Methods for inhibiting ifitm3 and treating bone marrow failure syndromes

Abstract

Described herein are genetically -modified hematopoietic stem cells (HSCs) comprising a disrupted, mutated, or deleted interferon-induced transmembrane protein 3 (IFITM3) or a deleted portion thereof, and compositions comprising the same. The disclosure also provides methods of generating populations of modified and genetically -modified HSCs and methods of treating a bone marrow failure syndrome with the same. The modified and genetically-modified HSCs can be quiescent and capable of self-renewal.

Description

[0001] Attorney’s Docket No.: 21101.0503P1

[0002] METHODS FOR INHIBITING IFITM3 AND TREATING BONE MARROW FAILURE SYNDROMES

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of the filing date of U. S. Provisional Application No.

[0004] 63 / 771,749, filed March 14, 2025. The content of this application is hereby incorporated herein by reference in its entirety.

[0005] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0006] This invention was made with government support under K24 HL155856 awarded by National Institutes of Health. The government has certain rights in the invention.

[0007] INCORPORATION OF THE SEQUENCE LISTING

[0008] The present application contains a sequence listing that is submitted concurrent with the filing of this application, containing the file name “21101_0503Pl_SL.xml” which is 12,288 bytes in size, created on January 28, 2026, and is herein incorporated by reference in its entirety.

[0009] BACKGROUND

[0010] In bone marrow failure (BMF) syndromes, hematopoietic stem cell (HSC) lose their ability to self-renew and generate blood cells due to chronic inflammation, inherited mutations, or external damage from chemotherapy or drugs. Current treatments, such as blood transfusions and bone marrow transplants, provide temporary relief or are limited by donor availability and high costs. Thus, new treatment strategies are needed.

[0011] SUMMARY

[0012] Disclosed herein are genetically-modified hematopoietic stem cells (HSCs) comprising a disrupted, mutated, or deleted interferon-induced transmembrane protein 3 (IFITM3) or a deleted portion thereof.

[0013] Disclosed herein are genetically-modified hematopoietic stem cells (HSCs) comprising a modified interferon-induced transmembrane protein 3 gene (Ifitm3).

[0014] Disclosed herein are genetically-modified hematopoietic stem cells (HSCs) comprising an artificially disrupted, mutated, or a partially or completely deleted interferon-induced transmembrane protein 3 gene Ifitm3).Attorney’s Docket No.: 21101.0503P1

[0015] Disclosed herein are genetically-modified HSCs, comprising an artificially disrupted, mutated, or a partially or completely deleted N-terminal domain of IFITM3, intramembrane domain of IFITM3, conserved intracellular loop of IFITM3, transmembrane domain of IFITM3 or the C-terminal domain of IFITM3.

[0016] Disclosed herein are methods of preserving or enhancing hematopoietic stem cell (HSC) function by disrupting, mutating, or a partially or completely deleting the interferon-induced transmembrane protein 3 gene (Ifitm3) in the HSC.

[0017] Disclosed herein are methods of preserving or enhancing hematopoietic stem cell (HSC) function by inhibiting IFITM3 in the HSCs.

[0018] Disclosed herein are methods of generating a population of modified human hematopoietic stem cells (HSCs), the method comprising: introducing into the HSCs a CRISPR system comprising a guide nucleic acid, wherein the CRISPR system produces an insertion and / or deletion in an endogenous Ifitm3 sequence, wherein the endogenous ifitm3 sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11. or a combination thereof, and wherein the insertion and / or deletion is capable of downregulating gene expression of Ifitm3, thereby generating the population of modified human HSCs.

[0019] Disclosed herein are populations of modified human hematopoietic stem cells (HSCs), wherein the modified HSCs comprise an insertion and / or deletion in an endogenous Ifitm3 sequence, wherein the endogenous Ifitm3 sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or a combination thereof, and wherein the insertion and / or deletion is mediated by a CRISPR system comprising a guide nucleic acid, and wherein the insertion and / or deletion is capable of downregulating gene expression of Ifitm3.

[0020] Other features and advantages of the present compositions and methods are illustrated in the description below, the drawings, and the claims.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 A-D show that IFITM3 is highly expressed in hematopoietic stem cells (HSCs) in the adult bone marrow (BM). FIG. 1A shows the relative expression level of Ifitm3 mRNA, as measured by qPCR, in Long-Term (LT)-HSC (labeled as Lin" Sca-1+cKit+CD150+CD48’ Flt3’), short-term (ST)-HSC (labeled as Lin Sca-1+cKit+CD150’ CD48’ Flt3’ ). multipotent progenitors, MPP2 (labeled as Lin’ Sca-1+cKit+CD150+CD48+Flt3 ), MPP3 (labeled as Lin" Sca-1+cKit+CD150’ CD48+Flt3‘), MPP4 (labeled as Lin’ Sca-1+cKit+Attorney’s Docket No.: 21101.0503P1

[0022] CD150’ CD48+Flt3+), relative to mononuclear cells (MNC) (CD45‘ CDllb+) harvested from the bone marrow of 12 weeks old WT mice (n=3 independent experiments). FIGS. 1B-C show the clustering analysis for RNA expression showing normalized counts of hepatic leukemia factor (hlf), the most specific marker to denote HSCs compared to normalized counts of ifitm3 in mouse HSCs. Data obtained from GSE6506. FIG. ID shows the deletion of Ifitm3 in BM cells in IFITM3 KO mice (where Ifitm3 has been deleted from the cells) relative to WT BM cells, as assessed by qPCR (n=4-5 mice / group; ****p<0.0001 by Welch’s two-tailed t-test).

[0023] FIGS. 2A-E show that loss of IFITM3 reduces HSC attrition during IFNa-induced stress hematopoiesis. FIG. 2A shows representative flow cytometry analysis identifying the long-term (LT)-HSCs (labeled as Lin’ Sca-1+cKit+CD150+CD48’ Flt3‘), and short-term (ST)-HSC (labeled as Lin’ Sca-1+cKit+CD 150’ CD48’ Flt3‘), harvested from the bone marrow of age-matched male WT and IFITM3 KO mice. FIG. 2B shows the frequency of LT-HSCs as a measure of the percent of LSK-LT-HSC cells in total BM cells and FIG. 2C shows the absolute numbers of total LSK-LT-HSCs per mouse were quantified via flow cytometry. FIG. 2D shows the frequency of ST-HSCs as a measure of the percent of LSK-ST-HSC cells in total BM cells and FIG. 2E shows the absolute numbers of total LSK-ST-HSCs per mouse were quantified via flow cytometry. (n=4-5 mice / group; *p<0.05: **p<0.01; ***p<0.001 by Welch’s two-tailed t-test).

[0024] FIGS. 3A-G show that loss of IFITM3 reduces multipotent Progenitor Differentiation during IFNa-induced stress hematopoiesis. FIG. 3A shows representative flow cytometry analysis identifying multipotent progenitors, MPP2 (labeled as Lin’ Sca-1+cKit+CD150+CD48+Flt3‘), MPP3 (labeled as Lin’ Sca-1+cKit+CD150’ CD48+Flt3’), MPP4 (labeled as Lin’ Sca-1+cKit+CD 150’ CD48+Flt3+) from BM cells harvested from WT and IFITM3 KO mice. FIG. 3B show the frequency of MPP2 as a measure of the percent of LSK-MPP2 cells in total BM cells and FIG. 3C shows the absolute numbers of total LSK-MPP2s per mouse were quantified via flow cytometry. FIG. 3D shows the frequency of MPP3s as a measure of the percent of LSK-MPP3 cells in total BM cells and FIG. 3E shows the absolute numbers of total LSK-MPP3s per mouse were quantified via flow cytometry. FIG. 3F shows the frequency of MPP4s as a measure of the percent of LSK-MPP4 cells in total BM cells and FIG. 3G shows the absolute numbers of total LSK-MPP4s per mouse were quantified via flow cytometry. (n=4-5 mice / group; *p<0.05; **p<0.01, by Welch’s two-tailed t-test).Attorney’s Docket No.: 21101.0503P1

[0025] FIGS. 4A-B show that the loss of IFITM3 protects HSCs from apoptosis during IFNa-induced stress hematopoiesis. Cells were stained with respective antibodies and Annexin V antibody to identify (FIG. 4A) Annexin V+LT-HSCs and (FIG. 4B) Annexin V+ST-HSCs. n=4-5 mice / group; *p<0.05; **p<0.01, by Welch’s two-tailed t-test).

[0026] FIGS. 5A-D show loss of IFITM3 preserves short-term engraftment potential in competitive primary bone marrow transplantation during IFNa-induced stress hematopoiesis. The levels of CD45.2 chimerism (FIG. 5A) and differentiated cell formation namely, B cells (FIG. 5B), T cells (FIG. 5C), and myeloid cells (FIG. 5D) was estimated every 4 weeks up to 16 weeks post-transplantation via flow cytometry. (n=10 mice / group; *p<0.05; **p<0.01; ***p<0.001 by Welch’s two-tailed t-test).

[0027] FIGS. 6A-D show that the loss of IFITM3 preserves long-term engraftment potential in competitive secondary bone marrow transplantation during IFNa-induced stress hematopoiesis. The levels of CD45.2 chimerism (FIG. 6A) and differentiated cell formation namely, B cells (FIG. 6B), T cells (FIG. 6C), and myeloid cells (FIG. 6D) was estimated every 4 weeks up to 16 weeks post-transplantation via flow cytometry. (n=10 mice / group; *p<0.05; **p<0.01; ***p<0.001 by Welch’s two-tailed t-test).

[0028] FIGS. 7A-D show that the loss of IFITM3 preserves short-term engraftment potential in competitive primary bone marrow transplantation during steady-state (non-IFNa) hematopoiesis. The levels of CD45.2 chimerism (FIG. 7A) and differentiated cell formation namely, B cells (FIG. 7B), T cells (FIG. 7C), and myeloid cells (FIG. 7D) was estimated every 4 weeks up to 16 weeks post-transplantation via flow cytometry’. (n=10 mice / group; *p<0.05; **p<0.01; ***p<0.001 by Welch’s two-tailed t-test).

[0029] FIGS. 8A-D show that the loss of IFITM3 preserves long-term engraftment potential in competitive secondary bone marrow transplantation during steady-state (non-IFNa) hematopoiesis. The levels of CD45.2 chimerism (FIG. 8A) and differentiated cell formation namely, B cells (FIG. 8B), T cells (FIG. 8C), and myeloid cells (FIG. 8D) was estimated every 4 weeks up to 16 weeks post-transplantation via flow cytometry. (n=10 mice / group; *p<0.05; **p<0.01; ***p<0.001 by Welch’s two-tailed t-test).

[0030] FIGS. 9A-D show that IFITM3-null HSCs have better engraftment potential in the adult BM during primary’ competitive LSK transplantation. The levels of CD45.2 chimerism (FIG. 9A) and differentiated cell formation namely, B cells (FIG. 9B), T cells (FIG. 9C), and myeloid cells (FIG. 9D) was estimated every' 4 weeks up to 16 weeks post-transplantation viaAttorney’s Docket No.: 21101.0503P1

[0031] flow cytometry. (n=10 mice / group; *p<0.05; **p<0.01; ***p<0.001 by Welch’s two-tailed t-test).

[0032] FIGS. 10A-D show that IFITM3-null HSCs have better engraftment potential in the adult BM during primary competitive LSK transplantation during IFNa-induced stress hematopoiesis. The levels of CD45.2 chimerism (FIG. 10A) and differentiated cell formation namely, B cells (FIG. 10B), T cells (FIG. 10C), and myeloid cells (FIG. 10D) was estimated every 4 weeks up to 16 weeks post-transplantation via flow cytometry. (n=10 mice / group; *p<0.05; **p<0.01 by Welch’s two-tailed t-test).

