Compositions and methods for treating x-linked sideroblastic anemia

Abstract

Methods and compositions for producing heme and / or treating sideroblastic anemia are disclosed.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING X-LINKED SIDEROBLASTIC ANEMIA

[0002] By Stefano Rivella Carlo Castruccio Castracani

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 733,591, filed December 13, 2024. The foregoing application is incorporated by reference herein.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to the field of hematology. More specifically, the invention provides compositions and methods for the treatment of anemia, particularly sideroblastic anemia or X-linked sideroblastic anemia.

[0006] BACKGROUND OF THE INVENTION

[0007] Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

[0008] Sideroblastic anemia is a group of congenital and acquired disorders that presents the characteristic bone marrow (BM) sideroblasts, a sign of excessive mitochondrial deposition of iron (Fujiwara, et al. (2019) Free Radic. Biol. Med., 133: 179-185; Ducamp, et al. (2019) Blood 133(l):59-69). The congenital forms (CSA: Congenital Sideroblastic Anemia) are rare and caused by mutations in the genes involved in heme biosynthesis, Fe-S cluster, and mitochondrial protein synthesis (Ducamp, et al. (2019) Blood 133(1): 59-69; Bottomley, et al. (2014) Hematol. Oncol. Clin. North. Am., 28(4):653-670). X-linked sideroblastic anemia (XLSA) is the most common form of CSA caused by germline mutations in the erythroid-specific 5-aminolevulinate synthase (4 / a.s2) gene, encoding for the enzyme ALAS2 (Fujiwara, et al. (2019) Free Radic. Biol. Med., 133: 179-185; Bottomley, et al. (2014) Hematol. Oncol. Clin. North. Am., 28(4):653-670). ALAS2 plays a crucial role in heme biosynthesis, specifically in erythroid cells (Liu, et al. (2018) Nat. Commun., 9(1 ):4386). Being the first and rate-limiting enzyme in the heme biosynthetic pathway, ALAS2 catalyzes the condensation of glycine and succinyl- CoA to form 5-aminolevulinic acid (ALA), which is the precursor for heme synthesis (Zhang, et al. (2017) Nucleic Acids Res., 45(2): 657-671 ). ALAS2 is an erythroid- specific isoform of ALAS, and it is expressed at high levels in erythroid precursor cells (Hoving, et al. (2023) Br. J. Haematol., 202(6): 1216-1219). Various factors, including GATA-1, a transcription factor essential for erythroid differentiation, regulate the expression of ALAS2. The GATA-1 -dependent genetic network controls ALAS2 expression and heme biosynthesis during erythroid differentiation (Tanimura, et al. (2016) EMBO Rep., 17(2):249-265). ALAS2 expression is not inhibited by high heme levels and is positively regulated by hypoxia (Khechaduri, et al. (2013) J. Am. Coll. Cardiol., 61(18): 1884-1893). Typically, patients affected by XLSA are hemizygous males who exhibit the disease before the age of 40, with hypochromic microcytic anemia and systemic iron overload (Fujiwara, et al. (2019) Free Radic. Biol. Med., 133: 179-185; Ducamp, et al. (2019) Blood 133(l):59-69; Bottomley, et al. (2014) Hematol. Oncol. Clin. North. Am., 28(4):653-670; Ohba, et al. (2013) Ann. Hematol., 92(1): 1-9). Heterozygous female carriers might develop the disease because of a non -well -defined mechanism, possibly related to skewed X-chromosome inactivation (Cotter, et al. (1995) J. Clin. Invest., 96(4):2090-2096; Cazzola, et al. (2000) Blood 96(13):4363-4365; Aivado, et al. (2006) Blood Cells Mol. Dis., 37(l):40-45; Rose, et al. (2017) Br. J. Haematol., 178(4):648-651 ; Ducamp, et al. (2011) Hum. Mutat., 32(6):590-597; Katsurada, et al. (2016) Int. J. Hematol., 103(6):713-717; Morimoto, et al. (2022) Blood Adv., 6(4): 1100-1114). Most mutations result in partial loss of function (Ducamp, et al. (2019) Blood 133(1): 59-69; Yoshida, et al. (2011) Nature 478(7367):64-69; Bekri, et al. (2003) Blood 102(2):698- 704; Kaneko, et al. (2014) Haematologica 99(2):252-261). Vitamin B6 is the first- line treatment for XLSA, but almost 50% of the cases are unresponsive (Bottomley, et al. (2014) Hematol. Oncol. Clin. North. Am., 28(4):653-670; Astner, et al. (2005) EMBO J., 24(18):3166-3177; May, et al. (1998) Haematologica 83(l):56-70). Oral ALA supplementation has failed as a second-line treatment in vitamin B6-refractory XLSA (Ishida, et al. (2018) Pediatr. Int., 60(5):496-497).

[0009] Gene therapy strategies for hemoglobinopathies, such as P-thalassemia and sickle cell disease, aim to introduce, by lentiviral vectors (LV), functional copies of the globin genes into patient hematopoietic stem cells (HSCs) to restore normal Hb production (Cavazzana-Calvo, et al. (2010) Nature 467(7313):318-322). One established approach involves employing erythroid-specific vectors, tailoring the expression of erythroid genes in RBC precursors. LVs have shown significant promise in the context of gene therapy due to their ability to efficiently transduce dividing and non-dividing cells, such as HSCs (Negre, et al. (2016) Hum. Gene Ther., 27(2): 148-165). LVs can accommodate larger gene inserts (such as the ALAS2 gene and erythroid regulatory sequences), essential for heme synthesis and typically active in late-stage erythropoiesis (Negre, et al. (2016) Hum. Gene Ther., 27(2): 148-165; Miccio, et al. (2008) Proc. Natl. Acad. Sci., 105(30): 10547-10552). Furthermore, more recent LVs have been extensively researched and modified to enhance safety and specificity (Negre, et al. (2015) Curr. Gene Ther., 15(l):64-81 ; Boulad, et al. (2014) Blood 123(10): 1483-1486).

[0010] Currently available therapies for sideroblastic anemia are limited and are largely drawn to only treating symptoms. Thus, there is an ongoing and unmet need for improved compositions and methods for treating anemias such as sideroblastic anemia.

[0011] SUMMARY OF THE INVENTION

[0012] In accordance with one aspect of the instant invention, nucleic acids and vectors, particularly viral vectors such as lentiviral vectors, are provided. In a particular embodiment, the nucleic acid or vector comprises a nucleic acid molecule comprising any one or more of: i) a 5’ long terminal repeat (LTR) and a 3’ LTR (e.g., at least one of the LTR may be self-inactivating); ii) at least one erythroid promoter; iii) a locus control region (e.g., a globin gene locus control region (LCR) (e.g., comprising HS2 and HS3)); iv) an insulator (e.g., an ankyrin insulator element (Ank)); v) an enhancer (e.g., beta globin 3’ enhancer; operably linked to the nucleic acid encoding the therapeutic protein); vi) a sequence encoding ALAS2; and vii) a Woodchuck Post-Regulatory Element (WPRE). The instant invention also encompasses cells (e.g., hematopoietic stem cells, hematopoietic progenitor cells, erythroid progenitor cells, or erythroid cells) and viral particles comprising the nucleic acid or vector (e.g., lentiviral vector) of the instant invention. Compositions comprising the nucleic acid or vector (e.g., lentiviral vector) or viral particles are also encompassed by the instant invention. The compositions may further comprise a pharmaceutically acceptable carrier.

[0013] In accordance with another aspect of the instant invention, methods of inhibiting, treating, and / or preventing anemias such as sideroblastic anemia (e.g., CSA or XLSA) in a subject are provided. In a particular embodiment, the method comprises administering a nucleic acid or viral vector or viral particle of the instant invention to a subject in need thereof. In a particular embodiment, the method comprises an ex vivo therapy utilizing a nucleic acid, viral vector, or viral particle of the instant invention. The nucleic acid, viral vector, or viral particle may be in a composition with a pharmaceutically acceptable carrier.

[0014] BRIEF DESCRIPTIONS OF THE DRAWINGS

[0015] Figure 1 A provides values of red blood cells (RBC), hemoglobin (Hb), and hematocrit (HCT), and reticulocytes (RET) in R26CreERT2and R26CreERT2-Alas2fl / Yanimals after administration of TAM. N = 4 for each group. Data are shown as mean ± standard error of the mean (SEM). Figure IB provides representative flow cytometry analysis of the erythroid populations in the BM and spleen in R26CreERT2and R26CreERT2-Alas2fl / Yanimals treated with TAM. Data are shown as FSC- A / CD44 subgated on Teri 19+ population. P1+P2 / P2 = Proerythroblasts / basophilic erythroblasts; P3 = Polychromatic erythroblasts; P4: Orthochromatic erythroblasts / reticulocytes; P5: Erythrocytes. Figure 1C provides the quantification of Alas2 allelic deletion (droplet digital polymerase chain reaction (ddPCR) analysis) of gDNA isolated from Lin' cells from Alas2a / YBM, after exposure to LNPCD117Cre. Data are shown as Mean ± SEM (N = 3 independent replicates). P-values are Tukey’s multiple comparison test after one-way ANOVA. **** p<0.0001. Figure ID provides amino acid sequences for functional (bottom; SEQ ID NO: 17) and nonfunctional (top; SEQ ID NO: 18) mouse ALAS2 protein, following Cre recombinase, created with SnapGene® software (from Dotmatics; available at snapgene.com). The sequence highlights the functional sequence, missing in the non-functional protein due to a stop codon at AA26. The stop codon is indicated by *.

[0016] Figure 2A shows the quantification of Alas2 allelic deletion (ddPCR analysis) of gDNA isolated from Lin cells from Alas2fl / YBM, after tamoxifen treatment (with or without BMT) or exposure to LNPCD117Cre. Data are shown as mean ± SEM (N = 3 independent replicates). P values are determined by the Tukey multiple-comparison test after the 1-way ANOVA. * P < .05, ** P < .001, **** p < .0001. Figure 2B provides X-ALAS2-LV VCN analysis 25 weeks post-transplantation in the BM and spleen (SPL) samples from TAM and LNPCD117Cre models (N = 6 in all experimental groups). Data are shown as mean ± SEM. Figures 2C and 2D provide the complete blood cell (CBC) panel at the end point in each experimental group. Each model includes animals treated (+) and not treated (-) with X-ALAS2-LV (N = 8 in all experimental groups). Data are shown as mean ± standard error of the mean (SEM). P values are determined by the Tukey multiple-comparison test after the 1-way analysis of variance (ANOVA). ** P < .01, *** P < .001, **** p < .0001. Although there is not a significant difference in the Alas2-KOBManimals treated with tamoxifen or LNPCI)I l 7Crc, the LNP -treated animals did show a reduced absolute reticulocyte count (ARC) compared with the tamoxifen model. Compared with tamoxifen, the LNP technology is more efficient in deleting Alas2 and the cells engrafted already lack the Alas2 gene. In the tamoxifen approach, the process to delete the target gene is slower (as this requires repeated administration of tamoxifen), and more undeleted cells may be present, which still possess the ability to regenerate and mature into reticulocytes. However, these cells are insufficient to support normal erythropoiesis and rescue the animals. Figure 2E provides a Kaplan-Meir analysis of experimental cohorts, in TAM and the LNPCD117Cre models. Each model includes animals treated (LV) and not treated (No LV) with X-ALAS2-LV (N = 9 in all experimental groups). Curve comparison in the TAM model: ***p<0.001, %2= 17.50 in the Mantel-Cox test. Curve comparison in the LNPCD117Cre model: **p<0.01, %2= 14.18 in the Mantel- Cox test. Figure 2F provides a schematic diagram of X-ALAS2 lentiviral vector.

[0017] Figure 3 A provides graphs of absolute cell count of erythroid populations in BM and spleen of animals treated (LV) or not treated (no LV) with X-ALAS2-LV (N = 9 in all experimental groups), using the TAM or LNPCD117Cre treatment at endpoint. P1+P2 / P2 = Proerythroblasts / basophilic erythroblasts; P3 = Polychromatic erythroblasts; P4: Orthochromatic erythroblasts / reticulocytes; P5: Erythrocytes. Data are shown as mean ± SEM. P-values are Tukey ’s multiple comparison test after oneway ANOVA. *p<0.05, ****p<0.0001. Figure 3B provides representative blood smears of each experimental group, in animals treated (+X-ALAS2-LV) or not treated (No Vector) with X-ALAS2-LV, from TAM and LNPCD117Cre cohorts. Figure 3C provides quantification of the spleen / body weight ratio in animals treated (LV) or not treated (no LV) with X-ALAS2-LV (N = 9 in all experimental groups) from TAM (TAM) and LNPCD117Cre (LNP) models at the endpoint of each experimental group. Data are shown as Mean ± SEM. P-values are Tukey ’s multiple comparison test after one-way ANOVA. * p <0.01, ** p <0.001, *** p <0.0001. Representative pictures of spleens were collected at the endpoint of each experimental group. Figure 3D provides RQ-PCR expression analysis of ALAS2, ALAS1, and Tfirc in isolated erythroid populations in the bone marrow. Pl = Proerythroblasts; P2 = Basophilic erythroblasts; P3 = Polychromatic erythroblasts; P4 = Orthochromatic erythroblasts. N = 4 C57 / BL6. Data are shown as mean ± SEM. P-values are Tukey’s multiple comparison test after one-way ANOVA. *** p<0.001, **** p<0.0001.

[0018] Figure 4A provides representative images of hematoxylin and eosin (H&E) and Prussian blue staining in spleen tissues were collected at the end point of each experimental group. Magnification * 10. Figure 4B provides representative images of H&E and Prussian blue staining in the bone marrow and liver tissues were collected at the end point of each experimental group. Magnification *4.

