Augmenting hematopoietic stem and progenitor function by intervening in peroxisomal beta-oxidation

Abstract

Compositions having, and methods for, enhanced stem cell or progenitor cell self-renewal, including use of an inhibitor of peroxisomal beta-oxidation.

Description

Docket: 93597 / 7647 / BJA / YXAUGMENTING HEMATOPOIETIC STEM AND PROGENITOR FUNCTION BY INTERVENING IN PEROXISOMAL BETA-OXIDATION

[0001] Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U. S. Provisional Application No. 63 / 808, 995 filed May 20, 2025 and U. S. Provisional Application No. 63 / 769,368 filed March 10, 2025, the contents of which are hereby incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under HL155868 and HL153487 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0004] Hematopoietic Stem Cells (HSCs) are the only cells within the hematopoietic system that possess the potential for both multi-potency and self-renewal. In the case of HSC, multipotency is the ability to differentiate into all functional blood cells, while self-renewal is the ability to give rise to identical daughter HSCs without differentiation (Seita 2010).

[0005] A major goal of stem cell research is to promote self-renewal for tissue regeneration. In the hematopoietic system, self-renewing hematopoietic stem cells (HSCs) and progenitors regenerate the blood and immune system upon transplantation into recipients. Single cell transplantation in vivo has revealed considerable heterogeneity in the self-renewal and lineage output capacity of HSCs (Dykstra B. et al. 2007). The most stringent test of HSC self-renewal is the serial transplantation assay where only HSCs with the highest durable reconstitution potential give robust reconstitution across multiple lineages upon repetitive transplantation into recipients.14938-3523-0789v.lWhile the most potent HSCs can be serially transplanted into recipients, their self-renewal and blood cell output are gradually depleted following each round of transplantation with minimal or no multilineage contribution from HSCs by tertiary transplantation (Purton et al. 2006 and Ema et al. 2005). However, it remains unclear what molecular mechanisms regulate HSCs with the most self-renewal potential and whether such mechanisms can be harnessed to promote the regeneration of the hematopoietic system.

[0006] Cellular metabolism critically regulates stem cells, including HSCs. While HSCs are initially thought to rely on glycolysis, recent evidence suggests that mitochondria activity and regulation are also required for the maintenance of HSCs (Filippi et al. 2019 and Nakamura-Ishizu et al, 2020). Besides mitochondria, peroxisome is another organelle important for cellular metabolism. Although often considered accessory organelles, peroxisomes have unique metabolic functions. For example, very long chain fatty acids are exclusively catabolized in peroxisomes (Cipolla et al. 2017). However, the role of peroxisomes in HSC regulation has not been investigated.24938-3523-0789v.lBRIEF SUMMARY OF THE INVENTION

[0007] The present disclosure provides a method of enhancing stem cell or progenitor cell selfrenewal, comprising administering to the stem cell or progenitor cell an inhibitor of peroxisomal beta-oxidation.

[0008] The present disclosure provides an ex vivo therapy method of enhancing self-renewal of stem cells or progenitor cells, comprising administering to a sample of stem cells and / or progenitor cell an inhibitor of peroxisomal beta-oxidation.

[0009] The present disclosure provides a composition comprising a population of stem cells or progenitor cells and an amount of a synthetic inhibitor of peroxisomal beta-oxidation.

[0010] The present disclosure provides a bone marrow sample comprising a population of stem cells or progenitor cells and an amount of a synthetic inhibitor of peroxisomal beta-oxidation in an amount sufficient to enhancing stem cell or progenitor cell self-renewal of stem cells or progenitor cells of the bone marrow.

[0011] The present disclosure provides a method of performing a bone marrow transplant comprising transplanting into a subject an amount of bone marrow that has been treated with an inhibitor of peroxisomal beta-oxidation as described herein in an amount sufficient to enhancing stem cell or progenitor cell self-renewal of stem cells or progenitor cells of the bone marrow. In some embodiments, the subject has a blood disorder or blood cancer, optionally, leukemia, lymphoma, aplastic anemia, or immune deficiency disorder.

[0012] The present disclosure provides a method of enhancing stem cell or progenitor cell selfrenewal in a subject, comprising administering to the subject an inhibitor of peroxisomal betaoxidation.

[0013] The present disclosure provides a method of enhancing hematopoietic stem and progenitor cells (HSPCs) self-renewal, comprising reducing the amount or the expression of Peroxisomal Biogenesis Factor 5 (PEX5) in the HSPCs.

[0014] The present disclosure provides a method of increasing the survivability of HSPCs and / or increasing the regenerability of HSPCs after treating the HSPCs with one or more34938-3523-0789v.lmyeloablative agents and / or irradiation, wherein the method comprises reducing the amount or the expression of PEX5 in the HSPCs.

[0015] The present disclosure provides a method of decreasing ferroptosis in HSPCs comprising reducing the amount or the expression of PEX5 in the HSPCs.

[0016] The present disclosure provides a method of increasing the plating capacity of bone marrow cells comprising reducing the amount or the expression of PEX5 in the bone marrow cells.

[0017] The present disclosure provides a method of increasing bone marrow cells’ contribution to HPSCs comprising reducing the amount or the expression of Peroxisomal Biogenesis Factor 5 (PEX5) in the bone marrow cells.

[0018] The present disclosure provides a method of enhancing multilineage reconstitution of bone marrow cells comprising reducing the amount or the expression of Peroxisomal Biogenesis Factor 5 (PEX5) in the bone marrow cells.44938-3523-0789v.lBRIEF DESCRIPTION OF THE DRAWINGS

[0019] Fig. 1: HSPCs preferentially express peroxisomal markers, a, Relative mRNA expression levels of Acoxl and Pex5 in HSPCs and whole bone marrow cells, b, Representative histogram and quantification of CAT staining in HSPCs including Lin'CD48‘, CD48’Lin' Scal+cKit+(CD48'LSK), MPP and HSC, and whole bone marrow cells, c, Representative histogram and quantification of PMP70 staining in HSPCs including Lin’CD48‘, CD48’Lin" Scal+cKit+(CD48 LSK), MPP and HSC, and whole bone marrow cells. MFI, Mean fluorescent intensity. Data are presented as mean ± SEM (* / ?<0.05, ** / ?<0.01). Statistical significance was assessed by RM one-way ANOVA followed by two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with a single pooled variance.

[0020] Fig. 2: Mxl-cre; Pex5^ mice have normal hematopoiesis at a steady state, a, Blood cell counts in Mxl-cre; Pex5^ and littermate control mice (n=9-10 per genotype); b, Bone marrow cellularity per hindlimb in Mxl-cre; PexS^ and littermate control mice (n=10 per genotype); c-i, Frequencies of HSCs (c); MPPs (d); LMPPs (e); MEP, GMP, and CMP (f); CLP (g); Grl+Macl+myeloid cells, Terll9+erythroid cells, and B220+B cells (h); CD3+T cells and CD41+megakaryocytes (i) in the bone marrow of Mxl-cre; Pex^ and littermate control mice (n=7-9 per genotype); j, Spleen cellularity in Mxl-cre; PexSflA and littermate control mice (n=9 per genotype); k, 1, Frequencies of HSCs (k) and MPPs (1) in the spleens of Mxl-cre; PexS^ and littermate control mice (n=9 per genotype); m, Percentage of HSCs in phase GO of cell cycle representing quiescent cells from bone marrow of Mx1-cre; Pex5fl / fland littermate control mice (n=4 per genotype); n, Percentage of Annexin V+HSCs from bone marrow of Mx1-cre; Pex5fl / fland littermate control mice (n=5 per genotype); o, p, MFI of TMRE (o) and ROS level (p) in HSCs from the bone marrow of Mxl-cre; Pex5^1and littermate control mice (n=3 per genotype); q, r, tSNE plots and population distribution in single cell sequencing using LK cells from bone marrow of Mx1-cre; Pex5fl / fland littermate control mice (data are from 2 independent experiments); Data presented as mean ± SEM (*?<0.05, **?<0.01). (a-p) Statistical significance was assessed by two-tailed, homoscedastic t-tests; (q-1) Statistical significance was assessed by ratio paired t-test.

[0021] Fig. 3: Bone marrow cells from Mxl-Cre; PexSf1^ mice give robust long-lasting reconstitution in serial transplantation, a, Procedure for primary (b, c) and serial bone marrow transplantation (d-i); b, c, Donor cell contributions to CD45+cells, Macl+myeloid cells, B220+B54938-3523-0789v.lcells, and CD3+T cells in the blood (b) as well as HSCs, MPPs, LK cells, LSK cells, and bone marrow cells in the bone marrow (c) of primary recipient mice competitively transplanted with Mxl-Cre; ex5,i nor littermate control bone marrow cells (4 donors per genotype were transplanted into a total of 16-18 recipients per genotype in 3-4 independent experiments); d, e, Donor cell contributions to the blood (d) and bone marrow (e) of secondary recipient mice transplanted with bone marrow cells from the primary recipient mice in (b) (13-14 recipients per genotype in 3 independent experiments); f, g, Donor cell contributions to the blood (f) and bone marrow (g) of tertiary recipient mice transplanted with bone marrow cells from the secondary recipient mice in (d) (5-9 recipients per genotype in 2 independent experiments); h, i, Donor cell contributions to the blood (h) and bone marrow (i) of quaternary recipient mice transplanted with bone marrow cells from the tertiary recipient mice in (f) (8-10 recipients per genotype in 2 independent experiments); Data presented as mean ± SEM (* <0.05, **j><0.01). Statistical significance was assessed by two-tailed, homoscedastic t-tests.

