Mitochondrial fusion peptidomimetics

Abstract

Disclosed herein are methods for preparing functional hematopoietic cells suitable for transplantation into a recipient comprising obtaining hematopoietic stem cells (HSC), culturing the HSC with a mitofusin 2 fusion peptidomimetic, and recovering hematopoietic cells suitable for transplantation into a recipient. Also disclosed herein are methods of treating diseases and preventing HSC exhaustion using the HSC produced by a method disclosed herein.

Description

MITOCHONDRIAL FUSION PEPTIDOMIMETICSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional patent application 63 / 425,989 filed November 16, 2022, the entire contents of which are incorporated by reference herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOP ENT

[0002] This invention was made with government support under Grant No. 5-R01- AG055910-04 awarded by the National Institute of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] This application contains a sequence listing having the filename 1958427- 00413_Sequence_Listing.xml, which is 4,000 bytes in size, and was created on November 16, 2023. The entire content of this sequence listing is incorporated herein by reference.SUMMARY

[0004] Disclosed herein are methods of culturing hematopoietic stem cells to produce cells suitable for transplantation with mitofusion 2 fusion peptidomimetic (MFP).

[0005] Disclosed herein are methods of preparing hematopoietic stem cells (HSC) for transplantation into a recipient comprising: obtaining hematopoietic stem cells (HSC); culturing the HSC with a mitofusin 2 fusion peptidomimetic (MFP); and recovering HSCs suitable for transplantation into a recipient.

[0006] In some embodiments, the HSC are cord blood HSCs. In some embodiments, the HSC are bone marrow HSCs.

[0007] In some embodiments, the MFP comprises Compound A:In some embodiments, the MFP comprises Compound B:

[0008] In some embodiments, the MFP comprises a peptide having the amino acid sequence of SEQ ID NO:1. In some embodiments, the MFP comprises a peptide having the amino acid sequence of SEQ ID NO:2. In some embodiments, the MFP comprises a peptide having the amino acid sequence of SEQ ID NO:3.

[0009] In some embodiments, the cord blood HSCs have the phenotype CD45|OWLim CD34+CD38’CD45RA’CD90+. In some embodiments, the HSC cells suitable for transplantation, after culture with the MFP, have the phenotype CD45l0WLin_CD34+. In some embodiments, the recovered HSCs do not exhibit HSC exhaustion when transplanted into the subject.

[0010] Also disclosed herein are methods of treating a malignant disorder in a subject in need thereof comprising administering hematopoietic cells produced by a method disclosed herein. In some embodiments, the malignant disorder is selected from the group consisting of multiple myeloma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), myelodysplastic dysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), myelofibrosis, essential thrombocytosis, and polycythemia vera.

[0011] Also disclosed herein are methods of treating a non-malignant disorder in a subject in need thereof comprising administering hematopoietic cells produced by a method disclosed herein. In some embodiments, the non-malignant disorder is selected from the group consisting of aplastic anemia, severe combined immune deficiency syndrome (SCID), a thalassemia, sickle cell disease, chronic granulomatous disease, leukocyte adhesion deficiency, Chediak-Higashi syndrome, Kostman syndrome, Fanconi anemia, Blackfan- Diamond anemia, and enzymatic disorders. In some embodiments, the non-malignant disorder is an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of systemic sclerosis, systemic lupus erythematosus, neuromyelitis optica, and relapsing-remitting multiple sclerosis. In some embodiments, the HSCs are allogeneic to the subject.

[0012] Also disclosed herein are methods of preventing HSC exhaustion in a subject receiving an HSC transplant, comprising culturing the HSC with a MFP according to a method disclosed herein before transplantation into the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 depicts the effect of mitochondrial fusion on hematopoietic stem cell (HSC) expansion in culture.

[0014] FIG. 2A-F depicts the expression of Prdm16 (FIG. 2A), Mfn2 (FIG. 2B) , Mfn1 (FIG. 2C), Opal (FIG. 2D), Drp1 (FIG. 2E), and Fis1 (FIG, 2F) in cord blood (CB) HSCs. HSPC (hematopoietic stem and progenitor cells); MPP (multipotent progenitors; Lin-CD34+CD38' CD45RACD90 ); CMP (common myeloid progenitors; Lin CD34+CD38 CD123+).