[0019] Figure 5 provides quantification of iron accumulation using the Aperio Versa 200 slide scanner and a positive pixel count algorithm to quantify the level of iron accumulation in the different organs. Data are shown as mean ± SEM. P-values are Tukey’s multiple comparison test after one-way ANOVA. *p< 0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0020] Figs. 6A-6D provide graphs of the iron metabolism and metabolomic analysis of the Alas2-KOBMmodel in presence or absence of X-ALAS2-LV. Fig. 6A: ERFE, HAMP, EPO, and iron values in blood serum of Alas2-KOBManimals transduced or not with + / - X-ALAS2-LV at the end point of each experimental group (N = 4-9 in all experimental groups). Data are shown as mean ± standard error of the mean (SEM). P values are determined by the Tukey multiple-comparison test after the 1- way analysis of variance (ANOVA). * P < .05, ** P < .01, *** P < .001, **** p < .0001. WT is a C57BL / 6J used as a reference. Fig. 6B: Metabolomic analysis of polychromatic erythroid cells, sorted from WT and Alas2-KOBM animals (N = 6 for each group). Data are shown as mean ± SEM. P values are determined by the Tukey multiple-comparison test after the 1-way ANOVA. * P < .05, ** P < .01, *** P < .001, **** P < .0001. Fig. 6C: Glycolysis analysis of polychromatic erythroid cells, sorted from WT and Alas2-KOBManimals (N = 6 for each group). Data are shown as mean ± SEM. P values are determined by the Tukey multiple-comparison test after the 1-way ANOVA. * P < .05, ** P < .01, *** P < .001, **** P < .0001. Fig. 6D: Relative gene expression analysis of glycolytic pathway (quantitative reverse transcription polymerase chain reaction [qRT-PCR]) in polychromatic erythroid cells, sorted from WT and Alas2-KOBManimals (N = 6 for each group). P values are determined by the Tukey multiple-comparison test after the 1-way ANOVA. * P < .05, ** P < .01. Figures 7A and 7B show the apoptosis and inflammation gene expression analysis in the Alas2-KOBMmodel and effect of the treatment with X-ALAS2-LV. Fig. 7A: Relative gene expression analysis of apoptosis pathway (qRT-PCR) in polychromatic erythroid cells, sorted from WT and Alas2-K0BManimals (N = 6 for each group). P values are determined by the Tukey multiple-comparison test after the 1-way analysis of variance (ANOVA). * P < .05. Fig. 7B: Relative gene expression analysis of Tnf, mil -6, and Hmoxl (qRT-PCR) in polychromatic erythroid cells, sorted from WT and Alas2-KOBManimals (N = 6 for each group). P values are determined by the Tukey multiple-comparison test after the 1-way ANOVA. ** P < .01.

[0021] Figure 8 provides VCN analysis of X-ALAS2-LV in NIH-3T3 and HUDEP-2 cells. N = 3 for each LV concentration. The results are shown as median ± SD.

[0022] Figure 9A provides a chimerism analysis post BM transplantation (N = 9 for each experimental group). Data are shown as mean ± SEM. Figure 9B shows the correlation of VCN (X-ALAS2-LV) and Hb (g / dL) values in the TAM and LNPCD117Cre models in X-ALAS2 treated mice. P-values, R-squared, and curve equations are obtained by simple linear regression analysis.

[0023] Figure 10A provides Hb, RBC, HTC, and reticulocytes (RET) values measured 4 and 25 weeks after BM secondary transplantation (N = 2 for donors group; N = 3 in secondary transplanted mice Group 1 and Group 2). Data are shown as mean ± SEM. P-values are Tukey ’s multiple comparison test after one-way ANOVA *p<0.05. Figure 10B provides VCN quantification at 4 and 25 weeks posttransplantation in bone marrow samples from secondary chimeras (N = 3 in Group 1 and Group 2). Secondary transplant mice show the original donors’ VCN at 25 weeks post-transplantation. Data are shown as mean ± SEM. Figure 10C shows donor chimerism in secondary chimeras at 25 weeks (N = 3 for each experimental group). Data are shown as mean ± SEM. Fig. 10D provides quantification of mAlas2 allelic deletion in BM samples 25 weeks after secondary transplant (N=3 in Group 1 and Group 2). Data are shown as mean ± SEM. P-values are Tukey’ s multiple comparison test after one-way ANOVA. *p<0.05, ** p<0.01.

[0024] Figures 11A and 11B provide schematics of X-ALAS2, X-ALAS2_M1 (D263N), X-ALAS2 M2 (R375C), X-ALAS2 M3 (R411H), X-ALAS2 U3, X- ALAS2_U5, and X-ALAS2_U3 / U5. Figs. 11C and 1 ID provide metal-free protoporphyrin (mf-PPIX) measurements in K562 cells. Figs. 1 IE and 1 IF provides mf-PPIX assessment in transplanted mice, transduced with the different LVs.

[0025] Figure 12A provides an example of a nucleotide sequence encoding human ALAS2 (SEQ ID NO: 19). Figure 12B provides an example of an amino acid sequence of human ALAS2 (SEQ ID NO: 20). Locations of point mutations are bolded and underlined. Figure 12C provides an example of a nucleotide sequence of an HBB promoter (SEQ ID NO: 21). Figure 12D provides an example of a nucleotide sequence of an HBB enhancer (HBB3) (SEQ ID NO: 22). Figure 12E provides an example of a nucleotide sequence of HS2 (SEQ ID NO: 23). Figure 12F provides an example of a nucleotide sequence of HS3 (SEQ ID NO: 24). Figure 12G provides an example of a nucleotide sequence of the ankyrin insulator (SEQ ID NO: 25).

[0026] Figures 13A-13E provide an example of the nucleotide sequence of X-ALAS2 with annotation (SEQ ID NO: 26).

[0027] DETAILED DESCRIPTION OF THE INVENTION

[0028] X-linked sideroblastic anemia (XLSA) is a congenital anemia caused by mutations in Alas2, a gene responsible for heme synthesis. Current treatment options are limited to pyridoxine supplements and blood transfusions, offering no definitive cure except for allogeneic hematopoietic stem cell transplantation, only accessible to a subset of patients (Fujiwara, et al. (2019) Free Radic Biol Med., 133: 179-185; Bottomley, et al. (2014) Hematol Oncol Clin North Am., 28:653-670; May, et al. (1998) Haematologica 83:56-70). The lack of a suitable animal model has hindered the development of gene therapy research for this disease. Considering the rarity of the disease, the lack of therapeutic options, and the elevated number of mutations causing the disease, it is important to establish a severe phenotype and study whether a therapeutic, such as an erythroid-specific ALAS2-LV, would be effective in reversing the most severe form of the disease.

[0029] Herein, a conditional A / is2-KO mouse model was engineered using two approaches: tamoxifen administration and treatment with lipid nanoparticles (LNP) carrying Cre-mRNA and conjugated to an anti-CDl 17 antibody. A / as2-KOBManimals displayed a severe anemic phenotype characterized by ineffective erythropoiesis (IE), leading to low numbers of red blood cells (RBC), hemoglobin (Hb), and hematocrit (HCT). In particular, erythropoiesis in these animals showed expansion of polychromatic erythroid cells, decreased activity in the electron transport chain and mitochondria’s function, and reduced activity of key Tricarboxylic Acid (TCA) cycle enzymes. The IE was also associated with marked splenomegaly and low hepcidin levels, leading to iron accumulation in the bone marrow (BM), liver, and spleen. To investigate the potential of a gene therapy approach for XLSA, a lentiviral vector (X-ALAS2-LV) was developed that exploits a globin promoter and globin-enhancer elements to direct human Alas2 tissue-specific expression in erythroid cells. Infusion of BM cells with at least 0.6 copies (e.g., 0.6- 1.4 copies) of the X-ALAS2-LV in Alas2 -KOBMmice rescued their phenotype by improving Hb, RBC, and HTC levels, tissue iron accumulation, and survival rates. These indicate that the vector is curative in XLAS patients. Therefore, gene therapy, such as ex-vivo HSC gene therapy, using an LV, such as an erythroid-driven LV, represents a therapeutic option, improving patients’ conditions by supplying a functional copy of the ALAS2 cDNA.

[0030] Patients with red blood cell (RBC) specific bone marrow failure (BMF) syndromes represent a particularly difficult cohort to treat. These patients can be transiently maintained on packed red blood cell transfusions but ultimately require curative therapy for which there are very limited options. BMF disorders include, without limitation, acquired aplastic anemia and inherited trilineage aplasia conditions including, for example, Fanconi Anemia and telomere biology disorders; diseases associated with specific failure of red blood cell (RBC) production including, for example, Diamond Blackfan Anemia and congenital sideroblastic anemia; and diseases associated with other single lineage cytopenias including severe congenital neutropenia and inherited thrombocytopenia syndromes (Parikh, et al., Curr. Opin. Pediatr. (2012) 24(l):23-32). While BMF associated with trilineage aplasia can now be cured in greater than 95% of cases by allogeneic stem cell transplantation (alloSCT) using a number of different donor sources and low intensity conditioning, single lineage BMF disorders remain difficult to cure (Peslak, et al., Curr. Treat. Options Oncol. (2017) 18(12):70; Dietz, et al., Curr. Opin. Pediatr. (2016) 28(1):3-11; Feffault de Latour, et al., Bone Marrow Transplant (2015) 50(9): 1168-72; Oved, et al., Biol. Blood Marrow Transplant (2019) 25(3):549-555). RBC-specific BMF diseases are particularly challenging to approach with alloSCT, as chronic RBC transfusion dependence often leads to human leukocyte antigen (HLA) alloimmunization that increases risk of graft failure / rej ection with reduced intensity preparative regimens, while transfusional iron overload leads to pre-SCT organ damage that limits the safety of myeloablative alloSCT conditioning approaches (Alter, B.P., Blood (2017) 130(21)2257-2264). Furthermore, many patients with RBC-specific BMF diseases will not have available fully HLA-matched donors for alloSCT, and use of alternative HLA-mismatched donors remains associated with high risk of debilitating graft-versus host disease (Gragert, et al., N. Engl. J. Med. (2014) 371(4):339-48). Additionally, hematopoietic stem cell transplant (HSCT) has offered limited curative potential for patients with CSA (Ayas, et al., Br. J. Haematol. (2001) 113(4):938-9).

[0031] In contrast, autologous hematopoietic stem cell (HSC) gene therapy based on lentiviral gene addition, performed with reduced toxicity mono-agent conditioning, is an attractive curative cell therapy approach for RBC-specific BMF diseases, as it eliminates risks of alloimmune complications, and has been associated with tolerable rates of organ toxicity when applied to patients with hemoglobin disorders (Thompson, et al., N. Engl. J. Med. (2018) 378(16): 1479-1493). Thus, lentiviral gene correction is a very promising curative modality for these patients.

[0032] In accordance with the instant invention, nucleic acid molecules, particularly viral vectors, are provided. Viral vectors of the instant invention comprise nucleic acid molecules. The viral vectors may be used to treat and or inhibit anemia, particularly sideroblastic anemia. In certain embodiments the sideroblastic anemia is congenital sideroblastic anemia (CSA). In certain embodiments the sideroblastic anemia is X-linked sideroblastic anemia (XLSA).

[0033] Viral vectors include, for example, retroviral vectors and lentiviral vectors. In certain embodiments, the viral vector is a lentiviral vector. In certain embodiments, the viral vector of the instant invention comprises the elements of the vectors set forth in Figures 2F, 11, or 13, particularly in the arrangement and / or orientation presented (e.g., in Fig. 2F).

[0034] In certain embodiments, the viral vector may comprise one or more (or all 6) of the elements listed below.

[0035] 1) The viral vectors of the instant invention may comprise a nucleic acid sequence encoding ALAS2 (5'-aminolevulinate synthase 2). In certain embodiments, the ALAS2 is human. In certain embodiments, the nucleic acid sequence encoding ALAS2 is in reverse or antisense orientation (e.g., compared to orientation of 5’LTR and 3’LTR). Examples of amino acid and nucleotide sequences of ALAS2 are provided in GenBank Gene ID: 212 and GenBank Accession Nos. NM_000032.5, NP_000023.2, NM_001037967.4, NP_001033056.1, NM_001037968.4, and NP_001033057.1. In certain embodiments, the nucleic acid sequence of ALAS2 is provided in GenBank Accession Nos. NM_000032.5 (variant 1). Figure 12B (SEQ ID NO: 20) provides an example of an amino acid sequence of human ALAS2 and Figure 12A (SEQ ID NO: 19) provides an example of a nucleotide sequence encoding human ALAS2. In certain embodiments, the amino acid sequence of ALAS2 has 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the sequence provided in Figure 12B or SEQ ID NO: 20. In certain embodiments, the nucelotide sequence of ALAS2 has 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the sequence provided in Figure 12A (SEQ ID NO: 19) or Figure 13 (SEQ ID NO: 26).

[0036] In certain embodiments, the ALAS2 comprises at least one mutation (e.g., a substitution mutation). In certain embodiments, the mutation prevents ALAS2 overexpression. In certain embodiments, the mutation reduces ALAS2 activity. In certain embodiments, the ALAS2 comprises a substitution mutation at E242 (e.g., E242K), D263 (e.g., D263N), P339 (e.g., P339L), R375 (e.g., R375C), and / or R411 (e.g., R411H). In certain embodiments, the ALAS2 comprises a substitution mutation at D263 (e.g., D263N), R375 (e.g., R375C), and / or R411 (e.g., R411H). In certain embodiments, the ALAS2 comprises a substitution mutation at D263 (e.g., D263N). In certain embodiments, the ALAS2 comprises a substitution mutation at R375 (e.g., R375C). In certain embodiments, the ALAS2 comprises a substitution mutation at R411 (e.g., R411H).

