Lentiviral vector for delivery of PKLR to treat pyruvate kinase deficiency

Abstract

To provide polynucleotide cassettes, expression vectors, and methods for expressing a gene in mammalian cells to provide gene therapy for pyruvate kinase deficiency (PKD).SOLUTION: Provided are: an expression cassette for PKD, in which a PGK promoter sequence is operably linked to a codon-optimized sequence having a specific sequence encoding a human PKLR gene product, the expression cassette comprising a woodchuck hepatitis virus posttranscriptional regulatory element (wPRE), a polypurine tract (PPT), and a polyadenylation (polyA) signal sequence; a lentiviral vector comprising the expression cassette; and treatment methods using vector-containing hematopoietic erythroid progenitor cells, viral vectors, or recombinant cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 573,037, filed October 16, 2017, which is incorporated herein by reference in its entirety.

[0002] Description of electronically submitted text files The sequence listing associated with this application is provided in text format in lieu of hard copy and is incorporated herein by reference. The filename of the text file containing the sequence listing is ROPA_004_01WO_SeqList_ST25.txt. This text file is 29 KB, was created on October 15, 2018, and has been submitted electronically via EFS-Web.

[0003] FIELD OF THE INVENTION The present invention relates to gene therapy for pyruvate kinase deficiency. [Background technology]

[0004] Background of the Invention Pyruvate kinase deficiency (PKD) is a metabolic monogenic disorder caused by mutations in the PKLR gene that results in variable hemolytic anemia and can be fatal in the neonatal period. The recessive inheritance of PKD and its curative treatment by allogeneic bone marrow transplantation provide an ideal scenario for the development of gene therapy approaches.

[0005] Among many other genetic enzyme defects affecting red blood cells, pyruvate kinase deficiency (PKD) is the most common, causing chronic nonspherocytic hemolytic anemia (CNSHA) (Zanella et al. 2007). PKD's onset and severity vary widely, ranging from mild to severe neonatal anemia, with the most severe cases being fatal during infancy (Pissard et al. 2006). Neonatal growth retardation, hydrops fetalis, and death have also been reported at low frequencies (Gilsanz et al. 1993). The prevalence of PKD is estimated at 1:20,000 in the general Caucasian population (Beutler et al. 2000), and more than 195 distinct mutations in the PKLR gene have been identified to date (http: / / www.lovd.nl / pklr).

[0006] Although allogeneic bone marrow transplantation (BMT) has been successfully used to cure patients with severe PKD (Tanphaichitr et al. 2000), the low availability of histocompatible donors and the severe complications associated with BMT in these patients (i.e., graft-versus-host disease, opportunistic infections, etc.) have made regular blood transfusions and splenectomy the primary treatment options for most severe forms of PKD (Zanella et al. 2005), dramatically increasing patient morbidity and mortality (Hilgard et al. 2005). The limited efficacy and side effects of these treatment options for patients with severe PKD, as well as the recessive inheritance of PKD, make PKD a suitable disease to be treated with gene therapy.

[0007] PKD is caused by a deficiency of the pyruvate kinase (PK) enzyme, which catalyzes the final ATP-producing reaction in the glycolytic pathway in all cells (Zanella 2005). PK becomes essential in mature red blood cells because RBCs express only the R-type specific isoform (RPK) under the control of an erythroid-specific alternative promoter for the PKLR locus (Noguchi et al. 1987) (Kanno et al. 1992). Therefore, any loss of RPK activity reduces RBC metabolism and lifespan (Zanella 2005) and leads to CNSHA.

[0008] The promising approach for treating and preventing hereditary and other diseases and disorders is to deliver therapeutic drugs, including gene therapy vectors.Currently, viral vectors show the highest efficiency in gene transfer, and for the cure of hereditary diseases that require continuous gene expression, the vectors based on herpesvirus, retrovirus, lentivirus, adenovirus or AAV are preferred due to the integration nature of viral life cycle.

[0009] Gene therapy for monogenic diseases, particularly those affecting the hematopoietic system, has provided compelling evidence that gene repair in autologous hematopoietic stem cells (HSCs) is an alternative therapeutic option to allogeneic HSCT, avoiding its major complications (Cartier et al., 2009; Cavazzana-Calvo et al., 2010; Cartier et al., 2012; Aiuti et al., 2013; Biffi et al., 2013). Gene repair for diseases affecting red blood cells, such as β-thalassemia and sickle cell disease, has been addressed in animal models (Pestina et al., 2009; Breda et al., 2012) and in humans (Cavazzana-Calvo et al., 2010). However, gene therapy approaches for inherited red blood cell metabolic deficiencies, such as PKD, remain limited.

[0010] The feasibility of HSC gene therapy for PKD has been demonstrated in both murine (Tani et al., 1994; Meza et al., 2009) and canine RPK-deficient experimental models (Trobridge et al., 2012), demonstrating that given the lack of selective advantage of gene-corrected HSCs, the level of donor chimerism or gene-modified cells is a critical point for achieving efficient correction of the hemolytic phenotype (Richard et al., 2004). Previous studies using PKD mouse models demonstrated that retroviral-derived human RPK expression can completely correct the PKD phenotype when more than 25% of gene-corrected cells are transplanted (Meza et al., 2009). A similar therapeutic threshold of repaired cells was recently reported in a PKD Basenji dog that was expanded in vivo and fused with HSCs repaired with a foamy vector (Trobridge et al., 2012).

[0011] There are still many problems remaining in the design of polynucleotide cassette and expression vector for use in gene therapy.One important problem is to obtain sufficient expression of transgene in target cell.The long-standing unmet need in the art is the sufficiently robust expression of transgene after gene transfer.In some cases, more efficient expression is necessary for the effectiveness of certain vectors, such as plasmid DNA vectors.In other cases, more efficient gene expression cassette is desired to enable lower therapeutic doses with a more favorable safety profile or less invasive administration route.

[0012] High levels of transgene expression can be achieved using gammaretroviral (gamma-RV) vectors due to the fact that therapeutic transgene expression is regulated by their long terminal repeats (LTRs). However, the first clinical trials based on this type of vector raised safety concerns because patients unexpectedly developed leukemia (Hacein-Bey-Abina et al., 2008). The strong promoter activity of LTRs can affect the regulation of surrounding genes by activating proto-oncogene promoters or by inhibiting tumor suppressor genes, resulting in insertional mutagenesis (Ott et al., 2006; Howe et al., 2008; Stein et al., 2010; Braun et al., 2014). These findings emphasize the need for the use of safer and more efficient vectors than gamma-RV vectors for PKD gene therapy. Summary of the Invention

[0013] In one embodiment, the invention provides an expression cassette comprising a polynucleotide sequence comprising: a) a promoter sequence; b) a sequence encoding a gene product; and c) a ribonucleic acid (RNA) export signal; wherein the promoter sequence is operably linked to a sequence encoding a pyruvate kinase polypeptide; and optionally, a) through c) are present in the expression cassette in 5' to 3' order. In certain embodiments, the promoter is a phosphoglycerate kinase (PGK) promoter. In some embodiments, the gene product is a therapeutic gene product. In some embodiments, the therapeutic gene product is a pyruvate kinase (PK) polypeptide, optionally a pyruvate kinase, liver and erythroid (PKLR) polypeptide. In certain embodiments, the sequence encoding the gene product is codon-optimized. In some embodiments, the sequence encoding the gene product is a codon-optimized version of human pyruvate kinase cDNA having at least 85% identity to SEQ ID NO:8. In certain embodiments, the RNA export signal is a mutated woodchuck hepatitis virus post-transcriptional regulatory element (wPRE).

[0014] In certain embodiments, the mutated wPRE is a chimeric wPRE comprising a sequence having at least 80% identity to SEQ ID NO:24. In some embodiments, the expression cassette further comprises one or more enhancer sequences. In some embodiments, the expression cassette further comprises a polypurine tract (PPT) or a polyadenylation (polyA) signal sequence. In some embodiments, the expression cassette further comprises one or more of the following sequences: i) a packing signal sequence, ii) a truncated Gag sequence, iii) a Rev response element (RRE), iv) a central polypurine tract (cPPT), v) a central terminal sequence (CTS), and vi) optionally, an upstream sequence element (USE) from simian virus 40 (SV40-USE). In some embodiments, the expression cassette further comprises 5' and 3' long terminal repeat (LTR) sequences.

[0015] In a related embodiment, the present invention provides a recombinant gene delivery vector comprising the expression cassette disclosed herein. In certain embodiments, the recombinant gene delivery vector is a virus or viral vector. In certain embodiments, the virus or viral vector is a lentivirus (LV).

[0016] In another related embodiment, the invention provides a cell comprising an expression cassette or gene delivery vector disclosed herein. In some embodiments, the cell is a blood cell. In some embodiments, the cell is an erythrocyte. In some embodiments, the cell is a bone marrow cell, e.g., a lineage-depleted bone marrow cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a CD34+ hematopoietic stem cell. In some embodiments, the cell is a committed hematopoietic erythroid progenitor cell.

[0017] In yet another related embodiment, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a recombinant gene delivery vector or cell disclosed herein.

[0018] In another embodiment, the present invention provides a method for treating or preventing a disease or disorder in a subject in need thereof, comprising providing to the subject an expression cassette, gene delivery vector, or pharmaceutical composition disclosed herein. In one embodiment, the disease or disorder is PKD, and the gene product is a PK polypeptide, optionally a PKLR polypeptide. In certain embodiments, the pharmaceutical composition comprises a recombinant gene delivery vector. In other embodiments, the pharmaceutical composition comprises cells. In one embodiment, the cells are autologous to the subject.

[0019] In a related embodiment, the present invention provides a method for expressing a transgene in red blood cells, comprising contacting one or more red blood cells with an effective amount of a recombinant viral vector comprising a human PGK promoter, a codon-optimized version of a human PKLR cDNA transgene, and a mutated wPRE, wherein after said contacting, PKLR is expressed at a detectable level in the one or more red blood cells. [The present invention 1001] 1. An expression cassette for pyruvate kinase deficiency (PKD), comprising: (a) a promoter sequence, and (b) Codon-optimized sequence encoding the human PKLR gene product a polynucleotide sequence comprising, in 5' to 3' order: the promoter sequence is operably linked to a codon-optimized sequence encoding the human PKLR gene product; the codon-optimized sequence encoding the human PKLR gene product shares at least 95% identity with SEQ ID NO:8; Expression cassette. [The present invention 1002] 1001. The expression cassette of the present invention, wherein the promoter is a phosphoglycerate kinase (PGK) promoter. [The present invention 1003] The expression cassette of invention 1001 or invention 1002, further comprising a mutated woodchuck hepatitis virus post-transcriptional regulatory element (wPRE). [The present invention 1004] 1003. The expression cassette of the present invention, wherein said mutated wPRE is a chimeric wPRE comprising a sequence having at least 99% identity to SEQ ID NO:24. [The present invention 1005] The expression cassette of any one of 1001 to 1004 of the present invention, further comprising a polypurine tract (PPT). [The present invention 1006] The expression cassette of any one of 1001 to 1005, further comprising a polyadenylation (polyA) signal sequence. [The present invention 1007] 1001-1006. An expression cassette of any of claims 1001-1006, which shares at least 95% identity with SEQ ID NO:26. [The present invention 1008] 1007 expression cassettes of the present invention which share at least 99% identity with SEQ ID NO:26. [The present invention 1009] A recombinant gene delivery vector which is a lentiviral vector and comprises any one of the expression cassettes of the present inventions 1001 to 1008. [The present invention 1010] The following array: (a) a 5'LTR, optionally a modified 5'LTR; (b) HIV-1ψ sequence; (c) RRE, (d) cPPT / CTS sequence; (e) a PGK promoter sequence, optionally a human PGK promoter sequence; (f) a codon-optimized sequence encoding a human PKLR gene product having at least 99% identity to SEQ ID NO:8; (g) a mutated wPRE sequence having at least 100% identity to SEQ ID NO:24; (i) modified 3'LTR, (j) SV40 poly(A) signal; (k) SV40 origin of replication (ori), (u) a CMV enhancer sequence, and (v) CMV promoter sequence 1009. A recombinant gene delivery vector of the present invention, comprising, in 5' to 3' order: [The present invention 1011] The recombinant gene delivery vector of the present invention 1009, which is a therapeutic gene delivery vector. [The present invention 1012] The recombinant gene delivery vector of the present invention 1011 has long-term stability and safety capability in clinical use. [The present invention 1013] A cell comprising any one of the expression cassettes of the present inventions 1001 to 1008 or any one of the recombinant gene delivery vectors of the present inventions 1009 to 1012. [The present invention 1014] The cell of the present invention 1013, which is a hematopoietic stem cell. [The present invention 1015] The cells of the present invention are committed hematopoietic erythroid progenitor cells. [The present invention 1016] A pharmaceutical composition comprising a pharmaceutically acceptable excipient and any one of the recombinant gene delivery vectors of the present inventions 1009 to 1012 or any one of the cells of the present inventions 1013 to 1015. [The present invention 1017] A method for treating or preventing pyruvate kinase deficiency (PKD) in a subject in need thereof, comprising providing to said subject a pharmaceutical composition of the present invention. [The present invention 1018] 1017. The method of claim 1017, wherein said pharmaceutical composition comprises said recombinant gene delivery vector. [The present invention 1019] 1017. The method of claim 1017, wherein said pharmaceutical composition comprises said cells. [The present invention 1020] 1020. The method of claim 1019, wherein said cells are autologous to said subject. [The present invention 1021] A method for expressing the human PKLR gene in red blood cells, comprising contacting one or more red blood cells with an effective amount of a recombinant viral vector, wherein the vector comprises a human phosphoglycerate kinase promoter, a codon-optimized PKLR transgene, and a mutated woodchuck hepatitis virus post-transcriptional regulatory element, wherein the codon-optimized PKLR transgene shares at least 95% identity with SEQ ID NO:8, and wherein after the contacting, PKLR is expressed at a detectable level in the one or more red blood cells. [Brief explanation of the drawings]

[0020] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

[0021] [Figure 1] A scheme reflecting the location of different elements present in the backbone of an exemplary LV vector is displayed. [Figure 2] Figure 2A is a schematic diagram of the exemplary self-inactivating (SIN) LV vectors used throughout the gene therapy experiments, with the human PGK promoter controlling expression of either the EGFP transgene in the control vector (top panel) or the codon-optimized sequence of the PKLR gene cDNA (coRPK) in the therapeutic vector (bottom panel). Figure 2B is a schematic diagram of the gene therapy protocol implemented to address the functionality of the developed PGK-coRPK LV vectors. [Figure 3A]Figures 3A-3D display data demonstrating the restoration of the PKD phenotype in the peripheral blood of primary recipients after gene repair. Figures 3A and 3B show RBC and reticulocyte levels in healthy mice (black bars, n = 5) and PKD anemic mice (gray bars, n = 6), as well as in PKD anemic mice transplanted with EGFP (white bars, n = 9) or coRPK-transduced cells (scratched bars, n = 17). Data are presented as mean ± SEM and analyzed by nonparametric Kruskal-Wallis tests. Figure 3C shows the flow cytometry strategy used to detect biotin-labeled RBCs over time, and Figure 3D shows the survival kinetics of RBCs in healthy mice (black line, n = 2), anemic mice (gray line, n = 2), and gene-repaired mice (discontinuous line, n = 4). Data are presented as mean ± SEM and analyzed by two-tailed ANOVA tests. Healthy non-transplanted control mice; PKD, non-transplanted PKD mice; coRPK, PKD mice expressing a therapeutic transgene. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 4A] Figures 4A-4C show multilineage hematopoietic reconstitution in secondary transplanted mice. Figure 4A is a diagram of the flow cytometry strategy used to identify different hematopoietic lineages by labeling with CD3-PE, B220-PE, B220-PE-Cy5, Gr1-biotin, and Mac1-biotin antibodies plus SAV-PE-Cy5. Figure 4B displays representative dot plots, and Figure 4C displays the proportion of each lineage in the PB at 140 days post-transplant. Bars represent the mean proportions ± SEM of healthy (n = 2, black bars) and PKD (n = 2, gray bars) controls and secondary transplanted mice expressing the coRPK therapeutic transgene (n = 4, scratched bars). [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 5A]Figures 5A-5D display the restoration of the PKD phenotype in secondary transplanted mice. Figure 5A shows brilliant cresyl blue staining of blood smears from non-transplanted mice and secondary recipients to identify reticulocytes (in blue). Figure 5B is a flow cytometry analysis of reticulocyte levels in peripheral blood. Figure 5C represents the ratio of RBCs, and Figure 5D represents the ratio of reticulocytes in secondary transplanted mice expressing the coRPK transgene (scratched bar, n = 4), healthy mice (black bar, n = 3), and anemic control mice (gray bar, n = 3). Data are presented as mean ± SEM and analyzed by the nonparametric two-tailed Mann-Whitney test. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6A] Figures 6A-6C show quantification of proviral integration. Figure 6A shows vector copy numbers per cell in BM CFU from individual transplanted mice at 120 and 170 days post-transplant. Transduction and chimerism rates are also shown. Figure 6B shows proviral copy numbers in cells from different hematopoietic compartments. Columns represent the mean ± SEM for different groups of transplanted mice. Figure 6C shows the kinetics of proviral integration in BM cells from individual transplanted EGFP-expressing mice (gray line) and mice carrying the coRPK transgene (black line). [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 7A]Figures 7A-7C show the normalization of erythroid differentiation patterns in gene-corrected mice. Figure 7A shows the ratios of different erythroid subpopulations in the bone marrow and spleen at 140 days after transplantation. Figure 7B shows a representative dot plot of the flow cytometry strategy used. The expression intensity of CD71 and Ter119 markers allows the identification of four erythroid subpopulations. Population I is early proerythroid (Ter119medCD71high), population II is basophilic erythroblasts (Ter119highCD71high), population III is late basophilic and polychromatic erythroblasts (Ter119highCD71med), and population IV is normochromatic erythroblasts, reticulocytes, and mature erythroid cells (Ter119highCD71low). Figure 7C shows plasma Epo levels measured by ELISA in non-transplanted and transplanted mice. Dots represent values ​​for individual mice. Lines represent mean ± SEM, which was analyzed by the nonparametric Kruskal-Wallis test. Healthy non-transplanted control mice; PKD, non-transplanted PKD mice; EGFP, PKD mice expressing the EGFP transgene; coRPK, PKD mice expressing the therapeutic transgene. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 8] Figures 8A-8B show hematopoietic progenitor cell assays in control mice and mice transplanted with transduced cells. Data illustrate total CFU from the spleen (Figure 8A) and bone marrow (Figure 8B) at 140 days post-transplant. Points represent the number of colonies per mouse analyzed, and lines represent the mean ± SEM for each group. Data were statistically analyzed by the nonparametric Kruskal-Wallis test. [Figure 9A]Figures 9A-9C illustrate the reversion of splenomegaly and organ pathology in gene-corrected mice at 140 days post-transplant. Figure 9A shows a representative spleen photograph, and Figure 9B shows the ratio of spleen weight to total body weight from primary and secondary transplanted PKD mice. Points represent individual mouse values. Lines represent group mean ± SEM. Data were analyzed by the nonparametric Kruskal-Wallis test. Figure 9C shows histological examination of spleens and livers from primary transplanted PKD mice. Columns 1 and 2 show representative tissue sections of spleens and livers stained with hematoxylin-eosin, photographed under a light microscope using 4x and 10x objectives, respectively. Arrows point to erythroid cell clusters, indicating extramedullary erythropoiesis. Column 3 shows Prussian blue staining (Fe) of liver sections to detect iron deposits, indicated by arrowheads. Photographs were taken using a 20x objective. Groups are as described in Figure 7. Secondary coRPK, secondary recipients. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 10A]Figures 10A-10G display metabolic profiling in RBC samples from mice transplanted with gene-repaired cells. Analysis of significant metabolic profile changes in healthy and transplanted mice by comparing PKD animals in two independent experiments. Figure 10A shows a complete RBC heatmap obtained by untargeted profiling, in which higher and lower metabolite levels are represented in red and blue, respectively. Listed metabolites have at least one comparison that is significant using the following criteria: absolute fold change >1.5; minimum signal >2000; adjusted p-value <0.01. Black boxes highlight clusters of metabolite changes with distinct profiles between groups. Figures 10B, 10C, and 10D display ATP, ADP, and pyruvate levels, respectively, measured by untargeted profiling by comparing PKD mice 140 days after transplantation. Assay 1: Healthy mice (black bars) n = 1, PKD (gray bars) n = 1, hPGK-EGFP (white bars) n = 2, hPGK-coRPK (scratched bars) n = 3. Assay 2: Healthy mice n = 2, PKD n = 2, hPGK-EGFP n = 6, hPGK-coRPK n = 10. Figures 10Ee, 10F, and 10G display RBC-targeted metabolic profiling of a selected number of metabolites involved in the glycolytic pathway (PEP, 3-phosphoglycerate, and D-lactate, respectively) at 280 days post-transplant. Points represent values ​​for individual mice. Lines represent mean ± SEM, which was analyzed by the nonparametric Kruskal-Wallis test. Assay 2: Healthy mice n = 7, PKD n = 5, hPGK-EGFP n = 3, hPGK-coRPK n = 5. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 10E] See legend to Figure 10A. [Figure 10F] See legend to Figure 10A. [Figure 10G] See legend to Figure 10A. [Figure 11] Figures 11A-11C show pyruvate kinase activity, hexokinase activity, and the ratio of pyruvate kinase to hexokinase enzyme activity in RBCs from control mice and mice transplanted with transduced cells, respectively. RBCs were purified from blood samples through a cellulose column to avoid contamination with leukocyte PK activity and subjected to enzyme activity assessment. Black bars represent healthy mice (n = 2), white bars represent mice transplanted with cells transduced with an EGFP-expressing vector (n = 3), and scratched bars represent mice transplanted with cells transduced with a coRPK-expressing vector (n = 3). Checkered bars represent values ​​from a healthy volunteer (n = 1). Data represent the mean ± SEM for each group. [Figure 12] Figures 12A-12D show metabolic profiling in WBC samples from mice transplanted with gene-corrected cells. Figure 12A shows principal component analysis of non-target metabolite profiles in RBCs (red dots, left and center) and WBCs (blue dots; right cluster) in control and transplanted mice. Figures 12B, 12C, and 12D show ATP, ADP, and pyruvate levels, respectively, in WBCs compared to PKD mice. Assay 1: Healthy mice (black bars) n = 1, PKD (gray bars) n = 1, hPGK-EGFP (white bars) n = 2, hPGK-coRPK (scratched bars) n = 3. Assay 2: Healthy mice n = 2, PKD n = 2, hPGK-EGFP n = 6, hPGK-coRPK n = 10. Data represent mean ± SEM per group and were analyzed by the nonparametric Kruskal-Wallis test. [Figure 13]Gel images of LAM-PCR products generated using the Tsp509I enzyme for samples collected from all mice at different time points and tissues are shown. The vector integration site was identified by LAM-PCR amplification of the 3' vector LTR-genome junction. A MultiNA automated system was used, which generated a pattern characterized by several bands. The Tsp509I internal control band (IC) from the vector backbone is indicated by an arrow. [Figure 14] Gel images of LAM-PCR products generated using the HpyCH4IV5 enzyme for samples collected from all mice at different time points and tissues are shown. The vector integration site was identified by LAM-PCR amplification of the 3' vector LTR-genome junction. A MultiNA automated system was used, which generated a pattern characterized by several bands. The HpyCH4IV5 IC from the vector backbone is indicated by an arrow. [Figure 15] A general scheme of the integration site mapping analysis performed in mice transplanted with genetically modified hematopoietic progenitor cells is shown. Bone marrow and leukocyte samples derived from transplanted mice belonging to two independent experiments (Table 3) and collected at different time points after transplantation were analyzed as described in supplementary methods, following the indicated route. [Figure 16A] Figures 16A-16B show the distribution of LV integration along the genome of transplanted mice. Figure 16A displays the integration site (IS) frequency distribution around the transcription start site (TSS) of the nearest RefSeq gene, spanning 500 kb upstream and downstream of the TSS. The numbers at the top are the number of ISs detected for all samples and time points. Figure 16B displays the chromosomal distribution of LV integration sites in transplanted mice expressing the EGFP transgene (black bars) or the coRPK therapeutic transgene (gray bars), demonstrating no bias toward any particular chromosome. [Figure 16B] See legend to Figure 16A. [Figure 17A]Figures 17A-17C illustrate clonal abundance analysis of coRPK-LV-transduced cells. These are dot plot representations of pooled clonal abundance of integrations in each mouse from assay 1 (Figure 17A) and assay 2 (Figures 17B and 17C). The relative proportion (y-axis) for each IS is relative to the total number of sequence reads obtained in each dataset. BM is bone marrow, PB is peripheral blood, and coRPK1-14 are mice transplanted with hematopoietic cells transduced with therapeutic vectors. [Figure 17B] See legend to Figure 17A. [Figure 17C] See legend to Figure 17A. [Figure 18] This figure presents tracked shared integrations between primary and secondary recipient mice carrying the therapeutic PGK-coRPK LV vector. Integrations detected in any organ and in any mouse at any time are pooled. Secondary recipients received pooled BM from transplanted mice coRPK11-14. The remainder of the detected ISs were detected in either primary or secondary recipients. Numbers in boxes indicate representative values ​​for the corresponding integration ratio in the mentioned mice. In addition to the ≥5% filter applied to integration analysis, all integrations with sequence counts <3 were excluded. [Figure 19] Figure 17 illustrates clonal abundance analysis of EGFP-LV-transduced cells. Figure 17 shows a dot plot representation of the pooled clonal abundance of integrations for each mouse in the bone marrow. The relative proportions (y-axis) for each IS are relative to the total number of sequence reads obtained in each dataset. Similar to co-RPK-transduced cells (Figure 17), the graph indicates that the vast majority of transplanted mice exhibit a polyclonal pattern of hematopoietic cell repopulation. [Figure 20]LV genome integration profiles are shown. Gene ontology (GO) analysis was performed using GREAT software on samples from transplanted mice. All integrations obtained from this study (N=2220) showed over-representation of the gene functions shown in the left portion of the figure. To address whether the most abundant integrations were enriched in specific gene classes, we selected all ISs (shown in Figure 17) with relative sequence counts >5% of the entire dataset and showed that no GO gene classes were over-represented. [Figure 21] A schematic diagram of the PGK-coRPK LV lentiviral vector is shown. [Figure 22] A diagram of the plasmid (CPcoRPKW-17) containing the PGK-coRPK LV lentiviral vector is shown. [Figure 23A] 23A-23F represent the nucleotide sequence of the PGK-coRPK LV lentiviral vector, annotated to indicate the location of various functional elements. [Figure 23B] See legend to Figure 23A. [Figure 23C] See legend to Figure 23A. [Figure 23D] See legend to Figure 23A. [Figure 23E] See legend to Figure 23A. [Figure 23F] See legend to Figure 23A. [Figure 24A]Figures 24A-24B show the mechanism of action of the PGK-coRPK LV lentiviral vector. Figure 24A shows that ectopic expression of the PGK-coRPK LV vector rescues the wild-type phenotype of PKD red blood cells, which otherwise cannot produce functional RPK protein and generate enough energy to perform their functions. Figure 24B shows a gene therapy strategy for PKD patients based on ex vivo transduction of RPK-deficient CD34+ hematopoietic progenitor cells with the PGK-coRPK LV lentiviral vector and subsequent transplantation into the patient. The developed PGK-coRPK LV lentiviral vector carrying the therapeutic human PKLR gene cDNA will integrate into the genome of the patient's CD34+ cells during ex vivo transduction. These gene-corrected cells will then be reintroduced into the patient's body, where they will produce RBCs expressing the therapeutic transgene and thus producing functional RPK protein, which will correct the PKD pathological phenotype. Figure modified from Boston Children's Hospital blog. [Figure 24B] See legend to Figure 24A. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description of the Invention definition As used herein, "vector" refers to a macromolecule or combination of macromolecules that contains or binds to a polynucleotide and can mediate delivery of the polynucleotide into a cell. Exemplary vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles.