[0015] FIG. 3 depicts exemplary mitochondrial peptidomimetics.

[0016] FIG. 4 depicts culture methodology with peptidomimetics and testing of expanded CB HSC for in vivo function.

[0017] FIG. 5A-C depicts increased mitochondrial fusion after mitofusin 2 fusion peptidomimetic (MFP) treatment with Compound A (FIG. 5A) or Compound B (FIG. 5B) and increased mitochondrial length (FIG. 5C) in phenotypic HSCs in vitro.

[0018] FIG. 6A-B depicts culture of MFP-treated cells. FIG. 6A: 7-day culture of resorted CD90+ cells. The scale bar = 2 micron. FIG. 6B: Seahorse extracellular flux analysis of mitochondrial respiration and ATP potential in 3-day cultures treated with vehicle, Compound A, or Compound B.

[0019] FIG. 7A-B depicts the effects of 7-day MFP treatment in expansion cultures (n>12 experiments) on frequency (FIG. 7A) and yield (FIG. 7B) of phenotypic HSCs.

[0020] FIG. 8A-B depicts significantly increased short-term (8 wk) human reconstitution in xenograft model of 7-day MFP-treated expansion cultures in two mice (FIG. 8A and 8B).

[0021] FIG. 9 depicts significantly increased long-term (15 wk) human reconstitution in xenograft model of 7-day MFP-treated expansion cultures.

[0022] FIG. 10 depicts long-term (17 wk) human reconstitution (chimerism) in a xenograft model.

[0023] FIG. 11A-B. FIG. 11A depicts 7-day MFP treatment of expansion cultures significantly increases long-term human reconstitution of phenotypic HSCs in recipient bone marrow and T cells in peripheral blood. FIG. 11 B depicts 7-day MFP treatment of expansioncultures significantly increases long-term human reconstitution of phenotypic HSCs in recipient bone marrow.

[0024] FIG. 12A-D. FIG. 12A depicts a schematic diagram describing primary and secondary xenograft transplant models. FIG. 12B-C depicts primary xenograft results 30 weeks post-transplantation of cord blood CD90+HSC cultures expanded with mitofusion agonist small molecules (FIG. 12B) or mitofusion agonist peptides (FIG. 12C). FIG. 12D depicts secondary xenograft results 30 weeks post-transplantation of primary xenograft bone marrow samples.

[0025] FIG. 13A-D. FIG. 13-B depict RNA sequence analysis of re-sorted CD90+HSCs after 7 days of expansion with mitofusion agonists. Principal component analysis (FIG. 13A) and differential gene expression (FIG. 13B) demonstrated differences between control (DMSO) and Compound B-treated CD90+HSCs. FIG. 13C-D depict gene ontology (GO) pathway analysis of differentially expressed genes showing upregulation of autophagy (FIG. 13C), ribosomal and stress granule (KEGG) pathways (FIG. 13D) after Compound B treatment.

[0026] FIG. 14A-F depicts results of OP-Puromycin protein rate synthesis assay showing representative flow cytometry plot and quantification (FIG. 14A and B), lysosome quantity (FIG. 14C), autophagosome formation (FIG. 14D), and transcription factor EP-green fluorescent protein (TFEB-GFP nuclear localization assay (FIG. 14F) from CD90+HSC cultures treated with DMSO or Compound B for 3 days.

[0027] FIG. 15 depicts the results of an immunoprecipitation assay using 293 cells transfected with His / Myc-tagged Mfn2 for 48 h.

