Polynucleotide construct for gene therapy of beta-hemoglobinopathies

Abstract

The present invention relates to a polynucleotide construct comprising a hemoglobin subunit alpha-hemoglobin subunit beta (HBA-HBB) hybrid gene construct for enhancing the potency of gene therapy for the β-hemoglobinopathies, particularly β-thalassemia and sickle cell disease (SCD). The polynucleotide construct comprising elements that are operatively connected to each other in the following sequence: HBB locus control region (HBB-LCR), HBA promoter, and β-like globin gene. Additionally, the HBA-HBB gene construct further comprises a microRNA-based short hairpin RNA (shRNAmir) targeting one of the endogenous HBA or HBB genes to increase the ratio of vector-encoded HBBT87Q to endogenous HBA and β-like globin chains while maintaining high vector titers.

Description

[0001] POLYNUCLEOTIDE CONSTRUCT FOR GENE THERAPY OF BETA-HEMOGLOBINOPATHIES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a polynucleotide construct for gene therapy of P- hemoglobinopathies.

[0004] BACKGROUND OF THE INVENTION -thalassemia arises from reduced or absent -globin chain synthesis due to mutations in human adult P-globin (hemoglobin subunit beta; HBB) gene or nearby regions. In the normal process of erythropoiesis, a-globin (hemoglobin subunit alpha; HBA) and P-globin are synthesized in equimolar quantities, forming tetrameric adult hemoglobin (HbA; a2p2). However, in p-thalassemia, a quantitative reduction or absence of p-globin chain synthesis disrupts the balance between a- and P-globin chains, leading to accumulation of unpaired a- globin in developing erythrocytes. Excess a-globin forms toxic aggregates, inducing apoptosis in immature red blood cells (RBCs). Severe forms of P-thalassemia (e.g., pO / pO-thalassemia or Cooley’s anemia) present as dyserythropoiesis and hemolytic anemia. Patients with transfusiondependent P-thalassemia (TDT), such as those with Cooley’s anemia and others similarly severe genotypes, face a fatal outcome if untreated, requiring regular blood transfusions every 3 to 4 weeks and lifelong iron chelation therapy. Sickle cell disease (SCD) arises from a single amino acid substitution (Glu6Val) caused by a single base substitution (A-T) in the first exon of the HBB gene. This leads to polymerization of sickle hemoglobin (HbS) upon deoxygenation. RBCs lose their flexibility, causing vaso-occlusive crises (VOCs), acute chest syndrome (ACS), chronic hemolytic anemia, and ultimately, multi-organ damage with early mortality. Palliative treatments for SCD comprise chronic transfusion, exchange transfusion, iron chelators, and disease -modifying agents.

[0005] Approximately 7% of the global population are carriers of hemoglobinopathies, particularly p-thalassemia and SCD. While allogeneic hematopoietic stem cell transplantation offers a curative option, its reliance on matched sibling donors limits accessibility. Alternatives like haploidentical or unrelated donor transplants pose immunological risks and have reduced efficacy in older patients or those with lesser donor-recipient histocompatibility. Consequently, gene therapy has emerged as a promising alternative to address these limitations.

[0006] There are several gene addition vectors that have been in clinical trials for gene therapy of HBB gene disorders, yet only one that has received market approval to date is LentiGlobin™ (bluebird bio), which has been commercialized under the names Zynteglo™ and Lyfgenia™ for the gene therapy of transfusion-dependent P-thalassemia and sickle cell disease, respectively. It comprises a lentiviral vector BB305 and relates the manufacturing process of ex vivo transduction of blood harvested CD34+ cells. LentiGlobin™’s BB305 is the lentiviral vector that comprises a therapeutic human HBB gene driven by the human HBB promoter and DNase I Hypersensitive Sites (HS) 2, 3 and 4 of the human HBB locus control regions (HBB-LCR). In addition, the human HBB gene in BB305 comprises a segmental deletion within its second intron and a mutation that results in expression of HBB protein with glutamine instead of threonine at amino acid position 87 (HBBT87Q) Efficacy of BB305 in treating TDT and SCD is hampered by limitations. In severe case of both diseases, many patients require multiple integrated vector copies to achieve therapeutic levels, yet some fail to attain a curative outcome. This limitation likely stems from variegated expression of therapeutic HBBT87Qgene in RBCs lineage due to random integration of vector copies in hematopoietic stem cells.

[0007] Previous efforts aimed at enhancing the potency of HBB construct and vectors have involved incorporating an shRNA against one of endogenous HBA RNA within a microRNA (mir- 30) embedded in the second intron of the human HBB187(2gene This modification aims to mimic the milder disease phenotype seen in patients inheriting a loss of 1 to 2 of the four HBA genes While this approach appeared promising, clinical trials with the BB305 vector have revealed unexpected complications in the context of natural deletions of the endogenous HBA genes. Two patients with a loss of two HBA genes exhibited persistent anemia and dyserythropoiesis despite reduced vaso-occlusive events. This outcome may be attributed to a massive excess of free P-like-globin, particularly pS-chain, which could damage RBC membranes, leading to dyserythropoiesis and hemolysis.

[0008] US12139720B2 describes a lentiviral vector design and its application for the treatment of sickle cell disease (SCD). The disclosed polynucleotide construct comprises the HBB locus control region (HBB-LCR), a hemoglobin subunit gamma (HBG) or a hemoglobin subunit delta (HBD) promoter, and a nucleotide sequence encoding a therapeutic protein, aiming to deliver corrective genes into hematopoietic stem cells. However, this document does not disclose the use of an HBA promoter, a P-like globin gene, or the incorporation of a microRNA-based short hairpin RNA (shRNAmir), which can enhance gene expression and increase vector titers - both critical factors for improving the efficacy of gene therapy.

[0009] Similarly, US6524851B1 discloses a hybrid nucleic acid molecule for gene therapy targeting erythroid lineage cells using various viral vectors, including retroviral, lentiviral, adenoviral, adeno-associated virus (AAV), and Semliki Forest virus. Nonetheless, it lacks details regarding the comparative design and optimization of multiple lentiviral vector constructs, controlled expression with low vector copy number (VCN), and normalization of gene expression relative to VCN, all of which are essential elements for effective and safe gene therapy.

[0010] Additionally, EP3737744B1 describes polynucleotide constructs and integrasedefective lentiviral vectors (IDLVs) for systemic expression of therapeutic proteins or RNAs. However, the system is not specifically designed to enhance gene therapy efficacy for the treatment of transfusion-dependent P-thalassemia (TDT) and SCD. Therefore, the present invention aims to provide a novel polynucleotide construct that enhances the potency of gene therapy for TDT and SCD through gene addition.