[0023] The term "LV" is an abbreviation for lentivirus and can refer to the virus itself or to its derivatives. The term encompasses all subtypes and both naturally occurring and recombinant forms, unless otherwise indicated.

[0024] As used herein, the term "gene" or "coding sequence" refers to an in vitro or in vivo nucleotide sequence that encodes a gene product. In some instances, a gene consists of, or consists essentially of, a coding sequence, i.e., a sequence that encodes a gene product. In other instances, a gene includes additional non-coding sequences. For example, a gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequences and 3'UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0025] As used herein, a "therapeutic gene" refers to a gene that, when expressed, confers a beneficial effect on the cell or tissue in which it is present or on the mammal in which it is expressed. Examples of beneficial effects include ameliorating the signs or symptoms of a condition or disease, preventing or inhibiting a condition or disease, or imparting a desired characteristic. Therapeutic genes include genes that repair a genetic defect in a cell or mammal.

[0026] As used herein, a transgene is a gene that has been delivered to a cell by a vector.

[0027] As used herein, the term "gene product" refers to the desired expression product of a polynucleotide sequence, such as a polynucleotide, peptide, protein, or interfering RNA (including short interfering RNA (siRNA), miRNA, or short hairpin RNA (shRNA)).

[0028] As used herein, the terms "polypeptide," "peptide," and "protein" refer to polymers of amino acids of any length. The terms also include amino acid polymers that have been modified, such as by disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling moiety.

[0029] "Comprising" means that the recited elements are essential, e.g., to a composition, method, kit, etc., but that other elements may be included within the scope of the claim to form, e.g., a composition, method, kit, etc. For example, an expression cassette "comprising" a gene encoding a therapeutic polypeptide operably linked to a promoter is an expression cassette that may include other elements in addition to the gene and promoter, e.g., polyadenylation sequences, enhancer elements, other genes, linker domains, etc.

[0030] "Consisting essentially of" refers to limiting the scope of a described, e.g., composition, method, kit, etc., to specified materials or steps that do not materially affect the basic and novel property(ies) of the composition, method, kit, etc. For example, an expression cassette "consisting essentially of" a gene encoding a therapeutic polypeptide operably linked to a promoter and an oriadenylation sequence may include additional sequences, e.g., linker sequences, so long as they do not materially affect the transcription or translation of the gene. As another example, a variant, or mutant polypeptide fragment, "consisting essentially of" a described sequence has an amino acid sequence of the described sequence plus or minus about 10 amino acid residues at the boundary of the sequence, based on the full-length native polypeptide from which it is derived, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue less than the described boundary amino acid residue, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues more than the described boundary amino acid residue.

[0031] "Consisting of" refers to the exclusion from a composition, method, or kit of any element, step, or ingredient not specified in the claim. For example, an expression cassette "consisting of" a gene encoding a therapeutic polypeptide operably linked to a promoter and post-transcriptional regulatory elements consists only of the promoter, a polynucleotide sequence encoding the therapeutic polypeptide, and post-transcriptional regulatory elements. As another example, a polypeptide "consisting of a recited sequence" contains only the recited sequence.

[0032] As used herein, "expression vector" includes vectors that contain a polynucleotide encoding a gene product of interest and result in expression of the gene product in an intended target cell, such as plasmids, minicircles, viral vectors, liposomes, and the like, as described above or known in the art. Expression vectors also contain control elements operably linked to the coding region to facilitate expression of the gene product in the target. The combination of control elements, such as promoters, enhancers, UTRs, miRNA targeting sequences, and the gene(s) to which they are operably linked for expression, is sometimes referred to as an "expression cassette." Many such control elements are known and available in the art and can be readily constructed from components available in the art.

[0033] As used herein, "promoter" refers to a DNA sequence that directs RNA polymerase binding and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Promoters and corresponding protein or polypeptide expression can be ubiquitous, meaning highly active in a wide range of cells, tissues, and species, or cell-, tissue-, or species-specific. Promoters can be "constitutive," meaning continuously active, or "inducible," meaning that the promoter can be activated or inactivated by the presence or absence of a biotic or abiotic factor. Enhancer sequences, which may or may not be adjacent to the promoter sequence, are also included in the nucleic acid constructs or vectors of the present invention. Enhancer sequences affect promoter-dependent gene expression and can be located within the 5' or 3' region of the native gene.

[0034] As used herein, "enhancer" encompasses cis-acting elements that stimulate or inhibit transcription of adjacent genes. Enhancers that inhibit transcription are also called "silencers." Enhancers can function in either orientation (i.e., associated with a coding sequence) over distances of up to several kilobase pairs from the coding sequence and from a position downstream of the transcribed region.

[0035] As used herein, a "termination signal sequence" includes any genetic element that causes RNA polymerase to terminate transcription, such as a polyadenylation signal sequence.

[0036] As used herein, the terms "operably linked" or "operably linked" refer to the juxtaposition of genetic elements, such as promoters, enhancers, termination signal sequences, polyadenylation sequences, etc., in a relationship that permits these elements to function in an expected manner. For example, a promoter is operably linked to a coding region if it helps initiate transcription of the coding sequence. There can be intervening bases between the promoter and the coding region, so long as this functional relationship is maintained.

[0037] As used herein, the term "heterologous" means derived from a genotypically different entity from that of the remainder of the entity being compared. For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques is a heterologous polynucleotide. As another example, a promoter removed from its native coding sequence and operably linked to a coding sequence to which it is not naturally linked is a heterologous promoter. Thus, for example, an LV vector containing a heterologous nucleic acid encoding a heterologous gene product is an LV vector containing a nucleic acid not normally contained in naturally occurring, wild-type LV, and the encoded heterologous gene product is a gene product not normally encoded by naturally occurring, wild-type LV.

[0038] The term "endogenous" as used herein with respect to a nucleotide molecule or gene product refers to a nucleic acid sequence, e.g., a gene or genetic element, or a gene product, e.g., RNA, protein, that naturally occurs in or is associated with a host virus or cell.

[0039] The term "native" as used herein refers to a nucleotide sequence, eg, a gene, a gene product, eg, RNA, a protein, that is present in a wild-type virus or cell.

[0040] As used herein, the term "variant" refers to a variant of a reference polynucleotide or polypeptide sequence, e.g., a naturally occurring polynucleotide or polypeptide sequence, i.e., one having less than 100% sequence identity with the reference polynucleotide or polypeptide sequence. Stated differently, a variant of a polynucleotide sequence contains at least one nucleotide difference (e.g., a nucleotide substitution, a nucleotide insertion, or a nucleotide deletion) relative to a reference polynucleotide sequence, e.g., a naturally occurring polynucleotide sequence. A variant of a polypeptide sequence contains at least one amino acid difference (e.g., an amino acid substitution, an amino acid insertion, or an amino acid deletion) relative to a reference polypeptide sequence, e.g., a naturally occurring polypeptide sequence. For example, a variant can be a polynucleotide having 70% or more sequence identity with a full-length naturally occurring polynucleotide sequence, e.g., 75% or 80% or more identity (e.g., 85%, 90%, or 95% or more), e.g., 98% or 99% identity, to a full-length naturally occurring polynucleotide sequence. As another example, a variant can be a polypeptide that has 70% or more sequence identity with a full-length, naturally occurring polypeptide sequence, e.g., 75% or 80% or more identity (such as 85%, 90%, or 95% or more), e.g., 98% or 99% identity to a full-length, naturally occurring polypeptide sequence. Variants can also include variant fragments of a reference, e.g., naturally occurring sequence, that share 70% or more sequence identity with a fragment of the reference, e.g., naturally occurring sequence, e.g., that share 75% or 80% or more identity (such as 85%, 90%, or 95% or more), e.g., 98% or 99% identity with the naturally occurring sequence.

[0041] As used herein, the terms "biological activity" and "biologically active" refer to activity attributable to a specific biological element in a cell. For example, the "biological activity" of an "immunoglobulin," "antibody," or a fragment or variant thereof, refers to its ability to bind to an antigenic determinant, thereby promoting immunological function. As another example, the biological activity of a polypeptide, or a functional fragment or variant thereof, refers to the ability of the polypeptide, or a functional fragment or variant thereof, to perform its natural function, such as binding, enzymatic activity, etc. As a third example, the biological activity of a gene regulatory element, such as a promoter, enhancer, Kozak sequence, etc., refers to the ability of the regulatory element, or a functional fragment or variant thereof, to regulate expression of a gene to which it is operably linked, i.e., to promote, enhance, or activate translation, respectively.

[0042] As used herein, the term "administering" or "introducing" refers to the delivery of a vector to a cell and / or organ of a subject, or to the delivery of a vector to a subject for recombinant protein expression. Such administration or introduction may be performed in vivo, in vitro, or ex vivo. A vector for expression of a gene product may be introduced into a cell by transfection, which typically refers to the insertion of heterologous DNA into a cell by physical means (e.g., calcium phosphate transfection, electroporation, microinjection, or lipofection); infection, which typically refers to introduction via an infectious agent, i.e., a virus; or transduction, which typically refers to stable infection of a cell by a virus or the transfer of genetic material from one microorganism to another via a viral agent (e.g., a bacteriophage).

[0043] "Transformation" is typically used to refer to bacteria containing heterologous DNA, or cells, such as tumor cells, that express an oncogene and have been converted into a continuous mode of growth. The vector used to "transform" a cell can be a plasmid, virus, or other vehicle.

[0044] Typically, cells are referred to as "transduced," "infected," "transfected," or "transformed" depending on the means used to administer, introduce, or insert heterologous DNA (i.e., a vector) into the cells. The terms "transduced," "transfected," and "transformed" may be used interchangeably herein, regardless of the method of introduction of the heterologous DNA.

[0045] As used herein, the term "host cell" refers to a cell that has been introduced, infected, transfected, or transformed with a vector. The vector may be a plasmid, a viral particle, a phage, etc. Culture conditions, e.g., temperature, pH, etc., will be those previously used with the host cell selected for expression and will be apparent to one of skill in the art. It will be understood that the term "host cell" refers to the originally introduced, infected, transfected, or transformed cell and its progeny.

[0046] "Treatment," "treating," and the like are used herein to generally mean achieving a desired pharmacological and / or physiological effect. This effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, e.g., reducing the likelihood that a disease or its symptoms will develop in a subject, and / or therapeutic, in terms of partially or completely curing the disease and / or the deleterious effects resulting from the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including (a) preventing the disease from occurring in a subject who may be susceptible to, but has not yet been diagnosed with, the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) alleviating the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of disease or injury. Treatment of ongoing disease, in which treatment stabilizes or reduces undesirable clinical symptoms in the patient, is of particular interest. Such treatment is desirably administered before complete loss of function in the affected tissue. The subject therapy is desirably administered during, and optionally after, the symptomatic stage of the disease.

[0047] "Individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans and non-human primates, including monkeys and humans; mammalian sports animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pet animals (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0048] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, when the terms "comprising," "including," "having," "having," "including," or variations thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0049] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, per practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. Where particular values ​​are described in this application and claims, unless otherwise specified, the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0050] Unless otherwise indicated, all terms used herein have the same meaning as those of ordinary skill in the art, and the present invention will employ conventional techniques of microbiology and recombinant DNA techniques within the knowledge of those skilled in the art.

[0051] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are fully explained in the literature, which may be found, for example, in "Molecular Cloning: A Laboratory Manual," second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M. Weir & C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction," (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J.E. Coligan et al., eds., 1996). al., eds., 1991), each of which is expressly incorporated herein by reference.

[0052] In certain embodiments, the present disclosure provides polynucleotides, polynucleotide cassettes, and expression vectors for expressing genes in cells. Also provided are pharmaceutical compositions and methods for using any of the compositions in promoting expression of genes in cells, e.g., in an individual, e.g., for the treatment or prevention of disorders. These and other objects, advantages, and features of the present invention will become apparent to those skilled in the art upon reading the details of the compositions and methods more fully described below.

[0053] The present invention relates generally to the fields of molecular biology and virology, and more particularly to gene expression cassettes and vectors containing them that are useful for the delivery of nucleic acid segments encoding selected therapeutic constructs (including, for example, peptides, polypeptides, ribozymes, and catalytic RNA molecules) to selected cells and tissues of vertebrates. In particular, these gene constructs are useful in the development of gene therapy vectors, including, for example, LV vectors, for the treatment of mammalian, particularly human, diseases, disorders, and dysfunctions.

[0054] The disclosed compositions can be utilized in a variety of research, diagnostic, and therapeutic regimens, including the prevention and treatment of a variety of human diseases. Various compositions and methods of the present invention are described below.

[0055] While particular compositions and methods are exemplified herein, it will be understood that any of a number of alternative compositions and methods are applicable and suitable for use in the practice of the present invention. It will also be understood that evaluation of the expression constructs and methods of the present invention can be carried out using procedures standard in the art.

[0056] In certain embodiments, methods and compositions are provided for the preparation of gene therapy vector compositions, e.g., viral vectors, comprising these gene expression cassettes for use in the preparation of medicaments useful for centrally targeted gene therapy of diseases, disorders, and dysfunctions in animals, particularly humans.

[0057] In some embodiments, the present invention provides gene therapy for PKD based on an LV vector carrying a PGK eukaryotic promoter driving expression of PKLR cDNA. This therapeutic vector can be used to transduce hematopoietic stem cells (HSCs), which can then be transplanted into a subject. Ectopic RPK expression normalizes the erythroid compartment, correcting the hematological phenotype and reversing organ pathology. Metabolomic studies have demonstrated that functional restoration of the glycolytic pathway in RBCs derived from gene-corrected PKD HSCs is not accompanied by metabolic disorders in leukocytes. Analysis of the LV IS in the genomes of transplanted hematopoietic cells showed no evidence of genotoxicity in any of the transplanted animals. Overall, these results highlight the therapeutic potential of the PGK-coRPK LV vector and hold great promise for gene therapy of PKD and other genetic diseases of erythrocyte metabolism.

[0058] In certain embodiments, the present invention provides a RPK LV vector for gene repair of PKD. Genetic modification of mouse PKD-HSCs with this vector can efficiently repair the hemolytic phenotype and RBC metabolic profile in transplanted PKD mice. No evidence of metabolic disorders or genotoxicity was observed in leukocytes derived from vector integration, supporting the therapeutic potential of the PGK-coRPK LV vector. Overall, these results provide promising evidence for the feasibility of gene therapy for PKD using LVs designed for clinical application.

[0059] Certain embodiments of the present invention include a self-inactivating (SIN) LV vector that expresses a codon-optimized version of the human PLKR gene. This expression vector includes a promoter region, a coding sequence, and post-transcriptional regulatory elements.

[0060] Certain embodiments of the polynucleotide cassette of the present invention comprise a promoter region comprising a promoter sequence, or a functional fragment thereof. In one embodiment, the promoter is the human PGK promoter.

[0061] Some embodiments of the present invention include a polynucleotide cassette for enhanced expression of pyruvate kinase (e.g., the human PLKR gene). In some embodiments, the polynucleotide cassette comprises a codon-optimized version of the human PKLR cDNA (coRPK) to increase mRNA stability during transcription. For optimization, GeneArt® software may be used to increase GC content and remove cryptic splice sites to avoid transcriptional silencing and thus increase transgene expression. The coRPK-optimized sequence showed 80.4% homology with the human PKLR gene, with no amino acid changes in the protein. Alternatively, any optimization method known in the art may be used.

[0062] In some embodiments, the polynucleotide cassette includes an RNA export signal downstream of the second enhancer. The RNA export signal may include a wPRE sequence. In some embodiments, a mutated wPRE lacking any remaining open reading frame (Schambach, Bohne et al. 2006) is also included to improve the level of expression and stability of the therapeutic gene. The term "RNA export signal" may refer to any of the "post-transcriptional regulatory elements" known in the art. The terms "RNA export signal" and "post-transcriptional regulatory element," and the like, are used interchangeably herein because of their common use in the field of vector engineering and development. Exemplary RNA export signals or post-transcriptional regulatory elements used in some embodiments of the present disclosure include, but are not limited to, the hepatitis B virus post-transcriptional regulatory element (HBVPRE) or the constitutive export element (CTE) derived from simian retroviruses, including simian retrovirus type 1 (SRV-1) and type 2 (SRV-2), and Mason-Pfizer (MPV). Post-transcriptional regulatory elements are provided by U.S. Patent No. 6,136,597; Donello et al. J. Virol. 72:5085-92 (1998); Hlavaty et al. Virology 341:1-11 (2005); and Oh et al. Retrovirology 4:38 (2007). Additional alternative wPRE sequences are provided by Zanta-Boussif et al. Gene Therapy 16:605-19 (2009). The disclosures of the foregoing references are incorporated herein by reference in their entireties.

[0063] In some aspects of the present invention, a gene delivery vector is provided comprising the polynucleotide cassette of the present invention. In some embodiments, the gene delivery vector is LV.

[0064] In some aspects of the present invention, pharmaceutical compositions are provided comprising a polynucleotide cassette of the present invention and a pharmaceutical excipient. In some embodiments, the pharmaceutical composition comprises a gene delivery vector of the present invention and a pharmaceutical excipient.

[0065] In some aspects of the present invention, methods for expressing a transgene in mammalian cells are provided. In some embodiments, the method comprises contacting one or more mammalian cells with an effective amount of a polynucleotide cassette or a gene delivery vector of the present invention, wherein the transgene is expressed at a detectable level in the one or more mammalian cells. In some embodiments, the method comprises contacting one or more mammalian cells with an effective amount of a polynucleotide cassette or a gene delivery vector of the present invention, wherein the transgene is expressed at a therapeutic level in the one or more mammalian cells. In some embodiments, the method is performed in vitro. In other embodiments, the method is performed in vivo.

[0066] In some aspects of the invention, methods are provided for treating or preventing a disease or disorder in a mammal in need thereof, hi some embodiments, the method comprises administering to the mammal an effective amount of a pharmaceutical composition of the invention, wherein the coding sequence encodes a therapeutic gene product.

[0067] composition In some aspects of the present disclosure, compositions are provided for expression of a transgene in a eukaryotic cell(s). In some aspects, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a hematopoietic stem cell. In some embodiments, the cell is a bone marrow cell, e.g., a lineage-depleted bone marrow cell. In some aspects, the mammalian cell is a committed hematopoietic erythroid progenitor cell.

[0068] In some embodiments of the present disclosure, the composition is a polynucleotide cassette. A "polynucleotide cassette" refers to two or more functional polynucleotide sequences, such as a regulatory element, a translation initiation sequence, a coding sequence, and / or a termination sequence, typically operably linked to each other. Similarly, a "polynucleotide cassette for expressing a transgene in a mammalian cell" refers to a combination of two or more functional polynucleotide sequences, such as a promoter, an enhancer, a 5'UTR, a translation initiation sequence, a coding sequence, and / or a termination sequence, that mediate the expression of a transgene in a cell.