[0033] FIGS. 11 A-E show that the loss of IFITM3 maintains quiescence and protects from cell cycling in HSCs and multipotent progenitors during IFNa-induced stress hematopoiesis. BrdU incorporation in LSK cells (FIG. 11 A), LT-HSCs (FIG. 1 IB), ST-HSCs (FIG. 11 C), MPP2 (FIG. 1 ID), and MPP3 (FIG. 1 IE) is denoted as the percentage of BrdU+cells in each phase of the cell cycle. N=4-5 mice / group, *p<0.05; **p<0.01; ***p<0.001 by Welch's two-tailed t-test).

[0034] FIGS. 12A-E show that the loss of IFITM3 is associated with upregulation of quiescence-enforcing pathways and downregulation of cell cycle regulators during IFNa-induced stress hematopoiesis. FIG. 12A shows the UMAP of 27 unbiased cKit clusters from WT and IFITM3 KO mice. FIG. 12B shows the relative expression level of Ifitm3 mRNA in the 27 clusters, red bars with arrows showing HSC clusters. FIG. 12C shows a Volcano plot depicting significantly upregulated (in red) and downregulated (in blue) genes within HSC cluster of IFITM3 KO cKit+BM cells. Colored dots annotated for candidate genes. Cell cycle regulators Ccnd2, Cdk4, Ccndl, and Cdk6, and quiescence-enforcing genes Egrl and Btg2. FIG. 12D show KEGG analyses of differentially expressed gene pathways upregulated in the IFITM3 within HSC cluster of IFITM3 KO cKit+BM cells including Apoptosis, TNF-alpha Signaling via NF-kB pathways. FIG. 12E shows KEGG analyses of differentially expressed gene pathways downregulated in the IFITM3 within HSC cluster of IFITM3 KO cKit+BM cells including DNA replication E2F targets and G2-M checkpoint pathways.

[0035] FIG. 13 shows a schematic representation elucidating the role of IFITM3 in hematopoietic stem cells regulating quiescence during IFNa-induced stress hematopoiesis.

[0036] FIGS. 14A-D show that the loss of IFITM3 is associated with reduced differentiation during steady-state (non-IFNa) hematopoiesis. The total number of (FIG. 14A) Colonyforming Units (CFU-C), (FIG. 14B) multilineage (CFU-GEMM), (FIG. 14C) granulocyte / macrophage (CFU-GM), (FIG. 14D) granulocytes (CFU-G), (FIG. 14E)Attorney’s Docket No.: 21101.0503P1

[0037] monocyte (CFU-M), and (FIG. 14F) blast-forming units-erythroid (BFU-E) colonies was quantified 14 days post-seeding. (WT and IFITM3 KO cells were pooled from n=5 mice / group, where each point in the graph denotes one of 15 technical replicates; *p<0.05; **p<0.01; ***p<0.001 by Welch’s two-tailed t-test).

[0038] FIGS. 15A-D show that the loss of IFITM3 is associated with reduced G0-G1 transition cell cycle regulators consistent with single-cell RNA Seq data during steady-state (non-IFNa) hematopoiesis. Total RNA was isolated using the QiagenRNAeasy Kit, and cDNA was prepared, reverse transcriptase (RT) PCR was performed, and mRNA levels were quantified for (FIG. 15A) Ccndl, (FIG. 15B) Ccnd2, (FIG. 15C) Cdk4, and (FIG. 15D) Cdk6. (n=3 mice / group; *p<0.05; **p<0.01; by Welch's two-tailed t-test).

[0039] FIGS. 16A-D show that loss of IFITM3 is associated with reduced G0-G1 transition cell cycle regulators consistent with single-cell RNA Seq data during IFNa-induced stress hematopoiesis. Total RNA was isolated using the Qiagen RNAeasy Kit, and cDNA was prepared, reverse transcriptase (RT) PCR was performed, and mRNA levels were quantified for (FIG. 16A) Ccndl, (FIG. 16B) Ccnd2, (FIG. 16C) Cdk4, and (FIG. 16D) Cdk6. (n=3 mice / group; *p<0.05; by Welch’s two-tailed t-test).

[0040] FIGS. 17A-B show that the loss of IFITM3 is associated with increased GO quiescence regulators consistent with single-cell RNA Seq data during steady-state (non-IFNa) hematopoiesis. Total RNA was isolated using the Qiagen RNAeasy Kit, and cDNA was prepared, reverse transcriptase (RT) PCR was performed, and mRNA levels were quantified for (FIG. 17A) Egrl and (FIG. 17B) Btg2. (n=3 mice / group; *p<0.05; **p<0.01; by Welch’s two-tailed t-test).

[0041] FIGS. 18A-B show that the loss of IFITM3 is associated with increased GO quiescence regulators consistent with single-cell RNA Seq data during IFNa-induced stress hematopoiesis. Total RNA was isolated using the Qiagen RNAeasy Kit, and cDNA was prepared, reverse transcriptase (RT) PCR was performed, and mRNA levels were quantified for (FIG. 18A) Egrl and (FIG. 18B) Btg2. (n=3 mice / group; **p<0.01; by Welch’s two-tailed t-test).

[0042] DETAILED DESCRIPTION

[0043] Many modifications and other embodiments of the present disclosure set forth herein will come to mind to one skilled in the art to which this disclosure pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings.

[0044] Therefore, it is to be understood that the present disclosure is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to beAttorney’s Docket No.: 21101.0503P1

[0045] included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0046] Before the present compositions and methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherw ise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, example methods and materials are now described.

[0047] Moreover, it is to be understood that unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, and the number or type of aspects described in the specification.

[0048] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosures. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0049] Definitions

[0050] As used in the specification and in the claims, the term “‘comprising” can include the aspects “consisting of” and “consisting essentially of.” “Comprising” can also mean “including but not limited to.”

[0051] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” can include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes mixtures of compounds; reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.Attorney’s Docket No.: 21101.0503P1

[0052] The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

[0053] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0054] As used herein, the term “sample” is meant a tissue or organ from a subject; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g., a polypeptide or nucleic acid), which is assayed as described herein. A sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells or cell components.

[0055] As used herein, the term “subject” refers to the target of administration, e.g., a human. The subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). In some aspects, a subject is a mammal. In some aspects, a subject is a human. The term does not denote a particular age or sex. Thus, adult, child, adolescent and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

[0056] As used herein, the term “patient” refers to a subject afflicted with a disease or disorder (e.g., a bone marrow failure syndrome). The term “patient” includes human and veterinary subjects. In some aspects of the disclosed methods, the “patient” has been diagnosed with a need for treatment for a bone marrow failure syndrome, such as, for example, prior to the administering step.

[0057] Ranges can be expressed herein as from “about” or “approximately” one particular value, and / or to “about” or “approximately” another particular value. When such a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” or “approximately,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example,Attorney’s Docket No.: 21101.0503P1

[0058] if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0059] “Inhibit,” “inhibiting” and “inhibition” mean to diminish or decrease an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% inhibition or reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, in an aspect, the inhibition or reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. In an aspect, the inhibition or reduction is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% as compared to native or control levels. In an aspect, the inhibition or reduction is 0-25, 25-50, 50-75, or 75-100% as compared to native or control levels.

[0060] “Modulate”, “modulating” and “modulation” as used herein mean a change in activity or function or number. The change may be an increase or a decrease, an enhancement or an inhibition of the activity, function or number.

[0061] As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting or slowing progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. Treatment can also be administered to a subject to ameliorate one more signs of symptoms of a disease, disorder, and / or condition. For example, the disease, disorder, and / or condition can be relating to a bone marrow failure syndrome.

[0062] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0063] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0064] The term “expand” as used herein refers to increasing in number, as in an increase in the number of cells. In some aspects, the cells that are expanded ex vivo increase in number relative to the number originally present in the culture. In some aspects, the cells that are expanded ex vivo increase in number relative to other cell types in the culture.Attorney’s Docket No.: 21101.0503P1

[0065] The term “ex vivo,” as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g.. in a culture dish, test tube, or bioreactor).

[0066] The term “hematopoietic stem cell” or “HSC” refers to an undifferentiated hematopoietic cell that is capable of differentiating into all blood cell types, myeloid and lymphoid cells. The HSC may reside in the bone marrow or be found elsewhere (e.g., peripheral blood).

[0067] The term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0068] The term “artificially” as used herein refers to a change or a material that is manmade rather than occurring in nature. In some aspects, artificially is the result of a human.

[0069] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0070] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, certain changes and modifications may be practiced within the scope of the appended claims.

[0071] Interferon-induced Transmembrane Protein 3 (IFITM3) has well-known roles in promoting innate immunity against viral infection, including Influenza A, West Nile virus, Yellow Fever virus, and SARS-CoV-2. The mechanisms by which IFITM3 protects against viral infections includes inhibiting the fusion of the viral membrane with the plasma membrane of host cells and facilitating the trafficking of viral particles into lysosomes for degradation.

[0072] As described herein, an unexpected new role for IFITM3 in HSCs was identified that is contrary to the mechanisms and role of IFITM3 described in Wurzer (Wurzer, Dissertation: Effects of Interferon a on Mouse and Human Hematopoietic Stem Cells, presented October 4, 2012). This surprising, unexpected activity of IFITM3 is also completely independent from the known antiviral immune mechanisms of IFITM3. As disclosed herein, a specific deletion of Ifitm3 was identified, using an Ifitm3 knockout (KO) mice, which preserves the sternness and quiescence of the long-term HSCs (LT-HSCs). LT-HSCs are the most primitive HSCs inAttorney’s Docket No.: 21101.0503P1

[0073] the bone marrow. Preserving the sternness of LT-HSCs is important for long-term engraftment for patients receiving bone marrow transplants or gene-edited HSC transplants. This finding shows that deleting IFITM3 in HSCs significantly promotes healthy bone marrow reconstitution, and identifies a completely new and unexpected function of IFITM3 in hematopoiesis with relevance in the treatment of bone marrow failure syndromes. As a summary, the following HSC phenotypes were identified when ifitm3 is knocked out:

[0074] • Significant retention of quiescent HSCs;

[0075] • Significantly reduced cycling and differentiation of HSCs to progenitor cells;

[0076] • Significantly improved preservation of LT-HSC sternness and quiescence;

[0077] • Increased expression of HSC quiescence-enforcing genes (Btg2 Egrl, others);

[0078] • Significantly decreased apoptosis in HSCs; and

[0079] • Increased chimerism and bone marrow engraftment at early and late time points following HSC transplantation.

[0080] The discovery that the specific deletion of Ifitm3 preserves the sternness and quiescence of long-term hematopoietic stem cells (LT-HSCs) is unexpected and surprising in light of previously published data on ifitm3. Prior studies have largely characterized ifitm3 as an important regulator of immune responses and antiviral defense, with some reports suggesting its involvement in cellular proliferation, differentiation, and stress responses. Notably, ifitm3 has been associated with promoting stem cell activation and mobilization, implying that its loss would likely impair HSC function or lead to their premature exhaustion. Contrary to these reports, the findings disclosed herein demonstrate that ifitm3 deletion surprisingly enhances LT-HSC quiescence and preserves their sternness, challenging the prevailing paradigm and revealing an unexpected role of ifitm3 in stem cell homeostasis.

[0081] Contrary to the compositions and methods disclosed herein, others have reported that deletion of the entire ifitm gene cluster (there are at least 6 ifitm genes in this cluster) does not affect HSC cell cycling, differentiation potential, engraftment potential, or self-renewal capacity of HSCs. Friedlova et al. (which is a review on the ifitm family; Front. Immunolo. Vol. 13, 2022) broadly summarizes some of the factors regulating the expression of various ifitm genes in stem cells and other compartments. Friedlova et al does not teach that ifitm3 enforces LT-HSC sternness or quiescence.

[0082] HSC gene therapy, where HSCs are ex vivo modified to contain corrective and functional copies of disease genes, is a viable therapeutic strategy for treating hematopoietic diseases, including immunodeficiencies and leukemias. Crispr-guides targeting specificAttorney’s Docket No.: 21101.0503P1

[0083] regions / domains of ifitm3 (Table 1) will be used to identify domains responsible for ifitm3 ’s unexpected functions in preserving HSC quiescence and design rationale therapeutic targets for commercial drug development.

