Compositions and methods for enhancing PKLR gene expression

Abstract

To provide gene therapy for pyruvate kinase deficiency.SOLUTION: In an aspect, 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 polynucleotide sequence comprising a ribonucleic acid (RNA) transport sequence, where in the expression cassette, the promoter sequence is functionally linked to a sequence encoding a pyruvate kinase polypeptide, optionally (a) to (c) are present in the expression cassette in an order of from 5' to 3'. In another aspect, the invention provides a recombination gene delivery vector including the expression cassette. In another aspect, the invention provides a method for treating or preventing a disease or disorder in a subject in need thereof, the method comprising providing the expression cassette, a gene delivery vector or a pharmaceutical composition to the subject in need thereof.SELECTED DRAWING: Figure 21

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under U.S. Provisional Application No. 62 / 325,397, filed on April 20, 2016, which is incorporated in its entirety by reference herein.

[0002] Field of Invention This invention relates to gene therapy for pyruvate kinase deficiency. [Background technology]

[0003] Background of the Invention Pyruvate kinase deficiency (PKD) is a monogenic metabolic disorder caused by mutations in the PKLR gene, resulting in variable-symptom hemolytic anemia that can be fatal in the neonatal period. The recessive inheritance trait of PKD and therapeutic treatment with allogeneic bone marrow transplantation provide an ideal scenario for developing gene therapy approaches.

[0004] Among the many hereditary enzyme deficiencies affecting red blood cells, pyruvate kinase deficiency (PKD) is the most frequent cause of chronic nonspherocytic hemolytic anemia (CNSHA) (Zanella et al. 2007). The onset and severity of PKD are highly variable, ranging from mild to severe neonatal anemia, and in the most severe cases, it can be fatal in childhood (Pissard et al. 2006). Growth retardation, fetal hydrops, and neonatal death have also been reported, albeit less frequently (Gilsanz et al. 1993). The prevalence of PKD is estimated at 1:20,000 in the general Caucasian population (Beutler et al. 2000), and to date, more than 195 different mutations have been identified in the PKLR gene (http: / / www.lovd.nl / pklr). Although allogeneic bone marrow transplantation (BMT) has been successfully used to treat patients with severe PKD (Tanphaichitr et al 2000), the low availability of tissue-matched donors and the serious BMT-related complications in these patients (i.e., graft-versus-host disease, opportunistic infections, etc.) have made routine blood transfusions and splenectomy the primary treatment option for the majority of severe PKD patients (Zanella et al 2005), dramatically increasing patient morbidity and mortality (Hilgard et al 2005). Due to the limited efficacy and side effects of treatment options for severe PKD patients, as well as its recessive genetic trait, PKD is a disease that is well-suited to be treated with gene therapy.

[0005] PKD is caused by a defect in the pyruvate kinase (PK) enzyme, which catalyzes the final ATP production reaction in the glycolysis pathway in all cells (Zanella 2005). In mature erythrocytes, PK is essential for the regulation of the erythrocyte-specific alternative promoter at the PKLR locus (Noguchi et al 1987), and because RBCs express only the R-type specific isoform (RPK) (Kanno et al 1992). Therefore, loss of RPK activity impairs the metabolism and lifespan of RBCs (Zanella 2005) and leads to CNSHA.

[0006] A promising approach for the treatment and prevention of hereditary and other diseases and disorders is the delivery of therapeutic agents via gene therapy vectors. Currently, viral vectors demonstrate the greatest efficacy in gene transfer, and for the correction of hereditary disorders requiring sustained gene expression, vectors based on herpesviruses, retroviruses, lentiviruses, adenoviruses, or AAVs are preferred due to the nature of their incorporation of the viral life cycle.

[0007] Genetherapy for monogenic disorders, specifically those affecting the hematopoietic system, provides conclusive evidence that genetic correction of autologous hematopoietic stem cells (HSCs) is an alternative treatment option to allogeneic HSCTs that avoids major complications (Cartier et al 2009; Cavazzana-Calvo et al 2010; Cartier et al 2012; Aiuti et al 2013; Biffi et al 2013). Genetic correction of erythrocyte-affecting disorders such as β-thalassemia and sickle cell disease has been pursued in animal models (Pestina et al 20091, Breda et al 2012) and in humans (Cavazzana-Calvo et al 2010). However, gene therapy approaches for hereditary erythrocyte metabolic deficiencies such as PKD remain limited. The feasibility of HSC gene therapy for PKD has been demonstrated in both mouse (Tani et al 1994; Meza et al 2009) and canine (Trobridge et al 2012) RPK-deficient experimental models, and due to the lack of selective advantage of donor gene-corrected HSCs, the level of donor chimerism and transduction has been shown to be key to achieving efficient correction of the hemolytic phenotype (Richard et al 2004). Previous studies using PKD mouse models have demonstrated that retrovirus-derived human RPK expression can completely correct the PKD phenotype when more than 25% of genetically corrected cells are transplanted (Meza et al 2009). A similar therapeutic threshold for corrected cells was recently reported in one PKD Basenji dog injected with HSCs corrected in vivo using a foamy vector (Trobridge et al 2012).

[0008] Numerous challenges remain in the design of polynucleotide cassettes and expression vectors for use in gene therapy. One major challenge is obtaining sufficient expression of transgenes in target cells. A long-standing unresolved need in this field is sufficiently robust expression of transgenes after gene transfer. In some cases, more efficient expression is required for the efficacy of certain vectors, such as plasmid DNA vectors. In other cases, more efficient gene expression cassettes are desirable to enable lower therapeutic doses with a more favorable safety profile, or to allow for less invasive administration routes.

[0009] Due to the fact that therapeutic transgene expression is controlled by LTR sequences, high levels of transgene expression can be achieved when gamma retrovirus (gamma RV) vectors are used. However, the first clinical trials based on this type of vector raised safety concerns, as several patients developed unexpected leukemia (Hacein-Bey-Abina et al 2008). The strong promoter activity of LTR sequences can affect the regulation of surrounding genes, leading to insertional mutagenesis through activation of proto-oncogene promoters or inhibition of tumor suppressor genes (Ott et al 2006; Howe et al 2008; Stein et al 2010; Braun et al 2014). These findings highlight the need for safer and more efficient vectors than gamma retrovirus vectors for PKD gene therapy. [Overview of the project]

[0010] In one embodiment, the present invention provides an expression cassette comprising (a) a promoter sequence; (b) a sequence encoding a gene product; and (c) a polynucleotide sequence comprising a ribonucleic acid (RNA) transport signal, wherein the promoter sequence is functionally linked to a sequence encoding a pyruvate kinase polypeptide, and optionally, (a) to (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 liver and red blood cell pyruvate kinase (PKLR) polypeptide. In certain embodiments, the sequence encoding the gene product is codon-optimized. In specific embodiments, the RNA transport signal is a mutant woodchuck hepatitis virus posttranscriptional regulatory element (Wpre). In certain embodiments, the mutant Wpre is a chimeric Wpre containing a sequence having at least 80% identity with SEQ ID NO:1. In some embodiments, the expression cassette further comprises one or more enhancer sequences. In some embodiments, the expression cassette further comprises a polyprint lacte (PPT) or polyadenylation (PolyA) signal sequence. In some embodiments, the expression cassette further comprises one or more of the following sequences: (i) packing signal sequence; (ii) truncated Gag sequence; (iii) Rev response element (RRE); (iv) central polyprint lacte (cPPT); (v) central terminal sequence (CTS); and (vi) upstream sequence element (USE), optionally derived from simian virus 40 (SV40-USE). In some embodiments, the expression cassette further comprises 5' and 3' long terminal repeat sequences.

[0011] In a related embodiment, the present invention provides a recombinant gene delivery vector comprising an 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).

[0012] In another related aspect, the present invention provides cells comprising an expression cassette or gene delivery vector disclosed herein. In some aspects, the cells are blood cells. In some aspects, the cells are erythrocytes. In some aspects, the cells are myeloid cells, e.g., lineage-depleted myeloid cells. In some aspects, the cells are hematopoietic stem cells. In some aspects, the cells are CD34+ hematopoietic stem cells. In some aspects, the cells are committed hematopoietic erythrocyte progenitor cells.

[0013] In yet another related embodiment, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a recombinant gene delivery vector or cell as disclosed herein.

[0014] In another embodiment, the present invention provides a method for treating or preventing a disease or disorder in a subject, comprising the step of providing an expression cassette, gene delivery vector, or pharmaceutical composition disclosed herein to a subject requiring such treatment. In one embodiment, the disease or disorder is pyruvate kinase deficiency (PKD), and the gene product is a pyruvate kinase (PK) polypeptide, optionally a hepatic erythrocyte pyruvate kinase (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 self to the subject. In another embodiment, the cells are homogeneous to the subject.

[0015] In a related embodiment, the present invention provides a method for expressing a transgene in erythrocytes, comprising the step of contacting one or more erythrocytes with an effective amount of recombinant viral vector, wherein the vector comprises a human phosphoglycerate kinase promoter, a codon-optimized version of human liver erythrocyte pyruvate kinase (PKLR) cDNA transgene, and a mutant woodchuck hepatitis virus post-transcriptional regulatory element, and after the contact step, PKLR is expressed at a detectable level in one or more erythrocytes. [Invention 1001] From 5' to 3' (a) Promoter sequence; (b) Sequences encoding gene products; and (c) Ribonucleic acid (RNA) transport signal An expression cassette comprising a polynucleotide sequence, wherein the promoter sequence is functionally linked to a sequence encoding a pyruvate kinase polypeptide. [Invention 1002] An expression cassette for the present invention 1001, wherein the promoter is a phosphoglycerate kinase (PGK) promoter. [Invention 1003] An expression cassette according to the present invention 1001 or 1002, wherein the gene product is a therapeutic gene product. [Invention 1004] An expression cassette of the present invention 1003, wherein the therapeutic gene product is a pyruvate kinase (PK) polypeptide, optionally a liver and red blood cell pyruvate kinase (PKLR) polypeptide. [Invention 1005] An expression cassette according to any of the present invention 1001 to 1004, wherein the sequence encoding the gene product is codon-optimized. [Invention 1006] An expression cassette according to any of the invention 1001 to 1005, wherein the RNA transport signal is a mutant woodchuck hepatitis virus posttranscriptional regulatory element (Wpre). [Invention 1007] The expression cassette of the present invention 1006, wherein the mutant Wpre is a chimeric Wpre containing a sequence having at least 80% identity with SEQ ID NO:1. [Invention 1008] An expression cassette according to any of the present invention 1001 to 1007, further comprising one or more enhancer sequences. [Invention 1009] An expression cassette according to any of the invention 1001 to 1008, further comprising a polyprint lactate (PPT) or polyadenylated (Poly-A) signal sequence. [Invention 1010] An expression cassette according to any of the present invention 1001 to 1009 further comprising one or more of the following sequences: (i) Packing signal sequence; (ii) Abbreviated Gag sequence; (iii) Rev response element (RRE); (iv) Central polyprint lactate (cPPT); (v) Central Terminal Array (CTS); and (vi) Upstream sequence element (USE), optional, derived from simian virus 40 (SV40-USE). [Invention 1011] An expression cassette according to any of the present invention 1001 to 1010, further comprising 5' and 3' long terminal repeat sequences. [Invention 1012] A recombinant gene delivery vector comprising any expression cassette described in invention 1001 to 1011. [Invention 1013] A recombinant gene delivery vector according to the present invention 1012, which is a virus or a viral vector. [Invention 1014] A recombinant gene delivery vector according to the present invention 1013, wherein the virus or viral vector is a lentivirus (LV). [Invention 1015] A cell comprising an expression cassette according to any of invention 1001 to 1011 or a gene delivery vector according to any of invention 1012 to 1014. [Invention 1016] Hematopoietic stem cells, according to the present invention 1015. [Invention 1017] The cells of the present invention 1015, which are committed hematopoietic erythrocyte progenitor cells. [Invention 1018] A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a recombinant gene delivery vector according to any of invention 1012 to 1014 or a cell according to any of invention 1015 to 1017. [Invention 1019] A method for treating or preventing a disease or disorder in a subject, comprising the step of providing the pharmaceutical composition of the present invention 1018 to a subject in need thereof. [Invention 1020] The method of the present invention 1019, wherein the disease or disorder is pyruvate kinase deficiency (PKD), and the gene product is pyruvate kinase (PK) polypeptide, optionally hepatic erythrocyte-type pyruvate kinase (PKLR) polypeptide. [Invention 1021] The method of the present invention 1019 or 1020, wherein the pharmaceutical composition comprises a recombinant gene delivery vector. [Invention 1022] The method of the present invention 1019 or 1020, wherein the pharmaceutical composition comprises cells. [Invention 1023] The method of the present invention 1022, wherein the cell is self to the target. [Invention 1024] The method of the present invention 1022, wherein the cells are homogeneous with respect to the target. [Invention 1025] A method for expressing a transgene in erythrocytes, comprising the step of contacting one or more erythrocytes with an effective amount of recombinant viral vector, wherein the vector comprises a human phosphoglycerate kinase promoter, a codon-optimized version of human liver-erythrocyte pyruvate kinase (PKLR) cDNA transgene, and a mutant woodchuck hepatitis virus post-transcriptional regulatory element (Wpre), wherein PKLR is expressed at a detectable level in one or more erythrocytes after the contact step. [Brief explanation of the drawing]

[0016] Novel features of this disclosure are specifically set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and appended drawings illustrating exemplary embodiments in which the principles of the present invention are utilized.