[0022] Fig. 4: Mxl-cre; Pex5^ mice have increased HSPC regeneration after chemotherapy, a, b, MFI of PMP70 (a) and CAT (b) in HSCs 7 days after 5FU treatment compared to control with PBS treatment or no treatment (n=6 per treatment); c, Procedure of serial 5FU treatment; d, Survival rate of Mxl-cre; PexS^ and littermate control mice in serial 5FU treatment (n=9-12 per genotype); e, White cell counts in Mxl-cre; PexSf1^ and littermate control mice 3-12 days after 5FU treatment (n=3-6 per genotype); f, Bone marrow cellularity per hindlimb in Mxl-cre; Pex5^ and littermate control mice 3-12 days after 5FU treatment (n=3-12 per genotype); g, h, HSC frequencies and numbers per hindlimb in Mxl-cre; PexP! nand littermate control mice at 3-12 days after 5FU treatment (n=3-l 1 per genotype); i-1, Frequencies of MPPs (i); CMP (j); GMP (k); and Grl+Mac-1+myeloid cells (1) in the bone marrow of Mxl-cre; Pex^1^ and littermate control mice 3-12 days after 5FU treatment (n=6-ll per genotype); m, Percentage of Annexin V+HSCs from bone marrow of Mxl-cre; PexS^ and littermate control mice 7 days after 5FU treatment (n=3 per genotype); n, Percentage of HSCs in phase GO of cell cycle representing quiescent cells from bone marrow of Mx1-cre; Pex5fl / fland littermate control mice 7 days after 5FU treatment (n=4 per genotype); o-q, MFI of TMRE (o), ROS level (p) and mitoSOX (q) in HSCs from bone marrow of Mxl-cre; Pexy1 nand littermate control mice 7 days after 5FU treatment (n=3-8 per genotype). Data presented as mean± SEM (* / ?<0.05, ** ><0.01). (a, b, e-1) Statistical significance was assessed by two-tailed, homoscedastic t-tests; (d) Statistical significance was assessed by64938-3523-0789v.lKaplan-Meyer survival analysis, followed by log rank comparison; (m-q) Statistical significance was assessed by ratio paired t-test.

[0023] Fig. 5: Inhibition of peroxisomal 0-oxidation promotes colony forming capacity of bone marrow cells, a, Procedure for serial plating of cells; b, The CFU colony number in primary and serial plating of bone marrow cells from Mxl-cre; Pex5^1and littermate control mice (n=3-5 per genotype); c, Images of representative colonies formed in serial plating by bone marrow cells from Mxl-cre; PexS^ and littermate control mice, d, e, Representative flow cytometry plots (d) and quantification (e) of LSK hematopoietic progenitor cells in secondary plating of bone marrow cells from Mxl-cre; PexS^ and littermate control mice, f, A diagram describing key pathways in peroxisomes; g, h, CFU colony numbers in primary and serial plating of bone marrow cells from wild type mice incubated in ELOVL1 inhibitor (n=3-6 per treatment); i, j, CFU colony numbers in primary and serial plating of bone marrow cells from wild type mice incubated in ACOX1 inhibitor (n=3-6 per treatment); k, Images of representative colonies from DMSO, ELOVL1 inhibitor, and ACOX1 inhibitor groups in serial plating (d, e); 1, m, Representative flow cytometry plots and quantification of LSK hematopoietic progenitors in the colonies from DMSO, ELOVL1 inhibitor, and ACOX1 inhibitor groups in secondary plating, n-q, CFU colony numbers in primary and serial plating of cells from primary plating CAT inhibitor (p, q) (n=3 per treatment); Data presented as mean ± SEM (* / ?<0.05, ** / ><0.01). Statistical significance was assessed by ratio paired t-test.

[0024] Fig. 6. Representative flow cytometry gating strategy used to identify hematopoietic stem and progenitor cell populations. These gating strategies were used for assessments of peroxisomal markers (CAT and PMP70) (a), mitochondrial membrane potential (TMRE), ROS (DCFDA), Mitochondrial Superoxide (mitoSOX) (b), cell cycle (KI67-DAPI) and apoptosis (AnnexinV-DAPI) (c).

[0025] Fig. 7. Pex5 is effectively recombined in Mxl-cre; Pex5'1mice after pIpC treatment, a, Procedure for Pex5 flox allele deletion efficiency in HSCs from the bone marrow of Mxl-cre; Pex 'fl and Pex^ mice; b, Representative genotyping results of colonies formed by doublesorted single HSCs after pIpC treatment of Mxl-cre; PexS^ and Pex5nmice. Experiment was repeated with biologically independent samples three times with similar results.74938-3523-0789v.l

[0026] Fig. 8. Mxl-cre; PexS^ mice have comparable HSC and MPP frequency in the spleen and liver, a, b, Representative image and mass of spleens in Mxl-cre; PexS^ and littermate control mice (n=9 per genotype); c, Frequencies of LSK hematopoietic progenitors in the spleens of Mx1-cre; Pex5fl / fland littermate control mice (n=9 per genotype); d, Differential expressed genes in HSCs from bone marrow of Mxl-cre; Pex^ and littermate control mice (data are from 2 independent experiments); e, f, Relative mRNA expression of Fos and Gdpd3 in HSCs from bone marrow of Mx1-cre; Pex5fl / fland littermate control mice (n=3 per genotype), g, Survival of all transplanted recipient mice, h-k, Myeloid cell population in the bone marrow of recipient mice from primary (h), secondary (i), tertiary (j) and quaternary transplantation (k). Data presented as mean ± SEM (* / ?<0.05, ** / ?<0.01). Statistical significance was assessed by two-tailed, homoscedastic t-tests.

[0027] Fig. 9. Accelerated hematopoietic recovery in Mxl-cre; PexS^ mice after 5FU treatment, a, Red blood cell and platelet counts in Mxl-cre; PexS and littermate control mice 3-12 days after 5FU treatment (n=3-6 per genotype); b-h, Frequencies of LMPPs (b), MEPs (c), CLP (d), Ter i 19+erythroid cells (e), and B220+B cells (f); CD3+T cells (g) and CD41+megakaryocytes (h) in the bone marrow of Mxl-cre; Pex5^ and littermate control mice 3-12 days after 5FU treatment (n=3-l 1 per genotype). Data presented as mean ± SEM (* / ?<0.05, ** ><0.01). Statistical significance was assessed by two-tailed, homoscedastic t-tests.

[0028] Fig. 10. Vav-cre; Pexf d1mice have normal hematopoiesis at steady state, a, Blood cell counts in Vav-cre; Pex5^ and littermate control mice (n=3 per genotype); b, Bone marrow cellularity per hindlimb in Vav-cre; Pex^ and littermate control mice (n=3 per genotype); c-i, Frequencies of HSCs (c); MPPs (d); LMPPs (e); MEP, GMP, and CMP (f); CLP (g); Grl+Macl+myeloid cells, Terll9+erythroid cells, and B220+B cells (h); CD3+T cells and CD41+megakaryocytes (i) in the bone marrow of Vav-cre; lJex5,l,iand littermate control mice (n=3 per genotype); j-1, Representative image (j), mass (k), and cellularity (1) of spleens in Vav-cre; PexS'1'' and littermate control mice (n=3 per genotype); m-o, Frequencies of HSCs (m) and MPPs (n) and LSK (o) in the spleen of Vav-cre; I’exy1" and littermate control mice (n=3 per genotype); Data presented as mean ± SEM (* / ?<0.05, ** / ?<0.01). Statistical significance was assessed by two-tailed, homoscedastic t-test.84938-3523-0789v.l

[0029] Fig. 11. Accelerated hematopoietic recovery in Vav-cre; PexM mice after 5FU treatment, a, Bone marrow cellularity per hindlimb in Vav-cre; Pex5^ mice and littermate controls after 5FU treatment (n=3-4 per genotype); b, c, Frequency and number of HSCs in Vav-cre; PexS^1mice and littermate controls after 5FU treatment (n=3-4 per genotype), d-i, Frequencies of MPPs (d); LMPPs (e); MEP, GMP, and CMP (f); CLP (g); Grl+Macl+myeloid cells, Teri 19+erythroid cells, and B220+B cells (h); CD3+T cells and CD41+megakaryocytes (i) in the bone marrow of Vav-cre; Pex5^ and littermate control mice after 5FU treatment (n=3-4 per genotype); j-1, Representative image (j), mass (k), and cellularity (1) of spleens in Vav-cre; Pex5nmice and littermate controls after 5FU treatment (n=3 per genotype); m-o, Frequencies of HSCs (m) and MPPs (n) and LSK (o) in the spleens of Vav-cre; Pex5'1*' mice and littermate controls (n=3-4 per genotype); Data presented as mean ± SEM (* / ?<0.05, ** / ?<0.01). Statistical significance was assessed by two-tailed, homoscedastic t-test.

