent (e.g., CXCR4 antagonist) and a gene editing system comprising one or more of a genome-targeting nucleic acid, a polynucleotide encoding a genome-targeting nucleic acid, a site-directed polypeptide, a polynucleotide encoding a site- directed polypeptide, and / or any nucleic acid or proteinaceous molecule necessary to carry out the aspects of the methods described herein, or any combination thereof. In preferred embodiments, the components of the gene editing system are formulated in LNPs.

[0360] In any of the above kits, the kit can comprise a single-molecule guide genometargeting nucleic acid. In any of the above kits, the kit can comprise a double-molecule genome-targeting nucleic acid. In any of the above kits, the kit can comprise two or more double-molecule guides or single-molecule guides. The kits can comprise a vector that encodes the nucleic acid targeting nucleic acid.

[0361] In any of the above kits, the kit can further comprise a polynucleotide to be inserted to effect the desired genetic modification.

[0362] Components of a kit can be in separate containers, or combined in a single container.

[0363] Any kit described above can further comprise one or more additional reagents, where such additional reagents are selected from a buffer, a buffer for introducing a polypeptide or polynucleotide into a cell, a wash buffer, a control reagent, a control vector, a control RNA polynucleotide, a reagent for in vitro production of the polypeptide from DNA, adaptors for sequencing and the like. A buffer can be a stabilization buffer, a reconstituting buffer, a diluting buffer, or the like. A kit can also comprise one or more components that can be used to facilitate or enhance the on -target binding or the cleavage of DNA by the endonuclease, or improve the specificity of targeting.

[0364] In addition to the above-mentioned components, a kit can further comprise instructions for using the components of the kit to practice the methods. The instructions for practicing the methods can be recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g. via the Internet), can be provided. An example of this case is a kit that comprises a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.EXAMPLES

[0365] The following are examples of the present invention and are not to be construed as limiting.EXAMPLE 1

[0366] The ability of plerixafor alone or in combination with G-CSF to mobilize HSPC to the peripheral blood was examined. C57BL / 6 mice received either Plerixafor alone at a doseof 10 mg / kg subcutaneously (SC, Selleckchem Cat# S8030), or they were pre-treated intraperitoneally (IP) with murine G-CSF (5 pg / mouse, Peprotech Cat#AF-250-05) daily for four days before Plerixafor administration. Blood samples were obtained via retro-orbital bleeding at 1 hour, 24 hours, and 6 days following the Plerixafor dose. The mobilization of hematopoietic stem progenitor cells into the peripheral blood was assessed by performing Colony-Forming Unit (CFU) assays using MethoCult™ M3234 (Stemcell Technologies, Cat#03234). The data is presented in FIG. 1 A-1B.

[0367] Next, the effect of plerixafor alone or in combination with G-CSF on in vivo editing of HSPC was examined. C57BL / 6 mice pretreated with or without Plerixafor and / or murine G-CSF as outlined in FIG. 1 A-1B, followed by administration of LNPs targeting the mouse BCL1 la gene at a dose level of 5 mg / kg. BCL1 la gene editing constructs are described in U.S. Pat. Publ. 20220211874, incorporated by reference herein its entirety. One week after LNP administration, the mice were euthanized, and bone marrow was obtained via bone crushing. Lineage-positive cells were then depleted using magnetic selection, and Lineage-negative cells were sorted via fluorescence-activated cell sorting (FACS) to isolate Lin-Sca+c-Kit+ cells (LSKs) that are enriched for hematopoietic stem and progenitor cells (HSPCs). DNA was extracted from LSK cells, and mBCLl la editing was evaluated using Amplicon sequencing (Amp-seq).

[0368] Next, the ability of plerixafor alone or in combination with G-CSF to increase gene editing over time in peripheral blood was examined. As described in FIG. 2A-2B, a separate cohort of mice treated with or without Plerixafor and / or murine G-CSF, followed by administration of LNPs targeting the mouse BCL1 la gene at a dose level of 5 mg / kg. Blood samples were obtained via retro-orbital bleeding at 1 week, 1 month, 2 months, and 3 months following LNP administration. DNA was extracted from peripheral blood, and mBCLl la editing was evaluated using Amp-seq. The data is presented in FIG. 3A-3D.

[0369] These data (FIG. 2A-2B) demonstrate that LNPs can deliver mRNA to bone marrow HSPC and that plerixafor can enhance gene editing when administered before LNP injection. Surprisingly, G-CSF administration along with plerixafor reduced the enhancing effects of plerixafor suggesting that HSPC mobilization is likely not the mechanism of the effect and that other plerixafor / CXCR4 chemokine responsive pathways may help with LNP mediated gene editing in HSPC.EXAMPLE 2

[0370] The ability of plerixafor alone or in combination with G-CSF to increase gene editing over time in peripheral blood was examined. Cohorts of immunocompetent mice were treated with Plerixafor and / or murine G-CSF, followed by administration of LNPs targeting the mouse BCL1 la gene at a dose level of 5 mg / kg. One cohort of mice was left untreated with only PBS administration and served as the control group. Blood samples were obtained via retro-orbital bleeding at 1 week, 1 month, 2 months, and 3 months, 4 months, and 5 months following LNP administration. The cells were sorted, DNA extracted, and mBCLl la editing was evaluated using Amp-seq. The data presented in FIG. 3 A-3D shows progressive increase in LNP-mediated editing rates of cells pre-treated with plerixafor alone compared to those pre-treated with the combination of plerixafor and G-CSF. FIGs. 3-6 and Tables 1 and 2 show data at 5 months and demonstrates increased editing of HSCs in bone marrow cells (Lin-Scal+c-Kit+CD45“ CD150+cells), myeloid progenitors (LKs; Lin-Scal+c-Kit+), and in multi-lineage cells after pre-treatment with plerixafor alone compared to delivery with LNP alone well as with cells pre-treated with plerixafor and G-CSF. The frequency of small ±1 bp indels was seen to go up over time in mice pre-treated with plerixafor alone (FIG. 6A-6B).Table 1 : Indel rate (%) in BM and peripheral blood cell at 5-month time-point (N=3)Table 2: Indel rate (%) in peripheral blood cell at 5-month time-point (N=3)EXAMPLE 3