[0011] As stated above, clinical benefits with current P-globin gene therapy vectors, including LentiGlobin™ (Zynteglo™ / Lyfgenia™), are conditional on the presence of several lentiviral integrants per hematopoietic stem cell in most patients, which is a requirement that increases insertional -oncogenesis risk and inflates manufacturing costs.

[0012] Pathophysiological evidence indicates that the timing of therapeutic P-like chain synthesis is decisive: in transfusion-dependent P-thalassaemia, free a-globin precipitates at the basophilic -to-polychromatic transition and diverts HSP70 from its nuclear reservoir, permitting caspase-3-mediated cleavage of GATA-1 and aborting erythroid maturation (Arlet et al., Nature 2014); in sickle-cell disease HbS polymer nuclei emerge while hemoglobinization is still incomplete, leading to premature clearance of sickled reticulocytes and a secondary wave of stress erythropoiesis (Zhang et al., Sci. Rep. 2023). Clinical marrow biopsies obtained three months after infusion of the classical P-promoter vector in the HGB-205 LentiGlobin™ study confirmed persistent dyserythropoiesis despite impending transfusion independence, underscoring the inadequacy of a promoter that activates relatively late in differentiation (Magrin et al., Nat. Med. 2022).

[0013] Vector-engineering attempts to advance the onset of expression without compromising viral titer or peak output have met only partial success. The ALS20 cassette, which incorporates a HBB promoter flanked by a HS1 / 2 / 3 / 4 mini-LCR and ankyrin insulators, accelerates transcription by approximately one cell division and reduces Annexin-V-positive precursors but delivers only 60% of the per-copy plateau achieved by the full HBB promoter / HBB-LCR configuration (Breda et al., Mol. Ther. 2021). A forward-oriented HS1-5 design restores full per-copy expression and yields higher titers, yet it exhibits intron-2 instability: 15 to 30% of proviruses delete intron-2 unless a Rev-responsive element (RRE) module is inserted within intron-2, since only transcripts that retain intron-2 (and thus the RRE) can bind Rev, be exported, packaged, and form vector particles. This RRE-based “self-selection” module and the extended LCR bring the vector genome close to the 9 Kb packaging limit, thereby precluding the addition of complementary therapeutic features, duplicates HIV cis elements, and produces heterogeneous manufacturing lots (Uchida et al., Nat. Commun. 2019).

[0014] Because endogenous a-globin transcripts precede P-globin by one to two days in definitive erythropoiesis (Georgolopoulos et al., Nat. Commun. 2021), HBA promoter derivatives have been explored as temporal accelerants; however, decades of reporter-gene, transgenic-mouse, and cell-line work have established an apparently invariant ceiling of 35% to 45% of the HBB promoter peak (Amrolia et al., J. Biol. Chem. 1998; Chae & Kim, Mol. Cells 2003; Ryan et al., PNAS 1989), rendering this strategy unattractive for clinical translation.

[0015] The present invention overturns that paradigm by coupling an HBB-LCR core comprising HS2, HS3, and HS4 directly to HBA promoter derivatives driving cis-linked HBB gene derivatives, within a self-inactivating lentiviral backbone. While this configuration aims at retaining the early-activation profile characteristic of HBA promoters, it also unexpectedly restores peak HBB expression to the level obtained with native HBB promoter constructs, all without a measurable reduction in viral titer. Terminally differentiated erythroid cells derived from transduced human CD34+ progenitors exhibit per-copy HBB output indistinguishable from benchmark HBB promoter vectors, and syngeneic, myeloablated mice reconstituted with vector-modified stem cells produce circulating red cells whose HBB content matches that of HBB promoter controls. Importantly, the overall length of the therapeutic payload is not increased, thereby allowing the addition of complementary therapeutic nucleotide sequences, which include short-hairpin-RNA (shRNA) cassettes embedded within a microRNA (miR).

[0016] Accordingly, the disclosed vectors simultaneously address the timing deficit that limits current therapies and the expression shortfall that has historically accompanied HBA promoter usage, thereby enabling efficient, lower-burden correction of P-haemoglobinopathies without introducing new manufacturing or safety liabilities.

[0017] Therefore, the present invention provides a novel polynucleotide construct and a vector derived therefrom, both of which enhance the potency of gene therapy for TDT and SCD through gene addition. The polynucleotide construct and the vector can be utilized to genetically modify hematopoietic stem cells (HSCs) for the gene therapy of p-hemoglobinopathies.

[0018] SUMMARY OF THE INVENTION

[0019] The present invention relates to a novel polynucleotide construct for the gene therapy of P-hemoglobinopathies. Increased potency is achieved through a combination of a polynucleotide encoding a P-like globin protein, a hemoglobin subunit alpha (HBA) promoter to drive its expression, and elements of the hemoglobin subunit beta locus control region (HBB-LCR).

[0020] In a first embodiment of the invention, the present invention relates to a polynucleotide construct comprising elements operatively connected to each other in a following sequence: a) a hemoglobin subunit beta locus control region (HBB-LCR); b) a hemoglobin subunit alpha (HBA) promoter; and c) a P-like globin gene wherein the P-like globin gene comprises exons 1 to 3 and at least one intron, the nucleotide sequences of exons 1 to 3 downstream of the initiator codon being, in the aggregate, at least 95% identical to the corresponding exons of hemoglobin subunit beta (HBB), hemoglobin subunit gamma (HBG), hemoglobin subunit delta (HBD), or a recombinant sequence thereof; wherein the HBB-LCR comprises at least two segments, each sharing at least 95% sequence identity with DNase I hypersensitivity (HS) site segments selected from HS2 (SEQ ID NO: 1), HS3 (SEQ ID NO: 2), or HS4 (SEQ ID NO: 3); and wherein the HBA promoter shares at least 95% sequence identity with a segment of a mammalian HBA promoter.

[0021] In a second embodiment of the invention, the present invention relates to a vector for genetically modifying hematopoietic stem cells (HSCs) comprising the polynucleotide construct according to the present invention.

[0022] In a third embodiment of the invention, an ex vivo method for producing P-like globin protein in erythroid cells after gene transfer to hematopoietic stem cells (HSCs), the method comprising steps of:

[0023] (a) providing a population of HSCs ex vivo-,

[0024] (b) delivering the vector according to the present invention to transduce the HSCs ex vivo under conditions that permit stable genomic integration;

[0025] (c) administering the transduced HSCs to a subject; and

[0026] (d) allowing the transduced HSCs to undergo erythroid differentiation, thereby producing P-like globin protein in the erythroid progeny.

[0027] In a fourth embodiment of the invention, an in vivo method for producing P-like globin protein in erythroid cells after gene transfer to hematopoietic stem cells (HSCs), the method comprising steps of:

[0028] (a) providing a population of HSCs within a subject;

[0029] (b) delivering the vector according to the present invention to the subject under conditions that permit stable genomic integration, such that the HSCs are transduced in vivo; and (c) allowing the transduced HSCs to undergo erythroid differentiation, thereby producing P-like globin protein in the erythroid progeny.