[0030] Fig. 12. Accelerated hematopoietic regeneration in Mx1-cre; Pex5fl / flmice after irradiation, a, Blood cell counts in Mxl-cre; Pexyt f!mice and littermate controls 5-7 weeks or 10- 15 weeks after 6Gy irradiation (n=18-19 per genotype); b, Bone marrow cellularity per hindlimb in Mxl-cre; PexS^ and littermate control mice 5-7 weeks after 6Gy irradiation (n=8 per genotype); c, d, HSC frequency (c) and number (d) per hindlimb in Mxl-cre; Pex^ mice and littermate controls 5-7 weeks after 6Gy irradiation (n=8 per genotype); e-j, Frequencies of MPPs (e); LMPPs (f); MEP, GMP, and CMP (g); CLP (h); Grl+Mac-1+myeloid cells, Teri 19+erythroid cells, and B220+B cells (i); CD3+T cells and CD41+megakaryocytes (j) in the bone marrow of Mxl-cre; PexM^ and littermate control mice 5-7 weeks after 6Gy irradiation (n=5-12 per genotype); k, Bone marrow cellularity per hindlimb in Mxl-cre; Pex5^1and littermate control mice 10-15 weeks after 6Gy irradiation (n=4-5 per genotype); 1, m, HSC frequency and number per hindlimb in Mxl-cre; Pexy1'land littermate control mice 10-15 weeks after 6Gy irradiation (n=4-5 per genotype); n-s, Frequencies of MPPs (n); LMPPs (o); MEP, GMP, and CMP (p); CLP (q); Grl+Mac-1+myeloid cells, Teri 19+erythroid cells, and B220+B cells (r); CD3+T cells and CD41+megakaryocytes (s) in the bone marrow of and Mxl-cre; P xP1*1and littermate control mice 10-15 weeks after 6Gy irradiation (n=4-5 per genotype); t, Percentage of Annexin V+HSCs from bone marrow of Mx1-cre; Pex5fl / fland littermate control mice 5-7 weeks after 6Gy irradiation (n=7 per genotype); u, Percentage of HSCs in phase GO of cell cycle representing quiescent cells from bone marrow of Mxl-cre; Pex5,i nand littermate control mice 5-7 weeks after 6Gy irradiation94938-3523-0789v.l(n=3 per genotype); v, MFI of TMRE (v) in HSCs from bone marrow of Mx1-cre; Pex5fl / fland littermate control mice 5-7 weeks after 6Gy irradiation (n=9 per genotype); w, MFI of ROS level in HSCs from bone marrow of Mxl-cre; Pex 'f1and littermate control mice 5 weeks after 6Gy irradiation (n=3-4 per genotype). Data presented as mean ± SEM (* / ?<0.05, ** ><0.01). (a-s) Statistical significance was assessed by two-tailed, homoscedastic t-tests; (t-w) Statistical significance was assessed by ratio paired t-test.

[0031] Fig. 13. Inhibition of AGPS in bone marrow cells may also significantly impact replating capacity. Acyl-CoA generated through peroxisomal b-oxidation can be used for ether lipid synthesis in the peroxisome. Therefore, whether inhibition of ether lipid synthesis also leads to enhanced serial plating was tested. Alkylglycerone phosphate synthase (AGPS) is a peroxisomal enzyme critical for the production of ether lipids, such as plasmalogen. Bone marrow cells from wild-type mice were incubated in methylcellulose with AGPS-IN-1, a specific and effective inhibitor for AGPS. Compared with controls, AGPS-IN-1 drastically enhanced the serial plating capacity of bone marrow cells (~60 fold in tertiary plating) compared with controls. These data suggest that the effect of peroxisomal b-oxidation on hematopoietic progenitor expansion in methylcellulose relies on ether lipid synthesis. A product of peroxisomal oxidation is ROS, which is scavenged by CAT in peroxisomes. Wild-type bone marrow cells were treated with a CAT inhibitor, 3-amino-l,2,4-triazole (3 AT), and performed colony formation assays in methylcellulose. Inhibition of CAT with 3AT significantly reduced the serial plating capacity of bone marrow cells, suggesting that CAT is required for robust serial colony formation by bone marrow cells.

[0032] Fig. 14. Inhibition of ELOVL1 significantly expands HSCs in culture. Acyl-CoA generated through peroxisomal b-oxidation can be used for ether lipid synthesis in the peroxisome. Therefore, whether inhibition of ether lipid synthesis also leads to enhanced serial plating was tested. Alkylglycerone phosphate synthase (AGPS) is a peroxisomal enzyme critical for the production of ether lipids, such as plasmalogen. Bone marrow cells from wild-type mice were incubated in methylcellulose with AGPS-IN-1, a specific and effective inhibitor for AGPS. Compared with controls, AGPS-IN-1 drastically enhanced the serial plating capacity of bone marrow cells (~60 fold in tertiary plating) compared with controls. These data suggest that the effect of peroxisomal b-oxidation on hematopoietic progenitor expansion in methylcellulose relies on ether lipid synthesis. A product of peroxisomal oxidation is ROS, which is scavenged by CAT 104938-3523-0789v.lin peroxisomes. Wild-type bone marrow cells were treated with a CAT inhibitor, 3-amino-l,2,4-triazole (3 AT), and performed colony formation assays in methylcellulose. Inhibition of CAT with 3 AT significantly reduced the serial plating capacity of bone marrow cells, suggesting that CAT is required for robust serial colony formation by bone marrow cells.

[0033] Fig. 15. Inhibition of ACOX1 significantly expands HSCs in culture. According to preliminary data, ACOX1 inhibitor TDYA promoted HSC expansion in culture. Thus, inhibition of peroxisomal b-oxidation promotes HSC expansion in culture.

[0034] Fig. 16. scRNA-seq reveals accelerated regeneration by Pex5-deficient HSCs. To further comprehensively understand the mechanisms by which Pex5-deficient HSCs and hematopoietic progenitors exhibit robust regenerative activity, single-cell RNA-seq (scRNA-seq) analysis on sorted Lin'cKit bone marrow cells from Mxl-cre; Pex5^^ and control mice was performed at both steady and 5FU-induced regenerating conditions. Cell populations corresponding to uncommitted HSC / multipotent progenitor 1 (MPP1), lineage-restricted MPPs, and more downstream progenitors committed to myeloid, lymphoid, erythroid, megakaryocyte, and mast / baso lineages were identified (Figure A and B). Consistent with flow cytometric analyses, scRNA-seq revealed normal cellular composition of the HSC and hematopoietic progenitor compartments from Mx1-cre; Pex5fl / flmice compared with controls (Figures C).

[0035] Fig. 17. Pex5-deficient HSCs are more resistant to ferroptosis. After 5FU treatment, the hematopoietic compartment regenerates significantly faster in Mxl-cre; PexS^ mice compared with controls (Figures C-D). Gene set enrichment analysis (GSEA) on HSCs from Mxl-cre; Pex5^ and control mice were performed after 5FU treatment. Consistent with the enhanced regenerative capacity, Eex5-deficient HSCs displayed enrichment of gene set of hematopoietic stem cell homeostasis compared with control HSCs (Figure E). Gene set enrichment of ferroptosis was observed, particularly negative regulation of ferroptosis in 7Yx5-deficient HSCs (Figure E). Several negative ferroptosis regulators, including Nfe2I2 Nrf2), Mboat2, Cd44, Gchl, w Aifm2 (FspP), are upregulated in 7Yx5-deficient HSCs compared with controls (Figure F). Lipid peroxidation levels in HSCs were directly accessed by staining with the lipid dye BODIPY Cl 1. Pcx5-deficient HSCs had significantly lower levels of lipid peroxidation (Figure G). Collectively, these data suggest that deletion of Pex5 leads to features of potent HSCs and diminished ferroptosis during regeneration.114938-3523-0789v.l

[0036] Fig. 18. Inhibition of peroxisomal b-oxidation promotes HSC regeneration in vivo. The effects of peroxisomal b-oxidation inhibition on HSC regeneration in vivo were tested. Mice were fed on chow containing TDYA for two weeks before 5FU challenge. One week later, hematopoietic regeneration was analyzed. Mice fed on TDYA chow had a 11 -fold increase of HSC frequency and number compared with controls. These data suggest that inhibition of peroxisomal b-oxidation promotes HSC and hematopoietic regeneration in vivo.124938-3523-0789v.lDETAILED DESCRIPTION OF THE INVENTION

[0037] The present disclosure provides a method of enhancing stem cell or progenitor cell selfrenewal, comprising administering to the stem cell or progenitor cell an inhibitor of peroxisomal beta-oxidation.

[0038] The present disclosure provides an ex vivo therapy method of enhancing self-renewal of stem cells or progenitor cells, comprising administering to a sample of stem cells and / or progenitor cell an inhibitor of peroxisomal beta-oxidation.

[0039] The present disclosure provides a method of enhancing stem cell or progenitor cell selfrenewal in a subject, comprising administering to the subject an inhibitor of peroxisomal betaoxidation.

[0040] In some embodiments, the inhibitor comprises an acyl-coenzyme A oxidase 1 inhibitor.

[0041] In some embodiments, the inhibitor comprises tricosadiynoic acid.

[0042] In some embodiments, the inhibitor comprises an inhibitor of ELOVL Fatty Acid Elongase 1 (ELOVL1).

[0043] In some embodiments, the inhibitor is a pyrazole amide or is a pyrimidine ether-based inhibitor.

[0044] In some embodiments, the inhibitor inhibits Pex5.

[0045] In some embodiments, the stem cell or progenitor cell is a hematopoietic stem cell or hematopoietic progenitor cell.

[0046] In some embodiments, the method further comprises subsequently administering the cells to a subject in need thereof or having a bone marrow transplant.