[0069] In some embodiments, the polynucleotide cassettes of the present disclosure provide enhanced expression of a transgene in mammalian cells. As used herein, "expression of a transgene" or "expressing a transgene" refers to both the transcription of the transgene into messenger RNA (mRNA) and the translation of that mRNA into a polypeptide (i.e., the transgene product) by a host cell. Thus, transgene expression can be measured either at the mRNA level (i.e., by sequence-specific quantification of the level of mRNA in the cell) or at the polypeptide level (i.e., by measuring the level of the polypeptide gene product using Western blot, enzyme-linked immunosorbent assay (ELISA), immunofluorescence microscopy, or other means of quantifying specific polypeptides). As demonstrated by the examples of the present disclosure, the inventors have discovered numerous polynucleotide elements, i.e., elements that are improved over those known in the art, that individually and synergistically provide enhanced expression of a transgene in mammalian cells. In certain embodiments, the arrangement of two or more functional polynucleotide sequences within the polynucleotide cassettes of the present disclosure provides enhanced expression of a transgene in mammalian cells. "Enhanced" means that the expression of a transgene is increased, enhanced, or stronger in cells carrying a polynucleotide cassette of the present disclosure compared to cells carrying a transgene operably linked to comparable regulatory elements, for example, as known in the art. In other words, the expression of a transgene is increased, enhanced, or stronger from a polynucleotide cassette of the present disclosure compared to expression from a polynucleotide cassette that does not contain one or more optimized elements of the present disclosure, i.e., a reference control. In certain embodiments, the enhanced expression is specific to or limited to one or more desired cell types.

[0070] For example, expression of a transgene is enhanced, increased, or increased in a cell comprising a polynucleotide cassette comprising a promoter disclosed herein than in a cell carrying a transgene operably linked to a different promoter, such as one known in the art. As another example, expression of a transgene is enhanced, increased, or increased, or stronger in a cell comprising a polynucleotide cassette comprising an enhancer sequence disclosed herein than in a cell carrying a transgene operably linked to a different enhancer sequence.

[0071] Without being bound by theory, enhanced expression of a transgene in a cell is believed to result from a more rapid assembly of the gene product in the cell or a more stable gene product in the cell. Therefore, enhanced expression of a transgene by the polynucleotide cassette of the subject disclosure can be observed in many ways. For example, enhanced expression can be observed by detecting transgene expression earlier, e.g., 2 days earlier, 7 days earlier, 2 weeks earlier, 3 weeks earlier, 4 weeks earlier, 8 weeks earlier, 12 weeks earlier or more, after contacting the polynucleotide cassette with the cell than the expression detected when the transgene is operably linked to a comparable regulatory element, such as one known in the art. Enhanced expression can also be observed as an increase in the amount of gene product per cell. For example, there can be a 2-fold or greater increase, e.g., a 3-fold or greater increase, a 4-fold or greater increase, a 5-fold or greater increase, or a 10-fold or greater increase in the amount of gene product per mammalian cell. Enhanced expression can also be observed as an increase in the number of mammalian cells expressing detectable levels of the transgene carried by the polynucleotide cassette. For example, there may be a 2-fold or greater increase, e.g., a 3-fold or greater increase, a 4-fold or greater increase, a 5-fold or greater increase, or a 10-fold or greater increase in the number of mammalian cells expressing detectable levels of the transgene.

[0072] As another example, the polynucleotides of the present invention can promote detectable levels of transgene expression in a higher percentage of cells than conventional polynucleotide cassettes; for example, if a conventional polynucleotide cassette promotes detectable levels of transgene expression in, for example, less than 5% of cells in a particular region, the polynucleotides of the present invention will promote detectable levels of expression in 5% or more of the cells in that region, such that 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, or 45% or more, and in some cases 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 75% or more, for example, 80% or more, 85% or more, 90% or more, or 95% or more of the contacted cells will express detectable levels of the gene product. Enhanced expression may also be observed as an alteration in cell viability and / or function.

[0073] The polynucleotide cassette of the present disclosure typically includes a promoter region. Any suitable promoter region or promoter sequence therein can be used in the subject polynucleotide, as long as the promoter region promotes the expression of the coding sequence in eukaryotic cells. In certain embodiments, the promoter region promotes the expression of the coding sequence in mammalian cells. In some cases, the promoter is a ubiquitous promoter, i.e., a promoter that is active in a wide range of cells, tissues, and major regions. In another example, the promoter is a human PGK promoter.

[0074] Promoter and enhancer elements can be tissue- or stage-specific. For example, a tissue-specific promoter or enhancer preferentially drives expression (or higher levels of expression) in one or more specific cell types. Examples of cell types include, but are not limited to, hematopoietic stem cells, long-term hematopoietic stem cells, short-term hematopoietic stem cells, multipotent progenitor cells, hematopoietic CD34+ cells, and any cluster of differentiated subpopulations within the CD34+ population. Stage-specific promoters or enhancers preferentially drive expression (or higher levels of expression) during one or more specific stages of the cell cycle or cell development. These include, but are not limited to, the beta-globin locus control region, the spectrin promoter, and erythroid-specific promoters.

[0075] In some embodiments, polynucleotides comprise one or more enhancers.Enhancers are nucleic acid elements known in the art for enhancing transcription, and can be located anywhere relative to the gene they regulate, for example, upstream, downstream, or within an intron.Any enhancer element can be used in the polynucleotide cassette and gene therapy vector of the present disclosure, as long as it enhances gene expression when used in combination with a promoter.

[0076] The coding sequence expressed in the cell can be any polynucleotide sequence, for example, a gene or cDNA encoding a gene product, such as a polypeptide or RNA-based therapeutic agent (e.g., siRNA, antisense, ribozyme, shRNA, etc.). The coding sequence can be heterologous to the promoter sequence to which it is operably linked, i.e., not naturally operably associated with it. Alternatively, the coding sequence can be endogenous to the promoter sequence to which it is operably linked, i.e., naturally associated with the promoter. The gene product can act constitutively or non-constitutively in mammalian cells; for example, the gene product can be secreted. For example, if the transgene is a therapeutic gene, the coding sequence can be any gene encoding a desired gene product, or a functional fragment or variant thereof, that can be used as a therapeutic agent to treat a disease or disorder. In various preferred embodiments, the transgene encodes human PKLR.

[0077] In one embodiment of the present invention, a transgene coding sequence is modified, or "codon-optimized," to enhance expression by replacing rarely occurring codons with more frequently occurring codons. The coding sequence is a portion of an mRNA sequence that encodes an amino acid for translation. During translation, each of the 61 trinucleotide codons is translated into one of 20 amino acids, resulting in the degeneracy or redundancy of the genetic code. However, different cell types and different animal species utilize tRNAs (each carrying an anticodon) that code the same amino acid at different frequencies. If a gene sequence contains a codon that is rarely represented by the corresponding tRNA, the ribosomal translation machinery can slow down and hinder efficient translation. For certain species, expression can be improved through "codon optimization," in which a coding sequence encodes the same protein sequence but utilizes highly expressed codons and / or codons utilized by highly expressed human proteins (Cid-Arregui et al., 2003; J. Virol. 77:4928). In one aspect of the present invention, the coding sequence of a transgene is modified to replace codons rarely expressed in mammals or primates with codons frequently expressed in primates. For example, in some embodiments, the coding sequence encoded by the transgene encodes a polypeptide having at least 85% sequence identity, e.g., at least 90% sequence identity, e.g., at least 95% sequence identity, at least 98% identity, at least 99% identity, to the polypeptide encoded by the sequence described above or disclosed herein, and at least one codon of the coding sequence has a higher tRNA frequency in humans than the corresponding codon in the sequence described above or disclosed herein.

[0078] In additional embodiments of the invention, the transgene coding sequence is modified to enhance expression by terminating or removing open reading frames (ORFs) that do not encode the desired transgene. An open reading frame (ORF) is a nucleic acid sequence following a start codon and that does not contain a stop codon. The ORF may be in forward or reverse orientation and may be "in frame" or "out of frame" relative to the gene of interest. Such open reading frames may be expressed in an expression cassette along with the gene of interest, which may result in undesirable adverse effects. In one embodiment of the present invention, the coding sequence of the transgene is modified to remove the open reading frame by further altering the codon usage. This was done by eliminating the start codon (ATG) and introducing a stop codon (TAG, TAA, or TGA) into the reverse or out-of-frame ORF, while conserving the amino acid sequence in the gene of interest and maintaining highly utilized codons (i.e., avoiding codons with a frequency of <20%). In the present invention, the transgene coding sequence may be optimized by using either or both techniques: codon optimization and removal of non-transgene ORFs. As will be apparent to one of skill in the art, it is preferable to remove or minimize non-transgene ORFs after codon optimization in order to remove introduced ORFs during codon optimization.

[0079] The RRE sequence improves the efficiency of gene transfer. In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the RRE sequence comprises or consists of the following sequence, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF0007817979000001.tif31144

[0080] The retroviral leader region contains a packaging signal (Ψ) that is responsible for packaging the retroviral genome into a viral capsid. LV vectors were thought to require approximately 300 bp of the Gag gene within this region. Currently, this Gag sequence has been reduced to just 40 bp (FIG. 1). In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the Ψ sequence is the HIV-1 Ψ sequence, or the Ψ sequence comprises or consists of the following sequence, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF0007817979000002.tif17146

[0081] In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the truncated HIV-1 5'LTR comprises or consists of the following sequence, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF0007817979000003.tif24146

[0082] In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the HIV-1 self-inactivating 3'LTR comprises or consists of the following sequence, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF0007817979000004.tif30145

[0083] In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the human cytomegalovirus (CMV) immediate early promoter comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF0007817979000005.tif24146

[0084] The cPPT, which facilitates nuclear import of the preintegration complex, along with the CTS, which is involved in the segregation of reverse transcriptase, have been shown to improve viral titers (Zennou, et al. 2000; Follenzi et al. 2000). In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the HIV-1 central polypurine tract and central termination sequence (cPPT / CTS) comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF0007817979000006.tif17144

[0085] In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the human phosphoglycerate kinase 1 (hPGK) promoter comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF0007817979000007.tif64146

[0086] In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the codon-optimized form of the human PKLR cDNA (coRPK) comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF0007817979000008.tif218146

[0087] In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the human CMV enhancer comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF0007817979000009.tif51146

[0088] In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the Simian Virus 40 (SV40) poly(A) signal comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF0007817979000010.tif17145

[0089] In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the SV40 origin of replication comprises or consists of the following sequence, a functional fragment thereof, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF0007817979000011.tif17144

[0090] In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the cPPT present in any of the expression cassettes and gene delivery vectors described herein has the following sequence: TIFF0007817979000012.tif4128, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:12.

[0091] In some embodiments of any of the expression cassettes and gene delivery vectors described herein, the dNEF present in any of the expression cassettes and gene delivery vectors described herein has the following sequence: TIFF0007817979000013.tif11145, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:13.

[0092] In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the NeoR / KanR sequence present in any of the expression cassettes and gene delivery vectors described herein has the following sequence: TIFF0007817979000014.tif98145, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:14.

[0093] In some embodiments of any of the expression cassettes and gene delivery vectors described herein, the rrnG terminator (the transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)) present in any of the expression cassettes and gene delivery vectors described herein has the following sequence: TIFF0007817979000015.tif17145, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:15.

[0094] In some embodiments of any of the expression cassettes and gene delivery vectors described herein, the ori (high copy number ColE1 / pMB1 / pBR322 / pUC origin of replication) present in any of the expression cassettes and gene delivery vectors described herein has the following sequence: TIFF0007817979000016.tif78145, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:16.

[0095] In some embodiments of any of the expression cassettes and gene delivery vectors described herein, the CAP binding site present in any of the expression cassettes and gene delivery vectors described herein has the following sequence: TIFF0007817979000017.tif4128, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:17.

[0096] In some embodiments of any of the expression cassettes and gene delivery vectors described herein, the E. coli lac promoter present in any of the expression cassettes and gene delivery vectors described herein has the following sequence: TIFF0007817979000018.tif4128, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:18.

[0097] In some embodiments of any of the expression cassettes and gene delivery vectors described herein, the lac operator present in any of the expression cassettes and gene delivery vectors described herein has the following sequence: TIFF0007817979000019.tif4128, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:19.

[0098] In some embodiments of any of the expression cassettes and gene delivery vectors described herein, the T3 promoter (the promoter for bacteriophage T3 RNA polymerase) present in any of the expression cassettes and gene delivery vectors described herein has the following sequence: TIFF0007817979000020.tif4128, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:20.

[0099] In some embodiments of any of the expression cassettes and gene delivery vectors described herein, the CMV enhancer present in any of the expression cassettes and gene delivery vectors described herein has the following sequence: TIFF0007817979000021.tif51146, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:21.

[0100] In some embodiments of any of the expression cassettes and gene delivery vectors described herein, the T7 promoter (the promoter for bacteriophage T7 RNA polymerase) present in any of the expression cassettes and gene delivery vectors described herein has the following sequence: TIFF0007817979000022.tif4128, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:22.

[0101] In some embodiments of any of the expression cassettes and gene delivery vectors described herein, the f1 ori (f1 bacteriophage origin of replication) present in any of the expression cassettes and gene delivery vectors described herein has the following sequence: TIFF0007817979000023.tif51145, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:23.

[0102] In some embodiments, the polynucleotide cassette of the present invention further comprises an RNA export signal. Exemplary RNA export signals include, but are not limited to, the wPRE. The wPRE significantly increases transgene expression in target cells by increasing RNA stability in the transgene in a promoter- and vector-dependent manner (Zuffrey et al., 1999). However, it can express a truncated 60-amino acid protein derived from the WHV X gene, which is involved in liver cancer (Kingsman et al., 2005). Therefore, most preclinical protocols and clinical trials include a mutant version of the wPRE element (Zanta-Boussif et al., 2009). On the other hand, the use of two SV40-USE elements in SIN-LV vectors has been found to be more efficient than the wPRE sequence in suppressing transcriptional readthrough (Schambach et al., 2007). More precisely, the wPRE disclosed herein is a chimeric wPRE (Zuffrey et al., 1999) that carries 589 nucleotides from the modified WPRE (nucleotides 1-589) (WO 2008136670 A2; [5]) and 88 nucleotides (nucleotides 590-677) from the former wPRE. The data disclosed herein show that this chimeric wPRE works better than the former wPRE. The chimeric wPRE sequence comprises the following sequence: TIFF0007817979000024.tif84146

[0103] In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the wPRE sequence comprises or consists of the sequence of SEQ ID NO:24 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO:24. TIFF0007817979000025.tif84146

[0104] Other combinations of both elements as disclosed herein or as known in the art will be readily apparent to those skilled in the art.

[0105] Additionally, as will be appreciated by those skilled in the art, the polynucleotide cassette may optionally contain other elements, including, but not limited to, restriction sites to facilitate cloning and regulatory elements for the particular gene expression vector.

[0106] In some aspects of the present invention, the subject polynucleotide cassettes are used to deliver genes to cells of an animal to treat cellular disorders, etc., and to determine the effect the gene has on cell viability and / or function, for example.

[0107] Any convenient gene therapy vector that can be used to deliver polynucleotide sequences to mammalian cells is included in the gene delivery vector of the present disclosure.For example, the vector can comprise single-stranded or double-stranded nucleic acid, for example, single-stranded or double-stranded DNA.For example, the gene delivery vector can be DNA, for example, naked DNA, for example, plasmid, minicircle, etc.The vector can comprise single-stranded or double-stranded RNA, including modified forms of RNA.In another example, the gene delivery vector can be RNA, for example, mRNA or modified mRNA.

[0108] As another example, the gene delivery vector may be a viral vector derived from a virus, such as an adenovirus, adeno-associated virus, LV, herpesvirus, alphavirus, or retrovirus, such as Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), or Rous sarcoma virus (RSV). While embodiments involving the use of LV are described in greater detail below, it is expected that those skilled in the art will understand that similar knowledge and techniques in the art can be applied to non-LV gene therapy approaches as well.

[0109] In some embodiments, the gene delivery vector is a self-limiting LV. In some embodiments, the gene delivery vector is a self-inactivating LV. In some embodiments, the gene delivery vector is a non-integrating LV. In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the self-limiting LV of the present disclosure (FIGS. 22 and 23) comprises or consists of the following sequence, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF0007817979000026.tif70146TIFF0007817979000027.tif224146TIFF0007817979000028.tif224146 TIFF0007817979000029.tif224146TIFF0007817979000030.tif224146TIFF0007817979000031.tif131146

[0110] In specific embodiments of the expression cassettes described herein, the expression comprises or consists of the following sequence, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the following sequence: TIFF0007817979000032.tif70146TIFF0007817979000033.tif224146TIFF0007817979000034.tif58146

[0111] In such embodiments, the subject polynucleotide cassette is flanked at the 5' and 3' ends by functional LTR sequences. In one embodiment, the locations of different elements present in the LV vector backbone are displayed in Figure 1. Both LTR sequences are modified to generate a SIN LV vector. The SIN vector has a 400-bp deletion in the 3'-LTR that covers the promoter / enhancer elements from the U3 region. This makes transgene expression dependent on the internal promoter, reducing the risk of RCL and reducing promoter interference (Ginn et al., 2003). This 3'-LTR deletion removes the TATA box, preventing transcription initiation (Miyoshi et al., 1998; Zuffrey et al., 1998), thus inactivating the vector. The U3 region of the 5'-LTR is replaced with another heterologous promoter sequence (i.e., CMV or RSV) to achieve Tat-independent transcription and increase genomic RNA synthesis, resulting in increased viral titers. Because the 5'-U3 region drives expression of the primary transcript, the modification will be absent in transduced cells (Schambach et al. 2009).

[0112] To reduce transcriptional read-through from internal promoters (Zaiss et al., 2002) or from remnants of the deleted U3 region of the SIN-LV vector (Almarza et al., 2011) and prevent possible transcriptional activation of downstream genes, exogenous elements such as β-globin or SV40 polyadenylation signals (Iwakuma et al., 1999) or the USE from SV40-USE (Schambach et al., 2007) have also been included in the R region of the viral 3' LTR.

[0113] In certain embodiments, the expression cassette or delivery vector, e.g., LV, comprises a polynucleotide sequence comprising the following sequences in 5' to 3' order: a) a PGK promoter sequence, optionally a human PGK promoter sequence; b) a sequence encoding a pyruvate kinase polypeptide, optionally a coRPK coding sequence or cDNA sequence, and c) Optionally, a mutant wPRE sequence comprising or consisting of the sequence of SEQ ID NO:24.

[0114] In certain embodiments, the expression cassette or delivery vector, e.g., LV, comprises a polynucleotide sequence comprising the following sequences in 5' to 3' order: a) cPPT sequence, b) a PGK promoter sequence, optionally a human PGK promoter sequence; c) a sequence encoding a pyruvate kinase polypeptide, optionally a coRPK coding sequence or cDNA sequence, and d) Optionally, a mutant wPRE sequence comprising or consisting of the sequence of SEQ ID NO:24.

[0115] In certain embodiments, the expression cassette or delivery vector, e.g., LV, comprises a polynucleotide sequence comprising the following sequences in 5' to 3' order: a) a 5'LTR, optionally a modified 5'LTR; b) cPPT sequence, c) a PGK promoter sequence, optionally a human PGK promoter sequence; d) a sequence encoding a pyruvate kinase polypeptide, optionally a coRPK coding sequence or a cDNA sequence; e) optionally, a mutant wPRE sequence comprising or consisting of the sequence of SEQ ID NO: 24, and f) 3'LTR, optionally a modified 3'LTR.

[0116] In certain embodiments, the expression cassette or delivery vector, e.g., LV, comprises a polynucleotide sequence comprising the following sequences in 5' to 3' order: a) a 5'LTR, optionally a modified 5'LTR; b) cPPT sequence, c) a PGK promoter sequence, optionally a human PGK promoter sequence; d) a sequence encoding a pyruvate kinase polypeptide, optionally a coRPK coding sequence or a cDNA sequence; e) optionally a mutant wPRE sequence comprising or consisting of the sequence of SEQ ID NO: 24; f) 3'LTR, optionally a modified 3'LTR; g) the SV40 poly(A) signal, and h) SV40 origin of replication (ori).

[0117] In certain embodiments, the expression cassette or delivery vector, e.g., LV, comprises a polynucleotide sequence comprising the following sequences in 5' to 3' order: a) a 5'LTR, optionally a modified 5'LTR; b) cPPT sequence, c) a PGK promoter sequence, optionally a human PGK promoter sequence; d) a sequence encoding a pyruvate kinase polypeptide, optionally a coRPK coding sequence or a cDNA sequence; e) optionally a mutant wPRE sequence comprising or consisting of the sequence of SEQ ID NO: 24; f) 3'LTR, optionally a modified 3'LTR; g) SV40 poly(A) signal; h) SV40 origin of replication (ori), i) T7 promoter, j)f1 ori, and k) NeoR / KanR sequence.

[0118] In certain embodiments, the expression cassette or delivery vector, e.g., LV, comprises a polynucleotide sequence comprising the following sequences in 5' to 3' order: a) a 5'LTR, optionally a modified 5'LTR; b) HIV-1 ψ sequence; c) RRE, d) cPPT / CTS sequence; e) a PGK promoter sequence, optionally a human PGK promoter sequence; f) a sequence encoding a pyruvate kinase polypeptide, optionally a coRPK coding sequence or a cDNA sequence; g) optionally a mutant wPRE sequence comprising or consisting of the sequence of SEQ ID NO: 24; h) dNEF signal; i) a 3'LTR, optionally a modified 3'LTR; j) SV40 poly(A) signal; k) SV40 origin of replication (ori), l) T7 promoter, m)f1 ori, n) NeoR / KanR sequence; o)rrnG Terminator p)ori array, q) CAP binding site, r) lac promoter sequence, s) lac promoter sequence, t) T3 promoter sequence; u) a CMV enhancer sequence, and v) CMV promoter sequence.

[0119] In certain embodiments, the gene delivery vector is PGK-coRPK LV or comprises the elements depicted in FIG.

[0120] In certain embodiments, the expression cassette or delivery vector, e.g., LV, comprises a polynucleotide sequence comprising the following sequences in 5' to 3' order: a) a 5'LTR, optionally a modified 5'LTR; b) HIV-1 ψ sequence; c) RRE, d) cPPT / CTS sequence; e) a PGK promoter sequence, optionally a human PGK promoter sequence; f) a sequence encoding a pyruvate kinase polypeptide, optionally a coRPK coding sequence or a cDNA sequence; g) optionally a mutant wPRE sequence comprising or consisting of the sequence of SEQ ID NO: 24; h) optionally, a dNEF signal; i) a 3'LTR, optionally a modified 3'LTR; j) SV40 poly(A) signal; k) SV40 origin of replication (ori), l) T7 promoter, m) pUC ori, u) a CMV enhancer sequence, and v) CMV promoter sequence.

[0121] In certain embodiments of any of the expression cassettes and gene delivery vectors described herein, the coRPK cDNA or coding sequence encodes a PKLR polypeptide comprising or consisting of a sequence disclosed in any of GenBank Accession Nos. XP_016856982.1, XP_011507942.1, XP_006711449.1, NP_870986.1, or NP_000289.1, or a functional fragment of any of these sequences, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any of these sequences.

[0122] Gene therapy vectors incorporating the polynucleotide cassettes of the present disclosure can be produced using standard methodologies. For example, in the case of LV virions, an LV expression vector according to the present invention can be introduced into producer cells, followed by an LV helper construct, which contains an LV coding region that can be expressed in the producer cells and complements LV helper functions not present in the LV vector. This is followed by the introduction of a helper virus and / or additional vectors into the producer cells, which provide additional functions that can support efficient LV virus production. The producer cells are then cultured, and LV is produced. These steps are carried out using standard methodologies.