[0030] BRIEF DESCRIPTION OF THE DRAWING

[0031] Figure 1 shows a schematic representation of the HBA and HBB loci, illustrating a- like (HBZ, HBA2, and HBA1) and p-like (HBE1, HBG2, HBG1, HBD, and HBB) globin genes. The corresponding globin chains encoded by each gene are shown below each box. Regulatory sites are indicated with grey boxes. Multispecies conserved sequences (MCS) with presumed cis-acting regulatory functions (Rl, R2, R3, and R4) of the HBA locus and the HBB- LCR are identified by DNase I hypersensitive sites (HS) in erythroid cells.

[0032] Figure 2 shows HBB-HBB construct of pBBBGl.

[0033] Figure 3 shows HBA-HBB construct of pBBBG6.

[0034] Figure 4 shows HBA-HBB construct of pBBBG8.

[0035] Figure 5 shows HBA-HBB construct of pBABG2.

[0036] Figure 6 shows HBA-HBB construct of pBABG5.

[0037] Figure 7 shows vector titers.

[0038] Figure 8 shows globin chain ratios in BBBG1 transduced erythroid cells.

[0039] Figure 9 shows a comparison of globin chain ratios in erythroid cells after transduction ofHSPCs with BBBG1 and BABG5 vector particles and subsequent liquid culture.

[0040] Figure 10 shows a comparison of globin chain ratios in erythroid cells after transduction of HSPCs with BBBG1 and BABG5 vector particles and culture in semi-solid medium for 7 days.

[0041] Figure 11 shows a comparison of globin chain ratios in erythroid cells after transduction ofHSPCs with BBBG1 and BABG5 vector particles and culture in semi-solid medium for 11 days. Figure 12 shows a comparison of globin chain ratios in erythroid cells after transduction of HSPCs with BBBG1 and BABG2 vector particles and culture in semi-solid medium for 12 days.

[0042] Figure 13 shows a comparison of HBA / HBB chain and mRNA ratios in erythroid cells after transduction of HSPCs with BBBG1 and BABG5 vector particles and culture in liquid medium for 12 days.

[0043] Figure 14 shows the level of HBBT87Qchains in red blood cells (RBCs) of mice that received hematopoietic stem cells transduced with BBBG1 and BABG2 vector particles.

[0044] Figure 15 shows the relative P-T87Q mRNA expression and P-T87Q mRNA kinetics.

[0045] DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention relates to a novel polynucleotide construct comprising a hybrid hemoglobin subunit alpha-hemoglobin subunit beta (HBA-HBB) gene construct. This is achieved by substituting an HBB promoter with an HBA promoter to drive the expression of HBBT87Qgene in the presence of HBB-LCR HS2, HS3, and HS4, resulting in vector titers equivalent compared to similar vector utilizing the HBB promoter. Notably, endogenous HBA gene expression precedes that of HBB in terminally differentiating erythroid cells. Furthermore, a microRNA-based short hairpin RNA (shRNAmir) cassette targeting one of the HBA genes is further comprised in said HBA-HBB gene construct. This addition aims to decrease the amount of toxic unbound a -globin - especially deleterious in P-thalassemia -, while maintaining vector titers. Similarly, a shRNAmir cassette targeting the HBB gene is further comprised in said HBA-HBB gene construct, while rendering the vector-encoded HBB gene derivative insensitive to it by means of 5 -point mutations in a known non-functional region of the 3’UTR. This complementary approach will be useful in the case of abnormal hemoglobins, which include HbS in sickle cell disease. The novel polynucleotide construct and the vector derived therefrom, according to the present invention, enhance the likelihood of achieving long-term cures in subjects with TDT or SCD. Additionally, the novel polynucleotide construct and the vector derived therefrom enable reaching a therapeutic threshold with few vector copies per hematopoietic stem cell. This approach may reduce the risk of residual dyserythropoiesis in TDT and residual tissue sickling in SCD, thereby mitigating potential long-term complications. Any embodiments depicted herein shall encompass modification to other aspects of this invention, unless stated otherwise.

[0047] Definitions

[0048] This invention contains a Sequence Listing which is presented in XML file format and submitted via the electronic filing system.

[0049] Technical terms or scientific terms used herein have definitions as understood by those having ordinary skills in the art unless stated otherwise.

[0050] Equipment, apparatus, methods, or chemicals mentioned here refer to those commonly operated or used by those skilled in the art, unless explicitly stated otherwise, that they are equipment, apparatus, methods, or chemicals specifically used in this invention.

[0051] The terms “a,” “an,” and “the” in the claims or the specification should be interpreted as “one” as well as “one or more,” “at least one,” and “one or more than one,” unless the context clearly dictates otherwise, and so forth.

[0052] The use of singular or plural nouns with the term “comprising” in the claims or the specification should be interpreted as “one” as well as “one or more,” “at least one,” and “one or more than one.”

[0053] All compositions and / or processes disclosed and claimed are intended to encompass aspects of the invention that involve actions, operation, modifications, or changes of any parameters without significantly deviating from experiments performed, examples described, or data shown in this invention, and obtaining similar objects with the same utilities and results as those described in the present invention by persons skilled in the art, even without specific mention in the claims. Therefore, substitutions or similar objects to the present invention, including minor modifications or changes that are apparent to persons skilled in the art, should be considered within the scope, and concept of the invention as defined by the appended claims.

[0054] Throughout this application, the term “about” and “approximately” are used to indicate that any value presented herein may potentially vary or deviate due to variety of factors, such as calculation errors, discrepancies in apparatus or methods, or differences between individual operators implementing the apparatus or methods.

[0055] The term “identity”, “homology”, or “similarity” between two different sequences refer to characteristics determine with reference to “aligned” sequences. “Aligned” sequences or “alignments” refer to multiple nucleic acid sequences or amino acid sequences, often adjusted for insertions or deletions (i.e., corrections for missing or additional bases or amino acids) when compared to a reference sequence.

[0056] The terms “sequence identity,” “percent sequence identity,” or “percent identical” in the context of nucleic acid sequences refer to the residues in the two sequences that are the same when aligned for maximum correspondence. Similarly, “percent sequence identity” may be readily determined for amino acid sequences, either over the full length of a protein, or a fragment thereof.

[0057] Alignments are performed using any of a variety of publicly or commercially available sequence alignment programs. Generally, such programs are used with default settings, although a person skilled in the art may alter these settings as needed. Alternatively, a person skilled in the art may utilize another algorithm or computer program that provides at least the same level of identity or alignment as the referenced algorithms and programs. Alignment algorithms may comprise a Needleman-Wunsch alignment, which performs a global alignment of two sequences (e.g., a sequence read and a reference sequence) using dynamic programming to compute an optimal score. The Needleman-Wunsch algorithm may also be used to detect rare variants and large insertions or deletions in a genome.

