This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional

Patent Application No. 62/868,224, filed June 28, 2019, and U.S. Provisional Patent Application No. 62/910,723, filed October 4, 2019. The foregoing applications are incorporated by reference herein. FIELD OF THE INVENTION

The present invention relates to the field of hematology. More specifically, the invention provides compositions and methods for the treatment of anemia.

BACKGROUND OF THE INVENTION

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.

Sideroblastic anemia occurs due to defects in the heme synthesis pathway. In addition, defects in iron sulfur pathways or other important pathways in the mitochondria of erythroblasts, which indirectly impair heme production, are responsible for the pathogenesis of sideroblastic anemia. A result of these abnormalities is decreased hemoglobin production and abnormal iron metabolism, leading to accumulation of iron in the nucleated immature erythroblasts. These erythroblasts have iron granule loaded mitochondria, which form rings around the nucleus, and are called ring sideroblasts. The exact mechanisms to explain why ring sideroblasts are produced in this type of anemia versus other types of anemia or disorders with iron overload (e.g., thalassemia or hemochromatosis) have not been clarified yet.

Sideroblastic anemia can be congenital or acquired with the latter being more common. There is a spectrum of severity on their effect on patient as well from mild life-long anemia to very severe transfusion-dependent anemia. Various types of sideroblastic anemias differ in terms of underlying mechanisms, symptoms and treatment. The unifying feature to all types is a defect in mitochondrial metabolisms related to iron utilization (Ducamp, et al., Blood (2019) 133(l):59-69; Bergmann, et ah, Pediatr. Blood Cancer (2010) 54(2):273-8). Another unifying feature is the ring sideroblasts around the nucleus, which are seen on bone marrow examination with Prussian blue stain and are the hallmark of sideroblastic anemia.

One of the congenital forms of sideroblastic anemia is due to defects in the gene SCL25A38 (Guernsey et al. (2009) Nature Genetics 41(6)651-653;

Kannenglessar et al. (2011) Haematologica 96(6):808-813). This is a gene for mitochondrial transporter, likely involved in bringing glycine into mitochondria, which is required for 5-aminolevulinic acid (ALA) production. This type is inherited in an autosomal recessive pattern and is usually a more severe anemia requiring chronic transfusion support.

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.

SUMMARY OF THE INVENTION

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 polyadenylation signal; iii) at least one promoter; iv) a locus control region (e.g., a globin gene locus control region (LCR)); v) an insulator (e.g., an ankyrin insulator element (Ank)); vi) a Woodchuck Post-Regulatory Element (WPRE) (e.g., wherein the WPRE is 3’ of the 3’LTR); vii) enhancer (e.g., beta globin 3’ enhancer; operably linked to the nucleic acid encoding the therapeutic protein); viii) a Rev response element (RRE) (e.g., from HIV); and/or ix) a sequence encoding a protein (e.g., a therapeutic protein). 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. In accordance with another aspect of the instant invention, methods of inhibiting, treating, and/or preventing bone marrow failure (BMF) syndromes (e.g., red blood cell (RBC) specific BMF syndromes) (see, e.g., Wegman-Ostrosky, et al., Br. J. Haematol. (2017) 177(4):526-542), dyserythropoietic syndromes, and/or anemias such as sideroblastic anemia (congenital or acquired sideroblastic anemia) in a subject are provided. In a particular embodiment, the method comprises

administering a nucleic acid or viral vector or viral paticle 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.

BRIEF DESCRIPTIONS OF THE DRAWINGS

Figure 1 provides schematics of an erythroid specific (Ery-SLC25A38) and constitutive (Con-SLC25A38; WPRE optional) lentiviral vectors backbones containing the SLC25A38 gene.