[0123] Any suitable method for producing viral particles for delivery of the subject polynucleotide cassettes can be used, including, but not limited to, those described in the Examples that follow. Any concentration of viral particles suitable for effectively transducing mammalian cells can be prepared for contacting mammalian cells in vitro or in vivo. For example, viral particles can be at a concentration of 10 per ml. 8 At a concentration of vector genomes, e.g., 5 x 10 per ml 8 10 vector genomes per ml 9 5 x 10 vector genomes per ml 9 10 vector genomes per ml 10 5 x 10 vector genomes per ml 10 10 vector genomes per ml 11 5 x 10 vector genomes per ml 11 10 vector genomes per ml 12 5 x 10 vector genomes per ml 12 10 vector genomes per ml 13 1.5 x 10 vector genomes per ml 13 3 x 10 vector genomes per ml 13 5 x 10 vector genomes per ml13 7.5 x 10 vector genomes per ml 13 9 x 10 vector genomes per ml 13 1 x 10 vector genomes per ml 14 5 x 10 vector genomes per ml 14 however, typically at a concentration of 1 x 10 vector genomes per ml 15 The vector may be formulated at a concentration of no more than 1 vector genome.

[0124] In preparing the subject LV compositions, any host cell may be utilized to produce LV virions, including, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell may also be a packaging cell, in which the LV rep and cap genes are stably maintained in the host or producer cell in which the LV vector genome is stably maintained and packaged. Exemplary packaging and producer cells are derived from SF-9, 293, A549, or HeLa cells. The LV vector is purified and formulated using standard techniques known in the art.

[0125] In certain embodiments, the invention provides cells comprising an expression cassette or gene delivery vector disclosed herein. In related embodiments, the cells comprise an expression cassette disclosed herein or have been transduced with a viral vector having an expression cassette disclosed herein integrated into the cellular genome. In certain embodiments, the cells are cells used to produce viral gene delivery vectors. In other embodiments, the cells are cells delivered to a subject to provide the subject with a gene product encoded by the expression cassette. Thus, in certain embodiments, the cells are autologous to the subject being treated or are cells obtained from the subject being treated. In other embodiments, the cells are allogeneic to the subject being treated or are cells obtained from a donor other than the subject being treated. In certain embodiments, the cells are mammalian cells, e.g., human cells. In certain embodiments, the cells are blood cells, red blood cells, hematopoietic progenitor cells, bone marrow cells, e.g., lineage-depleted bone marrow cells, hematopoietic stem cells (e.g., CD34+), or committed hematopoietic erythroid progenitor cells.

[0126] The present invention includes pharmaceutical compositions comprising the polynucleotide cassettes, gene delivery vectors, or cells described herein and pharmaceutically acceptable carriers, diluents, or excipients. The subject polynucleotide cassettes, gene delivery vectors, or cells can be combined with pharmaceutically acceptable carriers, diluents, and reagents useful in preparing formulations, including generally safe, non-toxic, and preferably primate-acceptable excipients. Such excipients can be solid, liquid, semisolid, or, in the case of aerosol compositions, gaseous. Examples of such excipients, carriers, or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Additional active compounds can also be incorporated into the formulation. The solutions or suspensions used in the formulation may include sterile diluents such as water for injection, saline, solidified oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate; detergents such as Tween 20 to prevent aggregation; and compounds for isotonicity such as sodium chloride or dextrose. pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. In certain embodiments, the pharmaceutical composition is sterile.

[0127] Pharmaceutical compositions suitable for use in the present invention further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.

[0128] Sterile solution can be prepared by incorporating active compound with one or combination of the components listed above in the required amount in suitable solvent, and then sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into sterile vehicle that contains basic dispersion medium and other components required from the components listed above.For the sterile powder that is used to prepare sterile injection solution, the method of preparation is vacuum drying and freeze-drying, which brings about the powder of active ingredient plus any additional desired components from the solution that has been previously sterilized and filtered.

[0129] In one embodiment, the composition is prepared with a carrier that protects the gene cassette or expression vector from rapid excretion from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used.Methods for preparing such formulations will be clear to those skilled in the art.Such materials can also be obtained commercially.

[0130] It is particularly advantageous to formulate oral, intraocular or parenteral compositions into unit dosage form for the sake of easy administration and uniformity of dosage.Unit dosage form as used herein refers to a physically separate unit suitable for single administration to the subject to be treated, each unit containing a predetermined amount of active ingredient calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification for unit dosage form of the present invention is determined by and directly depends on the specific characteristics of active compound and the therapeutic effect to be achieved, and the inherent limitations of the technical field of compounding such active compound for individual treatment.

[0131] The pharmaceutical compositions may be included in a dosage container, pack, or dispenser, eg, a syringe, eg, a pre-filled syringe, together with instructions for administration.

[0132] The pharmaceutical compositions of the present invention include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compounds that can provide (directly or indirectly) biologically active metabolites or residues thereof upon administration to an animal, including a human.

[0133] The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of the compound of the present invention, i.e., a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects thereto.Various pharmaceutically acceptable salts are known in the art and are described, for example, in "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and more recent editions); "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007; and J.Pharm.Sci. 66:2 (1977).In addition, for a discussion of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0134] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals, or organic amines. Metals used as cations include sodium, potassium, magnesium, calcium, etc. Amines include N-N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., "Pharmaceutical Salts," J. Pharma Sci., 1977, 66, 119). Base addition salts of acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in a conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acids for purposes of the present invention.

[0135] The subject polynucleotide cassettes, gene delivery vectors, e.g., recombinant viruses (virions), or cells (e.g., transduced with the gene delivery vectors disclosed herein) can be incorporated into pharmaceutical compositions for administration to mammalian patients, particularly primates, and more particularly humans. The subject polynucleotide cassettes, gene delivery vectors, e.g., virions, or cells can be formulated in a non-toxic, inert, pharmaceutically acceptable aqueous carrier, preferably at a pH in the range of 3 to 8, more preferably in the range of 6 to 8. Such sterile compositions will comprise vectors or virions containing nucleic acids encoding therapeutic molecules dissolved in an aqueous buffer having an acceptable pH upon reconstitution.

[0136] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of the cells, vectors, or virions disclosed herein in admixture with a pharmaceutically acceptable carrier and / or excipient, such as saline, phosphate-buffered saline, phosphate, and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary amino acids, polymers, and sugars include octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's solution and Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycols. Preferably, the formulation is stable at 4°C for at least 6 months.

[0137] In some embodiments, the pharmaceutical compositions provided herein contain a buffer such as phosphate-buffered saline (PBS) or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to those skilled in the art, such as those described in Good et al. (1966) Biochemistry 5:467. The pH of the buffer in which the pharmaceutical composition contains a tumor suppressor gene contained in an adenoviral vector delivery system can range from 6.5 to 7.75, preferably from 7 to 7.5, and most preferably from 7.2 to 7.4.

[0138] In one particular embodiment, 1×10 8 More than 1 x 10 vector genomes, e.g., 1 x 10 9 , 1×10 10 , 1×10 11 , 1×10 12 , or 1 × 10 13 More than 1 x 10 vector genomes, in one particular example, 14vector genomes, but typically 4 × 10 15 The vector may be formulated into any suitable unit dose, including, but not limited to, up to about 5 x 10 vector genomes. In some cases, the unit dose is up to about 5 x 10 15 vector genomes, e.g., 1 x 10 14 1 x 10 or less vector genomes, e.g., 1 x 10 13 , 1×10 12 , 1×10 11 , 1×10 10 , or 1 × 10 9 vector genomes, in one particular example, 1 x 10 8 vector genomes, typically 1 x 10 8 In some cases where the unit dose is greater than 1 x 10 vector genomes, 10 ~1×10 11 In some cases, the unit dose is 1 x 10 vector genomes. 10 ~3×10 12 In some cases, the unit dose is 1 x 10 vector genomes. 9 ~3×10 13 In some cases, the unit dose is 1 x 10 vector genomes. 8 ~3×10 14 In one embodiment, this range is about 5 x 10 vector genomes. 10 ~Approx. 1×10 11 In some embodiments, the range is about 1 x 10 vector genomes. 9 ~Approx. 1×10 10 This is a vector genome.

[0139] In some cases, the unit dose of the pharmaceutical composition can be measured using the multiplicity of infection (MOI). MOI refers to the ratio of vector or viral genome to cells to which nucleic acid can be delivered, or the fold ratio. In some cases, the MOI is 1 x 10 6 In some cases, the MOI can be 1 x 10 5 ~1×10 7 In some cases, the MOI can be 1 x 104 ~1×10 8 In some cases, the recombinant virus of the disclosure can be at least about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1 × 10 18 In some cases, the recombinant viruses of the present disclosure are administered at an MOI of 1x10 8 ~3x10 14 In some cases, the recombinant viruses of the present disclosure are at an MOI of up to about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1 × 10 18 In some embodiments, the range is an MOI of about 20 to about 400.

[0140] In some embodiments, the amount of pharmaceutical composition is about 1 x 10 8 ~Approx. 1×10 15 recombinant virus, approximately 1 x 10 9 ~Approx. 1×1014 recombinant virus, approximately 1 x 10 10 ~Approx. 1×10 13 recombinant virus, or approximately 1 x 10 11 ~Approx. 3×10 12 Contains recombinant viruses.

[0141] method As disclosed herein, the subject polynucleotide cassettes and gene delivery vectors, collectively referred to herein as "subject compositions," find use in expressing transgenes in animal cells. For example, the subject compositions may be used in research to, e.g., determine the effect of a gene on cell viability and / or function. As another example, the subject compositions may be used in medicine, e.g., to treat or prevent a disease or disorder. Thus, in some aspects of the present invention, methods for expressing a gene in a cell are provided, the methods comprising contacting the cell with a composition of the present disclosure. In some embodiments, the contacting occurs in vitro or ex vivo. In some embodiments, the contacting occurs in vivo, i.e., the subject composition is administered to a subject.

[0142] When mammalian cells are contacted in vitro or ex vivo with the subject polynucleotide cassette or a gene delivery vector containing the subject polynucleotide cassette, the cells can be derived from any mammalian species, for example, rodents (e.g., mice, rats, gerbils, squirrels), rabbits, cats, dogs, goats, sheep, pigs, horses, cows, primates, or humans. Cells can be derived from established cell lines, or they can be primary cells, where "primary cells," "primary cell lines," and "primary cultures" are used interchangeably herein to refer to cells and cell cultures derived from a subject and grown in vitro for a limited number of culture passages, i.e., for the division of the culture. For example, a primary culture is a culture that has been passaged 0, 1, 2, 4, 5, 10, or 15 times, but not enough times to pass through the crisis stage. Typically, the primary cell lines of the invention are maintained in vitro for fewer than 10 passages.

[0143] In certain embodiments, the cells contacted with the disclosed polynucleotide cassette or gene delivery vector are hematopoietic stem cells (HSCs) from a subject or donor, for example, an allogeneic donor. In some embodiments, a biological sample, for example, peripheral blood, is obtained from a subject after hematopoietic stem cell (HSC) mobilization. In one embodiment, HSCs and / or progenitor cells are mobilized by treating the subject with G-CSF or an analog thereof. HSCs and progenitor cells (HSPCs) in peripheral blood can be mobilized before collecting the biological sample. Peripheral blood HSCs and HSPCs can be mobilized by any method known in the art. Peripheral blood HSCs and HSPCs can be mobilized by treating the subject with any agent(s) described herein or known in the art that increases the number of HSPCs circulating in the subject's peripheral blood. For example, in certain embodiments, peripheral blood is mobilized by treating a subject with one or more cytokines or growth factors (e.g., G-CSF, Kit ligand (KL), IL-1, IL-7, IL-8, IL-11, Flt3 ligand, SCF, thrombopoietin, or GM-CSF (such as sargramostim)). Different types of G-CSF that can be used in peripheral blood mobilization methods include filgrastim and long-acting G-CSF: pegfilgrastim. In certain embodiments, peripheral blood is mobilized by treating a subject with one or more chemokines (e.g., macrophage inflammatory protein-1a (MIP1a / CCL3)), chemokine receptor ligands (e.g., chemokine receptor 2 ligands GROL3 and GR013M), chemokine receptor analogs (e.g., stromal cell-derived factor-1a (SDF-1a) protein analogs such as SDF-1a, such as CTCE-0021, CTCE-0214, or Met-SDF-113), or chemokine receptor antagonists (chemokine (C-X-C motif) receptor 4 (CXCR4) antagonists such as AMD3100). In certain embodiments, peripheral blood is mobilized by treating a subject with one or more anti-integrin signaling agents (e.g., anti-very late antigen 4 (VLA-4) antibodies or anti-vascular cell adhesion molecule 1 (VCAM-1) blocking antibodies).In certain embodiments, peripheral blood is mobilized by treating subject with one or more cytotoxic drugs such as cyclophosphamide, etoposide or paclitaxel.In certain embodiments, peripheral blood can be mobilized by administering one or more of the drugs listed above to subject for a certain period of time.For example, subject can be treated with one or more drugs (e.g., G-CSF) by injection (e.g., subcutaneous, intravenous, or intraperitoneal) once a day or twice a day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days before collecting HSPC.In specific embodiments, HSPC is collected within 1, 2, 3, 4, 5, 6, 7, 8, 12, 14, 16, 18, 20, or 24 hours after the last administration of the drug used to mobilize HSPC into peripheral blood. In certain embodiments, HSCs and HSPCs are mobilized by treating a subject with two or more different types of agents described above or known in the art, such as a growth factor (e.g., G-CSF), a chemokine receptor antagonist (e.g., a CXCR4 receptor antagonist such as AMD3100), or a growth factor (e.g., G-CSF or KL) and an anti-integrin agent (e.g., a function-blocking VLA-4 antibody). In one embodiment, HSCs and / or progenitor cells are mobilized by treating a subject with G-CSF or an analog thereof. In one embodiment, the G-CSF is filgrastim. In one embodiment, HSCs and / or progenitor cells are mobilized by treating a subject with plerixafor. In certain embodiments, HSCs and / or progenitor cells are mobilized using a combination of filgrastim and plerixafor, by filgrastim alone, or by plerixafor alone. In certain embodiments, different types of mobilizing agents are administered simultaneously or sequentially.For additional information regarding methods of peripheral blood mobilization, see, e.g., Craddock et al., 1997, Blood 90(12):4779-4788; Jin et al., 2008, Journal of Translational Medicine 6:39; Pelus, 2008, Curr. Opin. Hematol. 15(4):285-292; Papayannopoulou et al., 1998, Blood 91(7):2231-2239; Tricot et al., 2008, Haematologica 93(11):1739-1742; and Weaver et al., 2001, Bone Marrow Transplantation 27(2):S23-S29).

[0144] In some embodiments, cells are enriched before contacting the cells with the disclosed polynucleotide cassette or gene delivery vector. As used herein, "enriched" or "highly enriched" refers to a method of enriching a cell population that is intended to result in substantial enrichment of cells expressing a particular biological marker, e.g., CD34. For example, clinically used CD34 enrichment is characterized by the enrichment of cells expressing CD34. +This resulted in a mean cell yield of 61.6% and a median cell yield of 65.7%, and a mean relative purity of 88.5% and a median relative purity of 95.9% (N=166) (Clin Lab. 2016 Jul 1;62(7):1243-1248 (PMID: 28164638)). "Enrichment" refers to a process aimed at substantially enriching for CD34+ cells, a relatively rare cell type that typically comprises 0.2-2% of the cell product in mobilized leukapheresis or bone marrow collection. Enrichment of CD34+ cells from mobilized leukapheresis or bone marrow collection targets a final CD34+ percentage that increases from 0.2-2% to >80%. To achieve this, the initial application of the biological sample to the capture matrix is ​​followed by repeated buffer exchanges, referred to herein as "washes," to remove cells that are weakly or nonspecifically bound to the capture matrix. Typically, cells are removed from the capture matrix and reapplied after each wash cycle. Removal and reapplication can be done manually by pipetting from a tube, or automated using a pump and tubing system. For example, when using the Quad Technologies MagCloudz® coupled with the Dynabeads® magnetic cell separation system, cell-magnetic particle complexes are separated in a tube on a magnetic stand, and washing is performed manually. When using the Miltenyi Biotec CliniMACS® system, a pre-set automated program applies the cell-magnetic particle complexes to a magnetic column in a tubing set, and washing / reapplication is performed using a valve pump system.In certain embodiments, selection may be performed on a variety of instruments, including, but not limited to, the Miltenyi Biotec MACSQuant Tyto®, Quad Technologies MagCloudz®, GE Spax® Cell Separation System, TerumoElutra® Cell Separation System, COBE Spectra® Cell Separator, SynGenLAB® or WASH® System, Fresenius-KabiLovo®, Miltenyi Biotec CliniMACS® System, or CliniMACS Prodigy® System. Selection may be performed in the laboratory or at the point-of-care. Detailed methods for the preparation and enrichment of cells and cell populations, including exemplary methods for the selection of CD34+ cells, are described, for example, in International Patent Publication No. WO 2016 / 118790. Exemplary selection methods useful for high stringency selection are provided in U.S. Pat. No. 8,727,132.

[0145] In certain embodiments, peripheral blood is obtained via a syringe or catheter inserted into a subject's or donor's vein. For example, peripheral blood can be collected using an apheresis machine. Blood flows from the vein through a catheter into the apheresis machine, where white blood cells containing HSPCs are separated from the remaining blood, which is then returned to the subject's body. Apheresis can be performed for several hours over several consecutive days (e.g., 1-5 days) until sufficient HSPCs are collected.

[0146] In certain embodiments, bone marrow is obtained from the subject's posterior iliac crest by needle aspiration (see, eg, Koda et al., 1984, J. Clin Invest. 73:1377-1384).

[0147] In certain embodiments, the hematocrit level of a biological sample may be determined. The hematocrit level may be determined by centrifuging the sample in a processing chamber to separate the RBCs of the sample into layers so that the packed cell volume may be determined. It should be understood that the sample may be combined with an anticoagulant to aid in determining the hematocrit level, and such an anticoagulant may be added to the processing chamber before or during centrifugation. Alternatively, the hematocrit level may be determined by measuring the optical properties of the sample. For example, a spectrometer may be used to analyze the sample. It should be understood that any type of known spectroscopic method for determining hematocrit level may be used, such as, for example, Raman spectroscopy and / or light scattering techniques.

[0148] In certain embodiments, before preparing one or more cell populations enriched for CD34+ cells from the biological sample, the biological sample is depleted of red blood cells. In some embodiments, the cells remaining after the depletion procedure are washed. In another embodiment, a non-specific IgG is added to the washed cells. In some embodiments, the non-specific IgG is phlebogamma.

[0149] Embodiments of the present invention include mammalian cells (e.g., CD34+ cells) transduced with a viral delivery vector, e.g., an LV vector containing the human PKLR gene. Accordingly, the present invention includes methods of transducing mammalian cells, e.g., human hematopoietic stem cells or other cells described herein, comprising contacting the cells with a viral delivery vector, e.g., an LV vector, containing an expression cassette described herein. In certain embodiments, the cells are previously obtained from the subject to be treated or another donor. In certain embodiments, the subject is diagnosed with PKD, and the cells are transduced with an LV containing an expression cassette encoding pyruvate kinase, e.g., a coRPK coding region or cDNA. It is understood that the disclosed methods, e.g., methods used to deliver pyruvate kinase gene products to a subject using a coPRK cDNA sequence, can also be used to treat hemolytic anemia and / or normalize erythroid differentiation, increase the number of functional mature red blood cells, reduce extramedullary erythropoiesis, and reduce splenomegaly and other secondary effects of hemolytic anemia or PKD.

[0150] To promote expression of the transgene, a subject polynucleotide cassette or a gene delivery vector comprising a subject polynucleotide cassette is contacted with cells for about 30 minutes to 24 hours or more, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, 24 hours, etc.

[0151] A subject polynucleotide cassette or a gene delivery vector containing a subject polynucleotide cassette is provided to subject cells one or more times, e.g., one, two, three, or more than three times, and the cells are incubated with the agent(s) for some time after each contact event, e.g., 16-24 hours, after which the medium is replaced with fresh medium and the cells are further cultured. Contact with the cells can occur in any culture medium and under any culture conditions that promote cell survival. The culture may also contain growth factors to which the cells respond. As defined herein, growth factors are molecules that can promote cell survival, proliferation, and / or differentiation in culture or in intact tissues through specific effects on transmembrane receptors. Growth factors include polypeptide and non-polypeptide factors.

[0152] Typically, the subject polynucleotide cassette or a gene delivery vector comprising the subject polynucleotide cassette is provided to generate transgene expression in cells.As discussed elsewhere herein, the effective amount can be easily determined empirically, for example, by detecting the presence or level of the gene product of the transgene, and by detecting the effect on cell viability or function.Typically, an effective amount of the subject polynucleotide cassette or a gene delivery vector comprising the subject polynucleotide cassette will promote the expression of the transgene in cells more than the same amount of polynucleotide cassette known in the art.Typically, the expression will be enhanced by 2-fold or more, for example, as known in the art, for example, 3-fold, 4-fold, or 5-fold or more, and in some cases, 10-fold, 20-fold, or 50-fold or more, for example, 100-fold, compared to the expression from a reference or control polynucleotide cassette.

[0153] When cells are contacted in vivo with a subject polynucleotide cassette or a gene delivery vector comprising a subject polynucleotide cassette, the subject may be any mammal, such as a rodent (e.g., mouse, rat, gerbil), rabbit, cat, dog, goat, sheep, pig, horse, cow, or primate. In preferred embodiments, the primate is a human. In some embodiments, the cell is a CD34+ cell.

[0154] The methods and compositions of the disclosure find use, for example, in the treatment of pyruvate kinase deficiency.

[0155] In another embodiment, the present invention includes a method for treating a disease in a subject in need thereof, comprising providing to the subject an effective amount of cells transduced with a gene delivery vector, e.g., a viral vector, that expresses a therapeutic gene product within the cells. In certain embodiments, the cells are autologous to the subject. In certain embodiments, the cells are erythroid cells, e.g., hematopoietic stem cells or committed hematopoietic erythroid progenitor cells. In some embodiments, the cells are bone marrow cells, e.g., lineage-depleted bone marrow cells. In certain embodiments, the method is used to treat PKD, and the viral vector is an LV comprising an expression construct disclosed herein, comprising a human PGK promoter operably linked to a coRPK gene cDNA or coding sequence, and a mutated wPRE as disclosed herein. In certain embodiments, the cells are provided to the subject parenterally, e.g., via intravenous injection. In some embodiments, the cells are provided to the subject by transfusion.

[0156] In another embodiment, the present invention includes a method for treating PKD in a subject in need thereof, comprising providing to the subject an effective amount of autologous CD34+ stem cells transduced with an LV vector expressing a coRPK cDNA in the cells, the LV vector comprising a human PGK promoter operably linked to the coRPK cDNA or coding sequence and a mutated wPRE sequence as disclosed herein. In certain embodiments, the cells are hematopoietic stem cells or committed hematopoietic erythroid progenitor cells, e.g., bone marrow cells. In certain embodiments, the cells are provided to the subject parenterally, e.g., via intravenous injection.

[0157] In another embodiment, the present invention provides a method for treating a disease in a subject in need thereof, comprising providing to the subject an effective amount of a gene delivery vector, e.g., a viral vector, that expresses a therapeutic gene product in the subject. In certain embodiments, the method is used to treat PKD, and the viral vector is an LV comprising an expression construct disclosed herein, which comprises a human PGK promoter operably linked to a coRPK gene cDNA or coding sequence, and a mutated wPRE as disclosed herein. In certain embodiments, the gene delivery vector is provided to the subject parenterally, e.g., via intravenous injection.

[0158] In certain embodiments, the cells or gene delivery vector are provided to the subject in a pharmaceutical composition.

[0159] In some embodiments, the subject methods provide a therapeutic benefit, e.g., prevention of the onset of a disorder, halting the progression of a disorder, reversing the progression of a disorder, etc. In some embodiments, the subject methods include detecting that a therapeutic benefit has been achieved. Those skilled in the art will understand that such measurements of therapeutic efficacy will be applicable to the particular disease being modified and will recognize appropriate detection methods to use to measure therapeutic efficacy.