[0058] The description provided herein regarding particular nucleic acid and amino acid sequences, such as those detailed in the sequence listing, should be construed so as to also relate to modifications of said specific sequences resulting in sequences which are functionally equivalent to said specific sequences. For example, this includes amino acid sequences that have properties which are identical, similar, or share at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to those of the original sequences, as well as nucleic acid sequences encoding amino acid sequences with such properties or sequence identity.

[0059] The particular nucleic acid and amino acid sequences disclosed in the specification and the sequence listing were identified using sequence alignment programs. Nucleotide identity was determined using global Needleman-Wunsch alignment with optimized parameters, and amino acid identity was calculated using the same algorithm in combination with BLOSUM62 matrix.

[0060] Additionally, the materials, methods, and examples provided herein are for illustrative purposes only and are not intended to limit the scope of the present invention in any manner.

[0061] According to a first embodiment of the invention, a polynucleotide construct comprising elements operatively connected to each other in a following sequence: a) a hemoglobin subunit beta locus control region (HBB-LCR); b) a hemoglobin subunit alpha (HBA) promoter; and c) a P-like globin gene, wherein the P-like globin gene comprises exons 1 to 3 and at least one intron, the nucleotide sequences of exons 1 to 3 downstream of the initiator codon being, in the aggregate, at least 95% identical to the corresponding exons of hemoglobin subunit beta (HBB), hemoglobin subunit gamma (HBG), hemoglobin subunit delta (HBD), or a recombinant sequence thereof; wherein the HBB-LCR comprises at least two segments, each sharing at least 95% sequence identity with DNase I hypersensitivity (HS) site segments selected from HS2 (SEQ ID NO: 1), HS3 (SEQ ID NO: 2), or HS4 (SEQ ID NO: 3); and wherein the HBA promoter shares at least 95% sequence identity with a segment of a mammalian HBA promoter.

[0062] In an exemplary embodiment of the invention, the p-like globin gene comprises a deletion of at least 100 contiguous nucleotides within the second intron (IVS2).

[0063] In an exemplary embodiment of the invention, the p-like globin gene is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5. In an exemplary embodiment of the invention, the polynucleotide construct further comprises a 5' untranslated region (5' UTR) selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 7.

[0064] In an exemplary embodiment of the invention, the p-like globin gene comprises HBE1, HBG2, HBG1, HBD, and HBB.

[0065] In an exemplary embodiment of the invention, the HBB-LCR comprises segments of DNase I hypersensitive sites (HS) 2, HS3, and HS4 of the p-globin LCR.

[0066] In an exemplary embodiment of the invention, the HS2 is located on chromosome 11 at locations 8,228 to 8,865 (GenBank U01317.1), with a nucleotide sequence as shown in SEQ ID NO: 1.

[0067] In an exemplary embodiment of the invention, the HS3 is located on chromosome 11 at locations 4,280 to 5,121 (GenBank U01317.1), with a nucleotide sequence as shown in SEQ ID NO: 2.

[0068] In an exemplary embodiment of the invention, the HS4 is located on chromosome 11 at locations 1 to 1,145 (GenBank U01317.1), with a nucleotide sequence as shown in SEQ ID NO: 3.

[0069] In an exemplary embodiment of the invention, the HBA promoter further comprises one or more erythroid-specific transcription-factor binding sites (TFBS).

[0070] In an exemplary embodiment of the invention, the HBA promoter is selected from long HBA2 promoter or short HBA promoter with TFBS.

[0071] In an exemplary embodiment of the invention, the HBA promoter is selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9.

[0072] In an exemplary embodiment of the invention, the long HBA2 promoter is located on chromosome 16 at locations 33,329 to 33,738 (GenBank NG 000006.1), with a nucleotide sequence as shown in SEQ ID NO: 8. In an exemplary embodiment of the invention, the short HBA2 promoter with TFBS comprises a nucleotide sequence as shown in SEQ ID NO: 9.

[0073] In a preferred exemplary embodiment of the invention, the HBA promoter is a long HBA2 promoter.

[0074] In an exemplary embodiment of the invention, the deletion is located on chromosome 11 at locations 62,717 to 63,089 (GenBank U01317.1).

[0075] In an exemplary embodiment of the invention, the HBB protein comprises one or more amino acid substitutions from a group of T87Q, K82D, F41Y, G16D, or E22A.

[0076] In a preferred exemplary embodiment of the invention, the amino acid substitution is T87Q.

[0077] In a preferred exemplary embodiment of the invention, the P-like globin gene encodes a P-like globin polypeptide in which amino-acid residue 87 is glutamine (Gin).

[0078] In a preferred exemplary embodiment of the invention, the p-like globin gene comprises a nucleotide sequence as shown in SEQ ID NO: 4.

[0079] In an exemplary embodiment of the invention, said vector further comprises an HBA2- 5’ untranslated region which is located at locations 33,739 to 33,775 (GenBank NG 000006.1), with a nucleotide sequence as shown in SEQ ID NO: 6.

[0080] In an embodiment of the invention, said vector further comprises an HBB-5’ untranslated region as shown in SEQ ID NO: 7.

[0081] In an exemplary embodiment of the invention, the polynucleotide construct according to the present invention further comprises a microRNA-based short-hairpin RNA (shRNAmir) expression cassette.

[0082] In an exemplary embodiment of the invention, the shRNAmir expression cassette is inserted in place of the deletion within the second intron (IVS2) of the P-like globin gene. In an exemplary embodiment of the invention, the shRNAmir expression cassette is a miR30 derived shRNA expressing cassette selected from a group consisting of nucleotide sequences as shown in SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

[0083] In a preferred exemplary embodiment of the invention, the miR30 derived shRNA expressing cassette is the nucleotide sequence as shown in SEQ ID NO: 11.

[0084] In an exemplary embodiment of the invention, the P-like globin gene comprises a nucleotide sequence as shown in SEQ ID NO: 5.

[0085] According to a second embodiment of the invention, a vector for genetically modifying hematopoietic stem cells (HSCs) comprising the polynucleotide construct according to the present invention.

[0086] In an exemplary embodiment of the invention, said vector is selected from a group consisting of a type-C retroviral vector, a lentiviral vector, and a spumavirus vector.

[0087] In a preferred exemplary embodiment of the invention, said vector is the lentiviral vector.

[0088] In an exemplary embodiment of the invention, said vector further comprises a cytomegalovirus (CMV) promoter with a nucleotide sequence as shown in SEQ ID NO: 14.

[0089] In an exemplary embodiment of the invention, said vector further comprises a truncated HIV-1 5’ long terminal repeat (5’LTR) with a nucleotide sequence as shown in SEQ ID NO: 15.