[0028] FIG. 16 depicts a model for mitochondrial fusion agonist mechanism of action.DETAILED DESCRIPTION

[0029] Hematopoietic stem cells (HSCs), which sustain production of all blood cell lineages, rely on glycolysis for ATP production, yet little attention has been paid to the role of mitochondria. The short isoform of a critical regulator of HSCs, PR domain-containing 16 (Prdm16), induces mitofusin 2a protein involved in mitochondrial fusion and in tethering of mitochondria to the endoplasmic reticulum (ER) (FIG. 1). Mfn2 is specifically required for the maintenance of HSCs with extensive lymphoid potential but not, or less so, for the maintenance of myeloid-dominant HSCs. Mfn2 increases buffering of Cai2+, an effect mediated through its ER-mitochondria tethering activity, thereby negatively regulating nuclear translocation and transcriptional activity of Nuclear Factor of Activated T cells (NFAT). NFAT inhibition rescues the effects of Mfn2 deletion in HSCs, demonstrating that negative regulationof NFAT is the prime downstream mechanism of Mfn2 in the maintenance of HSCs with extensive lymphoid potential. Mitochondria therefore play an important role in HSCs.

[0030] Ex vivo manipulation of human HSCs is frequently accompanied by loss of their self-renewal potential due to the stress imposed by the ex vivo culture conditions in the presence of a combination of cytokines. Such systems frequently lead to HSC exhaustion due to increased HSC cycling and increased number of cell divisions. Regeneration-mediated stress induced by increased HSC divisions also skews HSC differentiation toward myeloid lineages. In turn, these events compromise HSC engraftment, sustained regeneration, and retention of their long term repopulating potential.

[0031] Developing improved cord blood (CB) HSC expansion methodologies that improve robust and durable HSC self-renewal and blood cell lineage output function in vivo are critical parameters for hematopoietic stem cell transplantation (HSCT) therapies. Effective methods to restore normal hematopoiesis in vivo and production of an abundant source of hematopoietic cells for transfusion therapies in vitro are currently lacking in transfusion / transplantation medicine. Mfn2 is highly expressed in cord blood HSCs as demonstrated in FIG. 2 and thus targeting Mfn2 is expected to improve production of HSCs suitable for transplantation.

[0032] Overexpressing Mfn2 improved HSC function in vivo and imparted lymphoid potency to HSCs (Luchsinger et al. Nature. 529:528-531 , 2016). Loss of Mfn2 expression resulted in myeloid-biased HSC function and loss of lymphoid output, which resembled the aging phenotype observed in both mouse and human hematopoiesis. In a recent manuscript, the investigation described Two classes of “peptidomimetics” (small molecules or membrane- permeable mini-peptides) have been described (Rocha et al. Science. 360:336-341 , 2018) which had the effect of facilitating HR1 & HR2 domain interaction between Mfn2 molecules expressed on neighboring mitochondrion to thereby induced mitochondrial fusion. This activity is impaired in several neurodegenerative disease, including Charcot Marie Tooth disease type 2A (CMT2A), which arises from autosomal dominant mutations of Mfn2.

[0033] Prdm16 is a 140 kDa zinc finger protein that is a physiologic regulator of HSCs. Prdm16 exists in two isoforms arising from distinct transcription start sites, full length (fl) and short (s) Prdm16, which lacks the N-terminal PR-domain. Only sPrdm16, but not flPrdm16, activated a Mfn2 promoter luciferase reporter, and induced Mfn2 mRNA in Prdm16~ / _mouse embryonic fibroblasts (MEFs). Consistent with these findings, chromatin immunoprecipitation in MEFs using FLAG-tagged isoforms of Prdm16 showed binding of sPrdm16, but not of flPrdm16, to the Mfn2 promoter. Mfn2 is therefore a direct target of sPrdm16. Although Prdm 16-deletion did not affect Mfn1, transduction of sPrdm16 did increase Mfn1 mRNAexpression. Mfn1 is therefore susceptible to regulation by sPrdm16, but with a higher and likely non-physiological threshold. Lentiviral transduction of Mfn2 does not rescue the competitive repopulation defect of Prdm16+I~ HSCs, however, indicating that multiple components of the sPrdm16 and flPrdm16 transcriptional program are required for HSC maintenance.