[0160] The expression of the transgene using the subject transgene is expected to be robust.Therefore, in some cases, the expression of the transgene, for example, by measuring the level of gene product, measuring therapeutic efficacy, etc., can be observed within 2 months after administration, for example, within 4, 3, or 2 weeks after administration, for example, within 1 week after administration of the subject composition.It is also expected that the expression of the transgene will continue over time.Therefore, in some cases, the expression of the transgene, for example, by measuring the level of gene product, measuring therapeutic efficacy, etc., can be observed within 2 months or more after administration of the subject composition, for example, within 4, 6, 8, or 10 months or more, and in some cases, for more than 1 year, for example, within 2, 3, 4, or 5 years, and in some cases, for more than 5 years.

[0161] In certain embodiments, the method includes detecting expression of the transgene in a cell or subject, wherein expression is enhanced compared to expression from a polynucleotide cassette that does not include one or more of the improved elements of the present disclosure. Typically, expression will be enhanced by 2-fold or more, e.g., 3-fold, 4-fold, or 5-fold or more, as known in the art, and in some cases 10-fold, 20-fold, or 50-fold or more, e.g., 100-fold, relative to expression from a reference, i.e., control, polynucleotide cassette, as evidenced, for example, by earlier detection, higher levels of gene product, stronger functional effect on the cell, etc.

[0162] Typically, when the subject composition is an LV comprising a subject polynucleotide cassette of the present disclosure, an effective amount to achieve the transformation is about 1 x 10 8 More than 1 x 10 vector genomes, in some cases 1 x 10 9 , 1×10 10 , 1×10 11 , 1×10 12 , or 1 × 10 13 More than 1 x 10 vector genomes, in one particular example, 14 vector genomes, and typically 1 x 10 15In some cases, the amount of vector genomes delivered will be up to about 1 x 10 15 vector genomes, e.g., 1 x 10 14 1 x 10 or less vector genomes, e.g., 1 x 10 13 , 1×10 12 , 1×10 11 , 1×10 10 , or 1 × 10 9 vector genomes, in one particular example, 1 x 10 8 vector genomes, typically 1 x 10 8 In some cases, the amount of vector genomes delivered is 1 x 10 or more. 10 ~1×10 11 In some cases, the amount of vector genome delivered is 1 x 10 10 ~3×10 12 In some cases, the amount of vector genome delivered is 1 x 10 9 ~3×10 13 In some cases, the amount of vector genome delivered is 1 x 10 8 ~3×10 14 This is a vector genome.

[0163] In some cases, the amount of pharmaceutical composition can be measured using multiplicity of infection (MOI). In some cases, MOI can refer to the ratio of vector or viral genome to cells to which nucleic acid can be delivered, or the ratio. In some cases, MOI is 1 x 10 6 In some cases, the MOI can be 1 x 10 5 ~1×10 7 In some cases, the MOI can be 1 x 10 4 ~1×10 8 In some cases, the recombinant viruses of the disclosure may be at least about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×105 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1 × 10 18 In some cases, the recombinant viruses of the present disclosure are administered at an MOI of 1 x 10 8 ~3×10 14 In some cases, the recombinant viruses of the present disclosure are at an MOI of up to about 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1 × 10 18 MOI is .

[0164] In some embodiments, the amount of pharmaceutical composition is about 1 x 10 8 ~Approx. 1×10 15 recombinant virus particles, approximately 1 x 10 9 ~Approx. 1×10 14 recombinant virus particles, approximately 1 x 10 10 ~Approx. 1×10 13 particles of recombinant virus, or approximately 1 x 10 11 ~Approx. 3×10 12 It contains recombinant virus particles.

[0165] Any total number of viral particles can be administered to a mammal suitable to provide adequate transduction of cells to impart a desired effect or to treat a disease. In various preferred embodiments, at least 10 8 ;5×10 8 ;10 9 ;5×10 9 ,10 10 ,5×10 10 ;10 11 ;5×10 11 ;10 12 ;5×10 12 ;10 13 ;1.5×10 13 ;3×10 13 ;5×10 13 ;7.5×10 13 ;9×10 13 , 1×10 14 virus particles, or 5 x 10 14 More than 1 x 10 virus particles, but typically 15 No more than 5 x 10 viral particles are injected. Any suitable number of vectors can be administered to the mammalian or primate eye. In one embodiment, the method involves a single administration, while in other embodiments, multiple administrations are administered over time as deemed appropriate by the attending clinician. In some embodiments, to achieve high transduction efficiency, 5 x 10 viral particles are injected in a single administration (24-hour transduction). 5 At least 2 x 10 cells / ml 8 VG / ml is required. Individual doses are typically at least the amount required to produce a measurable effect in a subject and can be determined based on the pharmacokinetics and pharmacology ("ADME") of the absorption, distribution, metabolism, and excretion of the subject composition or its by-products, and therefore based on the disposition of the composition in the subject's body. This includes consideration of the route of administration and dosage. Effective dosages and / or dosage regimens can be easily determined empirically from preclinical studies, safety and dose escalation studies, individual clinician-patient relationships, and in vitro and in vivo assays such as those described herein and illustrated in the Examples.

[0166] Some aspects of the present invention are described herein with reference to exemplary applications for illustration. It should be understood that numerous specific details, relationships, and methods are described to provide a thorough understanding of the present invention. However, those skilled in the relevant art will readily recognize that the present invention can be practiced without one or more of the specific details or in other ways. The present invention is not limited by the illustrated order of acts or events, as some acts may occur in different orders and / or simultaneously with other acts or events. Furthermore, not all illustrated acts or events are required to implement the methodology in accordance with the present invention.

[0167] It is further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a prior basis for using exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements, or the use of a "negative" limitation.

[0168] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0169] All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced herein and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety to disclose and describe, for example, the methods and / or materials in connection with which the publications are cited, and it is understood that the present disclosure supersedes any disclosure of the incorporated publications to the extent of any conflict.

[0170] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications can be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. [Example]

[0171] The following examples are put forward to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0172] Experimental Method Production of Vector LV Supernatant LVs were generated as described herein. The CoRPK sequence was designed using GeneArt® software to increase the GC content of the sequence and prevent ectopic splice sites. The vector was developed using the pCCL.sin.ppt.hPGK-EGFP-wPRE* construct as a backbone, generously provided by Dr. Naldini (HSR-TIGET, San Raffaele Telethon Institute, Milano, Italy). The original pCCL lentiviral transfer vector and its elements are described in Dull et al., J Virol, 1998;72(11):8463-8471, which is incorporated herein in its entirety. Vector stocks of VSV-G pseudotyped LV were prepared by three-plasmid calcium phosphate-mediated transfection in 293T cells (ATCC: CRL-1573, Rockeville, MD, USA) as previously described [Follenzi A, et al. (2000). Nat Genet 25:217-222]. Titers of infectious LV were determined by qPCR in HT1080 cells (ATCC: CCL-121) as previously described [Charrier S, et al. (2005). Gene Ther 12:597-606]. 7 ~10 8 LV stocks with titers of virus particles (vp) / mL were obtained according to conventional methods.

[0173] Purification and transduction of mouse HSCs. BM was collected from the leg bones of 8- to 14-week-old male PKD mice and transduced with Lin. - Lineage-negative cells (Lin) were isolated using a Cell Depletion kit (Miltenyi Biotec, Gladbach, Germany). - ) was purified to a purity of 70-90%. -Cells were pre-stimulated with 100 ng / mL recombinant mouse SCF (R&D Systems Inc., Minneapolis, MN) for 24 hours in IMDM-Glutamax medium supplemented with 20% FBS and 0.5% antibiotics (50 U / mL penicillin and 50 μg / mL streptomycin, Thermo Fisher Scientific, Waltham, MA). Then, they were transduced with EGFP- or coRPK-bearing LVs at an MOI of 1–10 vp / cell for two cycles. Each transduction was performed using CH-296 fibronectin fragments (2 μg / cm). 2 The incubation was carried out for 24 hours in the presence of the aforementioned cytokines on plates pre-coated overnight at 4°C with RetroNectin (Takara Shuzo, Otsu, Japan).

[0174] In vivo RBC survival. Transplanted mice carrying the coRPK transgene were injected with three consecutive intravenous injections (12-hour intervals) of biotin 3-sulfo-N-hydroxysuccinimide ester sodium salt (50 mg / kg) (Sigma Aldrich, Saint Louis, MO). 12 hours after the final injection, tail vein blood was collected and labeled with 2 μg / mL anti-mouse Ter119-PE (BD Bioscience, San Jose, CA) and streptavidin-FITC (50 μg / mL, BD Biosciences, San Jose, CA) for 30 minutes at 4°C. Samples were analyzed every 2–4 days for 40 days after injection using an EPICS XL flow cytometer (Beckman Coulter, Brea, CA). RBC survival kinetics was measured by the proportion of biotinylated cells within the total RBC population.

[0175] CFC assay. CFC assays were performed on the BM and spleen of control and transplanted mice using Methocult medium GF M3434 (Stem Cell Technologies, Vancouver, Canada) according to the manufacturer's protocol. BM cells were harvested from all groups of mice at different time points after transplantation, and CFUs (clusters of 30 or more cells) were counted using a Nikon Diaphot-TMD microscope 7 days after seeding.

[0176] Identification of hematopoietic lineages. PBMCs were obtained from the tail vein of transplanted animals and labeled with a panel of antibodies to detect different hematopoietic cell types. Myeloid cells were detected with anti-GR-1 and anti-Mac-1 biotinylated antibodies (BD Bioscience, San Jose, CA, 5 μg / mL), while lymphoid cells were detected using anti-CD3-PE antibodies for T cells and anti-B220-PE and anti-B220-PECy5 antibodies for B cells (BD Bioscience, San Jose, CA, 10 μg / mL), along with a SAV-TRC secondary antibody (Invitrogen, Thermo Fisher Scientific, Waltham, MA). Samples were analyzed on a BD LSR Fortessa Cytometer (BD Bioscience, San Jose, CA, USA) supplemented with DAPI (Boehringer, Ingelheim, Germany, 2 μg / mL) to exclude dead cells.

[0177] Structural and Histological Studies. Spleens were harvested, photographed, and weighed on a precision scale to determine the presence of splenomegaly. Histological studies were performed on spleen and liver sections obtained according to conventional histological methods and stained with hematoxylin (Gill-2 Hematoxylin, Thermo, Pittsburgh, USA) and eosin (Eosin Alcoholic, Thermo Fisher Scientific, Waltham, MA). Iron deposits were also studied in the spleen using Prussian blue or Perls staining (Sigma Aldrich, Saint Louis, MO) according to the manufacturer's instructions. All sections were examined using an Olympus BX40 light microscope and photographed with an Olympus DP21 camera at a final magnification of 100x or 200x.

[0178] Erythroid differentiation. Flow cytometry analysis of Ter119 and CD71 marker intensities in BM and spleen was performed as described elsewhere [Socolovsky M, et al. (2001). Blood 98:3261-3273] using 4 μg / mL anti-mouse Ter119-PE antibody (BD Bioscience, San Jose, CA), 10 μg / mL biotinylated anti-CD71 antibody (BD Bioscience, San Jose, CA), and streptavidin tricolor (Invitrogen, Thermo Fisher Scientific, Waltham, MA) to identify distinct erythroid subpopulations. Cells were then analyzed on an EPICS XL flow cytometer (Beckman Coulter, Brea, CA) using propidium iodide (IP, 2 μg / mL) to detect viable cells.

[0179] Provirus quantification. Detection and quantification of integrated provirus per cell was achieved using primers complementary to the packaging proviral sequence (Ψ) and the mouse Titin housekeeping gene. Total BM and peripheral blood samples were collected periodically, and genomic DNA from nucleated cells was isolated using the DNeasy Blood & Tissue Kit (Qiagen, Venlo, Limburg, The Netherlands). 20–50 ng of genomic DNA (gDNA) was amplified using a 7500 Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA) and primers and probes as previously described [Charrier S, et al. (2011). Gene Ther 18:479–487].

[0180] Chimerism. The presence of donor cells was quantified by qPCR detecting the Y-chromosome SRY gene and the mouse β-actin housekeeping gene. Genomic DNA from the PB of transplanted mice was amplified using the 7500 Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA) using previously described primers and probes [Navarro S et al (2006). Mol Ther 14:525-535]. A standard curve was generated using gDNA extracts from samples containing 0% to 100% BM cells from a male / female mouse mixture, and chimerism was calculated as % donor engraftment = 100 × 2. (CtβAct-CtSRY) It was calculated as follows.

[0181] LAM-PCR procedure. To identify the vector integration site, the 3' vector LTR-genome junction was amplified by LAM-PCR according to the method published by Schmidt et al. (2007) [Nat Methods 4:1051-1057]. An initial linear amplification (100 cycles) was performed using biotinylated LTR-specific primers and up to 100 ng of gDNA as template. The linear amplification product was purified using streptavidin magnetic beads, followed by complementary strand synthesis, parallel digestion with two different restriction enzymes (Tsp509I and HpyCH4IV), and two ligation reactions using linker cassettes complementary to the ends left by the enzyme cleavage. The resulting fragments were amplified by two additional exponential PCR steps. LAM-PCR products were separated and quantified by gel electrophoresis on a MultiNA automated system (Shimadzu).

[0182] Setting up LAM-PCR products for Illumina MiSeq sequencing. Following the method published by Parazynski et al. [Paruzynski A, et al. (2010). Nat Protoc 5:1379-1395], 40 ng of secondary exponential PCR products generated with the Tsp509I and HpyCH4IV enzymes were reamplified using fusion primers containing specific sequences that enable paired-end sequencing on an Illumina MiSeq sequencer. LAM-PCR samples were adapted for 454-pyrosequencing by fusion PCR, adding Roche 454 GS-FLX adapters: adapter A plus an 8-nucleotide barcode was added to the LTR end of the LAM-PCR amplicon; adapter B was added to the linker cassette side. In the 5' to 3' orientation, the final amplicon was constructed as follows: adapter A, barcode, LTR sequence, unknown genomic sequence, linker cassette sequence, and primer B. The purified fusion primer PCR products were run on a MultiNA automated electrophoresis system, quantified, and pooled together to obtain a final equimolar library of 10 nM. The final library was then requantified using the KAPA Library Quantification Kit for the Illumina Sequencing Platform (Kapa Biosystems, Wilmington, MA) on a Viia7 real-time PCR system (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA), yielding an estimated concentration of 16.35 nM. Finally, the library was sequenced using the Illumina MiSeq reagent kit.

[0183] Bioinformatics analysis. To extract vector ISs from high-throughput sequencing platforms, both Roche 454 and Illumina MiSeq / HiSeq, we designed a pipeline that takes raw data (typically in FastQ file format) as input and provides a list of reliable ISs and nearest genes. Fine-level analysis of clone abundance quantification and gene ontology enrichment was performed using Excel, GraphPad Prism™, and available online tools.

[0184] NGS Data Processing and Pipeline Use. The NGS data processing step deals with managing high-throughput data from the Illumina MiSeq sequencing platform, with the goal of identifying ISs that align all valid sequence reads to the reference genome. Data processing comprises two main activities: 1. Data quality check and analysis, where LV vector sequences and other contaminants are trimmed; and 2. Integration site identification, where all valid sequence reads are aligned to the reference genome to obtain valid ISs.

[0185] Data Quality Analysis. A bioinformatics pipeline was developed to identify ISs from Illumina MiSeq raw data. Standard LAM-PCR products contain long-transfer repeat (LTR) sequences, flanking human genome sequences, and linker cassette (LC) sequences. 459 technology enabled the acquisition of LAM-PCR sequences with lengths ranging from 10 bp to 900 bp. Similar results were obtained from Illumina MiSeq paired-end reads. These length boundaries are important parameters to consider in the quality analysis process, as they affect both the subsequent alignment procedure and the vector component identification algorithm. Sequences that were too short to properly align to the reference gene were discarded, as well as those exceeding the maximum size achievable by NSG technology to avoid missing part or all of the LC sequence. Upon completion of the pipeline for each pool, all integration sites were collected both in files (archived on TIGET network-attached file storage—NAS) and in an internal database, which was maintained on a storage server that tracks modified copies.

[0186] Identifying integration sites. To identify unique integration sites and extract an Excel file with all ISs in rows and each sample with the closest gene annotation in columns (IS matrix), the following steps were performed: 1. Creating an IS matrix using a program called create_matrix, which allows for collision detection between projects. This program generates a tab-separated value (TSV) file; 2. Annotating the IS matrix file using the annotate_bed program, which is called for each pool using the input TSV file: awk '{print "chr"$1"\t"$2"\t"$2}' TSV_FILE|tail-n+2>TSV_FILE.bed; annotate_bed-a / opt / genome / mouse / mm9 / annotation / mm9.refGene.TIGET.gtf-b TSV_FILE.bed-o TSV_FILE.annotated.bed; 3. Importing both the annotation and matrix files into a new Excel worksheet.

[0187] Collision detection. To obtain a reliable dataset of ISs from each transplanted mouse, data were filtered from potential contaminations / collisions and from false positives based on sequence counts. To combine integration sites obtained from different experiments, an additional step of data normalization was required.

[0188] The term "collision" is used to identify the presence of identical ISs in independent samples. In our experimental setting, vector integration at the exact same genomic location in different cells is an extremely low probability event. Therefore, detection of identical ISs in independent samples may result from contamination that may occur during different stages of the wet-lab procedures (sample purification, DNA extraction, LAM-PCR, and sequencing). Although our work pipeline is designed to minimize the occurrence of contact between samples, high-throughput analysis of ISs inherently involves a certain degree of background contamination. Because obtaining identical ISs in different samples obtained from the same mouse is used in subsequent steps to infer the biological properties of vector-marked hematopoietic cells (i.e., multilineage potential and persistent clonogenic activity), identifying the degree of contamination between samples is also important. Therefore, we must be able to distinguish the actual occurrence of the same IS in different samples (from the same mouse) from contamination / collision.

[0189] To address these issues, we measured the degree of collisions in the analysis and then designed rules to discard ISs that may be due to collisions from each mouse's dataset. We also evaluated the degree of shared ISs between samples from different test items and mice as a way to minimize the possibility of false positives when searching for shared ISs between samples from the same mouse. We designed a collision detection process that allows for validation of each integration locus. The overall result is that if a set of integration loci is I, then if integration locus i in I is classified as a collision, i is discarded from I. We applied the collision detection process to three independent transplant groups: 1. coPKR170s: Lin transduced with coRPK-expressing LV vectors (coRPK 1-3); - Mice from assay 1 were euthanized on day 170 after transplantation with cells. 2. EGFP: Lin carrying EGFP-expressing LV vectors (EGFP1-6) -Mice from assay 2 were transplanted with cells. 3. coPKR-TC: Secondary recipients were transplanted with pooled BM from a subgroup of primary transplanted mice (coRPK 11-14). Blood and BM were analyzed at different time points. - Mice from assay 2 transplanted with cells.

[0190] Each identical IS has different sequence reads (sequence counts) between different mice. Sequence counts can be used to determine whether a sample from one mouse contaminated another mouse's sample based on abundance criteria. Our rationale is that an integration found in two mice is assigned to the mouse with the highest abundance, while in the other mouse, it is considered a contaminant. Therefore, we were able to identify a differential sequence count threshold that allows us to assign a given collision to a mouse and eliminate other collisions. We obtained a data-derived threshold value of 10, which means that for each IS, if an IS obtains an abundance value (sequence count ratio) 10-fold lower than the highest abundance value (sequence count ratio) of any other TI among all TIs, then it is discarded from the current TI. We applied these rules both between TIs and between selected groups and calculated collision detection using an Excel file by applying the following rules (here we detail TI filtering, but the same rule applies to group filtering as well): 1. Isolate each TI and group all samples from the same TI by summing sequence counts. 2. For the three TIs obtained, for each IS, calculate the ratio of IS sequence counts to the total total reads for the TI. 3. Then, apply the following rules to calculate the threshold 10 that allows each IS to be assigned to a reliable TI:

[0191] When a deleted IS was detected, the reads with that IS were removed from that group so that they could no longer be assigned to that group. The above filters were applied between mice transplanted with different ex vivo transduced cell populations (one cohort of EGFP-expressing mice from Assay 2 and two cohorts of coPKR mice belonging to two independent transplantation experiments). Furthermore, for the coPKR-TC group (Assay 2), the above filtering method was modified in two ways: a) for clonal abundance analysis, to more clearly highlight integration sharing between time points, the following rule was added: if an integration is shared by more than one mouse, the integration is retained across all time points even if their sequence count is less than 10% of the maximum sequence count between mice; b) for lineage-tracing relationships, a stricter filter was applied by eliminating ISs with sequence counts less than 3 and a 10% sequence count filter for sharing between time points. This means that integrations shared between two time points are retained or discarded only if each is more or less abundant than the other.

[0192] Gene Ontology Analysis. All gene ontology analyses were performed using the GREAT online software (http: / / bejerano.stanford.edu / great / public / html / ). This webpage allows users to upload the genomic coordinates of integrations in each dataset and calculate the enrichment level for the tested datasets by correlating the location information (based on binomial distribution analysis for p-value calculation) with the annotated functions of the genes closest to the integration site (based on hypergeometric distribution analysis for p-value calculation) [Groeschel S, et al. (2011). J Inherit Metab Dis 34:1095-1102]. For enrichment analysis, biological processes and molecular functions from the GO database were selected. For both statistical analyses, only gene classes with a false discovery rate of <0.05 were considered (Figure 20).

[0193] Data storage. All data, both raw and results, are stored on TIGET network-attached file storage (NAS) in a root folder, where all alignments from the pipeline, as well as abundance matrices and plots, are available. The NAS storage is protected by authentication and authorization policies, built on a reliable, scalable infrastructure using a redundant array of disks RAID 5, and backed up with CrashPlan software, which is registered with TIGET.

[0194] Example 1 The PGK-coRPK therapeutic LV vector results in stable and long-term restoration of the anemic phenotype in genetically corrected PKD mice. The in vivo efficacy of PGK-coRPK LV (Fig. 2a) was demonstrated using lineage-depleted BM cells (Lin -The PKD phenotype was evaluated by transduction and transplantation of PKD cells. Figure 2a is a schematic diagram of the SIN LV vectors used throughout the gene therapy experiments, harboring the human PGK promoter, which regulates the expression of either the EGFP transgene in the control vector (top panel) or the coRPK cDNA in the treatment vector (bottom panel). The coRPK sequence exhibited 80.4% homology with the human PKLR cDNA and 76.5% homology with the mouse Pklr cDNA, with no changes in the amino acid sequence. Figure 2b is a schematic diagram of the gene therapy protocol implemented to address the functionality of the developed PGK-coRPK LV vector. Restoration of the PKD phenotype was studied 4–9 months after transplantation into the PB and BM through hematological analysis and metabolic profiling. Integration analysis was performed in different tissues and time points in all mice to address the safety of the LV vector. At 280 days post-transplant, total BM from primary recipient mice carrying the coRPK transgene was re-transplanted into lethally irradiated female PKD mice (secondary recipients) to test the stability and safety of engraftment. Lethally irradiated PKD mice transplanted with defective cells transduced with coRPK LVs showed significant improvements in all blood erythroid parameters tested compared with untransplanted PKD littermates or mice transplanted with cells transduced with EGFP LVs (Figure 3 and Table 2). Table 2. Hematological variables recorded in peripheral blood at 140 days after transplantation TIFF0007817979000035.tif55132Data represent mean ± SEM and were statistically analyzed by comparison with EGFP-expressing mice using the Kruskal-Wallis nonparametric test. *p<0.05; **p<0.01.