Figure 2 provides a Western blot of SLC25A38 expression in congenital sideroblastic anemia patient (3 patients) derived hematopoietic progenitor cells either untreated (patient 2) or treated with Con-SLC25A38 (patient 1, 2, or 3 + vector). CD34 ⁺ cells were harvested from three patients with SLC25A38 mediated congenital sideroblastic anemia (CSA). Transduction of CD34 ⁺ progenitor cells with constitutive expressing SLC25A38 vector showed expression of SLC25A38 at the expected protein size compared to no expression in the patient derived cells without vector. Superoxide dismutase 2 (SOD2) and calnexin are provided as controls.

Figure 3 provides a graph of the fold change in cell viability of congenital sideroblastic anemia patient derived hematopoietic progenitor cells either untreated or treated with Con-SLC25A38 (top). 48 hours after transduction, cells were cultured in erythroid differentiation media for 7 days after which they were analyzed for necrotic and apoptotic cells using Annexin-V and 7-AAE). There was a 12%, 36%, and 39% increase in the number of live cells in the vector transduced cells compared to non- transduced cells with the three patients. Figure 3 also provides an example of a fluorescence-activated cell sorting (FACS) analysis of cell viability using annexin and 7-aminoactinomycin D (7-AAD) (bottom). Figure 4 provides a Western blot of SLC25A38 expression in differentiating murine erythroleukemia (MEL) cells transduced with Ery-SLC25A38. The amount of vector is provided. Superoxide dismutase 2 (SOD2) is provided as a control.

Figure 5 A provides examples of amino acid sequences of human SLC25A38 (SEQ ID NO: 1 (top); SEQ ID NO: 2 (bottom)) and Figure 5B provides examples of nucleotide sequences encoding human SLC25A38 (SEQ ID NO: 3 (top); SEQ ID NO: 4 (bottom)). Figure 5C provides an example of an amino acid sequence of human ALAS2 (SEQ ID NO: 5) and Figure 5D provides an example of a nucleotide sequence encoding human ALAS2 (SEQ ID NO: 6).

Figure 6 provides an annotated sequence of ALS17 (SEQ ID NO: 7).

Figure 7A provides graphs of the proliferation rate (trypan blue assessment, top) and differentiation rate (benzidine staining assessment, bottom) of cord blood- derived 8E025A38 ^/_ hematopoietic progenitor cells (HPCs) versus control

SLC25A38 ^/+ HPCs. Cell size reduction, markers of differentiation (GPA) and maintenance of hematopoietic stem cells markers (c-KIT, and adhesion molecule CD44) were followed over time using flow cytometry analyses in SLC25A38 ^/ and control cells (Figure 7B). Apoptosis in hemoglobinized SLC25A38 ^/ and control erythroid cells were measured by annexin5 and 7AAD staining (Figure 7C).

Asterisks identify differentiation peaks. Morphological assessment of SLC25A38 ^/ erythroid cells on differentiation day 7 showed some iron deposition but not ringed sideroblasts by Prussian blue staining (Figure 7D).

Figure 8 provides an image of a Western Blot showing SLC25A38 expression in K562 cells, K562 SLC25A38 ^/ cells, and then a dose dependent expression in K562 SLC25A38 ^/ cells transduced with lx or 4x of constitutively expressing

SLC25A38 vector. Superoxide dismutase 2 (SOD2) is provided as a control.

Figure 9A provides an image of a Western Blot showing SLC25A38, HbA, and HbG expression in K562 cells (right four columns) and K562 SLC25A38 ^/ cells (left four columns) optionally transduced with the indicated amount of constitutively expressing SLC25A38 vector. Glyceraldehyde 3 -phosphate dehydrogenase

(GAPDH) is provided as a control. Figure 9B provides an image of a Western Blot showing SLC25A38, and ALAS2 expression in K562 cells (right four columns) and K562 SLC25A38 ^/_ cells (left four columns) optionally transduced with the indicated amount of constitutively expressing SLC25A38 vector. Glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) is provided as a control. Figure 9C provides reverse phase HPLC graphs of FASC standards (hemoglobin standards containing approximately equal amounts of HbA, HbF, HbS, and HbC; top left), parental K562 cells (bottom left), and K562 SLC25A38 cells optionally transduced with the indicated amount of constitutively expressing SLC25A38 vector (right). Figure 9D provides reverse phase HPLC graphs of FASC standards (top left), parental K562 cells (bottom left), and K562 SLC25A38 cells, K562 SLC25A38 cells treated with glycine, and parental K562 cells treated with glycine (right).