[0084] As used herein, the nucleic acid sequences disclosed in Table 1 can be a sequence to which a guide sequence (e g. gRNA) can be designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. In some aspects, the degree of complementarity between a guide sequence (e.g. gRNA) and its corresponding target sequence is about or more than about 50%, 60%, 70%, 75%. 80%. 85%. 90%. 95%. 99%. or more. In some aspects, the nucleic acid sequences provided in Table 1 can serve as a target sequence.

[0085] Table 1. ifitm3 domains and corresponding amino acid and nucleic acid sequences, features, and functional relevance.

[0086] Sequence Domain / Regio Features Functional n Relevance 1-57 amino acids N-terminal Located in the Y20 within N- MNHTVQTFFSPVNSGQPPN Domain (NTD) cytosol, contains terminal YEMLKEEHEVAVLGAPHNP (variable) Tyrosine at 20 Domain APPTSTVIHIRSETSVPDH residue (Y20) regulates (SEQ ID NO: 1) and the YEML intracellular ATGAACCACACCGTGCAG (SEQ ID NO: 2) trafficking ACCTTCTTCTCCCCGGTG A sorting motif for (phosphorylatio ACAGCGGCCAGCCGCCGA ACTACGAGATGAAGGAGG binding to n at Y20 AGCACGAGGTGGCCGTGC Clathrin- restrains TGGGCGCCCCGCACAACC

[0087] mediated IFITM3 to the CGGCCCCGCCGACCAGCA CCGTGATCCACATCCGCAG Endocytosis plasma CGAGACCAGCGTGCCGGA membrane) CCAC (SEQ ID NO: 3)

[0088] 58-78 amino acids Intramembrane Contains an Important for VVWSLFNTLFMNPCCLGFI Domain (IMD) amphipathic antiviral activity AF (SEQ ID NO: 4) (conserved) helix, which

[0089]

[0090] Attorney’s Docket No.: 21101.0503P1 GTGGTGTGGAGCCTGTTC interacts with AACACCCTGTTCATGAAC membrane CCCTGCTGCCTGGGCTTC cholesterol, part

[0091] ATCGCCTTC (SEQ ID NO: 5) of the highly

[0092] conserved

[0093] CD225 domain

[0094] 79-105 amino acids Conserved Lysine-rich Important for AYSVKSRDRKMVGDVTGA Intracellular domain, antiviral QAYASTAKC (SEQ ID NO: Loop (CIL) conserved across activity.

[0095] 6) (conserved) species, part of Mutating the GCCTACAGCGTGAAGAGC the highly lysine residues CGGGACCGCAAGATGGTG conserved to arginine GGCGACGTGACCGGCGCC CD225 domain abolishes the CAGGCCTACGCCAGCACC and link the antiviral activity GCCAAGTGC (SEQ ID NO: Transmembrane ofIFITM3, 7) Domain to the involved in Intramembrane protein-protein Domain, interactions contains sites for

[0096] post-translational

[0097] modifications

[0098] like

[0099] ubiquitination

[0100] and methylation

[0101] 106-129 amino acids Transmembran Spans the Important for LNNIWALILGILMTILLIVIP e Domain membrane, membrane VLI (SEQ ID NO: 8) (TMD) highly anchoring and CTGAACAACATCTGGGCC (conserved) hydrophobic fusion, interacts CTGATCCTGGGCATCCTG with HA2 ATGACCATCCTGCTGATC subunit of GTGATCCCGGTGCTGATC Influenza A (SEQ ID NO: 9) virus

[0102]

[0103] Attorney’s Docket No.: 21101.0503P1

[0104] 130-134 amino acids C-terminal Located Important for FQAYG (SEQ ID NO: 10) Domain (CTD) extracellularly intracellular (variable) trafficking and TTCCAGGCCTACGGC (SEQ protein turnover ID NO: 11)

[0105]

[0106] Disclosed herein are methods of inhibiting Interferon-Induced Transmembrane Protein 3 (IFITM3) as a strategy for preserving hematopoietic stem cell (HSC) function in bone marrow failure (BMF) syndromes. The compositions and methods disclosed herein specifically target the role of IFITM3 in regulating HSC quiescence, self-renewal, and differentiation, providing an approach to treat conditions characterized by HSC dysfunction.

[0107] As described herein, IFITM3 acts as an important regulator of HSC quiescence, a state in which stem cells remain dormant and retain their ability to self-renew over long periods. Under normal and inflammatory conditions, IFITM3 pushes HSCs out of quiescence and into rapid cycling, leading to their premature differentiation and depletion. By inhibiting IFITM3, the early activation of HSCs can be prevented, preserving their sternness and prolonging their regenerative capacity.

[0108] Also disclosed herein is IFITM3's role in disrupting the balance between HSC quiescence and activation. IFITM3 promotes cell cycle entry by inducing important cell cycle regulators such as cyclins (e.g., Ccndl, Cdk4, Cdk6), which drive HSCs into active division. The results show that inhibiting Ifitm3, HSC quiescence can be maintained by downregulating these cell cycle activators and enhancing the expression of quiescence enforcing genes such as Egrl and Btg2. This feature allows the long-term viability of HSCs to be maintained, which is important for sustainable blood cell production over the patient's lifetime.

[0109] One of the defining features of the compositions and methods disclosed herein is the ability to enhance HSC engraftment in bone marrow transplantation (BMT). The inhibition of ifitm3 as described herein can improve donor HSC engraftment and chimerism by approximately 15% in both steady-state (non-inflammatory) and stress-induced hematopoiesis conditions. This enhanced engraftment is observed as early as four weeks posttransplantation and is sustained over a period of 16 weeks. In mouse models, ifitm3-n\x\\ HSCs demonstrate superior engraftment and differentiation into blood cell lineages, includingAttorney’s Docket No.: 21101.0503P1

[0110] B cells. T cells, and myeloid cells, compared to wild-type HSCs. This feature is important for improving the success rates of BMT and reducing complications related to graft failure.

[0111] The compositions and methods disclosed herein are also superior in competitive bone marrow transplantation, where donor HSCs lacking ifitm3 outperform their wild-type counterparts in terms of engraftment potential. This characteristic is particularly important for patients who undergo bone marrow transplants, as it ensures more efficient and robust blood cell production post-transplantation. The ability to enhance engraftment under both steady state and stress-induced conditions sets this method apart from other HSC-modulating therapies that do not address inflammation-induced hematopoietic stress.

[0112] The compositions and methods disclosed herein demonstrate that ifitm3 inhibition protects HSCs from the deleterious effects of inflammatory stress, which typically drives HSCs into rapid cycling and differentiation. The administration of IFN (interferon) in experimental models mimics the inflammatory stress observed in BMF patients. Under these conditions, HSCs from ifitm3-null\ mice show reduced apoptosis and a greater ability to remain in quiescence, preventing early depletion of the HSC pool. This is an important advantage over existing treatments, which do not specifically protect HSCs from inflammatory-induced cycling and exhaustion

[0113] The compositions and methods disclosed herein also maintain long-term HSC function by inhibiting ifitm3, which prevents the onset of HSC exhaustion. This is particularly important for managing chronic conditions where prolonged inflammation exacerbates the depletion of HSCs. By targeting ifitm3, the compositions and methods disclosed herein provide a protective mechanism that extends the life of HSCs, reducing the need for frequent therapeutic interventions such as blood transfusions or stem cell transplants.

[0114] While the compositions and methods disclosed herein can be used to treat bone marrow failure syndromes, they can be used to treat other diseases associated with increased levels or expression of IFITM3 are found. IFITM3 is highly expressed in various cancers, including hematologic malignancies (e.g., leukemias) and solid tumors (e.g., hepatocellular carcinoma and prostate cancer). High IFITM3 expression is often associated with poor prognosis in these cancers, making it a valuable target for therapeutic intervention. By inhibiting IFITM3, the compositions and methods disclosed herein can also be used as a treatment for patients with BMF who are at increased risk of developing secondary malignancies. The ability to target ifitm3 across multiple disease states distinguishes this method from other technologies that are limited to BMF alone.Attorney’s Docket No.: 21101.0503P1

[0115] Current treatments for BMF, such as immunosuppressants and corticosteroids, have broad and often deleterious effects on the immune system, leading to a range of side effects. In contrast, IFITM3 -specific inhibitors would provide a more targeted therapeutic approach with fewer systemic side effects. This specificity makes the invention particularly appealing for drug development, offering a safer and more effective alternative to current treatments. The compositions and methods disclosed herein can be used in combination with existing BMF treatments, such as bone marrow transplants and immunosuppressants, to enhance patient outcomes. For example, IFITM3 inhibition could improve engraftment success rates in patients undergoing stem cell transplantation, reducing the need for immunosuppressive drugs and minimizing the risk of graft-versus-host disease (GVHD).

[0116] One of the major advantages of the compositions and methods disclosed herein is that it offers a therapeutic solution that could reduce or eliminate the need for invasive procedures such as bone marrow transplants. By preserving the patient's own HSC function and preventing HSC exhaustion, ifitm3 inhibition could obviate the need for transplantation in a subset of patients. This reduces the physical and psychological burden on patients, improving their quality of life while decreasing healthcare costs.

[0117] Unlike bone marrow transplants, which are limited by donor availability and require highly specialized facilities, IFITM3 inhibitors could be produced at scale and administered more easily across diverse healthcare environments. This scalability makes this method highly attractive for global implementation, particularly in regions with limited access to advanced transplantation services.

[0118] Unlike existing treatments that focus on managing the symptoms of BMF (e.g., blood transfusions or immunosuppressive drugs), the compositions and methods disclosed herein target the root cause of HSC exhaustion by directly regulating quiescence. It is the first technology to identify ifitm3 as an important regulator of HSC cycling and differentiation.

[0119] Existing therapies do not address the role of inflammation in driving HSC exhaustion. IFITM3 inhibition stands out by protecting HSCs from inflammatory stress, which is a major contributor to BMF progression, making this method particularly effective in managing chronic BMF conditions exacerbated by persistent inflammation.

[0120] While bone marrow transplants are the current gold standard for treating BMF syndromes, the compositions and methods disclosed herein can reduce or even eliminate the need for such invasive procedures in many patients by preserving the function of their native HSCs. This feature has an advantage over current technologies, which rely heavily on donor transplants for long-term treatment.Attorney’s Docket No.: 21101.0503P1

[0121] The compositions and methods described herein for inhibiting ifitm3 presents significant advantages over current solutions for treating bone marrow failure (BMF) syndromes, by addressing several limitations in existing therapies. This approach not only meets the unmet needs in BMF treatment but also offers cost savings and improved long-term patient outcomes. Current therapies for treating BMF syndromes are focused on mitigating symptoms rather than addressing the underlying cause — HSC dysfunction. Most existing treatments fail to preserve or enhance the intrinsic ability of hematopoietic stem cells (HSCs) to self-renew and generate new blood cells, a core function that is compromised in BMF syndromes. The compositions and methods disclosed herein meet this need by inhibiting ifitm3 and preserving sternness and quiescence. For example, the compositions and methods disclosed herein directly targets the regulation of HSC quiescence, which is important for maintaining their self-renewal capacity. HSCs in a quiescent state are important for sustaining long-term hematopoiesis. Current therapies, like immunosuppressants and corticosteroids, do not preserve this quiescence and can lead to the premature activation of HSCs, causing exhaustion over time. By inhibiting ifltm3, the compositions and methods disclosed herein ensure that HSCs remain in a quiescent state longer, preventing early depletion and sustaining their regenerative capacity.