[0047] The present disclosure provides a composition comprising a population of stem cells or progenitor cells and an amount of a synthetic inhibitor of peroxisomal beta-oxidation.

[0048] In some embodiments, the inhibitor comprises an acyl-coenzyme A oxidase 1 inhibitor.

[0049] In some embodiments, the inhibitor comprises tricosadiynoic acid.

[0050] In some embodiments, the inhibitor comprises an inhibitor of ELOVL Fatty Acid Elongase 1 (ELOVL1).134938-3523-0789v.l

[0051] In some embodiments, the inhibitor is a pyrazole amide or is a pyrimidine ether-based inhibitor.

[0052] In some embodiments, the inhibitor inhibits Pex5.

[0053] In some embodiments, the stem cell or progenitor cell is a hematopoietic stem cell or hematopoietic progenitor cell.

[0054] In some embodiments, the cells are for administration to or transplant into a subject in need thereof.

[0055] In some embodiments, the cells of the composition have enhanced self-renewal relative to an otherwise identical composition which does not contain any inhibitor of peroxisomal betaoxidation.

[0056] In some embodiments, the subject has a blood disorder or blood cancer, optionally, leukemia, lymphoma, aplastic anemia, or immune deficiency disorder.

[0057] In some embodiments, the stem cells or progenitor cells are part of a bone marrow sample.

[0058] In some embodiments, the bone marrow sample has been previously removed from a donor subject.

[0059] In some embodiments, the bone marrow sample is treated with the inhibitor of peroxisomal beta-oxidation prior to transplant or administration to a recipient subject.

[0060] In some embodiments, the inhibitor does not inhibit mitochondrial fatty acid oxidation.

[0061] The present disclosure provides a method of performing a bone marrow transplant comprising transplanting into a subject an amount of bone marrow that has been treated with an inhibitor of peroxisomal beta-oxidation as described herein in an amount sufficient to enhancing stem cell or progenitor cell self-renewal of stem cells or progenitor cells of the bone marrow.

[0062] In some embodiments, the subject has blood disorder or blood cancer.

[0063] In some embodiments, the blood disorder or blood cancer is leukemia, lymphoma, aplastic anemia, or immune deficiency disorder.144938-3523-0789v.l

[0064] In some embodiments, the method further comprises treating the bone marrow sample with the inhibitor of peroxisomal beta-oxidation.

[0065] The present disclosure provides a method of enhancing hematopoietic stem and progenitor cells (HSPCs) self-renewal, comprising reducing the amount or the expression of Peroxisomal Biogenesis Factor 5 (PEX5) in the HSPCs.

[0066] The present disclosure provides a method of increasing the survivability of HSPCs and / or increasing the regenerability of HSPCs after treating the HSPCs with one or more myeloablative agents and / or irradiation, wherein the method comprises reducing the amount or the expression of PEX5 in the HSPCs.

[0067] The present disclosure provides a method of delaying ferroptosis in HSPCs comprising reducing the amount or the expression of PEX5 in the HSPCs.

[0068] In some embodiments, the myeloablative agent is 5 -fluorouracil (5-FU), busulfan, or cyclophosphamide.

[0069] In some embodiments, the irradiation is X-ray.

[0070] In some embodiments, the HSPCs are hematopoietic stem cells (HSCs); lymphoid-primed multipotent progenitors (LMPPs), common myeloid progenitors (CMPs), megakaryocytic / erythroid progenitors (MEPs), granulocyte / macrophage progenitors (GMPs), common myeloid progenitors (CLPs), Grl+Macl+myeloid cells, Teri 19+erythroid cells, B220+B cells, CD3+T cells, and CD41+megakaryocytic cells.

[0071] In some embodiments, the HSPCs are HSCs.

[0072] The present disclosure provides a method of increasing the plating capacity of bone marrow cells comprising reducing the amount or the expression of PEX5 in the bone marrow cells.

[0073] In some embodiments, the HSPCs are part of a bone marrow sample.

[0074] In some embodiments, the bone marrow sample has been previously removed from a donor subject.

[0075] In some embodiments, the bone marrow sample is treated with the inhibitor of peroxisomal beta-oxidation prior to transplant or administration to a recipient subject.

[0076] In some embodiments, the inhibitor does not inhibit mitochondrial fatty acid oxidation.154938-3523-0789v.l

[0077] The present disclosure provides a method of increasing bone marrow cells’ contribution to HPSCs comprising reducing the amount or the expression of Peroxisomal Biogenesis Factor 5 (PEX5) in the bone marrow cells.

[0078] The present disclosure provides a method of enhancing multilineage reconstitution of bone marrow cells comprising reducing the amount or the expression of Peroxisomal Biogenesis Factor 5 (PEX5) in the bone marrow cells.

[0079] In some embodiments, the reducing the amount or the expression of PEX5 is achieved by contacting the HSPCs or bone marrow cells with at least one inhibitor.

[0080] In some embodiments, the inhibitor is an alkylglycerone phosphate synthase (AGPS) inhibitor.

[0081] In some embodiments, the inhibitor comprises tricosadiynoic acid.

[0082] In some embodiments, the inhibitor comprises an inhibitor of ELOVL Fatty Acid Elongase 1 (ELOVL1).

[0083] In some embodiments, the inhibitor is a pyrazole amide or is a pyrimidine ether-based inhibitor.

[0084] In some embodiments, the inhibitor is an inhibitor of peroxisomal beta-oxidation.

[0085] In some embodiments, the inhibitor is a Pex5 inhibitor.

[0086] In some embodiments, the method further comprising contacting the bone marrow cells with an Alkylglycerone phosphate synthase (AGPS) inhibitor.

[0087] In some embodiments, the method comprises subsequently administering the HSPCs or bone marrow cells to a subject in need thereof or having a bone marrow transplant.

[0088] In some embodiments, the subject has a blood disorder or blood cancer.

[0089] In some embodiments, the subject has leukemia, lymphoma, aplastic anemia, or immune deficiency disorder.

[0090] The present disclosure provides a method of promoting HSPC regeneration capacity in a subject, wherein the method comprises administering to the subject one or more peroxisomal beta-oxidation inhibitors.164938-3523-0789v.l

[0091] The present disclosure provides a method of enhancing bone marrow transplantation efficiency in a subject, wherein the method comprises administering to the subject one or more peroxisomal beta-oxidation inhibitors.

[0092] In some embodiments,(a) the peroxisomal beta-oxidation inhibitor is an alkylglycerone phosphate synthase (AGPS) inhibitor; preferably, the inhibitor comprises tricosadiynoic acid;(b) the peroxisomal beta-oxidation inhibitor is an inhibitor comprises an inhibitor of ELOVL Fatty Acid Elongase 1 (ELOVL1); preferably, the inhibitor is a pyrazole amide or is a pyrimidine ether-based inhibitor;(c) the subject is mammal; preferably the mammal is human; and / or(d) the administration is administered daily, bi-monthly or monthly.Definitions

[0093] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0094] “And / or” as used herein, for example with option A and / or option B, encompasses the separate embodiments of (i) option A, (ii) option B, and (iii) option A plus option B.

[0095] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about includes the specified value. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of and any combination of items it conjoins.174938-3523-0789v.l

[0096] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application.

[0097] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0098] In the description and claims of the present application, each of the verbs, “comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb. Other terms as used herein are meant to be defined by their well-known meanings in the art.General

[0099] For the foregoing embodiments, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments.

[0100] Examples of pyrazole amide and pyrimidine ether-based inhibitor can be found in, inter alai, e.g., Boyd et al., J. Med. Chem. 2021, 64, 24, 17777-17794, and also e.g., Come et al. J Med Chem. 2021 Dec 23;64(24):17753-17776, both of which are hereby incorporated by reference.

[0101] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections. All combinations of the various elements disclosed herein are within the scope of the invention.

[0102] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which184938-3523-0789v.lare not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0103] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0104] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.EXAMPLESMethods

[0105] Mice. Mxl-cre31and PexS^19mice were obtained from the Jackson Laboratory and maintained on C57BL / 6 background. The Cre activity in Mxl-cre mice was induced by intraperitoneal pIpC (Invivogen) injection. Young adult mice were treated with 20-50ug pIpC every other day for 5 doses. Both male and female mice were included for analysis and no differences were notice. All mice were housed in specific pathogen-free, Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC)-approved unit at Columbia University Medical Center. All protocols were approved by Columbia University Committee on the Institute Animal Care and Use.

[0106] Genotyping PCR Primers. The following primers were used for genotyping. Mxl-cre-. 5’-CATGTGTCTTGGTGGGCTGAG-3’ and 5’-CGCATAACCAGTGAAACAGCAT-3’; Pex5fl: 5’- GTGGGGAAAGAAGGTGGAAG-3’,and 5’- CCTGCTTCGCTACTGTTTGG-3’; Pex5: 5’-CAGTCCCTTCAGGGCATTAC-3’ and 5 ’-CCTGCTTCGCTACTGTTTGG-3’.194938-3523-0789v.l

[0107] Irradiation and 5FU treatment. Sublethal irradiation (6 Gy) of mice was performed using a MultiRad 225 X-ray irradiator (Precision X-Ray). Fluorouracil (5-FU, 150 mg / kg, Fresenius Kabi) was administered intraperitoneally to mice.