[0017] [Figure 1] Figure 1 shows a scheme that reflects the locations of different described elements present in the lentiviral vector skeleton. [Figure 2] Figure 2A is a schematic diagram of a self-inactivating lentiviral vector used in gene therapy experiments, which contains a human PGK promoter that controls the expression of an EGFP transgene in the control vector (top figure) or the expression of a codon-optimized sequence (coRPK) of the PKLR gene cDNA in the therapeutic vector (bottom figure). Figure 2b is a schematic diagram of the gene therapy protocol performed to elucidate the functionality of the developed PGK-coRPK lentiviral vector. [Figure 3a]Figures 3a–d show data illustrating the correction of the PKD phenotype in the peripheral blood of primary recipients after genetic correction. Figure 3a shows the levels of RBCs in healthy mice (black bars, n=5) and PKD anemia mice (gray bars, n=6), as well as in PKD anemia mice transplanted with cells transduced by EGFP (white bars, n=9) or coRPK (shaded bars, n=17). Figure 3b shows the levels of reticulocytes. Data are expressed as mean ± SEM and analyzed by the nonparametric Kruskal-Wallis test. Figure 3c shows the flow cytometry strategy used to detect biotin-labeled RBCs over time, and Figure 3d shows the RBC survival kinetics in healthy mice (black line, n=2), anemic mice (gray line, n=2), and genetically corrected mice (discontinuous line, n=4). Data are expressed as mean ± SEM and analyzed by the two-way ANOVA test. Healthy, untransplanted control mice; PKD, untransplanted PKD mice; coRPK, PKD mice expressing therapeutic transgenes. [Figure 3b] See the explanation in Figure 3a. [Figure 3c] See the explanation in Figure 3a. [Figure 3d] See the explanation in Figure 3a. [Figure 4a] Figures 4a-4c show multi-lineage hematopoietic reconstitution in secondary transplant mice. Figure 4a is a diagram of the flow cytometry strategies used to identify different hematopoietic lineages by labeling with CD3-PE, B220-PE, B220-PECy5, Gr1-biotin, and Mac1-biotin antibodies + SAV-PE-Cy5. Figure 4b shows representative dot plots of each lineage in PB at 140 days post-transplant, and Figure 4c shows percentages. The bars represent the mean ± SEM percentages for healthy mouse controls (n=2, black bars) and PKD mouse controls (n=2, gray bars), as well as secondary transplant mice expressing coRPK therapeutic transgenes (n=4, shaded bars). [Figure 4b] See the explanation in Figure 4a. [Figure 4c] See the explanation in Figure 4a. [Figure 5-1] Figures 5a–d show PKD phenotypic correction in secondary transplant mice. Figure 5a shows brilliant cresil blue staining of blood smears from non-transplant mice and secondary recipients for identification of reticulocyte populations (blue). Figure 5b is flow cytometry analysis of reticulocyte levels in peripheral blood. Figure 5c shows the percentage of RBCs in secondary transplant mice expressing coRPK transgenes (shaded bars, n=4), healthy mice (black bars, n=3), and anemic control mice (gray bars, n=3), and Figure 5d shows the percentage of reticulocytes. Data are expressed as mean ± SEM and analyzed by nonparametric two-sided Mann-Whitney test. [Figure 5-2] See the explanation in Figure 5-1. [Figure 6a] Figures 6a–c show the quantification of proviral integration. Figure 6a shows the vector copy number per cell in BM CFUs derived from individual transplanted mice at 120–170 days post-transplant. Percentages of transduction and chimerism are also shown. Figure 6b shows the proviral copy number in cells from different hematopoietic compartments. Columns represent the mean ± SEM values ​​for different groups of transplanted mice. Figure 6c shows the kinetics of proviral integration in BM cells derived from individual transplanted EGFP-expressing mice (gray line) and mice retaining coRPK transgenes (black line). [Figure 6b] See the explanation in Figure 6a. [Figure 6c] See the explanation in Figure 6a. [Figure 7a]Figures 7a–c show the normalization of erythrocyte differentiation patterns in genetically corrected mice. Figure 7a shows the percentage of different erythrocyte subpopulations in the bone marrow and spleen 140 days post-transplant. Figure 7b shows a representative dot plot of the flow cytometry strategy used. The expression intensities of the CD71 and Ter119 markers allow for the identification of four erythrocyte subpopulations: Population I: Early proerythroblasts (high CD71, medium Ter119), Population II: Basophilic erythroblasts (high CD71, high Ter119), Population III: Late basophilic polychromatic erythroblasts (high CD71, medium Ter119), and Population IV: Orthochromatic erythroblasts, reticulocytes, and mature erythrocytes (high CD71, high Ter119). Figure 7c shows plasma Epo levels measured by ELISA in non-transplanted and transplanted mice. Dots represent values ​​for individual mice. The lines represent the mean ± SEM and were analyzed by the non-parametric 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 the explanation for Figure 7a. [Figure 7c] See the explanation for Figure 7a. [Figure 8] Figures 8a and 8b show hematopoietic progenitor cell assays in control mice and mice transplanted with transducers. Data show total CFUs from the spleen (Figure 8a) and bone marrow (Figure 8b) at 140 days post-transplantation. Dots represent the number of colonies in each 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–c demonstrate the reversal of splenomegaly and organopathy in genetically corrected mice 140 days post-transplant. Figure 9a is a representative spleen photograph, and Figure 9b shows the ratio of spleen weight to total body weight in primary and secondary transplanted PKD mice. Dots represent values ​​for individual mice. Lines represent the mean ± SEM for each group. Data were analyzed by the non-parametric Kruskal-Wallis test. Figure 9c shows histological studies of the spleen and liver derived from primary transplanted PKD mice. The first and second columns show representative histological sections of the spleen and liver stained with hematoxylin-eosin and photographed using 4x and 10x objective lenses, respectively, under a light microscope. Arrows point to erythrocyte clusters indicating extramedullary erythropoiesis. The third column shows Prussian blue (Fe) staining of liver sections to detect iron deposits indicated by arrowheads. Photographs were obtained using a 20x objective lens. Group legends similar to those in Figure 7. Secondary coRPK, secondary recipient. [Figure 9b] See the explanation in Figure 9a. [Figure 9c] See the explanation in Figure 9a. [Figure 10a]Figures 10a–g show metabolic profiling in RBC samples derived from genetically modified cell-transplanted mice. Analysis of significant metabolic profiling changes in healthy mice and transplanted mice compared to PKD animals in two independent experiments. Figure 10a shows the complete RBC heatmap obtained by untargeted profiling. Higher metabolic levels are shown in red, and lower metabolic levels are shown in blue. Listed metabolites have at least one comparison that is significant using the following criteria: absolute change factor > 1.5; minimum signal > 2000; adjusted p-value < 0.01. Black boxes highlight clusters of metabolite changes that have different profiles between groups. Figures 10b, 10c, and 10d show the levels of ATP, ADP, and pyruvate in RBCs measured by untargeted profiling, compared to PKD mice, at 140 days post-transplant, respectively. Assay 1: Healthy mice (black bars) n=1, PKD (gray bars) n=1, hPGK-EGFP (white bars) n=2, hPGK-coRPK (slashed bars) n=3. Assay 2: Healthy mice n=2, PKD n=2, hPGK-EGFP n=6, hPGK-coRPK n=10. Figures 10e, 10f, and 10g show RBC-targeted metabolic profiling of a selected number of metabolites involved in the glycolysis pathway (PEP, 3-phosphoglycerate, and D-lactate, respectively) at 280 days post-transplant. Dots represent individual mouse values. Lines represent mean ± SEM and were analyzed by the non-parametric Kruskal-Wallis test. Assay 2: Healthy mice n=7, PKD n=5, hPGK-EGFP n=3, hPGK-coRPK n=5. [Figure 10b] See the explanation in Figure 10a. [Figure 10c] See the explanation in Figure 10a. [Figure 10d] See the explanation in Figure 10a. [Figure 10e] See the explanation in Figure 10a. [Figure 10f] See the explanation in Figure 10a. [Figure 10g] See the explanation in Figure 10a. [Figure 11] Figures 11a-c show 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 enzyme activity to hexokinase enzyme activity. RBCs were purified from blood samples via a cellulose column to avoid contamination with leukocyte PK activity and then used for enzyme activity evaluation. Black bars represent healthy mice (n=2); white bars represent mice transplanted with cells transduced by an EGFP expression vector (n=3); and diagonally lined bars represent mice transplanted with cells transduced by a coRPK expression 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–d show non-targeted metabolic profiling in WBC samples derived from mice transplanted with genetically modified cells. Figure 12a shows principal component analysis of non-targeted 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 the levels of ATP, ADP, and pyruvate in WBCs compared to PKD mice, respectively. Assay 1: Healthy mice (black bars) n=1, PKD (gray bars) n=1, hPGK-EGFP (white bars) n=2, hPGK-coRPK (slashed bars) n=3. Assay 2: Healthy mice n=2, PKD n=2, hPGK-EGFP n=6, hPGK-coRPK n=10. Data represent the mean ± SEM for each group and were analyzed by the non-parametric Kruskal-Wallis test. [Figure 13]Figure 13 shows gel images of LAM-PCR products generated by the Tsp509I enzyme for samples collected from all mice at different time points and tissues. Vector integration sites were identified by LAM-PCR amplification of the 3' vector LTR genomic junction. A MultiNA automated system was used to generate patterns characterized by several bands. The Tsp509I internal control band (IC) derived from the vector skeleton is indicated by the arrow. [Figure 14] Figure 14 shows gel images of LAM-PCR products generated by the HypyCH4IV5 enzyme for samples collected from all mice at different time points and tissues. Vector integration sites were identified by LAM-PCR amplification of the 3' vector LTR genomic junction. A MultiNA automated system was used to generate patterns characterized by several bands. The HypyCH4IV5 internal control band (IC) derived from the vector skeleton is indicated by the arrow. [Figure 15] Figure 15 shows a general scheme for the analysis of integration site mapping performed in mice transplanted with genetically modified hematopoietic progenitor cells. Bone marrow and leukocyte samples from transplanted mice, collected at different time points after transplantation and belonging to two independent experiments (Table 3), were analyzed according to the pipeline shown and as described in the supplementary methods. [Figure 16a] Figures 16a and 16b show the distribution of LV integrations along the genome of transplanted mice. Figure 16a shows the frequency distribution of integration sites (ISs) around the nearest RefSeq gene transcription start site (TSS), extending 500Kb upstream and downstream of the TSS. The numbers at the top represent the number of ISs detected for all samples and time points. Figure 16b shows the chromosomal distribution of LV integration sites in transplanted mice expressing EGFP transgenes (black bars) or coRPK therapeutic transgenes (gray bars) that do not show distortion to specific chromosomes. [Figure 16b] See the explanation in Figure 16a. [Figure 17a]Figures 17a–c demonstrate the clonal abundance analysis of coRPK-LV transduced cells. Dot plots show the pooled clonal abundance of inclusions in each mouse from Assay 1 (Figure 17a) and 2 (Figures 17b and 17c). The relative percentage (y-axis) for each inclusion site is relative to the total number of sequence reads obtained in each dataset. IS, inclusion site; BM, bone marrow; PB, peripheral blood; coRPK1–14, mice transplanted with hematopoietic cells transduced with therapeutic vectors. [Figure 17b] See the explanation in Figure 17a. [Figure 17c] See the explanation in Figure 17a. [Figure 18] Figure 18 shows tracked shared inclusions between primary and secondary recipient mice carrying the therapeutic PGK-coRPK LV vector. Inclusions detected in either mouse at all organs and at all time points are pooled. Secondary recipients received pooled BMs derived from transplanted mice coRPK11-14. Any remaining detected ISs were detected in either the primary or secondary recipient. The numbers in boxes indicate the percentage representationality of the corresponding inclusion in the mice mentioned. In addition to the ≥5% filter applied to the inclusion analysis, all inclusions with a sequence count <3 were excluded. [Figure 19] Figure 19 demonstrates the clonal abundance analysis of EGFP-LV transdescent cells. A dot plot shows the pooled clonal abundance of integrations in each mouse in the bone marrow. The relative percentage (y-axis) for each integration site is relative to the total number of sequence reads obtained in each dataset. Similar to co-RPK transdescent cells (Figure 17), the graph shows that the majority of transplanted mice exhibit a polyclonal pattern of hematopoietic repopulation IS, integration site. [Figure 20]Figure 20 shows the LV genome integration profile. Gene ontology (GO) analysis was performed using GREAT software on samples derived from transplanted mice. All integrations (N=2220) retrieved from this study showed overexpression of gene function, as shown in the left portion of the figure. To determine whether the highest abundance of integrations was enriched in specific gene classes, all integration sites with a relative sequence count of >5% of the entire dataset (shown in Figure 17) were selected, and no overexpressed GO gene classes were identified. [Figure 21] Figure 21 shows a schematic diagram of the medical product (PGK-coRPK LV). [Figure 22a] Figures 22a and 22b illustrate the mechanism of action of the medical product. Figure 22a shows that ectopic expression of the PGK-coRPK LV medical product would rescue the wild-type phenotype of PKD erythrocytes, which otherwise would be unable to produce functional RPK protein to generate sufficient energy to perform its function. Figure 22b illustrates a gene therapy strategy for PKD patients based on ex vivo transduction of RPK-deficient CD34+ hematopoietic progenitor cells with the medical product and subsequent transplantation into the patient. The developed medical product, which holds the therapeutic human PKLR gene cDNA, will be incorporated into the patient's CD34+ cell genome by ex vivo transduction. These genetically corrected cells will then be reinjected into the patient, where they will produce RBCs expressing the therapeutic transgene and thus produce functional RPK protein that corrects the PKD pathological phenotype. Figures adapted from the Boston Children's Hospital blog. [Figure 22b] See the explanation in Figure 22a. [Modes for carrying out the invention]

[0018] Detailed description of the invention definition "Vector" as used herein means a polymer or a polymer association containing or associated with polynucleotides, which can be used to mediate the delivery of polynucleotides to cells. Exemplary vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery media.

[0019] The term "LV" is an abbreviation for lentivirus and can be used to refer to the virus itself or its derivatives. The term covers all subtypes unless otherwise required, and covers both naturally occurring and recombinant types.

[0020] As used herein, the terms “gene” or “coding sequence” refer to an in vitro or in vivo nucleotide sequence that codes for a gene product. In some cases, a gene consists of, or is essentially, a coding sequence, i.e., a sequence that codes for a gene product. In other cases, a gene includes additional non-coding sequences. For example, a gene may or may not include regions preceding and succeeding the coding region, such as a 5' untranslated (5'UTR) or “leader” sequence and a 3'UTR or “trailer” sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0021] As used herein, “therapeutic gene” means a gene that, when expressed, confers a beneficial effect to the cells or tissues in which it exists, or to the mammal in which it is expressed. Examples of beneficial effects include improvement of signs or symptoms of a condition or disease, prevention or inhibition of a condition or disease, or conferring a desired characteristic. Therapeutic genes include genes that correct gene deficiencies in cells or mammals.

[0022] As used herein, a transgene is a gene that is delivered to a cell by a vector.

[0023] As used herein, the term “gene product” refers to a desired expression product of a polynucleotide sequence, such as a polypeptide, peptide, protein, or interfering RNA including small interfering RNA (siRNA), miRNA, or small hairpin RNA (shRNA).

[0024] As used herein, the terms “polypeptide,” “peptide,” and “protein” refer to polymers of amino acids of any length. These terms also include modified amino acid polymers; for example, disulfide bond formation, glycosylation, lipid addition, phosphorylation, or conjugation with labeling components.

[0025] "Contains" means that, for example, in a composition, method, kit, etc., the specified elements are required, but other elements may be included within the scope of the claims, for example, to form the composition, method, kit, etc. For example, an expression cassette "contains" a gene encoding a therapeutic polypeptide functionally linked to a promoter is an expression cassette that, in addition to the gene and promoter, may contain other elements, such as polyadenylated sequences, enhancer elements, other genes, linker domains, etc.

[0026] "Essentially derived from" means that the scope of the described, for example, composition, method, kit, etc., is limited to the specified materials or processes that do not substantially affect the basic novel features of the composition, method, kit, etc. For example, an expression cassette "essentially derived from" a gene encoding a therapeutic polypeptide functionally linked to a promoter and a polyadenylation sequence may include additional sequences, such as linker sequences, as long as they do not substantially affect the transcription or translation of the gene. As another example, a variant or mutant, polypeptide fragment "essentially derived from" a specified sequence has an amino acid sequence of the specified sequence with approximately 10 amino acid residues added or removed at the boundary of the sequence based on the full-length untreated polypeptide from which it is derived, for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue fewer than the specified boundary amino acid residues, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues more than the specified boundary amino acid residues.

[0027] "Consists of" means that any elements, processes, or components not expressly stated in the claims are excluded from the composition, method, or kit. For example, an expression cassette "consists of" a gene encoding a therapeutic polypeptide functionally linked to a promoter and a post-transcriptional regulatory element would consist only of the promoter, the polynucleotide sequence encoding the therapeutic polypeptide, and the post-transcriptional regulatory element. As another example, a polypeptide "consists of a specified sequence" contains only the specified sequence.

[0028] An "expression vector" includes, as used herein, a polynucleotide encoding a gene product of interest and used to achieve the expression of the gene product in intended target cells, such as plasmids, minicircles, viral vectors, liposomes, etc. Expression vectors also include regulatory elements effectively ligated to the coding region to facilitate the expression of the gene product in the target. The combination of regulatory elements, such as promoters, enhancers, UTRs, miRNA targeting sequences, etc., and a gene functionally ligated for expression is sometimes referred to as an "expression cassette." Many such regulatory elements are known and available in the art or can be readily constructed from components available in the art.

[0029] The “promoter” includes, as used herein, a DNA sequence that directs binding to RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. The expression of the promoter and the corresponding protein or polypeptide may be eccentric, i.e., strongly active in a wide range of cells, tissues, and species, or it may be cell type-specific, tissue-specific, or species-specific. The promoter may be “constitutive,” i.e., constantly active, or “inducible,” i.e., activated or inactivated by the presence or absence of biotic or non-biotic factors. Enhancer sequences, which may or may not be adjacent to the promoter sequence, are also included in the nucleic acid construct or vector of the present invention. The enhancer sequence affects promoter-dependent gene expression and may be located in the 5' or 3' region of the native gene.

[0030] An "enhancer" includes cis-acting elements that stimulate or inhibit the transcription of an adjacent gene, as used herein. An enhancer that inhibits transcription is also called a "silencer." Enhancers can function in either direction (i.e., relative to the coding sequence) at a distance of several kilobase pairs (Kb) downstream of the coding sequence and the region being transcribed.

[0031] The “termination signal sequence” includes, as used herein, a genetic element that causes the termination of RNA polymerase transcription, such as a polyadenylation signal sequence.

[0032] As used herein, the terms “effectively linked” or “functionally linked” refer to the juxtaposition of gene elements, such as promoters, enhancers, termination signal sequences, polyadenylation sequences, etc., in a relationship that enables the elements to function in the expected manner. For example, if a promoter helps initiate transcription of a coding sequence, then that promoter is effectively linked to its coding region. Intervening residues may exist between the promoter and the coding region, as long as this functional relationship is maintained.

[0033] As used herein, the term “heterogeneous” means that it originates from an entity whose genotype differs from the rest of the entities being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector originating from a different species is a heterogeneous polynucleotide. Another example is a promoter that has been removed from a native coding sequence and effectively ligated to a coding sequence that is not found to be ligated in nature is a heterogeneous promoter. Thus, for example, an LV vector containing a heterogeneous nucleic acid encoding a heterogeneous gene product is an LV vector containing nucleic acids not typically found in naturally occurring wild-type LV, and the encoded heterogeneous gene product is a gene product not typically encoded by naturally occurring wild-type LV.

[0034] With respect to nucleotide molecules or gene products, as used herein, the term “endogenous” means nucleic acid sequences, such as genes or gene elements, or gene products, such as RNA or proteins, that are naturally present in or associated with a host virus or host cell.

[0035] The term “native” means, as used herein, a nucleotide sequence, e.g., a gene, or a gene product, e.g., RNA, or protein, that is present in a wild-type virus or wild-type cell.

[0036] The term "variant," as used herein, refers to a variant of a reference polynucleotide sequence or reference polypeptide sequence, e.g., a native polynucleotide sequence or native polypeptide sequence, i.e., one having less than 100% sequence identity with the reference polynucleotide sequence or reference polypeptide sequence. In other words, a variant contains at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) compared to a reference polynucleotide sequence, e.g., a native polynucleotide sequence or native polypeptide sequence. For example, a variant may be a polynucleotide having 70% or more sequence identity with the full-length native polynucleotide sequence, e.g., 75%, 80%, or more, e.g., 85%, 90%, or 95%, or more, e.g., 98%, or 99% identity with the full-length native polynucleotide sequence. As another example, a variant may be a polypeptide having 70% or more sequence identity with the full-length native polypeptide sequence, for example, 75% or 80% or more, for example, 85%, 90%, or 95%, or more, for example, 98% or 99% identity with the full-length native polypeptide sequence. A variant may also include a reference sequence, for example, a variant fragment of the native sequence, that shares 70% or more sequence identity with the reference sequence, for example, a fragment of the native sequence, for example, 75% or 80% or more, for example, 85%, 90%, or 95%, or more, for example, 98% or 99% identity with the native sequence.

[0037] As used herein, the terms “biological activity” and “having biological activity” refer to activity attributable to a particular 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 and thereby facilitate immunological function. As another example, the biological activity of a polypeptide or a functional fragment or variant thereof refers to its ability to perform its native function, such as binding or enzymatic activity. As a third example, the biological activity of a gene regulatory element, such as a promoter, enhancer, or Kozak sequence, refers to its ability to control the expression of a functionally linked gene, i.e., its ability to promote, enhance, or activate its translation, respectively.

[0038] The terms “administration” or “introduction” as used herein refer to the delivery of a vector for recombinant protein expression to a cell, a cell and / or organ of interest, or to an interest. Such administration or introduction may be performed in vivo, in vitro, or ex vivo. A vector for gene product expression may be introduced into a cell typically by transfection, which means 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 by an infection vector, 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 by a viral vector (e.g., a bacteriophage).

[0039] "Transformation" is typically used to refer to cells that express oncogenes, such as bacteria containing heterologous DNA or tumor cells, and are therefore converted into a mode of sustained growth. The vectors used to "transform" cells can be plasmids, viruses, or other media.

[0040] Typically, cells are referred to as “transduced,” “infected,” “transfected,” or “transformed” by means used for the administration, introduction, or insertion of heterologous DNA (i.e., a vector) into the cell. The terms “transduced,” “transfected,” and “transformed” may be used interchangeably herein, regardless of the method of heterologous DNA introduction.

[0041] The term “host cell” means, as used herein, a cell that has been transduced, infected, transfected, or transformed by a vector. The vector may be a plasmid, viral particle, phage, etc. Culture conditions such as temperature, pH, etc., are those conventionally used in host cells selected for expression and will be apparent to those skilled in the art. The term “host cell” will be understood to mean the initial transduced, infected, transfected, or transformed cell and their offspring.