[0122] The compositions and methods disclosed herein also reduce the impact of inflammation. For example, BMF syndromes are often exacerbated by chronic inflammation, which accelerates HSC proliferation and exhaustion. The compositions and methods disclosed herein address the inflammatory stress that further drives HSC dysfunction. By inhibiting ifitm3, the compositions and methods disclosed herein protects HSCs from inflammation-induced cycling and differentiation, thereby offering a protective mechanism that is currently missing in existing treatments.

[0123] The compositions and methods disclosed herein also reduce costs. For example, the cost of treating BMF syndromes is substantial due to the need for recurring blood transfusions, immunosuppressive therapies, and, in severe cases, bone marrow transplants. These treatments not only involve significant financial costs but also place a heavy burden on healthcare systems and patients alike. The compositions and methods disclosed herein can reduce costs by minimizing the need for repeated blood transfusions. Blood transfusions are one of the most common treatments for BMF patients to alleviate cytopenias. However, transfusions provide temporary relief and must be repeated, leading to high cumulative costs. Transfusion-related complications, such as iron overload, also require additional treatments. Using the compositions and methods disclosed herein that inhibit ifitm3 preserve HSCAttorney’s Docket No.: 21101.0503P1

[0124] function and promote self-renewal, and, thus, reduce the need for frequent transfusions by enhancing the body’s ability to produce its own blood cells, significantly lowering both direct and indirect costs associated with transfusion dependence. Bone marrow transplantation is currently the only curative option for many BMF patients, but it is associated with high costs, ranging from an estimated $350,000 to $800,000 per procedure, depending on the type and complexity. Moreover, transplants carry risks of graft rejection, requiring lifelong immunosuppressive therapy, which adds further costs and health risks. The compositions and methods disclosed herein to inhibit ifitm3 can reduce the need for these transplants by enhancing the regenerative capacity of the patient’s own HSCs, thus eliminating or delaying the need for a donor transplant. Even in patients who still require a transplant, the compositions and methods disclosed herein can improve transplant outcomes, reducing complications and associated healthcare costs.

[0125] The compositions and methods disclosed herein can also lower drug dependency. For example, many BMF patients are dependent on long-term corticosteroids and immunosuppressive drugs to maintain HSC function and control symptoms. These drugs can be costly and are associated with a range of side effects that require additional treatments. By providing a targeted approach that directly addresses the loss of HSC sternness, ifitm3 inhibition can reduce the dependency on these drugs, lowering long-term medication costs and minimizing the need for managing side effects.

[0126] The compositions and methods disclosed herein can further improve the quality’ of care and patient outcomes. The quality of care for BMF patients is currently limited by the transient nature of most treatments and the invasive nature of bone marrow transplants.

[0127] Existing solutions focus on short-term symptom relief rather than long-term preservation of HSC function, leading to recurrent hospitalizations, decreased quality of life, and, in some cases, progression to secondary malignancies. ifitm3 inhibition improves quality of care by leading to long-term preservation of HSC function. By targeting ifitmS, the compositions and methods disclosed herein provide an advantage in preserving the long-term function of HSCs. Current treatments do not prevent the early exhaustion of HSCs, leading to disease relapse and the eventual need for bone marrow transplantation. ifitm3 inhibition ensures that HSCs remain in a quiescent, self-renewing state, prolonging their ability to regenerate and support blood cell production. This leads to improved patient outcomes, with fewer relapses and better overall health. For patients who still require bone marrow' transplants, ifitm3 inhibition can significantly improve engraftment rates.Attorney’s Docket No.: 21101.0503P1

[0128] Preclinical studies disclosed herein have demonstrated that ifitm3-null HSCs show better engraftment and chimerism compared to wild-type cells. This enhanced engraftment leads to faster and more complete recovery of blood cell populations post-transplant, reducing the risk of graft failure and improving the overall success rate of the procedure.

[0129] Immunosuppressive therapies and corticosteroids, often used to manage BMF, come with a host of side effects, including increased risk of infections, osteoporosis, and metabolic disorders. ifitm3 inhibition, being a targeted therapy, is expected to have fewer systemic side effects compared to these broader-acting drugs. Patients can avoid the long-term complications associated with current treatments, leading to a better quality of life and reduced hospital admissions for treatment-related complications.

[0130] Beyond BMF syndromes, ifitm3 inhibition can be used in other hematologic disorders where HSC exhaustion or dysfunction is a problem, including but not limited to leukemias and other cancers where HSC preservation is important. By protecting HSC function across a range of conditions, the compositions and method disclosed herein can improve outcomes for a broader patient population.

[0131] The long-term impact of the compositions and methods disclosed herein goes beyond individual patients, affecting the healthcare system as a whole. By reducing the need for expensive and invasive procedures like bone marrow transplants and minimizing the use of long-term medications with severe side effects, the compositions and methods disclosed herein can lower the overall burden on healthcare systems. Additionally, the enhanced quality of life and reduced relapse rates for patients can decrease hospitalizations and healthcare resource usage, further contributing to cost savings and improved healthcare efficiency.

[0132] The current treatments for bone marrow failure syndromes include: 1) Blood Transfusions: Frequently administered to increase red blood cells (RBCs), white blood cells (WBCs), and platelets. However, transfusions are limited to offering temporary relief, requiring repeated interventions; 2) Immunosuppressants: Commonly used to prevent the immune system from attacking bone marrow cells. Biologies like Humira (AbbVie) and Xeljanz (Pfizer), as well as Sirolimus (Rapamune: Pfizer), are used in some cases to suppress immune attacks in BMF patients, however, these can result in significant side effects, including infections and organ damage; 3) Corticosteroids: Used to stimulate blood cell production, but often come with long-term side effects such as weakened bones, infections, and hormonal imbalances. Medications like prednisone are often used; and 4) Bone Marrow Transplants (Stem Cell Transplants): The most definitive treatment, but limited by matched donor availability and the risks of graft rejection and graft-versus-host disease (GVHD).Attorney’s Docket No.: 21101.0503P1

[0133] These approaches either provide temporary relief (e.g., blood transfusions) or come with significant risks and challenges (e.g., bone marrow transplants and immunosuppressants). Thus, there is a clear unmet need for more sustainable and less invasive treatment options.

[0134] In conclusion, the inhibition of ifitm3 provides a breakthrough in the treatment of bone marrow failure syndromes by addressing the root cause — HSC dysfunction. It offers a cost-effective, long-term solution that reduces the need for frequent blood transfusions and invasive bone marrow transplants, while also improving patient outcomes by preserving HSC function and minimizing treatment-related side effects. The compositions and methods disclosed herein meet an unmet need in the current treatment landscape as well as providing a broader range of applications in hematologic and solid tumors, positioning it as a versatile and impactful treatment strategy in modem medicine.

[0135] Compositions and Pharmaceutical Compositions

[0136] Disclosed herein are genetically-modified hematopoietic stem cells (HSCs) useful in the methods described herein. In some aspects, the disclosed HSCs comprise a disrupted, mutated, or deleted interferon-induced transmembrane protein 3 (IFITM3) or a deleted portion thereof. In some aspects, the disruption can be a deletion or a substitution in the amino acid sequence of SEQ ID NO: 1 (N-terminal domain of IFITM3). In some aspects, the disruption can be a deletion or a substitution in the amino acid sequence of SEQ ID NO: 4 (intramembrane domain of IFITM3). In some aspects, the disruption can be a deletion or a substitution in the amino acid sequence of SEQ ID NO: 6 (conserved intracellular loop of IFITM3). In some aspects, the disruption can be a deletion or a substitution in the amino acid sequence of SEQ ID NO: 8 (the transmembrane domain of IFITM3). In some aspects, the disruption can be a deletion or a substitution in the amino acid sequence of SEQ ID NO: 10 (the C-terminal domain of IFITM3). In some aspects, ifitm3 can be completely deleted. In some aspects, the genetically-modified HSC can be a long-term HSC. In some aspects, the genetically-modified HSC: exhibits reduced cycling and differentiation of HSCs to progenitor cells as compared to wild-type HSCs; exhibits improved preservation of LT-HSC sternness and quiescence as compared to wild-type HSCs; has increased expression of HSC quiescence-enforcing genes as compared to wild-type HSCs; exhibits decreased apoptosis as compared to wild-type HSCs; or exhibits increased chimerism and bone marrow engraftment at early and late time points following HSC transplantation as compared to wild-ty pe HSCs. In some aspects, the modified HSCs can be autologous human cells obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen.Attorney’s Docket No.: 21101.0503P1

[0137] Also disclosed herein are genetically-modified hematopoietic stem cells (HSCs) comprising modified interferon-induced transmembrane protein 3 gene (Ifitm3). In some aspects, ifitm3 can be completely deleted. In some aspects, the genetically-modified HSC can be a long-term HSC. In some aspects, the genetically-modified HSC: exhibits reduced cycling and differentiation of HSCs to progenitor cells as compared to wild-type HSCs; exhibits improved preservation of LT-HSC sternness and quiescence as compared to wildtype HSCs; has increased expression of HSC quiescence-enforcing genes as compared to wild-type HSCs; exhibits decreased apoptosis as compared to wild-type HSCs; or exhibits increased chimerism and bone marrow engraftment at early and late time points following HSC transplantation as compared to wild-type HSCs. In some aspects, the modified HSCs can be autologous human cells obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen.

[0138] Further disclosed herein are genetically-modified hematopoietic stem cells (HSCs) comprising an artificially disrupted, mutated, or a partially or completely deleted interferon-induced transmembrane protein 3 gene (Ifltm3). In some aspects, ifitm3 can be completely deleted. In some aspects, the genetically-modified HSC can be a long-term HSC. In some aspects, the genetically-modified HSC: exhibits reduced cycling and differentiation of HSCs to progenitor cells as compared to wild-type HSCs; exhibits improved preservation of LT-HSC sternness and quiescence as compared to wild-type HSCs; has increased expression of HSC quiescence-enforcing genes as compared to wild-type HSCs; exhibits decreased apoptosis as compared to wild-type HSCs; or exhibits increased chimerism and bone marrow engraftment at early and late time points following HSC transplantation as compared to wildtype HSCs. In some aspects, the modified HSCs can be autologous human cells obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen.

[0139] Disclosed herein are genetically-modified HSCs, comprising an artificially disrupted, mutated, or a partially or completely deleted N-terminal domain of IFITM3, intramembrane domain of IFITM3, conserved intracellular loop of IFITM3, transmembrane domain of IFITM3 or the C-terminal domain of IFITM3. In some aspects, the disruption, mutation or deletion can result in a deletion or a substitution of the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 8, or the amino acid sequence of SEQ ID NO: 10. In some aspects, the genetically-modified HSC can be a long-term HSC. In some aspects, the genetically-modified HSC: exhibits reduced cycling and differentiation of HSCs to progenitorAttorney’s Docket No.: 21101.0503P1

[0140] cells as compared to wild-type HSCs; exhibits improved preservation of LT-HSC sternness and quiescence as compared to wild-type HSCs; has increased expression of HSC quiescence-enforcing genes as compared to wild-type HSCs; exhibits decreased apoptosis as compared to wild-type HSCs; or exhibits increased chimerism and bone marrow engraftment at early and late time points following HSC transplantation as compared to wild-ty pe HSCs.

[0141] Table 1 provides the ifitm3 domains and corresponding amino acid and nucleic acid sequences, features, and functional relevance.

[0142] Disclosed herein are populations of modified human hematopoietic stem cells (HSCs). In some aspects, the modified HSCs can comprise an insertion and / or deletion in an endogenous ifltm3 sequence. In some aspects, the endogenous ifitm3 sequence can comprise the nucleic acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9. SEQ ID NO: 11, or a combination thereof. In some aspects, the insertion and / or deletion can be mediated by a CRISPR system comprising a guide nucleic acid. In some aspects, the insertion and / or deletion is capable of downregulating gene expression of ifitm3. In some aspects, the modified HSCs can be autologous human cells obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen. In some aspects, the modified HSCs can be autologous human cells obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen. In some aspects, the HSCs can be quiescent and capable of self-renewal.