DETAILED DESCRIPTION OF THE INVENTION

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 ak, 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 ak, Curr. Treat. Options Oncol. (2017) 18(12):70; Dietz, et ak, Curr. Opin. Pediatr. (2016) 28(1):3-11; Feffault de Latour, et ak, Bone Marrow Transplant (2015) 50(9): 1168-72; Oved, et ak, 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/rejection 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).

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. However, the scarcity of each disease in isolation makes this less feasible. Herein, a novel lentiviral vector backbone with an interchangeable transfer gene cassette is provided so that it can be used in multiple RBC specific BMF syndromes.

Specifically, SLC25A38 mediated sideroblastic anemia (particularly congenital sideroblastic anemia (CSA)), an RBC BMF syndrome caused by a defect in heme biosynthesis, was utilized as a proof of principle for the vector. SLC25A38 mediated CSA is the most common autosomal recessive form of the disease.

SLC25A38 mutations, which are generally missense or indel changes located in splicing or coding regions, manifest with severe pyridoxine refractory CSA and require chronic RBC transfusions (Kannegiesser, et al., Haematol ogica (2011)

96(6): 808- 13). However, the protein is still not well characterized. SLC25A38 is known to provide glycine as a substrate for ALAS2 (5'-aminolevulinate synthase 2 or erythroid ALA-synthase) mediated heme biosynthesis and thus X-linked CSA associated with ALAS2 specific mutations is a target for curative therapy with the optimized vector backbone (Peoc’h et al., Mol. Genet. Metab. (2019) S1096- 7192(18)30632-2). Studies in zebrafish and yeast have shown that SLC25A38 ^/ cells exhibit severely impaired glycine transport (Fernandez -Murray, et al., PLoS Genet. (2016) 12(l):el005783; LeBlanc, et al., Pediatr. Blood Cancer (2016) 63(7): 1307-9; Dufay, et al., G3 (2017) 7(6): 1861-1873; Lunetti, et al., J. Biol. Chem. (2016)

291(38): 19746-59). However downstream pathophysiologic consequences resulting from this impairment that lead to erythropoietic failure remain poorly defined.