[0371] The effect of plerixafor on LNP-mediated delivery of a gene editing system to human CD34+ cells was investigated in vitro and in vivo.In vitro dataTo examine the effect of plerixafor pre-treatment on gene editing in vitro, human CD34+ hematopoietic stem and progenitor cells (HSPCs) were treated with 100 ng of LNP in the presence or absence of 1-100 nM of plerixafor (AMD3100, Sigma cat no 5602). Cryopreserved mobilized peripheral blood derived human CD34+ cells were obtained from Charles River and cultured for 48hrs in SCGM media (CellGenix Cat no 20802-0500) containing thrombopoietin (R&D systems 288-TP-200 / CF), human Flt3 ligand (R&D systems Cat no 308-GMP-01M), human stem cell factor (R&D systems 255-SC / CF). Human recombinant apolipoprotein E3 (ApoE3, Sigma cat no SRP4696) was added to culture media (1 ug / ml concentration) for LNP transfection. LNP was formulated with SpCas9 mRNA and human Bell la sgRNA. Genomic DNA was extracted from cultured CD34+ cells 48hr post LNP + / - plerixafor treatment, and Bell la editing was evaluated using Amp-seq assay. The data shown in FIG. 7 demonstrates that plerixafor at only 10 nM concentration can enhance gene editing.In vivo data

[0372] JAX CD34+ hu-NSG mice were divided into 7 cohorts of 5 mice each, as shown in Table 3 below. The goal of the study is to evaluate the effect of plerixafor pre-treatment before LNP delivery of a gene editing system, as well as the effect of plerixafor conjugated to 0.5% DSPE. JAX CD34+ hu-NSG mice were pre-treated with or without plerixafor 10 mg / kg subcutaneously followed by administration of LNP targeting human Bell la at 2 mg / kg intravenously with 1 hour interval between plerixafor and LNP. For each cohort, the gene editing system was Cas9 mRNA and a human Bell la gene-targeting sgRNA.Table 3: Study Details

[0373] The results are presented in FIG. 8. In FIG. 8A, it is seen that plerixafor pretreatment increased LNP-mediated gene editing in short term (12 days post dose). There was an about two- to four-fold increase in editing with plerixafor increased editing with plerixafor pre-treatment. We also observed up long term editing with plerixafor pre-treatment in hu- NSG mice (FIG. 8B).

[0374] Overall, the data supports our hypothesis of plerixafor as a mechanism leading to direct GO to G1 transition of the cells. The data supports a method of increasing editing efficiency by administration of plerixafor.EXAMPLE 4

[0375] The effect of redosing with LNPs containing a gene editing system with plerixafor pretreatment was evaluated.

[0376] C57BL / 6 mice were separated at 10 mice per group. One group was the control group administered PBS alone. The other two groups were both pre-treated with plerixafor at 10 mg / kg s.c. An LNP-delivered gene editing system targeting a murine BCL1 la gene was administered 1 hour after the pre-treatment. The LNP dose was 5 mg / kg i.v. On day 12, half the mice of each group were euthanized for BM and blood collection. As shown in FIG. 9A- 9C, pre-treatment with plerixafor boosted LNP-mediated editing of HSCs, progenitor cell, and total BM cells compared to the controls. HSC cells are Lin“Sca+cKit+CD48- CD150+; LK cells are Lin“Sca“ cKit+. Editing levels in peripheral blood cells in all mice of the pretreated group showed an increase after plerixafor pre-treatment, as seen in FIG. 10.

[0377] Some of the mice were then re-dosed with plerixafor and LNP-mediated gene editing system 3 months after the first administration. The plerixafor re-dose was 10 mg / kg s.c. and the LNP re-dose was 10 mg / kg i.v. Mice selected from all groups were euthanized 10-14 days after the re-dosing time-point. As can be seen in FIGs 11-13, plerixafor pretreatment reproducibly increased LNP-mediated gene editing of HSCs and peripheral blood cells in vivo. It is seen that redosing with plerixafor and LNP-delivered gene editing system resulted in a further increase in editing of phenotypic HSC cells as well as peripheral blood cells. Editing rates in HSCs, total BM cells, and LK cells were measured 14 days after the redose which was approximately 3.5 months after initial treatment. Editing rates in peripheral blood cells are seen in FIG. 12A-12D to continue to increase at 3.5 months to 10-months as well. There was also a boost in editing rates in mature cells in the bone marrow after redosing. FIGs. 12E-12G show mBCLl la editing in HCSs, LKs and total bone marrow in all groups (PBS control group, LNP group and PLX+LNP group) at 2-weeks and 6-months.

[0378] FIG. 13A-13D show that redosing with plerixafor and LNP leads to increased editing of mBCLl la in selected lineages (T cells (FIG. 13 A), B cells (FIG. 13B), myeloid cells (FIG. 13C) and erythroid cells (FIG. 13D)) in bone marrow.

[0379] Overall, the data demonstrates the novel method of boosting editing efficiency in LNP-mediated gene editing of HSPCs, BM progenitor cells, as well as mature cells by redosing with plerixafor and the LNP-mediated gene editing system.EXAMPLE 5

[0380] The ability of edited HSCs to engraft into recipient mice and retain editing efficiency was studied. Gene-edited and control CD45.2 HSCs were prepared by administering to C57BL / 6 (donor) mice: (a) PBS as a control, (b) LNP-mediated gene editing system alone, (c) LNP-mediated gene editing system after pretreatment with Plerixafor, and (d) LNP-mediated gene editing system after pretreatment with Plerixafor and murine G-CSF as described in Example 2 above. Bone marrow cells were collected from donor mice using a crushing method. Following euthanasia, femurs, tibias and sternum bones were crushed with a mortar and pestle to collect the bone marrow. The resulting cells suspension was passed through a 40-micron cell strainer to remove debris, then the cells were washed and counted. The editing rate of donor HSCs was 0% in the PBS group, 0.1% in the LNP-only group, 17.2% in the Plerixafor pretreated group, and 14.3% in the Plerixafor+GCSF pretreated group.