[0042] Terms such as “treatment” and “to treat” are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in the sense of complete or partial prevention of a disease or its symptoms, e.g., a reduction in the likelihood of the disease or its symptoms occurring in a subject, and / or therapeutic in the sense of partial or complete treatment of the disease and / or adverse effects resulting from the disease. “Treatment,” as used herein, covers the treatment of a disease in mammals and includes: (a) prevention of the onset of the disease in a subject predisposed to the disease but not yet diagnosed as having it; (b) inhibition of the disease, i.e., prevention of its development; or (c) reduction of the disease, i.e., induction of disease regression. Therapeutic agents may be administered before, during, or after the onset of a disease or injury. Treatment of an ongoing disease is particularly important if the treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment is preferably carried out before complete loss of function in the affected tissue. The treatment of the present invention will preferably be administered during the symptomatic phase of the disease, and in some cases, after the symptomatic phase of the disease.

[0043] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein and refer to mammals, including but not limited to humans and non-human primates, e.g., monkeys and humans; mammalian athletic animals (e.g., horses); mammalian domestic animals (e.g., sheep, goats, etc.); mammalian pets (e.g., dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0044] The terminology used herein is for the purpose of describing specific embodiments and not to limit the invention. As used herein, the singular forms "(a)", "(an)", and "(the)" are to include the plural form unless the context expressly indicates otherwise. Furthermore, where the terms "including", "includes", "having", "has", and "with", or their variations thereof, are used in any part of the detailed description and / or claims, such terms are to be as comprehensive as the term "comprising".

[0045] The terms “approximately” or “about” mean within the range of acceptable error for a particular value as determined by a person skilled in the art, which depends in part on the method by which the value is measured or determined, i.e., on the limits of the measuring system. For example, “approximately” can mean within or greater than one standard deviation, in accordance with the practice in the art. Alternatively, “approximately” can mean within 20%, preferably 10%, more preferably 5%, and even more preferably 1% of a given value. Or, particularly with respect to biological systems or processes, the term can mean within one order of magnitude of a given value, preferably within five times, and more preferably within two times. Where a particular value is described in this application and claims, unless otherwise stated, the term “approximately” should be assumed to mean within the range of acceptable error for that particular value.

[0046] Unless otherwise indicated, all terms used herein have the same meaning as they have to those skilled in the art, and the implementation of the present invention will utilize the prior art of microbiology and recombinant DNA technology that is within the scope of the knowledge of those skilled in the art.

[0047] The implementation of this invention will utilize, unless otherwise indicated, the prior art of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology that is within the scope of the art. Such techniques are explicitly incorporated herein by reference: "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al, 1989); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Animal Cell Culture" (RI Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (DM Weir & CC Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (JMMiller & MPCalos, eds., 1987); "Current Protocols in Molecular Biology" (FMAusubel et al, eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (JEColigan et al.). This is fully explained in literature such as al., eds., 1991).

[0048] In certain embodiments, this disclosure provides polynucleotides, polynucleotide cassettes, and expression vectors for gene expression in cells. Also provided are pharmaceutical compositions and methods of using any of the compositions in promoting gene expression in cells, e.g., cells of an organism, for the treatment or prevention of a disorder. These and other objectives, advantages, and features of the present invention will become more apparent to those skilled in the art by referring more fully to the details of the compositions and methods described below.

[0049] The present invention generally relates to gene expression cassettes and vectors comprising the same that are useful for the delivery of selected therapeutic constructs (including, for example, peptides, polypeptides, ribozymes, and catalytic RNA molecules) of nucleic acid segments to selected cells and tissues of vertebrates, in the fields of molecular biology and virology. Specifically, these gene constructs are useful in the development of gene therapy vectors, including, for example, lentiviral vectors, for the treatment of diseases, disorders, and functional impairments in mammals, specifically humans.

[0050] The disclosed compositions can be used in a variety of investigation, diagnostic, and therapeutic planning, including the prevention and treatment of various human diseases. Various compositions and methods of the present invention are described below.

[0051] While specific compositions and methods are illustrated herein, it will be understood that any of the numerous alternative compositions and methods are applicable and suitable for use in carrying out 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 standard methods in the art.

[0052] In certain embodiments, gene therapy vector compositions comprising these gene expression cassettes, for example, methods and compositions for the preparation of viral vectors, are provided for use in the preparation of pharmaceuticals useful in central and targeted gene therapies for diseases, disorders, and functional impairments in animals, specifically humans.

[0053] In some embodiments, the present invention provides gene therapy for PKD based on a lentiviral vector possessing an hPGK eukaryotic promoter that drives the expression of PKLR cDNA. This therapeutic vector can be used to transduce mouse PKD hematopoietic stem cells (HSCs) and subsequently transplanted into myelosophically disrupted PKD mice. Ectopic RPK expression normalizes the erythrocyte compartment, correcting the hematological phenotype and reversing organopathy. Metabolome studies demonstrate functional correction of the glycolytic pathway in RBCs derived from genetically corrected PKD HSCs without metabolic disruption in leukocytes. Analysis of lentiviral insertion sites in the genomes of transplanted hematopoietic cells shows no evidence of genotoxicity in any of the transplanted animals. Overall, the results clearly demonstrate the therapeutic potential of the hPGK-coRPK lentiviral vector and offer high expectations for gene therapy for PKD and other hereditary erythrocyte metabolic disorders.

[0054] In certain embodiments, the present invention provides an RPK lentiviral vector (LV) for the genetic correction of PKD. Genetic modification of mouse PKD-HSCs with this vector can efficiently correct the hemolytic phenotype and RBC metabolite profile in transplanted PKD mice. Notably, no evidence of metabolic interference in leukocytes or genotoxicity derived from vector incorporation was observed, which supports the therapeutic potential of the PGK-coRPK LV vector. Overall, the results provide strong evidence for the viability of gene therapy for PKD with LV designed for clinical application.

[0055] Certain aspects of the present invention include a self-inactivating lentiviral vector expressing a codon-optimized version of the human PLKR gene. This expression vector comprises a promoter region, a coding sequence, and post-transcriptional regulatory elements.

[0056] Certain embodiments of the polynucleotide cassette of the present invention include a promoter region comprising a promoter sequence or a functional fragment thereof. In one embodiment, the promoter is a human phosphoglycerate kinase (PGK) promoter.

[0057] Some aspects of the present invention include a polynucleotide cassette for enhancing pyruvate kinase expression. In some aspects, the polynucleotide cassette includes a codon-optimized version (coRPK) of human PKLR cDNA to increase mRNA stability during transcription. For optimization, GeneArt® software can be used to increase the GC content and remove potential splice sites to avoid transcriptional silencing and thus increase transgene expression. The coRPK-optimized sequence exhibits 80.4% homology to the human PKLR gene without altering the amino acid sequence of the protein. Alternatively, any optimization method known in the art may be used.

[0058] In some embodiments, the polynucleotide cassette includes an RNA transport signal downstream of a second enhancer. The RNA transport signal may include a woodchuck hepatitis virus posttranscriptional element (WPRE) sequence. In some embodiments, a variant woodchuck hepatitis virus posttranscriptional regulatory element (Wpre) lacking a residual open reading frame (Schambach, Bohne et al. 2006) is also included to improve the level of expression and stability of the therapeutic gene.

[0059] In some aspects of the present invention, a gene delivery vector comprising the polynucleotide cassette of the present invention is provided. In some embodiments, the gene delivery vector is a lentivirus.

[0060] In some aspects of the present invention, a pharmaceutical composition comprising the polynucleotide cassette of the present invention and a pharmaceutical excipient is provided. In some embodiments, the pharmaceutical composition comprises the gene delivery vector of the present invention and a pharmaceutical excipient.

[0061] In some aspects of the present invention, a method for expressing a transgene in mammalian cells is provided. In some embodiments, the method comprises contacting one or more mammalian cells with an effective amount of the polynucleotide cassette or gene delivery vector of the present invention, thereby expressing the transgene at a detectable level in one or more mammalian cells. In some embodiments, the method comprises contacting one or more mammalian cells with an effective amount of the polynucleotide cassette or gene delivery vector of the present invention, thereby expressing the transgene at a therapeutic level in one or more mammalian cells. In some embodiments, the method is in vitro. In other embodiments, the method is in vivo.

[0062] In some aspects of the present invention, a method for treating or preventing a disease or disorder in a mammal in need of treatment or prevention of the disease or disorder is provided. In some embodiments, the method comprises the step of administering an effective amount of a pharmaceutical composition of the present invention, the coding sequence of which encodes a therapeutic gene product, to a mammal.

[0063] composition In some aspects of this disclosure, compositions for the expression of transgenes in eukaryotic cells are provided. In some aspects, eukaryotic cells are mammalian cells. In some aspects, mammalian cells are hematopoietic stem cells. In some aspects, cells are myeloid cells, e.g., lineage-depleted myeloid cells. In some aspects, mammalian cells are fate-determined hematopoietic erythrocyte progenitor cells.

[0064] In some aspects of this disclosure, the composition is a polynucleotide cassette. A “polynucleotide cassette” typically means a polynucleotide sequence comprising two or more functional polynucleotide sequences that are functionally linked to each other, e.g., a regulatory element, a translation initiation sequence, a coding sequence, and / or a termination sequence. Similarly, a “polynucleotide cassette for transgene expression in mammalian cells” means a combination of two or more functional polynucleotide sequences that promote transgene expression in cells, e.g., a promoter, an enhancer, a 5'UTR, a translation initiation sequence, a coding sequence, and / or a termination sequence.

[0065] For example, in some embodiments, the polynucleotide cassette includes a human phosphoglycerate kinase (PGK) promoter, a codon-optimized version of human PKLR cDNA (coRPK), and a mutant woodchuck hepatitis virus post-transcriptional regulatory element (Wpre).

[0066] In any specific embodiment of the expression cassettes and gene delivery vectors described herein, the human PKLR promoter comprises or consists of the following sequences, functional fragments thereof, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the following sequences. TIFF0007842149000001.tif73149

[0067] In any specific embodiment of the expression cassettes and gene delivery vectors described herein, the human PKLR promoter comprises or consists of the following sequences, functional fragments thereof, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the following sequences. TIFF0007842149000002.tif66150

[0068] In any specific embodiment of the expression cassettes and gene delivery vectors described herein, the RPE sequence comprises or consists of the following sequences, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the following sequences. TIFF0007842149000003.tif25150

[0069] In any specific embodiment of the expression cassettes and gene delivery vectors described herein, the psi sequence is either an HIV-1 psi sequence, or the psi sequence comprises or consists of the following sequences, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the following sequences. TIFF0007842149000004.tif18150

[0070] In any specific embodiment of the expression cassettes and gene delivery vectors described herein, the 5'LTR comprises or consists of the following sequences, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the following sequences. TIFF0007842149000005.tif32149

[0071] In any specific embodiment of the expression cassettes and gene delivery vectors described herein, the 3'LTR comprises or consists of the following sequences, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the following sequences. TIFF0007842149000006.tif32149

[0072] In some embodiments, the polynucleotide cassettes of this disclosure provide enhancement of transgene expression in mammalian cells. As demonstrated by the examples of this disclosure, the inventors have discovered a number of polynucleotide elements, i.e., elements that are improved compared to those known in the art, that individually and collectively provide enhancement of transgene expression in mammalian cells. In certain embodiments, the arrangement of two or more functional polynucleotide sequences within the polynucleotide cassette of this disclosure provides enhancement of transgene expression in mammalian cells. "Enhancement" means that transgene expression is increased, strengthened, or more potent in cells holding the polynucleotide cassette of this disclosure compared to cells holding transgenes functionally linked to comparable regulatory elements, for example, known in the art. In other words, transgene expression from the polynucleotide cassette of this disclosure is increased, strengthened, or more potent compared to expression from a polynucleotide cassette that does not contain one or more of the optimized elements of this disclosure, i.e., a reference control. In certain embodiments, the enhancement of expression is specific to or limited to one or more desired cell types.

[0073] For example, transgene expression may be enhanced, strengthened, or more potent in cells containing a polynucleotide cassette containing the promoter disclosed herein than in cells containing transgenes functionally linked to different promoters, for example, known in the art. As another example, transgene expression may be enhanced, increased, strengthened, or more potent in cells containing a polynucleotide cassette containing the enhancer sequence disclosed herein than in cells containing transgenes functionally linked to different enhancer sequences.

[0074] Promoter and enhancer elements can be tissue-specific or stage-specific. For example, tissue-specific promoters or enhancers preferentially drive 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, pluripotent progenitor cells, hematopoietic CD34+ cells, and cluster-differentiated subpopulations within the CD34+ population. Stage-specific promoters or enhancers preferentially drive expression (or higher levels of expression) in one or more specific stages of the cell cycle or development. These include, but are not limited to, β-globin locus regulatory regions, spectrin promoters, and erythrocyte-specific promoters.

[0075] While we do not wish to be constrained by theory, the enhancement of transgene expression in cells is thought to be due to faster assembly of the gene product in the cell or a more stable gene product in the cell. Therefore, the enhancement of transgene expression by the polynucleotide cassette of this disclosure can be observed in numerous ways. For example, the enhancement of expression can be observed by detecting transgene expression earlier than when the transgene is functionally linked to a comparable regulatory element, such as those known in the art, after contact of the polynucleotide cassette with the cell, for example, two days earlier, seven days earlier, two weeks earlier, three weeks earlier, four weeks earlier, eight weeks earlier, twelve weeks earlier, or earlier. The enhancement of expression can also be observed as an increase in the amount of gene product per cell. For example, there may be a doubling or greater increase in the amount of gene product per mammalian cell, for example, a tripling, quadrupling, quintuple, or tenfold increase. The enhancement of expression can also be observed as an increase in the number of mammalian cells expressing the transgene held by the polynucleotide cassette at a detectable level. For example, there may be a doubling or greater increase in the number of mammalian cells expressing a detectable level of transgene, e.g., a tripling, quadrupling, quintuple, or even a tenfold increase. As another example, the polynucleotides of the present invention can promote a detectable level of transgene in a larger percentage of cells compared to conventional polynucleotide cassettes; for example, if a conventional cassette promotes a detectable level of transgene expression in, for example, less than 5% of cells in a certain region, the polynucleotides of the present invention will promote a detectable level of expression in 5% or more of cells in that region; for example, 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 of the contacted cells, and in some cases, 50% or more, 55% or more; 60% or more, 65% or more, 70% or more, or 75% or more, e.g., 80% or more, 85% or more, 90% or more, or 95% or more will express a detectable level of the gene product.Enhanced expression may also be observed as changes in cell viability and / or function.

[0076] The polynucleotide cassettes of this disclosure typically include a promoter region. Any suitable promoter region or promoter sequence within it may be used in the polynucleotide cassette of the present invention, insofar as the promoter region promotes the expression of a coding sequence in eukaryotic cells. In certain embodiments, the promoter region promotes the expression of a coding sequence in mammalian cells. In some cases, the promoter is an eccentric promoter, i.e., a promoter that is active in a wide range of cells, tissues, and species. In other cases, the promoter is a human phosphoglycerate kinase (PGK) promoter.

[0077] In some embodiments, the polynucleotide comprises one or more enhancers. The enhancers are nucleic acid elements known in the art to enhance transcription and may be located at any site, for example, upstream, downstream, or within an intron, in association with the gene they control. Any enhancer element can be used in the polynucleotide cassettes and gene therapy vectors of this disclosure, insofar as it enhances gene expression when used in combination with a promoter.

[0078] The coding sequence expressed in a cell may be any polynucleotide sequence, such as a gene product, such as a gene or cDNA encoding a polypeptide, or an RNA-based therapeutic agent (siRNA, antisense, ribozyme, shRNA, etc.). The coding sequence may be heterologous to a functionally linked promoter sequence, i.e., not functionally associated in nature. Alternatively, the coding sequence may be endogenous to a functionally linked promoter sequence, i.e., associated with its promoter in nature. The gene product may act endogenously or exogenously in mammalian cells, for example, by secretion. For example, when the transgene is a therapeutic gene, the coding sequence may be a 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.

[0079] In one embodiment of the present invention, a transgene-coding sequence is modified or “codon-optimized” to enhance expression by replacing low-frequency-presented codons with higher-frequency-presented codons. The coding sequence is the portion of the mRNA sequence that codes for amino acids for translation. During translation, each of the 61 trinucleotide codons is translated to one of 20 amino acids, resulting in degeneracy or duplication of the genetic code. However, different cell types and different animal species utilize tRNAs that code for the same amino acids (each carrying an anticodon) at different frequencies. When a gene sequence contains codons that are low-frequency-presented by the corresponding tRNA, the ribosome translation mechanism can become slow, hindering efficient translation. Expression can be improved through “codon optimization” for specific species, where the coding sequence is modified to code for the same protein sequence but utilize codons that are highly presented and / or 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 a codon that is infrequently expressed in mammals or primates with a codon that is highly 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, or at least 99% identity with a polypeptide encoded by a sequence disclosed herein, the polypeptide having a higher tRNA frequency in humans than the corresponding codon in the sequence disclosed herein.

[0080] In an additional aspect of the present invention, the sequence encoding a transgene is modified to enhance expression by terminating or removing an open reading frame (ORF) that does not encode the desired transgene. An open reading frame (ORF) is a nucleic acid sequence that follows a start codon and does not contain a stop codon. The ORF may be forward or reversed, and may be "in-frame" or "out-of-frame" relative to the gene of interest. Such an open reading frame may be expressed in the expression cassette alongside the gene of interest and may result in unwanted adverse effects. In one aspect of the present invention, the transgene encoding sequence is modified to remove an open reading frame by further altering codon usage. This was done by eliminating the start codon (ATG) and introducing a stop codon (TAG, TAA, or TGA) into a reversed or out-of-frame ORF, while preserving the amino acid sequence and maintaining codons that are highly utilized in the gene of interest (i.e., avoiding codons with a frequency of <20%). In the present invention, the sequence encoding the transgene can be optimized by either codon optimization and / or by removing non-transgene ORFs. As will be apparent to those skilled in the art, it is preferable to remove or minimize non-transgene ORFs after codon optimization in order to remove ORFs introduced in codon optimization.