[0195] RBC counts increased as soon as 40 days post-transplant (Figure 3a), and constitutive reticulocytosis, one of the most common signs of PKD, was significantly restored in mice carrying the PGK-coRPK transgene, reaching levels close to those observed in healthy controls for at least 9 months post-transplant (Figure 3b). Conversely, PKD animals transplanted with EGFP LV-transduced cells exhibited anemia and marked reticulocytosis, paralleling those observed in PKD mice at all time points analyzed. Hemoglobin levels (HGB), hematocrit index (HTC), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) values ​​were also restored in mice transplanted with LV repair cells compared to non-transplanted PKD littermates (Table 2). This hematological restoration was achieved with donor chimerism of 63.66 ± 4.45% and transduction efficiencies ranging from 60% to 90% (Table 3).

[0196] Table 3. Relevant molecular parameters of mice transplanted with genetically modified cells TIFF0007817979000036.tif133138 Data represent mean ± SEM, and nd indicates not determined. a Estimated transduction ratio (X-axis: VCN / WBC, Y-axis: provirus) obtained by interpolation in the linear regression constructed from Experiment 1. + % of CFU.

[0197] Transduced cells showed an average of 1.65 ± 0.08 integrated vector copies per cell, indicating that the PGK-coRPK LV-vector provided sufficient human RPK transgene expression to reverse hemolytic anemia. Notably, expression of the coRPK transgene resulted in an extended red blood cell half-life compared with non-transplanted PKD mice (Figure 3c, d). On average, PKD mice exhibited an RBC half-life of 19 days, whereas gene-repaired mice experienced an RBC half-life of 25 days (a 6-day extension), reaching a value close to the wild-type RBC half-life (Figure 3d). Thus, RBCs from coRPK-expressing mice exhibited survival kinetics intermediate between those of healthy and deficient controls (Figure 3c), likely due to the lack of complete chimerism in these animals (Table 3).

[0198] Nine months after transplantation, hematopoietic progenitor cells from primary recipients were transplanted into secondary recipients, maintaining engraftment levels (62.89 ± 5.61%) and VCN (1.44 ± 0.08 copies) (Table 3). Secondary transplant recipients demonstrated multilineage hematopoietic reconstitution up to 5 months posttransplant (Figure 4) and significant improvement in all PB erythroid parameters (Figure 5 and Table 2). Figure 4a is a diagram of the flow cytometry strategy used to identify different hematopoietic lineages by labeling with CD3-PE, B220-PE, B220-PE-Cy5, Gr1-biotin, and Mac1-biotin antibodies + SAV-PE-Cy5. Figure 4b displays representative dot plots showing the proportion of each lineage in the PB at 140 days posttransplant (Figure 4c). Bars represent the mean ratio ± SEM of healthy (n = 2, black bars) and PKD (n = 2, gray bars) controls and secondary transplanted mice expressing the coRPK therapeutic transgene (n = 4, scratched bars). Additionally, proviral integration was detected in different committed hematopoietic progenitor cells (Figure 6a, b), the number of which remained constant over time (Figure 6c), demonstrating the stability of gene repair and highlighting the safety of PGK-coRPK LV. Figure 6a shows the vector copy number per cell in BM CFU from individual transplanted mice at days 120 and 170 posttransplant. Transduction and chimerism rates are also shown. Figure 6b shows the proviral copy number in cells from different hematopoietic compartments. Columns represent the mean ± SEM of different groups of transplanted mice. Figure 6c shows the kinetics of proviral integration in BM cells from individual transplanted EGFP-expressing mice (gray line) and mice carrying the coRPK transgene (black line).

[0199] Example 2 LV-derived RPK expression normalizes erythroid differentiation, allowing the production of functional mature erythrocytes. PKD mice exhibit a distinctive expansion of the erythroid compartment, driven by a compensatory erythropoietic mechanism (Min-oo et al., 2004). Study of the erythroid differentiation pattern in transplanted mice showed that ectopic RPK expression restored this mechanism (Figure 7a, b). PKD and EGFP-expressing mice exhibited a predominance of immature erythroid progenitors (subpopulation I: proerythroblasts, and subpopulation II: basophilic erythroblasts) in the BM and spleen, and a significant reduction in late erythroid cells (population IV: reticulocytes and mature erythrocytes). Meanwhile, mice transplanted with cells transduced with coRPK LVs exhibited a significant reduction in immature erythroid progenitors (subpopulations I and II) in the BM and spleen, and a significant increase in the late erythroid compartment (subpopulation IV), comparable to that of healthy mice (Figure 7a, b). Additionally, unlike PKD and EGFP-expressing mice, mice carrying the coRPK transgene showed significantly reduced plasma erythropoietin (Epo) levels (Figure 7c). Figure 8a shows total CFUs from the spleen, and Figure 8b shows bone marrow at 140 days post-transplant. Points represent the number of colonies analyzed per mouse, and lines represent the mean ± SEM in each group. Data were statistically analyzed using the nonparametric Kruskal-Wallis test. Normalization of erythropoiesis in PKD mice treated with the therapeutic vector was achieved by a reduction in splenic progenitor cell content to normal levels (Figure 8a), but no change in BM CFU content was observed (Figure 8b).

[0200] Example 3 Transplantation of cells transduced with the coRPK LV vector restores extramedullary erythropoiesis and organ pathology. Due to the active destruction of RPK-deficient red blood cells, PKD and EGFP-expressing mice exhibited acute splenomegaly, with spleen weight and size exceeding 200% compared to healthy controls (Figure 9a, b). Disorganized splenic tissue structure and enlargement of the splenic red pulp were also observed in these animals, indicating robust extramedullary erythropoiesis, supported by the presence of red blood cell clusters in PKD and EGFP-expressing liver sections (Figure 9c). Notably, ectopic expression of the coRPK transgene completely reversed the splenic and liver pathology of gene-corrected mice, reducing RBC accumulation and normalizing the splenic histological structure and size (Figure 9). Furthermore, histological studies revealed the complete absence of iron deposits in the livers of gene-corrected mice, whereas PKD mice, either untransplanted or transplanted with HSCs transduced with an EGFP-carrying vector, exhibited severe iron overload due to the ongoing hemolytic process (Figure 9c). Overall, transplantation of gene-corrected HSCs in PKD mice restored normal erythropoiesis and all secondary effects caused by hemolytic anemia.

[0201] Example 4 LV-derived expression of PGK-coRPK restores the glycolytic pathway in RBCs without altering the WBC metabolic balance. Next, we performed extensive metabolomic analysis of all transplanted and control mice to study functional correction of RPK enzyme activity. Following an untargeted profiling strategy, we observed significant changes in RBC glycolytic intermediates between different groups and identified three broad clusters of metabolite patterns with distinct trends (Figure 10a). RBCs from coRPK-expressing mice showed increases in metabolites from cluster 1, similar to healthy controls but distinct from transplanted mice carrying the EGFP transgene. Similarly, cluster 3 reflected a trend toward decreased metabolites in gene-repaired mice, similar to wild-type mice and distinct from EGFP-expressing mice. Nevertheless, cluster 2 from assay 1 showed no differences in metabolite profiles between transplanted mouse groups (mice expressing EGFP and coRPK) (Figure 10a). Untargeted metabolic profiling showed that the genetic modification altered several key glycolytic intermediates, achieving increased levels of ATP (Figure 10b), ADP (Figure 10c), and pyruvate (Figure 10d) in erythrocytes isolated from mice transplanted with PGK-coRPK LV-transduced HSCs. Given these metabolic trends, we used targeted profiling techniques to analyze other metabolites located near the PK-catalyzed reaction. Levels of the direct PK substrates phosphoenolpyruvate (PEP) (Figure 10e) and 3-phosphoglycerate (3-PG) (Figure 10f), located upstream of the PK-catalyzed reaction, approached those of healthy control mice. Defective erythrocytes expressing the coRPK transgene also resulted in increased levels of D-lactate (Figure 10g), the end product of anaerobic glycolysis, compared with PKD and EGFP-expressing mice. To test whether compensation with glycolytic metabolites resulted in normalization of PK activity in mature RBCs, we measured the activity of this enzyme and normalized it relative to hexokinase activity to avoid the effects of high reticulocyte abundance in deficient animals. To prevent contamination with leukocyte PK activity, RBCs were purified through a cellulose column. Complete compensation of PK activity was observed in animals expressing coRPK, reaching ratios similar to those obtained from wild-type healthy animals and from normal, healthy blood donor volunteers (Figure 11).Figure 11a shows pyruvate kinase activity in RBCs from control mice and mice transplanted with transduced cells, Figure 11b shows hexokinase activity, and Figure 11c shows the ratio of pyruvate kinase to hexokinase enzyme activity. RBCs were purified from blood samples through a cellulose column to avoid contamination with leukocyte PK activity and subjected to enzyme activity assessment. Black bars represent healthy mice (n = 2), white bars represent mice transplanted with cells transduced with an EGFP-expressing vector (n = 3), and scratched bars represent mice transplanted with cells transduced with a coRPK-expressing vector (n = 3). Checkered bars represent values ​​from a healthy volunteer (n = 1). Data represent the mean ± SEM for each group.

[0202] Principal component analysis (PCA) showed that RBC metabolite patterns varied across groups and were significantly different from WBC profiles (Figure 12a). In contrast, WBC subgroups clustered with little variation between clusters, indicating no change in the metabolic balance of leukocytes upon ectopic coRPK expression (Figure 12a). Furthermore, specific metabolite changes observed in untargeted profiling of RBCs were absent in WBCs (Figures 12b-d).

[0203] Example 5 PGK-coRPK LV-transduced cells undergo polyclonal hematopoietic reconstitution without evidence of vector genotoxicity. The integration profiles of LVs carrying either the coRPK or EGFP transgene were analyzed in transplanted mice. Results from genome-wide integration profiles of LVs indicated that each insertion created a unique genetic mark that could be used to track the clonal behavior of individual transduced cells. Genomic DNA (gDNA) was collected from WBCs, BM cells, and primary and secondary transplanted mice, as well as from transduced cell pools (Lin et al., 2014). -The vector IS was obtained from 1000 ribosomal RNA (1000 ribosomal RNA) and 1000 ribosomal RNA (1000 ribosomal RNA). Linear amplification-mediated PCR (LAM-PCR) (Figures 13 and 14) was used to amplify the vector / genome junction and identify the vector IS. Figure 13 demonstrates that the vector IS was identified by LAM-PCR amplification of the 3' vector LTR-genome junction. A MultiNA automated system was used, which generated a pattern characterized by several bands. The Tsp509I internal control band (IC) derived from the vector backbone is indicated by an arrow. Figure 14 demonstrates that the vector IS was identified by LAM-PCR amplification of the 3' vector LTR-genome junction. A MultiNA automated system was used, which generated a pattern characterized by several bands. The HpyCH4IV5 IC derived from the vector backbone is indicated by an arrow.

[0204] The PCR products were sequenced using the MiSeq Illumina platform, and the resulting sequences were mapped to the mouse genome using a bioinformatics pipeline and collisions were filtered as described in the methods section above (Figure 15). Figure 15 shows the general scheme of the IS mapping analysis performed in mice transplanted with genetically modified hematopoietic progenitor cells. Bone marrow and leukocyte samples derived from transplanted mice belonging to two independent experiments (Table 3) and collected at different time points after transplantation were analyzed as described in the supplementary methods, following the indicated route.

[0205] Overall, we mapped 5,173,892 sequencing reads across the transplanted mouse genome, yielding 2,220 unique vector integration sites. The genomic distribution of ISs obtained from two independent experiments was consistent with the previously reported LV preference for integration within transcription units (specifically, within the first 50 kb downstream of the transcription start site—TSS—) (Figure 16a) and showed no bias toward any particular chromosome in the mouse genome (Figure 16b). Figure 16a shows the IS frequency distribution around the TSS of the nearest RefSeq gene, spanning 500 kb upstream and downstream of the TSS. The numbers on top are the number of ISs detected for all samples and time points. Figure 16b shows the chromosomal distribution of LV ISs in transplanted mice expressing an EGFP transgene (black bars) or a coRPK therapeutic transgene (gray bars), demonstrating no bias toward any particular chromosome.

[0206] The safety of PGK-coRPK LV-based gene therapy was investigated by clonal abundance estimation, which calculates the ratio of the sequence count of each IS (clonal mark) to the total number of sequences in the dataset. Dot plot and heat map representations of the relative abundance of each IS obtained in each mouse (Figures 17, 18, and 19) are shown for different mice and in vitro-cultured Lin -The clonal composition of cells showed strong variation. Figure 17 shows a chart of tracked shared integrations between primary and secondary recipient mice carrying the therapeutic PGK-coRPK LV vector. Integrations detected in any mouse at any organ and any time are pooled. Secondary recipients received pooled BM from transplanted mice coRPK11-14. The remainder of the detected ISs were detected in either the primary or secondary recipient. Numbers in boxes indicate representative values ​​for the corresponding integration ratio in the referenced mouse. In addition to the ≥5% filter applied to integration analysis, all integrations with sequence counts <3 were excluded. Figure 19 presents a dot plot representation of the clonal abundance of pooled integrations for each mouse in the bone marrow. The relative ratio (y-axis) for each IS is relative to the total number of sequence reads obtained in each dataset. Similar to the co-RPK-transduced cells (Figure 17), the graph indicates that the overwhelming majority of transplanted mice exhibited a polyclonal pattern of hematopoietic cell repopulation.

[0207] It was also possible to observe that in some samples, a small number of integrations contributed to a large number of sequence reads (Figures 17 and 18), revealing a polyclonal pattern of repopulation of transduced HSCs. In addition, tracking shared integrations between primary mice bearing therapeutic PGK-coRPK LVs and subsequently transplanted secondary mice did not show strong sharing of integrations between groups, confirming the absence of clonal dominance (Figure 18).

[0208] To determine whether features of insertional mutagenesis were present in transplanted mice, we assessed the occurrence of common insertion sites (CIS), similar to the ongoing LV-mediated clinical trial. CIS are insertion hotspots that can result from integration bias during transduction or in vivo selection of clones harboring vector integrations that confer a growth advantage. CIS were identified using an algorithm based on Abel and cols and a Grubbs test for outliers that do not detect CIS; therefore, this readout does not signal genotoxicity. Furthermore, GO analysis revealed no deviations in gene classes involved in regulating cancer, cell proliferation, or apoptosis in any of the integration datasets sorted by tissue distribution, time point, or abundance of repopulating hematopoietic cell clones (Figure 20). Figure 20 depicts the LV genomic integration profile. GO analysis was performed using GREAT software on samples from transplanted mice. All integrations obtained from this study (N = 2220) showed overrepresentation of the gene functions shown in the left portion of the figure. To address whether the most abundant integrations were enriched in specific gene classes, we selected all ISs (shown in Figure 17) with relative sequence counts >5% of the entire dataset, indicating that no GO gene class was overrepresented.

[0209] These results suggest neutrality of vector integration and demonstrate the safety of PGK-coRPK LV in a preclinical setting.

[0210] Example 6 Human Clinical Trials A clinical trial will be conducted to evaluate the safety and preliminary efficacy of autologous hematopoietic stem cell transplantation (HSCT) using the EU / 3 / 14 / 1130 medicinal product (autologous CD34+ hematopoietic stem cells transduced with an LV vector containing the RPK gene) in patients with PKD and a history of severe, transfusion-dependent anemia refractory to splenectomy.

[0211] ODD EU / 3 / 14 / 1130 contains a SIN LV vector expressing the coRPK gene (FIG. 21).

[0212] SIN LV vectors provide more robust expression (Ellis 2005) and are less susceptible to transcriptional silencing than gamma-RV vectors (Pfeifer, Ikawa et al. 2002). They also demonstrate a much safer integration profile (Schroder, Shinn et al. 2002) (Mitchell, Beitzel et al. 2004) (Wu, Li et al. 2003). Due to the 400-bp deletion they carry in the 3' LTR sequence (Miyoshi, Blomer et al. 1998) (Zufferey, Dull et al. 1998), transgene expression is regulated by an internal promoter, increasing the safety of LV-based gene modification.

[0213] The accepted LV vector sequence also contains several modifications to improve transgene expression and safety in target cells.

[0214] One modification is the use of the human PGK promoter, which has already been characterized for its stable in vivo activity and improved safety compared to other promoters used in gene therapy (Montini, Cesana et al. 2006; Modlich, Navarro et al. 2009; Montini, Cesana et al. 2009; Biffi, Montini et al. 2013). Incorporation of the PGK promoter results in physiological expression of the transgene and lower susceptibility to transcriptional silencing (Gerolami, Uch et al. 2000; Zychlinski, Schambach et al. 2008).

[0215] Another modification was the coRPK gene to increase mRNA stability during transcription. Optimization was performed using GeneArt® software, which increased the GC content and removed hidden splice sites to avoid transcriptional silencing and thus increase transgene expression. The coRPK optimized sequence showed 80.4% homology with the human PKLR gene, with no changes in the amino acids of the protein.

[0216] Another modification is the inclusion of a mutated wPRE (Schambach, Bohne et al. 2006) lacking any remaining open reading frame to improve the level of expression and stability of the therapeutic gene. The backbone, promoter, and wPRE* sequence of this LV vector (PGK-coRPK LV) are identical to those corresponding to those used in the pharmaceutical "Lentiviral Vector Containing the Fanconi Anemia A (FANCA) Gene for the Treatment of Patients with Fanconi Anemia Type A" (Ref. 141 / 2000) and the vector backbone used in the ongoing clinical trial of metachromatic leukodystrophy (MLD) (Biffi, Montini et al. 2013).

[0217] Mode of action CD34 cells from PKD patients, either from bone marrow (BM) or mobilized peripheral blood cells, were isolated. + After harvesting the progenitor cells, they are transduced ex vivo with a therapeutic agent, resulting in the integration of the therapeutic vector into the cell's genome. Once integrated, the therapeutic human gene (coRPK) is transcribed and translated within the defective cells to produce the therapeutic RPK protein that is missing or reduced in PKD mature red blood cells. The transduced PKD hematopoietic progenitor cells are then genetically repaired, enabling them to produce red blood cells with sufficient ATP to fulfill their function (Figure 22). These genetically repaired hematopoietic progenitor cells (which would constitute the therapeutic agent) are then transplanted into the patient, and once engrafted, they generate normal red blood cells, providing a lifelong cure for the disease.

[0218] The active ingredient is hematopoietic stem cells (CD34 + The new drug, consisting of a cell suspension of PKD cells, has been designated as an orphan drug by the European Commission (ODD EU / 3 / 14 / 1130) for the treatment of PKD. This new drug should be included in the group of advanced therapeutic developments within the gene therapy subclass.

[0219] The active ingredient is at least 2 x 10 cells containing at least 0.1 copies of the therapeutic vector per cell. 6 CD34 + It consists of a gene-corrected cell suspension at 100 cells / kg body weight. The cells are suspended in saline buffer containing 2% HSA.

[0220] The final therapeutic agent will be manufactured in accordance with GMP regulations, and therefore product requirements for its release and infusion into patients will be related to product quality. In this context, these specifications will include cell viability ≥ 30%, sterility (Gram test and sterility according to the Pharmacopoeia), absence of mycoplasma, absence of replication-competent LV particles, and demonstration of therapeutic efficacy by detecting the presence of at least 0.1 vector copies per cell by quantitative PCR. Furthermore, investigations and studies of hematopoietic progenitor cell content and vector copy number will be performed. To ensure that the procedure meets the above requirements, three independent validations will be performed on healthy control cells.

[0221] The final product will be packaged in a heat-sealed transport bag for freezing and storage prior to its infusion into the patient, and samples will be collected in advance for correspondingly rigorous quality control.

[0222] mobilization Patients will be mobilized at their respective hospitals, with the first two patients mobilized at Hospital del Nino Jesus, Madrid (Spain). The mobilization process includes recombinant granulocyte colony stimulating factor (G-CSF, Neupogen, Amgen, Thousand Oaks, CA, USA) administered at 12 mg / kg twice daily for up to 8 days from birth, and plerixafor (Mozobil®, Genzyme Europe BV, Naarden, Netherlands) administered subcutaneously for four consecutive days at 240 mg / kg / day on day 4. Hematopoietic progenitor cells from peripheral blood will be collected by leukapheresis, and a large volume from day 5 of mobilization will be passed through a cell separator according to standard protocols at Hospital del Nino Jesus, Madrid. All equipment and solutions are CE-marked and meet the specifications of medical device legislation.

[0223] CD34 + Cell purification Consistent with the mobilization process, apheresis is performed at the hospital where the patient is mobilized. The apheresis product is then sorted into hematopoietic progenitor cells (CD34) via MACS (Magnetic Cell Sorting) technology (Miltenyi Biotec, Germany), which allows for the separation of cells by high-field gradient through a separation column equipped with a strong permanent magnet and a ferromagnetic matrix. + The CliniMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) system is a computer-assisted (CliniMACS® plus Instrument) instrument that detects specific CD34 +The equipment consists of selection software, a set of sterile tubes (CliniMACS Tubing Sets), a magnetically controlled reaction sterilizer (CliniMACS CD34 Reagent), and a sterile buffer solution (CliniMACS PBS / EDTA buffer). The instruments and reagents used are CE-marked and meet the specifications of medical device legislation. This stage and subsequent washes utilize nonspecific immunoglobulins (intravenous Flebogamma 5% 0.5gr, Grifols) and human albumin (human albumin Grifols® 20%; Grifols), which are subsequently removed by washing after centrifugation. CD34+ cells are then quantified. Microbiological control of the resulting product is performed by taking standard fungal, aerobic, and anaerobic samples for culture according to specific protocols.

[0224] CD34 + Transduction CD34 purified by ODD EU / 3 / 14 / 1130 +Cell transduction will be performed under GMP conditions within a 48-hour time window from the extraction (apheresis) of cells from the patient. Ex vivo cell culture will last less than 48 hours and will be cultured according to established standards, including the use of appropriately formulated medium X-vivo-20 (Lonza), hematopoietic growth factors (100 ng / ml hrSCF, 100 ng / ml hrFlt-3, 100 ng / ml TPO, and 20 ng / ml IL-3 (all from Prepotech), the addition of 1 μg / ml Pulmozyme, and a controlled 5% O2 concentration. Transduction will be performed under GMP conditions according to ODD EU / 3 / 14 / 1130, manufactured by VIVEbiotech (San Sebastian, Spain). The transduction process is carried out using LV batches. After transduction, the cells are washed with X-vivo-20 (Lonza) and finally packaged in transport bags suitable for cryopreservation. Specific samples are collected to determine whether the final product meets all previously mentioned specifications for its final release. Three independent validations are performed to evaluate the stability of the product. All products and solutions containing vectors meet the legal specifications for medical devices and clinical use. Prior to production, all raw materials (including consumables, biological reagents, and chemical powders) will be inspected by CliniStem's quality control (QC) unit according to standard operating procedures (SOPs).

[0225] Conditioning Patients are pretreated according to a standardized specific protocol that is considered for testing. To be considered as a substitute for patient pretreatment, 2 × 10 cells are used for use in cases where the prepared product does not completely reconstitute hematopoiesis in the treated patient. 6 Unmanipulated CD34 + A backup of cells / kg is kept frozen.

[0226] injection Prior to infusion, patient eligibility will be checked to ensure they meet the study requirements. On the day of infusion, any pre- and prophylactic medications used will be recorded.