[0037] While the ALAS2 coding sequence may be flanked by its natural untranslated regions (UTRs), the ALAS2 coding sequence may be flanked by a 3’UTR and / or 5’UTR that are not from the ALAS2 gene. In certain embodiments, the ALAS2 coding sequence is flanked by a 3’ UTR and / or 5’ UTR from the HBB gene. In certain embodiments, the ALAS2 coding sequence is flanked by a HBB 3’ UTR and an ALAS2 5’ UTR. In certain embodiments, the ALAS2 coding sequence is flanked by an ALAS2 3’ UTR and a HBB 5’ UTR. In certain embodiments, the ALAS2 coding sequence is flanked by a HBB 3’ UTR and a HBB 5’ UTR. In certain embodiments, the ALAS2 coding sequence is flanked by an ALAS2 3’ UTR and an ALAS2 5’ UTR. Examples of HBB UTRs are provided in Figure 13 (SEQ ID NO: 26). In certain embodiments, the nucleic acid sequence of the HBB UTR (3’ UTR and / or 5’UTR) has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the sequence provided in Figure 13 (SEQ ID NO: 26).

[0038] In certain embodiments, the ALAS2 coding sequence is flanked by a 3’ UTR and / or 5’ UTR from ALAS1. In certain embodiments, the ALAS2 coding sequence is flanked by an ALAS1 3’ UTR and an ALAS2 5’ UTR. In certain embodiments, the ALAS2 coding sequence is flanked by an ALAS1 3’ UTR and a HBB 5’ UTR. In certain embodiments, the ALAS2 coding sequence is flanked by a HBB 3’ UTR and an ALAS1 5’ UTR. In certain embodiments, the ALAS2 coding sequence is flanked by an ALAS2 3’ UTR and an ALAS1 5’ UTR. Examples of amino acid and nucleotide sequences of ALAS1 are provided in GenBank Gene ID: 211 and GenBank Accession Nos. NM_000688.6, NP_000679.1, NM_001304443.1, NP-001291372.1, NM_001304444.1, NP_001291373.1, NMJ99166.2, and NP 954635.1. The inclusion of ALAS1 UTRs can allow regulation of ALAS2 expression based on heme concentration, a unique characteristic of ALAS1 but not of ALAS2 (Roberts et al. (2005) FEBS Letters 579: 1873-3468; Cable, et al. (2001) Hepatology 34: 356A-Abstract 737; Roberts et al. (2001) Biochim Biophys Acta., 1518:95-105; each reference incorporated by reference herein).

[0039] In certain embodiments, the ALAS2 coding sequence is modified to contain ALAS1 domains (e.g., from human ALAS1) regulated by heme catalysis (e.g., heme regulatory motif or CP motif). In certain embodiments, one or both of the UTRs are also from ALAS1. In certain embodiments, the ALAS1 domains are inserted into the corresponding positions in ALAS2 (e.g., replace the same domain / region of ALAS2). In certain embodiments, one, two, or three ALAS1 domains regulated by heme catalysis are incorporated into the ALAS2 coding sequence. In certain embodiments, the ALAS1 domains are as described in Kubota et al. (J. Biol. Chem. (2016) 291(39):20516-20529) or Lathrop, et al. (Science (1993) 259:522-525), each incorporated by reference herein.

[0040] 2) The viral vectors of the instant invention may comprise an erythroid- specific promoter. The erythroid-specific promoter may control the expression of ALAS2 (e.g., is operably linked to the nucleic acid sequence encoding ALAS2). In certain embodiments, the promoter is an HBB promoter (hemoglobin subunit beta promoter). In certain embodiments, the promoter is a human promoter. In certain embodiments, the promoter is a HBB 200bp promoter. Figure 12C (SEQ ID NO: 21) provides an example of the HBB promoter. In certain embodiments, the nucleic acid sequence of the HBB promoter has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the sequence provided in Figure 12C (SEQ ID NO: 21) or Figure 13 (SEQ ID NO: 26).

[0041] 3) The viral vectors of the instant invention may comprise an enhancer. The enhancer may be 3’ of the nucleic acid sequence encoding of ALAS2, particularly including UTRs (e.g., is operably linked to the nucleic acid sequence encoding ALAS2). In certain embodiments, the enhancer is an HBB enhancer. In certain embodiments, the enhancer is a 3’ enhancer (e.g., the 3’ HBB enhancer). In certain embodiments, the enhancer is a human enhancer. Figure 12D (SEQ ID NO: 22) provides an example of the HBB enhancer. Figure 13 (SEQ ID NO: 26) provides an example of the HBB enhancer (e.g., nucleotides 1-205 of the sequence provided in Figure 12D). In certain embodiments, the nucleic acid sequence of the HBB enhancer has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the sequence provided in Figure 12D (SEQ ID NO: 22) or Figure 13 (SEQ ID NO: 26).

[0042] 4) The viral vectors of the instant invention may comprise a locus control region (LCR). The LCR may be 3’ of the erythroid-specific promoter (e.g., the HBB promoter) and / or nucleic acid sequence encoding of ALAS2 (e.g., is operably linked to the HBB promoter and / or nucleic acid sequence encoding ALAS2). In certain embodiments, the LCR is a globin gene locus control region (LCR). In certain embodiments, the globin gene locus control region is a beta-globin gene locus control region. In certain embodiments, the LCR comprises at least two, at least three, or all four of HS1, HS2, HS3, and HS4. In certain embodiments, the LCR comprises HS2 and HS3. The LCR may be in reverse or antisense orientation. In certain embodiments, the LCR (e.g., HS2 and HS3) is operably linked to the nucleic acid sequence encoding ALAS2 and / or the promoter. Figure 12E (SEQ ID NO: 23) provides an example of HS2 and Figure 12F (SEQ ID NO: 24) provides an example of HS3. In certain embodiments, the nucleic acid sequence of HS2 has 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the sequence provided in Figure 12E (SEQ ID NO: 23) or Figure 13 (SEQ ID NO: 26). In certain embodiments, the nucleic acid sequence of HS3 has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the sequence provided in Figure 12F (SEQ ID NO: 24) or Figure 13 (SEQ ID NO: 26). 5) The viral vectors of the instant invention may comprise an insulator. Insulators can shelter the transgenic cassette from the silencing effect of non- permissive chromatin sites and, at the same time, protect the genomic environment from the enhancer effect mediated by active regulatory elements (like the LCR) introduced with the vector. In certain embodiments, the insulator is within the 3’UTR. Examples of insulators are known. The 1.2 Kb cHS4 insulator has been used to rescue the phenotype of thalassemic CD34+ BM-derived cells (Puthenveetil, et al. (2004) Blood, 104(12):3445-53). Further, fetal hemoglobin can be synthesized in human CD34+-derived cells after treatment with a lentiviral vector encoding the gamma-globin gene, either in association with the 400bp core of the cHS4 insulator or with a lentiviral vector carrying an shRNA targeting the gamma-globin gene repressor protein BCL 11 A (Wilber, et al. (2011) Blood, 117(10):2817-26). The HS2 enhancer of the GATA1 gene has also been used to achieve high beta-globin gene expression in human cells from patients with beta-thalassemia (Miccio, et al. (2011) PLoS One, 6(12):e27955). The use of an insulator derived from the promoter of the ankyrin gene, resulted in a significant amelioration of the thalassemic phenotype in mice and high level of expression was reached in both human thalassemic and SCD cells (Breda, et al. (2012) PloS one 7(3):e32345). The foamy virus has a 36-bp insulator located in its long terminal repeat (LTR) which reduces its genotoxic potential (Goodman, et al. (2018) J. Virol., 92:e01639-17). In certain embodiments, the insulator is an ankyrin insulator. Figure 12G (SEQ ID NO: 25) provides an example of the ankyrin insulator. In certain embodiments, the nucleic acid sequence of the ankyrin insulator has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the sequence provided in Figure 12G (SEQ ID NO: 25) or Figure 13 (SEQ ID NO: 26).

[0043] 6) The viral vectors of the instant invention may comprise a 5’ long terminal repeat (LTR) and a 3’ LTR. In certain embodiments, the 5’ LTR and / or 3’ LTR are from HIV (e.g., HIV-1). In certain embodiments, at least one of the LTRs (e.g., at least the 3’ LTR) is self-inactivating. A self-inactivating LTR comprises a deletion, addition, or mutation relative to its native sequence that results in it being replication incompetent. In certain embodiments, the self-inactivating LTR (e.g., a selfinactivating 3’ LTR) comprises an insulator element (e.g., ankyrin insulator and / or a foamy virus insulator). Figure 13 (SEQ ID NO: 26) provides examples of the nucleotide sequences of LTRs. In certain embodiment, the nucleic acid sequence of the LTR (5’ LTR and / or 3’ LTR) has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,

[0044] 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to an LTR nucleotide sequence of Figure 13 (SEQ ID NO: 26). In certain embodiment, the nucleic acid sequence of the LTR (5’ LTR and / or 3’ LTR) has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,

[0045] 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to an LTR nucleotide sequence of a viral vector in U.S. Patent Application Publication 2021 / 0222200, incorporated herein by reference, (see, e.g., Figure 8 or 9) or in U.S. Patent Application Publication 2023 / 0287449, incorporated herein by reference, (see, e.g., Figure 4B).

[0046] In certain embodiments, the viral vector further comprises a Woodchuck Post- Regulatory Element (WPRE). The WPRE can be placed outside the integrating sequence to increase the safety of the vector. An example of a nucleotide sequence of the WPRE is provided in Figure 13 (SEQ ID NO: 26). In a particular embodiment, the WPRE is 3’ of the 3 ’LTR. The WPRE increases the titer of the lentivirus, but it can undergo chromosomal rearrangement upon integration. In order to preserve the ability of WPRE to increase viral titers without having this viral element in the integrating sequence, the WPRE can be removed from the integrating portion and added, for example, after the 3 ’LTR. In addition, a polyadenylation signal (e.g., a bovine growth hormone polyA tail) can be inserted after the WPRE region to increase lentiviral titers (Zaiss, et al. (2002) J. Virol., 76(14):7209-19). In certain embodiment, the nucleic acid sequence of the WPRE has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the WPRE sequence provided in Figure 13 (SEQ ID NO: 26). In certain embodiment, the nucleic acid sequence of the WPRE has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,

[0047] 93, 94, 95, 96, 97, 98, 99, or 100% identity to an WPRE nucleotide sequence of a viral vector in U.S. Patent Application Publication 2021 / 0222200, incorporated herein by reference, (see, e.g., Figure 8 or 9) or in U.S. Patent Application Publication 2023 / 0287449, incorporated herein by reference, (see, e.g., Figure 4B).

[0048] In certain embodiments, the viral vector of the instant invention has a nucleotide sequence identical to a viral vector presented herein (see, e.g., Figure 13 (SEQ ID NO: 26)) or they can have least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to the nucleotide sequence of a viral vector disclosed herein (see, e.g., Figure 13 (SEQ ID NO: 26)). In certain embodiments, an element (e.g., as set forth above) of the viral vector of the instant invention has a nucleotide sequence identical to an element presented herein (see, e.g., Figure 12 (SEQ ID NOs: 19 and 21-25) and Figure 13 (SEQ ID NO: 26)) or they can have least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to the nucleotide sequence of an element disclosed herein (see, e.g., Figure 12 (SEQ ID NOs: 19 and 21-25) and Figure 13 (SEQ ID NO: 26)). In certain embodiment, an element (e.g., as set forth above) of the viral vector of the instant invention has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to an element sequence of a viral vector in U.S. Patent Application Publication 2021 / 0222200, incorporated herein by reference, (see, e.g., Figure 8 or 9) or in U.S. Patent Application Publication 2023 / 0287449, incorporated herein by reference, (see, e.g., Figure 4B).

[0049] In a particular embodiment, the viral vector comprises: i) a 5’ long terminal repeat (LTR) and a 3’ LTR, wherein at least one of the LTR is self-inactivating; ii) at least one promoter (e.g., a cell (e.g., erythroid) promoter (e.g., beta-globin promoter (e.g., the 200 bp beta globin promoter)); and iii) a sequence encoding an ALAS2.

[0050] In a particular embodiment, the viral vector comprises: i) a 5’ long terminal repeat (LTR) and a 3’ LTR, wherein at least one of the LTR is self-inactivating; ii) at least one promoter (e.g., a cell (e.g., erythroid) promoter (e.g., beta-globin promoter (e.g., the 200 bp beta globin promoter)); iii) a sequence encoding an ALAS2; and iv) an LCR (e.g., HS2 and HS3).

[0051] In a particular embodiment, the viral vector comprises: i) a 5’ long terminal repeat (LTR) and a 3’ LTR, wherein at least one of the LTR is self-inactivating; ii) at least one promoter (e.g., a cell (e.g., erythroid) promoter (e.g., beta-globin promoter (e.g., the 200 bp beta globin promoter)); iii) a sequence encoding an ALAS2; and iv) an enhancer (e.g., a 3’ enhancer (e.g., the HBB 3’ enhancer)).

[0052] In a particular embodiment, the viral vector comprises: i) a 5’ long terminal repeat (LTR) and a 3’ LTR, wherein at least one of the LTR is self-inactivating; ii) at least one promoter (e.g., a cell (e.g., erythroid) promoter (e.g., beta-globin promoter (e.g., the 200 bp beta globin promoter)); iii) a sequence encoding an ALAS2; iv) an LCR (e.g., HS2 and HS3); and v) an enhancer (e.g., a 3’ enhancer (e.g., the HBB 3’ enhancer)).

[0053] In a particular embodiment, the viral vector comprises: i) a 5’ long terminal repeat (LTR) and a 3’ LTR, wherein at least one of the LTR is self-inactivating; ii) at least one promoter (e.g., a cell (e.g., erythroid) promoter (e.g., beta-globin promoter (e.g., the 200 bp beta globin promoter)); iii) a sequence encoding an ALAS2; and iv) an insulator (e.g., an ankyrin insulator (e.g., within an LTR).