[0081] In some embodiments, the polynucleotide cassette of the present invention further comprises an RN transport signal. Exemplary RNA transport sequences include, but are not limited to, sequences derived from the woodchuck hepatitis virus posttranscriptional element (WPRE). The woodchuck hepatitis virus (WHV) posttranscriptional regulatory element (Wpre) significantly increases transgene expression in target cells by increasing RNA stability in transgenes, independently of promoters and vectors (Zuffrey et al, 1999). However, it can express a truncated 60-amino acid protein derived from the WHV X gene involved in liver cancer (Kingsman et al, 2005). Therefore, most preclinical protocols and clinical trials include variant versions of the Wpre element (Zanta-Boussif et al, 2009). On the other hand, the use of two SV40-USE elements in the SIN-LV vector has been shown to be more efficient than the WPRE sequence in repressing transcriptional readthrough (Schambach et al, 2007). More precisely, the WPRE disclosed herein is a chimeric WPRE that retains 589 nucleotides (nucleotides 1-589) derived from a modified WPRE performed by Axel Schambach (WO 2008136670 A2;[5]) and 88 nucleotides (nucleotides 590-677) derived from the original WPRE (Zuffrey et al, 1999). The data disclosed herein demonstrate that this chimeric wpre functions better than the original WPRE. The chimeric WPRE sequence includes the sequences listed in the table below.

[0082] (Table 1) Modified WPRE sequences TIFF0007842149000007.tif45154

[0083] The present invention also includes nucleic acids, such as polynucleotide sequences, that have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO:1 or SEQ ID NO:9. In a specific embodiment, the polynucleotide sequence includes the sequence shown in SEQ ID NO:1 or SEQ ID NO:9.

[0084] In any specific embodiment of the expression cassettes and gene delivery vectors described herein, the Wpre sequence comprises or consists of the sequence of SEQ ID NO:1, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO:1.

[0085] In any specific embodiment of the expression cassettes and gene delivery vectors described herein, the Wpre sequence comprises or consists of the following sequences, or sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the following sequences. TIFF0007842149000008.tif73150

[0086] In certain embodiments, an expression cassette or gene delivery vector, such as a lentivirus, contains a polynucleotide sequence comprising the following sequence in 5' to 3' order: (a) PGK promoter sequence, optionally, human PGK promoter sequence; (b) A sequence encoding a pyruvate kinase polypeptide, optionally a codon-optimized RPK encoding sequence or cDNA sequence; and (c) Optionally, a variant Wpre sequence containing or consisting of the sequence of SEQ ID NO:1.

[0087] In certain embodiments, an expression cassette or gene delivery vector, such as a lentivirus, contains a polynucleotide sequence comprising the following sequence in 5' to 3' order: (a) cPPT sequence; (b) PGK promoter sequence, optionally, human PGK promoter sequence; (c) A sequence encoding a pyruvate kinase polypeptide, optionally a codon-optimized RPK encoding sequence or cDNA sequence; and (d) Optionally, a variant Wpre sequence containing or consisting of the sequence of SEQ ID NO:1.

[0088] In certain embodiments, an expression cassette or gene delivery vector, such as a lentivirus, contains a polynucleotide sequence comprising the following sequence in 5' to 3' order: (a) 5'LTR, optional, modified 5'LTR; (b) cPPT sequence; (c) PGK promoter sequence, optionally, human PGK promoter sequence; (d) A sequence encoding a pyruvate kinase polypeptide, optionally a codon-optimized RPK encoding sequence or cDNA sequence; (e) Optionally, a variant Wpre sequence containing or consisting of the sequence of SEQ ID NO:1; and (f) 3'LTR, optional, modified 3'LTR.

[0089] In certain embodiments, the gene delivery vector is either PGK-coRPK LV or contains the elements shown in Figure 21.

[0090] In any specific embodiment of the expression cassettes and gene delivery vectors described herein, the codon-optimized RPK cDNA sequence or coding sequence codes a PKLR polypeptide comprising or consisting of a sequence disclosed in any of the GenBank accession numbers XP_016856982.1, XP_011507942.1, XP_006711449.1, NP_870986.1, or NP_000289.1, 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 with any of these sequences.

[0091] Other combinations of both the elements disclosed herein and those known in the art will be readily understood by those skilled in the art.

[0092] Furthermore, as will be recognized by those skilled in the art, polynucleotide cassettes include, but are not limited to, restriction sites and regulatory elements for specific gene expression vectors, and may optionally include other elements.

[0093] In some aspects of the present invention, the polynucleotide cassette of the present invention is used to deliver genes to animal cells, for example, to determine the effect of a gene on cell viability and / or function, to treat cytotoxicity, etc. Accordingly, in some aspects of the present invention, a composition that provides transgene expression in mammalian cells is a gene delivery vector comprising the polynucleotide cassette of the present disclosure.

[0094] Any convenient gene therapy vector useful for delivering polynucleotide sequences to mammalian cells is included in the gene delivery vectors of this disclosure. For example, a vector may contain single-stranded or double-stranded nucleic acid, e.g., single-stranded or double-stranded DNA. For example, a gene delivery vector may be DNA, e.g., naked DNA, e.g., plasmids, minicircles, etc. A vector may contain single-stranded or double-stranded RNA, including modified forms of RNA. In another example, a gene delivery vector may be RNA, e.g., mRNA or modified mRNA.

[0095] As another example, the gene delivery vector may be a viral vector derived from a virus, such as an adenovirus, adeno-associated virus, lentivirus (LV), herpesvirus, alphavirus, or retrovirus, such as Moloney's mouse leukemia virus (M-MuLV), Moloney's mouse sarcoma virus (MoMSV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary cancer virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, friend mouse leukemia virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV), or lentivirus. While embodiments encompassing the use of lentiviruses are described in more detail below, those skilled in the art will understand that similar knowledge and techniques in the art may be applied to non-LV gene therapy vectors. In some embodiments, the gene delivery vector is a self-healing lentivirus.

[0096] In such embodiments, the 5' and 3' ends of the polynucleotide cassette of the present invention are flanked by functional long terminal repeat (LTR) sequences. In one embodiment, the positions of different elements present in the lentiviral vector backbone are shown in Figure 1. Both LTR sequences are modified to generate a self-inactivating (SIN) LV vector. The SIN vector has a 400 bp deletion in the 3'-LTR that covers a promoter / enhancer element derived from the U3 region. Thus, transgene expression is dependent on the internal promoter, reducing the risk of RCL and decreasing 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); and thus inactivating the vector. The U3 region of the 5'-LTR was replaced with other heterologous promotion sequences (i.e., CMV or RSV) to achieve Tat-independent transcription, increase genomic RNA synthesis, and result in increased viral titer. Since the 5'-U3 region drives primary transcript expression, its modification would not be present in transduced cells (Schambach et al. 2009). Exogenous elements such as β-globin or SV40 polyadenylation signals (Iwakuma et al., 1999) or upstream sequence elements from simian virus 40 (USE) (SV40-USE) (Schambach et al., 2007) were also included in the R region of the viral 3'LTR to reduce transcriptional readthrough from the internal promoter (Zaiss et al., 2002) or the residue of the deleted U3 region of the SIN-LV vector (Almarza et al. 2011), preventing potential transcriptional activation of downstream genes. The leader region contains the packaging signal (Ψ), and the LV vector was thought to require approximately 300 bp of the Gag gene in this region. Currently, this Gag sequence has been reduced to just 40 bp (Figure 1). The Rev response element (RRE) also contains surrounding Env residues, but it was included to improve the efficiency of gene transfer.The central polyprint lactate (cPPT), which facilitates nuclear translocation of the pre-integration complex, has been shown to improve viral titer together with the central terminal sequence (CTS) involved in the separation of reverse transcriptase (Zennou, et al. 2000; Follenzi et al. 2000). In a specific embodiment, the cPPT present in either the expression cassette or gene delivery vector described herein is the following sequence: It contains or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with TIFF0007842149000009.tif4128 or SEQ ID NO:2.

[0097] The dNEF / PPT signal is essential for reverse transcription, and its inclusion significantly improves LV vector preparation.

[0098] Genetherapy vectors encapsulating the polynucleotide cassettes of this disclosure can be prepared using standard methodologies. For example, in the case of LV virions, an LV expression vector according to the present invention is introduced into a producing cell, followed by the introduction of an LV helper construct. The helper construct can be expressed in the producing cell and contains an LV coding region that complements the LV helper function not present in the LV vector. Subsequently, a helper virus and / or an additional vector providing auxiliary functions that can support efficient LV virus production is introduced into the producing cell. The producing cell is then cultured to produce LVs. These steps are carried out using standard methodologies.

[0099] Any suitable method, including but not limited to those described in the following examples, can be used to prepare viral particles for the delivery of the polynucleotide cassette of the present invention. A suitable concentration of viral particles for efficient transduction of mammalian cells can be prepared for contact with mammalian cells in vitro or in vivo. For example, the viral particles may be 10 per ml.8 Above the vector genome, for example, 5×10 per ml 8 vector genome; 10 per ml 9 vector genome; 5×10 per ml 9 vector genome, 10 per ml 10 vector genome, 5×10 per ml 10 vector genome; 10 per ml 11 vector genome; 5×10 per ml 11 vector genome; 10 per ml 12 vector genome; 5×10 per ml 12 vector genome; 10 per ml 13 vector genome; 1.5×10 per ml 13 vector genome; 3×10 per ml 13 vector genome; 5×10 per ml 13 vector genome; 7.5×10 per ml 13 vector genome; 9×10 per ml 13 vector genome; 1×10 per ml 14 vector genome, 5×10 per ml 14 vector genome, or more, typically 1×10 per ml 15 It can be formulated at a concentration below the vector genome.

[0100] In the preparation of the LV composition of the present invention, for example, any host cell including mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeasts can be used to produce LV virions. The host cell may be a packaging cell in which the rep gene and gap gene of LV are stably maintained in the host cell, or a production cell in which the LV vector genome is stably maintained and packaged. Exemplary packaging cells and production cells are derived from SF-9 cells, 293 cells, A549 cells, or HeLa cells. The LV vector is purified and formulated using standard techniques known in the art.

[0101] In certain embodiments, the present invention includes cells containing an expression cassette or gene delivery vector disclosed herein. In relevant embodiments, cells are transduced by a viral vector containing an expression cassette disclosed herein, or have an expression cassette disclosed herein incorporated into the genome of the cell. In certain embodiments, cells are cells used to construct a viral gene delivery vector. In other embodiments, cells are cells delivered to a subject to deliver a gene product encoded by an expression cassette to the subject. Thus, in certain embodiments, cells are self to the subject being treated or obtained from the subject being treated. In other embodiments, cells are homogeneous to the subject being treated or obtained from a donor other than the subject being treated. In specific embodiments, cells are mammalian cells, e.g., human cells. In certain embodiments, cells are blood cells, erythrocytes, hematopoietic progenitor cells, bone marrow cells, e.g., lineage-depleted bone marrow cells, hematopoietic stem cells (e.g., CD34+), or fate-determined hematopoietic erythrocyte progenitor cells.

[0102] The present invention comprises a pharmaceutical composition comprising a polynucleotide cassette, gene delivery vector, or cell as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient. The polynucleotide cassette, gene delivery vector, or cell of the present invention may be combined with pharmaceutically acceptable carriers, diluents, and reagents useful in the preparation of formulations, which generally include excipients that are safe, non-toxic, preferably acceptable for use in primates. Such excipients may be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. Examples of such excipients, carriers, or diluents include, but are not limited to, water, physiological saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Complementary active compounds may be incorporated into the formulation. The solution or suspension used for the formulation may contain a sterile diluent such as water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent; an antimicrobial compound such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating compound such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetic acid, citric acid, or phosphoric acid; a surfactant such as Tween 20 to prevent aggregation; and a compound for adjusting osmotic pressure such as sodium chloride or dextrose. The pH may be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. In specific embodiments, the pharmaceutical composition is sterile.

[0103] Suitable pharmaceutical compositions for use in the present invention further include sterile aqueous solutions or dispersions, as well as sterile powders for the immediate preparation of sterile injectable solutions or dispersions.

[0104] Sterile solutions can be prepared by incorporating the required amount of the active compound, along with one or a combination of the aforementioned components, into a suitable solvent, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and other necessary components from the aforementioned. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which yield a powder of the active component + additional desired components from a pre-filtered solution.

[0105] In one embodiment, the composition is prepared with a carrier that protects the gene cassette or expression vector from rapid elimination from the body, such as a controlled-release formulation including implantation and microencapsulation delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials may be commercially available.

[0106] Formulating oral, ophthalmic, or parenteral compositions in drug unit form is particularly advantageous because it facilitates administration and ensures uniform dosage. A drug unit form, as used herein, refers to a physically discontinuous unit suitable as a unit dosage for the subject being treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in conjunction with the required pharmaceutical carrier. The details of the drug unit forms of the present invention are indicated by and directly depend on the unique characteristics of the active compound, the specific therapeutic effect to be achieved, and the limitations inherent in the field of compounding such active compounds for the treatment of an individual.

[0107] The pharmaceutical composition may be contained in a container, pack, or dispenser, such as a syringe, such as a pre-filled syringe, along with instructions for administration.

[0108] The pharmaceutical compositions of the present invention include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compounds that can (directly or indirectly) provide a metabolite or residue thereof that has biological activity when administered to an animal, including a human.

[0109] The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of the compound of the present invention: that is, a salt that retains the desired biological activity of the parent compound and does not impart any unwanted toxicological effects. A variety of 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). For an overview of suitable salts, see also Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0110] Pharmaceutically acceptable base addition salts are formed by metals or amines, such as alkali metals 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, e.g., Berge et al, "Pharmaceutical Salts", "J. Pharma Sci., 1977, 66, 119). Base addition salts of acidic compounds are prepared in the conventional manner by contacting the free acid form with a sufficient amount of the desired base to produce the salt. The free acid form can be regenerated in the conventional manner by contacting the salt form with the acid and isolating the free acid. The free acid form differs somewhat from the respective salt form in certain physical properties, such as solubility in polar solvents, but in other respects, the salt is equivalent to the respective free acid for the purposes of this invention.

[0111] The polynucleotide cassettes, gene delivery vectors, e.g., recombinant viruses (virions), or cells (e.g., transduced by gene delivery vectors disclosed herein) of the present invention can be incorporated into pharmaceutical compositions for administration to mammalian patients, specifically primates, and more specifically, humans. The polynucleotide cassettes, gene delivery vectors, e.g., virions, or cells of the present invention may 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 would contain a vector or virion containing nucleic acids encoding a therapeutic molecule dissolved in an aqueous buffer having a pH acceptable upon regeneration.

[0112] In some embodiments, the pharmaceutical compositions provided herein include a therapeutically effective amount of cells, vectors, or virions disclosed herein, mixed with pharmaceutically acceptable carriers and / or excipients, such as saline, phosphate-buffered saline, phosphoric acid, 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, citric acid, acetic acid, Ringer's solution, Hanks' solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycols. Preferably, this formulation is stable at 4°C for at least 6 months.

[0113] In some embodiments, the pharmaceutical compositions provided herein include buffers 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 the pharmaceutical composition containing the tumor suppressor gene contained in the adenovirus vector delivery system may be in the range of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

[0114] In certain embodiments, the viral vector is, without limitation, 1 × 10⁻⁶ 8 Vector genomes larger than, for example, 1 × 10⁻¹⁶ 9 Vector genome, 1 × 10 10 Vector genome, 1 × 10 11 Vector genome, 1 × 10 12 Vector genome, or 1 × 10⁻⁶ 13A vector genome, or more, in certain cases, 1 × 10 14 Vector genomes are typically 4 × 10⁶ 15 It can be formulated into any appropriate unit dose, including the vector genome. In some cases, the unit dose is at most about 5 × 10⁻⁶. 15 Vector genome, for example, 1 × 10⁶ 14 Below the vector genome, for example, 1 × 10 13 Vector genome, 1 × 10 12 Vector genome, 1 × 10 11 Vector genome, 1 × 10 10 Vector genome, or 1 × 10⁻⁶ 9 A vector genome, or smaller, in certain cases, 1 × 10 8 Below the vector genome, typically 1 × 10 8 It is greater than a vector genome. In some cases, the unit dose is 1 × 10⁻⁶ 10 ~1 × 10 11 It is a vector genome. In some cases, the unit dose is 1 × 10⁻⁶. 10 ~3×10 12 It is a vector genome. In some cases, the unit dose is 1 × 10⁻⁶. 9 ~3×10 13 It is a vector genome. In some cases, the unit dose is 1 × 10⁻⁶. 8 ~3×10 14 It is a vector genome. In one embodiment, the range is approximately 5 × 10⁻⁶. 10 ~Approx. 1×10 11 It is a vector genome. In some embodiments, the range is approximately 1 × 10⁻⁶. 9 ~Approx. 1×10 10 It is a vector genome.

[0115] In some cases, the unit dose of a pharmaceutical composition can be measured using the multiplicity of infection (MOI). MOI refers to the ratio or multiple of the vector or viral genome to the cells to which the nucleic acid can be delivered. In some cases, the MOI is 1 × 10⁻⁶ 6 It is possible. In some cases, the MOI is 1 × 10⁻⁶.5 ~1×10 7 may be. In some cases, the MOI is 1×10 4 ~1×10 8 may be. In some cases, the recombinant virus of the present disclosure is at least about 1×10 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. In some cases, the recombinant virus of the present disclosure is 1×10 8 ~3×10 14 MOI. In some cases, the recombinant virus of the present disclosure is at most 1×10 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 and 1×10 14 、1×10 15 、1×10 16 、1×10 17 、and 1×10 18 MOI. In some embodiments, the range is from about 20 to about 400 MOI.

[0116] [[ID=******]]In some aspects, the amount of the pharmaceutical composition is about 1×10 8~about 1×10 15 recombinant virus, about 1×10 9 ~about 1×10 14 recombinant virus, about 1×10 10 ~about 1×10 13 recombinant virus, or about 1×10 11 ~about 3×10 12 comprises a recombinant virus.

[0117] Method As disclosed herein, the polynucleotide cassettes and gene delivery vectors of the invention, collectively referred to herein as "the compositions of the invention", are useful in the expression of transgenes in animal cells. For example, the compositions of the invention can be used in research, for example, to determine the effect of a gene on cell viability and / or function. As another example, the compositions of the invention can be used in medicine, for example, to treat or prevent a disease or disorder. Accordingly, in some aspects of the invention, there is provided a method of gene expression in a cell, comprising the step of contacting the cell with a composition of the present disclosure. In some embodiments, the contacting is performed in vitro or ex vivo. In some embodiments, the contacting is performed in vivo, i.e., the composition of the invention is administered to a subject.