[0227] Example 7 Non-clinical Development Previous studies have demonstrated the feasibility of HSC gene therapy for PKD in mice when >25% gene-corrected cells were transplanted. These results suggest that a significant number of donor gene-corrected HSCs (Zaucha, Yu et al. 2001) and high levels of transgene expression are required to achieve therapeutic efficacy in PKD. A novel therapeutic LV vector carrying the hPGK eukaryotic promoter driving PKLR cDNA expression was developed and proposed for clinical trials, and in August 2014, it was designated as an orphan drug (EU / 3 / 14 / 1130). Using this vector, we conducted a preclinical gene therapy protocol for PKD in a mouse model of the disease. Using LV dosing based on clinical criteria, ectopic RPK expression was able to normalize the erythroid compartment, restore the hematological phenotype, and reverse organ pathology. Metabolomic studies demonstrated that functional restoration of the glycolytic pathway in gene-corrected RBCs was not accompanied by metabolic disorders in leukocytes. Notably, leukocytes analyzed in parallel showed no changes in the metabolic balance of leukocytes when RPK was ectopically expressed under the activity of a ubiquitous promoter such as PGK, eliminating the benefit of leukocyte metabolism as a possible safety concern and enhancing the therapeutic efficacy of the EU / 3 / 14 / 1130 vector.

[0228] The lack of any leukemic events or clonal expansion in multilineage reconstitution and secondary recipients after the proliferative stress induced by BM retransplantation demonstrates the long-term stability and safety of the PGK-coRPK LV vector-based protocol. The use of the human PGK eukaryotic promoter, which i) is likely to result in more physiological expression of the RPK transgene, ii) has proven to be a weak transactivator, and iii) is currently being used in clinical trials for metachromatic leukodystrophy (MLD), may also explain the safety of the overall procedure.

[0229] To assess the long-term safety of HSC gene therapy through analysis of vector ISs, we used next-generation sequencing to predict the risk of insertion-induced oncogenicity in HSCs. Over 5,173,892 sequence reads were mapped to a total of 2,220 unique vector ISs across the mouse genome, and no evidence of in vivo expansion or selection of IS-harboring clones was found. Rather, our data demonstrate the clonal composition and dynamics of hematopoiesis following transplantation of transduced HSCs in mice, suggesting true, stable in vivo genetic modification of HSCs over time. Overall, analysis of vector integration patterns highlights the safety profile of the PGK-coRPK LV vector, which provides PKD genetic repair without evidence of genotoxicity.

[0230] Example 8 Clinical Development Clinical trial protocol support will be requested from regulatory authorities. Our goal is to execute a clinical trial sponsored by the European Commission. The ForGeTPKD Consortium, comprised of various European clinicians and basic researchers, was established to focus on PKD research and the development of novel therapeutic strategies. The ForGeTPKD clinical trial will be the first in humans to administer this drug. It is designed as an international, multicenter, open-label, phase I / II study to evaluate the safety and efficacy of transplantation of autologous CD34+ cells transduced ex vivo with a lentiviral vector containing the erythroid pyruvate kinase (RPK) gene (EU / 3 / 14 / 1130) in patients with severe pyruvate kinase deficiency.

[0231] Regulatory Status The drug does not currently have marketing approval. The PKD Consortium's goal is to advance the drug's clinical development with a view to ultimately obtaining marketing approval.

[0232] The mentioned final product will be manufactured by LV vectors that have received orphan drug designation related to:

[0233] Indications: Treatment of pyruvate kinase deficiency

[0234] Criteria: The only curative treatment for PKD is allogeneic BMT, which is used in patients with transfusion-dependent severe anemia refractory to other treatment options. However, allogeneic BMT is not a widely accepted treatment for PKD because it is associated with severe complications related to intensive pre-allo-BMT conditioning with chemotherapy or chemoradiotherapy, as well as acute and chronic graft-versus-host disease (GVHD) (only one patient has been reported in the literature (Tanphaichitr, Suvatte et al. 2000)). Our hypothesis is that gene therapy using autologous hematopoietic stem cells transduced with a viral vector containing a wild-type version of the gene provided by ODD EU / 3 / 14 / 1130 may represent a potential cure for these patients while avoiding the risk of GVHD, the main cause of hematopoietic progenitor cell transplant failure.

[0235] Active substance: autologous CD34 transduced with a lentiviral vector containing the erythroid pyruvate kinase (RPK) gene (ODD EU / 3 / 14 / 1130) + Hematopoietic stem cells express the wild-type version of the protein.

[0236] Final product: The frozen bag contains at least 2 x 10 cells suspended in saline buffer containing 2% HAS. 6 of active substance / kg patient's body weight.

[0237] Example 9 Pharmacology Completed trials: The developed drug contains several modifications in its sequence that offer several advantages for gene therapy of PKD. 1) The use of a SIN-LV vector design allows for relatively easy and safe production of viral stocks and efficient transduction of HSCs; 2) The use of a weak eukaryotic promoter, such as hPGK, which is less susceptible to silencing by methylation (Gerolami, Uch et al. 2000), results in physiological transgene expression, achieving therapeutic levels with a viral dose (1.65 VCN) within clinical standards (Matrai, Chuah et al. 2010); and 3) the presence of a codon-optimized transgene sequence and a mutated wPRE sequence increases the stability of the transgene mRNA. The therapeutic vector sequence does not contain a reporter gene, avoiding potential immunogenicity issues (Morris, Conerly et al. 2004); (Stripecke, Carmen, Villacres et al. 1999).

[0238] The developed hPGK-coRPK LV drug product efficiently reversed PKD pathology in primary and secondary deficiency mice transplanted with progenitor cells transduced and repaired with ODD EU / 3 / 14 / 1130. Repair was achieved in cells carrying an average of 1.65 copies per cell of the therapeutic transgene.

[0239] The human PGK promoter was strong enough to restore the hemolytic phenotype of transplanted mice and to express clinically relevant levels of coRPK protein.

[0240] Genetic repair extended RBC half-life, normalized hematological variables and reticulocyte levels, restored constitutively activated compensatory erythropoiesis in PKD mice, rescued splenic and liver pathology, and significantly reduced iron overload, one of the life-threatening complications of PKD. Additionally, ectopic expression of human RPK repaired RBC energy defects without altering WBC metabolic balance, highlighting the efficacy and safety of the drug.

[0241] Example 10 Ongoing trials We performed transduction of human hematopoietic progenitor cells from healthy donors and PKD patients for the following studies: 1) to determine the transduction efficiency of ODD EU / 3 / 14 / 1130 in human cells; 2) to define the optimal vector copy number / cell to obtain efficient and therapeutic expression of RPK therapeutic proteins; and 3) to define the optimal conditions to obtain therapeutic transduction levels without losing hematopoietic stem cell potential.

[0242] Planned studies include setting conditions for large-scale transduction in a GMP facility, a pre-confirmatory study to set optimal conditions to reach the required specifications defined for the final therapeutic product, and three confirmatory studies.

[0243] toxicology Completed study results include: 1) ectopic expression of human RPK corrects RBC energy defects without altering the metabolic balance of leukocytes; and 2) genomic integration analysis of the vector demonstrated that (i) analysis of the relative abundance of specific cell clones revealed oligoclonal hematopoietic reconstitution in some mice and did not reveal clonal dominance in any of the primary and secondary transplanted mice. (ii) CIS analysis, considered a hallmark of insertional mutagenesis, did not show any signs of genotoxicity or abnormal enrichment of CIS over time, because the detected CIS from two independent gene therapy experiments performed in mice were not represented by high sequence counts and did not preferentially target oncogenes. (iii) GO analysis of genes targeted by LV integration and analysis of the location of vector integration in specific regions of the genome demonstrated no bias toward gene classes involved in regulating cancer, cell proliferation, or apoptosis. Overall, the drug integration analysis demonstrated no evidence of genotoxicity.

[0244] The planned studies include 1) analysis of recombinant competent lentivirus (RCL) production. Human T lymphocytes from healthy donors and from PKD patients will be transduced with ODD EU / 3 / 14 / 1130 and cultured long-term in vitro. The presence of viral p24 protein will be analyzed by ELISA in the supernatant to assess the potential for RCL generation. 2) Drug biodistribution: Murine hematopoietic progenitor cells will be transduced with ODD EU / 3 / 14 / 1130 and transplanted into lethally irradiated recipients. One month after transplantation, the animals will be sacrificed and different organs (gonads, liver, kidneys, brain, bone marrow, spleen, and peripheral blood) will be analyzed for the presence of vector DNA and 3) for vector integrome in human cells: hematopoietic progenitor cells from healthy donors and from PKD patients will be transduced with ODD EU / 3 / 14 / 1130 and transplanted into severely immunodeficient mice to allow engraftment and expansion of human hematopoietic cells. At various time points (1, 2, and 3 months after transplantation), blood and BM grafts will be harvested, sorted for human cells, and subjected to vector integrome analysis as already performed with mouse cells.

[0245] Example 11 Human Clinical Trials To test its clinical efficacy, the ForGeTPKD trial will be conducted. The proposed clinical trial will evaluate the efficacy of the EU / 3 / 14 / 1130 medicinal product (autologous CD34 transduced with a lentiviral vector containing the erythroid pyruvate kinase (RPK) gene) in PKD patients with a history of severe, transfusion-dependent anemia refractory to splenectomy. + The aim of this study is to evaluate the safety and preliminary efficacy of autologous HSCT using hematopoietic stem cells.

[0246] The primary objective is to evaluate treatment, safety, and tolerability / feasibility. Therefore, the following endpoints will be measured: 1) occurrence and characterization of adverse events (AEs), including AEs related to transduced cell infusion, AEs resulting from conditioning treatments prior to cell infusion, and AEs resulting from clonal evolution related to the transduced cells; and 2) number of patients with stem cell engraftment at 30 days post-transplant.

[0247] Secondary objectives are to evaluate the efficacy of the preliminary treatment. Therefore, the following endpoints will be measured: 1) the number of patients who become "transfusion independent" at the end of the study and who still require transfusions after treatment; 2) the ratio of the average number of transfusions required within the study period (1 year) to the average number of transfusions in the previous 1.5 years before the baseline assessment; 3) a clinically significant reduction in anemia, defined as the number of patients with a 2 gr / dL increase in hemoglobin level from baseline at the end of the study; 4) a clinically significant reduction in reticulocytosis, defined as the number of patients showing a 50% reduction from baseline assessment at the end of the study; and 5) the number of patients with stem cell engraftment at the end of the study, where 1% of transduced cells can be detected 6 and 12 months after cell infusion.

[0248] An exploratory objective is to assess the impact of treatment on patients' quality of life. The following endpoints will be measured according to the improvement in quality of life from baseline at the end of the study using quality of life questionnaires (SF-36 for adults and PEDSQL for children) and their validated versions translated into the languages ​​of the participating countries (Italian, Dutch, Spanish).

[0249] The ForGetPKD trial is a multi-center, international collaborative study conducted in three EU member states: Spain, Italy, and the Netherlands. Participating centers include Reference National Investigators and Institutions for PKD diagnosis and treatment.

[0250] This study will represent the first administration of the described product in humans. It is designed as an uncontrolled, open-label, single-dose, Phase I / II study.

[0251] The global trial duration will be 2 years, from the first visit of the first patient to the last visit of the last patient. This includes a 1-year recruitment period, a 1-year treatment period, and an early (immediate) follow-up period. After the trial ends, subjects included in the trial will be asked to participate in subsequent follow-up studies monitoring safety and efficacy for up to 5 years after transplantation.

[0252] The study procedures include a screening period, a treatment period, and a follow-up period. Details of each phase of the rounds and associated study procedures are detailed below and summarized in Table 4. (Table 4) Test procedure TIFF0007817979000037.tif170136TIFF0007817979000038.tif197136

[0253] Screening Period Rounds-1: Pre-screening rounds Potential candidates will be informed about the purpose and characteristics of the study, and two written informed consent forms will be obtained, executed, and signed by the patient (or their legal representative if they are a minor). To be eligible for the study, patients must meet all inclusion criteria and none of the exclusion criteria. This will allow for the mobilization of viable CD34+ cells, 2 x 10, to serve as a backup in case of non-engraftment. 6 CD34 + B. Save at least 6 x 10 cells / kg body weight 6 CD34+ cells / kg body weight are transduced with the EU / 3 / 14 / 1130 vector to produce the drug product, including pre-treatment steps to perform all the quality control steps required for drug product release. Enough transduced cells (2 x 10) are available after drug product release. 6 Transduced CD34 +Only patients with a ≥ 1000kJ / kg body weight (1000kJ / kg body weight) will be enrolled in the study.

[0254] The following procedures will also be performed during this study round: - Registration of relevant medical or surgical history. -Registration of demographic data and clinically relevant physical examination findings -Registration of relevant concomitant medications. -Peripheral blood testing for routine complete blood count (CBC), biochemistry, coagulation measurements, and serology. -Echocardiogram, pulmonary function tests, chest x-ray. - Quality of Life Questionnaire (SF-36 or PEDSQL). - Genetic diagnosis of PKD.

[0255] To be eligible for the study, patients must meet all of the following inclusion criteria and none of the exclusion criteria.

[0256] Inclusion criteria were willingness to sign informed consent (for children under 18 years of age, a parent or legal guardian must sign), a previous diagnosis of PKD confirmed by genetic testing, a history of severe transfusion-dependent anemia, no response to splenectomy, and a candidate for autologous hematopoietic stem cell transplantation, with a mean age of ≥ 2 × 10 6 Transduced CD34 + Male or female patients, over 2 years of age at the time of recruitment, who have been treated and followed for at least the past 2 years in specialized centers where cells / kg body weight are available and who maintain detailed medical records, including transfusion history.

[0257] Exclusion criteria were positive for the presence of human immunodeficiency virus type 1 or 2 (HIV 1 and HIV 2), unresolved bleeding disorders, the presence of other causes of hemolysis, any previous or current malignancy, or myeloproliferative or immunodeficiency disorders, known or suspected immediate family members with familial cancer syndromes (including, but not limited to, hereditary breast and ovarian cancer syndrome, hereditary nonpolypoid colorectal cancer syndrome, and familial adenomatous polyposis), previous allogeneic transplant recipients, the presence of donor-derived residual cells, grade III / IV cardiac, pulmonary, hepatic, or renal dysfunction after medical evaluation, uncontrolled seizure disorder, and diffusing capacity for carbon monoxide (DLco) below predicted (He). The exclusion criteria were: severe comorbidities considered to result in less than 50% of the total iron intake (repaired to IgG1), patients with any other evidence of severe iron overload that in the opinion of the investigator justified exclusion, participation in another clinical trial using the investigational drug within 30 days of screening, availability of an HLA-matched sibling donor for allogeneic bone marrow transplantation, pregnant or lactating women, inability to understand the study objectives, benefits, and risks and / or inability to comply with study procedures according to the investigator's criteria, and poor functional status as evidenced by a Karnofsky index of 80 or less in adults or a Lansky index of 80 or less in children.

[0258] Statistical analysis for this study will be descriptive. Qualitative endpoints will be described using frequencies and proportions. Qualitative endpoints include adverse events, the number of patients with stem cell engraftment, the number of patients who are "transfusion-independent," the number of patients with a clinically significant reduction in anemia, the number of patients with a clinically significant reduction in reticulocytosis, the number of patients with stem cell engraftment in whom the presence of transduced cells can be detected, and improvement in quality of life from baseline as measured by the SF-36 or PEDSQL questionnaire. Quantitative endpoints will be described using means and standard deviations or medians and quartiles. Quantitative endpoints include reductions in anemia and reticulocytosis from baseline, the number of transfusions required during the study period relative to the number of transfusions in the past year before the baseline assessment, and vector copy numbers in peripheral blood and bone marrow. All endpoints will be described at the end of the study.

[0259] Previous studies have demonstrated the feasibility of HSC gene therapy for PKD in mice when >25% gene-corrected cells were transplanted. These results suggest that a significant number of donor gene-corrected HSCs (Zaucha, Yu et al. 2001) and high levels of transgene expression are required to achieve therapeutic efficacy in PKD. A novel therapeutic LV vector carrying the hPGK eukaryotic promoter driving PKLR cDNA expression was developed and proposed for clinical trials, and in August 2014, it was designated as an orphan drug (EU / 3 / 14 / 1130). Using this vector, we conducted a preclinical gene therapy protocol for PKD in a mouse model of the disease. Using LV dosing based on clinical criteria, ectopic RPK expression was able to normalize the erythroid compartment, restore the hematological phenotype, and reverse organ pathology. Metabolomic studies demonstrated that functional restoration of the glycolytic pathway in gene-corrected RBCs was not accompanied by metabolic disorders in leukocytes. Notably, leukocytes analyzed in parallel showed no changes in the metabolic balance of leukocytes when RPK was ectopically expressed under the activity of a ubiquitous promoter such as PGK, eliminating the benefit of leukocyte metabolism as a possible safety concern and enhancing the therapeutic efficacy of the EU / 3 / 14 / 1130 vector.

[0260] The lack of any leukemic events or clonal expansion in the multilineage reconstitution and secondary recipients after the proliferative stress induced by BM retransplantation demonstrates the long-term stability and safety of the PGK-coRPK LV vector-based protocol. The use of the human PGK eukaryotic promoter, which likely results in more physiological expression of the RPK transgene, has proven to be a weak transactivator, and is currently being used in clinical trials for metachromatic leukodystrophy (MLD), may also explain the safety of the overall procedure.

[0261] To assess the long-term safety of HSC gene therapy through analysis of vector ISs, we used next-generation sequencing to predict the risk of insertion-induced oncogenicity in HSCs. Over 5,173,892 sequence reads were mapped to a total of 2,220 unique vector ISs across the mouse genome, and no evidence of in vivo expansion or selection of IS-harboring clones was found. Rather, our data demonstrate the clonal composition and dynamics of hematopoiesis following transplantation of transduced HSCs in mice, suggesting true, stable in vivo genetic modification of HSCs over time. Overall, analysis of vector integration patterns highlights the safety profile of the PGK-coRPK LV vector, which provides PKD genetic repair without evidence of genotoxicity.

[0262] The invention can be further defined by reference to the following exemplary items. 1. An expression cassette for pyruvate kinase deficiency (PKD), comprising: a) a promoter sequence; b) a sequence encoding a gene product, and c) a polynucleotide sequence comprising, in 5' to 3' order, a ribonucleic acid (RNA) transport signal; An expression cassette in which a promoter sequence is operably linked to a sequence encoding a gene product. 2. The expression cassette according to item 1, wherein the promoter is a phosphoglycerate kinase (PGK) promoter. 3. The expression cassette according to item 1 or 2, wherein the gene product is a therapeutic gene product. 4. The expression cassette of item 3, wherein the therapeutic gene product is a pyruvate kinase (PK) polypeptide, optionally a pyruvate kinase, liver and erythrocyte (PKLR) polypeptide. 5. The expression cassette according to any one of items 1 to 4, wherein the sequence encoding the gene product is codon-optimized. 6. The expression cassette according to item 5, wherein the codon-optimized sequence comprises a sequence having at least 85% identity with SEQ ID NO:8. 7. The expression cassette of any one of items 1 to 6, wherein the RNA export signal is a mutated woodchuck hepatitis virus post-transcriptional regulatory element (wPRE). 8. The expression cassette of item 7, wherein the mutated wPRE is a chimeric wPRE comprising a sequence having at least 80% identity to SEQ ID NO:24. 9. The expression cassette according to any one of items 1 to 8, further comprising one or more enhancer sequences. 10. The expression cassette of any one of items 1 to 9, further comprising a polypurine tract (PPT) or polyadenylation (polyA) signal sequence. 11. The following sequence: i) a packaging signal sequence; ii) a truncated Gag sequence; iii) Rev response element (RRE); iv) central polypurine tract (cPPT); v) central terminal sequence (CTS), and vi) The expression cassette of any one of items 1 to 10, optionally further comprising one or more upstream sequence elements from Simian Virus 40 (SV40-USE). 12. The expression cassette of any one of items 1 to 11, further comprising 5' and 3' long terminal repeat sequences. 13. A recombinant gene delivery vector comprising the expression cassette according to any one of items 1 to 12. 14. The recombinant gene delivery vector according to item 13, which is a virus or viral vector. 15. The recombinant gene delivery vector of item 14, wherein the virus or viral vector is a lentivirus (LV). 16. A cell comprising the expression cassette according to any one of items 1 to 12 or the recombinant gene delivery vector according to any one of items 13 to 15. 17. The cell according to item 16, which is a hematopoietic stem cell. 18. The cells according to item 16, which are committed hematopoietic erythroid progenitor cells. 19. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the recombinant gene delivery vector according to any one of items 13 to 15 or the cell according to any one of items 16 to 18. 20. A method for treating or preventing a disease or disorder in a subject in need thereof, comprising providing to the subject the pharmaceutical composition according to item 19. 21. The method of item 20, wherein the disease or disorder is pyruvate kinase deficiency (PKD) and the gene product is a pyruvate kinase (PK) polypeptide, optionally a pyruvate kinase, liver and erythrocyte (PKLR) polypeptide. 22. The method of item 20 or 21, wherein the pharmaceutical composition comprises a recombinant gene delivery vector. 23. The method according to item 20 or 21, wherein the pharmaceutical composition comprises cells. 24. The method of item 23, wherein the cells are autologous to the subject. 25. A method for expressing a transgene in red blood cells, comprising contacting one or more red blood cells with an effective amount of a recombinant viral vector, wherein the vector comprises a human phosphoglycerate kinase promoter, a codon-optimized human pyruvate kinase liver and erythroid (PKLR) cDNA transgene, and a mutated woodchuck hepatitis virus post-transcriptional regulatory element, and wherein after said contacting, PKLR is expressed at a detectable level in the one or more red blood cells.