[0381] The harvested bone marrow cells from the donor mice were administered at a dose of 0.5 x 106cells per mouse to recipient C57BL / 6J mice, native CD45.1 HSCs, that had been treated with either sublethal irradiation or lethal irradiation. Results are shown in FIG. 14A- 14D. The percent of CD45.2 cells in the recipient mice treated with sublethal irradiation (FIG. 14A) or lethal irradiation (FIG. 14B) are depicted. The engrafted CD45.2 cells increase over time and become predominant in all recipient mice. The percent of mBCLl la gene editing in peripheral blood cells of the recipient mice treated with sublethal irradiation (FIG. 14C) or lethal irradiation (FIG. 14D) are depicted. The percent of gene-edited cells increase over time in the mice treated with lethal irradiation.EXAMPLE 6

[0382] The effect of agents that may be instrumental in indirectly transitioning cells from GO to G1 was studied. 5 -fluoro-uracil and cyclophosphamide are typically used as chemotherapeutic agents and may be used to deplete the bone marrow of committed progenitor cells. Poly-Inosinic-poly-Cytidylic (pIpC) is a viral mimic that can induce the activation of hematopoietic stem cells (HSCs) in bone marrow.

[0383] The effect of pre-treatment of these agents alone on LNP -mediated editing was studied. Some cohorts of immunocompetent mice were pretreated with PIPC (Polyinosinic- poly cytidylic acid) at 100 pg / mouse, IP 3 days and 1 day prior to LNP administration), 5-FU (5 Fluorouracil) 100 mg / Kg, IP 4 days prior to LNP administration or Cyclophosphamide 150 mg / Kg, IP 2 days prior to LNP administration, followed by administration of LNPs targeting the mouse BCL1 la gene at a dose level of 5 mg / kg. One cohort of mice was left untreated with only PBS administration and served as the control group. 10 days post LNP administration, some mice from each group were sacrificed, and total bone marrow (BM) was collected. HSCs (Lin-Scal+ c-Kit+CD48-CD150+) and LKs (Lin-Seal- c-Kit+) were sorted, DNA extracted, and mBCLl la editing was evaluated using Amp-seq. As seen in FIG. 15A- 15C, 5-FU and cyclophosphamide increased editing in HSCs, LK cells, total bone marrow and peripheral blood cells in the short term (10 days) with continued increase in editing up to 6-months. FIGS 15A-C show effect of the pretreatment with PIPC, 5-FU and cyclophosphamide on editing of mBCLl la in sorted HSCs (FIG. 15 A), LKs (FIG. 15B) and total bone marrow (FIG. 15C) at 10-days compared to 6-months post administration. Further, FIG. 15D shows effect of 5-FU and cyclophosphamide agents in peripheral blood at 10-days, 1-month, 2-months, 3-months, 4-months, 5-months and 6-months post administration. Theseresults indicate that 5-FU and cyclophosphamide (in the absence of plerixafor) boost in vivo editing of HSCs by LNP. Additionally, variability in LNP dosing also affects editing levels.EXAMPLE 7

[0384] A combo treatment study was designed in vivo in C57B1 / 6 mice, as illustrated in FIG. 16. The study was to test the effect of the administration of various cycling agents, 5- FU, or Cyclophosphamide, in combination with Plerixafor, as well as various mobilizing agents, Gro-0, BIO5192, or Mavorixafor, in combination with Plerixafor. A conjugate of plerixafor and LNP was also studied. The conjugate was administered alone or in combination with Mavorixafor.

[0385] Plerixafor, Gro-0, and BIO5192 were subcutaneously administered; 5-FU and Cyclophosphamide were intraperitoneally administered; Mavorixafor was orally administered. Plerixafor was given 1 hr prior to LNP administration on Day 5 of the study or in a three-day regimen at Days 3, 4, and 5. Mavorixafor, BIO5192 and Gro-0 were given prior to LNP administration on Day 5. 5 -Fluorouracil was given on Day 1.Cyclophosphamide was given on Day3.The doses of each agent are as follows: BIO5192: ~1 mg / kg 30 minutes prior to administration of the LNP; Mavorixafor: -2 mg / kg 1 hour prior to administration of the LNP; Gro-0: ~2.5 mg / kg 15 minutes prior to administration of the LNP; Plerixafor: -10 mg / kg; 5 -Fluorouracil: -100 mg / kg; Cyclophosphamide: -150 mg / kg.

[0386] The result of the study is presented in FIG 17 and show a remarkable boost in HSC editing at 2 weeks continuing through to 1.5 months (FIG. 17A-17B, respectively), in bone marrow editing at 2 weeks continuing through to 1.5 months (FIGS. 17C-D, respectively), in peripheral blood editing continuing through to 4 months (FIGS. 17E-F), in LK editing 2 weeks continuing through to 1.5 months (FIG. 17G-H), erythroid cell editing at 2 weeks (FIG.171), myeloid cell editing at 2 weeks (FIG.17 J) and B cell editing at 2 weeks (FIG.17K) after pre-treatment of plerixafor in combination with either of the cycling agents 5-FU or cyclophosphamide. The effect of combination of plerixafor and 5-FU or cyclophosphamide is a synergistic effect which is an unexpected finding.

[0387] In contrast, the combination of plerixafor with mobilization agents like Gro-0, BIO5192, and Mavorixafor had no effect on editing efficiency in peripheral blood over and above the effect produced by plerixafor alone (FIG. 18B-18D). Each agent was able to mobilize cells as shown by the increase in CFU count (FIG. 18 A). This data confirms that the effect on editing efficiency is not because of mobilization of cells to the periphery but is aresult of transition of the cells from a GO quiescent state to a more active metabolic or proliferative state.EXAMPLE 8

[0388] The effect of plerixafor pre-treatment on LNP -mediated delivery to human CD34+ cells of a gene editing system targeting a different gene, CCR5, was investigated in vivo.

[0389] JAX CD34+ hu-NSG mice were divided into 5 cohorts of 6 mice each, as shown in Table 4 below. The mice were pre-treated with or without plerixafor 10 mg / kg subcutaneously followed by administration of two different doses of LNP targeting CCR5, 2 mg / kg or 4 mg / kg intravenously with 1 hour interval between plerixafor and LNP. For each cohort, the gene editing system was Cas9 mRNA and a human CCR5 gene-targeting sgRNA.