Herein, nucleic acids and viral vectors for the inhibition or treatment of bone marrow failure (BMF) syndromes (e.g., red blood cell (RBC) specific BMF syndromes), dyserythropoietic syndromes, and/or anemias such as sideroblastic anemia (particularly congenital sideroblastic anemia (CSA)) are provided. In a particular embodiment, the viral vector comprises: i) a 5’ long terminal repeat (LTR) and a 3’ LTR (particularly, at least one of the LTR (at least the 3’LTR) is self inactivating; a self-inactivating LTR comprises a deletion or mutation relative to its native sequence that results in it being replication incompetent); 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)) or a constitutive promoter (e.g., PGK promoter); the promoter may be in antisense orientation); and iii) a sequence encoding a therapeutic protein (optionally a sequence that is a reverse complement to a sequence encoding a therapeutic protein). In a particular embodiment, the promoter is an erythroid promoter. Since SLC25A38 is upregulated during early erythroid differentiation, a lentiviral vector with an erythroid specific promoter can correct the CSA phenotype more efficiently and with less off target effects than a vector with a constitutive promoter. In a particular embodiment, the vector further comprises one or more of: at least one polyadenylation signal (e.g., a strong bovine growth hormone polyA tail (e.g., inserted after the WPRE region) increases lentiviral titers (Zaiss, et al. (2002) J. Virol., 76(14):7209-19)); an enhancer (e.g., a beta globin 3’ enhancer; operably linked to the nucleic acid encoding the therapeutic protein); a locus control region (e.g., a globin gene locus control region (LCR)); at least one insulator element (e.g., an ankyrin insulator element (Ank) and/or foamy virus insulator; particularly, the insulator is within the 3’ LTR); a Woodchuck Post-Regulatory Element (WPRE) (e.g., configured such that the WPRE does not integrate into a target genome); and/or rev response element (RRE) (particularly, the RRE is from HIV; the RRE may be located between the 5’ LTR and the sequence encoding the therapeutic protein). In a particular embodiment, the viral vector comprises: i) a 5’ long terminal repeat (LTR) and a self-inactivating 3’ LTR; ii) a rev response element (RRE); iii) at least one promoter (e.g., operably linked or controlling expression of the therapeutic protein; optionally in reverse or antisense orientation); iv) a sequence encoding a therapeutic protein (optionally a sequence that is a reverse complement to a sequence encoding a therapeutic protein); v) a globin gene locus control region (LCR); vi) at least one insulator element (e.g., an ankyrin insulator element (Ank) and/or foamy insulator); vii) an enhancer (e.g., operably linked to the nucleic acid encoding the therapeutic protein); and, optionally, at least one polyadenylation signal. In a particular embodiment, the viral vector comprises: i) a 5’ long terminal repeat (LTR) and a self inactivating 3’ LTR; ii) a rev response element (RRE); iii) at least one promoter (e.g., operably linked or controlling expression of the therapeutic protein); iv) a sequence encoding a therapeutic protein (optionally a sequence that is a reverse complement to a sequence encoding a therapeutic protein); v) at least one insulator element (e.g., an ankyrin insulator element (Ank) and/or foamy insulator); vi) a Woodchuck Post- Regulatory Element (WPRE); and, optionally, at least one polyadenylation signal.

U.S. Patent Application Publication 2018/0008725 and PCT/US2019/029787, both applications are incorporated by reference herein in their entirety, provide viral vectors as well as sequences for certain of the above elements. Figure 6 provides the sequence of vector ALS17 (SEQ ID NO: 7) which provides an example of nucleotide sequences for certain of the above elements.

In a particular embodiment, the viral vector is Ery-SLC25A38 or Con- SLC25A38 (see, e.g., Figure 1), optionally wherein the SLC25A38 encoding nucleic acid is replaced with a sequence encoding a different therapeutic protein.

Viral vectors include, for example, retroviruses and lentiviruses. In a particular embodiment, the viral vector is a lentiviral vector. The viral vector may comprise one or more (or all) of the modifications listed below. In a particular embodiment, either Ery-SLC25A38 or Con-SLC25A38 (optionally with a sequence encoding a therapeutic protein other than SLC25A38) comprises one or more (or all) of the modifications listed below.

First, in certain embodiments of the instant invention, the therapeutic protein is SLC25A38 (solute carrier family 25 member 38) or ALAS2 (5'-aminolevulinate synthase 2). In a particular embodiment, the therapeutic protein is human. Examples of amino acid and nucleotide sequences of SLC25A38 are provided in GenBank Gene ID: 54977 and GenBank Accession Nos. NM_001354798.2, NP_001341727.1, NM_017875.4, and NP_060345.2. Figure 5A provides examples of amino acid sequences of human SLC25A38 and Figure 5B provides examples of nucleotide sequences encoding human SLC25A38. 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 a particular embodiment, the amino acid sequence of human SLC25A38 is SEQ ID NO: 2. In a particular embodiment, the nucleotide sequence encoding human SLC25A38 is SEQ ID NO: 4. In a particular embodiment, the amino acid sequence of SLC25A38 has 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 2. Figure 5C provides an example of an amino acid sequence of human ALAS2 and Figure 5D provides an example of a nucleotide sequence encoding human ALAS2. In a particular embodiment, 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, or 99% identity with SEQ ID NO: 5.