[0108] Long-term competitive reconstitution assay. Adult recipient mice (B6. SJL-PtprcaPepcb / BoyJ mice from the Jackson Laboratory) were lethally irradiated by a Multi Rad 225 X-ray irradiator (Precision) with a total of 1,050 rads with two doses delivered at least 2 hr apart. Cells were transplanted by retro-orbital venous sinus injection of anesthetized mice. In primary transplantation, donor bone marrow cells (5 x 105cells) were transplanted along with recipient bone marrow cells (5 x 105cells) into lethally irradiated recipient mice by retro-orbital venous sinus injection. In secondary to quaternary transplantation, equal numbers of bone marrow cells from recipient mice in the previous transplantation were mixed and 5-7xl06cells were transplanted into lethally irradiated recipient mice. Mice were maintained on antibiotic water (Baytril 0.17 g / L, Bayer) for two weeks. Recipient mice were bled every four weeks for 16 weeks to assess the level of donor-derived blood lineages, including myeloid, B and T cells. Blood cells were treated with ammonium chloride potassium for red cell lysis before antibody staining. The antibodies were all from Biolegend, including anti-CD45.2 (104), anti-CD45.1 (A20), anti-CD3 (17A2), anti-B220 (6B2), anti-Grl (8C5), and anti-Macl (MI / 70).

[0109] Colony formation. Bone marrow cells or double-sorted HSCs were plated in methyl cellulose culture medium (3434, STEMCELL Technologies) and incubated at 37°C with 5% CO2 and constant humidity. Colonies were counted 12-14 days after plating and scored as granulocyte-macrophage progenitor cells [colony-forming unit (CFU)-GM], multipotent GEMM progenitor cells (CFU-GEMM), erythroid progenitor cells (BFU-E), and megakaryocyte progenitor cells (CFU-MK). After counting, the cells from colonies were collected by FACS buffer for DNA analysis, serial plating or flow cytometry analysis after HSC staining.

[0110] Complete blood count. Peripheral blood was collected to EDTA-coated tubes (Microvette CB300, Sarstedt) and mixed well immediately. Blood samples were then analyzed on a complete blood count analyzer (Genesis, Oxford Science).

[0111] Flow cytometry. Bone marrow cells were isolated by flushing long bones or crushing long bones, pelvis, and vertebrae with mortar and pestle. Spleen cells were isolated by crushing the whole spleen between two glass slides. The cells were passed through 25G syringes at least204938-3523-0789v.ltwo times with Ca2+and Mg2+free HBSS with 2% heat-inactivated bovine serum for single cell suspension. The cells were through filtered through a 70-pm nylon mesh. The following antibodies were used for staining HSCs (all from Biolegend): lineage markers (anti-Terl 19, anti-B220 (6B2), anti-Grl (8C5), anti-CD2 (RM2-5), anti-CD3 (17A2), anti-CD5 (53-7.3) and anti-CD8 (53-6.7)), anti-Sca-1 (E13-161.7), anti-cKit (2B8), anti-CD150 (TC15-12F12.2), and anti-CD48 (HM48-1). The following antibodies were used for staining hematopoietic progenitors: lineage markers (anti-Terl 19, anti-B220 (6B2), anti-Grl (8C5), anti-CD2 (RM2-5), anti-CD3 (17A2), anti-CD5 (53-7.3) and anti-CD8 (53-6.7)), anti-Sca-1 (D7), anti-cKit (2B8), anti-Flt3 (A2F10), CD16 / 32 (93), CD 127 (A7R34) (all from BioLegend), and anti-CD34 (RAM34, BD Biosciences). The following antibodies were used for staining mature cells (all from BioLegend): anti-CD3 (17A2), anti-B220 (6B2), anti-Grl (8C5), anti-Macl (MI / 70), anti-Terl 19 (TER-119) and anti-CD41 (MWReg30). DAPI (lug / ml) was used to exclude dead cells. Samples were run on FACSAria II, or FACSCelesta flow cytometers. FACSDiva (BD, v.9) or FlowJo (FlowJo LLC) software was used for data analysis.

[0112] Intracellular staining. BD Cytofix / Cytoperm buffer (BD Biosciences) was used to fix bone marrow cells for 20 min on ice. The cells were washed in 1× BD Perm / Wash buffer and then incubated with the following primary antibodies: anti-PMP70 (EPR5614, Abeam), anti-Catalase (EPR20198, Abeam), or Rabbit mAb Isotype Control (3900, Cell Signaling Technology). Cells were washed in Perm / Wash buffer and incubated with anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific). Then cells were washed in Perm / Wash buffer after staining and resuspended in Perm / Wash buffer with DAPI, followed by flow cytometry analysis.

[0113] For cell cycle analysis, followed by fixation and wash, cells were incubated in Perm / Wash buffer with Ki-67 FITC (11F6, BioLegend). After staining, cells were washed in Perm / Wash buffer and resuspended in Perm / Wash buffer with DAPI for flow cytometry analysis.

[0114] Reactive oxygen species and mitochondrial membrane potential analysis. For analysis of reactive-oxygen-species levels, cells were incubated with 10 pM DCFDA (29-79-dichlorofluorescein diacetate; Thermo Fisher Scientific) for 30 min at 37 °C after antibody staining. For analysis of superoxide levels in mitochondria, cells were incubated with MitoSOX Red (5 pM; MedChemExpress) in the presence of verapamil (50 pM; Sigma-Aldrich). For analysis of mitochondrial membrane potential, cells were incubated with tetramethylrhodamine ethyl ester214938-3523-0789v.l(TMRE; 2 nM; Thermo Fisher) in the presence of verapamil for 30 min at 37 °C after antibody staining. The cells were analyzed by flow cytometry.

[0115] scRNA-seq. DAPI−lineage−cKit+cells from bone marrow were double-sorted in a yield-purity order of sorting modes on BD SORP FACSAria. Immediately following FACS isolation, 10x Genomics Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 were used to prepare the library according to the manufacturer’s instructions. Individual libraries were pooled for sequencing on the NovaSeq 6000 Sequencing System (Illumina).

[0116] Quantitative PCR. Cells were FACS sorted directly into Trizol (Ambion). Total RNA was purified according to the manufacturer’s instructions and was used for reverse transcription using GoScript Reverse Transcriptase (Promega). Gene expression was assessed by quantitative real-time PCR using GoTaq qPCR Master Mix (Promega) on a Bio-Rad CFX Connect Real-Time PCR machine (Bio-Rad). β-Actin was used to normalize the expression of genes across samples and relative gene expression was quantified using the 2−ΔΔCTmethod. Primers used in this study were: p-actnr. 5’-GCTCTTTTCCAGCCTTCCTT-3’ and 5’-CTTCTGCATCCTGTCAGCAA-3’; Pex5-. 5 -ATCGACTGGAGGAGGGAGAC-3’ and 5’-TAGTTCCAGACACCTCCGCA-3’; Acox J, 5’-TTCAAGACAGAGCCGTGCAA-3’ and 5’-GCATCCACCAAAGCCACAG-3’.

[0117] Quantification and statistical analysis. Statistical analyses were performed using GraphPad Prism 9 or Excel, unless otherwise stated. For Kaplan-Meyer survival analysis, p value was calculated by log rank. For all other comparisons, p value was calculated by two-tailed unpaired t test or ratio-paired t test as stated in the figure legends. In all figures, dots on bar graphs represent biological replicates and error bars represent standard error of the mean.DISCUSSION

[0118] Hematopoietic stem and progenitor cells preferentially express peroxisome markers

[0119] The present disclosure assessed the expression pattern of peroxisome markers in the hematopoietic system. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) revealed that HSCs and multipotent progenitors (MPPs) express significantly higher levels of peroxisomal markers, Acoxl and Pex5, than whole bone marrow cells or more restricted Lin progenitors (Fig. la). Flow cytometry data showed that HSCs, MPPs and hematopoietic progenitors (see Fig. 6 for flow cytometry gating) express significantly higher levels of224938-3523-0789v.lperoxisomal proteins, such as CAT and PMP70, than whole bone marrow cells (Fig. lb and c). These data raised that possibility that peroxisomes may regulate HSC function.

[0120] Peroxisomes are not required for steady-state hematopoiesis and HSC maintenance

[0121] To study the role of peroxisomes in HSCs, a floxed allele of Pex5 was obtained, an essential biogenesis gene for peroxisomes (Platta et al. 2024, Skowyra et al, 2024, Baes et al. 2002, and Santos 1998). Deletion of Pex5 abolishes peroxisome biogenesis in cells, allowing the assessment of the function of peroxisomes in vivo (waterham 2012, Nagotu, 2012 and Kocherlakota, 2023). Mx1-cre; Pex5fl / flmice were generated. Treatment of adult Mx1-cre; Pex5fl / flmice with polyinosinic-polycytidylic acid (pIpC) five times efficiently deleted Pex5 from HSCs (Fig- 7).