[0054] In a particular embodiment, the viral vector comprises: i) a 5’ long terminal repeat (LTR) and a 3’ LTR, wherein at least one of the LTR is self-inactivating; ii) at least one promoter (e.g., a cell (e.g., erythroid) promoter (e.g., beta-globin promoter (e.g., the 200 bp beta globin promoter)); iii) a sequence encoding an ALAS2; iv) an LCR (e.g., HS2 and HS3); v) an enhancer (e.g., a 3’ enhancer (e.g., the HBB 3’ enhancer)); and vi) an insulator (e.g., an ankyrin insulator (e.g., within an LTR).

[0055] In a particular embodiment, the viral vector comprises: i) a 5’ long terminal repeat (LTR) and a 3’ LTR, wherein at least one of the LTR is self-inactivating; ii) at least one promoter (e.g., a cell (e.g., erythroid) promoter (e.g., beta-globin promoter (e.g., the 200 bp beta globin promoter)); iii) a sequence encoding an ALAS2; iv) an LCR (e.g., HS2 and HS3); and v) an insulator (e.g., an ankyrin insulator (e.g., within an LTR).

[0056] In a particular embodiment, the viral vector comprises: i) a 5’ long terminal repeat (LTR) and a 3’ LTR, wherein at least one of the LTR is self-inactivating; ii) at least one promoter (e.g., a cell (e.g., erythroid) promoter (e.g., beta-globin promoter (e.g., the 200 bp beta globin promoter)); iii) a sequence encoding an ALAS2; iv) an enhancer (e.g., a 3’ enhancer (e.g., the HBB 3’ enhancer)); and v) an insulator (e.g., an ankyrin insulator (e.g., within an LTR).

[0057] In certain embodiments, the viral vector of any of the above may have a sequence encoding ALAS2 comprising a 3’UTR and / or 5’UTR from a different gene (e.g., ALAS1 and / or HBB), as explained hereinabove.

[0058] In certain embodiments, the viral vector of any of the above may further comprise a WPRE. In certain embodiments, the WPRE is outside of the LTRs.

[0059] The present disclosure provides compositions and methods for the inhibition, prevention, and / or treatment of anemia, particularly sideroblastic anemia. In certain embodiments the sideroblastic anemia is congenital sideroblastic anemia (CSA). In certain embodiments the sideroblastic anemia is X-linked sideroblastic anemia (XLSA). In particular, the present disclosure provides novel nucleic acids and viral vectors for the inhibition, prevention, and / or treatment of anemia, particularly sideroblastic anemia. In certain embodiments the sideroblastic anemia is congenital sideroblastic anemia (CSA). In certain embodiments the sideroblastic anemia is X- linked sideroblastic anemia (XLSA). In a particular embodiment, the methods of the instant invention can be used to inhibit, treat, and / or prevent a disease or disorder characterized by a mutant or defective ALAS2 gene.

[0060] The viral vectors may be transduced into cell by any method known in the art. In certain embodiments, the cell is an hematopoietic stem cell, erythroid precursor cell or erythroid cell (e.g., CD34+ cell). In certain embodiments, the cell is contacted with a virus or virus particle comprising the viral vector. In a particular embodiment, the transduction is performed with the adjuvant / enhancer LentiBoost™ or cyclosporine H. In a particular embodiment, the viral vector is pseudotyped with Cocal envelope. In a particular embodiment, the transduction is performed by prestimulating for 24 hours and using a 2-hit transduction (e.g., a MOI 10 / 10 at 16 and 8 hours).

[0061] In accordance with the instant invention, compositions and methods are provided for increasing heme and / or hemoglobin production in a cell or subject. The method comprises administering a viral vector of the instant invention to the cell, particularly a hematopoietic stem cell, erythroid precursor cell or erythroid cell (e.g., CD34+ cell), or subject. In a particular embodiment, the subject has anemia, particularly sideroblastic anemia. In certain embodiments the sideroblastic anemia is congenital sideroblastic anemia (CSA). In certain embodiments the sideroblastic anemia is X-linked sideroblastic anemia (XLSA). The viral vector may be administered in a composition further comprising at least one pharmaceutically acceptable carrier.

[0062] In accordance with another aspect of the instant invention, compositions and methods for inhibiting (e.g., reducing or slowing), treating, and / or preventing anemia in a subject are provided. In certain embodiments, the anemia is sideroblastic anemia. In certain embodiments, the sideroblastic anemia is congenital sideroblastic anemia (CSA). In certain embodiments, the sideroblastic anemia is X-linked sideroblastic anemia (XLSA). In a particular embodiment, the methods comprise administering to a subject in need thereof a viral vector of the instant invention. The viral vector may be administered in a composition further comprising at least one pharmaceutically acceptable carrier. The viral vector may be administered via an ex vivo methods wherein the viral vector is delivered to a hematopoietic stem cell, erythroid precursor cell or erythroid cell (e.g., CD34+ cell), particularly autologous ones, and then the cells are administered to the subject. In a particular embodiment, the method comprises isolating hematopoietic cells (e.g., erythroid precursor cells) or erythroid cells from a subject, delivering (e.g., transducing) a viral vector of the instant invention to the cells, and administering the treated cells to the subject. In certain embodiments, the cells are administered to the bone marrow (e.g., bone marrow transplant). The cells may be administered as a composition with a pharmaceutically acceptable carrier. The methods of the instant invention may further comprise monitoring the disease or disorder in the subject after administration of the cells and / or composition(s) of the instant invention to monitor the efficacy of the method. For example, the subject may be monitored for characteristics of anemia, particularly sideroblastic anemia (e.g., CSA or XLSA).

[0063] As explained hereinabove, the compositions and methods of the instant invention are useful for increasing heme and / or hemoglobin production and for treating anemia, particularly sideroblastic anemia (e.g., CSA or XLSA). A therapeutically effective amount of the cells or compositions may be administered to a subject in need thereof. The dosages, methods, and times of administration are readily determinable by persons skilled in the art, given the teachings provided herein.

[0064] The components as described herein will generally be administered to a patient as a pharmaceutical preparation. The term “patient” or “subject” as used herein refers to human or animal subjects. The components of the instant invention may be employed therapeutically, under the guidance of a physician for the treatment of the indicated disease or disorder.

[0065] The pharmaceutical preparation comprising the components of the invention may be conveniently formulated for administration with an acceptable medium (e.g., pharmaceutically acceptable carrier) such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. The concentration of the agents in the chosen medium may be varied and the medium may be chosen based on the desired route of administration of the pharmaceutical preparation. Except insofar as any conventional media or agent is incompatible with the agents to be administered, its use in the pharmaceutical preparation is contemplated.

[0066] The compositions of the present invention can be administered by any suitable route, for example, by injection (e.g., for local (direct) or systemic administration), oral, pulmonary, topical, nasal or other modes of administration. The composition may be administered by any suitable means, including parenteral, intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous, topical, inhalatory, transdermal, intrapulmonary, intraareterial, intrarectal, intramuscular, and intranasal administration. In a particular embodiment, the composition is administered directly to the blood stream (e.g., intravenously). In a particular embodiment, the composition is administered directly to the bone marrow. In general, the pharmaceutically acceptable carrier of the composition is selected from the group of diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. The compositions can include diluents of various buffer content (e.g., Tris HC1, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). The compositions can also be incorporated into particulate preparations of polymeric compounds such as polyesters, polyamino acids, hydrogels, polylactide / glycolide copolymers, ethylenevinylacetate copolymers, polylactic acid, polyglycolic acid, etc., or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of components of a pharmaceutical composition of the present invention. See, e.g., Remington: The Science and Practice of Pharmacy, 21st edition, Philadelphia, PA. Lippincott Williams & Wilkins. The pharmaceutical composition of the present invention can be prepared, for example, in liquid form, or can be in dried powder form (e.g., lyophilized for later reconstitution).

[0067] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation, as exemplified in the preceding paragraph. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the molecules to be administered, its use in the pharmaceutical preparation is contemplated.

[0068] Pharmaceutical compositions containing an agent of the present invention as the active ingredient in intimate admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous. Injectable suspensions may be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. Pharmaceutical preparations for injection are known in the art. If injection is selected as a method for administering the therapy, steps should be taken to ensure that sufficient amounts of the molecules reach their target cells to exert a biological effect.

[0069] A pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. Dosage units may be proportionately increased or decreased based on the weight of the patient. Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art. The appropriate dosage unit for the administration of the molecules of the instant invention may be determined by evaluating the toxicity of the molecules in animal models. Various concentrations of pharmaceutical preparations may be administered to mice with transplanted human tumors, and the minimal and maximal dosages may be determined based on the results of significant reduction of tumor size and side effects as a result of the treatment. Appropriate dosage unit may also be determined by assessing the efficacy of the treatment in combination with other standard therapies.

[0070] The pharmaceutical preparation comprising the molecules of the instant invention may be administered at appropriate intervals, for example, at least twice a day or more until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level. The appropriate interval in a particular case would normally depend on the condition of the patient.

[0071] Definitions

[0072] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0073] The terms “isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers. “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0074] A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers. Suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Rowe, et al., Eds., Handbook of Pharmaceutical Excipients, Pharmaceutical Pr.

[0075] The term “treat” as used herein refers to any type of treatment that imparts a benefit to a patient suffering from a disease or disorder, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.

[0076] As used herein, the term “prevent” refers to the prophylactic treatment of a subject who is at risk of developing a condition and / or sustaining a disease or disorder, resulting in a decrease in the probability that the subject will develop conditions associated with the hemoglobinopathy or thalassemia.

[0077] A “therapeutically effective amount" of a compound or a pharmaceutical composition refers to an amount effective to prevent, inhibit, or treat a particular injury and / or the symptoms thereof. For example, “therapeutically effective amount” may refer to an amount sufficient to modulate the pathology associated with an anemias such as sideroblastic anemia (e.g., CSA or XLSA).

[0078] As used herein, the term “subject” refers to an animal, particularly a mammal, particularly a human.

[0079] A “vector” is a genetic element, such as a plasmid, cosmid, bacmid, phage or virus. A vector may be either RNA or DNA and may be single or double stranded. A vector may comprise expression operons or elements such as, without limitation, transcriptional and translational control sequences, such as promoters, enhancers, translational start signals, polyadenylation signals, terminators, and the like, and which facilitate the expression of a polynucleotide or a polypeptide coding sequence in a host cell or organism.

[0080] The following example is provided to illustrate various embodiments of the present invention. It is not intended to limit the invention in any way.

[0081] EXAMPLE

[0082] Materials and Methods

[0083] Mouse lines and breeding conditions.

[0084] Alas2n" transgenic animals were generated as described herein. B6.129- Gt(ROSA)26Sortml(cre / ERT2)Ty]U (R26-CreERT2- RRID: IMSR_JAX:008463), B6.SJL- PtprcaPepcb / BoyJ / U CVBoyJ CD45.1 - RRID: IMSR_JAX:002014) and C57BL / 6- Tg(UBC-GFP)30Scha / J (B6-GFP - RRID: IMSR_JAX:004353) mice were purchased from The Jackson Laboratory (Bar Harbor, ME).

[0085] Institutional Animal Care and Use Committee (IACUC) regulation.

[0086] All experiments performed in mice were approved by the IACUC (protocol #1173) at the Children’s Hospital of Philadelphia. The animals have been housed in the Children’s Hospital of Philadelphia animal facility according to the standards provided by the OLAW and AAALAC.

[0087] Mouse screening

[0088] The genotyping primers to identify the LoxP sites and the R26-CreERT2sequence are shown in Table 2.

[0089] 'able 2: Primer sequences for mA!as2 and R26CreERT2.

[0090] Cell sorting for isolation of polychromatic erythroid population

[0091] Bone marrow cells extracted from one femur have been washed in IX PBS. Subsequently, Teri 19+cells selection was obtained using MACS LS Columns (Cat. 130-042-401), and Anti-Terl l9 Microbeads (Cat. 130-042-401) (Miltenyi Biotec, Auburn, CA). Isolated Teri 19+cells were then incubated with FITC anti-mouse Ter- 119 (116206) and APC anti-mouse / human CD44 (103012) antibodies and Propidium Iodide (PI) (421301) (Biolegend, San Diego, CA) and polychromatic erythroid population (P3) was sorted using a BD FACSMelody™ Cell Sorter and BD FACSChorus software (BD Biosciences, Franklin Lakes, NJ), using a 100pm nozzle at 23 PSI, drop frequency 34 kHz. P3 sorted cells were centrifuged at 400xg for 5 minutes at 4°C and immediately frozen and stored at -80°C until processing for analysis.

[0092] Mitochondria isolation

[0093] Cells were washed twice in ice-cold 0.1M phosphate-buffered saline (PBS), then lysed in 0.5mL buffer A (50mmol / L Tris, lOOmmol / L KC1, 5mmol / L MgCh, 1.8mmol / L ATP, Immol / L EDTA, pH 7.2), supplemented with protease inhibitor cocktail III [100 mmol / L AEBSF, 80mmol / L aprotinin, 5mmol / L bestatin, 1.5mmol / L E-64, 2mmol / L leupeptin and Immol / L pepstatin (Merck KGaA, Darmstadt, DE)], Immol / L phenylmethyl sulfonyl fluoride (PMSF), 250mmol / L NaF (Xu, et al. (2021) EMBO Mol. Med., 13(7):el4133). Samples were clarified by centrifuging at 650 x g for 3 minutes at 4°C, and the supernatant was collected and centrifuged at 13,000 x g for 5 minutes at 4°C. The new supernatant was discarded, the pellet containing mitochondria was washed in 0.5mL buffer A and re-suspended in 0.25mL buffer B (250mmol / L sucrose, 15pmol / L K2HPO4, 2mmol / L MgCh, 0.5mmol / L EDTA, 5% w / v bovine serum albumin). A 50 pL aliquot was sonicated and used for the measurement of protein content. To confirm the presence of mitochondrial proteins in the extracts, 10 pg of each sonicated sample were subjected to SDS-PAGE and probed with an anti-porin antibody (Abeam, Cambridge, UK). The sonicated samples were used to measure the enzymatic activities of 5'-Aminolevulinate synthase 1, citrate synthase, a-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase. The remaining not-sonicated part was used to measure the electron transport chain (ETC) complexes I-IV activities.