Second, the Woodchuck Post-Regulatory Element, or 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 6 (e.g., nucleotides 11190- 11805). 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).

Third, in certain embodiments of the instant invention, the vector may comprise insulators to maximize therapeutic protein expression at a random site of integration and to protect the host genome from possible genotoxicity. 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.

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 a 200bp 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). An example of a nucleotide sequence of the ankyrin insulator is provided in Figure 6 (e.g., nucleotides 10956-10768). 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).

Fourth, the LCR may be a globin gene locus control region (LCR). In a particular embodiment, the globin gene locus control region is a beta-globin gene locus control region. In a particular embodiment, the LCR comprises at least two, at least three, or all four of HS1, HS2, HS3, and HS4. In a particular embodiment, the LCR comprises HS2 and HS3. In a particular embodiment, the LCR comprises HS2, HS3, and HS4. In a particular embodiment, the LCR comprises HS1, HS2, HS3, and HS4. In a particular embodiment, the LCR is in antisense orientation. In a particular embodiment, only HS2, HS3, and HS4 of the LCR are in antisense orientation. An example of a nucleotide sequence of the LCR is provided in Figure 6 (e.g., nucleotides 6766-10641). In a particular embodiment, the LCR is operably linked to the promoter of the nucleic acid encoding the therapeutic protein.

Fifth, in certain embodiments of the instant invention, the vector comprises the Rev response element (RRE) from HIV (e.g., located near an LTR). The Rev response element (RRE) of HIV facilitates nucleo-cytoplasmic export of viral mRNAs (Sherpa et al. (2015) Nucleic Acids Res., 43(9):4676-86; incorporated by reference herein).

In certain embodiment, the nucleic acid or viral vector of the instant invention has a nucleotide sequence identical to those presented herein 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 or to an element of a nucleotide sequence of a viral vector disclosed herein (e.g., the sequences provided in U.S. Patent Application Publication 2018/0008725 and

PCT/US2019/029787 or Figure 5 or 6). In a particular embodiment, the nucleic acid or viral vector of the instant invention comprises the elements of either vector set forth in Figure 1, particularly in the orientation presented.

The present disclosure provides compositions and methods for the inhibition, prevention, and/or treatment of bone marrow failure (BMF) syndromes (e.g., red blood cell (RBC) specific BMF syndromes), dyserythropoietic syndromes, and/or anemias such as sideroblastic anemia (particularly congenital sideroblastic anemia (CSA)). In particular, the present disclosure provides novel nucleic acids and viral vectors for the inhibition, prevention, and/or treatment of bone marrow failure (BMF) syndromes (e.g., red blood cell (RBC) specific BMF syndromes), dyserythropoietic syndromes, and/or anemias such as sideroblastic anemia (particularly congenital sideroblastic anemia (CSA)). 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 SLC25A38 and/or ALAS2 gene.

In accordance with another aspect of the instant invention, methods of transducing cells with a nucleic acid or viral vector of the instant invention are provided. 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).

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 nucleic acid or 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 a bone marrow failure (BMF) syndrome (e.g., red blood cell (RBC) specific BMF syndrome), dyserythropoietic syndrome, and/or anemia such as sideroblastic anemia (particularly congenital sideroblastic anemia (CSA)). In a particular embodiment, the subject has congenital sideroblastic anemia (CSA). The viral vector may be administered in a composition further comprising at least one pharmaceutically acceptable carrier.