[0118] When mammalian cells are brought into contact in vitro or ex vivo with the polynucleotide cassette of the present invention or a gene delivery vector containing the polynucleotide cassette of the present invention, the cells may be derived from any mammalian species, e.g., rodents (e.g., mice, rats, gerbils, squirrels), rabbits, cats, dogs, goats, sheep, pigs, horses, cattle, primates, or humans. The cells may be derived from an established cell line or primary cells, and “primary cells,” “primary cell line,” and “primary culture” are used interchangeably herein to refer to cells and cell cultures derived from the subject and having been subcultured a limited number of times, i.e., grown in vitro between divisions. For example, a primary culture may be a culture that has been subcultured 0, 1, 2, 4, 5, 10, or 15 times, or a number not sufficient to go through a crisis period. Typically, the primary cell line of the present invention is maintained in vitro for fewer than 10 passages.

[0119] Aspects of the present invention include mammalian cells (e.g., CD34+ cells) transduced by a viral delivery vector containing the human liver-erythrocyte pyruvate kinase (PKLR) gene, such as a lentiviral vector. Accordingly, the present invention includes a method for transducing mammalian cells, such as human hematopoietic stem cells or other cells, as described herein, comprising the step of contacting a viral delivery vector, such as a lentiviral vector, containing an expression cassette as described herein, with the cells. In certain embodiments, the cells are obtained in advance from the subject to be treated or from another donor. In specific embodiments, the subject has been diagnosed with PKD, and the cells are transduced by an expression cassette encoding pyruvate kinase, such as an LV containing a codon-optimized RPK coding region or cDNA. It is understood that the disclosed method, for example, the method used to deliver, for example, the pyruvate kinase gene product to a subject using the coPRK cDNA sequence, may also be used to treat hemolytic anemia and / or normalize erythrocyte differentiation, to increase the number of functionally mature erythrocytes, to reduce extramedullary erythropoiesis, and to reduce splenomegaly and other secondary effects of hemolytic anemia or PKD.

[0120] To promote transgene expression, the polynucleotide cassette of the present invention or a gene delivery vector containing the polynucleotide cassette of the present invention may be exposed to cells for approximately 30 minutes to 24 hours or longer, for example, 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.

[0121] The polynucleotide cassette of the present invention or a gene delivery vector containing the polynucleotide cassette of the present invention may be delivered to the cells of the present invention once or more times, for example, once, twice, three times, or more than three times, and the cells are incubated with the drug for some amount of time after each contact event, for example, 16 to 24 hours, after which the medium is replaced with fresh medium and the cells are further cultured. Cell contact may be carried out in any culture medium under any culture conditions that promote cell survival. The culture may contain growth factors to which the cells are responsive. Growth factors are molecules that can promote cell survival, proliferation, and / or differentiation in either a culture or a complete tissue through specific effects on transmembrane receptors, as defined herein. Growth factors include polypeptides and non-polypeptide factors.

[0122] Typically, the polynucleotide cassette of the present invention or a gene delivery vector containing the polynucleotide cassette of the present invention is provided in an amount effective to produce transgene expression in cells. As described elsewhere herein, the effective amount can be readily determined empirically, for example, by detecting the presence or level of the transgene gene product, detecting its effect on cell viability or function, etc. Typically, an effective amount of the polynucleotide cassette of the present invention or a gene delivery vector containing the polynucleotide cassette of the present invention will promote transgene expression in cells more than an equivalent amount of polynucleotide cassette known in the art. Typically, the expression will be enhanced by more than 2 times, for example, 3 times, 4 times, or 5 times, or more, and in some cases by 10 times, 20 times, or 50 times, or more, for example, 100 times, compared to expression from a reference polynucleotide cassette or control polynucleotide cassette known in the art.

[0123] When cells are brought into contact in vivo with the polynucleotide cassette of the present invention or a gene delivery vector containing the polynucleotide cassette of the present invention, the subject may be any mammal, e.g., rodents (e.g., mice, rats, gerbils), rabbits, cats, dogs, goats, sheep, pigs, horses, cattle, or primates. In a further preferred embodiment, the primate is a human. In a further embodiment, the cells are CD34+ cells.

[0124] The methods and compositions disclosed herein are useful, for example, in the treatment of pyruvate kinase deficiency.

[0125] In another embodiment, the present invention includes a method for treating a disease in a subject, comprising the step of delivering an effective amount of cells transduced by a gene delivery vector expressing a therapeutic gene product in cells, such as a viral vector, to a subject in need. In specific embodiments, the cells are self to the subject. In certain embodiments, the cells are erythrocytes, such as hematopoietic stem cells or fate-determined hematopoietic erythrocyte progenitor cells. In some embodiments, the cells are myeloid cells, such as lineage-depleted myeloid cells. In specific embodiments, the method is used to treat PKD, and the viral vector is an LV comprising a human PGK promoter functionally linked to the codon-optimized human PKLR gene cDNA or coding sequence and an expression construct disclosed herein, including the mutant Wpre disclosed herein. In specific embodiments, the cells are delivered to the subject parenterally, for example, by intravenous injection.

[0126] In another embodiment, the present invention includes a method for treating PKD in a subject, comprising the step of providing to a subject in need an effective amount of autologous C34+ stem cells transduced by a lentiviral vector expressing codon-optimized PKLR cDNA in cells, the lentiviral vector comprising a human PGK promoter functionally linked to codon-optimized human PKLR cDNA or coding sequence and a mutant Wpre sequence disclosed herein. In a specific embodiment, the cells are hematopoietic stem cells or fate-determined erythropoiesis progenitor cells, such as bone marrow cells. In a specific embodiment, the cells are provided to the subject parenterally, for example, by intravenous injection.

[0127] In another embodiment, the present invention provides a method for treating a disease in a subject, comprising the step of providing to the subject an effective amount of a gene delivery vector, such as a viral vector, that expresses a therapeutic gene product in the subject. In a specific embodiment, the method is used to treat PKD, and the viral vector is an LV comprising a human PGK promoter functionally ligated to the codon-optimized human PKLR gene cDNA or coding sequence and an expression construct disclosed herein, including a mutant Wpre disclosed herein. In a specific embodiment, the gene delivery vector is provided to the subject parenterally, for example, by intravenous injection.

[0128] In a specific embodiment, cells or gene delivery vectors are included in a pharmaceutical composition and provided to a target.

[0129] In some embodiments, the methods of the present invention result in therapeutic benefits, such as prevention of the onset of a disability, cessation of the progression of a disability, or reversal of the progression of a disability. In some embodiments, the methods of the present invention include a step of detecting that a therapeutic benefit has been achieved. Those skilled in the art will understand that such measures of therapeutic efficacy are applicable to the specific disease being modified, and will recognize appropriate detection methods for use in measuring therapeutic efficacy.

[0130] Transgene expression using the transgene of the present invention is expected to be robust. Therefore, in some cases, transgene expression, as detected by, for example, measuring the level of the gene product or measuring therapeutic efficacy, may be observed within two months after administration of the composition of the present invention, for example, within four weeks, three weeks, or two weeks after administration, or even earlier, for example, within one week after administration. Transgene expression is expected to persist over time. Therefore, in some cases, transgene expression, as detected by, for example, measuring the level of the gene product or measuring therapeutic efficacy, may be observed more than two months after administration of the composition of the present invention, for example, four months, six months, eight months, or ten months after administration, or even later, and in some cases more than one year after administration, for example, two years, three years, four years, or five years after administration, and in certain cases more than five years after administration.

[0131] In certain embodiments, the method includes a step of detecting the expression of a transgene in a cell or subject, wherein the expression is enhanced compared to expression from a polynucleotide cassette that does not contain one or more of the improved elements of the Disclosure. Typically, the expression will be enhanced by two or more, for example, three, four, or five times, or more, and in some cases by ten, twenty, or fifty times, or more, for example, 100 times, compared to expression from a reference known in the Art, i.e., a control polynucleotide cassette, as evidenced, for example, by earlier detection, higher levels of the gene product, stronger functional effects on the cell, etc.

[0132] Typically, if the composition of the present invention is an LV containing the polynucleotide cassette of the present disclosure, the effective amount to achieve the change is about 1 × 10⁻⁶ 8 In some cases, beyond the vector genome, 1 × 10 9 Vector genome, 1 × 10 10 Vector genome, 1 × 10 11 Vector genome, 1 × 10 12 Vector genome, or 1 × 10⁶ 13A vector genome, or more, in certain cases, 1 × 10 14 Generally, for vector genomes larger than 1 × 10⁻¹⁶ 15 It will likely be less than or equal to the vector genome. In some cases, the amount of vector genome delivered is at most about 1 × 10⁻⁶. 15 Vector genome, for example, 1 × 10⁶ 14 Below the vector genome, for example, 1 × 10 13 Vector genome, 1 × 10 12 Vector genome, 1 × 10 11 Vector genome, 1 × 10 10 Vector genome, or 1 × 10⁶ 9 A vector genome, or smaller, in certain cases, 1 × 10 8 A vector genome is typically 1 × 10⁶ 8 It is greater than the vector genome. In some cases, the amount of vector genome delivered is 1 × 10⁻⁶ 10 ~1 × 10 11 It is a vector genome. In some cases, the amount of vector genome delivered is 1 × 10⁻⁶. 10 ~3×10 12 It is a vector genome. In some cases, the amount of vector genome delivered is 1 × 10⁻⁶. 9 ~3×10 13 It is a vector genome. In some cases, the amount of vector genome delivered is 1 × 10⁻⁶. 8 ~3×10 14 It is a vector genome.

[0133] In some cases, the amount of pharmaceutical composition administered can be measured using the Multiplicity of Infection (MOI). In some cases, the MOI may refer to the ratio or multiple of the vector or viral genome to the cells to which the nucleic acid is delivered. In some cases, the MOI may be 1 × 10⁻⁶ 6 It is possible. In some cases, the MOI is 1 × 10⁻⁶. 5 ~1 × 10 7 It is possible. In some cases, the MOI is 1 × 10⁻⁶. 4 ~1 × 10 8It is possible that in some cases the recombinant viruses of this disclosure may be at least about 1 × 10⁻⁶. 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 , 1 x 10 16 , 1 x 10 17 , and 1 × 10 18 It is MOI. In some cases, the recombinant viruses of this disclosure are 1 × 10⁻⁶. 8 ~3×10 14 The MOI is at most about 1 × 10⁻⁶. In some cases, the recombinant viruses of this disclosure have a MOI of at most about 1 × 10⁻⁶. 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 , 1 x 10 16 , 1 x 10 17 , and 1 × 10 18 It is MOI.

[0134] In some aspects, the amount of the pharmaceutical composition is approximately 1 × 10⁻⁶. 8 ~Approx. 1×10 15 A recombinant virus particle, approximately 1 x 10⁻⁶ 9 ~Approx. 1×10 14 A recombinant virus particle, approximately 1 x 10⁻⁶ 10 ~Approx. 1×10 13A number of recombinant virus particles, or approximately 1 x 10⁶ 11 ~Approx. 3×10 12 Contains recombinant virus particles.

[0135] Any total number of appropriate viral particles can be administered to a mammal to provide appropriate transduction of cells in order to impart a desired effect or treat a disease. In various preferred embodiments, at least 10 8 pieces; 5×10 8 pieces;10 9 pieces; 5×10 9 pieces, 10 10 pieces, 5×10 10 pieces;10 11 pieces; 5×10 11 pieces;10 12 pieces; 5×10 12 pieces;10 13 pieces; 1.5×10 13 pieces; 3×10 13 pieces; 5×10 13 pieces;7.5×10 13 pieces; 9×10 13 pieces, 1×10 14 individual virus particles, or 5 x 10⁶ 14 A number of virus particles, or more, typically 1 × 10⁶ 15 A number of virus particles or less is injected. An appropriate number of doses of the vector can be administered to the eye of a mammal or primate. In one embodiment, the method comprises a single dose; in other embodiments, multiple doses are administered over time as deemed appropriate by the attending physician. In some embodiments, at least 2 × 10⁶ doses are administered to result in a high transduction efficiency. 8 VG / ml or 5×10 5 Cells / ml are required for a single dose (24-hour transduction).

[0136] Individual doses are typically greater than or equal to the amount required to produce a measurable effect on the subject and can be determined based on the pharmacokinetics and pharmacology of the absorption, distribution, metabolism, and excretion ("ADME") of the composition of the present invention or its by-products, and therefore based on the distribution of the composition in the subject. This includes consideration of the route of administration as well as the dosage. Effective doses and / or dose plans can be readily determined empirically from preclinical assays, safety and escalation and dose-range studies, individual physician-patient relationships, and in vitro and in vivo assays such as those described herein and illustrated in the examples.

[0137] Several aspects of the present invention are described herein with respect to illustrative examples of applications. It should be understood that numerous specific details, relationships, and methods are provided to offer a complete understanding of the invention. However, those skilled in the art will readily recognize that the invention may be carried out without one or more of the specific details, or in other ways. Since some actions may be performed in different orders and / or concurrently with other actions or events, the present invention is not limited to the illustrated order of actions or events. Furthermore, not all illustrated actions or events are necessarily required to carry out the methodology according to the present invention.

[0138] It is further noteworthy that claims may be drafted in order to exclude optional elements. Accordingly, this statement shall serve as a basis for the use of exclusive terminology such as “solely” and “only” with respect to the use of references or “negative” limitations of claim elements.

[0139] The publications described herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an acknowledgment that the present invention does not qualify as prior to such publications due to prior art. Furthermore, the dates of the provided publications may differ from the actual publication dates and may require independent verification.

[0140] All of the aforementioned U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the application datasheet are all incorporated herein in their entirety by reference, for example, to disclose and describe the methods and / or materials cited in those publications. In the event of any conflict, it is understood that this disclosure supersedes the disclosure of the incorporated publication.

[0141] For illustrative purposes, specific embodiments of the present invention have been described herein, but it will be recognized that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the present invention is not limited to the appended claims. [Examples]

[0142] The following examples are provided to the art to provide a complete disclosure and explanation of how to create and use the present invention, and are not intended to limit the scope of what the inventors consider to be the present invention, nor to represent that the following experiments are all or only experiments performed. With respect to the figures used (e.g., quantities, temperatures, etc.), efforts have been made to ensure accuracy, but some experimental error and deviation should be taken into account. Unless otherwise indicated, parts are weight parts, molecular weights are weight-average molecular weights, temperatures are degrees Celsius, and pressures are atmospheric pressure or approximately atmospheric pressure.

[0143] Experimental method Vector and lentiviral supernatant preparation. LVs were generated as described herein. The coRPK sequence was designed using GeneArt® software to increase the GC content of the sequence and prevent latent splice sites. The vector was developed using the pCCL.sin.ppt.hPGK-EGFP-Wpre* construct, generously provided by Dr. Naldini (HSR-TIGET, San Raffaele Telethon Institute, Milano, Italy), as the backbone. The vector stock of VSV-G pseudotyped LV was prepared by 3 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]. The titer of infective LV was determined by qPCR in HT1080 cells (ATCC:CCL-121), as described elsewhere [Charrier S, et al. (2005). Gene Ther 12:597-606]. 10 7 ~10 8 Lentiviral stocks with a titer of virus particles (vp) / mL were routinely obtained.

[0144] Purification and transduction of mouse HSCs. BMs from 8-14 week old male PKD mice were collected from the leg bones and Lin - Using a cell depletion kit (Miltenyi Biotec, Gladbach, Germany), lineage-negative cells (Lin - The ) was purified to obtain a purity of 70-90%. -Cells were pre-stimulated for 24 hours with 100 ng / mL recombinant human IL-11 (Peprotech EC Ltd., London, UK) and 100 ng / mL recombinant mouse SCF (R&D Systems Inc., Minneapolis, MN) 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)) and 100 ng / mL recombinant mouse SCF (R&D Systems Inc., Minneapolis, MN). Subsequently, they were transduced with LV containing EGFP or coRPK in two transduction cycles at an MOI of 1–10 vp / cell. Each transduction was followed by overnight at 4°C with CH-296 fibronectin fragment (2 μg / cm³). 2 The procedure was performed for 24 hours in plates pre-coated with Retronectin (Takara Shuzo, Otsu, Japan) in the presence of the cytokine.

[0145] In vivo RBC survival. Transplanted mice carrying the coRPK transgene were injected intravenously with biotin 3-sulfo-N-hydroxysuccinimid sodium salt (50 mg / kg) (Sigma Aldrich, Saint Louis, MO) three times in succession (12-hour intervals). Twelve hours after the last 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) at 4°C for 30 minutes. Samples were analyzed every 2–4 days for 40 days post-injection using an EPICS XL flow cytometer (Beckman Coulter, Brea, CA). RBC survival kinetics were measured by the percentage of biotinylated cells in the total RBC population.

[0146] CFC assay. The CFC assay was performed in BM and spleen from control and transplanted mice according to the manufacturer's method using Methocult medium GF M3434 (Stem Cell Technologies, Vancouver, Canada). BM cells were collected from all mouse groups at different post-transplant time points and scored as CFUs (clusters of 30 or more cells) 7 days after seeding on a Nikon Diaphot-TMD microscope.

[0147] Identification of hematopoietic lineages. PBMCs were obtained from the tail veins of transplant animals and labeled with a panel of antibodies to detect different hematopoietic cells. Myeloid cells were detected with anti-GR-1 biotinylated antibody and anti-Mac-1 biotinylated antibody (BD Bioscience, San Jose, CA, 5 μg / mL). Lymphoid cells were detected using anti-CD3-PE antibody for T cells and anti-B220-PE antibody and anti-B220-PECy5 antibody (BD Bioscience, San Jose, CA, 10 μg / mL) for B cells, together with SAV-TRC secondary antibody (Invitrogen, Thermo Fisher Scientific, Waltham, MA). To eliminate dead cells, DAPI (Boehringer, Ingelheim, Germany, 2 μg / mL) was added, and samples were analyzed using a BD LSR Fortessa Cytometer (BD Bioscience, San Jose, CA, USA).

[0148] Structural and histological studies were conducted. The spleen was collected, 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 Haematoxylin, Thermo, Pittsburgh, USA) and eosin (Eosin Alcoholic, Thermo Fisher Scientific, Waltham, MA). Iron deposits in the spleen were also studied by Prussian blue or Perlus staining (Sigma Aldrich, Saint Louis, MO) according to the manufacturer's instructions. All sections were examined using an Olympus BX40 optical microscope and photographed with an Olympus DP21 camera at a final magnification of 100x or 200x.