[0094] Citrate synthase, a-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase activities

[0095] The enzymatic activities of citrate synthase, a-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase were measured on lOpg mitochondrial proteins using the Citrate Synthase Assay Kit (Sigma Aldrich, St. Louis, MO, catalog n° MAK193), Alpha Ketoglutarate (alpha KG) Assay Kit (Abeam, Cambridge, UK, catalog n° ab83431), Malate Dehydrogenase Assay Kit (Sigma Aldrich, St. Louis, MO, catalog n° MAKI 96), Succinate Dehydrogenase Activity Colorimetric Assay Kit (BioVision, Milpitas, CA, catalog n° K660), as per manufacturer’s instructions. Results were expressed as mU / mg mitochondrial proteins.

[0096] Activity of mitochondrial ETC complexes I-IV.

[0097] The activity of mitochondrial electron transport chain complexes was measured (Xu, et al. (2021) EMBO Mol. Med., 13(7):el4133). Specifically, to measure complex I activity, 20 pg of non-sonicated mitochondrial samples were resuspended in 0.2 ml buffer 1 A (5 mM KH2PO4, 5 mM MgCh, 5% w / v bovine serum albumin), incubated 1 minute at RT followed by 7 minutes in 0.1 ml buffer IB (25% w / v saponin, 50 mM KH2PO4, 5 mM MgCh, 5% w / v bovine serum albumin, 0.12 mM oxidized ubiquinone, which acts as electrons shuttle from complex I to complex III, 2.5 mM antimycin A, which inhibits complex III, 0.2 mMNaNs, which blocks complex IV; pH 7.5). 1.5 mM NADH, as an electron donor, was added to the mix. The rate of NADH oxidation was followed for 5 minutes at 37°C, reading the absorbance at 340 nm. The results were expressed as NAD+ / min / mg mitochondrial protein nanomoles. Complex II activity was measured as the electron transfer rate between complex II and III. 20 pg of non-sonicated mitochondrial samples was resuspended in 0.1 ml buffer 2A (50 mM KH2PO4, 7.5 mM MgCh, 25% w / v saponin, 20 mM succinic acid; pH 7.2) and incubated for 30 minutes at room temperature. 0.2 ml buffer 2B (50 mM KH2PO4, 7.5 mM MgCh, 5% w / v bovine serum albumin, 30 mM succinic acid as substrate of complex II, 0.12 mM oxidized ubiquinone as electrons shuttle from complex II to complex III, 0.12 mM oxidized cytochrome c as acceptor of electrons flowing from complex II to complex III, 5 mM rotenone to prevent electron flux from complex I, 0.2 mM NaNs, to block complex IV) was added. The reduction rate of cytochrome c was measured for 5 minutes at 37°C, reading the absorbance at 550 nm. The results were expressed as nanomoles of reduced cytochrome c / min / mg mitochondrial proteins.

[0098] The activity of complex III was measured in 50 pg of non-sonicated mitochondrial samples re-suspended in 0.2 ml buffer A (5 mM KH2PO4, 5 mM MgCh, 5% w / v bovine serum albumin, bovine serum albumin; pH 7.2) to which 0.1 ml buffer B (25% w / v saponin, 50 mM KH2PO4, 5 mM MgCh, 5% w / v bovine serum albumin, 0.12 mM oxidized cytochrome c, 0.2 mM NaNs, which blocks complex IV allowing the accumulation of reduced cytochrome c; pH 7.5) was added for 5 minutes at room temperature. The cytochrome c reduction reaction was started by adding 0.15 mM NADH. After 1 minute from the addition of NADH, as an inducer of electron flow, 5 mM rotenone, which blocks the activity of complex I, was added. The reduction rate of cytochrome c, dependent on the activity of complex III only in the presence of rotenone, was followed for 5 minutes at 37°C, reading the absorbance at 550 nm. The results were expressed as nanomoles of reduced cytochrome c / min / mg mitochondrial proteins.

[0099] The rate of oxidation of cytochrome c (reduced form, generated by complex III) was measured to measure the activity of complex IV. 20 pg of non-sonicated mitochondrial samples was resuspended in 0.1 ml buffer 4A (50 mM KH2PO4, 20 mM succinic acid, 25% w / v saponin; pH 7.2) and incubated for 30 minutes at room temperature. 0.2 ml buffer 4B (50 mM KH2PO4, 5 mM rotenone, which prevents electron flux from complex I to complex III, 30 mM succinic acid as a substrate of complex II and electrons generator, 0.03 mM reduced cytochrome c as an acceptor of electrons flowing from complex III to complex IV) was added. The oxidation rate of cytochrome c was followed for 5 minutes at 37°C, reading the absorbance at 550 nm. The results were expressed as nanomoles of oxidized cytochrome c / min / mg mitochondrial proteins. 5'-Aminolevulinate synthase activity

[0100] ALAS activity was measured using 10 pg of isolated mitochondrial proteins. 10 pl of the reaction product were injected into a Waters Acquity™ ultra-performance liquid chromatography (UPLC), equipped with a binary solvent manager, sample manager, photodiode array detector (PDA), fluorescence detector, column heater, and an Acquity™ UPLC BEH Cl 8, 1.7 pM, 2.1 x 100 mm column. ALA-derivative was detected by setting the detector with excitation 1 = 370 nm and emission 1 = 460 nm and the range of the PDA scanner between 210 and 500 nm. The results were converted into nmol / min according to a previously set titration curve and expressed as nmol / min / mg mitochondrial proteins (Bergonia, et al. (2015) Clin. Biochem., 48(12):788-795).

[0101] ATP levels in mitochondria

[0102] The ATP Bioluminescent Assay Kit was used to assess ATP levels in mitochondria extracts (Sigma-Aldrich, St. Louis, MO, catalog n° FLAA). In this case, ATP was quantified as relative light units (RLU) and converted into nmol ATP / mg mitochondrial proteins, according to the calibration curve indicated by the kit.

[0103] Vector production and characterization of X-ALAS2-LV

[0104] To create viral stocks, the human ALAS2 gene transfer plasmid was cotransfected with the envelope plasmid (VSV-G), the packaging plasmid (pMDLg / pRRE), and the pRSVREV vector into HEK293T cells (Breda, et al. (2021) Mol. Then, 29(4): 1625-1638). Briefly, the concentrated virus was obtained after ultracentrifugation, and serial dilutions of the virus were used to infect 5 104NIH 3T3 cells (ATCC, Manassas, VA) in 1 mL of transfection media (10% fetal bovine serum, 1% Penicillin / Streptomycin, IMDM) complemented with 1% polybrene (Millipore, Billerica, MA) at a final concentration of 8 pg / mL. The cells were incubated for three days, and genomic DNA was extracted using a Qiagen kit (Valencia, CA). The multiplicity of infection (MOI) was calculated using the following formula: number of cells (5xl04) X dilution factor (1 mL / pL viral preparation) X VCN (measured using a ddPCR probe specific for the Psi sequence found in lentiviral vectors). X-ALAS2-LV was characterized in vitro using NH4-3T3 and HUDEP-2 cells. To determine the optimal multiplicity of infection (MOI) of X- ALAS2-LV for ex vivo transduction of HSC, serial dilutions of concentrated viral preparations were utilized to transduce NIH-3T3 and HUDEP-2 cells. Vector copy number (VCN) was evaluated by ddPCR. The titer was 1.10E+09 infection particle / mL in NIH-3T3 cells and 3.7E+10 infection particle / mL in HUDEP-2 cells.

[0105] Glycolytic enzyme activities and intracellular lactate

[0106] Cells were washed with fresh medium, detached with trypsin / EDTA, resuspended at 1 x 105cells / ml in 200 pL of 100 mM TRIS 10 mM / EDTA 1 mM (pH 7.4), and sonicated on ice with two 10 second bursts. Enzymatic activities were measured on 10 pl cell lysates, incubated for 5 minutes at 37°C. The protein content was measured using the BCA1 kit (Sigma Aldrich, St. Louis, MO, catalog n° 71285- M). Aldolase and hexokinase (HK) activity were measured using the Aldolase Activity Colorimetric Assay Kit (Bio-Vision, Waltham, MA, catalog n° K665) and the Hexokinase Activity Assay kit (Abeam, Cambridge, UK, catalog n° ab 136957), respectively. The activity of phosphofructokinase- 1 (PFK) was measured spectrophotometrically (Sharma et al. (2011) Enzyme Res., 2011 :939472). The activities of glyceraldehyde 3 -phosphate dehydrogenase (GAPDH), enolase, and lactate dehydrogenase (LDH) were measured spectrophotometrically (Riganti, et al. (2002) Free Radic. Biol. Med., 32(9):938-949; Capello, et al. (2016) Oncotarget 7(5):5598-5612). For GAPDH, tissue lysate was incubated with 5 mM 3- phosphogly ceric acid, 1 U phosphoglycerate 3 -kinase, 5 mM ATP, and 2.5 mM NADH. For enolase, cell lysate was incubated with 10 mM MgCh, 100 mM KC1, 1 mM 2-phosphogly ceric acid, 0.4 mM ADP, 6.8 U / mL pyruvate kinase (PK), 9.9 U / mL LDH, 0.2 mM NADH. For LDH, cell lysate was incubated with 5 mM NADH and 20 mM pyruvic acid. For all assays of glycolytic enzymes, the activities were monitored by measuring the absorbance variation at 340 nm using a Synergy HTX 96-well microplate reader (Bio-Tek Instruments, Winooski, VT). The kinetics were linear throughout the measurement. To measure the intracellular lactate, 100 pg of whole cell lysates were analyzed using the Lactate Assay Kit (Sigma Aldrich, St. Louis, MO, catalog n° MAK-064), as per manufacturer’s instructions. The amount of lactate was measured spectrophotometrically at 570 nm with a Synergy HTX 96-well microplate reader.

[0107] Bone marrow isolation and Lin selection. Bone marrow cells were extracted from animal femurs employing mechanical crushing to optimize cell recovery. BM cells were then suspended in 4% FBS from Hyclone (Logan, UT) in PBS solution from Gibco (Waltham, MA). Following the manufacturer's guidelines, red blood cells were lysed using ACK lysis buffer from Gibco (Waltham, MA) and filtered through a 40 mM sterile strainer from Corning (Glendale, AZ). Cells were washed with a 4% FBS PBS solution. Following lysis, cell number, and viability were assessed using AOPI staining on a Cellometer® Auto 2000 cell viability counter from Nexcelom Bioscience (Lawrence, MA). Cells were seeded at 1.5E+06 / mL concentration in StemSpan™ SFEM from Stem Cell Technologies (Vancouver, BC, Canada). The culture medium was supplemented with 50 ng / mL mSCF, 6 ng / mL mIL-3, and 10 ng / mL mIL-6, all provided by Peprotech (Cranbury, NJ). Lin' HSCs were isolated by immunobeading using the mouse lineage cell depletion kit (130-110-470) from Miltenyi Biotech (Auburn, CA).

[0108] HSCs transduction with LV and transfection with LNP

[0109] 1.5E+06 / mL Lin' HSCs isolated from the BM of donor mice (8-12 weeks old) (as described above) were transduced overnight using a range of 100-150 MOEcell in StemSpan™ Serum Free Expansion Medium (SFEM) culture medium from StemCell Technologies (Vancouver, Canada). The culture medium was supplemented with 50 ng / mL recombinant murine stem cell factor (mSCF), 10 ng / mL recombinant murine interleukin-6 (IL-6), 6 ng / mL IL-3 from PeproTech (Rocky Hill, NJ), 200 mM L- glutamine, 100 U / mL penicillin / streptomycin from Gibco (Thermo Fisher Scientific), 0.2 mg / mL Poloxamer 338 from Sigma-Aldrich (St. Louis, MO), and 2 pL / mL Lentiblast Premium from OZ Biosciences (San Diego, CA). For the Alas2-\<dfv'[experimental group, BM cells isolated from the femurs of 2 mice (depleted of RBC as described above) were seeded at a concentration of 1.5E+06 / mL in StemSpan™ SFEM from Stem Cell Technologies (Vancouver, BC, Canada). The culture medium was supplemented with 50 ng / mL mSCF, 6 ng / mL mIL-3, and 10 ng / mL mIL-6 and incubated with l.Opg of the LNPCD117Cre formulation. A fraction of the cells was retained for 24 and 72 hours for analysis of allelic deletion. For the X-ALAS2-LV experimental group, 1.5E+06 / mL Lin cells were incubated with L0pg / lE+06 of LNPCD117Cre for 2 hours, followed by overnight transduction with X-ALAS2 LV using a range of 100-150 MOEcell. Bone marrow transplantation

[0110] 300,000 viable untreated or treated Lin donor cells were injected intravenously into lethally irradiated (two doses of 550cGy each, administered 4 hours apart) B6.SJL-PtprcaPepcb / BoyJ or C57BL / 6-Tg(UBC-GFP)30Scha / J recipient mice (8-12 weeks old, sourced from The Jackson Laboratory, Bar Harbor, ME). Cell injections took place no less than 1 hour after the second irradiation dose. Secondary transplants were performed by injecting 106whole BM cells into lethally irradiated secondary recipient mice following the same protocol as the primary transplants listed above.

[0111] Tamoxifen preparation and administration.