[0390] Table 4: Study Details

[0391] The results are presented in FIG. 19A-C. FIG 19A shows the percent of CCR5 gene editing at 2 weeks after LNP administration in bone marrow cells, CD34+ cells, CD34+ / CD38- / CD90- cells, and CD34+ / CD38- / CD90+ cells. FIG. 19B shows the percent of CCR5 gene editing at 6 weeks in peripheral blood. FIG. 19C shows the percent of CCR5 gene editing at 8 weeks after LNP administration in bone marrow cells, CD34+ cells, CD34+ / CD38- / CD90- cells, and CD34+ / CD38- / CD90+ cells.EXAMPLE 9

[0392] The effect of the timing of plerixafor pre-treatment on LNP -mediated delivery t of a gene editing system targeting Bel 1 was investigated in vivo.

[0393] Plerixafor 10 mg / kg was given s.c. (a) 1 hr prior to LNP administration on Day 5 of the study or (b) in a three-day regimen of 10 mg / kg s.c. at Days 3, 4, and 5. The LNP doseas 5 mg / kg i.v. On day 10, some mice from each group were euthanized for BM and blood collection. Blood collection from mice at 1 month, 1.5 months, 3 months and 4 months showed that the average editing rates with plerixafor pre-treatment were at least 2-fold higher than (increasing nearly 3 -fold higher by 4 months) the editing rates of LNP without plerixafor pre-treatment, and there was no significant difference between the 1-hr or 3 -day pretreatment. These results indicate a 3 -day pre-treatment with plerixafor may not be needed for efficient gene editing. See Table 5 below. The results of this study suggest that 1 hour pretreatment with Plerixafor is enough to boost editing and produce similar results to 3 consecutive days of pretreatment.

[0394] The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention.

[0395] Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

CLAIMSWhat is claimed is:

1. An in vivo method of editing the genome of one or more hematopoietic stem and progenitor cells (HSPCs) in a subject in need thereof comprising: administering a CXCR4 antagonist to the subject; and administering a gene editing system to the subject so that the genome(s) of HSPCs in the subject are edited.

2. A compound comprising a CXCR4 antagonist for use in increasing the efficiency of in vivo gene editing of one or more hematopoietic stem and progenitor cells (HSPCs) in a subject in need thereof.

3. An ex vivo method of editing the genome of one or more hematopoietic stem and progenitor cells (HSPCs) from a subject comprising: obtaining HSPCs from a subject; contacting the HSPCs with a CXCR4 antagonist; and introducing a gene editing system into the HSPCs so that the genome(s) of HSPCs are edited.

4. A compound comprising a CXCR4 antagonist for use in increasing the efficiency of ex vivo gene editing of one or more hematopoietic stem and progenitor cells (HSPCs) from a subject in need thereof.

5. The method or use of any one of claims 3 to 4, wherein the HSPCs are infused back into the subject.

6. The method or use of any one of claims 3 to 5, wherein the patient is not subjected to a conditioning treatment.

7. The method or use of any one of claims 1 to 6, wherein the CRCX4 antagonist is plerixafor or a functional derivative thereof.

8. The method or use of any one of claims 1 to 7, wherein the subject has or is at risk of having a disease or condition selected from the group consisting of a hemoglobinopathy, a primary immunodeficiency and a congenital cytopenia.

9. The method or use of any one of claims 1 to 8, wherein the subject has or is at risk of having a disease or condition selected from the group consisting of Sickle cell disease, Thalassemia, Pyruvate kinase deficiency, Hemophilia A, Hemophilia B, Factor X deficiency,Wiskott-Aldrich syndrome, Diamond-Blackfan anemia, Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease, Kostmann’s syndrome, X-linked adrenoleukodystrophy, Metachromatic leukodystrophy, Friedreich’s ataxia, Gaucher disease, Hunter syndrome, Mucopolysaccharidosis type 1, Osteopetrosis, X-linked agammaglobulinemia, X-linked hyper IgM syndrome, Immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, Early-onset inflammatory disease, Hemophagocytic lymphohistiocytosis, VEXAS (vacuoles, El enzyme, X-linked, autoinflammatory, somatic) syndrome, and erythropoietic protoporphyria.

10. The method of use of claim 9, wherein the SCID is selected from the group consisting of Adenosine deaminase-deficient SCID, Artemis SCID, X-linked SCID, and Recombination activating gene SCID.

11. The method or use of any one of claims 1 to 10, wherein the gene editing system is selected from the group consisting of a zinc finger nuclease (ZFN) gene editing system, a transcription activator-like effector nuclease (TALEN) gene editing system, a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease gene editing system, a base or prime editing system, a reverse transcriptase (RT) editing system, a non-LTR retrotransposon editing system, and a retron gene editing system.

12. The method or use of claim 11, wherein the gene editing system is a CRISPR / Cas nuclease gene editing system.

13. The method or use of claim 12, wherein the CRISPR / Cas nuclease gene editing system comprises a gRNA that targets a sequence in the genome of the one or more HSCs.

14. The method or use of claim 13, wherein the gRNA is a single-molecule guide RNA (sgRNA).

15. The method or use of any one of claims 12 to 14, wherein the CRISPR / Cas nuclease gene editing system comprises a Cas nuclease selected from the group consisting of Casl , CasI B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Casl 00, Csyl , Csy2, Csy3, Csel , Cse2, Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl , Cmr3, Cmr4, Cmr5, Cmr6, Csbl , Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl , Csxl5, Csfl , Csf2, Csf3, Csf4, Cpfl, or SluCas9 endonuclease; a homolog thereof, a recombination of the naturally occurring molecule thereof, codon-optimized thereof, or modified versions thereof, and combinations thereof.

16. The method or use of claim 15, wherein the Cas nuclease is encoded by an nucleic acid molecule.

17. The method or use of any one of claims 1 to 16, wherein the gene editing system effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within a target gene in the genomes of the one or more the subject HSCs.

18. The method or use of any one of claims 1 to 17, wherein the gene editing system introduces an indel mutation into a target gene in the genomes of the one or more subject HSPCs.