In accordance with another aspect of the instant invention, compositions and methods for inhibiting (e.g., reducing or slowing), treating, and/or preventing a bone marrow failure (BMF) syndrome (e.g., red blood cell (RBC) specific BMF syndrome), dyserythropoietic syndrome, and/or anemia such as sideroblastic anemia (particularly congenital sideroblastic anemia (CSA)) in a subject are provided. In a particular embodiment, the disease is congenital sideroblastic anemia (CSA). In a particular embodiment, the methods comprise administering to a subject in need thereof a nucleic acid or viral vector of the instant invention. The viral vector may be administered in a composition further comprising at least one pharmaceutically acceptable carrier. The nucleic acid or viral vector may be administered via an ex vivo methods wherein the nucleic acid or 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 a nucleic acid or viral vector of the instant invention to the cells, and administering the treated cells to the subject. The methods of the instant invention may further comprise monitoring the disease or disorder in the subject after administration of the composition(s) of the instant invention to monitor the efficacy of the method. For example, the subject may be monitored for characteristics of low heme or a bone marrow failure (BMF) syndrome (e.g., red blood cell (RBC) specific BMF syndrome), dyserythropoietic syndrome, and/or anemia such as sideroblastic anemia (particularly congenital sideroblastic anemia (CSA)).

The methods of the instant invention may further comprise the administration of another therapeutic regimen. For example, the methods may further comprise administering glycine, optionally with folate (WO 2014/108812), vitamin B6

(pyridoxine), and or an iron chelating agent (e.g., deferoxamine). In a particular embodiment, the method further comprises giving the subject a blood transfusion.

As explained hereinabove, the compositions of the instant invention are useful for increasing heme and/or hemoglobin production and for treating a bone marrow failure (BMF) syndrome (e.g., red blood cell (RBC) specific BMF syndrome), dyserythropoietic syndrome, and/or anemia such as sideroblastic anemia (particularly congenital sideroblastic anemia (CSA)). A therapeutically effective amount of the composition 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.

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. 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.

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 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 metabi sulfite), 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). 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.

Pharmaceutical compositions containing a compound 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.

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.

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.

Definitions

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

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.

A“carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabi sulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxilliary 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 ah, Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Rowe, et al., Eds., Handbook of Pharmaceutical Excipients,

Pharmaceutical Pr.

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.

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.

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 a bone marrow failure (BMF) syndromes (e.g., red blood cell (RBC) specific BMF syndromes), dyserythropoietic syndromes, and/or anemias such as sideroblastic anemia (particularly congenital sideroblastic anemia (CSA).

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

A“vector” is a genetic element, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication and/ or expression of the attached sequence or element. 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.

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

EXAMPLE

Figure 1 provides schematics of Ery-SLC25A38 and Con-SLC25A38. Ery- SLC25A38 is a lentiviral vector that carries the SLC25A38 gene under the regulation of the b-globin promoter and the hypersensitive sites HS2, HS3 of the Locus Control Region (LCR). It also contains an ankyrin insulator to prevent repression of

SLC25A38 expression. Con-SLC25A38 is a lentiviral vector that carries the SLC25A38 gene under the regulation of a mild constitutive 3 -phosphogly cerate kinase (PGK) promoter. It also included a foamy and ankyrin insulator to assist with protein expression.

The transduction of the Con-SLC25A38 vector (0.5 mΐ) into human cells led to the constitutive expression of SLC25A38 protein. Specifically, SLC25A38 protein expression was observed in transduced 3T3 and HUDEP-2 cells. Notably, the anti- SLC25A38 antibody from Abeam (Cambridge, United Kingdom) is specific to human SLC25A38 whereas an anti-SLC25A38 antibody from Sigma (St. Louis, MO) recognizes both human and murine protein. As seen in Figure 2, constitutive

SLC25A38 protein expression was observed in congenital sideroblastic anemia patient derived hematopoietic progenitor cells with human-specific anti-SLC25A38 antibody from Abeam. Specifically, cells from 3 patients were treated with virus at 3, 4, or 5 vector copy number (VCN), respectively. Increasing VCN led to greater expression compared to controls.

Con-SLC25A38 also increases cell viability of patient derived cells in a dose dependent manner. As seen in Figure 3, Con-SLC25A38 transduced congenital sideroblastic anemia patient derived hematopoietic progenitor cells have increased viability during erythroid differentiation in a dose dependent manner.