[0143] In some aspects, the disruption of the disclosed genetically modified HSCs can be made using CRISPR. In some aspects, the disruption, mutation, or deletion of the disclosed genetically modified HSCs can be made using CRISPR. In some aspects, the disruption of the disclosed genetically modified HSCs can be made using Cas-CLOVER gene editing techniques. In some aspects, the disruption of the disclosed genetically modified HSCs can be made using NICER gene editing techniques. In some aspects, the disruption of the disclosed genetically modified HSCs can be made using any of the gene editing techniques described herein as they are effective in targeting specific regions while reducing off-target effects. In some aspects, the disruption of the disclosed genetically modified HSCs can be made using site-directed mutagenesis to delete specific nucleotides of desired regions of the gene.

[0144] In some aspects, the activity of a polypeptide (IFITM3) can be decreased or eliminated by disrupting the gene encoding the polypeptide. The gene ifitm3) encoding the polypeptide can be disrupted by any method known in the art, for example, by genome editing, or random or targeted mutagenesis and selecting for cells that have decreasedAttorney’s Docket No.: 21101.0503P1

[0145] activity. In some aspects, when the mutations are made within the coding sequence of a gene, they can render the gene and its subsequent protein product non-functional. creating a targeted gene disruption or “knockout” of the gene. In certain aspects, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% or more of the HSCs comprise a genetic disruption of at least one the N-terminal domain of ifitm3, the intramembrane domain of ifitm3, the conserved intracellular loop of ifitm3, the transmembrane domain of ifitm3 or the C-terminal domain of ifitm3. In some aspects, the gene disruption is by CRISPR.

[0146] In some aspects, the disclosed genetically modified HSCs can be a long-term HSC. As disclosed herein, long-term hematopoietic stem cells (LT-HSCs) are a specific subpopulation of bone marrow HSCs that can self-renew indefinitely and maintain long-term hematopoiesis due to their ability to differentiate into all types of blood cells, including but not limited to myeloid and lymphoid lineages, unlike other short-term HSCs or multipotent progenitors. In some aspects, the LT-HSCs exhibit superior engraftment, and reconstitute to functional, multi-lineage adult blood in the recipient. In some aspects, genetically modified HSCs include Lin- / Scal+ / c-kit+ cells. LT-HSCs self-renew to sustain the stem cell pool or differentiate into short-term HSCs (ST-HSCs) or lineage-restricted progenitors that undergo extensive proliferation and differentiation to produce terminally differentiated, functional hematopoietic cells. ST-HSCs or multipotent progenitors (MPPs) are able to sustain hematopoiesis in the short term, while the LT-HSCs can persist for the lifespan of the organism to perpetually replenish the hematopoietic system. In some aspects, the genetically modified HSCs disclosed herein comprise at least about 0.0001 % HSCs, or at least about 0.001% LT-HSCs, or at least about 0.01% LT-HSCs, or at least about 0.1% LT-HSCs, or at least about 1% LT-HSCs. In some aspect, subpopulations of cells (e.g., LT- HSCs) can be isolated or enriched using, for example, cell sorting approaches. For example, in some aspects, the genetically modified HSCs disclosed herein can be selected or enriched for CD34+ cells. In some aspects, the genetically modified HSCs disclosed herein can be enriched or sorted based on the expression of one or more of CD34, CD45, CD38, CD90, CD49L and GPI-80. In some aspects, the genetically modified HSCs disclosed herein can be enriched or sorted for cells having one or more of the following phenotypic markers:

[0147] CD110+, or CD135+.

[0148] In some aspects, the disclosed genetically modified HSCs are useful in preserving or enhancing (HSC) function. In some aspects, the disclosed genetically modified HSCs are useful in treating a bone marrow failure syndrome. In some aspects, the disclosed geneticallyAttorney’s Docket No.: 21101.0503P1

[0149] modified HSCs are useful in ameliorating one or more symptoms of a bone marrow failure svndrome.

[0150] Further disclosed herein are compositions comprising a genetically-modified HSC population described herein.

[0151] Also, disclosed herein are pharmaceutical compositions, comprising the genetically-modified HSCs disclosed herein.

[0152] In some aspects, a composition for cellular therapy can be prepared that comprises a population of genetically-modified HSCs prepared by the methods described herein, and a pharmaceutically acceptable vehicle. In some aspects, the pharmaceutical composition can comprise at least about 102genetically-modified HSCs, or at least about 103genetically-modified HSCs, or at least about 104genetically-modified HSCs, or at least about 105genetically-modified HSCs, or at least about 106genetically-modified HSCs, or at least about 107genetically-modified HSCs, or at least 108genetically -modified HSCs. For example, in some aspects, the pharmaceutical composition can be administered, comprising from about 100,000 to about 400,000 (CD34+) HSCs per kilogram (e.g., about 200,000 cells / kg) of a recipient’s body weight.

[0153] In some aspects, the composition or cell composition can comprise a pharmaceutically acceptable carrier or vehicle suitable for intravenous infusion or other administration route, and may include a suitable cryoprotectant. In some aspects, the pharmaceutical compositions can further comprise a pharmaceutically acceptable carrier.

[0154] As used herein, the term '‘pharmaceutically acceptable carrier” refers to solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants that can be used as media for a pharmaceutically acceptable substance. The pharmaceutically acceptable carriers can be lipid-based or a polymer-based colloid. Examples of colloids include liposomes, hydrogels, microparticles, nanoparticles and micelles. The compositions can be formulated for administration by any of a variety of routes of administration, and can include one or more physiologically acceptable excipients, which can vary depending on the route of administration. Any of the genetically modified HSCs or compositions described herein can be administered in the form of a pharmaceutical composition.

[0155] As used herein, the term “excipient” means any compound or substance, including those that can also be referred to as “carriers” or “diluents.” Preparing pharmaceutical and physiologically acceptable compositions is considered routine in the art, and thus, one ofAttorney’s Docket No.: 21101.0503P1

[0156] ordinary skill in the art can consult numerous authorities for guidance if needed. The compositions can also include additional agents (e.g., preservatives).

[0157] In some aspects, the pharmaceutically acceptable carrier can be DMSO.

[0158] In some aspects, the compositions or cell compositions can be provided in unit vials or bags, and stored frozen until use. In some aspects, the volume of the composition is from about one fluid ounce to one pint.

[0159] The pharmaceutical compositions as disclosed herein can be prepared for oral or parenteral administration. Pharmaceutical compositions prepared for parenteral administration include those prepared for intravenous (or intra-arterial), intramuscular, subcutaneous, intrathecal or intraperitoneal administration. Paternal administration can be in the form of a single bolus dose, or may be, for example, by a continuous pump. In some aspects, the compositions can be prepared for parenteral administration that includes dissolving or suspending the disclosed compound or genetically modified HSCs in an acceptable carrier, including but not limited to an aqueous carrier, such as water, buffered water, saline, buffered saline (e.g.. PBS), and the like. One or more of the excipients included can help approximate physiological conditions, such as pH adjusting and buffenng agents, tonicity adjusting agents, wetting agents, detergents, and the like. Where the compositions include a solid component (as they may for oral administration), one or more of the excipients can act as a binder or filler (e g., for the formulation of a tablet, a capsule, and the like). Where the compositions are formulated for application to the skin or to a mucosal surface, one or more of the excipients can be a solvent or emulsifier for the formulation of a cream, an ointment, and the like.

[0160] The compositions and cell compositions disclosed herein can be formulated and administered according to many factors, for example, the particular the type and severity of the BMF. In some aspects, the disclosed genetically modified HSCs can be contained within a pharmaceutical formulation. In some aspects, the pharmaceutical formulation can be a unit dosage formulation.

[0161] In some aspects, the disclosed genetically modified HSC can be formulated parental administration. In some aspects, the parenteral administration can be intravenous, subcutaneous, intramuscular or direct injection.

[0162] The pharmaceutical compositions can be sterile and sterilized by conventional sterilization techniques or sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation, which is encompassed by the present disclosure, can be combined with a sterile aqueous carrier prior to administration. The pH of theAttorney’s Docket No.: 21101.0503P1

[0163] pharmaceutical compositions typically will be between 3 and 11 (e.g., between about 5 and 9) or between 6 and 8 (e.g.. between about 7 and 8). The resulting compositions in solid form can be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment. The compositions can also be formulated as powders, elixirs, suspensions, emulsions, solutions, syrups, aerosols, lotions, creams, ointments, gels, suppositories, sterile injectable solutions and sterile packaged powders. The active ingredient can be any of the disclosed compounds or genetically modified HSCs described herein in combination with one or more pharmaceutically acceptable carriers. As used herein “pharmaceutically acceptable” means molecules and compositions that do not produce or lead to an untoward reaction (i.e., adverse, negative or allergic reaction) when administered to a subject as intended (i.e., as appropriate).

[0164] The therapeutically effective amount or dosage of any of the disclosed compositions described herein, and any of the chemotherapeutic agents, used in the methods as disclosed herein, applied to mammals (e g., humans) can be determined by one of ordinary skill in the art with consideration of individual differences in age, weight, sex, other drugs administered and the judgment of the attending clinician. Variations in the needed dosage may be expected. Variations in dosage levels can be adjusted using standard empirical routes for optimization. The particular dosage of a pharmaceutical composition to be administered to the patient will depend on a variety of considerations (e.g., the severity of the BMF symptoms), the age and physical characteristics of the subject and other considerations known to those of ordinary skill in the art. Dosages can be established using clinical approaches know n to one of ordinary skill in the art.

[0165] The duration of treatment with any composition provided herein can be any length of time from as short as one day to as long as the life span of the host (e.g., many years). For example, the compositions can be administered once a week (for, for example, 4 weeks to many months or years); once a month (for, for example, three to twelve months or for many years); or once a year for a period of 5 years, ten years, or longer. It is also noted that the frequency of treatment can be variable. For example, the present compositions can be administered once (or twice, three times, etc.) daily, weekly, monthly, or yearly.

[0166] The total effective amount of the compositions or genetically modified HSCs as disclosed herein can be administered to a subject as a single dose, either as a bolus or byAttorney’s Docket No.: 21101.0503P1

[0167] infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol in which multiple doses are administered over a more prolonged period of time. Alternatively, continuous intravenous infusions sufficient to maintain therapeutically effective concentrations in the blood are also within the scope of the present disclosure.

[0168] The compositions described herein can be administered in conjunction with other therapeutic modalities to a subject in need of therapy. The present genetically modified HSCs, compositions, or cell compositions can be given to prior to, simultaneously with or after treatment with other agents or regimes. For example, any of the compositions or genetically modified HSCs disclosed herein alone or with any of the therapeutic agents, or therapies disclosed herein can be administered in conjunction with standard therapies used to treat BMF (e.g., a chemotherapy and / or radiotherapy). In some aspects, any of the compositions or genetically modified HSCs disclosed herein can be co-formulated with a chemotherapeutic agent. In some aspects, any of the compositions, cell compositions, or genetically modified HSCs disclosed herein can be co-formulated with an immunosuppressive agent, a cell cycle inhibitor, an mTOR inhibitor, plasma exchange, IVIG, or an antiproliferative agent.

[0169] Any of the compositions or genetically modified HSCs described herein can be administered as a “combination.” It is to be understood that, for example, any of the compounds, compositions, cell compositions, or genetically modified HSCs disclosed herein can be provided to the subject in need, either prior to administration a chemotherapeutic agent or radiotherapy or any combination thereof, concomitant with administration of said chemotherapeutic agent, radiotherapy, or any combination thereof (co-administration) or shortly thereafter.