[0090] In an exemplary embodiment of the invention, said vector further comprises a truncated lentiviral gag region with a nucleotide sequence as shown in SEQ ID NO: 16.

[0091] In an exemplary embodiment of the invention, said vector further comprises an HIV-1 env splice acceptor with a nucleotide sequence as shown in SEQ ID NO: 17.

[0092] In an exemplary embodiment of the invention, said vector further comprises an HIV-1 REV response element (RRE) with a nucleotide sequence as shown in SEQ ID NO: 18. In an exemplary embodiment of the invention, said vector further comprises an HIV-1 central polypurine tract / central termination signal sequence (cPPT / CTS) with a nucleotide sequence as shown in SEQ ID NO: 19.

[0093] In an exemplary embodiment of the invention, said vector further comprises a terminal polypurine tract (PPT) and a self-inactivating HIV-1 3’LTR, wherein said 3’LTR comprises a 400 base pairs deletion of the U3 with a nucleotide sequence as shown in SEQ ID NO: 20, an HIV R with a nucleotide sequence as shown in SEQ ID NO: 21, and a synthetic polyadenylation / termination sequence replacing the 3' U5 region with a nucleotide sequence as shown in SEQ ID NO: 22.

[0094] In an exemplary embodiment of the invention, said vector for use in a treatment of P-hemoglobinopathies by systemic administration, thereby producing, in erythroid cells of a subject, a P-like globin polypeptide encoded by the vector.

[0095] According to a third embodiment of the invention, an ex vivo method for producing P- like globin protein in erythroid cells after gene transfer to hematopoietic stem cells (HSCs), the method comprising steps of:

[0096] (a) providing a population of HSCs ex vivo-,

[0097] (b) delivering the vector according to the present invention to transduce the HSCs ex vivo under conditions that permit stable genomic integration;

[0098] (c) administering the transduced HSCs to a subject; and

[0099] (d) allowing the transduced HSCs to undergo erythroid differentiation, thereby producing P-like globin protein in the erythroid progeny.

[0100] According to a fourth embodiment of the invention, an in vivo method for producing P- like globin protein in erythroid cells after gene transfer to hematopoietic stem cells (HSCs), the method comprising steps of:

[0101] (a) providing a population of HSCs within a subject; (b) delivering the vector according to the present invention to the subject under conditions that permit stable genomic integration, such that the HSCs are transduced in vivo; and

[0102] (c) allowing the transduced HSCs to undergo erythroid differentiation, thereby producing P-like globin protein in the erythroid progeny.

[0103] In an exemplary embodiment of the invention, the HSCs comprise one or more surface phenotypes from a group of CD34+, CD133+, CD38Lo, CD90+, and CD45RA-.

[0104] In an exemplary embodiment of the invention, the HSCs are a P-thalassemia or SCD genotype HSCs.

[0105] EXPERIMENTS AND EXAMPLES

[0106] List of Abbreviations and Descriptions of Plasmids Used Herein o “BBBG1” refers to pBBBGl, the plasmid constructed by introducing a synthetic fragment SF85 between the Notl and Kpnl sites of pBLV. The pBLV contains a cytomegalovirus (CMV) promoter (SEQ ID NO: 14), a truncated HIV-1 5’ long terminal repeat (5'LTR) (SEQ ID NO: 15), a truncated lentiviral gag region (SEQ ID NO: 16), an HIV-1 env splice acceptor (SEQ ID NO: 17), an HIV-1 REV response element (RRE) (SEQ ID NO: 18), an HIV-1 central polypurine tract / central termination sequence (cPPT / CTS) (SEQ ID NO: 19), a terminal polypurine tract (PPT) followed by a self-inactivating HIV-1 3' LTR (SEQ ID NO: 20), an HIV R (SEQ ID NO: 21), and a synthetic polyadenylation / termination sequence replacing the 3’ U5 region (SEQ ID NO: 22). The synthetic fragment SF85 contains DNase I hypersensitive sites (HS) 4 (SEQ ID NO: 3), HS3 (SEQ ID NO: 2) and HS2 (SEQ ID NO: 1) of the HBB-LCR, HBB promoter (SEQ ID NO: 10), HBB-5 ’untranslated region (5’UTR) (SEQ ID NO: 7), HBB exon 1, intron 1, exon 2, intron 2 (deleted at the Rsal site), exon 3, and approximately 0.9 kb of 3’ non-transcribed HBB region. Exon 2 is mutated in codon 2 to produce HBBT87Qwith threonine at position 87 (HBBT87Q). o “BBBG6” refers to pBBBG6, the plasmid constructed by introducing a shRNAmir directed against human HBA1 (SEQ ID NO: 11), at the Rsal restriction site of HBB intron 2 in pBBBGl. o “BBBG8” refers to pBBBG8, the plasmid constructed by introducing the shRNAmir directed against human HBA2 (SEQ ID NO: 12), at the Rsal restriction site of HBB intron 2 in pBBBGl. o “BABG2” refers to pBABG2, the plasmid constructed by replacing HBB promoter (SEQ ID NO: 10) and HBB-5’UTR (SEQ ID NO: 7) in pBBBGl with HBA2 promoter (SEQ ID NO: 8) and HBA2-5’UTR (SEQ ID NO: 6), respectively. o “BABG2(short-TFBS)” refers to pBABG2(short-TFBS), the plasmid constructed by introducing the short HBA2 promoter with cis-linked TFBS (SEQ ID NO: 9) in pBABG2 in place of the long HBA2 promoter (SEQ ID NO: 8). o “BABG5” refers to pBABG5, the plasmid constructed by introducing the shRNAmir directed against human HBA1 (SEQ ID NO: 11), at the Rsal restriction site of HBB intron 2 in pBABG2.

[0107] Vector productions and vector titrations

[0108] Lentiviral vector particles are produced by co-transfecting a transfer vector with a packaging plasmid system including HPV275, p633, and TN 15 into HEK293T cells using PEIpro (Polyplus). Viral supernatants are harvested in approximately 1 mb, 7.5 mb, or 75 mb of culture medium, corresponding to the transfection vessel sizes of 6-well plates, 10cm petri dishes, or 22cm square dishes, respectively. The viral supernatants can be concentrated using a precipitation solution (Alston) or purified by ion exchange chromatography using Mustang™Q chromatography membrane, followed by concentration through precipitation.

[0109] While a lentiviral vector is selected for use in the following examples due to its well- established efficiency in transducing both dividing and non-dividing cells, it should be understood that other types of vectors may also be suitable for achieving similar results. These include, but are not limited to, type C retroviral vectors, spumaviral vectors, and alternative lentiviral vectors with different configurations. The choice of vector may vary depending on factors such as target cell type, desired duration of expression, payload capacity, and safety profile. Therefore, the invention is not limited to the use of lentiviral vectors and encompasses any vector system capable of delivering the desired genetic material effectively.