[0149] Erythrocyte differentiation. Flow cytometry analysis of the intensity of Ter119 and CD71 markers in BM and spleen was used to identify different erythrocyte subpopulations, 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). Cells were then analyzed using an EPICS XL flow cytometer (Beckman Coulter, Brea, CA) with propidium iodide (IP, 2 μg / mL) to detect viable cells.

[0150] Provirus quantification. Detection and quantification of integrated proviruses per cell were achieved using primers complementary to the packaging provirus sequence (Ψ) and the mouse titin housekeeping gene. All BM and peripheral blood samples were collected periodically, and genomic DNA was isolated from nucleated cells using the DNeasy Blood & Tissue Kit (Qiagen, Venlo, Limburg, The Netherlands). 20–50 ng of genomic DNA (gDNA) was amplified by multiplex qPCR using the 7500 Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA) and previously described primers and probes [Charrier S, et al. (2011). Gene Ther 18:479-487].

[0151] 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 derived from transplanted mouse PB was amplified using a 7500 Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA) with previously described primers and probes [Navarro S et al (2006). Mol Ther 14:525-535]. Standard curves were prepared using gDNA extracts from samples containing 0% to 100% BM cells derived from male / female mouse mixtures, and chimerism was calculated as follows: Donor engraftment % = 100 × 2 (CtβAct-CtSRY) .

[0152] LAM-PCR method. To identify the vector integration site, the 3' vector LTR-genomic junction was amplified by LAM-PCR according to the method published by Schmidt et al. 2007 [Nat Methods 4:1051-1057]. Initial linear amplification (100 cycles) was performed using biotinylated LTR-specific primers and up to 100 ng of gDNA as a template. After purifying the linear amplification product using streptavidin magnetic beads, complementary chain synthesis, parallel digestion with two different restriction enzymes (Tsp509I and HyCH4IV), and two ligation reactions using a linker cassette complementary to the end remaining after enzymatic cleavage were performed. The generated fragments were amplified by two additional exponential PCR steps. LAM-PCR products were separated and quantified by gel electrophoresis in a MultiNA automated system (Shimadzu).

[0153] Setup of 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 a second exponential PCR product generated by the Tsp509I and HyPCH4IV enzymes was re-amplified using fusion primers containing specific sequences to enable paired-end sequencing on an Illumina MiSeq sequencer. The LAM-PCR sample was adapted for 454 pyrosequencing by fusion PCR to attach Roche 454 GS-FLX adapters: adapter A + 8 nucleotide barcode was attached to the LTR end of the LAM-PCR amplicon; adapter B was attached to the linker cassette side. In the 5'-3' direction, the final amplicon was configured as follows: adapter A, barcode, LTR sequence, unknown genome 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 10 nM equimolar library. The final library was then requantified using the KAPA Library Quantification Kit for Illumina Sequencing Platform (Kapa Biosystems, Wilmington, MA) on a Viia7 real-time PCR system (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA) to obtain an estimated concentration of 16.35 nM. Finally, the library was sequenced using the Illumina MiSeq Reagent Kit.

[0154] Bioinformatics analysis. A pipeline was designed to incorporate raw data (typically in FastQ file format) to extract vector integration sites (ISs) from both high-throughput sequencing platforms, Roche 454 and Illumina MiSeq / HiSeq, providing a reliable list of ISs and closest genes. High-level analyses of clonal abundance quantification and gene ontology enrichment were performed using Excel, GraphPad Prism™, and available online tools.

[0155] NGS Data Processing and Pipeline Usage. The NGS data processing process, which aligns all valid sequence reads with the reference genome, addresses the management of high-throughput data from the Illumina MiSeq sequencing platform and aims to identify embedded sites (ISs). Data processing includes two main activities: 1. Data quality inspection and analysis, where lentiviral vector sequences and other contaminants are trimmed. 2. Integration site identification, where all valid sequence reads are aligned with the reference genome and valid ISs are searched for.

[0156] Data quality analysis. To identify IS from Illumina MiSeq raw data, a bioinformatics pipeline was developed. Standard LAM-PCR products contain LTR sequences, adjacent human genomic sequences, and linker cassette (LC) sequences. The 459 technology enabled the search for LAM-PCR sequences with lengths ranging from 10 bp to 900 bp. Similar results were retrieved from Illumina MiSeq paired-end reads. These length boundaries are important parameters to consider in the quality analysis process because they affect both subsequent alignment methods and the algorithm for vector component identification. To avoid partial or complete loss of the LC sequence, sequences that were too short to align accurately with the reference gene were discarded, as were those that exceeded the maximum size achievable by the NSG technology. After the pipeline for each pool was completed, all integration sites were collected in both a file (archived on the TIGET network-connected file storage NAS) and an internal database, and maintained on a storage server that tracks modified copies.

[0157] Integration site identification. To identify unique integration sites and extract an excel file (IS matrix) that includes all IS with the closest gene annotation in rows and each sample in columns, the following steps are performed: 1. Creation of the IS matrix using a program called create_matrix that enables collision detection between projects. This program will create a tab-separated file (TSV); 2. Annotation of the IS matrix file using the program annotate_bed, which will be called as follows, for each pool using the input TSV file: TIFF0007842149000010.tif24132; 3. Import of both the annotation and matrix files into a new Excel worksheet called XLS in this specification.

[0158] Collision detection. To obtain a reliable dataset of IS from each transplanted mouse, the inventors filtered out data from potential contamination / collisions and false positives based on sequence counts. An additional step of data standardization was required to combine implantation sites obtained from different experiments.

[0159] The term "collision" is used to identify the presence of identical IS in independent samples. In our experimental context, vector integration at the exact same genomic location in different cells is an extremely low-probability event. Therefore, the detection of identical IS in independent samples is likely to be due to contamination, which can occur at different stages of wet lab techniques (sample purification, DNA extraction, LAM-PCR, and sequencing). Although our working pipeline is designed to minimize the occurrence of inter-sample contact, high-throughput analysis of IS inherently retains some degree of background contamination. Identifying the degree of inter-sample contamination is important, as the search for identical IS in different samples obtained from the same mouse is used in subsequent steps to infer the biological properties of the vector-marked hematopoietic cells (i.e., polyphyletic potential and sustained clonal activity). Therefore, we must be able to distinguish the actual occurrence of identical IS in different samples (from the same mouse) from contamination / collision. To address these issues, the inventors measured the degree of collision in their analysis, then designed rules for discarding potentially collision-caused IS from each mouse dataset, and assessed the degree of shared IS between samples from different test items and mice as a means to minimize the possibility of false positives when searching for shared IS between samples from the same mouse. The inventors designed a collision detection process that enables validation of each embedded locus. The overall result is that, given a set of embedded loci I, if embedded locus i in I is classified as a collision, i is discarded from I. The inventors applied the collision detection process between three independent transplantation groups: 1. Lin transduced by coPKR170:coRPK expression LV vector - Mice from Assay 1 (coRPK1-3) that were euthanized 170 days after cell transplantation. 2. EGFP: Lin carrying an EGFP-expressing LV vector -Mice (EGFP1-6) from assay 2 transplanted with cells. 3. coPKR-TC: Lin transduced by coPKR-expressing LV vector, including secondary recipients transplanted with pooled BM derived from a subgroup of primary transplanted mice (coRPK11-14), where blood and BM were analyzed at different time points - Mice (coRPK1-14) from assay 2 transplanted with cells.

[0160] Each identical IS has different sequence reads (sequence counts) among different mice. The sequence count can be used to determine whether a sample from one mouse has contaminated the samples of other mice based on abundance criteria. In the inventors' rationale, integrations found in two mice will be assigned to the mouse showing the highest abundance, and in the other mouse it will be considered a contaminant. Thus, the inventors can identify a differential sequence count threshold that allows the assignment of a given hit to one mouse and the removal from others. The inventors searched for the threshold from the data and obtained a value of 10. That is, for each IS, if, among all TIs, the IS obtained an abundance value (percent sequence count ratio) 10 times lower than the highest abundance value (percent sequence count) of other TIs, it was discarded from that TI. The inventors applied these rules both among TIs and among selected groups (although detailed herein for TI filtering, it is similarly extended to group filtering), and used an Excel file to calculate collision detection by applying the following rules: 1. Group all samples of the same TI together by isolating each TI and summing the sequence counts. 2. For each IS, calculate the percentage ratio of the IS sequence count to the total of the reads for the TI for the 3 TIs obtained. 3. Then apply the following rule to calculate the determination step with a threshold of 10 that allows each IS to be assigned to a reliable TI.

[0161] After it was detected that an IS should be removed, the lead of that IS was removed from the group and therefore was no longer assigned to that group. The above filter was applied between mice transplanted with different ex vivo transdextrin 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) with respect to clonal abundance analysis, the following rule was added to better emphasize the sharing of inclusions between time points: if an inclusion is shared among one or more mice, that inclusion is maintained for all time points, even if the sequence count is less than 10% of the maximum sequence count among the mice; (b) for lineage tracking of relationships, a more stringent filter was applied by excluding IS with a sequence count of less than 3 and by a 10% sequence count filter for sharing between time points. That is, an inclusion shared between two time points is maintained or discarded only if each is more or less than the other.

[0162] Gene ontology analysis. All gene ontology analyses were performed using GREAT online software (http: / / bejerano.stanford.edu / great / public / html / ). The webpage allows uploading the embedded genomic coordinates for each dataset and calculating the enrichment level in the tested datasets by correlating the location information (based on binomial distribution analysis for p-value calculation) with the annotated function of the gene closest to the embedded site (based on hypergeometric distribution analysis for p-value calculation) [Groeschel S, et al. (2011). J Inherit Metab Dis 34:1095-1102]. Biological processes and molecular functions from the gene ontology database were selected for enrichment analysis. Only gene classes with a false discovery rate <0.05 were considered for both statistical analyses (Figure 20).

[0163] Data Storage. All data, both raw data and results, are stored in TIGET Network Attached File Storage (NAS) within the root folder, where alignment from the pipeline is available, and abundance matrices and plots are also available. The NAS storage is guaranteed by authentication and authorization policies, built on a reliable and scalable infrastructure using a disk RAID 5 redundant array, and backed up by the inventors' CrashPlan software, registered with TIGET.

[0164] Example 1 The PGK-coRPK therapeutic lentiviral vector provides stable and long-term correction of the anemia phenotype in genetically corrected PKD mice. The in vivo efficacy of PGK-coRPK LV (Figure 2a) was demonstrated using strain-depleted BM cells (Lin) derived from PKD mice. -The assays were performed by transduction and transplantation of cells (Figure 2b). Figure 2a is a schematic diagram of the self-inactivated lentiviral vectors used throughout the gene therapy experiments, each containing a human PGK promoter that controls the expression of the EGFP transgene in the control vector (top) or the codon-optimized sequence (coRPK) of the PKLR gene cDNA in the therapeutic vector (bottom). The coRPK sequence showed 80.4% homology to human PKLR cDNA and 76.5% homology to mouse Pklr cDNA without amino acid sequence changes. Figure 2b is a schematic diagram of the gene therapy protocol performed to elucidate the functionality of the developed PGK-coRPK lentiviral vector. Correction of the PKD phenotype was studied for 4–9 months after transplantation in PB and BM mice through hematological analysis and metabolic profiling. Integration analysis was performed at different tissues and time points derived from all mice to elucidate the safety of the LV vector. On day 280 post-transplantation, to test the stability and safety of engraftment, whole erythrocytes (BMs) derived from primary transplant mice carrying coRPK transgenes were re-transplanted into lethally irradiated female PKD mice (secondary recipients). Lethally irradiated PKD mice transplanted with coRPK LV-transduced deficient cells showed significant improvements in all tested serum erythrocyte parameters compared to non-transplanted PKD littermates or mice transplanted with EGFP LV-transduced cells (Figure 3 and Table 2).

[0165] (Table 2) Hematological variables recorded in peripheral blood 140 days post-transplant TIFF0007842149000011.tif57135

[0166] Data 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.

[0167] In mice carrying the PGK-coRPK transgene, the RBC count increased as early as 40 days post-transplant (Figure 3a), and constitutive reticulocytes, one of the most common signs of PKD, were significantly reversed, 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 transdextrins exhibited anemia and marked reticulocytes at all time points analyzed, in parallel with PKD mice. Hemoglobin levels (HGB), hematocrit (HTC), mean cytology (MCV), and mean corpuscular hemoglobin (MCH) levels were also corrected in mice transplanted with lentivirus-corrected cells when compared to non-transplanted PKD littermates (Table 2). This hematological correction was achieved with a donor chimerism of 63.66 ± 4.45% and a transdextrin efficacy ranging from 60% to 90% (Table 3).

[0168] (Table 3) Relevant molecular parameters in mice transplanted with genetically modified cells TIFF0007842149000012.tif139142

[0169] The data represents the mean ± SEM, and "nd" indicates undetermined. a Estimated trait introduction percentage obtained by interpolation in linear regression constructed from Experiment 1 (X axis: VCN / WBC, Y axis: % provirus) + CFU).

[0170] Transduced cells showed an average of 1.65 ± 0.08 incorporated vector copies per cell, indicating that the PGK-coRPK LV vector provided sufficient human RPK transgenic expression to reverse hemolytic anemia. Notably, coRPK transgene expression resulted in an extension of the erythrocyte half-life compared to non-transplanted PKD mice (Figure 3c, d). On average, PKD mice showed an RBC half-life of 19 days, but in genetically corrected mice, this was extended to 25 days (an extension of 6 days), reaching a value close to that of wild-type RBCs (Figure 3d). Thus, RBCs in coRPK-expressing mice showed an intermediate survival kinetics between healthy and deficient control mice (Figure 3c), most likely because complete chimerism was not achieved in these animals (Table 3).

[0171] Nine months after transplantation, hematopoietic progenitor cells derived from primary recipients that maintained engraftment levels (62.89 ± 5.61%) and VCN (1.44 ± 0.08 copies) (Table 3) were transplanted into secondary recipients. Secondary transplant recipients showed significant improvements in multi-lineage hematopoietic reconstitution (Figure 4) and all PB erythrocyte parameters up to five months post-transplant (Figure 5 and Table 2). Figure 4a shows the flow cytometry strategies used to identify different hematopoietic lineages by labeling with CD3-PE, B220-PE, B220-PECy5, Gr1-biotin, and Mac1-biotin antibody + SAV-PE-Cy5. Figure 4b shows representative dot plots and percentages of each lineage in PB at 140 days post-transplant (Figure 4c). The bars represent the mean percentage ± SEM for healthy mouse controls (n=2, black bars), PKD mouse controls (n=2, gray bars), and secondary transplant mice expressing the coRPK therapeutic transgene (n=4, shaded bars). Furthermore, proviral integration was detected in differently fate-determined hematopoietic progenitor cells (Figure 6a, b), and its number remained constant over time (Figure 6c), demonstrating the stability of genetic correction and highlighting the safety of PGK-coRPK LV. Figure 6a shows the vector copy number per cell in BM CFU derived from individual transplant mice 120–170 days post-transplant. Percentages of transduction and chimerism are also shown. Figure 6b shows the proviral copy number in cells from different hematopoietic compartments. The columns represent the mean ± SEM for different groups of transplant mice. Figure 6c shows the kinetics of proviral integration in BM cells derived from individual transplanted EGFP-expressing mice (gray lines) and mice harboring coRPK transgenes (black lines).

[0172] Example 2 Lentivirus-derived RPK expression normalizes erythrocyte differentiation and enables the production of functionally mature erythrocytes. PKD mice exhibit a characteristic enlargement of the erythrocyte compartment, triggered by a compensatory erythropoiesis mechanism (Min-oo et al 2004). Studies of erythrocyte differentiation patterns in transplanted mice have shown that ectopic RPK expression reverses this mechanism (Figure 7a, b). EGFP-expressing PKD mice showed a dominance of immature erythrocyte progenitor cells (subpopulation I: proerythroblasts and subpopulation II: basophilic erythroblasts) and a significant decrease in late erythrocyte cells (population IV: reticulocytes and mature erythrocytes) in the BM and spleen. However, mice transplanted with cells transduced by coRPK LV showed a significant decrease in immature erythrocyte progenitor cells (subpopulations I and II) and a significant increase in the terminal erythrocyte compartment (subpopulation IV) in the BM and spleen, similar to healthy mice (Figure 7a, b). Furthermore, unlike EGFP-expressing PKD mice, those retaining the coRPK transgene showed a significant decrease in plasma erythropoietin (Epo) levels (Figure 7c). Figure 8a shows the total CFU from the spleen, and Figure 8b shows the bone marrow 140 days post-transplant. Dots represent the number of colonies in each mouse analyzed, and lines represent the mean ± SEM in each group. Data were statistically analyzed by the non-parametric Kruskal-Wallis test. Although no change in BM CFU content was observed (Figure 8b), normalization of erythropoiesis in PKD mice treated with the therapeutic vector was accompanied by a decrease in spleen progenitor cell count to normal levels (Figure 8a).

[0173] Example 3 Transplantation of cells transduced by the coRPK lentiviral vector reverses extramedullary erythropoiesis and organopathy. Due to the active destruction of RPK-deficient erythrocytes, EGFP-expressing PKD mice exhibited acute splenomegaly with a more than 200% increase in spleen weight and size compared to healthy controls (Figure 9a, b). Structural breakdown of splenic tissue and enlargement of the red pulp, indicating extreme extramedullary erythropoiesis, also supported by the presence of erythrocyte clusters in EGFP-expressing PKD liver sections, were also observed in these animals (Figure 9c). Notably, ectopic expression of coRPK transgenes completely reversed splenic and hepatic pathology in genetically corrected mice, reducing RBC accumulation and normalizing the histological structure and size of the spleen (Figure 9). Furthermore, histological studies revealed a complete absence of iron deposits in the liver of genetically corrected mice, while PKD mice from either the non-transplant group or the group transplanted with HSCs transduced with an EGFP-retaining vector showed extreme iron overload due to a continuous hemolytic process (Figure 9c). Overall, transplantation of genetically corrected HSCs in PKD mice restored normal erythropoiesis and all secondary effects caused by hemolytic anemia.