In addition to the above, it was determined that Ery-SLC25A38 expresses SLC25A38 in differentiating murine erythroleukemia (MEL) cells (Figure 4). Cells were transduced with virus and incubated for 48 hours. They were then induced to differentiate with hexamethylene bisacetamide (HMBA) and 5 days later cell lysates were collected for Western blot analysis.

SLC25A38 mediated CSA causes transfusion dependent anemia due to a defect in erythrocyte differentiation. SLC25A38 has not been well characterized in humans, though its expression has been shown to be increased in erythrocytes. Using established protocols, CD34 ⁺ hematopoietic stem cells were isolated from healthy donors, patients heterozygous for loss-of-function SLC25A38 mutations and patients with severe homozygous SLC25A38 457-lG>T CSA. Cells were cultured and maintained in an undifferentiated state to proliferate for 14 days in glycine containing media. They were then transferred to erythroid differentiation media for 7 days. During the proliferative state in which media is supplemented with human serum, plasma, transferrin, IL3, and human stem cell factor (hSCF), the cells from patients with CSA divided at the same rate as control cells (derived from a family member heterozygous for the 457-lG>T mutation), and even surpassed the proliferation rate of control cells at day 13 (Figure 7A, top). These cells, however, had a significantly slower proliferation rate (0.37x compared to control cells) when cultured in erythroid differentiation media. Benzidine staining showed decreased hemoglobinized cells in CSA versus control -derived cell cultures, indicating that SLC25A38 function is critical to enable completion of erythroid differentiation (Figure 7A, bottom). A flow cytometry analysis was used to analyze erythroid surface markers taken at 3 time points during proliferation and 2 time points during differentiation. Compared with control cells, the riZC25A3S-mutant CD34 ⁺ cells exhibited lower expression of glycophorin A (GPA) and retained higher expression of CD117 (c-KIT) out to day 7 in differentiation media, indicating less efficient erythroid differentiation (Figure 7B).

Using reverse-phase High Performance Liquid Chromatography (HPLC) and spectrophotometry on the hemolysate from 0.5 x 10 ⁶ benzidine positive SLC25A38- mutant erythroid cells, 50% of the hemoglobin content was observed in the hemolysate of CSA-derived cells compared to that seen in an equal number of control erythroid cells. Hemoglobinized CSA cells had a significantly higher rate of apoptosis (80%) compared to controls (30%) (Figure 7C). CSA-derived cells also exhibited iron deposition when stained with Prussian Blue (Figure 7D). Taken together, these data indicate that the SLC25A38 channel is necessary in late stages of erythroid differentiation and establish robust culture conditions.

In order to create a more versatile and rapid in vitro system for experiments aimed at characterizing and also correcting the CSA phenotype, K562 cells are being utilized, which are a myeloid leukemia cell line with the ability to differentiate to erythrocytes when stimulated with sodium butyrate. Three unique SLC25A38 ^/_ K562 clones have been generated with CRISPR/Cas9. These clones are unable to differentiate into erythrocytes or produce functional hemoglobin when exposed to sodium butyrate thus making them an ideal and versatile system for vector

optimization. K562 SLC25A38 knockout (KO) cells can be partially rescued by glycine (1.3 mM). These cells have been transduced with the vectors of the instant invention and protein expression has been observed (Figure 8).

SLC25A38 addition was also found to correct HbA but not HbG production in K562 KO cells (Figure 9A). Notably, SLC25A38 expression does not significantly alter ALAS2 expression (Figure 9B). Reverse-phase High Performance Liquid Chromatography (HPLC) was used to analyze the hemoglobin in SLC25A38 K562 knockout cells. As seen in Figure 9C, SLC25A38 K562 knockout cells have an additional tetramer that is corrected with SLC25A38 gene addition. Supratherapeutic glycine was also found to correct the SLC25A38 KO tetramer changes (Figure 9D).

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.