[0034] Induction of Mfn2 by Prdm 16 suggests a role for Mfn2 in HSC function. HSCs display clonal heterogeneity in their differentiation potential ranging from rare lymphoid-biased HSCs, to balanced myeloid / lymphoid and myeloid-dominant HSCs with low lymphoid potential. Myeloid-dominant HSCs are enriched in the CD150hi, while HSCs with extensive lymphoid potential are enriched in the CD15010fraction. HSCs expressed more Mfn2 mRNA and protein than more mature populations. Within the HSC compartment, CD15O10HSCs expressed more Mfn2 mRNA and protein than did CD150hiHSCs. In contrast, Mfn1 did not show HSC-selective expression, and its expression in CD15O10HSCs was tenfold lower than that of Mfn2. In accordance with Mfn2 induction by sPrdm16, sPrdm16 was the predominant Prdm16 isoform in CD15O10but not in CD150hiHSCs. Using mice expressing a mitochondrially- targeted Dendra2 fluorescent protein (Pham mice), we observed longer mitochondria in HSCs compared to other hematopoietic populations, and within the HSC compartment, in CD15O10than in CD150hicells. Mitochondrial length therefore paralleled Mfn2 expression.

[0035] Thus, disclosed herein are methods for preparing increased numbers of functional HSCs suitable for transplantation into a subject from cord blood HSCs comprising culturing the CB HSCs with a compound which induces mitochondrial fusion via an Mfn2 protein. The HSCs expanded according to the disclosed methods retain their long-term hematopoietic potentiality and self renewal characteristics after transplantation in contrast to untreated HSCs which undergo HSC exhaustion. As used herein, the term “functional” when used regarding HSCs, refers to HSCs which maintain their hematopoietic potentiality and do not undergo HSC exhaustion after transplantation into a subject.

[0036] Peptidomimetic compounds (FIG. 3) have been described to induce mitochondrial fusion via Mfn2 proteins (Pelay-Gimeno et al. Angew Chem Int Ed Engl. 54:8896-927, 2015). Supplementation by mitochondrial fusion peptidomimetics in CB HSC cultures may improve engraftment of expanded CB HSC cultures in immunocompromised xenotransplantation models of human hematopoiesis. Addition of mitofusin 2 fusion peptidomimetics (MFPs) significantly improves the function of 7-day CB HSC expansion cultures in vivo using NSG (NOD-Pr^dc86'^^™^'') recipient mice. This could constitute a significant advancement in the technology available to reliably expand functional HSCs in vitro for clinical and cell therapy use.Mfn2 fusion peptidomimetics

[0037] There are several types of peptidomimetics:• Class A (modified peptides) - peptides mainly formed of a-amino acids with minor side chain or backbone modification;• Class B (modified peptides / foldamers) - peptides with various backbone and side chain alterations including foldamers;• Class C (structural mimetics) - small molecule-like scaffolds that project substituents in analogy to peptide side chains; and• Class D (mechanistic mimetics) - molecules that mimic the mode of action of a peptide without a direct link to its side chains.

[0038] Non-limiting examples of Class D small molecule MFPs include but are not limited to Compounds A and B (below and FIG. 3).

[0039] Non-limiting examples of Class B modified peptide MFPs include, but are not limited to, G, S, and D peptides depicted below and in FIG. 3.GIMDSLHAAARGGYGRKKRRQRRR Peptide S (SEQ ID NO:1)GIMDDLHAAARGGYGRKKRRQRRR Peptide D (SEQ ID NO:2)GIMDSLHAAARGGYGRKKRRQRRR Peptide G (SEQ ID NO:3)

[0040] Hematopoietic stem cells can be isolated from numerous sources including, but not limited to, peripheral blood, bone marrow, umbilical cord blood, and embryonic stem cells. In some embodiments, the hematopoietic stem cells are isolated from cord blood.