[0112] Tamoxifen was purchased by Millipore- Sigma (Burlington, MA) and dispersed in Corn Oil (Millipore-Sigma - Burlington, MA) at the 10 mg / mL stock concentration. A 1 mL syringe with a 21 -gauge 5 / 8 needle was used for IP administration in the lower abdomen of 1 mg / mouse. Tamoxifen irradiated chow was purchased by Inotiv (Chicago, IL) at a 500 mg / Kg diet concentration. It has been provided ad libitum, considering a daily intake of 80 mg / kg body weight.

[0113] RNA synthesis and preparation of targeted LNP-mRNA

[0114] The gene coding sequence for ere recombinase was cloned into an IVT- mRNA production template plasmid containing a T7 promoter, 5’ and 3’ UTR elements, Kozak consensus sequence, and 101 poly(A) tail. DNA synthesis, cloning, and industrial-grade endotoxin-free plasmid preparation service were provided by GenScript (Piscataway, NJ). IVT-mRNA was produced using linearized IVT template plasmid and the MEGAScript T7 kit (Thermo Fisher Scientific, AMB13345, Waltham, MA) and formulated with nucleoside-modified m I -5 ’-triphosphate (TriLink, N-1081, San Diego, CA). 5’ Capping of the IVT-mRNAs was performed co-transcriptionally using the trinucleotide capl analog, CleanCap® Reagent AG (3’ OMe) (TriLink, N-7413, San Diego, CA). Single-stranded IVT-mRNA was purified by cellulose purification (Vlatkovic, et al. (2022) Pharmaceutics 14(2) : 328). All mRNAs were analyzed by agarose gel electrophoresis and were stored at -20°C. Cellulose-purified ml'P-containing RNAs were encapsulated in LNP using a selfassembly process (Maier, et al. (2013) Mol. Ther., 21(8): 1570-1578). Briefly, an ethanolic lipid mixture of ionizable cationic lipid, phosphatidylcholine, cholesterol, and polyethylene glycol-lipid was rapidly mixed with an aqueous solution containing the mRNA at acidic pH. The RNA-loaded particles were characterized by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) and a Ribogreen assay. The mean hydrodynamic diameter of these LNP- mRNAs was approximately 80 nm with a poly dispersity index of 0.02-0.06 and an encapsulation efficiency of -95%. LNPs used in this study are proprietary to Acuitas Therapeutics (Vancouver, BC, Canada). The ionizable cationic lipid and LNP composition are described in US Patent No. 10,221, 127. To prepare antibody -targeted LNP-mRNA, LNP-mRNA were conjugated with purified rat anti -mouse CD117 (c-kit), clone 2B8 (BioLegend, 93235, San Diego, CA) via SATA-maleimide chemistry (Breda, et al. (2023) Science 381 (6656):436-443). Briefly, LNPs were modified with maleimide functioning groups (DSPE-PEG-mal) by a post-insertion technique. The antibody was functionalized with SATA (N-succinimidyl S-acetylthioacetate, 26102) from Thermo Fisher (Burlington, MA) to introduce sulfhydryl groups allowing conjugation to maleimide. SATA was deprotected using 0.5 M hydroxylamine, followed by removal of the unreacted components by Zeba spin desalting columns (Thermo Fisher Scientific, 89890, Waltham, MA). The reactive sulfhydryl group on the antibody was then conjugated to maleimide moieties using thioether conjugation chemistry. Purification was carried out using Sepharose CL-4B gel filtration columns (MilliporeSigma, GE17-0150-01, Burlington, MA). mRNA content was calculated by performing a modified Quant-iT™ RiboGreen RNA assay (Thermo Fisher Scientific, R11490, Waltham, MA). After adding the targeting ligand, all the targeted and non-targeted LNP preparations were kept at 4°C and used within three days of preparation.

[0115] RAL Diff-Quik Stain

[0116] Peripheral blood smears were created using a drop of freshly collected blood on a standard glass slide. Slides were allowed to air dry and then stained with the RAL Diff-Quik® stain (Siemens, Munich, Germany) according to the manufacturer’s instructions. Slides were placed in Diff-Quik® solution I for 5 seconds, blotted, and placed in Diff-Quik® solution II for 5 seconds and in Diff-Quik® solution III for 5 seconds before being washed in distilled water and allowed to air dry and imaged using an Olympus BX60 Microscope (EVIDENT, Olympus Scientific Solutions Americas DBA, Waltham, MA) and an Infinity2 Lumenera camera and Lumenera Capture software (Lumenera Corporation).

[0117] Prussian Blue staining

[0118] Bone marrow smears were created using a Cytospin™ 4 Cytocentrifuge (Thermo Fisher Scientific, Waltham, MA) using 100,000 cells / slide at 400xg for 5 minutes. The smears were subsequently stained for iron detection using an Iron Stain Kit (Prussian Blue Stain) (ab 150674) (Abeam, Cambridge, UK), following the manufacturer’s instructions. Imaging has been conducted using a Leica DM4000B upright imaging scope with a Leica DFC7000 T Camera and LAS X Life Science Microscope Software Platform (Leica Microsystems, Wetzlar, Germany).

[0119] Droplet Digital PCR (ddPCR) for Analysis of VCN and Alas2 deletion

[0120] Droplet Digital PCR (ddPCR) was performed using a BioRad QX200 Droplet Digital PCR System (BioRad, Hercules, CA). The Vector Copy Number analysis was performed with a double-probe assay, using Psi to detect the lentiviral Psi packaging sequence and a portion of the PCBP2 gene intron conserved among 17 species as the housekeeping gene (Makeyev, et al. (1999) J. Biol. Chem., 274(35):24849-24857; Christodoulou, et al. (2016) Gene Ther., 23(1): 113-118). Viral potency was calculated using the formula MOI = VCN x number of cells x Dilution Factor. The Alas2 allelic deletion was performed with a probe / primer set specific for the region between exon 5 and exon 6 o Alas2.

[0121] RNA Extraction and Quantitative Real-Time PCR Analysis

[0122] RNA extraction and quantitative real-time PCR (qRT-PCR) analyses were performed (Petrillo, et al. (2023) Angiogenesis 26(3):365-384). Briefly, total RNA was extracted using PureLink™ RNA Mini Kit (Thermo Fisher Scientific, Waltham, MA), and 0.5-1 ug of total RNA was transcribed into complementary DNA (cDNA) by High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA). qRT-PCR was performed using gene-specific TaqMan™ Gene Expression Assays (Thermo Fisher Scientific Waltham, MA). qRT-PCR was performed on a 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA), and the analyses were done using RQ Manager software. Transcript abundance, normalized to 18s messenger ribonucleic acid (mRNA) expression, is expressed as a fold increase over a calibrator sample.

[0123] Flow cytometry

[0124] Flow cytometry was performed using a BD FACSCalibur (BD Biosciences, Franklin Lakes, NJ) instrument. Erythroid populations were characterized using FITC Anti-Mouse CD71 (113806) and APC Anti-Mouse Teri 19 (116212), PE Anti- Human / Mouse CD44 (103008), as well as 7-AAD Viability Staining Solution (420404) (Biolegend, San Diego, CA). Donor chimerism was determined using PE Anti-Mouse CD45.2 (12-0454-82) (ThermoFisher, Waltham, MA) antibody for the donor and APC Anti -Mouse CD45.1 (110714) (Biolegend, San Diego, CA) antibody for the recipient. The flow cytometry results were analyzed using FlowJo™ vl0.8 Software (BD Life Sciences).

[0125] Electron microscopy

[0126] Tissues for electron microscopic examination were fixed with 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1M sodium cacodylate buffer, pH 7.4, overnight at 4°C. After subsequent buffer washes, the samples were post-fixed in 2.0% osmium tetroxide for 1 hour at room temperature and rinsed in DH2O before en bloc staining with 2% uranyl acetate. After dehydration through a graded ethanol series, the tissue was infiltrated and embedded in EMbed-812 (Electron Microscopy Sciences, Fort Washington, PA). Thin sections were stained with uranyl acetate and lead citrate and examined with a JEOL 1010 electron microscope fitted with a Hamamatsu digital camera and AMT Advantage image capture software.

[0127] Pathology assessment.

[0128] Four-micron-thick liver, spleen, and bone marrow sections were stained with hematoxylin and eosin for morphological assessment and Prussian blue stain to detect iron. The slides were scanned using the Aperio Versa 200 slide scanner, and a positive pixel count algorithm was used to quantify the level of iron accumulation in organs.

[0129] Enzyme-Linked Immunosorbent Assays (ELISAs) and Colorimetric Assays. Mouse Erythroferrone (ERFE) and Hepcidin were measured in the serum using Hepcidin Murine-Compete™ ELISA Kit (ERF-200) and Hepcidin Murine- Compete™ ELISA Kit (HMC-001), respectively (Intrinsic LifeSciences, La Jolla, CA). Transferrin was evaluated in the serum using a Mouse Transferrin ELISA Kit (ab 157724) (Abeam, Cambridge, UK). Erythropoietin (EPO) was measured in the serum using a Mouse Erythropoietin / EPO Quantikine ELISA Kit (MEPOOB) (R&D Systems, Inc., Minneapolis, MN). Iron, UIBC, and TIBC were evaluated using Iron / TIBC Reagents (1750460) (Pointe Scientific, Canton, MI). All the described tests were conducted according to manufacturers’ instructions.

[0130] Mitochondrial cristae analysis

[0131] The evaluation of the mitochondrial cristae has been performed using the open-source platform for biological-image analysis Fiji and the Trainable Weka Segmentation plug-in (Schindelin, et al. (2012) Nat Methods 9(7):676-682; Arganda- Carreras, et al. (2017) Bioinformatics 33(15):2424-2426).

[0132] Data analysis

[0133] Data analysis was performed using GraphPad Prism 10 Version 10.1.0 for MacOS, GraphPad Software, Boston, Massachusetts (graphpad.com).

[0134] Results

[0135] Generation of a conditional Alas2-KO transgenic line

[0136] It is expected that a complete knock-out of the Alas2 gene would be lethal (Nakajima, et al. (1999) EMBO J., 18(22):6282-6289; Peoc'h, et al. (2019) Mol. Genet. Metab., 128(3): 190-197). Therefore, an inducible conditional knockout (KO) model was pursued as a feasible alternative to reproduce a complete Alas2 -KO model. To this end, transgenic Alas2 mice were generated, which were designed with LoxP sites flanking a critical segment of the Alas2 mouse gene upstream and downstream of exon 5, as deletion of this exon is expected to result in the formation of a stop codon at the beginning of exon 6 and the loss of functional ALAS2 (Fig. ID). The mice carrying the LoxP sites were then crossed with the well -characterized B6.129- Gt(ROSA)26Sortml(cre / ERT2)Tyj / J(or R26-CreERT2) Cre strain (Feil, et al. (2009) Methods Mol. Biol., 530:343-363). In these mice, the Cre-ERT2(Cre recombinase - estrogen receptor T2) cassette was introduced into the ROSA locus. Tamoxifen (TAM) administration induces the translocation of Cre recombinase from the cytoplasm to the nucleus from the ERT2moiety by permitting the recombination of genomic LoxP sites. The Fl generation consisted of R26-CreERl 2-4 / a.s211mice, which were further crossed to obtain the R26-CreERT2-d / a.s211 Ygenotype or F2 generation. The screening process was carried out using primers identifying the LoxP sites and the R26-CreERT2sequences (Table 2).

[0137] Administration of TAM determined the development of anemia (Fig. 1A) and IE (Fig. IB). The IE in the BM and the spleen of the R b-CreER^ +M / a ^ Y mice was characterized by the expansion of the proerythroblast / basophilic erythroblast population (P1 / P2) and the polychromatic erythroid population (P3). In contrast, the orthochromatic / reticulocyte (P4) and RBC populations (P5) were reduced. KO mice were sacrificed or succumbed due to severe anemia. To rigorously establish that the observed phenotype was not solely attributed to TAM administration, TAM was administered in R26-CreI R I 2-4 / a.s'2 ' mice, which lack the flox sites. Upon treatment, only a temporary reduction in RBC (P5) was observed with concurrent expansion of the orthochromatic / reticulocyte population (P4), an effect that was reversed after 21 days. These alterations were consistent with the known effects of TAM usage and led to mild but transient anemia, as evidenced by subsequent recovery (Fig. 1 A). This controlled experiment reinforces the conclusion, demonstrating that the persistence of disease phenotype was independent of TAM administration. Having observed that complete deletion of Alas2 in mice showed erythroid-specific pathophysiology, deleting the Alas2 gene only in the hematopoietic compartment became the focus.

[0138] Alas2-K0 in the BM leads to IE and severe anemia

[0139] The first approach was based on the engraftment of R26-CreERT2-4 / a.s2 / 7' Lin" HSCs into lethally irradiated WT mice, namely B6.SJL-PtprcaPepcb / BoyJ or C57BL / 6-Tg(UBC-GFP)30Scha / J mice. Eight weeks post-transplantation, the mice were treated with the same dose of TAM used for the previous experiments. As an alternative approach, LNP particles embedded with a Cre mRNA and decorated with an antibody targeting the CD117 receptor (LNPCD117Cre) was used to induce the deletion of Ax A!as2 gene ex vivo in HSC (Breda, et al. (2023) Science 381(6656):436-443). Lin" HSCs A!as2nto LNPCD117Cre cultured cells showed high levels of Alas2 deletion after 24-hour and a complete absence of mA!as2 after 72-hour incubation with LNPCD117Cre (Fig. 1C). Using this approach, lethally irradiated wildtype (WT) mice were injected with Lin' HSCs Alas2 freshly treated ex vivo by LNPCD117Cre. To assess the extent of Alas2 gene deletion following either TAM administration or infusion of HSC treated with LNPCD117Cre, the abundance of the vaAlas2 allele in mouse BM was quantified by digital droplet PCR assay using a probe / primer set specifically designed to amplify the region between exon 5 and exon 6 of Alas2 (Table 3). The data showed nearly complete deletion of the floxed allele in the BM (92.2-96.3%) in both experimental cohorts. (Fig. 2A). Compared with TAM, the LNP -mediated delivery of Cre recombinase was more effective and faster in deleting the target gene.