[0110] Following transfection, titrations are performed using diluted solutions of lentiviral vector particles in the presence of approximately IxlO6cells of NIH3T3 or HEK293T cell lines and about 8 pg / mL of protamine sulfate in 6-well plates for about 6 hours. The cells are then cultured and passaged for approximately 10 days. Vector copy number (VCN) is measured using quantitative polymerase chain reaction (qPCR), by comparison with a reference range of genomic DNA from NIH3T3 or KG1A cell lines containing one copy of the vector per haploid genome. Titer is calculated by multiplying the VCN by the number of cells present in the well at the time of transduction, then dividing by the volume of the vector used for transduction.

[0111] In the following examples, for any experiments involving BABG2, BABG2(short- TFBS) was also consistently used in parallel. Even if not explicitly stated, the use of BABG2 in the examples should be understood to include the use of BABG2(short- TFBS) as well.

[0112] Example 1

[0113] BBBG1 vector particles were produced by co-transfecting the BBBG1 vector (Figure 2) with a packaging plasmid system into HEK293T cells in three 22-cm square dishes. The viral supernatant, approximately 225 mL in volume, was collected from the cell culture, then purified using an ion exchange chromatography, precipitated, and resuspended to an approximate initial volume of about 1 mL. Titration on NIH3T3 cells was performed twice, both before and after purification / concentration, as shown in Figure 7A. The titers demonstrated that the BBBG1 vector particles exhibited a higher titer after purification / concentration than before.

[0114] Example 2

[0115] BABG5 vector particles were produced by co-transfecting the BABG5 vector (Figure 5) with a packaging plasmid system into HEK293T cells in three 22-cm square dishes. The viral supernatant, approximately 225 mL in volume, was collected from the cell culture, then purified using an ion exchange chromatography, precipitated, and resuspended in approximately 1 mL of the initial volume. Titration on NIH3T3 cells was performed twice after purification and concentration. The results from comparing the titers of BBBG1 and BABG5 vector particles are shown in Figure 7B, indicated that the BBBG1 vector particles exhibited a higher titer than the BABG5 vector particles.

[0116] Example 3

[0117] BBBG1, BABG2 (Figure 5), and BABG5 vector particles were produced by cotransfecting each vector with a packaging plasmid system into HEK293T cells in two wells of 6 well-plates for each vector. The viral supernatants from the cell culture were directly titered. Titration on NIH3T3 cells was performed twice. As shown in Figure 7C, the titers demonstrated that the BBBG1 vector particles exhibited the highest titer, followed by the BABG2 vector particles, although not significantly different from BBBG1, and then the BABG5 vector particles, which exhibited a gradual decrease in titer.

[0118] Example 4

[0119] BABG2 vector particles were produced by co-transfecting the BABG2 vector with a packaging plasmid system into HEK293T cells in three 22-cm square dishes. The viral supernatant, approximately 225 mb in volume, was collected from the cell culture, then purified using an ion exchange chromatography, precipitated, and resuspended in approximately one- thousandth of the initial volume. Titration on NIH3T3 cells was performed twice before purification / concentration (non-concentrated) and three times after purification / concentration (concentrated), as shown in Figures 7D. The titers demonstrated that the BABG2 vector particles exhibited a higher titer after purification / concentration than before .

[0120] Example 5

[0121] BBBG1, BBBG6 (Figure 3), BBBG8 (Figure 4) vector particles were produced by cotransfecting each vector with a packaging plasmid system into HEK293T cells in three wells of 6 well-plates for each vector. The viral supernatants from the cell culture were directly titered. Titration on HEK293T cells was performed three times (titration 1, titration 2, and titration 3). The titer results for BBBG1, BBBG6, and BBBG8 vector particles are shown in Figure 7E. The BBBG6 and BBBG8 vector particles have titers at least as high as, if not slightly higher than, that of BBBG1. Example 6

[0122] BBBG6 and BBBG8 vector particles were produced by co-transfecting each vector with a packaging plasmid system into HEK293T cells in three wells of 6 well -plates for each vector. The viral supernatants from the cell culture were directly titered. Titration on HEK293T cells was performed once. The results comparing the titers of BBBG6 and BBBG8 vector particles are shown in Figures 7F. The titers demonstrated that the BBBG6 vector particles exhibited a higher titer than the BBBG8 vector particles.

[0123] HBBT87Qgene expression in human erythroid cells

[0124] Example 7

[0125] Cord blood CD34+ cells were thawed and cultured in IMDM medium supplemented with approximately 15% BIT (Stemcell Technologies), approximately 1% PSG, approximately 100 ng / mL hSCF, approximately 5 ng / mL hIL3, and approximately 3U / mL hEpo (Peprotech). On Day 3, the CD34+ cells were transduced with each vector particle (BBBG1 or BABG5) at multiplicities of infection (MOIs) of 1 and 5 in the presence of protamine sulfate (about 8 pg / mL). On Day 4, the transduced cells were transferred in triplicate to the same medium without IL3. On Day 8, the transduced cells were transferred in the same medium without IL3 and SCF. After 12 days, erythroid cells were harvested, washed, and stored at about -80°C for subsequent DNA and globin chain analyses, performed using a high-performance liquid chromatography (HPLC).

[0126] The analysis of globin chain ratios as a function of VCN in erythroid cells transduced with BBBG1 vector particles is presented in Figure 8. The globin chain ratios in erythroid cells transduced with BABG5 vector particles were higher than those in erythroid cells transduced with BBBG1 vector particles, as shown in Figure 9, suggesting that under conditions of limited HBA availability, the exogenous HBBT87Qsubunit has a competitive advantage over endogenous HBB for pairing with HBA.

[0127] Curve fitting in Figure 8 was performed using data from the erythroid cells transduced with BBBG1 vector particles and the mean values obtained from non-transduced cells (NT). The mathematical model and constraints utilized included linear regression with Y0=0 (Figure 8A and 8B), hyperbola with Y0=0 and plateau < 1 (Figure 8C and 8D), and exponential decay with Y0 equal to the mean NT value and plateau > 0 (Figure 8E and 8F). This approach will also be employed in Figure 9 and further examples.

[0128] Example 8

[0129] Cord blood CD34+ cells were thawed and cultured as mentioned in Example 7. On Day 3, the CD34+ cells were transduced with each vector particle (BBBG1 or BABG5) at MOIs of 1, 2.5 and 5 for BBBG1, and at MOIs of 2, 4, 8 and 10 for BABG5, in the presence of protamine sulfate (about 8 pg / mL). On Day 4, the transduced cells were transferred in triplicate to methylcellulose-based medium HSC-CFU lite+Epo (Miltenyi Biotech) and to methylcellulose- based medium H4434 (Stemcell Technologies). On Day 7 and Day 11 after plating, erythroid cells were harvested, washed, and stored at about -80°C for DNA and globin chain analyses. Globin chain analysis was performed using the HPLC.