[0041] The HSCs are cultured under standard culture conditions with the inclusion of a mitofusion 2 fusion peptidomimetic (MFP). In some embodiments, the MFP is a small molecule such as, but not limited to, Compound A or Compound B disclosed herein. The small molecule MFP is included in the culture at a concentration of 1 nM to 10 nM. In some embodiments, the concentration is 2.5 nM to 5 nM. In some embodiments, the concentration of small molecule MFP is 5 nM. In some embodiments, the small molecule MFP is Compound B.

[0042] In some embodiments, the MFP is a peptide such as, but not limited to Peptide S, Peptide D, or Peptide G disclosed herein. The peptide MFP is included in the culture at a concentration of 0.5-1.0 pM. In some embodiments, the peptide MFP is Peptide S.

[0043] In some embodiments, the MFP is present in the culture medium for 3-14 days. In some embodiments, the HSCs are cultured with the MFP for 7-10 days.

[0044] The HSCs generated by culture with MFPs as disclosed herein are suitable for transplantation into subjects with a variety of conditions. The HSCs produced by the methods disclosed herein can be used to treat malignant and non-malignant conditions. In some embodiments, the malignant condition includes, but is not limited to, multiple myeloma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), myelodysplastic dyndrome (MDS), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), myelofibrosis, essential thrombocytosis, and polycythemia vera. HSC transplantation can also be useful in treatment of solid tumors. In some embodiments, the non-malignant condition includes, but is not limited to, aplastic anemia, severe combined immune deficiency syndrome (SCID), a thalassemia, sickle cell disease, chronic granulomatous disease, leukocyte adhesion deficiency, Chediak-Higashi syndrome, Kostman syndrome, Fanconi anemia, Blackfan-Diamond anemia, and enzymatic disorders. Additionally, HSC transplantation may be useful in treating autoimmune diseases, including, but not limited to, systemic sclerosis, systemic lupus erythematosus, neuromyelitis optica, and relapsing-remitting multiple sclerosis.

[0045] Typically, cord blood units are cryopreserved after collection and expansion of HSCs is performed on previously cropreserved cells. In some embodiments, the HSCs are expanded in the presence of an MFP as disclosed herein and then cryopreserved prior to administration to a subject. In some embodiments, the HSCs are expanded in culture in the presence of an MFP as disclosed herein and administered to a subject after the desired number of days in culture without cryopreservation.

[0046] In some embodiments, a bank of expanded HSCs is generated by culturing HSC from individual CB units with an MFP as disclosed herein, and the HSCs cryopreserved as an off-the-shelf HSC preparation.

[0047] In some embodiments, the HSCs are autologous to the intended recipient. However, in most embodiments, the HSCs are allogenic to the intended recipient. If the HSCs are allogeneic, they are HLA matched at one or more of HLA-A, HLA-B, HLA-C, and HLA- DRB1 alleles.EXAMPLESExample 1.

[0048] Cord blood HSCs were cultured as in FIG. 4 with peptidomimetics and the expanded CB HSC were tested for in vivo function.

[0049] Studies were conducted in both fresh and frozen CB units. Phenotypic CB HSC population (CD45lowLin_CD34+CD38'CD45RA'CD90+) were isolated to initiate cultures and standard tissue culture materials were used to expand HSCs (StemSpan from SCT® + standard human recombinant cytokines) and treated with either mitofusin 2 fusion peptidomimetics (MFPs; Compound A or Compound B) or vehicle control (DMSO). After 7 days of expansion, we confirmed that phenotypic HSCs in vitro treated with Class D MFPs significantly increased mitochondrial fusion after 7 days in culture (FIG. 5A-B) and the mitochondrial length was increased (FIG. 5C).

[0050] We confirmed that mitochondrial respiration was not significantly changed in sorted phenotypic HSCs after 3 days in culture with MFPs, suggesting ATP metabolism is not affected by mitochondrial fusion agonists (FIG. 6A-B).

[0051] After 7 days of expansion, neither the frequency (FIG. 7A) or yield of phenotypic HSCs (FIG. 7B) or total nucleated cell counts were significantly changed as a function of MFP treatment.