[0140] 'able 3: Probes and primer sets used for vector copy number analysis (PSI) and

[0141] Alas2 allelic deletion analysis (KO). *PCBP2 has been used as a housekeeping probe / primer set.

[0142] Both experimental groups, engrafted with LNPCD117Cre treated Alas2'nHSC or R26-CreERl 2M / a.s2 / 7' HSC, showed that the deletion of the Alas2 gene led to the development of severe anemia. Of note, in animals transplanted with LNPCD117Cre treated Alas2'nHSC, the recipient mice received HSC already deleted of the Alas2 gene. In the animals engrafted with R26-CreERT2-d / a.s2 / 7' HSC, the deletion was induced with TAM 8 weeks post-engraftment. Regardless, the endpoints were comparable (severe lethal anemia, Figs. 2C and 2D). Importantly, the animals transplanted with R26-CreERl 2-d / a.s2 / 7' HSC and treated with TAM recapitulated the phenotype observed in animals were total body deletion of Alas2 was induced (Fig. 1A).

[0143] The KO animals exhibited pronounced anemia in all models, marked by diminished Hb levels and RBC count. Additionally, they exhibited an elevated reticulocyte (RET) count and decreased HCT levels (Figs. 2C and 2D, Table 1). Eventually, no animal survived without expression of Alas2. However, a difference in the onset of the anemia was observed between the two cohorts, given the faster development of the phenotype and earlier death observed in the LNPCD117Cre model (deletion induced in HSC in vitro, before engraftment) vs. the TAM model (deletion induced ~8 weeks post-BMT) (Fig. 2E). each experimental group. Each model includes animals treated (X-ALAS2-LV) and not treated (d / a.s2-KOB I) with X-ALAS2-LV (N = 8 in all experimental groups).

[0144] The BM and spleen cells were characterized by flow cytometry using specific Teri 19, CD71, and CD44 markers (Liu, et al. (2013) Blood 121(8):e43-49). The Alas2AAOBUphenotype was associated with a strong reduction in RBC, with an elevated number of polychromatic erythroblasts (P3) in the BM and spleen. (Fig. 3 A). Blood smears from these mice revealed a heterogenous population of RBC, including hypochromic RBC and not fully mature reticulocytes (Fig. 3B). The analysis of expression of A!as2, Alasl, and Tfrc in the erythroid populations showed a solid correlation between the expression of these genes and differentiation of the erythroid populations. In fact, the expression of Alas2 is directly correlated with the progressive differentiation of the erythroid populations, with the peak of expression in the orthochromatic erythroblasts (P4) (Fig. 3D). On the contrary, the expression of Alasl, the ubiquitous isoform ofd / a.s, is almost suppressed across the differentiation from proerythroblasts (Pl) to basophilic erythroblasts (P2). The Tfrc levels also correlate with the progression of differentiation of the erythroid population under partial regulation of erythropoietin, ensuring the survival and proliferation of erythroblasts, leading to the restoration of oxygenation through RBC production (Fig. 3D) (Richard, et al. (2020) Int. J. Mol. Sci., 21(24): 9713). These findings highlight the significance of Alas2, in contrast to Alasl, during this critical stage of erythroid development. These observations indicate that erythroid populations at this stage initiate the accumulation of iron, underscoring the importance of Alas2 in this iron homeostasis regulation. Furthermore, these results provide compelling support for the Alas2-KOBMphenotype, wherein the absence of Alas2 disrupts the differentiation of polychromatic erythroblasts (P3) into orthochromatic erythroblasts (P4) and, consequently, impedes the production of functional RBCs (Fig. 3D).

[0145] Alas2-KOBMmice showed severe splenomegaly, associated with abnormal color and consistency (Fig. 3C). The paler color (pink vs. red in WT mice) in the spleen of Alas2AAOBUmice likely indicated decreased heme synthesis and iron accumulation. The histological analysis showed extramedullary hematopoiesis in the liver and brownish pigment accumulation, consistent with hemosiderin formation. The most consistent alterations in the spleen were extramedullary hematopoiesis and lymphoid hyperplasia. In both organs, the hematopoiesis had a strong erythroid component. The most consistent alterations in the BM were erythroid hyperplasia and a similar brown pigmentation accumulation. In both organs, hematopoiesis is strongly skewed toward the erythroid lineage. In all organs of most mice, blood vessels frequently contained smaller erythrocytes and erythrocytes with a basophilic cytoplasm. This change was most prominent in the BM. In the liver, clusters of varying-sized cells are distributed randomly throughout the hepatic parenchyma, containing abundant cytoplasmic iron accumulation. These cells likely consist of Kupffer cells and other macrophages. Abnormal iron accumulation was observed in several tissues of mice lacking the Alas2 gene (Figs. 4-5). Notably, the iron staining of bone marrow tissues and smears confirmed the presence of ring sideroblasts, and the electron microscopy analysis revealed iron deposits in the mitochondria. To confirm the dysregulation of iron homeostasis, erythroferrone (ERFE) and hepcidin (HAMP) serum levels were evaluated. HAMP is the hormone controlling dietary iron intake, as well as plasma and total body iron levels, while ERFE is the negative erythroid regulator of HAMP and is expressed in erythroid cells under conditions of high erythropoietin (EPO) levels (Nicolas, et al. (2001) Proc. Natl. Acad. Sci., 98(15):8780-8785; Kautz, et al. (2014) Nat. Genet., 46(7):678-684). Diseases characterized by IE are generally associated with high levels of EPO and ERFE, and low levels of HAMP, leading to increased iron absorption and organ iron overload (Guerra, et al. (2023) Haematologica 108(10):2582-2593). Alas2- Q)BManimals showed increased ERFE and decreased HAMP levels, consistent with increased EPO and serum iron levels and organ iron overload (Figs. 6A-6B). Increased ferritin levels and transferrin saturation due to IE, increased gastrointestinal iron absorption, and subsequent systemic iron overload due to compensation for defective heme synthesis were also observed, leading to excess iron in both storage and circulating forms.

[0146] Alas2 Knock-Out causes a shutdown of the mitochondria in the polychromatic erythroid population

[0147] To further characterize the impact of Alas2-KO on the erythroid population and the consequent effect on RBC differentiation and production, metabolic assays focusing on isolated polychromatic erythroid cells in R26-CreERT2+ / +-zl / a52fl / Ymice that underwent TAM treatment (P3) were performed. P3 was focused on as this was the most abnormally elevated erythroid population (Fig. 3 A), which simultaneously showed a high proportion of apoptotic or dead cells (Fig. 7B). At the onset of the anemia (induced by TAM administration), the live P3 population in the BM was sorted using the FSC and the CD44 marker on Teri 19+ gated cells. Isolated P3 cells were then analyzed by ultra-performance liquid chromatography (UPLC). Moreover, the activity of ALASs, the key enzymes in the TCA cycle, the electron transport chain (ETC) complexes, was measured as well as the level of mitochondrial ATP (mtATP). As expected, Alas2 -KOBMP3 cells presented a significant and strong reduction of ALASs activity compared to the P3 population isolated from WT animals. Notably, the diminished ALASs activity correlated with the shutdown of oxidative metabolism, as indicated by the substantially lowered activity of pivotal TCA cycle enzymes, such as citrate synthase, a-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase, as well as by a considerable reduction in the activity of mitochondrial ETC complexes (Fig. 6B). In line with this, mtATP levels were also significantly decreased in the Alas2 -KOBMP3 population (Fig. 6B). The data are further supported by the electron microscopy analysis of the mitochondrial cristae, showing an increased area (p < 0.01) and perimeter (p < 0.05) with a typical perturbed unfolded shape in the Alas2-KOBMP3 population. In this nonphy si ologi cal scenario, the ETC super-complexes and dimers of adenosine triphosphate synthase disassemble, decreasing mitochondrial respiratory performance (Cogliati, et al. (2016) Trends Biochem Sci., 41(3):261-273). The analysis of the glycolytic profile in Alas2-KOBMP3 cells showed increased activity of glycolytic enzymes compared with the control, supported by transcriptional reprogramming.

[0148] Interestingly, the expression of some components of the respiratory chain complexes yielded opposite results compared with the enzymatic activity (Figs. 6B- 6D). This indicates a compensatory response to the reduced activity, possibly indicating an increase in gene expression related to the oxidative phosphorylation (OXPHOS) malfunction. These results further highlighted the altered erythropoiesis observed in the BM (Fig. IB, Fig. 3 A), underscoring the crucial role of ALAS2 in the differentiation and maturation of polychromatic erythroid cells to orthochromatic cells. The inability of Alas2-KOBMcells to robustly differentiate is likely to promote cell death, as indicated by the relative increase of proapoptotic genes in the P3 population (Fig. 7A). A further assessment showed an increased tendency toward inflammation in the P3 population (Fig. 7B). All these observations shed light on how the reduction of ALAS2 activity in hypomorphic patients disrupts the erythropoietic development, leading to diminished production of RBCs (Peoc'h, et al. (2019) Mol. Genet. Metab., 128(3): 190-197).

[0149] Generation of an erythroid-specific lentiviral vector to express the human Alas2 cDNA

[0150] To develop a definitive curative therapeutic option for severe forms of XLSA, a novel gene therapy lentiviral vector expressing the human ALAS2 was generated. The vector X-ALAS2-LV was designed to express the human ALAS2 gene, specifically the Homo sapiens 5'-aminolevulinate synthase 2, transcript variant 1 (NM_000032.5). To allow exclusive erythroid expression, the ALAS2 gene was cloned under the control of an erythroid-specific promoter (HBB promoter, 200 bp) into a lentiviral backbone (Breda, et al. (2021) Mol. Then, 29(4): 1625-1638). To ensure controlled expression of human ALAS2 cDNA in the erythroid compartment, the vector was designed to include additional sequences used to express the P-globin gene, such as the 3’ HBB enhancer (876 bp) and the hypersensitive sites 2 (HS2) (1435 bp) and 3 (HS3) (1202 bp) of the locus control region (LCR) (Fig. 2E) (Breda, et al. (2012) PLoS One 7(3):e32345; Roselli, et al. (2010) EMBO Mol. Med., 2(8):315-328). These sequences have been extensively studied and proven effective in achieving elevated levels of erythroid-specific expression both in vitro and in vivo (Roselli, et al. (2010) EMBO Mol. Med., 2(8):315-328). Moreover, the Ankyrin insulator (190 bp) was embedded in the vector’s 3’ self-inactivating long terminal repeat (sinLTR, Fig. 2F), as including this element reduces integration position effects on expression and genome toxicity, resulting in a safer vector overall (Breda, et al. (2012) PLoS One 7(3):e32345; Romero, et al. (2015) Mol. Ther. Methods Clin. Dev., 2: 15012; Burgess, et al. (2015) Nat. Rev. Genet., 16(3): 130-131; Rivella, et al. (1998) Semin. Hematol., 35(2): 112-125; Browning, et al. (2016) Biomedicines 4(1):4; Zhou, et al. (2017) Cytogenet. Genome Res., 151(2):72-81 ; Goodman, et al. (2018) J. Virol. 92(l):e01639-17; Rivella, et al. (2000) J. Virol., 74(10):4679-4687). To characterize and determine the optimal concentration of X-ALAS2-LV for in vivo application, vector potency was measured by transducing NH4-3T3 and HUDEP-2 cells, as shown in Fig. 8.

[0151] X-ALAS2 lentivirus rescues Alas2-KOBM

[0152] The X-ALAS2-LV vector’s ability to reverse the anemia caused in the Alas2- KOBMmurine models was tested. For the TAM model, R26-CreERl 2M / a.s2 / 7' Lin' HSCs were isolated. Half of the cells were exposed to 100-150 MOI of X-ALAS2- LV. Transduced and non-transduced cells were then, separately, infused into lethally irradiated B6.SJL-PtprcaPepcb / BoyJ or C57BL / 6-Tg(UBC-GFP)30Scha / J mice. As controls, untreated R26-CreERT2Lin' HSCs were transplanted into recipient animals. For the LNP model, Alas2" ' Lin' HSCs were isolated and incubated with LNPCD117Cre for 2 hours before transduction with X-ALAS2-LV (100-150 MOI). As controls, untreated Alas2'1' Lin' HSCs were transplanted into (lethally irradiated) recipient animals. Donor chimerism was evaluated eight weeks post-transplantation. In the TAM model, donor chimerism reached 90% (± 2.0) and 89.8% (± 3.1) in the treated and untreated animals, respectively, while in the LNPCD117Cre model, donor chimerism levels were 86.2% (± 2.7) and 89.0% (± 3.8) in the treated and untreated animals, respectively (Fig. 9A). The vector copy number (VCN) in BM and spleen in both the TAM and LNPCD117Cre experimental settings was stable up to 25 weeks posttransplantation (Fig. 2B). In the TAM settings, the VCN in the BM of X-ALAS2-LV treated animals treated with ranged between 0.5 and 1.5, while in the LNPCD117Cre counterpart, the VCN ranged between 0.5 and 1.1. Animals with VCNs lower than 0.6 showed extended lifespan compared to untreated animals, yet the expression of Alas2 produced by the transgene was insufficient to rescue them from anemia; hence, these animals were euthanized for analyses when Hb levels reached ~7.0 g / dL (Fig. 2E and Fig. 9B). In these animals, an expansion of the polychromatic erythroid (P3) population and mild splenomegaly was observed, with a phenotype intermediate between the complete KO and animals rescued with VCNs >0.6. This indicated that this limited amount of integrated copies of ALAS2 was insufficient to prevent the development of the Alas2 -KO erythroid cells and was unable to support the generation of sufficient RBCs to reverse the anemia. Conversely, animals with VCN exceeding 0.6 were successfully rescued from the severe anemic phenotype, a recovery sustained at the 25 weeks post-BMT pre-established endpoint (Fig. 2E). Only animals treated with X-ALAS2-LV showed high levels of ALAS2 expression. A direct correlation between VCN and Hb levels was observed (Fig. 9B).