[0170] The dosage to be administered depends on many factors including, for example, the route of administration, the formulation, the severity of the patient's condition / disease, previous treatments, the patient's size, weight, surface area, age, and gender, other drugs being administered, and the overall general health of the patient including the presence or absence of other diseases, disorders or illnesses. Dosage levels can be adjusted using standard empirical methods for optimization known by one skilled in the art. Administrations of the compositions described herein can be single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). Further, encapsulation of the compositions in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) can improve the efficiency of delivery.Attorney’s Docket No.: 21101.0503P1

[0171] Methods and Methods of Treatment

[0172] The methods disclosed herein can be useful for the treatment of a subject with a bone marrow failure syndrome. In some aspects, the bone marrow failure syndrome can be Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, congenital amegakary ocytic thrombocytopenia (reduced or absent megakaryocytes in the BM, severe thrombocytopenia), severe congenital neutropenia, aplastic anemia, idiopathic BMF, or myelodysplastic syndromes. In some aspects, the genetically-modified HSCs disclosed herein can preserve or enhance hematopoietic stem cell (HSC) function in a subject. In some aspects, the genetically -modified HSCs disclosed herein can ameliorating one or more symptoms of a bone marrow failure syndrome in a subject.

[0173] Disclosed herein are methods for generating a population of modified human hematopoietic stem cells (HSCs). In some aspects, the methods can comprise: introducing into the HSCs a CRISPR system comprising a guide nucleic acid. In some aspects, the CRISPR system can produces an insertion and / or deletion in an endogenous ifitm3 sequence. In some aspects, the endogenous ifitm3 sequence can comprise the nucleic acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or a combination thereof, and wherein the insertion and / or deletion is capable of downregulating gene expression of ifitm3, thereby generating the population of modified human HSCs. In some aspects, the modified HSCs can be human cells obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen. In some aspects, the methods can be carried out ex vivo. In some aspects, the HSCs can be maintained ex vivo. In some aspects, HSCs can be quiescent and capable of self-renewal.

[0174] Disclosed herein are methods of preserving or enhancing hematopoietic stem cell (HSC) function. In some aspects, the methods of preserving or enhancing HSC function can be by disrupting, mutating, or a partially or completely deleting the interferon-induced transmembrane protein 3 gene (ifitm3) in the HSC. In some aspects, the methods can be carried out ex vivo. In some aspects, the HSCs can be maintained ex vivo. In some aspects, the methods can be carried out in vitro. In some aspects, the HSCs can be maintained in vitro. In some aspects, the HSC comprises an artificially disrupted, mutated, or a partially or completely deleted N-terminal domain of ifitm3, intramembrane domain of ifitm3, conserved intracellular loop of ifitm3, transmembrane domain of ifitm3 or the C-terminal domain of ifitm3. In some aspects, the disruption, mutation or deletion can result in a deletion or aAttorney’s Docket No.: 21101.0503P1

[0175] substitution of the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 6. the amino acid sequence of SEQ ID NO: 8, or the amino acid sequence of SEQ ID NO: 10.

[0176] Disclosed herein are methods of preserving or enhancing hematopoietic stem cell (HSC) function. In some aspects, the methods of preserving or enhancing HSC function can be by inhibiting ifitm3 in the HSCs. In some aspects, the methods can be carried out ex vivo. In some aspects, the HSCs can be maintained ex vivo. In some aspects, the methods can be carried out in vitro. In some aspects, the HSCs can be maintained in vitro. In some aspects, the HSC comprises an artificially disrupted, mutated, or a partially or completely deleted N-terminal domain of ifitm3, intramembrane domain of ifitm3, conserved intracellular loop of ifitm3, transmembrane domain of ifitm3 or the C-terminal domain of ifitm3. In some aspects, the disruption, mutation or deletion can result in a deletion or a substitution of the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 8, or the amino acid sequence of SEQ ID NO: 10.

[0177] Disclosed herein are methods of preserving or enhancing hematopoietic stem cell (HSC) function in a subject. In some aspects, the methods can comprise: administering to the mammalian subject a genetically -modified HSC, wherein the genetically-modified HSC comprises a disrupted, mutated, or deleted interferon-induced transmembrane protein 3 (IFITM3) or a deleted portion thereof. In some aspects, the subject can be a human subject. In some aspects, the subject can be a recipient of a bone marrow transplant. In some aspects, the subject has anemia or blood loss. In some aspects, the subject has depleted bone marrow. In some aspects, the subject has a decreased blood cell level or is at risk for developing a decreased blood cell levels as compared to a control blood cell level. In some aspects, the genetically modified HSC can be purified prior to administration. In some aspects, the methods to generate the genetically-modified HSCs can be carried out ex vivo. In some aspects, the methods can be carried out in vitro. In some aspects, the HSCs can be maintained in vitro. In some aspects, the HSCs can be maintained ex vivo prior to the administration step. In some aspects, the HSCs can be maintained in vitro prior to the administration step.

[0178] Disclosed herein are methods of ameliorating one or more symptoms of a bone marrow failure syndrome in a subject. In some aspects, the methods can comprise: administering to the subject a therapeutically effective amount of a genetically modified HSC. wherein the genetically-modified HSC comprises a disrupted, mutated, or deleted interferon-induced transmembrane protein 3 (ifitm3) or a deleted portion thereof, therebyAttorney’s Docket No.: 21101.0503P1

[0179] ameliorating one or more symptoms of the bone marrow failure syndrome in the mammalian subject. In some aspects, the subject can be a human subject. In some aspects, the subject can be a recipient of a bone marrow transplant. In some aspects, the subject has anemia or blood loss. In some aspects, the subject has depleted bone marrow. In some aspects, the subject has a decreased blood cell level or is at risk for developing a decreased blood cell levels as compared to a control blood cell level. In some aspects, the genetically modified HSC can be purified prior to administration. In some aspects, the one or more symptoms of the bone marrow failure syndrome can be fatigue, pale skin, easy bruising, easy bleeding, infections, fever, bone pain, or shortness of breath. In some aspects, the methods to generate the genetically -modified HSCs can be carried out ex vivo. In some aspects, the HSCs can be maintained ex vivo prior to the administration step.

[0180] Disclosed herein are methods treating a bone marrow failure syndrome, the method comprising: administering to a subject a therapeutically effective amount of the genetically modified HSC, wherein the genetically -modified HSC comprises a disrupted, mutated, or deleted interferon-induced transmembrane protein 3 (ifitm3) or a deleted portion thereof, thereby treating the bone marrow failure syndrome in the subject. In some aspects, the subject can be a human subject. In some aspects, the subject can be a recipient of a bone marrow transplant. In some aspects, the subject has anemia or blood loss. In some aspects, the subject has depleted bone marrow. In some aspects, the subject has a decreased blood cell level or is at risk for developing a decreased blood cell levels as compared to a control blood cell level. In some aspects, the genetically modified HSC can be purified prior to administration. In some aspects, the methods to generate the genetically-modified HSCs can be carried out ex vivo. In some aspects, the HSCs can be maintained ex vivo prior to the administration step.

[0181] Disclosed herein are methods of treating bone marrow failure syndrome in a subject in need thereof. In some aspects, the methods can comprise: administering to the subject a population of modified human HSCs that are quiescent and capable of self-renewal. In some aspects, the modified human HSCs can be any of the genetically-modified HSC disclosed herein or any of the modified human HSCs disclosed herein, thereby treating bone marrow failure syndrome in the subject. In some aspects, the modified human HSCs can be autologous to the subject and obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen. In some aspects, the population of modified human HSCs can be purified prior to administration. In some aspects, the population of modified human HSCs can be administered intravenously to the subject. In some aspects, the population of modified human HSCs can beAttorney’s Docket No.: 21101.0503P1

[0182] administered intravenously to the subject once after conditioning chemotherapy and / or radiotherapy.

[0183] In some aspects, the bone marrow failure syndrome can be inherited or acquired. In some aspects, the inherited bone marrow failure syndrome can be Fanconi anemia (progressive BM failure, increased cancer predisposition), dyskeratosis congenita, Shwachman-Diamond syndrome (BM failure, developmental defects), Diamond-Blackfan anemia (severe anemia, repeated blood transfusions, congenital defects), congenital amegakaryocytic thrombocytopenia (reduced or absent megakaryocytes in the BM, severe thrombocytopenia), or severe congenital neutropenia.

[0184] In some aspects, the acquired bone marrow failure (BMF) syndrome can be acquired aplastic anemia (most common type characterized by pancytopenia and reduced BM cellularity), idiopathic BMF, or myelodysplastic syndromes.

[0185] In some aspects, the methods can further include the step of identifying a subject (e.g., a human patient) as being in need of treatment before the administration step. In some aspects, the subject has been diagnosed with a bone marrow syndrome prior to the administering step. In some aspects, the subject has been identified as having anemia or blood loss. In some aspects, the subject has been identified as having depleted bone marrow. In some aspects, the subject has been identified as being a recipient of bone marrow transplant. In some aspects, the methods can further include the step of identifying a subject who in need of treatment and then providing to the subject: subject a therapeutically effective amount of the genetically modified HSC, wherein the genetically-modified HSC comprises a disrupted, mutated, or deleted interferon-induced transmembrane protein 3 ifitm3) or a deleted portion thereof.

[0186] In some aspects, the subject has a bone marrow failure syndrome. In some aspects, the bone marrow failure syndrome can be Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, congenital amegakaryocytic thrombocytopenia, severe congenital neutropenia, aplastic anemia, idiopathic BMF, or myelodysplastic syndromes. In some aspects, the subject has anemia or blood loss. In some aspects, the subject has depleted bone marrow. In some aspects, the subject can be experiencing one or more symptoms of a bone marrow failure syndrome. In some aspects, the one or more symptoms of the bone marrow failure syndrome can be fatigue, pale skin, easy bruising, easy bleeding, infections, fever, bone pain, or shortness of breath. In some aspects, the subject can be a recipient of a bone marrow transplant.Attorney’s Docket No.: 21101.0503P1

[0187] In any of the methods described herein, the modified HSCs, the modified human HSCs or the genetically-modified HSCs can be autologous to the subject and obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen.

[0188] Disclosed herein are methods, comprising: a) obtaining or having obtained a sample comprising blood cells from a subject; b) measuring the blood cell levels in the sample; c) identifying the subject as having a decreased blood cell level or at risk for developing a decreased blood cell level as compared to a control blood cell level. In some aspects, the methods further identify the subject as a suitable candidate for treatment with a therapeutically effective amount of the genetically modified HSC, wherein the genetically modified HSC comprises a disrupted, mutated, or deleted interferon-induced transmembrane protein 3 (ifitm3) or a deleted portion thereof. For example, a patient having a decreased blood cell level could have an absolute neutrophil count (ANC) of less than 500 / pL, platelets less than 20,000 / pL, bone marrow cellularity of less than 25%, or hemoglobin less than 8g / dL.

[0189] In some aspects, the method can further comprise administering a therapeutically effective amount of a second therapeutic agent or therapy. In some aspects, the second therapeutic agent or therapy can be an immunosuppressive agent, a cell cycle inhibitor, an mTOR inhibitor, plasma exchange, IVIG, or an antiproliferative agent.

[0190] The therapeutically effective amount can be the amount of the composition or cell composition administered to a subject that leads to a full resolution of the symptoms of the condition or disease, a reduction in the severity of the symptoms of the condition or disease, or a slowing of the progression of symptoms of the condition or disease. The methods described herein can also include a monitoring step to optimize dosing. The compositions or cell compositions described herein can be administered as a preventive treatment or to delay or slow the progression of the condition or disease (e.g., BMF).

[0191] The compositions and cell compositions disclosed herein can be formulated in a variety of combinations. The particular combination of the compositions or cell compositions disclosed herein with one or more second therapeutic agents or therapies can vary according to many factors, for example, the particular the type and severity of the BMF. The compositions or cell compositions described herein can be formulated to include a therapeutically effective amount of the genetically modified HSCs disclosed herein alone or in combination with a second therapeutic agent or therapy. In some aspects, the geneticallyAttorney’s Docket No.: 21101.0503P1

[0192] modified HSCs disclosed herein can be contained within a pharmaceutical formulation. In some aspects, the pharmaceutical formulation can be a unit dosage formulation.