19. The method or use of any one of claims 8 to 18, wherein the CRISPR / Cas nuclease gene editing system further comprises a donor template.

20. The method or use of claim 19, wherein the donor template is a DNA molecule.

21. The method or use of any one of claims 1 to 20, wherein the gene editing system is administered via a lipid nanoparticle (LNP) or adeno-associated virus (AAV) vector or combination thereof.

22. The method or use of claim 21, wherein the gene editing system is administered via a lipid nanoparticle (LNP).

23. The method or use of claim 22, wherein the LNP comprises ionizable lipid.

24. The method or use of any one of claims 1-18 and 21-23, wherein the editing step further comprises introducing into the subject HSCs one or more deoxyribonucleic acid (DNA) endonucleases to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the BCL11A gene or other DNA sequence that encodes a regulatory element of the BCL11A gene that results in a permanent deletion, modulation, or inactivation of a transcriptional control sequence of the BCL11A gene.

25. The method or use of any one of claims 19 to 23, wherein the method further comprises introducing into the subject HSCs one guide ribonucleic acid (gRNA) and a polynucleotide donor template comprising a wild-type BCL11A gene or cDNA comprising a modified transcriptional control sequence and one or more DNA endonucleases that effect one single-strand break (SSB) or double-strand break (DSB), at a locus within or near the BCL11A gene or other DNA sequence that encodes a regulatory element of the BCL11A gene, that facilitates insertion of a new sequence from the polynucleotide donor template intothe chromosomal DNA at the locus that results in a permanent insertion, modulation, or inactivation of the transcriptional control sequence of the chromosomal DNA proximal to the locus, and wherein the gRNA comprises a spacer sequence that is complementary to a segment of the locus.

26. The method or use of any one of claims 19 to 23, wherein the method further comprises introducing into the cell one or more guide ribonucleic acid (gRNAs) and a polynucleotide donor template comprising a wild-type BCL11A gene or cDNA comprising a modified transcriptional control sequence and one or more DNA endonucleases that effect effect or create a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs), the first break at a 5' locus and the second break at a 3' locus, within or near the BCL11A gene or other DNA sequence that encodes a regulatory element of the BCL11A gene, that facilitates insertion of a new sequence from the polynucleotide donor template into the chromosomal DNA between the 5' locus and the 3' locus that results in a permanent insertion, modulation, or inactivation of the transcriptional control sequence of the chromosomal DNA between the 5' locus and the 3' locus.

27. The method or use of any one of claims 19 to 23, wherein the method further comprises introducing into the cell one or more guide ribonucleic acid (gRNAs and one or more DNA endonucleases that effect or create a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs), a first SSB or DSB at a 5' locus and a second SSB or DSB at a 3' locus, within or near the BCL11A gene or other DNA sequence that encodes a regulatory element of the BCL11A gene that causes a deletion of the chromosomal DNA between the 5' locus and the 3' locus that results in a permanent deletion, modulation, or inactivation of the transcriptional control sequence of the chromosomal DNA between the 5' locus and the 3' locus.

28. A kit comprising: a container containing a CXCR4 antagonist; and a container containing a formulation comprising one or more components of a gene editing system sufficient for editing a gene or nucleic acid sequence associated with a disease or condition selected from the group consisting of a hemoglobinopathy, a primary immunodeficiency and a congenital cytopenia.

29. The kit of claim 28, wherein the CRCX4 antagonist is plerixafor or a functional derivative thereof.

30. The kit of any one of claims 28 to 29, wherein the subject has or is at risk of having a disease or condition selected from the group consisting of Sickle cell disease, Thalassemia, Pyruvate kinase deficiency, Hemophilia A, Hemophilia B, Factor X deficiency, Wiskott-Aldrich syndrome, Diamond-Blackfan anemia, Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease, Kostmann’s syndrome, X-linked adrenoleukodystrophy, Metachromatic leukodystrophy, Friedreich’s ataxia, Gaucher disease, Hunter syndrome, Mucopolysaccharidosis type 1, Osteopetrosis, X-linked agammaglobulinemia, X-linked hyper IgM syndrome, Immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, Early-onset inflammatory disease, Hemophagocytic lymphohistiocytosis, VEXAS (vacuoles, El enzyme, X-linked, autoinflammatory, somatic) syndrome, and erythropoietic protoporphyria.

31. The kit of claim 30, wherein the SCID is selected from the group consisting of Adenosine deaminase-deficient SCID, Artemis SCID, X-linked SCID, and Recombination activating gene SCID.

32. The kit of any one of claims 28 to 31, wherein the gene editing system is selected from the group consisting of a zinc finger nuclease (ZFN) gene editing system, a transcription activator-like effector nuclease (TALEN) gene editing system, a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease gene editing system, a base or prime editing system, a reverse transcriptase (RT) editing system, a non-LTR retrotransposon editing system, and a retron gene editing system.

33. The kit of claim 32, wherein the gene editing system is a CRISPR / Cas nuclease gene editing system.

34. The kit of claim 33, wherein the CRISPR / Cas nuclease gene editing system comprises a gRNA that targets a sequence in the genome of the one or more HSCs.

35. The kit of claim 34, wherein the gRNA is a single-molecule guide RNA (sgRNA).

36. The kit of any one of claims 33 to 35, wherein the CRISPR / Cas nuclease gene editing system comprises a Cas nuclease selected from the group consisting of Casl , CasI B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Casl 00, Csyl , Csy2, Csy3, Csel , Cse2, Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl , Cmr3, Cmr4, Cmr5, Cmr6, Csbl , Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl , Csxl5, Csfl , Csf2, Csf3, Csf4, Cpfl, or SluCas9 endonuclease; a homologthereof, a recombination of the naturally occurring molecule thereof, codon-optimized thereof, or modified versions thereof, and combinations thereof.

37. The kit of claim 36, wherein the Cas nuclease is encoded by an nucleic acid molecule.

38. The kit of any one of claims 33 to 37, wherein the CRISPR / Cas nuclease gene editing system further comprises a donor template.

39. The kit of claim 38, wherein the donor template is a DNA molecule.

40. The kit of any one of claims 28 to 39, wherein the one or more components of the gene editing system are formulated in a LNP.