[0153] X-ALAS2 significantly improves the IE and iron overload of the Alas2-KOBMmice X-ALAS2-LV Treated mice with VCN >0.6 exhibited increased levels of Hb and HCT, improved RBC count (Fig. 1C, Table 1), and significantly reduced reticulocyte levels (Fig. 1C, Table 1), averaging levels comparable with what would be considered transfusion-free survival in humans. Both treated models showed a decreased expansion of the polychromatic erythroid (P3) population and an increased number of RBC (P5). RBCs of treated mice were also morphologically more similar to those of WT (Figs. 3A-3B). The percentage of viable cells in P3 was also improved. Treated animals showed a remarkable improvement in Hb (10.3±0.5), RBC (6.5±0.3), and HCT (33.6±1.0) levels, reaching near-normal ranges.

[0154] Animals rescued with X-ALAS2-LV treatment had improved spleen / body- weight ratio, and their spleen color or consistency was comparable to those of WT mice (Fig. 3C). The histological analysis further supported these findings, where the spleens showed improved tissue architecture and definition between red and white pulp (Fig. 4A). Additionally, no iron was detected in the liver or the BM, indicating improvement in iron metabolism, while the spleen exhibited a slightly higher iron concentration, likely due to some levels of compensatory anemia (Fig. 4B; Fig. 5). Animals treated with X-ALAS2-LV also showed improvement in ERFE, HAMP, and EPO measures, trending closer to the parameters in WT animals (Fig. 6A). Moreover, no iron accumulation was detected in the mitochondria of the animals treated with X- ALAS2-LV. Animals treated with X-ALAS2-LV also showed an improved iron metabolism, trending closer to the parameters in WT animals. The mitochondrial and glycolytic metabolism in the treated animals showed improvement toward the WT phenotype (Figs. 6B-6C). Ring sideroblasts were also prevented. Interestingly, although the expression of glycolytic enzymes aligned with the restored activity, the transcriptional levels of specific critical components of the electron transport chain did not match their enzymatic activity, likely indicating specific and different compensatory responses to changes in metabolism (Fig. 6D). Nevertheless, a sensible improvement in the shape and area of the mitochondrial cristae was observed. The animals also exhibited a decreased expression of proapoptotic and inflammatory genes, as the reintroduction of ALAS2 conferred antiapoptotic and anti-inflammatory effects (Fig. 7A-7B).

[0155] Secondary BMTs confirmed integration of X-ALAS2-LV in long-term HSC. Notably, Hb, RBC, HCT, and reticulocyte levels in secondary recipients were comparable to those of the primary donors (Fig. 10A). Additionally, both groups (TAM and LNP cohorts) exhibited stable VCN levels, ranging from 1.2 to 1.4 in the bone marrow at 25 weeks post-transplantation (Fig. 10B). Animals’ survival and the efficacy of X-ALAS2-LV were corroborated by engraftment of the majority of donor (4 / a.s2-I<OB+X-ALAS2-LV) cells (Group 1 : 81.3±2.7%; Group 2: 74.2±5.0%) and the low levels of endogenous Alas2 in the recipient animals (Fig. 10C-10D). Secondary recipients showed improved and effective erythropoiesis and RBC production comparable to WT animals. These results highlight the persistent correction of the ALAS2-K0BMphenotype over multiple transplantation cycles. Altogether, this study demonstrated the remarkable efficacy of X-ALAS2-LV in mitigating, if not rescuing, the severe phenotype of Alas2AAOBUmice.

[0156] Production of metal-free protoporphyrin IX (mfPPIX) in K562 cells (clone Bl 1) transduced with certain vectors after erythroid differentiation is shown in Figure 11C. PPIX is the precursor molecule to heme. Transduction was performed to achieve pancellular expression - i.e., all the cells carried at least one copy of each vector. Table 4 provides values of mfPPIX presented as mean fluorescence intensity (MFI) or the positive fluorescent cell ratio (fluorocyte ratio, FR%). Figure 1 ID shows RBC of an XLSA animal rescued with X-ALAS2-LV (~1 VCN) showing production of mfPPIX compared to RBC from a WT animal. Figures 1 IE and 1 IF show the production of mfPPIX in XLSA animals. The control is transplanted wildtype without LV treatment. Different levels of mfPPIX produced are produced by each vector. Additional assessments both in K562 lines and mouse models can be performed to identify the most effective vectors to express ALAS2 under different genomic backgrounds.

[0157] Table 4

[0158] This study characterized the pathophysiology of a new murine model of XLSA and explored the therapeutic potential of ex-vivo HSC gene therapy using a novel lentiviral vector expressing the human ALAS2 gene. In contrast to the murine model, patients affected by XLAS usually carry missense mutations or in regulatory regions with partial loss of function or decreased ALAS2 expression, respectively (Fujiwara, et al. (2019) Free Radic. Biol. Med., 133: 179-185; Bekri, et al. (2003) Blood 102(2):698-704; Kaneko, et al. (2014) Haematologica 99(2):252-261; Camaschella, C. (2009) Semin. Hematol., 46(4):371-377; Furuyama, et al. (2002) Cell Mol. Biol., 48(1): 5- 10; Bergmann, et al. (2010) Pediatr. Blood Cancer 54(2):273-278; Camaschella, C. (2008) Br. J. Haematol., 143 (1): 27-38). In fact, except for nonsense mutations in clinically affected female carriers, in families with lethal ALAS2 mutations, no affected males were generally identified in the pedigree (Rose, et al. (2017) Br. J. Haematol., 178(4):648-651). In general, patients are male and become symptomatic at variable age, and typically before the age of 40 (Kaneko, et al. (2014) Haematologica 99(2):252-261; Fleming, M.D. (2011) Hematology Am. Soc. Hematol. Educ. Program 2011 :525-531; Furuyama, et al. (2003) Blood 101(11):4623-4624). XLSA is generally a relatively late-onset disease, while the model recapitulates the most rapid and severe hypothetical form of this disorder. Therefore, a vector that rescues a more severe model of the disease will be curative in patients with some ALAS2 activity.

[0159] To generate a severe Alas2-¥AA model, the deletion of the murine Alas2 gene was successfully induced by two independent approaches. Notably, the findings demonstrated comparable phenotypes between the TAM and the LNP models. However, the use of targeted LNP encapsulating mRNA encoding the Cre achieves similar results to that of TAM treatment, with the advantage of a more rapid and efficient process. This underscores the potential of targeted LNP -mediated Cre- mRNA delivery as an effective method for accelerating the study of hematological phenotypes and diseases.

[0160] Irrespective of the approach utilized, the study’s significant findings include establishing a robust anemic phenotype in mice through induced Alas2 gene deletion, characterized by reduced Hb levels, diminished RBC count, and IE. Interestingly, this is the first study elucidating the importance of ALAS2 in the polychromatic erythroblast population, highlighting the step of erythroid development in which ALAS2 activity and heme synthesis are most required. The lack of ALAS2 in this population was associated with an expansion in the number of cells at this stage of differentiation, with a high proportion of cells undergoing cell death. Importantly, it is worth noting that within these cells, a significant disruption in the electron transport chain was observed, resulting in low levels of mitochondrial ATP (mtATP). The diminished oxidative metabolism in the P3 population might be responsible for the hindered differentiation and maturation of these cells into orthochromatic ones. Indeed, it is well-established that HSCs, committed progenitors, and fully differentiated blood cells exhibit significant differences in both their metabolic characteristics and mitochondrial functions and that alterations in mitochondrial metabolism and activity are not merely passive consequences but active drivers of progenitors’ fate decisions (Papa, et al. (2019) Stem Cells Int., 2019:4067162; Vannini, et al. (2016) Nat. Commun., 7: 13125). More precisely, mitochondrial function and oxidative phosphorylation are critical to sustain the terminal phases of erythroid differentiation (Sen, et al. (2020) Exp. Hematol., 88:28-41). Finally, these findings are consistent with the importance of heme synthesis in triggering metabolic rewiring in various non-erythroid tissues (Fiorito, et al. (2021) Cell Rep., 35(11): 109252; Petrillo, et al. (2021) Biomedicines 9(11): 1557).

[0161] Furthermore, the study showed a strong correlation between anemia and altered iron metabolism, as it reported in a few XLSA patients in the absence of blood transfusions (Lira Zidanes, et al. (2020) Front. Physiol., 11 : 581386). In the mice, expansion in the polychromatic erythroblast population was associated with high ERFE levels, likely responsible for low HAMP synthesis. The histological analysis provided further insights, revealing consistent alterations in the liver, spleen, and BM of 4 / a.s2-KOB Imice, including extramedullary hematopoiesis and iron accumulation. This mouse model, along with transgenic lines, provides a comprehensive view of XLSA, capturing the cellular and physiological effects and enabling therapeutic intervention studies (Ducamp, et al. (2024) Blood 144(13): 1418-1432). Human cell lines offer detailed insights into cellular mechanisms specific to human erythropoiesis, but they lack the ability to model the disease’s systemic consequences (Ono, et al. (2022) Sci. Rep., 12(l):9024; Kaneko, et al. (2018) Exp Hematol., 65:57- 68. e2). Together, these models provide complementary value: the mouse model is more suitable for studying overall disease progression and therapy, whereas the human cell lines are ideal for investigating specific human cellular responses.

[0162] The fatal anemia caused by the complete KO of the Alas2 gene can be improved or corrected by X-ALAS2-LV. Treatment with X-ALAS2-LV was able to extend the survival of animals with VCN<0.6, although these mice eventually showed progressive anemia that required their sacrifice. In animals with VCN>0.6 instead, treatment with X-ALAS2-LV rescued disease phenotype, leading to medium-high levels of Hb synthesis (proportional to VCN), with improvement of splenomegaly and extra-medullary hematopoiesis. These animals also presented improved glycolysis and mitochondrial activity.

[0163] In animals with lower VCN, a good proportion of cells were likely still generating erythroid cells with absent ALAS2 activity, limiting the therapeutic potential of X- ALAS2-L V. Another possibility is that the human ALAS2 may not be as efficient as the mouse orthologue, hence hindering the therapeutic potential of X- ALAS2-LV in mice. Nevertheless, in animals with VCN>0.6, the improvement of EPO, ERFE, and HAMP levels, coupled with improved serum iron levels, highlighted the therapeutic effect of X-ALAS2-LV in ameliorating both erythropoiesis and iron homeostasis. This is crucial, as iron overload is a prominent feature of XLSA. Notably, animals rescued by X-ALAS2-LV showed VCN in the range of 0.6-1.4, indicating that a relatively small number of integrations were sufficient to rescue Alas2 -KOBManimals. These findings indicate that HSCs carrying relatively low VCN of the vector can be curative in XLSA patients who exhibit hypomorphic or residual expression of the Alas2 gene.

[0164] While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. A lentiviral vector comprising a nucleic acid molecule comprising: i) a 5’ long terminal repeat (LTR) and a 3’ LTR, wherein at least one of said LTR is self-inactivating; ii) an erythroid-specific promoter; iii) an insulator, wherein said insulator is within one of said LTR; and iv) a nucleic acid sequence encoding ALAS2.

2. The lentiviral vector of claim 1, further comprising an enhancer.

3. The lentiviral vector of claim 2, wherein said enhancer is a hemoglobin subunit beta (HBB) enhancer.

4. The lentiviral vector of any one of claims 1-3, further comprising a globin gene locus control region (LCR).

5. The lentiviral vector of claim 4, wherein said LCR comprises HS2 and HS3.

6. The lentiviral vector of any one of claims 1-5, further comprising a Woodchuck Post-Regulatory Element (WPRE).

7. The lentiviral vector of any one of claims 1-6, wherein said insulator is an ankyrin insulator.

8. The lentiviral vector of any one of claims 1-7, wherein said ALAS2 comprises a substitution mutation.

9. The lentiviral vector of claim 8, wherein the substitution mutation is selected from the group consisting of E242K, D263N, P339L, R375C, and R411H.

10. The lentiviral vector of any one of claims 1-7, wherein said ALAS2 comprises an ALAS1 heme regulatory motif.

11. The lentiviral vector of any one of claims 1-7, wherein said nucleic acid sequence encoding ALAS2 comprises at least one 5’UTR or 3’UTR from a different gene.

12. The lentiviral vector of claim 11, wherein said nucleic acid sequence encoding ALAS2 comprises at least one 5’UTR or 3’UTR from ALAS1 and / or HBB.

13. An isolated hematopoietic stem cell or CD34+ cell comprising a lentiviral vector of any one of claims 1-12.

14. A composition comprising the lentiviral vector of any one of the preceding claims and a pharmaceutically acceptable carrier.

15. A composition comprising viral particles, wherein the viral particles comprise the lentiviral vector of any one of the preceding claims.

16. A method of inhibiting, treating, and / or preventing anemia in a subject, said method comprising administering the lentiviral vector of any one of claims 1-12 to the subject or introducing the lentiviral vector of any one of claims 1-12 into hematopoietic stem cells or erythrocyte progenitor cells and delivering the hematopoietic stem cells or erythrocyte progenitor cells to the subject.

17. The method of claim 16, wherein the hematopoietic stem cells or erythrocyte progenitor cells are isolated from the subject to be treated.

18. The method of claim 16, wherein said anemia is congenital sideroblastic anemia.

19. The method of claim 16, wherein said anemia is X-linked sideroblastic anemia.

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