[0193] In some aspects, the genetically-modified HSCs disclosed herein can be administered intravenously to the subject. In some aspects, the genetically-modified HSCs can be administered intravenously to the subject once after conditioning chemotherapy and / or radiotherapy.

[0194] Kits

[0195] Disclosed herein are kits that comprise any combination of the compositions or genetically modified HSCs described above and suitable instructions (e.g., written and / or provided as audio-, visual-, or audiovisual material). Disclosed herein are kits that comprise any combination of the pharmaceutical compositions described above and suitable instructions (e.g., written and / or provided as audio-, visual-, or audiovisual material). In some aspects, the kit comprises a predetermined amount of a composition or pharmaceutical composition comprising the genetically modified HSCs as disclose herein. The kit can further comprise one or more of the following: instructions, sterile fluid, syringes, a sterile container, delivery devices, and buffers or other control reagents.

[0196] EXAMPLES

[0197] Example 1.

[0198] FIG. 1 shows IFITM3 is highly expressed in hematopoietic stem cells (HSCs) in the adult bone marrow (BM).

[0199] Age-matched and sex-matched WT and ifitm3 KO mice were injected with IFNa at 25000 Units / mouse for three consecutive days, once daily, via intraperitoneal injection. Mice were rested for 10 days with no injections. On Day 11, mice were sacrificed to harvest BM cells. Cells were stained with respective antibodies to identify LT-HSCs and ST-HSCs. FIGS.

[0200] 2A-E show that loss of ifitm3 reduces HSC attrition during IFNa-induced stress hematopoiesis.

[0201] Age-matched and sex-matched WT and ifitm3 KO mice were injected with IFNα at 25000 Units / mouse for three consecutive days, once daily, via intraperitoneal injection. Mice were rested for 10 days with no injections. On Day 11, mice were sacrificed to harvest BM cells. Cells were stained with respective antibodies to identify MPP2s, MPP3s, and MPP4s. FIGS. 3A-G show that loss of ifitm3 reduces multipotent Progenitor Differentiation during IFNα-induced stress hematopoiesis.Attorney’s Docket No.: 21101.0503P1

[0202] FIGS. 4A-B show that the loss of ifitmS protects HSCs from apoptosis during IFNa-induced stress hematopoiesis. Age-matched and sex-matched WT and ifitm3 KO mice were injected with IFNα at 25000 Units / mouse for three consecutive days, once daily, via intraperitoneal injection. Mice were rested for 10 days with no injections. On Day 11, mice were sacrificed to harvest BM cells. Cells were stained with respective antibodies and Annexin V antibody to identify (FIG. 4A) Annexin V+LT-HSCs and (FIG. 4B) Annexin V+ST-HSCs. n=4-5 mice / group; *p<0.05; **p<0.01, by Welch’s two-tailed t-test).

[0203] Age-matched and sex-matched WT and ifitm3 KO mice were injected with IFN at 25000 Units / mouse for three consecutive days, once daily, via intraperitoneal injection. Mice were rested for 10 days with no injections. On Day 11, WT and ifitm3 KO mice were sacrificed to harvest donor BM cells. WT B6. SJL-Ptprca Pep3b / BoyJ (CD45.1) mice at the age of 8-10 weeks were used as the recipients. 2 million Donor CD45.2+BM cells were mixed with 2 million recipient CD45.1+BM cells (rescue cells) at a 1: 1 ratio, and intravenously transplanted into lethally irradiated (10 Gy, x-irradiation, given in two split doses of 5 Gy at 3 hours apart) BoyJ (CD45.1) mice. FIGS. 5A-D show loss of ifitm3 preserves short-term engraftment potential in competitive primary bone marrow transplantation during IFNα-induced stress hematopoiesis.

[0204] Engraftment potential was examined at 16 weeks by performing BM analysis, at the end of the primary transplantation. For secondary transplantations, engrafted 2 million CD45.2+cells were sort-purified from the primary’ recipients’ bone marrow and mixed with 2 million recipient BoyJ CD45.1 BM cells at a ratio of 1: 1, and intravenously injected into BoyJ mice. FIGS. 6A-D show that the loss of ifitm3 preserves long-term engraftment potential in competitive secondary bone marrow transplantation during IFNα-induced stress hematopoiesis.

[0205] Age-matched and sex-matched WT and ifitmS KO mice were sacrificed to harvest donor BM cells. WT B6. SJL-Ptprca Pep3b / BoyJ (CD45.1) mice at the age of 8-10 weeks were used as the recipients. 2 million Donor CD45.2+BM cells were mixed with 2 million recipient CD45.1+BM cells (rescue cells) at a 1:1 ratio, and intravenously transplanted into lethally irradiated (10 Gy, x-irradiation, given in two split doses of 5 Gy at 3 hours apart) BoyJ (CD45.1) mice. FIGS. 7A-D show that the loss of ifitm3 preserves short-term engraftment potential in competitive primary bone marrow transplantation during steady-state (non-IFNα) hematopoiesis.Attorney’s Docket No.: 21101.0503P1

[0206] The engraftment potential was examined at 16 weeks by performing BM analysis, at the end of the primary transplantation. For secondary transplantations, engrafted 2 million CD45.2+cells were sort-purified from the primary’ recipients’ bone marrow and mixed with 2 million recipient BoyJ CD45.1 BM cells at a ratio of 1: 1, and intravenously injected into Boy J mice. FIGS. 8A-D show that the loss of ifitm3 preserves long-term engraftment potential in competitive secondary bone marrow transplantation during steady-state (non-IFNa) hematopoiesis.

[0207] Age-matched and sex-matched WT and ifitm3 KO mice were sacrificed to harvest donor BM cells. WT B6. SJL-Ptprca Pep3b / BoyJ (CD45.1) mice at the age of 8-10 weeks were used as the recipients. 1000 Lin-Sca-1+cKit+(LSK) cells were sort-purified from either donor WT or IFITM3 KO mice and mixed with 1000 LSK cells sort-purified from recipient BoyJ mice and mixed at a ratio of 1: 1, and intravenously injected into recipient BoyJ mice. FIGS. 9A-D show that IFITM3-null HSCs have better engraftment potential in the adult BM during primary competitive LSK transplantation.

[0208] Age-matched and sex-matched WT and ifitm.3 KO mice were injected with IFNa at 25000 Units / mouse for three consecutive days, once daily, via intraperitoneal injection. Mice were rested for 10 days with no injections. On Day 11, WT and ifitm.3 KO mice were sacrificed to harvest donor BM cells. WT B6. SJL-Ptprca Pep3b / BoyJ (CD45.1) mice at the age of 8-10 weeks were used as the recipients. 1000 Lin-Sca-1+cKit+(LSK) cells were sort-purified from either donor WT or ifitm3 KO mice and mixed with 1000 LSK cells sort-purified from recipient BoyJ mice and mixed at a ratio of 1: 1, and intravenously injected into recipient BoyJ mice. FIGS. 10A-D show that ifitm3-null HSCs have better engraftment potential in the adult BM during primary competitive LSK transplantation during IFNa-induced stress hematopoiesis.

[0209] Bone Marrow (BM) cells from age-matched and sex-matched IFNα-treated WT and ifitm3 KO mice (injected daily for 3 consecutive days with 25000Units / mouse, rest for 10 days, harvest at Day 11), were stained with various HSC and MPP markers and anti-BrdU antibodies by flow cytometry to show the percentages of BrdU incorporation across the different stages of cell cycle. G0-G1 denotes quiescence, S denotes DNA synthesis, and G2-M denotes active cell division stages of the cell cycle. FIGS. 11A-E show that the loss of ifltm3 maintains quiescence and protects from cell cycling in HSCs and multipotent progenitors during IFNa-induced stress hematopoiesis.Attorney’s Docket No.: 21101.0503P1

[0210] Age-matched and sex-matched WT and ifitm3 KO mice were injected with IFN at 25000 Units / mouse for three consecutive days, once daily, via intraperitoneal injection. Mice were rested for 10 days with no injections. On Day 11, WT and ifitm3 KO mice were sacrificed to harvest donor BM cells. cKit+cells were isolated using cKit-Dynabeads magnetic separation kit as per manufacturer’s instructions. The cells from WT and ifitm3 KO mice were then resuspended at 1000cells / μL and single-cell RNA sequencing was performed using the 10X Genomics 3’ gene expression kit (v3.1 dual indexed). The manufacturer’s instructions were followed for reverse transcription, cDNA amplification, and library preparation according to the v.3.1 3 '-single-cell RNA-seq protocol. Sequencing was performed on an Illumina NovaSeq 6000 instrument to generate paired-end sequencing reads, and FASTQ files were generated using Illumina bcl2fastq software. 10x Genomics CellRanger software suite was used following the default parameters, to perform data preprocessing including alignment, filtering, barcode counting, and UMI counting. Downstream analyses were performed using Seurat V3 for R (pubmed.ncbi.nlm.nih.gov / 31178118 / ). Briefly, the feature-barcode matrices were log-normalized, and top variable genes across single cells were identified using the FindVariableGenes function. Dimensionality reduction was performed using principal component analysis (PCA) and then the distance matrix was organized into a K-nearest neighbor graph (KNN), partitioned into clusters using the Louvain algorithm, and clusters were visualized on a UMAP or t-SNE plot. Top differentially expressed genes for each cluster were computed using the FindMarkers function, and cell types were annotated based on the expression of known markers. FIGS. 12A-E show that the loss of ifitm3 is associated with upregulation of quiescence-enforcing pathways and downregulation of cell cycle regulators during IFNa-induced stress hematopoiesis.

[0211] During inflammatory stress, ifitm3 positively regulates the exit of HSCs from quiescence by inducing cell cycle activation and subsequent proliferation and differentiation to generate blood cells (WT: wild type, KO: ifitm.3 knock out). FIG. 13 shows a schematic representation elucidating the role of ifitm3 in hematopoietic stem cells regulating quiescence during IFNα-induced stress hematopoiesis.

[0212] FIGS. 14-18 demonstrate that loss of ifitm3 is associated with increased quiescence (candidate markers for quiescence and cell cycle, identified via single-cell RNA Seq. have now been validated by RT-PCR), reduced cell cycling, and, consequently, reduced differentiation.Attorney’s Docket No.: 21101.0503P1

[0213] FIG. 14 shows that the loss of ifitm3 is associated with reduced differentiation during steady-state (non-IFNa) hematopoiesis. 20,000 BM cells from age-matched WT and ifitm3 KO mice were mixed with ImL of MethoCult™ GF3434 (Stemcell Technologies) and plated onto 35mm culture dishes (Coming) and cultured for 12 days.

[0214] FIG. 15 shows that the loss of ifltm3 is associated with reduced G0-G1 transition cell cycle regulators consistent with single-cell RNA Seq data during steady-state (non-IFNa) hematopoiesis. BM cells were isolated from age-matched WT and ifitm3 KO mice and sorted for the long term (LT)-HSCs (labeled as Lin- Sca-1+ cKit+ CD150+ CD48- Flt3‘), and shortterm (ST)-HSC (labeled as Lin- Sca-1+ cKit+ CD150- CD48- Flt3-). Total RNA was isolated using the QiagenRNAeasy Kit, and cDNA was prepared, reverse transcriptase (RT) PCR was performed, and mRNA levels were quantified for (FIG. 15 A) Ccndl, (FIG. 15B) Ccnd2, (FIG. 15C) Cdk4, and (FIG. 15D) Cdk6.