41. The kit of claim 40, wherein the LNP comprises an ionizable lipid.

42. The kit of any one of claims 28 to 40, wherein the one or more components of the gene editing system edit BCL11A gene or control sequence of the BCL11A gene.

43. The kit of any one of claims 28 to 40 for use in treating a with a disease or condition selected from the group consisting of a hemoglobinopathy, a primary immunodeficiency and a congenital cytopenia.

44. The use of claim 43, wherein the subject has or is at risk of having a disease or condition selected from the group consisting of Sickle cell disease, Thalassemia, Pyruvate kinase deficiency, Hemophilia A, Hemophilia B, Factor X deficiency, Wiskott-Aldrich syndrome, Diamond-Blackfan anemia, Severe Combined Immunodeficiency (SCID), X- linked chronic granulomatous disease, Kostmann’s syndrome, X-linked adrenoleukodystrophy, Metachromatic leukodystrophy, Friedrich’s ataxia, Gaucher disease, Hunter syndrome, Mucopolysaccharidosis type 1, Osteopetrosis, X-linked agammaglobulinemia, X-linked hyper IgM syndrome, Immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, Early-onset inflammatory disease, Hemophagocytic lymphohistiocytosis, VEXAS (vacuoles, El enzyme, X-linked, autoinflammatory, somatic) syndrome, and erythropoietic protoporphyria.

45. The use of claim 44, wherein the SCID is selected from the group consisting of Adenosine deaminase-deficient SCID, Artemis SCID, X-linked SCID, and Recombination activating gene SCID.

46. The method of any of claims 1-27, the kit of any of claims 28-43 or the use of any of claims 44-45, wherein the CXCR4 antagonist is plerixafor and is administered in theabsence of administering G-CSF or in the absence of administrating other mobilizing agents that have primarily mobilizing activity.

47. The method of any of claims 1-27, the kit of any of claims 28-43 or the use of any of claims 44-46, wherein the subject does not undergo myeloablative conditioning, and / or wherein the subject does not undergo HSC harvest.

48. An in vivo method of editing the genome of one or more hematopoietic stem and progenitor cells (HSPCs) in a subject in need thereof comprising: administering a cycling agent; and administering a gene editing system to the subject so that the genome(s) of one or more HSPCs in the subject are edited.

49. The method of claim 48, wherein the cycling agent is administered in an amount effective to increase the gene editing efficiency of a gene editing system.

50. The method of claim 48, wherein the cycling agent is administered in an amount effective to increase the percentage of HSPCs in a more active metabolic state or proliferative state.

51. The method of claim 48 wherein the administration of the cycling agent occurs within about six hours of administering the gene editing system, optionally within about an hour before administering the gene editing system.

52. A cycling agent for use in increasing the efficiency of in vivo gene editing of one or more hematopoietic stem and progenitor cells (HSPCs) in a subject in need thereof.

53. An ex vivo method of editing the genome of one or more hematopoietic stem and progenitor cells (HSPCs) from a subject comprising: obtaining HSPCs from the subject; contacting the HSPCs with a cycling agent; and introducing a gene editing system into the HSPCs so that the genome(s) of the HSPCs are edited.

54. The method of claim 53, wherein the cycling agent is administered in an amount effective to increase the gene editing efficiency of the gene editing system.

55. The method of claim 53, wherein the cycling agent is administered in an amount effective to increase the percentage of HSPCs in a more active metabolic state or proliferative state.

56. A cycling agent for use in increasing the efficiency of ex vivo gene editing of one or more hematopoietic stem and progenitor cells (HSPCs) from a subject in need thereof.

57. The method or use of any one of claims 1-56, wherein the HSPCs are infused back into the subject.

58. The method or use of any one of claims 1-57, wherein the subject is not subjected to a myeloablative conditioning treatment.

59. The method or use of any one of claims 48-58, wherein the subject has or is at risk of having an HSPC-treatable disease, optionally a disease or condition selected from the group consisting of a hemoglobinopathy, immunodeficiency, hematological malignancies, cancer, anemias, autoimmune diseases, and cytopenias.

60. The method or use of any one of claims 48-59 , wherein the subject has or is at risk of having a disease or condition selected from the group consisting of Sickle cell disease, Thalassemia, Pyruvate kinase deficiency, metabolic disorders such as hypophosphatasia, Hemophilia A, Hemophilia B, Factor X deficiency, Wiskott-Aldrich syndrome (WAS), Diamond-Blackfan anemia, primary immunodeficiency disorder (PIDs) including Severe Combined Immunodeficiency (SCID), chronic granulomatous disease (CGD), etc., X-linked chronic granulomatous disease, Kostmann’s syndrome, X-linked adrenoleukodystrophy, Metachromatic leukodystrophy, CNS diseases such as Friedreich’s ataxia and Alzheimer’s diseases, lysosomal storage disease (LSD), such as Gaucher disease, Fabry’s disease, Pompe disease, Mucopolysaccharidosis Type I (MPSOI) and MPS-II; autoimmune conditions such as lupus (SLE / LN), scleroderma, myositis, myasthenia gravis, multiple sclerosis, Crohn’s disease, and stiff-persons-syndrome; Hunter syndrome, Mucopolysaccharidosis type 1, Osteopetrosis, X-linked agammaglobulinemia, X-linked hyper IgM syndrome, Immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, Early-onset inflammatory disease, Hemophagocytic lymphohistiocytosis, VEXAS (vacuoles, El enzyme, X-linked, autoinflammatory, somatic) syndrome, and erythropoietic protoporphyria.

61. The method or use of claim 60, wherein the SCID is selected from the group consisting of Adenosine deaminase-deficient SCID, Artemis SCID, X-linked SCID, and Recombination activating gene SCID.

62. The method or use of any one of claims 48-61, wherein the gene editing system is selected from the group consisting of a zinc finger nuclease (ZFN) gene editing system, a transcription activator-like effector nuclease (TALEN) gene editing system, aclustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease gene editing system, a base or prime editing system, a reverse transcriptase (RT) editing system, a non-LTR retrotransposon editing system, and a retron gene editing system.