[0122] Mx1-cre; Pex5fl / flmice were analyzed and littermate controls at least three weeks after the last pIpC treatment. Deletion of Pex5 from HSCs and the hematopoietic system did not lead to significant alterations in the white blood cell, red blood cell, and platelet counts in Mx1-cre; Pex5fl / flmice compared with controls (Fig. 2a). In the bone marrow, Mx1-cre; Pex5fl / flmice had normal bone marrow cellularity, HSC frequency, and MPP frequency (Fig. 2b-d). More restricted hematopoietic progenitors were examined and mature cells. Mx1-cre; Pex5fl / flmice had compatible frequencies of lymphoid-primed multipotent progenitors (LMPPs), common myeloid progenitors (CMPs), megakaryocytic / erythroid progenitors (MEPs), granulocyte / macrophage progenitors (GMPs), common myeloid progenitors (CLPs), Grl+Macl+myeloid cells, Teri 19+erythroid cells, B220+B cells, CD3+T cells, and CD41+megakaryocytic cells compared with controls (Fig. 2e-i). Although their size is mildly reduced, the spleens from Mx1-cre; Pex5fl / flmice had normal cellularity, HSC frequency, MPP frequency, and LSK frequency (Fig. 8a-c, Fig. 2j-k). HSCs from Mx1-cre; Pex5fl / flmice maintained their quiescence, cell death status, mitochondrial transmembrane potential revealed by tetramethylrhodamine ethyl ester (TMRE) staining, and normal reactive oxygen species (ROS) levels revealed by 2’,7’-dichlorofluorescin diacetate (DCFDA) staining (Fig. 2m-p). Consistent with flow cytometric analyses, single-cell RNA-seq (scRNA-seq) revealed normal cellular composition of HSPC compartment (Fig. 2q-r) and normal gene expression in HSCs from Mx1-cre; Pex5fl / flmice (Fig. 8d-f). Overall, these data suggest that peroxisomes are not required for steady-state hematopoiesis and HSC maintenance.

[0123] Pex5-deficient regenerating HSCs exhibit robust and extensive self-renewal activity234938-3523-0789v.l

[0124] Competitive reconstitution assay was used to test the function of HSCs. 500,000 bone marrow cells from Mx1-cre; Pex5fl / flor control mice along with 500,000 competitor bone marrow cells were transplanted into lethally irradiated recipient mice (Fig. 3a). / Ax5-deficient bone marrow cells gave rise to normal levels of overall, myeloid, B, and T lineage cells in the peripheral blood compared with controls (Fig. 3b). At 16 weeks after the bone marrow transplantation, the bone marrow in the recipient mice were analyzed. Consistent with the data from the peripheral blood, / Ax5-deficient bone marrow also gave rise to normal levels of whole bone marrow cells, LKs, LSKs, MPPs, and HSCs (Fig. 3c).

[0125] To test the self-renewal potential of HSCs, secondary bone marrow transplantation was performed after the primary transplantation assay. Interestingly, / Ax5-deficient bone marrow cells gave rise to significantly higher multilineage reconstitution in the peripheral blood compared with controls (-80% vs -30%) (Fig. 3d). / Ax5-deficient bone marrow cells also gave rise to significantly higher contribution to hematopoietic progenitors and HSCs in the bone marrow of recipient mice (Fig. 3e), suggesting that deletion of Pex5 promotes self-renewal of HSCs.

[0126] Tertiary transplantation was performed at the end of secondary transplantation. Strikingly, while control cells exhausted and did not give discernable reconstitution, Pex5-deficient bone marrow cells continued to give robust multilineage reconstitution (Fig. 3f). Nearly all donor-derived peripheral blood and bone marrow cells were from PexJ-deficient bone marrow cells (Fig. 3g). To further test the extent to which ex5-deficient HSCs self-renew, quaternary transplantation was performed. Consistent with the tertiary transplantation data, ex5-deficient bone marrow cells still gave robust reconstitution across multiple lineages, including myeloid, B, and T cells, suggesting that loss of Pex5 confers extensive self-renewal to HSCs (Fig. 3h-i). Importantly, no signs of leukemic transformation were observed in the recipient mice transplanted with TAxd-deficient bone marrow cells, including normal health status (46 out of 46 survived for Pex5 mutants vs 32 out of 37 survived for controls across four rounds of transplantation) and the absence of leukemic blasts in the bone marrow (Fig. 9g-k), ruling out a leukemic transformation after Pex5 deletion.[ 0127 / Mxl-cre; Pex5fl / flmice have robust hematopoietic regeneration after 5FU challenge

[0128] The manifestation of HSC phenotypes during regeneration but not at steady state raised the possibility that regenerating HSCs use more peroxisomes. Indeed, flow cytometry analysis244938-3523-0789v.lrevealed increased PMP70 and CAT expression in regenerating HSCs induced by 5FU treatment compared with steady-state HSCs (Fig. 4a-b). To test the role of peroxisomes during hematopoietic regeneration after chemotherapy stress, Mx1-cre; Pex5fl / fland control mice was treated with multiple doses of a myeloablative agent, 5 -fluorouracil (5FU) (Fig. 4c). Mx1-cre; Pex5fl / flsurvived significantly better compared with control mice (Fig. 4d). Consistent with a better survival, Mx1-cre; Pex5fl / flmice had a faster recovery of white and red blood cell counts after 5FU treatment compared with controls (Fig. 4e). In the bone marrow, Mx1-cre; Pex5fl / flmice had higher cellularity, HSC frequency, MPP frequency and frequencies of other restricted progenitors, including LMPPs, MEPs, GMPs, CMPs, and Grl+Macl+myeloid cells compared with controls (Fig. 4f-n). Thus, augmented HSC function after Pex5 deletion confers better survival and faster hematopoietic regeneration after 5FU-mediated stress.

[0129] Pex5-deflcient HSCs have reduced ROS and are more quiescent during regeneration after 5FU treatment

[0130] The impact of Pex5 deletion on regenerating HSCs was investigated. No significant differences in Annexin V staining were observed in PexJ-deficient regenerating HSCs compared with controls (Fig. 4m), suggesting that loss of Pex5 does not promote HSC function by suppressing cell death. Lower mitochondria activity, lower levels of reactive oxygen species, and quiescence in cell cycle are associated with HSCs with higher stem cell function (Filippi et al.2019, and Nakamura-Ishizu et al. 2020). Consistently, during regeneration after 5FU treatment, Pex5-deficient HSCs displayed features of enhanced function, including more quiescent (Fig. 4n), lower TMRE staining (Fig. 4o), and lower total ROS levels (Fig. 4p) compared with controls. Mitochondria are a major ROS generator in the cell (Murphy, 2009). However, assessing mitochondria ROS with MitoSOX staining did not reveal any differences between Pex5-defi cient HSCs and controls (Fig. 4q), suggesting the total ROS reduction in CexJ-deficient HSCs is caused by other cellular mechanisms, likely peroxisomes. Therefore, impeding the biogenesis of peroxisomes enhances HSC function during regeneration. These data are consistent with the robust reconstitution activity by / Ax5-deficient HSCs in serial transplantation assays.

[0131] Vavl-cre; P ex mice also have enhanced hematopoietic regeneration after 5FU challenge254938-3523-0789v.lTreatment with high amounts of pTpC impacts HSCs, although the effects are largely resolved within one to two weeks (Sugiyama et al. 2006). Nonetheless, to rule out the potential effects of pIpC on the role of Pex5 in HSCs, Vav1-cre; Pex5fl / flmice was generated. Young adult Vav1-cre; Pex5fl / flmice had normal blood cell counts compared with controls (Fig. 10a). Within the bone marrow, these mice had normal cellularity, HSC frequency, and frequencies of other restricted hematopoietic progenitors and maturing hematopoietic cells (Fig. lOb-i). Hematopoiesis in the spleens was normal as well (Fig. lOj-o). Thus, consistent with Mx1-cre; Pex5fl / flmodel, deletion of Pex5 does not impact normal hematopoiesis in Vav1-cre; Pex5fl / flmice.

[0132] Vav1-cre; Pex5fl / flmice was then subjected to hematopoietic regeneration condition induced by 5FU treatment. Vav1-cre; Pex5fl / flmice had a significant higher HSC frequency and numbers compared with controls after 5FU treatment (Fig. lla-c). Restricted progenitors and maturing hematopoietic cells were also significantly higher in Vav1-cre; Pex5fl / flmice compared with controls (Fig. 11d-i). Spleen hematopoiesis was largely normal in Vav1-cre; Pex5fl / flmice compared with controls after 5FU treatment (Fig. 11j-o). Thus, deletion of Pex5 leads to accelerated HSC regeneration after 5FU challenge.

[0133] P ex 5 -deficient HSCs also regenerate faster after irradiation challenge

[0134] Beside 5FU treatment, HSCs also regenerate after irradiation challenge. HSC and hematopoietic regeneration capacity were assessed after irradiation using Mx1-cre; Pex5fl / flmice. By 5-7 weeks after the irradiation, peripheral blood counts and bone marrow cellularity in Mx1-cre; Pex5fl / flmice recovered similarly to controls (Fig. 12a-b). However, HSC frequency and number in Mx1-cre; Pex5fl / flmice regenerated significantly faster compared with controls (Fig.12c-d). Most hematopoietic progenitors recovered similarly between Mx1-cre; Pex5fl / fland controls mice, except MEPs and CMPs which showed higher frequencies in Mx1-cre; Pex5fl / flmice (Fig. 12e-j). By 10-15 weeks after irradiation, hematopoiesis largely recovered with no significant differences between Mx1-cre; Pex5fl / fland controls mice (Fig. 12k-s). Similar to the 5FU challenge, HSCs from Mx1-cre; Pex5fl / flmice at 5-7 weeks after irradiation showed normal Annexin V staining, but high quiescence, lower TMRE levels, and lower ROS levels compared with controls (Fig. 12t-w). Overall, these data suggest that enhanced regeneration of 7Yx5-deficient HSCs is a common response to stress induced by either 5FU treatment or irradiation.