[0263] Sequence information SEQUENCE LISTING <110> CENTRO DE INVESTIGACIONES ENERGETICAS, MEDIOAMBIENTALES Y TECNOLOGICAS, OA, MP FUNDACION INSTITUTO DE INVESTIGACION SANITARIA FUNDACION JIMENEZ DIAZ CONSORTIO CENTER OF BIOMEDICAL RESEARCH NETWORK <120> LENTIVIRAL VECTORS FOR DELIVERY OF PKLR TO TREAT PYRUVATE KINASE DEFICIENCY <150> US 62 / 573,037 <151> 2017-10-16 <160> 26 <170> PatentIn version 3.5 <210> 1 <211> 234 <212> DNA <213> Artificial Sequence <220> <223> Rev responsive element (RRE) <400> 1 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat 60 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt 120 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca 180 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcct 234 <210> 2 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> HIV-1 packaging signal (psi) <400> 2 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagaga tgggtgcgag 120 agcgtc 126 <210> 3 <211> 181 <212> DNA <213> Artificial Sequence <220> <223> HIV-1 5' LTR <400> 3 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact aggaccca 120. ctgcttaagc ctcaataag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc a 181 <210> 4 <211> 234 <212> DNA <213> Artificial Sequence <220> <223> HIV-1 self-inactivating 3' LTR <400> 4 60. tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 180 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agca 234 <210> 5 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> human cytomegalovirus (CMV) immediate early promoter <400> 5 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 60 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 120 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 180 tgggaggtct atataagcag agct 204 <210> 6 <211> 118 <212> DNA <213> Artificial Sequence <220> <223> central polypurine tract and central termination sequence of HIV-1 (cPPT / CTS) <400> 6 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat 60 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaatttt 118 <210> 7 <211> 511 <212> DNA <213> Artificial Sequence <220> <223> human phosphoglycerate kinase 1 (hPGK) promoter <400> 7 ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc 60 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc 120 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc 180 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac 240 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc 300 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag 360 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct 420 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct 480 cgttgaccga atcaccgacc tctctcccca g 511 <210> 8 <211> 1725 <212> DNA <213> Artificial Sequence <220> <223> codon-optimized version of the human PKLR cDNA (coRPK) <400> 8 atgagcatcc aggaaaatat cagctctctg cagctgcggt cctgggtgtc caagagccag 60 agagacctgg ccaagagcat cctgatcgga gcccctggcg gaccagccgg atacctgaga 120 agggctagcg tggcccagct gacccaggaa ctgggcaccg cctttttcca gcagcagcag 180 ctgccagccg ccatggccga cacctttctg gaacacctgt gcctgctgga catcgactct 240 gagcccgtgg ccgccagaag caccagcatc attgccacca tcggccctgc cagcagaagc 300 gtggagcggc tgaaagagat gatcaaggcc ggcatgaata tcgcccggct gaacttctcc 360 cacggcagcc acgagtacca cgcagagagc attgccaacg tccgggaggc cgtggagagc 420 tttgccggca gccccctgag ctacagaccc gtggccattg ccctggacac caagggcccc 480 gagatcagaa caggaattct gcagggaggg cctgagagcg aggtggagct ggtgaagggc 540 agccaagtgc tggtgaccgt ggaccccgcc ttcagaacca gaggcaacgc caacacagtg 600 tgggtggact accccaacat cgtgcgggtg gtgcctgtgg gcggcagaat ctacatcgac 660 gacggcctga tcagcctggt ggtgcagaag atcggacctg agggcctggt gacccaggtc 720 gagaatggcg gcgtgctggg cagcagaaag ggcgtgaatc tgccaggcgc ccaggtggac 780 ctgcctggcc tgtctgagca ggacgtgaga gacctgagat ttggcgtgga gcacggcgtg 840 gacatcgtgt tcgccagctt cgtgcggaag gcctctgatg tggccgccgt gagagccgct 900 ctgggccctg aaggccacgg catcaagatc atcagcaaga tcgagaacca cgagggcgtg 960 aagcggttcg acgagatcct ggaagtgtcc gacggcatca tggtggccag aggcgacctg 1020 ggcatcgaga tccccgccga gaaggtgttc ctggcccaga aaatgatgat cggacggtgc 1080 aacctggccg gcaaacctgt ggtgtgcgcc acccagatgc tggaaagcat gatcaccaag 1140 cccagaccca ccagagccga gacaagcgac gtggccaacg ccgtgctgga tggcgctgac 1200 tgcatcatgc tgtccggcga gacagccaag ggcaacttcc ccgtggaggc cgtgaagatg 1260 cagcacgcca ttgccagaga agccgaggcc gccgtgtacc accggcagct gttcgaggaa 1320 ctgcggagag ccgcccctct gagcagagat cccaccgaag tgaccgccat cggagccgtg 1380 gaagccgcct tcaagtgctg cgccgctgca atcatcgtgc tgaccaccac aggcagaagc 1440 gcccagctgc tgtccagata cagacccaga gccgccgtga tcgccgtgac aagatccgcc 1500 caggccgcta gacaggtcca cctgtgcaga ggcgtgttcc ccctgctgta ccgggagcct 1560 cccgaggcca tctgggccga cgacgtggac agacgggtgc agttcggcat cgagagcggc 1620 aagctgcggg gcttcctgag agtgggcgac ctggtgatcg tggtgacagg ctggcggcct 1680 ggcagcggct acaccaacat catgagggtg ctgtccatca gctga 1725 <210> 9 <211> 380 <212> DNA <213> Artificial Sequence <220> <223> human CMV enhancer <400> 9 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240 aagtgtatca tatgccaagt acgccccta ttgacgtcaa tgacggtaaa tggcccgcct 300 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360 tagtcatcgc tattaccatg 380 <210> 10 <211> 122 <212> DNA <213> Artificial Sequence <220> <223> simian virus 40 (SV40) poly(A) signal <400> 10 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120 is 122 <210> 11 <211> 136 <212> DNA <213> Artificial Sequence <220> <223> SV40 origin of replication <400> 11 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 60 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga 120 ggcttttttg gaggcc 136 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> central polypurine tract (cPPT) <400> 12 tttaaaagaa aaggggggat tggggggt 28 <210> 13 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> dNEF signal sequence <400> 13 gaattcgagc tcggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca 60 ctttttaaaa gaaaaggggg gac 83 <210> 14 <211> 795 <212> DNA <213> Artificial Sequence <220> <223> NeoR / KanR sequence <400> 14 atgattgaac aagatggatt gcacgcaggt tctccggcgg cttgggtgga gaggctattc 60 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120 gcgcaggggc gtccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180 caagacgagg cagcgcggct atcgtggctg gcgacgacgg gcgttccttg cgcggctgtg 240 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgtc tatgcccgac 540 ggcgaggatc tcgtcgtgac ccacggcgat gcctgcttgc cgaatatcat ggtggaaaat 600 ggccgctttt ctggattcat cgactgtggc cgtctgggtg tggcggaccg ctatcaggac 660 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720 cttgtgcttt acggtatcgc cgcgcccgat tcgcagcgca tcgccttcta tcgccttctt 780 gacgagttct tctga 795 <210> 15 <211> 137 <212> DNA <213> Artificial Sequence <220> <223> rrnG terminator (transcription terminator from the E.coli ribosomal RNA rrnG operon) <400> 15 gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc 60 cagattcagc aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac 120 cagaaattta tccttaa 137 <210> 16 <211> 589 <212> DNA <213> Artificial Sequence <220> <223> ori (high-copy-number ColE1 / pMB1 / pBR322 / pUC origin of replication) <400> 16 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 60 agcggtggtt tgtttgccgg atcaagagct accaactctt ttccgaagg taactggctt 120 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt 180 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 240 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 300 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 360 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 420 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 480 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggttcgcc acctctgact 540 tgagcgtcga ttttgtgat gctcgtcagg ggggcggagc ctatggaaa 589 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CAP binding site sequence <400> 17 taatgtgagt tagctcactc at 22 <210> 18 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> E.coli lac promote <400> 18 tttacacttt atgcttccgg ctcgtatgtt g <210> 19 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> lac operator sequence <400> 19 ttgtgagcgg attack <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> T3 promoter (promoter for bacteriophage T3 RNA polymerase) <400> 20 aattaaccct cactaagg <210> 21 <211> 380 <212> DNA <213> Artificial Sequence <220> <223> CMV enhancer <400> 21 60. gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360 tagtcatcgc tattaccatg 380 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> T7 promoter (promoter for bacteriophage T7 RNA polymerase) <400> 22 cctatagtga gtcgtatta 19 <210> 23 <211> 429 <212> DNA <213> Artificial Sequence <220> <223> f1 ori (f1 bacteriophage origin of replication) <400> 23 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 60 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 120 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 180 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 240 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 300 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 360 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 420 acgcgaatt 429 <210> 24 <211> 677 <212> DNA <213> Artificial Sequence <220> <223> chimeric wPRE RNA export signal <400> 24 cgagcatctt accgccattt attcccatat ttgttctgtt tttcttgatt tgggtataca 60 tttaaatgtt aataaaacaa aatggtgggg caatcattta catttttagg gatatgtaat 120 tactagttca ggtgtattgc cacaagacaa acatgttaag aaactttccc gttatttacg 180 ctctgttcct gttaatcaac ctctggatta caaaatttgt gaaagattga ctgatattct 240 taactatgtt gctcctttta cgctgtgtgg atatgctgct ttaatgcctc tgtatcatgc 300 tattgcttcc cgtacggctt tcgttttctc ctccttgtat aaatcctggt tgctgtctct 360 ttatgaggag ttgtggcccg ttgtccgtca acgtggcgtg gtgtgctctg tgtttgctga 420 cgcaaccccc actggctggg gcattgccac cacctgtcaa ctcctttctg ggactttcgc 480 tttccccctc ccgatcgcca cggcagaact catcgccgcc tgccttgccc gctgctggac 540 aggggctagg ttgctgggca ctgataattc cgtggtgttg tcggggaagg gcctgctgcc 600 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg 660 ggccgcctcc ccgcctg 677 <210> 25 <211> 9087 <212> DNA <213> Artificial Sequence <220> <223> self-limiting lentivirus gene delivery vector <400> 25 gtgtttaaac ctagatattg atagtctgat cggtcaacgt ataatcgagt cctagctttt 60 gcaaacatct atcaagagac aggatcagca ggaggctttc gcatgattga acaagatgga 120 ttgcacgcag gttctccggc ggcttgggtg gagaggctat tcggctatga ctgggcacaa 180 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgtccggtt 240 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg 300 ctatcgtggc tggcgacgac gggcgttcct tgcgcggctg tgctcgacgt tgtcactgaa 360 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac 420 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt 480 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact 540 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg 600 ccagccgaac tgttcgccag gctcaaggcg tctatgcccg acggcgagga tctcgtcgtg 660 acccacggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc 720 atcgactgtg gccgtctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt 780 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tccttgtgct ttacggtatc 840 gccgcgcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaccg 900 attctaggtg cattggcgca gaaaaaaatg cctgatgcga cgctgcgcgt cttatactcc 960 handicapatgcc agattcagca acggatacgg cttccccaac ttgcccactt ccatacgtgt 1020 cctccttacc agaaatttat ccttaacgat cggacgggga gtcaggcaac tatggatgaa 1080 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 1140 caagtttact entatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 1200 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 1260 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 1320 cgcgtaatct gctgcttgca aacaaaaaa ccaccgctac cagcggtggt ttgtttgccg 1380 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 1440 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 1500 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 1560 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 1620 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 1680 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 1740 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 1800 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 1860 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 1920 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg 1980 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag 2040 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc 2100 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc 2160 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttggcc attgcatacg ttgtatccat atcataatat gtacatttat attggctcat gtccaacatt accgccatgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgacccc gcccattgac gtcaataatg 2580. acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagttgttt tggcaccaaa atcaacggga ctttccaaaa 2940 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 3000 tatataagca gagctcgttt agtgaccgg gtctctctg gttagaccag atctgagcct 3060 gggagctctc tgctaacta gggaacccac tgcttaagcc tcataaagc tgccttgag 3120 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac 3180 ccttttagtc agtgtggaa atctctagca gtggccccg aacagggact tgaagccgaa 3240 agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag 3300 aggcgagggg cggcgactgg tgagtacgcc aaaaatttg actagcggag gctagaagga 3360 gagagatgggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa 3420 aattcggtta aggccagggg gaagaaaaaaaatta aaacatatag tatggggcag 3480 cagggagcta gaacgatcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag 3540 acaaatactg ggacagctac aaccatccct tcagacagga tcagagaac ttagatcatt 3600 atatataca gtagcaaccc tctattgtgt gcatcaagg atagataa aagacaccaa 3660 ggaagcttta ggagatag gggagag AAAAAAAGt AAGACCG GACAGCAG 3720 ggccgctgat cttcagacct gggaggag attgaggga cattggag attgaatt 3780 aataata agtagtaaaa attgaaccat taggagtagc acccaccaag gcaagagaa 3840 gagtggtgca gagagaaaaa agagcagtgg gataggagc ttgttcctt gggttctttgg 3900 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat 3960 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc 4020 atctgttgca accacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg 4080 aaagatacct aaaggatcaa cagctcctgg ggatttgggg tgctctgga aaactcattt 4140 gcaccactgc tgtgccttgg atgctagtt ggagtaataa atctctggaa cagatttgga 4200 atcacacgac ctggatggag tgggacag aattachaa ttacacagc ttatacact 4260 ccttaattga agaatcgca aaccagcaag aaagaatga acagaatta ttggattag 4320 aaatgggc aagtttgtgg aattggttta acatacaaa ttggctgtgg tataaaat 4380 tattcataat gatagtagga ggcttggtag gtttaagaat agttttgct gtactttcta 4440 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc 4500 cgagggacc cgaggggccc gagagaagg gagagaagg gagagagagagagagaa 4560 gatccattcg attagtgac ggatctcgac ggattcggtt aacttttaaa agaaaagggg 4620 ggattggggg gtacagtgca ggggaaaaa tagtagacat atagcaa gacatacaaa 4680 ctaaagaatt aaaaaaaaaaaaa ttcaaattt tatcgatcac gagactagcc 4740 tcgagaagct tgatatcgaa ttccacgggg tggggttgc gccttttcca aggcagccct 4800 gggtttgcgc agggaccgg ctgctctggg cgtggttccg ggaacgcag cggcgccgac 4860 cctgggtctc gcacattctt cacgtccgtt cgcagcgtca cccgatctt cgccgctacc 4920 cttgtgggcc ccccggcgac gcttcctgct ccgccctaa gtcgggaagg ttccttgcgg 4980 ttcgcggcgt gccggacgtg aaacggaa gccgcacgtc tcactagtac cctcgcagac 5040 ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc 5100 tgctcagcag ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt 5160 ggggcggtag tgtgggccct gttcctgccc gcggcggtgtt ccgcattctg caagcctccg 5220 gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagggg 5280 5340. gatccgtcga caccggtgcc accatgagca tccaggaaaa tatcagctct ctgcagctgc ggtcctgggt gtccaagagc cagagagacc tggccaagag catcctgatc ggagcccctg 5400. gcggaccagc cggatacctg agaagggcta gcgtggccca gctgacccag gaactgggca 5460 ccgcctttttt ccgcagcag cagctgccag ccgccatggc cgacaccttt ctggaacacc 5520 tgtgcctgct ggacatcgac tctgagcccg tggccgccag aagcaccagc atcattgcca 5580 ccatcggccc tgccagcaga agcgtggagc ggctgaaaga gatgatcaag gccggcatga 5640. atatcgcccg gctgaacttc tcccacggca gccacgagta ccacgcagag agcattgcca 5700. acgtccggga ggccgtggag agctttgccg gcagccccct gagctacaga cccgtggcca 5760 ttgccctgga caccaagggc cccgagatca gaacaggaat tctgcaggga gggcctgaga 5820 gcgaggtgga gctggtgaag ggcagccaag tgctggtgac cgtggacccc gccttcagaa 5880 ccagaggcaa cgccaacaca gtgtgggtgg actaccccaa catcgtgcgg gtggtgcctg 5940 tgggcggcag aatctacatc gacgacggcc tgatcagcct ggtggtgcag aagatcggac 6000 ctgagggcct ggtgacccag gtcgagaatg gcggcgtgct gggcagcaga aagggcgtga 6060 atctgccagg cgcccaggtg gacctgcctg gcctgtctga gcaggacgtg agagacctga 6120 gatttggcgt ggagcacggc gtggacatcg tgttcgccag cttcgtgcgg aaggcctctg 6180 atgtggccgc cgtgagagcc gctctgggcc ctgaaggcca cggcatcaag atcatcagca 6240 agatcgagaa ccacgagggc gtgaagcggt tcgacgagat cctggaagtg tccgacggca 6300 tcatggtggc cagaggcgac ctgggcatcg agatccccgc cgagaaggtg ttcctggccc 6360 agaaaatgat gatcggacgg tgcaacctgg ccggcaaacc tgtggtgtgc gccacccaga 6420 tgctggaaag catgatcacc aagcccagac ccaccagagc cgagacaagc gacgtggcca 6480 acgccgtgct ggatggcgct gactgcatca tgctgtccgg cgagacagcc aagggcaact 6540 tccccgtgga ggccgtgaag atgcagcacg ccattgccag agaagccgag gccgccgtgt 6600 accaccggca gctgttcgag gaactgcgga gagccgcccc tctgagcaga gatcccaccg 6660 aagtgaccgc catcggagcc gtggaagccg ccttcaagtg ctgcgccgct gcaatcatcg 6720 tgctgaccac cacaggcaga agcgcccagc tgctgtccag atacagaccc agagccgccg 6780 tgatcgccgt gacaagatcc gcccaggccg ctagacaggt ccacctgtgc agaggcgtgt 6840 tccccctgct gtaccgggag cctcccgagg ccatctgggc cgacgacgtg gacagacggg 6900 tgcagttcgg catcgagagc ggcaagctgc ggggcttcct gagagtgggc gacctggtga 6960 tcgtggtgac aggctggcgg cctggcagcg gctacaccaa catcatgagg gtgctgtcca 7020 tcagctgacc gcggtctaga ggatcccccg ggctgcagga attcgagcat cttaccgcca 7080 tttattccca tatttgttct gtttttcttg atttgggtat acatttaaat gttaataaaa 7140 caaaatggtg gggcaatcat ttacattttt agggatatgt aattactagt tcaggtgtat 7200 tgccacaaga caaacatgtt aagaaacttt cccgttattt acgctctgtt cctgttaatc 7260 aacctctgga ttacaaaatt tgtgaaagat tgactgatat tcttaactat gttgctcctt 7320 ttacgctgtg tggatatgct gctttaatgc ctctgtatca tgctattgct tcccgtacgg 7380 ctttcgtttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc 7440 ccgttgtccg tcaacgtggc gtggtgtgct ctgtgtttgc tgacgcaacc cccactggct 7500 ggggcattgc caccacctgt caactccttt ctgggacttt cgctttcccc ctcccgatcg 7560 ccacggcaga actcatcgcc gcctgccttg cccgctgctg gacaggggct aggttgctgg 7620 gcactgataa ttccgtggtg ttgtcgggga agggcctgct gccggctctg cggcctcttc 7680 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg 7740 gaattcgagc tcggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca 7800 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct 7860 gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg 7920 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt 7980 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt 8040 gtggaaaatc tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc 8100 aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa 8160 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 8220 tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta 8280 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 8340 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag 8400 tagtgaggag gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt 8460 attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta 8520 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg 8580 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct 8640 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 8700 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 8760 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac 8820 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct 8880 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 8940 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt 9000 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 9060 ttaacaaaat cgttccctca ggacgtc 9087 <210> 26 <211> 2890 <212> DNA <213> Artificial Sequence <220> <223> PKD Expression Cassette Sequence (5`-3`) <400> 26 ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc 60 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc 120 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc 180 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac 240 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc 300 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag 360 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct 420 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct 480 cgttgaccga atcaccgacc tctctcccca gggggatccg tcgacaccgg tgccaccatg 540 agcatccagg aaaatatcag ctctctgcag ctgcggtcct gggtgtccaa gagccagaga 600 gacctggcca agagcatcct gatcggagcc cctggcggac cagccggata cctgagaagg 660 gctagcgtgg cccagctgac ccaggaactg ggcaccgcct ttttccagca gcagcagctg 720 ccagccgcca tggccgacac ctttctggaa cacctgtgcc tgctggacat cgactctgag 780 cccgtggccg ccagaagcac cagcatcatt gccaccatcg gccctgccag cagaagcgtg 840 gagcggctga aagagatgat caagccggc atgaatatcg cccggctgaa cttctcccac 900 ggcagccacg agtaccacgc agagagcatt gccaacgtcc gggaggccgt ggagagcttt 960 gccggcagcc ccctgagcta cagacccgtg gccattgccc tggacaccaa gggccccgag 1020 atcagaacag gaattctgca gggagggcct gagagcgagg tggagctggt gaagggcagc 1080 caagtgctgg tgaccgtgga ccccgcctt agaaccagag gcaacgccaa cacagtgtgg 1140 gtggactacc ccaacatcgt gcgggtggtg cctgtgggcg gcagaatcta catcgacgac 1200 ggcctgatca gcctggtggt gcagaagatc ggacctgagg gcctggtgac ccaggtcgag 1260 aatggcggcg tgctggggcag cagaaagggc gtgaatctgc caggcgccca ggtggacctg 1320 cctggcctgt ctgagcagga cgtgagagac ctgagatttg gcgtggagca cggcgtggac 1380 atcgtgttcg ccagcttcgt gcggaaggcc tctgatgtgg ccgccgtgag agccgctctg 1440 ggccctgaag gccacggcat caagatcatc agaagatcg agaacacga gggcgtgaag 1500 cggttcgacg agatcctgga agtgtccgac ggcatcatgg tggccagagg cgacctgggc 1560 atcgagatcc ccgccgagaa ggtgttcctg gcccagaaaa tgatgatcgg acggtgcaac 1620 ctggccggca aacctgtggt gtgcgccacc cagatgctgg aaagcatgat caccaagccc 1680 agacccacca gagccgagac aagcgacgtg gccaacgccg tgctggatgg cgctgactgc 1740 atcatgctgt ccggcgagac agccaagggc aacttccccg tggaggccgt gaagatgcag 1800 cacgccattg ccagagaagc cgaggccgcc gtgtaccacc ggcagctgtt cgaggaactg 1860 cggagagccg cccctctgag cagagatccc accgaagtga ccgccatcgg agccgtggaa 1920 gccgccttca agtgctgcgc cgctgcaatc atcgtgctga ccaccacagg cagaagcgcc 1980 cagctgctgt ccagatacag acccagagcc gccgtgatcg ccgtgacaag atccgcccag 2040 gccgctagac aggtccacct gtgcagaggc gtgttccccc tgctgtaccg ggagcctccc 2100 gaggccatct gggccgacga cgtggacaga cgggtgcagt tcggcatcga gagcggcaag 2160 ctgcggggct tcctgagagt gggcgacctg gtgatcgtgg tgacaggctg gcggcctggc 2220 agcggctaca ccaacatcat gagggtgctg tccatcagct gaccgcggtc tagaggatcc 2280 cccgggctgc aggaattcga gcatcttacc gccatttatt cccatatttg ttctgttttt 2340 cttgatttgg gtatacattt aaatgttaat aaaacaaaat ggtggggcaa tcatttacat 2400 ttttagggat atgtaattac tagttcaggt gtattgccac aagacaaaca tgttaagaaa 2460 ctttcccgtt atttacgctc tgttcctgtt aatcaacctc tggattacaa aatttgtgaa 2520 agattgactg atattcttaa ctatgttgct ccttttacgc tgtgtggata tgctgcttta 2580 atgcctctgt atcatgctat tgcttcccgt acggctttcg ttttctcctc cttgtataaa 2640 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tccgtcaacg tggcgtggtg 2700 tgctctgtgt ttgctgacgc aacccccact ggctggggca ttgccaccac ctgtcaactc 2760 ctttctggga ctttcgcttt ccccctcccg atcgccacgg cagaactcat cgccgcctgc 2820 cttgcccgct gctggacagg ggctaggttg ctgggcactg ataattccgt ggtgttgtcg 2880 gggaagggcc 2890

Claims

1. (a) a promoter sequence; (b) a codon-optimized sequence encoding the human PKLR gene product, and (c) Mutated woodchuck hepatitis virus post-transcriptional regulatory element (wPRE) and a polynucleotide sequence comprising, in 5' to 3' order: the promoter sequence is operably linked to a codon-optimized sequence encoding the human PKLR gene product; and The codon-optimized sequence encoding the human PKLR gene product comprises a sequence having at least 95% identity to SEQ ID NO: 8; and The mutated wPRE is a chimeric wPRE having a sequence having at least 99% identity with SEQ ID NO: 24; A pharmaceutical composition for treating or preventing a disease or disorder, comprising a cell containing the expression cassette.

2. 2. The pharmaceutical composition of claim 1, wherein the promoter is a phosphoglycerate kinase (PGK) promoter.

3. The pharmaceutical composition of claim 1 or 2, wherein the mutated wPRE comprises the sequence of SEQ ID NO:

24.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the expression cassette further comprises a polypurine tract (PPT).

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the expression cassette further comprises a polyadenylation (polyA) signal sequence.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the expression cassette comprises a sequence having at least 95% identity to SEQ ID NO:

26.

7. 7. The pharmaceutical composition of claim 6, wherein the expression cassette comprises a sequence having at least 99% identity to SEQ ID NO:

26.

8. The pharmaceutical composition of any one of claims 1 to 6, wherein the cells are transduced with a recombinant gene delivery vector.

9. The pharmaceutical composition of claim 8 , wherein the recombinant gene delivery vector is a lentiviral vector.

10. The pharmaceutical composition of claim 8 , wherein the recombinant gene delivery vector is a therapeutic gene delivery vector.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the cells are hematopoietic stem cells.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the cells are committed hematopoietic erythroid progenitor cells.

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