[0130] The analysis of globin chain ratios as a function of VCN in erythroid cells transduced with BBBG1 and BABG5 vector particles is presented in Figure 10 (Day 7) and Figure 11 (Day 11), which confirms that globin chain ratios are higher in erythroid cells transduced with BABG5 vector particles compared to those transduced with BBBG1.

[0131] Example 9

[0132] Cord blood CD34+ cells were thawed and cultured as described in Example 7. On Day 3, the CD34+ cells were transduced with each vector particle (BBBG1 or BABG2) at MOIs of 1, 5, and 20 in the presence of protamine sulfate (about 8 pg / mL). On Day 4, the transduced cells were transferred in triplicate to methylcellulose-based medium H4230 (Stemcell Technologies) supplemented with hSCF (50 ng / mL), hIL3 (10 ng / mL), and hEpo (3 U / mL) for 12 days. On Day 12 after plating, erythroid cells were harvested, washed, and stored at about -80°C for DNA and globin chain analyses. Globin chain analysis was performed using the HPLC and HBA / HBB mRNA ratios were analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

[0133] The analysis of globin chain ratios as a function of VCN in erythroid cells transduced with BBBG1 and BABG2 vectors is presented in Figure 12 and Figure 13A. The results demonstrate that the erythroid cells transduced with BBBG1 and BABG2 vector particles, after normalization to values measured in NT exhibited comparable globin chain relative to VCN. These results were confirmed at the transcriptional level, as there was no difference in the relative levels of expression of the HBA genes (HBA1 + HBA2) and the HBB genes (endogenous and exogenous) as shown in Figure 13B.

[0134] Curve fitting was performed using data from vector-transduced cells and the mean values obtained from NT, which were set at 1. The mathematical model and constraints utilized included an exponential decay with Y0=l plateau > 0.

[0135] Example 10

[0136] Hematopoietic cells from p-thalassemic mice (HBBthall) previously treated with 5- fluorouracil were transduced with the BBBG1 or BABG2 vector particles at MOIs of 1 or 5 in StemSpan medium with mIL3 (6.25 ng / mL), mIL6 (10 ng / mL), and mSCF (100 ng / mL). C57BL6 / J CD45.1 mice, which had been irradiated the previous day with two times 5.5 Gray (Gy), were administered by injection with approximately 5xl06transduced cells each. The VCN and globin chain expression were determined after three months by qPCR and HPLC, respectively. The residual HBB chain pSl""lefrom the C57BL6 / J CD45.1 mice, the HBB™nchain from HBBthall mice, and the HBBT87Qchain from the BBBG1 and BABG2 vector particles can be differentiated by the HPLC.

[0137] Analysis of HBBT87Qchain in donor red blood cells (RBCs) demonstrated that the C57BL6 / J CD45.1 mice from the BABG2 group exhibited lower VCNs compared to the BBBG1 group. However, statistical analysis of the HBBT87Qchain expression in donor RBCs as a function of VCN revealed no significant difference between the groups (Figure 14A and Figure 14B).

[0138] Curve fitting was performed using data from vector-transduced cells and the mean values obtained from NT. The mathematical model and constraints utilized hyperbola with Y0=0 and plateau < 100. After fitting each dataset using hyperbolic regression, the best-fit values of selected unshared parameters did not differ between datasets (p=0. 1898).

[0139] Example 11

[0140] The following working example was conducted to demonstrates the earlier onset of P-T87Q mRNA expression from an LCR / HBA promoter lentivector versus an LCR / HBB promoter lentivector control in transduced human CD34+-derived erythroid cultures. While it is widely established that the HBA promoter reproducibly drives expression of a variety of cislinked genes at least 1 cell cycle earlier than the HBB promoter during erythroid differentiation, and preliminary experiments confirm this contention.

[0141] The test vectors were BABG2 and BABG2(short-TFBS) comprising: (i) mini-LCR HS2, HS3, HS4 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3), (ii) long HBA2 promoter (SEQ ID NO: 8) or short HBA2 promoter with TFBS (SEQ ID NO: 9), (iii) HBA2 5'UTR (SEQ ID NO: 6), and (iv) HBB gene bearing the T87Q mutation, a native first intron, a second intron with a deletion of 374 contiguous nucleotides (SEQ ID NO: 4) or HBB gene bearing the T87Q mutation, a native first intron, a second intron with a deletion of 374 contiguous nucleotides, and a mutated 5'UTR (SEQ ID NO: 5). The BBBG1 vector was the control vector, identical to BABG2, except the HBA promoter was replaced by the HBB promoter (SEQ ID NO: 10) and its native HBB-5'UTR (SEQ ID NO: 7). Test cells were mobilized peripheral-blood CD34+ cells from three healthy donors (from local hospital after informed consent).

[0142] The culture reagents included phase I expansion medium (from Day 1): IMDM medium supplemented with approximately 15% BIT (Stemcell Technologies), approximately 1% PSG, approximately 50 ng / mL hSCF, approximately 10 ng / mL hIL3, and approximately 2U / mL hEpo (Peprotech); transduction (on Day 3) at several multiplicities of infection (MOIs) in the presence of protamine sulfate (about 8 pg / mL); phase II differentiation medium (from Day 4): same medium as phase I without IL3; phase III differentiation medium (from Day 8) same medium as phase I without IL3 or SCF. RT-qPCR reagents include RNeasy Mini kit (Qiagen) and TaqMan™ One-Step RT-qPCR Master Mix (Thermo Fisher). Primer / probe sets were as follows: T87Q-specific (spanning codon 87) Primer 1: 5'-TAGTGATGGCCTGGCT-3'; Primer 2: 5'-GATGGGCCAGCACACAG-3'; Probe: 5'-FAM-ACCTTTGCCCAGCTGAGT-BHQl- 3', and commercially available GAPDH primer / probe set (endogenous control).

[0143] Methods and experimental design were as follows: (1) About 1 x 10sCD34+ cells were amplified in Phase I medium. Cells were transduced at several MOIs (matched for both vectors) in the presence of about 8 pg / mL polybrene between Day 1 and Day 3, then wash and continued in fresh phase I medium for a total of 4 days; (2) Differentiation (Day 4 to 14): Cells were switched to phase II and phase III medium as described. About 2x 105cells were harvested two times on Days 4, 6, 8, 10, 12, and 14; (3) Vector Copy Number (VCN): Genomic DNA was extracted, and qPCR was performed using LTR and GAPDH primers to confirm equivalent VCN (target = 1 ± 0.2 copies / cell); and (4) RNA Isolation and RT-qPCR: Total RNA was extracted (RNeasy) from 2x l05cells at each timepoint. On-column DNase treatment was included. One-step RT-qPCR was performed in triplicate using the following cycling conditions: (i) about 50 °C for 15 min (RT), (ii) about 95 °C for 10 min (denature), and (iii) 40 cycles: about 95 °C for 15 s and about 60 °C for 1 min. T87Q Ct values were normalized to GAPDH (ACt), then to Day 2 control (AACt); and relative T87Q mRNA (2A-AACt) were plotted versus day for each vector.