[0135] Pex5-deficient bone marrow cells have enhanced serial plating capacity264938-3523-0789v.l

[0136] 10,000 bone marrow cells were plated from Mx1-cre; Pex5fl / fland control mice into methylcellulose (Fig. 5a). Bone marrow cells from Mx1-cre; Pex5fl / flmice formed modestly more colonies in methylcellulose compared with controls (Fig. 5b). However, the difference did not reach statistical significance. Secondary plating using cells from the first plating was then performed. Strikingly, colonies formed by bone marrow cells from Mx1-cre; Pex5fl / flmice increased more than 25 fold compared with controls (Fig. 5b). Bone marrow cells from Mx1-cre; Pex5fl / flmice formed even more colonies in tertiary plating compared with controls (—116 fold) (Fig. 5b). The colonies formed by bone marrow cells from Mx1-cre; Pex5fl / flmice were larger and contained more cells compared with controls (Fig. 5c). Consistent with the capacity to form more colonies, flow cytometry analysis revealed more (-104 fold) LSK hematopoietic progenitors in the colonies formed in secondary plating by bone marrow cells from Mx1-cre; Pex5fl / flmice compared with controls (Fig. 5d-e). Thus, consistent with the augmented HSC and hematopoietic function in vivo (Figs. 3 and 4), disruption of peroxisomal biogenesis leads to enhanced serial plating and colony formation capacity of bone marrow cells.

[0137] Inhibiting peroxisomal b-oxidation leads to progenitor expansion and enhanced serial plating

[0138] To investigate the mechanisms mediating the effects on Pex5 deletion, several peroxisomal pathways were perturbed that may be the consequence of defects in peroxisomal biogenesis (Fig. 5f). Very long chain fatty acids (VLCFAs) can only be broken down by peroxisomes through b-oxidation(Cipolla et al. 2017). Cellular VLCFAs are primarily generated by elongation of very long chain fatty acids-like 1 (ELOVLl)(Ofman et al. 2010). The input of VLCFAs to the peroxisomes was impeded by lowering the cellular levels of VLCFAs using an ELOVL1 inhibitor (ELOVL1-IN-1) ex vivo (Boyd et al. 2021 and Come et al. 2021). The number of colonies formed in methylcellulose during primary plating was not significantly changed by ELOVL-IN-1 treatment (Fig. 5g). However, bone marrow cells treated with ELOVL-IN-1 formed significantly more colonies (-7 fold) at the secondary plating compared with controls (Fig. 5h). By the tertiary plating, bone marrow cells treated with ELOVL-IN-1 formed a large number of colonies (-26 fold) in methylcellulose compared with controls (Fig. 5h). Given the similarity of colony formation phenotypes between Pex5 deletion and ELOVL-IN-1 treatment, these data suggest that VLCFA oxidation may mediate the effects of Pex5 deletion.274938-3523-0789v.l

[0139] Whether inhibition of peroxisomal b-oxidation could also lead to enhanced serial plating capacity and progenitor expansion was further tested. Acyl-coenzyme A oxidase 1 (ACOX1) is the first and rate-limiting enzyme of peroxisomal b-oxidation (Van Veldhoven 2010). Its activity was inhibited by treating bone marrow cells from wild-type mice with an ACOX1 inhibitor, 10,12-Tricosadiynoic acid, or vehicle control, in methylcellulose culture. Similar to ELOVL-IN-1, 10,12-Tricosadiynoic acid treatment drastically increased serial plating efficiency of bone marrow cells (~8 fold in secondary plating and -284 fold in tertiary plating compared with controls, respectively) (Fig. 5i-j). These data suggest that inhibiting peroxisomal b-oxidation augments hematopoietic progenitor expansion and serial plating efficacy of bone marrow cells.

[0140] Similar to Pex5 deletion (Fig. 5c), colonies formed with bone marrow cells with the presence of ELOVL-IN or 10,12 Tricosadiynoic acid were larger and contained more cells (Fig.5k). Flow cytometry analysis were performed on these cells (Fig. 5l). There was a striking expansion of LSK hematopoietic progenitors in secondary plating with bone marrow cells treated with ELOVL-IN-1 (-16 fold), or 10,12 Tricosadiynoic acid (-37 fold) compared with controls (Fig. 5m). Thus, reducing cellular VLCFA levels in bone marrow cells with ELOVL-IN-1 leads to expansion of hematopoietic progenitors.

[0141] Acyl-CoA generated through b-oxidation can be used for ether lipid synthesis in the peroxisome (Cipolla et al. 2017). Therefore, whether inhibition of ether lipid synthesis also leads to enhanced serial plating was being tested. Alkylglycerone phosphate synthase (AGPS) is a peroxisomal enzyme critical for the production of ether lipids, such as plasmalogen. Bone marrow cells from wild-type mice in methylcellulose with AGPS-IN-1 was incubated, an effective inhibitor for AGPS (Stazi et al. 2019). These data suggest that the effect of b-oxidation on hematopoietic progenitor expansion does not rely on either lipid synthesis.

[0142] A product of peroxisomal oxidation is the generation of ROS, which is scavenged by catalase (CAT) in peroxisomes. Bone marrow cells were treated with a CAT inhibitor, 3-amino-1,2,4-triazole (3 AT), and performed colony formation assays in methylcellulose. Inhibition of CAT with 3 AT significantly reduced the serial plating capacity of bone marrow cells (Fig. 5p-q), suggesting that CAT is required for robust colony formation by bone marrow cells.

[0143] Peroxisomes are present in all eukaryotic cells. Although they presumably play important metabolic functions, peroxisomes are considered as a secondary metabolic organelle284938-3523-0789v.lthat complements mitochondrial function. Consistently, patients with peroxisomal biogenesis defects display phenotypes in specific tissues. The present disclosure suggests that peroxisomes are dispensable for steady state and regenerative hematopoiesis. Given that VLCFAs can only be metabolized in the peroxisome through b-oxidation, it is possible that peroxisomes in HSCs may be remnant of a mechanism for using VLCFAs as fuel from the past when food supply is scarce. Detailed study of the function of peroxisomes in other cell types is needed in the future.

[0144] The lipid metabolic function of peroxisome and mitochondria is closely linked. Shortened fatty acids in the peroxisomes can be further oxidized in the mitochondria(Cipolla et al.2017, Kumar et al. 2024 and Wanders, 2023). The role of mitochondria as an organelle for fatty acid oxidation has been investigated. Lipid metabolism, particularly fatty acid oxidation, is required for HSC self-renewal. Pharmacological inhibition of mitochondrial fatty acid oxidation with Etomoxir, an inhibitor of carnitine palmitoyltransferase la (CPTla), exhausts HSCs (Ito et al. 2012). Similarly, deletion of Cpt2 leads to HSC depletion (Bonora et al. 2024). Normal HSC function when Cpt2 was deleted was also reported (Pizzato et al. 2023). A recent study suggests that Cptla or hydroxyacyl-CoA dehydrogenase (HADHA) is not required for HSC self-renewal under normal conditions in young adult mice (Merchant et al. 2024), while another reported that deletion of Cptla leads to HSC depletion (Li et al. 2025).. Nonetheless, given the distinct impact by peroxisomal vs mitochondrial fatty acid oxidation on HSCs, peroxisomal lipid b-oxidation likely plays a unique role in HSCs.

[0145] Extensive self-renewal potential preserves stem cells and sustains tissue regeneration. However, the molecular mechanisms promoting stem cell self-renewal during regeneration remain unclear. The present disclosure investigates the role of peroxisomes, a key metabolic organelle, in regulating hematopoietic stem cell (HSC) self-renewal. It was found that disrupting peroxisome biogenesis by deleting Pex5 does not significantly impact HSC number or hematopoiesis at steady state. Strikingly, ex5-deficient HSCs self-renew extensively and give rise to robust multilineage reconstitution upon multiple rounds of serial transplantation into recipients. / AxJ-deficient HSCs are more resistant to stress and have enhanced capacity to regenerate the hematopoietic system following 5FU treatment or irradiation. Deletion of Pex5 promotes expansion of HSCs and hematopoietic progenitors with extensive serial ex vivo plating capacity. Mechanistically, the present disclosure discovers that the effect is largely through perturbation of peroxisomal b-oxidation. Pharmacological inhibition of peroxisomal b-oxidation also promotes extensive self- 294938-3523-0789v.lrenewal in HSCs and hematopoietic progenitors. Intervening in peroxisomal b-oxidation can be exploited to promote HSC self-renewal for regenerative medicine.304938-3523-0789v.lREFERENCES1 Dykstra, B. et al. Long-term propagation of distinct hematopoietic differentiation programs in vivo. Cell stem cell 1, 218-229 (2007).https: / / doi.org:10.1016 / j.stem.2007.05.015Purton, L. E. et al. RARgamma is critical for maintaining a balance between hematopoietic stem cell self-renewal and differentiation. The Journal of experimental medicine 203, 1283-1293 (2006). https: / / doi.org:10.1084 / jem.20052105Ema, H. et al. Quantification of self-renewal capacity in single hematopoietic stem cells from normal and Lnk-deficient mice. 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Proliferation inhibition of murine pluripotent haemopoietic stem cells by interferon or poly I: C. Cell Tissue Kinet 14, 1-7 (1981). https: / / doi.org:10.1111 / j.1365-2184.1981.tb00505.x18 Velasco-Hernandez, T., Sawen, P., Bryder, D. & Cammenga, J. Potential Pitfalls of the Mxl-Cre System: Implications for Experimental Modeling of Normal and Malignant Hematopoiesis. Stem Cell Reports 7, 11-18 (2016).https: / / doi.org:10.1016 / j.stemcr.2016.06.00219 Ofman, R. etal. The role of ELOVL1 in very long-chain fatty acid homeostasis and X- linked adrenoleukodystrophy. EMBO Mol Med 2, 90-97 (2010).https: / / doi.org:10.1002 / emmm.20100006120 Boyd, M. J. et al. Discovery of Novel, Orally Bioavailable Pyrimidine Ether-Based Inhibitors of ELOVL1. JMedChem 64, 17777-17794 (2021).https: / / doi.org:10.1021 / acs.jmedchem.1c0094821 Come, J. H. etal. Discovery and Optimization of Pyrazole Amides as Inhibitors of ELOVL1. 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Claims

CLAIMS1. A method of enhancing stem cell or progenitor cell self-renewal, comprising administering to the stem cell or progenitor cell an inhibitor of peroxisomal beta-oxidation.