[0215] FIG. 16 shows that loss of ifitm3 is associated with reduced G0-G1 transition cell cycle regulators consistent with single-cell RNA Seq data during IFNa-induced stress hematopoiesis. Bone Marrow (BM) cells from age-matched and sex-matched IFNa-treated WT and ifltm3 KO mice (injected daily for 3 consecutive days with 25000Units / mouse, rest for 10 days, harvest at Day 11) were isolated from age-matched WT and ifitm3 KO mice and sorted for the long-term (LT)-HSCs (labeled as Lin- Sca-1+ cKit+ CD150+ CD48- Flt3-), and short-term (ST)-HSC (labeled as Lin- Sca-1+ cKit+ CD150- CD48- Flt3-). Total RNA was isolated using the Qiagen RNAeasy Kit, and cDNA was prepared, reverse transcriptase (RT) PCR was performed, and mRNA levels were quantified for (FIG. 16A) Ccndl, (FIG.

[0216] 16B) Ccnd2, (FIG. 16C) Cdk4, and (FIG. 16D) Cdk6.

[0217] FIG. 17 shows that the loss of ifitm3 is associated with increased GO quiescence regulators consistent with single-cell RNA Seq data during steady-state (non-IFNa) hematopoiesis. BM cells were isolated from age-matched WT and ifitm3 KO mice and sorted for the long term (LT)-HSCs (labeled as Lin- Sca-1+ cKit+ CD150+ CD48- Flt3-), and shortterm (ST)-HSC (labeled as Lin- Sca-1+ cKit+ CD150- CD48- Flt3-). Total RNA was isolated using the Qiagen RNAeasy Kit, and cDNA was prepared, reverse transcriptase (RT) PCR was performed, and mRNA levels were quantified for (FIG. 17A) Egrl and (FIG. 17B) Btg2.

[0218] FIG. 18 shows that the loss of ifitm3 is associated wi th increased GO quiescence regulators consistent with single-cell RNA Seq data during IFNa-induced stress hematopoiesis. Bone Marrow (BM) cells from age-matched and sex-matched IFNa-treated WT and ifitm3 KO mice (injected daily for 3 consecutive days with 25000Units / mouse, rest for 10 days, harvest at Day 11) were isolated from age-matched WT and ifitm3 KO mice andAttorney’s Docket No.: 21101.0503P1

[0219] sorted for the long-term (LT)-HSCs (labeled as Lin- Sca-1+ cKit+ CD150+ CD48- Flt3-), and short-term (ST)-HSC (labeled as Lin- Sca-1+ cKit+ CD150- CD48- Flt3-). Total RNA was isolated using the Qiagen RNAeasy Kit, and cDNA was prepared, reverse transcriptase (RT) PCR was performed, and mRNA levels were quantified for (FIG. 18A) Egrl and (FIG.

[0220] 18B) Btg2.

Claims

Attorney’s Docket No.: 21101.0503P1CLAIMSWHAT IS CLAIMED IS:

1. A genetically-modified hematopoietic stem cell (HSC) comprising a modified interferon-induced transmembrane protein 3 gene (ifitm3).

2. A genetically-modified hematopoietic stem cell (HSC) comprising an artificially disrupted, mutated, or a partially or completely deleted interferon-induced transmembrane protein 3 gene (ifitm3).

3. The genetically-modified HSC of any one of the preceding claims, wherein Ifitm3 is completely deleted.

4. A genetically-modified HSC, comprising an artificially disrupted, mutated, or a partially or completely deleted N-terminal domain of ifitm3, intramembrane domain of ifitm3, conserved intracellular loop of ifitm3, transmembrane domain of ifitm3 or the C-terminal domain of ifitm3.

5. The genetically-modified HSC of claim 4, wherein the disruption, mutation or deletion results in a deletion or a substitution of the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 8, or the amino acid sequence of SEQ ID NO: 10.

6. The genetically-modified HSC of any one of the preceding claims, wherein the genetically -modified HSC is a long-term HSC.

7. The genetically-modified HSC of any one of the preceding claims, wherein the genetically -modified HSC:a) exhibits reduced cycling and differentiation of HSCs to progenitor cells as compared to wild-type HSCs;Attorney’s Docket No.: 21101.0503P1b) exhibits improved preservation of LT-HSC sternness and quiescence as compared to wild-type HSCs;c) has increased expression of HSC quiescence-enforcing genes as compared to wild-type HSCs;d) exhibits decreased apoptosis as compared to wild-type HSCs; or e) exhibits increased chimerism and bone marrow engraftment at early and late time points following HSC transplantation as compared to wild-type HSCs.

8. A method of preserving or enhancing hematopoietic stem cell (HSC) function by disrupting, mutating, or a partially or completely deleting the interferon-induced transmembrane protein 3 gene (ifitm3) in the HSC.

9. The method of claim 8, wherein the HSC comprises an artificially disrupted, mutated, or a partially or completely deleted N-terminal domain of ifitm3, intramembrane domain of ifitm3, conserved intracellular loop of ifitm3, transmembrane domain of ifitm3 or the C-terminal domain of ifitm3.

10. The method of claim 9, wherein the disruption, mutation or deletion results in a deletion or a substitution of the amino acid sequence of SEQ ID NO:

1. the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 8, or the amino acid sequence of SEQ ID NO: 10.

11. A method of preserving or enhancing hematopoietic stem cell (HSC) function by inhibiting ifitm3 in the HSC.

12. A method of preserving or enhancing hematopoietic stem cell (HSC) function in a subject, the method comprising: administering to the mammalian subject a genetically-modified HSC of any one of claims 1 to 7.

13. The method of claim 8, wherein the subject is a human subject.

14. The method of claim 8, wherein the subject is a recipient of a bone marrow transplant.Attorney’s Docket No.: 21101.0503P115. The method of claim 8, wherein the subject has anemia or blood loss.

16. The method of claim 8, wherein the subject has depleted bone marrow.

17. The method of claim 8, wherein the subject has a decreased blood cell level or is at risk for developing a decreased blood cell levels as compared to a control blood cell level.

18. The method of claim 8, wherein the genetically modified HSC is purified prior to administration.

19. A method of ameliorating one or more symptoms of a bone marrow failure syndrome in a subject, the method comprising: administering to the subject a therapeutically effective amount of the genetically modified HSC of any one of claims 1 to 7, thereby ameliorating one or more symptoms of the bone marrow failure syndrome in the subject.

20. The method of claim 19, wherein the subject is a human subject.

21. The method of claim 19, wherein the subject is a recipient of a bone marrow transplant.

22. The method of claim 19, wherein the subject has anemia or blood loss.

23. The method of claim 19, wherein the subject has depleted bone marrow.

24. The method of claim 19, wherein the subject has a decreased blood cell level or is at risk for developing a decreased blood cell levels as compared to a control blood cell level.

25. The method of claim 19, wherein the genetically modified HSC is purified prior to administration.Attorney’s Docket No.: 21101.0503P126. The method of claim 19, wherein the one or more symptoms of the bone marrow failure syndrome is fatigue, pale skin, easy bruising, easy bleeding, infections, fever, bone pain, or shortness of breath.

27. A method of treating a bone marrow failure syndrome, the method comprising:administering to a subject a therapeutically effective amount of the genetically modified HSC of any one of claims 1 to 7, thereby treating the bone marrow failure syndrome in the subject.

28. The method of claim 27, wherein the subject is a human subject.

29. The method of claim 27, wherein the subject is a recipient of bone marrow transplant.

30. The method of claim 27, wherein the subject has anemia or blood loss.

31. The method of claim 27, wherein the subject has depleted bone marrow.

32. The method of claim 27, wherein the subject has a decreased blood cell level or is at risk for developing a decreased blood cell levels as compared to a control blood cell level.

33. The method of claim 27, wherein the genetically-modified HSC is purified prior to administration.

34. The method of claim 27, wherein the bone marrow failure syndrome is inherited or acquired.

35. The method of claim 34, wherein the inherited bone marrow failure syndrome is Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, Diamond- Blackfan anemia, congenital amegakaryocytic thrombocytopenia (reduced or absent megakary ocytes in the BM, severe thrombocytopenia), or severe congenital neutropenia.Attorney’s Docket No.: 21101.0503P136. The method of claim 34, wherein the acquired bone marrow failure syndrome is aplastic anemia, idiopathic BMF, or myelodysplastic syndromes.

37. The method of any of the preceding claims, further comprising administering a therapeutically effective amount of a second therapeutic agent or therapy.

38. The method of claim 37, wherein the second therapeutic agent or therapy is an immunosuppressive agent, a cell cycle inhibitor, an mTOR inhibitor, plasma exchange, IVIG, or an antiproliferative agent.

39. The method of any one of the preceding claims, wherein the subject is identified as being in need of treatment before the administration step.

40. The method of any one of the preceding claims, the subject has been diagnosed with a bone marrow syndrome prior to the administering step.

41. The method of any one of the preceding claims, wherein the genetically modified HSC is administered intravenously to the subject.

42. The method of claim 41. wherein the genetically -modified HSC is administered intravenously to the subject once after conditioning chemotherapy and / or radiotherapy.

43. A method for generating a population of modified human hematopoietic stem cells (HSCs), the method comprising: introducing into the HSCs a CRISPR system comprising a guide nucleic acid, wherein the CRISPR system produces an insertion and / or deletion in an endogenous ifitm3 sequence, wherein the endogenous ifitm3 sequence comprises the nucleic acid sequence set forth in SEQ ID NO:

5. SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or a combination thereof, and wherein the insertion and / or deletion is capable of downregulating gene expression of ifitm3, thereby generating the population of modified human HSCs.Attorney’s Docket No.: 21101.0503P144. The method of claim 43, wherein the modified HSCs are human cells obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen.

45. The method of any one of claims 43-44, wherein the modified HSCs are quiescent and capable of self-renewal.

46. A population of modified human hematopoietic stem cells (HSCs), wherein the modified HSCs comprise an insertion and / or deletion in an endogenous ifitm3 sequence, wherein the endogenous Ifitm3 sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or a combination thereof, and wherein the insertion and / or deletion is mediated by a CRISPR system comprising a guide nucleic acid, and wherein the insertion and / or deletion is capable of downregulating gene expression of ifitm3.

47. The population of modified human HSCs of claim 45, wherein the modified HSCs are autologous human cells obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen.

48. The method of any one of claims 46-47, wherein the population of modified HSCs is quiescent and capable of self-renewal.

49. A method of treating bone marrow failure syndrome in a subject in need thereof, the method comprising: administering to the subject a population of modified human HSCs that are quiescent and capable of self-renewal, wherein the modified human HSCs are the genetically -modified HSC of any one of claims 1-7 or the modified human HSCs of any one of claims 46-47, thereby treating bone marrow failure syndrome in the subject.

50. The method of claim 49, wherein the modified human HSCs are autologous to the subject and obtained from a source selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph node, and spleen.Attorney’s Docket No.: 21101.0503P151. The method of claim 49, wherein the bone marrow failure syndrome is inherited or acquired.

52. The method of claim 49, wherein the inherited bone marrow failure syndrome is Fanconi anemia, dyskeratosis congenita. Shwachman-Diamond syndrome, Diamond- Blackfan anemia, congenital amegakaryocytic thrombocytopenia (reduced or absent megakaryocytes in the BM, severe thrombocytopenia), or severe congenital neutropenia.

53. The method of claim 49, wherein the acquired bone marrow failure syndrome is aplastic anemia, idiopathic BMF, or myelodysplastic syndromes.

54. The method of claim 49, wherein the population of modified human HSCs is purified prior to administration.

55. The method of claim 49, further comprising administering a therapeutically effective amount of a second therapeutic agent or therapy.

56. The method of claim 55. wherein the second therapeutic agent or therapy is an immunosuppressive agent, a cell cycle inhibitor, an mTOR inhibitor, plasma exchange, IVIG, or an antiproliferative agent.

57. The method of claim 49, wherein the subject is identified as being in need of treatment before the administration step.

58. The method of claim 49, the subject has been diagnosed with a bone marrow syndrome prior to the administering step.

59. The method of claim 49, wherein the population of modified human HSCs is administered intravenously to the subject.Attorney’s Docket No.: 21101.0503P160. The method of claim 49, wherein the population of modified human HSCs is administered intravenously to the subject once after conditioning chemotherapy and / or radiotherapy.

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