[0144] The expected results are as follows: (1) Onset: The BABG2 vector and derivative are expected to exhibit a > 2-fold increase in T87Q mRNA by Day 4, whereas the BBBG1 control is expected to reach the same threshold by Day 6 (i.e. approximately 1 cell cycle earlier onset), and (2) Peak expression: Both vectors are expected to achieve comparable maximal T87Q mRNA levels by Day 10 (within 5% of each other when normalized to VCN).

[0145] These results confirm that incorporation of the human HBA promoter in place of the HBB promoter yields earlier transgene activation without compromising peak mRNA output, confirming the utility of the LCR / HBA promoter / p-T87Q design for rapid and robust HBB expression in therapeutic contexts.

[0146] In summary, the comparative analysis of the three lentiviral constructs according to this invention reveals a hierarchy of HBB transgene performance. The BABG5 vector, which couples the HBA promoter to the HBB cassette and simultaneously knocks down HBA via an HBA-targeting shRNAmir, yields the highest P-T87Q-to-endogenous HBB ratios. Although the HBA-targeting shRNAmir in BABG5 yields the highest P-T87Q expression, alternative constructs may optionally employ shRNAmir against HBB (SEQ ID NO: 13) instead, depending on the intended application. This enhancement is best explained by an earlier onset of P-T87Q expression driven by the HBA promoter, allowing the transgene to pair with the scarce pool of a-chains when they are most limiting, thereby outcompeting the endogenous HBB. The BABG2 vector (HBA promoter without shRNA) and BBBG1 vector (HBB promoter) produce indistinguishable globin-chain ratios within the timeframe sampled. It is inferred that any kinetic advantage conferred by the HBA promoter in BABG2 likely occurs at an earlier differentiation stage than the one captured in Day 7 and Day 11 measurements; once HBA becomes abundant, the competitive landscape equalizes, masking subtle temporal differences in transgene initiation. Since it is well established from an abundant literature that the HBA promoter confers an earlier expression pattern of a variety of cis-linked genes and reporters, as also anticipated in Figure 15 with the HBB gene, the entire family of the HBA- HBB vectors described in this application (i.e., HBB gene derivatives driven by the HBA promoter in the presence of the HBB-LCR) maintain vector titers, peak HBB expression levels, while affording earlier and beneficial initiation of HBB expression during erythroid differentiation.

[0147] The use of the polynucleotide construct and the vector derived therefrom, according to the present invention enhances the potency of gene therapy. Alternatively, the construct optionally incorporates a shRNAmir element, which can be included in the polynucleotide or its vector to further improve the genetic modification of HSCs. This modification holds promise for enhancing the efficacy of gene therapy and improving the likelihood of achieving long-term cures in subjects with TDT or SCD.

[0148] BEST MODE OF THE INVENTION

[0149] Best mode of the invention is as provided in the description of the invention.

Claims

CLAIMS1. A polynucleotide construct comprising elements operatively connected to each other in a following sequence: a) a hemoglobin subunit beta locus control region (HBB-LCR); b) a hemoglobin subunit alpha (HBA) promoter; and c) a P-like globin gene, wherein the P-like globin gene comprises exons 1 to 3 and at least one intron, the nucleotide sequences of exons 1 to 3 downstream of the initiator codon being, in the aggregate, at least 95% identical to the corresponding exons of hemoglobin subunit beta (HBB), hemoglobin subunit gamma (HBG), hemoglobin subunit delta (HBD), or a recombinant sequence thereof; wherein the HBB-LCR comprises at least two segments, each sharing at least 95% sequence identity with DNase I hypersensitivity (HS) site segments selected from HS2 (SEQ ID NO: 1), HS3 (SEQ ID NO: 2), or HS4 (SEQ ID NO: 3); and wherein the HBA promoter shares at least 95% sequence identity with a segment of a mammalian HBA promoter.

2. The polynucleotide construct of claim 1, wherein the P-like globin gene encodes a P-like globin polypeptide in which amino-acid residue 87 is glutamine (Gin).

3. The polynucleotide construct of claim 1, wherein the P-like globin gene comprises a deletion of at least 100 contiguous nucleotides within the second intron (IVS2).

4. The polynucleotide construct of claim 1, wherein the P-like globin gene is selected from the group consisting of SEQ IDNO: 4 and SEQ ID NO: 5.

5. The polynucleotide construct of claim 1, wherein the construct further comprises a 5' untranslated region (5' UTR) selected from the group consisting of SEQ IDNO: 6 and SEQ ID NO: 7.

6. The polynucleotide construct of claim 3, further comprising a microRNA-based short-hairpin RNA (shRNAmir) expression cassette.

7. The polynucleotide construct of claim 6. wherein the shRNAmir expression cassette is inserted in place of the deletion within the second intron (IVS2) of the P-like globin gene.

8. The polynucleotide construct of claim 1, wherein the HBA promoter further comprises one or more erythroid-specific transcription-factor binding sites (TFBS).

9. The polynucleotide construct of claim 1, wherein the HBA promoter is selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9.

10. A vector for genetically modifying hematopoietic stem cells (HSCs) comprising the polynucleotide construct of claim 1.

11. The vector of claim 10, wherein the vector is selected from the group consisting of a type-C retroviral vector, a lentiviral vector, and a spumaviral vector.

12. The vector of claim 10 for use in a treatment of P-hemoglobinopathies by systemic administration, thereby producing, in erythroid cells of a subject, a P-like globin polypeptide encoded by the vector.

13. An ex vivo method for producing P-like globin protein in erythroid cells after gene transfer to hematopoietic stem cells (HSCs), the method comprising steps of:(a) providing a population of HSCs ex vivo-,(b) delivering the vector of claim 10 to transduce the HSCs ex vivo under conditions that permit stable genomic integration;(c) administering the transduced HSCs to a subject; and(d) allowing the transduced HSCs to undergo erythroid differentiation, thereby producing P-like globin protein in the erythroid progeny.

14. An in vivo method for producing P-like globin protein in erythroid cells after gene transfer to hematopoietic stem cells (HSCs), the method comprising steps of:(a) providing a population of HSCs within a subject;(b) delivering the vector of claim 10 to the subject under conditions that permit stable genomic integration, such that the HSCs are transduced in vivo,' and(c) allowing the transduced HSCs to undergo erythroid differentiation, thereby producing P-like globin protein in the erythroid progeny.

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