[0174] Example 4 Expression derived from PGK-coRPK LV restores the glycolytic pathway in red cell backbone (RBCs) without altering the WBC metabolic balance. Next, to study the functional correction of RPK enzyme activity, the inventors performed extensive metabolome analysis on all transplanted and control mice. Following a non-targeted profiling strategy, the inventors observed significant changes in glycolytic intermediates within RBCs between different groups and identified three broad clusters of metabolite patterns with distinct trends (Figure 10a). RBCs from coRPK-expressing mice showed increased metabolites from cluster 1, similar to healthy controls and different from transplanted mice carrying the EGFP transgene. Similarly, cluster 3 reflected a reduced metabolite trend in genetically corrected mice, similar to wild-type mice and different from EGFP-expressing mice. Nevertheless, cluster 2 from assay 1 did not show any difference in metabolite profiles between the transplanted mouse groups (EGFP-expressing mice and coRPK-expressing mice) (Figure 10a). Non-targeted metabolic profiling also showed that genetic modification can modify 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 transdextrin HSCs. Considering these metabolic trends, we then used a targeted profiling approach to analyze other metabolites located closer to 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. When deficient erythrocytes expressing the coRPK transgene were compared with EGFP-expressing PKD mice, an increase in D-lactate, the end product of anaerobic glycolysis, also occurred (Figure 10g). To test whether the compensation for glycolytic metabolites is a result of the normalization of PK activity in mature red blood cells, the inventors measured the activity of this enzyme and standardized it against hexokinase activity to avoid the influence of a large amount of reticulocytes in deficient animals. To prevent contamination with leukocyte PK activity, RBCs were purified through a cellulose column.A complete compensation of PK activity, reaching a ratio similar to that obtained from wild-type healthy animals and normal healthy blood donor volunteers, was observed in animals expressing coRPK (Figure 11). Figure 11a shows pyruvate kinase activity in RBCs derived from control mice and mice transplanted with transduced cells, Figure 11b shows hexokinase activity, and Figure 11c shows the ratio of pyruvate kinase and hexokinase enzyme activities. RBCs were purified from blood samples through a cellulose column and subjected to enzyme activity evaluation to avoid contamination of leukocyte PK activity. Black bars, healthy mice (n = 2); white bars, mice transplanted with cells transduced with the EGFP expression vector (n = 3); striped bars, mice transplanted with cells transduced with the coRPK expression vector (n = 3). The barred bars represent values from healthy volunteers (n = 1). Data represent the mean ± SEM for each group.

[0175] Principal component analysis showed that the metabolite patterns of RBCs differed among groups and were significantly different from the WBC profile (Figure 12a). In contrast, the WBC subgroups, with very small differences between groups, clustered together and did not show changes in the metabolic balance of leukocytes when expressing ectopic coRPK (Figure 12a). Furthermore, the changes in specific metabolites observed in RBC non-target profiling were not present in WBCs (Figures 12b - d).

[0176] Example 5 PGK-coRPK LV transduced cells give polyclonal hematopoietic reconstitution without evidence of vector genotoxicity. The integration profile of LV carrying either the coRPK transgene or the EGFP transgene was analyzed in transplanted mice. Due to the genome-wide integration profile of LV, each insertion creates a unique genetic marker that can be used to track the clonal behavior in individual transduced cells. Genomic DNA (gDNA) was obtained from WBCs and BM cells derived from primary and secondary transplanted mice, and the transduced cell pool before transplantation (Lin -Cells were also obtained. Linear amplification-mediated PCR (LAM-PCR) was used to amplify the vector / genomic junction and identify the vector insertion site (IS) (Figures 13 and 14). Figure 13 demonstrates that the vector integration site was identified by LAM-PCR amplification of the 3' vector LTR-genomic junction. A MultiNA automated system was used to generate a pattern featuring several bands. The Tsp509I internal control band (IC) derived from the vector skeleton is indicated by the arrow. Figure 14 demonstrates that the vector integration site was identified by LAM-PCR amplification of the 3' vector LTR-genomic junction. A MultiNA automated system was used to generate a pattern featuring several bands. The HypyCH4IV5 internal control band (IC) derived from the vector skeleton is indicated by the arrow.

[0177] PCR products were sequenced using the MiSeq Illumina platform, and the obtained sequences were mapped to the mouse genome using a bioinformatics pipeline and filtered for collisions as described in the Methods section above (Figure 15). Figure 15 shows a general scheme for the analysis of integration site mapping performed in mice transplanted with genetically modified hematopoietic progenitor cells. Following the pipeline shown, bone marrow and leukocyte samples from transplanted mice belonging to two independent experiments (Table 3), collected at different post-transplant time points, were analyzed as described in the Supplemental Methods.

[0178] Overall, we mapped 5,173,892 sequencing reads onto the transplanted mouse genome, resulting in 2,220 unique vector integration sites. The genomic distribution of ISs from two independent experiments matched previously reported LV preferences for integration within transcription units (specifically, within the first 50 kb downstream of the transcription start site TSS) (Figure 16a) and did not show any distortion to specific chromosomes in the mouse genome (Figure 16b). Figure 16a shows the frequency distribution of integration sites (ISs) around the transcription start site (TSS) of the nearest RefSeq gene, extending 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 shows the chromosomal distribution of LV integration sites in transplanted mice expressing EGFP transgenes (black bars) or coRPK therapeutic transgenes (gray bars), showing no distortion to specific chromosomes.

[0179] The safety of gene therapy based on PGK-coRPK LV was studied by clonal abundance estimation, which calculates the percentage of sequence counts for each IS (clonal mark) relative to the total number of sequences in the dataset. Dot plots and heatmaps of the relative abundance of each IS searched for for each mouse (Figures 17, 18, and 19) are shown for different mice and in vitro cultured Lin. -This demonstrated strong variability in the clonal composition of cells. Figure 17 is a chart of tracked shared inclusions between primary and secondary recipient mice holding the therapeutic PGK-coRPK LV vector. Inclusions detected in either mouse at any organ and time point are pooled. Secondary recipients received pooled BM derived from transplanted mice coRPK11-14. The remainder of detected IS were detected in either the primary or secondary recipient. The numbers in the boxes indicate the presentation rate (%) of the corresponding inclusion in the mentioned mouse. In addition to the ≥5% filter applied to the inclusion analysis, all inclusions with a sequence count <3 were excluded. Figure 19 presents a dot plot representation of the clonal abundance of pooled inclusions in each mouse in the bone marrow. The relative percentage (y-axis) of each inclusion site is relative to the total number of sequence reads obtained in each dataset. Similar to co-RPK transdependents (Figure 17), the graph shows that the majority of transplanted mice exhibit a polyclonal pattern of hematopoietic repopulation.

[0180] Furthermore, for some samples, it was possible to understand that a small number of inclusions contributed to a large number of sequence reads (Figures 17 and 18), revealing a polyclonal pattern in the repopulation of transduced HSCs. In addition, tracked shared inclusions between primary mice carrying therapeutic PGK-coRPK LV and subsequently transplanted secondary mice did not show strong sharing of inclusions between the groups, confirming the lack of clonal dominance (Figure 18).

[0181] To determine whether insertion mutagenic hallmarks were present in transplanted mice, we assessed the occurrence of common insertion sites (CIS) similar to those in ongoing LV-mediated clinical trials. CIS are insertion hotspots that may result from insertion bias during transduction or in vivo selection of clones possessing vector insertions that confer proliferation advantage. We identified CIS using an algorithm based on Abel et al. and Grubbs test for outliers, but no CIS were found, and therefore no warning signs of genotoxicity from this readout were found. Furthermore, gene ontology (GO) analysis did not reveal any distortion to gene classes involved in the regulation of cancer, cell proliferation, or apoptosis in any of the insertion datasets sorted by tissue distribution, time, or abundance of regrowing hematopoietic cell clones (Figure 20). Figure 20 represents the LV genome insertion profile. Gene ontology (GO) analysis was performed using GREAT software on samples derived from transplanted mice. All integrations (N=2220) retrieved from this study showed overexpression of gene function, as shown in the left portion of the figure. To determine whether the highest abundance of integrations was enriched in a particular gene class, all integration sites with a relative sequence count >5% of the entire dataset (shown in Figure 17) were selected, and no overexpressed GO gene classes were identified.

[0182] These results suggest vector integration neutrality and demonstrate the safety of PGK-coRPK LV in preclinical settings.

[0183] Example 6 Human clinical trials The clinical trial will evaluate the safety and preliminary efficacy of autologous hematopoietic stem cell transplantation (HSCT) using EU / 3 / 14 / 1130 medical product (autologous CD34+ hematopoietic stem cells transduced by a lentiviral vector containing the RPK gene) in patients with pyruvate kinase deficiency who have a history of severe transfusion-dependent anemia resistant to splenectomy.

[0184] ODD EU / 3 / 14 / 1130 contains a self-inactivating lentiviral vector expressing a codon-optimized version of the therapeutic human PKLR gene (Figure 21).

[0185] Self-inactivating lentiviral vectors (SIN-LVs) offer more robust expression (Ellis 2005) and are less susceptible to transcriptional silencing than gamma retroviral vectors (Pfeifer, Ikawa et al. 2002). They also exhibit a much safer integration profile (Schroder, Shinn et al. 2002) (Mitchell, Beitzel et al. 2004) (Wu, Li et al. 2003), and due to a 400bp deletion within the 3'LTR sequence (Miyoshi, Blomer et al. 1998) (Zufferey, Dull et al. 1998), transgene expression is regulated by an internal promoter, which increases the safety of LV-based genetic modification.

[0186] The vector sequence of the accepted lentiviral vector also includes several modifications to improve transgene expression and safety in target cells.

[0187] One modification is the use of the human phosphoglycerate kinase (PGK) promoter, which already features stable in vivo activity and improved safety characteristics 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). PGK results in more physiological expression of transgenes and lower sensitivity to transcriptional silencing (Gerolami, Uch et al. 2000, Zychlinski, Schambach et al. 2008).

[0188] Another modification is a codon-optimized version (coRPK) of human PKLR cDNA that increases mRNA stability during transcription. For optimization, GeneArt® software was used to increase GC content and remove latent splice sites to avoid transcriptional silencing and thus increase transgene expression. The optimized coRPK sequence showed 80.4% homology to the human PKLR gene without any changes to the protein's amino acid sequence.

[0189] Another modification is the inclusion of a mutant woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) (Schambach, Bohne et al. 2006) lacking the residual open reading frame, in order to improve the level of expression and stability of the therapeutic gene. The backbone, promoter, and Wpre of this lentiviral vector (PGK-coRPK LV) are also included. * The sequence is the same as that of the medical product "Lentiviral vector containing the Fanconi anemia type A (FANCA) gene for the treatment of patients with Fanconi anemia type A" (Ref 141 / 2000), and the same vector skeleton used in ongoing clinical trials for metachromatic leukodystrophy (MLD) (Biffi, Montini et al. 2013).

[0190] Mode of action CD34 derived from PKD patients, either from bone marrow (BM) or mobilized peripheral blood cells. + After collecting progenitor cells, they are transduced ex vivo with a medical product, and the therapeutic vector is incorporated into the cell genome. Following incorporation, the therapeutic human gene (coRPK) is transcribed and translated in the deficient cells to produce the therapeutic RPK protein that is deficient or reduced in PKD mature erythrocytes. The transduced PKD hematopoietic progenitor cells are then genetically corrected and can therefore produce RBCs with sufficient ATP to achieve function (Figure 22). These genetically corrected hematopoietic progenitor cells (which constitute the medical product) are then re-transplanted into the patient, and after engraftment, they produce normal erythrocytes for life and treat the disease.

[0191] The active ingredient is hematopoietic stem cells (CD34) corrected by the therapeutic lentiviral vector PGK.coRPK.wpre (ODD EU / 3 / 14 / 1130), which has been designated by the European Commission as an orphan drug for the treatment of pyruvate kinase deficiency. + It would likely be present in the cellular suspension of the cells. Therefore, this new drug should be included in the group of cutting-edge therapeutic developments within the gene therapy subclass.

[0192] The active ingredient contains at least 2 × 10⁶ cells per kg of body weight, each containing at least 0.1 copies of a therapeutic vector. 6 CD34 + It consists of genetically modified cell suspensions. The cells are suspended in a physiological salt buffer containing 2% HSA.

[0193] The final therapeutic product will be prepared according to GMP rules, and therefore the required amount of the product for liberation and injection in the patient will depend on the quality of the product. In this regard, these specifications include demonstrating the efficacy of therapeutic potential by detecting the presence of at least 0.1 vector copies per cell by quantitative PCR, cell viability ≥ 30%, sterility (gram test and sterility according to pharmacopoeia), absence of mycoplasma, absence of competitive lentiviral particles, and quantitative PCR. Furthermore, investigations and studies of hematopoietic progenitor cell content and the number of vector copies in these cells will be conducted. To ensure that the method reaches the aforementioned required amount, three independent validations will be performed on healthy control cells.

[0194] The final product is ultimately packaged in heat-sealed transport bags, particularly for storage until freezing and injection into the patient; prior to this, samples will be collected for precise corresponding quality control.

[0195] mobilization Patients will be mobilized at their respective hospitals, but the first two patients will be mobilized at Hospital del Nino Jesus (Madrid, Spain). The mobilization process will involve the administration of recombinant stimulating factor granulocyte colonies (G-CSF, Neupogen, Amgen, Thousand Oaks, CA, USA) at a dose of 12 mg / kg twice daily until the first eight days of life, followed by four subcutaneous doses of 240 mg / kg / d of prelixafor (Mozobil®, Genzyme Europe BV, Naarden, Netherlands) for four consecutive days starting from day four. Peripheral blood-derived hematopoietic progenitor cells will be collected in large quantities from day five of mobilization at Hospital Nino Jesus in Madrid via leukocyte apheresis, following a standard protocol and cell separation equipment. All equipment and solutions are CE marked and comply with the requirements of medical device regulations.

[0196] CD34 + cell purification In line with the mobilization process, apheresis will be performed at the hospital where the patient is mobilized. Apheresis will be performed using MACS "magnetic cell sorting" technology (Miltenyi Biotec, Germany), which enables the separation of hematopoietic progenitor cells (CD34) through a separation column containing a powerful permanent magnet and a ferromagnetic matrix with a high magnetic field gradient. + Cells will be processed immediately to select them. The CliniMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) system uses a computer (CliniMACS® plus Instrument) to select specific CD34 + The system consists of selection software, a set of sterile tubing (CliniMACS Tubing Sets), a magnetically controlled reactive sterile apparatus (CliniMACS CD34 Reagent), and a sterile buffer (CliniMACS PBS / EDTA Buffer). The apparatus and reagents used are CE marked and comply with the requirements of medical device regulations. Nonspecific immunoglobulins (intravenous phlebogamma 5% 0.5g, Grifols) and human albumin (human albumin Grifols® 20%, Grifols) are used in this phase and subsequent washing, and are subsequently removed by washing after centrifugation. CD34+ cells are then quantified. Microbiological preparation of the obtained product is carried out by taking samples of standard fungi, aerobic bacteria, and anaerobic organisms for culture according to specific protocols.

[0197] CD34 + Trait introduction Refined CD34 +Transduction of cells using ODD EU / 3 / 14 / 1130 is performed under GMP conditions within a 48-hour timeframe following cell extraction (apheresis) from the patient. Ex vivo culture of cells is continued for less than 48 hours and cultured according to established standards, including the use of appropriately formulated medium X-vivo-20 (Lonza), hematopoietic growth factors (10 ng / ml hrSCF, 10 ng / ml hrFlt-3, 10 ng / ml TPO, and 20 ng / mL IL-3 (all from Prepotech)), 1 μg / mL Pulmozyme, and the addition of a controlled 5% O2 concentration. Transduction is performed using a GMP lentiviral batch of ODD EU / 3 / 14 / 1130 prepared by VIVEbiotech (San Sebastian, Spain). After transduction, the cells are washed with X-vivo-20 (Lonza) and finally packaged in appropriate transport bags for cryopreservation. Specific samples are collected to determine whether the final product meets all the previously mentioned specifications for final release. Three independent validations are performed to assess the stability of the product. All products and solutions, including the vector, meet the specifications of the law for medical devices and clinical use. Prior to preparation, all raw materials (including consumables, biological reagents, and chemical powders) are inspected by the Quality Control (QC) Unit of CliniStem according to Standard Operating Procedures (SOPs).

[0198] conditioning Patients are conditioned according to a standardized, specific protocol considered for the clinical trial. To consider alternative conditioning for patients, 2 × 10⁶ products are used in cases where the prepared product does not completely reconstitute the hematopoiesis of the treated patient. 6 Unused CD34 + Freeze a backup of cells / kg.

[0199] injection Before infusion, check patient eligibility to ensure that the study requirements are met. Record premedication and prophylactic medication used on the day of infusion.

[0200] Example 7 Nonclinical development Previous studies have demonstrated that HSC gene therapy for PKD in mice is viable when more than 25% of the cells are genetically corrected. These results suggest that a significant number of donor gene-corrected HSCs (Zaucha, Yu et al. 2001) and high levels of transgene expression are necessary to achieve therapeutic efficacy in PKD. We developed a novel therapeutic lentiviral vector proposed for this clinical trial, possessing an hPGK eukaryotic promoter that drives PKLR cDNA expression, which was designated as an orphan drug in August 2014 (EU / 3 / 14 / 1130). Using this vector, we implemented a preclinical gene therapy protocol for PKD in a mouse model of the disease. At clinical standard lentiviral doses, ectopic RPK expression was able to normalize the erythrocyte compartment, correct the hematological phenotype, and reverse organopathy. Metabolome studies demonstrated functional correction of the glycolytic pathway in genetically corrected RBCs, with no metabolic disruption observed in leukocytes. Notably, when RPK was ectopically expressed under the activity of an eccentric promoter such as PGK, parallel-analyzed WBCs showed no alteration of metabolic balance in leukocytes, thus ruling out leukocyte metabolic dominance as a potential safety issue and enhancing the therapeutic potential of the EU / 3 / 14 / 1130 vector.

[0201] The absence of multi-lineage rearrangements and leukemia events or clonal growth in secondary recipients after proliferative stress induced by BM re-transplantation demonstrates the long-term stability and safety of the PGK-coRPK LV vector-based protocol. The use of a human PGK eukaryotic promoter, which (i) is likely to result in more physiological expression of RPK transgenes, (ii) has been proven to be a weak transactivator, and (iii) is currently used in clinical trials for metachromatic leukodystrophy (MLD), can also explain the safety of the overall methodology.