2. An ex vivo therapy method of enhancing self-renewal of stem cells or progenitor cells, comprising administering to a sample of stem cells and / or progenitor cell an inhibitor of peroxisomal beta-oxidation.

3. The method of claim 1 or 2, wherein the inhibitor comprises an acyl-coenzyme A oxidase 1 inhibitor.

4. The method of claim 3, wherein the inhibitor comprises tricosadiynoic acid.

5. The method of claim 1 or 2, wherein the inhibitor comprises an inhibitor of ELOVL Fatty AcidElongase 1 (ELOVL 1).

6. The method of claim 5, wherein the inhibitor is a pyrazole amide or is a pyrimidine ether- based inhibitor.

7. The method of claim 1 or 2, wherein the inhibitor inhibits Pex5.

8. The method of any one of claims 1-7, wherein the stem cell or progenitor cell is a hematopoietic stem cell or hematopoietic progenitor cell.

9. The method of any one of claims 1-8, further comprising subsequently administering the cells to a subject in need thereof or having a bone marrow transplant.

10. A composition comprising a population of stem cells or progenitor cells and an amount of a synthetic inhibitor of peroxisomal beta-oxidation effective to enhance stem cell or progenitor cell self-renewal.344938-3523-0789v.l11. The composition of claim 10, wherein the inhibitor comprises an acyl-coenzyme A oxidase 1 inhibitor.

12. The composition of claim 11, wherein the inhibitor comprises tricosadiynoic acid.

13. The composition of claim 10, wherein the inhibitor comprises an inhibitor of ELOVL Fatty AcidElongase 1 (ELOVL 1).

14. The composition of claim 13, wherein the inhibitor is a pyrazole amide or is a pyrimidine ether-based inhibitor.

15. The composition of claim 10, wherein the inhibitor inhibits Pex5.

16. The composition of claim 10-15, wherein the stem cell or progenitor cell is a hematopoietic stem cell or hematopoietic progenitor cell.

17. The composition of any one of claims 10-16, wherein the cells are for administration to or transplant into a subject in need thereof.

18. The composition of any of claims 10-17, wherein the cells of the composition have enhanced self-renewal relative to an otherwise identical composition which does not contain any inhibitor of peroxisomal beta-oxidation.

19. The method of claim 9, wherein the subject has a blood disorder or blood cancer, optionally, a leukemia, lymphoma, aplastic anemia, or immune deficiency disorder.

20. The method of claim 9 or 19, wherein the stem cells or progenitor cells are part of a bone marrow sample.

21. The method of claim 20, wherein the bone marrow sample has been previously removed from a donor subj ect.354938-3523-0789v.l22. The method of claim 21, wherein the bone marrow sample is treated with the inhibitor of peroxisomal beta-oxidation prior to transplant or administration to a recipient subject.

23. The method of any of one claims 1-9 or 19-22, wherein the inhibitor does not inhibit mitochondrial fatty acid oxidation.

24. A bone marrow sample comprising a population of stem cells or progenitor cells and an amount of a synthetic inhibitor of peroxisomal beta-oxidation in an amount sufficient to enhancing stem cell or progenitor cell self-renewal of stem cells or progenitor cells of the bone marrow.

25. A method of performing a bone marrow transplant comprising transplanting into a subject an amount of bone marrow that has been treated with an inhibitor of peroxisomal betaoxidation in an amount sufficient to enhancing stem cell or progenitor cell self-renewal of stem cells or progenitor cells of the bone marrow optionally, the subject has a blood disorder or blood cancer, optionally, a leukemia, lymphoma, aplastic anemia, or immune deficiency disorder.

26. A method of enhancing stem cell or progenitor cell self-renewal in a subject, comprising administering to the subject an inhibitor of peroxisomal beta-oxidation.

27. The method of claim 26, wherein the inhibitor comprises an acyl-coenzyme A oxidase 1 inhibitor.

28. The method of claim 27, wherein the inhibitor comprises tricosadiynoic acid.

29. The method of claim 26, wherein the inhibitor comprises an inhibitor of ELOVL Fatty AcidElongase 1 (EL0VL1).

30. The method of claim 29, wherein the inhibitor is a pyrazole amide or is a pyrimidine ether- based inhibitor.364938-3523-0789v.l31. The method of claim 26, wherein the inhibitor inhibits Pex5.

32. The method of any one of claims 26-31, wherein the inhibitor does not inhibit mitochondrial fatty acid oxidation.

33. A method of enhancing hematopoietic stem and progenitor cells (HSPCs) self-renewal, comprising reducing the amount or the expression of Peroxisomal Biogenesis Factor 5 (PEX5) in the HSPCs.

34. A method of increasing the survivability of HSPCs and / or increasing the regenerability of HSPCs after treating the HSPCs with one or more myeloablative agents and / or irradiation, wherein the method comprises reducing the amount or the expression of PEX5 in the HSPCs.

35. A method of decreasing ferroptosis in HSPCs comprising reducing the amount or the expression of PEX5 in the HSPCs.

36. The method of claim 34, wherein the myeloablative agent is 5 -fluorouracil (5-FU), busulfan, or cyclophosphamide.

37. The method of any one of claims 33-36, wherein(a) the HSPCs are hematopoietic stem cells (HSCs); lymphoid-primed multipotent progenitors (LMPPs), common myeloid progenitors (CMPs), megakaryocytic / erythroid progenitors (MEPs), granulocyte / macrophage progenitors (GMPs), common myeloid progenitors (CLPs), Grl+MacE myeloid cells, Terll9+erythroid cells, B220+B cells, CD3+T cells, or CD41+megakaryocytic cells; preferably, the HSPCs are HSCs; and / or(b) the HSPCs are part of a bone marrow sample, preferably, the bone marrow sample has been previously removed from a donor subject; more preferably, the bone marrow sample is treated with the inhibitor of peroxisomal beta-oxidation prior to374938-3523-0789v.ltransplant or administration to a recipient subject, more preferably, the inhibitor does not inhibit mitochondrial fatty acid oxidation.

38. A method of increasing the plating capacity of bone marrow cells comprising reducing the amount or the expression of PEX5 in the bone marrow cells.

39. A method of increasing bone marrow cells’ contribution to HPSCs comprising reducing the amount or the expression of PEX5 in the bone marrow cells.

40. A method of enhancing multilineage reconstitution of bone marrow cells comprising reducing the amount or the expression of PEX5 in the bone marrow cells.

41. The method of any one of claims 33-40, wherein the reduction of the amount or the expression of PEX5 is achieved by contacting the HSPCs or bone marrow cells with at least one inhibitor.

42. The method of claim 41, wherein the inhibitor is(a) an alkylglycerone phosphate synthase (AGPS) inhibitor; preferably, the inhibitor comprises tricosadiynoic acid;(b) an inhibitor comprises an inhibitor of ELOVL Fatty Acid Elongase 1 (ELOVL1);preferably, the inhibitor is a pyrazole amide or is a pyrimidine ether-based inhibitor; (c) an inhibitor of peroxisomal beta-oxidation; or(d) a Pex5 inhibitor.

43. The method of any one of claims 33-42 further comprising subsequently administering the HSPCs or bone marrow cells to a subject in need thereof or having a bone marrow transplant, optionally, the subject has a blood disorder or blood cancer, more optionally, the subject has leukemia, lymphoma, aplastic anemia, or immune deficiency disorder.384938-3523-0789v.l44. A method of promoting HSPC regeneration capacity in a subject, wherein the method comprises administering to the subject one or more peroxisomal beta-oxidation inhibitors.

45. A method of enhancing bone marrow transplantation efficiency in a subject, wherein the method comprises administering to the subject one or more peroxisomal beta-oxidation inhibitors.

46. The method of any one of claims 44-45, wherein(a) the peroxisomal beta-oxidation inhibitor is an alkylglycerone phosphate synthase (AGPS) inhibitor; preferably, the inhibitor comprises tricosadiynoic acid;(b) the peroxisomal beta-oxidation inhibitor is an inhibitor comprises an inhibitor of ELOVL Fatty Acid Elongase 1 (EL0VL1); preferably, the inhibitor is a pyrazole amide or is a pyrimidine ether-based inhibitor;(c) the subject is mammal; preferably the mammal is human; and / or(d) the administration is administered daily, bi-monthly or monthly.394938-3523-0789v.l

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