63. The method or use of claim 62, wherein the gene editing system is a CRISPR / Cas nuclease gene editing system.

64. The method or use of claim 48-61, wherein the gene editing system is a reverse transcriptase (RT) editing system.

65. The method or use of claims 48-61, wherein the gene editing system is a base editing system.

66. The method or use of any one of claims 63-65, wherein the gene editing system comprises a Cas nuclease selected from the group consisting of Casl , Cast B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Casl 00, Csyl , Csy2, Csy3, Csel , Cse2, Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl , Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl , Csxl5, Csfl , Csf2, Csf3, Csf4, Cpfl, or SluCas9 endonuclease; a homolog thereof, a recombination of the naturally occurring molecule thereof, codon-optimized thereof, or modified versions thereof, and combinations thereof.

67. The method or use of any of claims 63-66, wherein the gene editing system comprises(a) (i) a nucleic acid molecule encoding the Cas nuclease, optionally an mRNA or DNA encoding the Cas nuclease and (ii) one or two or more guide RNAs or nucleic acid(s) encoding the guide RNA;(b) (i) an mRNA encoding the Cas nuclease and (ii) one or two or more guide RNAs;(c) (i) an mRNA encoding the Cas nuclease and (ii) a DNA encoding one or two or more guide RNAs, optionally wherein the DNA is in an expression vector comprising one or more regulatory sequences;(d) (i) a DNA encoding the Cas nuclease, optionally wherein the DNA is in an expression vector comprising one or more regulatory sequences, and (ii) a DNA encoding one or two or more guide RNAs, optionally wherein the DNA is in an expression vector comprising one or more regulatory sequences; or(e) (i) a Cas nuclease polypeptide and (ii) one or two or more guide RNAs, optionallypre-complexed.

68. The method or use of any one of claims 48-67, wherein the gene editing system effects one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within a target gene in the genomes of one or more HSPCs in the subject.

69. The method or use of any one of claims 48-68, wherein the gene editing system introduces an indel mutation into a target gene in the genomes of the one or more HSPCs of the subject.

70. The method or use of any one of claims 48-68, wherein the gene editing system further comprises a donor template.

71. The method or use of claim 70, wherein the donor template is a DNA molecule.

72. The method or use of any one of claims 48-71, wherein the gene editing system is administered via a lipid nanoparticle (LNP), and wherein components of the gene editing system are delivered in the same LNP or in separate LNPs.

73. A method or use of increasing gene editing efficiency of HSPCs comprising a repeated administration of a cycling agent and a gene editing system, wherein the repeated administration is repeated one, two, three, four or more times, or up to five times.

74. An in vivo method of editing the genome of one or more hematopoietic stem and progenitor cells (HSPCs) in a subject in need thereof comprising:(a) a first step of administering a cycling agent prior to or in conjunction with administering a gene editing system to the subject so that the genome(s) of HSPCs in the subject are edited, and(b) a second step of administering a cycling agent prior to or in conjunction with administering a gene editing system to the subject so that the genome(s) of HSPCs in the subject are edited.

75. The method of claim 74, wherein the cycling agent is administered in an amount effective to increase the gene editing efficiency of the gene editing system.

76. The method of claim 74, wherein the cycling agent is administered in an amount effective to increase the percentage of HSPCs in a more active metabolic state of proliferative state.

77. The method of claim 74 wherein the second step is performed about 3 monthsafter the first step, or about 3 to 6 months after the first step, or about 3 to 9 months after the first step.

78. An in vivo method of editing the genome of HSPCs comprising administering to a subject in need thereof a second dose of a cycling agent and a second dose of a gene editing system, wherein the subject has previously received a first dose of a cycling agent and a first dose of a gene editing system, and wherein the first dose and second dose of cycling agent are of the same compound or different compounds.

79. The method or use of any one of claims 48-78, wherein the percentage of HSPCs in the bone marrow that maintain editing five months after the first administration of the gene editing system is at least 10%.

80. The method or use of any of claims 48-79, wherein the percentage of HSPCs in the bone marrow that maintain editing five months after the first administration of the gene editing system is at least 15%, at least 20%, at least 25%, at least between 10-50%, at least between 15-40%, at least between 20-30%.

81. The method or use of any of claims 48-80, wherein one or more cell lineages of the HSPCs are selected from myeloid cells, erythroid cells, B cells and T cells and combinations thereof exhibit editing at least five months after the first administration of the gene editing system.

82. The method or use of claim 81, wherein the cell lineage is myeloid cells and the percent of editing is greater than 10%.

83. The method or use of claim 81, wherein the cell lineage is erythroid cells and the percent of editing is greater than 10%.

84. The method or use of claim 81, wherein the cell lineage is B cells and the percent of editing is greater than 10%.

85. The method or use of claim 81, wherein the cell lineage is T cells and the percent of editing is greater than 7%.

86. An in vivo method of editing the genome of one or more hematopoietic stem and progenitor cells (HSPCs) in a subject in need thereof comprising:(a) administering two or more cycling agents, in amounts effective to increase the gene editing efficiency of a gene editing system, and(b) administering a gene editing system to the subject so that the genome(s) of HSPCsin the subject are edited.

87. The method of claim 86, wherein the administration of step (a) is before the administration of step (b).

88. The method of claim 86, wherein the administration of step (a) and (b) are done at the same time.

89. The method of any one of claims 86-88, wherein the two or more cycling agents are administered as a single composition.

90. The method or use of any one of claims 48-86, wherein the cycling agent is a CXCR4 antagonist, antimetabolite, alkylating agent, or cytotoxic agent or other chemotherapeutic agent, optionally plerixafor, 5-fluorouracil, and / or cyclophosphamide.

91. The method of any of claims 86-90, wherein the gene editing system is administered in an LNP formulation.

92. The method of any one of claims 86-91, wherein the editing efficiency of the gene editing system is higher after the administration of the two or more cycling agents than that of the editing efficiency of the gene editing system after the administration of one of the cycling agents alone.