[0052] To test in vivo function, 10,000 phenotypic HSCs were expanded in vitro for 7 days with MFP treatment and 25% of the culture’s total nucleated cell count (5x103CD34' CD38+CD45RA CD90+HSCs cultured in 1x StemSpan) was transplanted into preconditioned NSG recipient mice to perform xenotransplantation studies. Short-term engraftment was assessed at 8 weeks post-transplant and the results showed a significant increase in human chimerism from donor cell cultures treated with Class D MFPs compared to vehicle control in two mice (FIG. 8A-B).

[0053] Initial long-term engraftment was assessed at 15 weeks post-transplant and the results showed an approximate 10-fold increase in human chimerism from donor cells cultured with Compound B as opposed to Compound A or vehicle control (FIG. 9). These data suggest functional differences in long-term reconstitution of expanded HSCs are produced between Compounds A and B, which may underlie a mechanism involving mitophagy associated with Pinkl kinase activity.

[0054] Nevertheless, examination of recipients showed a high degree of correlation in human chimerism between peripheral blood (PB) and bone marrow (BM) (FIG. 10), suggesting human engraftment was likely stable at the time of analysis. Strikingly, we observed significant reconstitution of phenotypic HSCs in the bone marrow of recipients transplanted with expanded HSC cultures treated with Class D MFPs compared to vehicle controls. In fact, while peripheral blood counts showed positive chimerism for all treatments (FIG. 11 A), FACS analysis of BM from recipients transplanted with vehicle-treated cultures showed almost no CD90+HSCs, suggesting long-term engraftment was not achieved (FIG. 11 B). These data support the conclusion that Class D MFPs can dramatically improve the functional in vivo performance of HSC after a 7-day expansion in vitro. Furthermore, peripheral T-cell reconstitution (CD3+) was observed only in recipients transplanted with expanded HSC cultures treated with Class D MFPs (FIG. 11A-B). These data suggest lymphoid potency is enhanced in expanded HSC cultures treated with Class D MFPs.

[0055] The MFP-expanded HSC were evaluated in primary and secondary xenografts transplant models as depicted in FIG. 12A. Two small molecule MFPs (Compounds A and B; FIG. 12C) and three peptide MFPs (Peptides G, D, and S; FIG. 12D) were evaluated in the primary xenograft model 30 weeks post-transplantation of cord blood CD90+HSC cultures expanded with the MFPs. Compound B was also evaluated in the secondary xenograft model 30 weeks post-transplantation of primary xenograft bone marrow cells from primary NSG recipients (FIG. 12E). The results show that long-term engraftable HSCs were maintained in Compound B treated cultures in both primary and secondary transplants, while vehicle treatment alone showed eventual exhaustion of donor HSCs. These data suggest MFPs are effective at maintaining long-term HSCs during in vitro expansion cultures and could improve safety and efficacy of expanded CB units for adult transplant applications.

[0056] RNA was isolated from the CD90+HSCs, after 7 days of expansion with MFPs, and the RNA analyzed by principal component analysis (FIG. 13A) and differential gene expression (FIG. 13B) showing differences between DMSO and Compound B treated CD90+HSCs. Gene Ontology pathway analysis (FIG. 13C-D) of differentially expressed genes showing upregulation of autophagy, ribosomal and stress granule pathways with CompoundB treatment. Heatmaps calling out specific genes of interest in the GO pathway analysis were also performed. These data agree with previous studies showing that precise regulation of protein synthesis, mRNA processing and autophagy formation is a crucial feature of long-term repopulating HSC function.

[0057] The CD90+HSCs treated with Compound B, or DMSO for 3 days were then assays for 1 hr with OP-Puromycin-AF488 to measure protein synthesis rates. These data showed protein synthesis was decreased in Compound B-treated cells (FIG. 14A-E) and consistent with previous studying describing low protein synthesis as a feature of long-term engraftable HSCs.

[0058] The association of Mfn2 and MTORC1 (mammalian target of rapamycin complex 1) proteins was then evaluated (FIG. 15). 293 cells were transfected with his / Myc-tagged Mfn2 for 24 hr followed by treatment with Compound B or vehicle for an additional 24 hr. The cells were lysed and incubated with Ni / NTA beads and imidazole-eluted extracts were analyzed by SDS-PAGE. Immunoblotting with anti-Mfn2 or anti-MTORC1 demonstrated a positive association between the Mfn2 and MTORC1 protein, which increased in the presence of mitofusion agonist Compound B.