93. The method of claim 92, wherein the effect of the two or more the cycling agents is synergistic.

94. The method of claim 92, wherein the effect of the two or more the cycling agents is not additive.

95. The method or use or kit of any of the preceding claims wherein the HSPC is an HSC.

96. The method or use or kit of any of the proceeding claims wherein the HSPC is a progenitor cell.

97. The method or use or kit of any of the preceding claims, wherein the gene editing system comprises(a) (i) a nucleic acid molecule encoding the Cas nuclease, optionally an mRNA or DNA encoding the Cas nuclease and (ii) one or two or more guide RNAs or nucleic acid(s) encoding the guide RNA;(b) (i) an mRNA encoding the Cas nuclease and (ii) one or two or more guide RNAs;(c) (i) an mRNA encoding the Cas nuclease and (ii) a DNA encoding one or two or more guide RNAs, optionally wherein the DNA is in an expression vector comprising one or more regulatory sequences;(d) (i) a DNA encoding the Cas nuclease, optionally wherein the DNA is in an expression vector comprising one or more regulatory sequences, and (ii) a DNA encoding one or two or more guide RNAs, optionally wherein the DNA is in an expression vector comprising one or more regulatory sequences; or(e) (i) a Cas nuclease polypeptide and (ii) one or two or more guide RNAs, optionally pre-complexed.

98. The method or use or kit of claim 97, wherein the mRNA encoding the Cas nuclease is chemically modified, optionally with a polyA tail, a 5' cap analog, modified 5' or 3' untranslated regions (UTRs), or modified bases, optionally Pseudo-U, N6-Methyl-A, 2- Thio-U and / or 5-Methyl-C modifications.

99. The method or use or kit of claim 98 wherein the guide RNA is chemically modified, optionally with a modified backbone, one or more substituted sugar moieties, and / or nucleobase modifications or substitutions, optionally a combination of (a) PS backbone modifications and (b) 2’ substitutions, optionally 2'-O-Methyl, 2'-Fluoro, or 2'- Hydro.

100. The method or use or kit of any of the preceding claims, wherein the gene editing system is administered via a lipid nanoparticle (LNP), and wherein the components of the gene editing system are delivered in the same LNP or in separate LNPs.

101. The method or use or kit of any of the preceding claims, wherein the cycling agent is a CXCR4 antagonist, Thrombopoietin Receptor Agonist, CXCR2 agonist, VLA-4 antagonist, antimetabolite, alkylating agent, cytotoxic agent or other chemotherapeutic agent.

102. The method or use or kit of any of the preceding claims, wherein the cycling agent is a CXCR4 antagonist, optionally AMD-3100 (Plerixafor), AMD-11070 (Mavorixafor), Motixafortide, AMD-3465, BMS-936564 / MDX-1338, LY2510924, N, N- dipropyl-N-[4-({[(lH-imidazol-2-yl)methyl)benzyl][(l-methyl-lH-imidazol-2-yl) methyl]amino]methyl)benzyl]-N-methylbutane-l, 4-diamine tri(2R, 3R)-tartrate (KRH- 3955), and ([5-(4-methyl-l-piperazinyl)-2-({methyl[(8S)-5,6,7,8-tetrahydro-8- quinolinyl]amino}methyl)imidazo[l,2-a]pyridin-3-yl]methanol) (GSK812397), an antibody or fragment thereof that binds CXCR4, or ALB408-423.

103. The method or use or kit of any of the preceding claims, wherein the cycling agent is an antimetabolite, alkylating agent, cytotoxic agent or other chemotherapeutic agent, optionally camptothecin, cisplatin, carboplatin, oxaliplatin, bleomycin, mitomycin C, calicheamicins, maytansinoids, geldanamycin, doxorubicin, idarubicin, daunorubicin, epirubicin, busulfan, carmustine (BCNU), lomustine (CCNU), semustine, thalidomide, lenalidomide, methotrexate, azathioprine, 6-mercaptopurine, fludarabine, 5-azacytidine, pentostatin (2'-deoxycoformycin), cytarabine (cytosine arabinoside), gemcitabine, 5- fluorouracil, hydroxyurea, elesclomol, etoposide, teniposide, amsacrine, topotecan, irinotecan, chlorambucil, cyclophosphamide, ifosfamide, melphalan, bortezomib, vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, amphotericin B, rifampicin, pentamidine, cyclosporine A, tacrolimus (FK506), sirolimus (rapamycin), everolimus, temsirolimus, zotarolimus, biolimus, oxaliplatin or mitomycin, 5 -fluorouracil (5-FU), hydroxyurea, gemcitabine, mercaptopurine, thioguanine, cladribine, fludarabine phosphate, fluorouracil (5- FU), floxuridine, cytarabine, pentostatin, methotrexate, azathioprine, acyclovir, adenine p-1- D-arabinoside, amethopterin, aminopterin, 2-aminopurine, aphidicolin, 8 azaguanine, azaserine, 6-azauracil, 2'-azido-2'-deoxynucleosides, 5-bromodeoxycytidine, cytosine P— 1-D- arabinoside, diazooxynorleucine, dideoxynucleosides, 5-fluorodeoxycytidine, 5 fluorodeoxyuridine, hydroxyurea, cyclophosphamide, alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as a benzodizepa, carboquone, meturedepa, and uredepa; ehylenimines and methylmelamines, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylol melamine; nitrogen mustards such as chlorambucil, chlomaphazine, cyclophosphamide, estramustine, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, and uracil mustard; and nitroso ureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, or ranimustine.

104. The method or use or kit of any of the preceding claims, wherein the cycling agent is plerixafor, 5 -fluorouracil, and / or cyclophosphamide.

105. The method or use or kit of any of the preceding claims, wherein the cycling agent is plerixafor administered at a dose ranging from about 0.06 mg / kg to about 0.72 mg / kg.

106. The method or use or kit of any of the preceding claims, wherein the cycling agent is 5 -fluorouracil administered parenterally at a dose ranging from about 200 to about 1200 mg / m2.

107. The method or use or kit of any of the preceding claims, wherein the cycling agent is cyclophosphamide administered orally or parenterally at a dose ranging from about 1 mg / kg to about 10 mg / kg.

108. The method or use or kit of any of the preceding claims, wherein the gene editing system is a CRISPR / Cas nuclease gene editing system administered via an LNP at a dose of about 0.1 mg / kg to about 10 mg / kg.

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