[0059] FIG. 16 depicts a model for the mitochondrial fusion agonist mechanism of action. Under tissue culture conditions, high nutrient availability and proliferative demand force HSCs to activate anabolic MTORC1 signaling pathways, including high protein synthesis and low autophagy, which have been previously shown to induce HSC exhaustion. Mitofusion agonist Compound B induced the open conformation of Mfn2, which leads to direct interaction with MTORC1 and negative regulation of MTORC1 activity, increased autophagy and reduced protein synthesis. These pathways converge to maintain repopulating HSCs during in vitro expansion and the observed increase in xenograft transplants without a substantial increase in the yield of phenotypic HSCs in vitro.

[0060] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein the terms "about" and “approximately” means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed considering the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that thenumerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0061] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0062] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0063] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0064] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially o limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0065] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

[0066] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

What is claimed is:1 . A method of preparing hematopoietic stem cells (HSC) for transplantation into a recipient comprising: obtaining hematopoietic stem cells (HSC); culturing the HSC with a mitofusin 2 fusion peptidomimetic (MFP); and recovering HSCs suitable for transplantation into a recipient.

2. The method of claim 1 , wherein the HSC are cord blood HSCs.

3. The method of claim 1 , wherein the HSC are bone marrow HSCs.

4. The method of any one of claims 1-3, wherein the MFP comprises CompoundA:

5. The method of any one of claims 1-3, wherein the MFP comprises Compound B:

6. The method of any one of claims 1-3, wherein the MFP comprises a peptide having the amino acid sequence of SEQ ID NO:1.

7. The method of any one of claims 1-3, wherein the MFP comprises a peptide having the amino acid sequence of SEQ ID NO:2.

8. The method of any one of claims 1-3, wherein the MFP comprises a peptide having the amino acid sequence of SEQ ID NO:3.

9. The method of any one of claims 1-8, wherein the cord blood HSCs have the phenotype CD45|OWLin CD34+CD38 CD45RACD90+.

10. The method of any one of claims 1-9, wherein the HSC cells suitable for transplantation, after culture with the MFP, have the phenotype CD45lowLin'CD34+.

11. The method of any one of claims 1-10, wherein the recovered HSCs do not exhibit HSC exhaustion when transplanted into the subject.

12. A method of treating a malignant disorder in a subject in need thereof comprising administering hematopoietic cells produced by the method of claims 1-11.

13. The method of claim 12, wherein the malignant disorder is selected from the group consisting of multiple myeloma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), myelodysplastic dysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), myelofibrosis, essential thrombocytosis, and polycythemia vera.

14. A method of treating a non-malignant disorder in a subject in need thereof comprising administering hematopoietic cells produced by the method of claims 1-11.

15. The method of clam 14, wherein the non-malignant disorder is selected from the group consisting of aplastic anemia, severe combined immune deficiency syndrome (SCID), a thalassemia, sickle cell disease, chronic granulomatous disease, leukocyte adhesion deficiency, Chediak-Higashi syndrome, Kostman syndrome, Fanconi anemia, Blackfan-Diamond anemia, and enzymatic disorders.

16. The method of claim 14, wherein the non-malignant disorder is an autoimmune disease.

17. The method of claim 16, wherein the autoimmune disease is selected from the group consisting of systemic sclerosis, systemic lupus erythematosus, neuromyelitis optica, and relapsing-remitting multiple sclerosis.

18. The method of any one of claims 12-17, wherein the HSC are autologous to the subject.

19. The method of any one of claims 12-17, wherein the HSC are allogeneic to the subject.

20. A method of preventing HSC exhaustion in a subject receiving an HSC transplant, comprising culturing the HSC with a MFP according to any one of claims 1-10 before transplantation into the subject.

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