[0202] Next-generation sequencing was used to assess the long-term safety of HSC gene therapy through analysis of vector integration sites and to predict the risk of carcinogenesis due to insertions in HSCs. Over 5,173,892 sequence reads were mapped to a total of 2,220 unique vector ISs on the mouse genome, but no evidence of in vivo augmentation or selection of clones carrying ISs was found. Rather, our data showed the clonal composition and dynamics of hematopoiesis after transplantation of transduced HSCs in mice, suggesting true stable genetic in vivo modification of HSCs over time. Overall, the analysis of vector integration patterns strongly demonstrates the safety characteristics of the PGK-coRPK LV vector, which provides genetic correction of PKD without evidence of genotoxicity.

[0203] Example 8 Clinical development To date, no clinical studies using the medical product have been conducted. This will be the first time that protocol assistance is required from regulatory authorities. The inventors' goal is to conduct a clinical trial supported by the European Commission. The ForGeTPKD Consortium, comprised of various clinicians and basic researchers from Europe, has been established to focus on PKD research and the development of new therapeutic strategies. The ForGeTPKD clinical trial will be the first administration of this medical product in humans. It will be 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 by a lentiviral vector (EU / 3 / 14 / 1130) containing the erythrocyte pyruvate kinase (RPK) gene in patients with severe pyruvate kinase deficiency.

[0204] Regulated state The medical product does not currently have marketing authorization. The PKD Consortium's objective is to move forward with clinical development of the medical product in order to eventually obtain marketing authorization.

[0205] The final product mentioned is produced by a lentiviral vector designated as an orphan drug, related to the following:

[0206] Indications Treatment for pyruvate kinase deficiency.

[0207] standardThe only therapeutic treatment for PKD is allogeneic BMT, used in patients with transfusion-dependent severe anemia resistant to other treatments. However, allogeneic BMT is not a widely accepted treatment for PKD because it is associated with severe complications related to intensive prealo-BMT conditioning with chemotherapy or chemoradiotherapy, and is also associated with 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 by a viral vector containing the wild-type version of the gene provided by ODD EU / 3 / 14 / 1130 may represent a potential therapeutic opportunity for these patients to avoid the risk of GVHD, a major cause of hematopoietic progenitor cell transplantation failure.

[0208] active substance : Auto-CD34 transduced by a lentiviral vector (ODD EU / 3 / 14 / 1130) containing the erythrocyte-type pyruvate kinase (RPK) gene expressing the wild-type version of the protein. + Hematopoietic stem cells.

[0209] Finished product : Suspended in saline buffer containing 2% HAS, at least 2 × 10⁶ units per kg of patient body weight. 6 A freezing bag containing this active substance.

[0210] Example 9 Pharmacology Completed researchThe developed therapeutic product contains several modifications within its sequence that offer several advantages for gene therapy of PKD: (1) The use of a SIN-LV vector design enabled the relatively easy and safe production of a viral stock that can efficiently transduce 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), resulted in more physiological expression of the transgene, achieving therapeutic levels at a clinically standard viral dose (1.65 VCN) (Matrai, Chuah et al. 2010); (3) The presence of a codon-optimized transgene sequence and a mutant Wpre sequence increased transgene mRNA stability; the reporter gene was not included in the therapeutic vector sequence, avoiding potential immunogenicity issues (Morris, Conerly et al. 2004); (Stripecke, Carmen Villacres et al. 1999).

[0211] The developed hPGK-coRPK LV therapeutic product efficiently reversed PKD pathology in both primary and secondary knockout mice transplanted with progenitor cells transduced and corrected by ODD EU / 3 / 14 / 1130. Correction was achieved in cells harboring an average of 1.65 copies of therapeutic transgenes per cell.

[0212] The human PGK promoter was potent enough to express clinically appropriate levels of coRPK protein, restoring the hemolytic phenotype in transplanted mice.

[0213] Genetic correction extended the RBC half-life; normalized hematological variables and reticulocyte levels; reversed constitutively activated compensatory erythropoiesis in PKD mice; relieved pathology in the spleen and liver; and, notably, reduced iron overload, one of the life-threatening complications of PKD. Furthermore, ectopic expression of human RPK corrected the energetic deficiency in RBCs without altering the metabolic balance in WBCs, thus strongly demonstrating the efficacy and safety of the medicinal product.

[0214] Example 10 Ongoing research Transduction of human hematopoietic progenitor cells derived from healthy donors and PKD patients for the following purposes: (1) studying the efficiency of transduction of ODD EU / 3 / 14 / 1130 in human cells; (2) clarifying the optimal vector copy number / cell for obtaining efficient therapeutic expression of RPK therapeutic protein; and (3) clarifying the optimal conditions for obtaining therapeutic transduction levels without loss of hematopoietic stem cell capacity.

[0215] The planned research includes setting up conditions for large-scale transfection in a GMP facility, as well as pre-validation studies and three validation studies to set up optimal conditions to reach the required specifications for the final therapeutic product.

[0216] toxicology Completed researchThis includes the following: (1) Ectopic expression of human RPK corrected the energetic deficiency in RBCs without altering the metabolic balance in WBCs; (2) Genomic integration analysis of the vector demonstrated the following: (i) Analysis of the relative abundance of specific cell clones revealed oligoclonal hematopoietic rearrangements in several mice and did not show clonal dominance for primary and secondary transplant mice; (ii) Preferential integration sites (CIS, high-density clusters of vector integrations at defined genomic intervals), considered hallmarks of insertional mutagenesis, did not show signs of genotoxicity, nor were there any abnormal enrichments of CIS over time, nor were CIS detected from two independent gene therapy experiments conducted in mice presented by high sequence counts, and did not preferentially target oncogenes; (iii) Gene ontology (GO) analysis of genes targeted by lentiviral integration and studies of the location of vector integrations in specific regions of the genome did not show any distortion to gene classes involved in the regulation of cancer, cell proliferation, or apoptosis; (iv) Overall, medical product integration analysis did not show evidence of genotoxicity.

[0217] Planned researchThis includes the following: (1) Analysis of recombinant competent lentivirus (RCL) production: Human T lymphocytes derived from healthy donors and PKD patients are transduced using ODD EU / 3 / 14 / 1130 and cultured in vitro for an extended period. The presence of viral p24 protein in the supernatant is analyzed by ELISA to assess the potential generation of RCLs. (2) Distribution of medical products in the body: Mouse hematopoietic progenitor cells are transduced using ODD EU / 3 / 14 / 1130 and transplanted into lethally irradiated recipients. One month after transplantation, the animals are sacrificed and different organs (gonads, liver, kidneys, brain, bone marrow, spleen, and peripheral blood) are analyzed for the presence of vector DNA; (3) Vector integrome in human cells: Hematopoietic progenitor cells derived from healthy donors and PKD patients are transduced using ODD EU / 3 / 14 / 1130 and transplanted into severely immunodeficient mice to enable engraftment and proliferation of human hematopoietic cells. Blood and BM grafts will be collected at different time points (1 month, 2 months, and 3 months post-transplant), human cells will be sorted, and subjected to vector integrome analysis, similar to that already performed with mouse cells.

[0218] Example 11 Human clinical trials To test clinical efficacy, the ForgetPKD clinical trial will be conducted. The proposed clinical trial will involve patients with pyruvate kinase deficiency who have a history of severe transfusion-dependent anemia resistant to splenectomy, and who have been transduced by a lentiviral vector containing the EU / 3 / 14 / 1130 medical product (erythrocyte pyruvate kinase (RPK) gene). + The goal is to evaluate the safety and preliminary efficacy of autologous hematopoietic stem cell transplantation (HSCT) using hematopoietic stem cells.

[0219] The primary objective is to evaluate the safety and tolerability / feasibility of the procedure. Therefore, the following endpoints are measured: (1) the incidence and characterization of adverse events (AEs), including AEs associated with transdextrin injection, AEs resulting from pre-transdextrin conditioning, and AEs resulting from transdextrin-related clonal evolution; and (2) the number of patients with stem cell engraftment at 30 days post-transplant.

[0220] A secondary objective is to evaluate the preliminary efficacy of the treatment. Therefore, the following endpoints will be measured: the number of patients who became “transfusion-independent” after the study; the ratio of the average number of transfusions required during the study period (1 year) to the average number of transfusions in the last 1.5 years prior to baseline assessment for patients who still required transfusions after treatment; a clinically significant reduction in anemia after the study, defined as the number of patients with a 2 gr / dL increase in hemoglobin levels from baseline; a clinically significant reduction in polyretic erythrocytosis after the study, defined as the number of patients with a 50% decrease from baseline assessment; and the number of patients with 1% detectable stem cell engraftment at 6 and 12 months after cell infusion and after the study.

[0221] The objective of the study is to evaluate the impact of treatment on patients' quality of life. Therefore, the following endpoints are measured: improvement from baseline in quality of life after the study, using a quality of life questionnaire (SF-36 for adults or PEDSQL for children) and validated versions translated into the languages ​​of the relevant countries (Italian, Dutch, and Spanish).

[0222] The ForGetPKD clinical trial is a multicenter international trial conducted in three EU member states: Spain, Italy, and the Netherlands. Participating sites include the Reference National Investigators and Institutions for PKD diagnosis and treatment.

[0223] This clinical trial represents the first administration of the described product to humans. It is designed as a non-comparative, open-label, single-dose Phase I / II study.

[0224] The global study period will be two years, from the first patient's first consultation to the last patient's final consultation. This includes a one-year recruitment period and a one-year treatment period and initial (immediate) follow-up. After the completion of the trial, included subjects will be asked to participate in a subsequent follow-up study to monitor safety and efficacy for up to five years post-transplant.

[0225] Based on disease prevalence and study design, the study is planned to include six patients per year. This estimate was determined considering the presence of three potential participants already identified.

[0226] The research methodology includes a screening period, a treatment period, and a follow-up period. Details of the examinations and related research methodologies in each phase are detailed below and summarized in Table 4.

[0227] (Table 4) TIFF0007842149000013.tif168143TIFF0007842149000014.tif160143

[0228] Screening period Initial consultation: Pre-screening consultation Potential candidates will be notified of the purpose and characteristics of the clinical trial, and two copies of the informed consent document will be obtained, completed, and signed by the patient (or legal guardian if a minor). To be eligible for the study, patients must meet all selection criteria and not meet any exclusion criteria, which will be checked. This serves as a backup in case of non-engraftment (A. 2 x 10⁶ per kg of body weight). 6 Store individual CD34+ cells, generate the medical product, and implement all quality control necessary for the release of the medical product, at least 6 × 10⁶ cells per kg of body weight. 6To transduce individual CD34+ cells using the EU / 3 / 14 / 1130 vector, live CD34 + This includes pretreatment techniques for mobilizing and obtaining cells. After the release of the medical product, sufficient transduced cells (2 × 10⁶ cells per kg of body weight) are required. 6 Individual trait introduction CD34 + Only patients whose cells are available will be included in the study.

[0229] The following techniques will also be used in this examination: - Registration of relevant medical and surgical history - Registration of demographic data and clinically relevant physical examination findings - Registration of related concomitant medications - Peripheral blood tests for routine hematological counting (CBC), biochemistry, coagulation determination, and serology. - Echocardiography, pulmonary function tests, and chest X-ray - Questionnaire on quality of life (SF-36 or PEDSQL) - Genetic diagnosis of PKD

[0230] To be eligible for the study, patients must meet all of the following selection criteria and not meet any exclusion criteria.

[0231] The selection criteria are: being a male or female patient aged >2 years at the time of mobilization; willingness to give signed informed consent (signed by a parent or legal guardian in the case of a child under 18 years); prior diagnosis of PKD confirmed by genetic testing; a history of severe transfusion-dependent anemia unresponsive to splenectomy; being a candidate for autologous hematopoietic stem cell transplantation; and having ≥2 × 10⁻¹⁰�⁻¹⁰⁰⁻¹⁰⁰⁻¹⁰⁰⁻¹⁰⁰⁻¹⁰⁰ 6 Individual trait introduction CD34 + The cells must be available and have been treated and followed up for at least the past two years at a specialized facility that maintains detailed medical records, including transfusion history.

[0232] Exclusion criteria include being positive for human immunodeficiency virus type 1 or 2 (HIV 1 and HIV 2), having an uncorrected hemorrhagic disorder, having other causes of hemolysis, having a history of or present malignancy or spinal proliferative disorder or immunodeficiency disorder, having a close relative with a known or suspected familial cancer syndrome (including, but not limited to, hereditary breast and ovarian cancer syndrome, hereditary nonpolyposis colorectal cancer syndrome, and familial adenomatous polyposis), having previously received an allogeneic transplant, having residual cells of donor origin, having severe complications considered to be grade III / IV cardiac, pulmonary, hepatic, or renal dysfunction after medical evaluation, having an uncontrolled seizure disorder, (corrected for hemoglobin) This includes having the ability to diffuse carbon monoxide (DLco) at a rate of <50% of the expected value, having other evidence of severe iron overload that guarantees exclusion in the investigator's view, participating in another clinical study with the investigational drug within 30 days of screening, having an HLA-matched family donor available for allogeneic bone marrow transplantation, being a pregnant or breastfeeding woman, being a patient who, according to investigator criteria, would not be able to understand and / or adhere to the objectives, benefits, and risks of the study, or to comply with the study methodology, and having an inadequate functional state evidenced by a Karnofsky index ≤ 80 in adults or Lansky ≤ 80 in children.

[0233] The statistical analysis of the study is descriptive. Qualitative endpoints are described by frequency and percentage. Qualitative endpoints include adverse events, the number of patients with stem cell engraftment, the number of patients becoming "transfusion-independent," the number of patients with a clinically significant reduction in anemia, the number of patients with a clinically significant reduction in reticulosis, the number of patients with stem cell engraftment in which the presence of transduced cells could be detected, and improvements in quality of life from baseline as measured by the SF-36 or PEDSQL questionnaire. Quantitative endpoints are described by mean and standard deviation, or median and quartiles. Quantitative endpoints include reductions from baseline in anemia and reticulosis, the number of transfusions required during the study period compared to the number of transfusions in the past year prior to baseline assessment, and vector copy counts in peripheral blood and bone marrow. All endpoints are described after the study.

[0234] Previous studies have demonstrated that HSC gene therapy for PKD in mice is viable when more than 25% of genetically corrected cells are 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. We have developed a novel therapeutic lentiviral vector proposed for this clinical trial, possessing an hPGK eukaryotic promoter that drives PKLR cDNA expression, which was designated as an orphan drug in August 2014 (EU / 3 / 14 / 1130). Using this vector, we implemented a preclinical gene therapy protocol for PKD in a mouse model of the disease. At clinical standard lentiviral doses, ectopic RPK expression was able to normalize the erythrocyte compartment, correct the hematological phenotype, and reverse organ pathology. Metabolome studies demonstrated functional correction of the glycolytic pathway in genetically corrected RBCs, with no metabolic disruption observed in leukocytes. Notably, when RPK was ectopically expressed under the activity of an eccentric promoter such as PGK, parallel-analyzed WBCs showed no alteration of metabolic balance in leukocytes, thus ruling out leukocyte metabolic dominance as a potential safety issue and enhancing the therapeutic potential of the EU / 3 / 14 / 1130 vector.

[0235] The absence of multi-lineage rearrangements and leukemia events or clonal growth in secondary recipients after proliferative stress induced by BM re-transplantation demonstrates the long-term stability and safety of the PGK-coRPK LV vector-based protocol. The use of a human PGK eukaryotic promoter, which has been proven to be a weak transactivator and is currently used in clinical trials for metachromatic leukodystrophy (MLD), likely resulting in more physiological expression of the RPK transgene, can also explain the overall safety of the methodology.

[0236] Next-generation sequencing was used to assess the long-term safety of HSC gene therapy through analysis of vector integration sites and to predict the risk of carcinogenesis due to insertions in HSCs. Over 5,173,892 sequence reads were mapped to a total of 2,220 unique vector ISs on the mouse genome, but no evidence of in vivo augmentation or selection of clones carrying ISs was found. Rather, our data show the clonal composition and dynamics of hematopoiesis after transplantation of transduced HSCs in mice, suggesting true stable genetic in vivo modification of HSCs over time. Overall, the analysis of vector integration patterns strongly demonstrates the safety characteristics of the PGK-coRPK LV vector, which provides genetic correction of PKD without evidence of genotoxicity.

[0237] Sequence information SEQUENCE LISTING <110> CENTRO DE INVESTIGACIONES ENERGETICAS, MEDIOAMBIENTALES Y TECNOLOGICAS, OA, MP FUNDACION INSTITUTO DE INVESTIGACION SANITARIA FUNDACION JIMENEZ DIAZ CONSORCIO CENTRO DE INVESTIGACION BIOMEDICA EN RED <120> COMPOSITIONS AND METHODS FOR ENHANCED GENE EXPRESSION OF PKLR <150> US 62 / 325,397 <151> 2016-04-20 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 677 <212> DNA <213> Artificial Sequence <220> <223> Chimeric modified Woodchuck hepatitis virus posttranscriptional regulatory element (Wpre) <400> 1 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> 2 <211> 28 <212> DNA <213> Human immunodeficiency virus 1 <400> 2 tttaaaagaa aaggggggat tggggggt 28 <210> 3 <211> 205 <212> DNA <213> Human immunodeficiency virus 1 <400> 3 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg 60 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta 120 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa 180 gaatcctggc tgtggaaaga tacct 205 <210> 4 <211> 566 <212> DNA <213> Homo sapiens <400> 4 attatggtaa atccacttac tgtctgccct cgtagccatc gagataaacc ctaccgggta 60 ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact 120 tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa 180 ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag 240 ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc 300 actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc 360 agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg 420 tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga 480 ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca 540 tctccgggcc tttcgacctg cagccc 566 <210> 5 <211> 138 <212> DNA <213> Human immunodeficiency virus 1 <400> 5 tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt 60 gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt 120 cagtattaag cgggggag 138 <210> 6 <211> 236 <212> DNA <213> Artificial Sequence <220> <223> Modified LTR nucleic acid sequence <400> 6 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc 60 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 120 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 180 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagt 236 <210> 7 <211> 235 <212> DNA <213> Artificial Sequence <220> <223> Modified LTR nucleic acid sequence <400> 7 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc 60 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 120 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 180 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcag 235 <210> 8 <211> 516 <212> DNA <213> Artificial Sequence <220> <223> Synthesized codon optimized human PKLR promoter sequence <400> 8 tccacggggt tggggttgcg ccttttccaa ggcagccctg ggtttgcgca gggacgcggc 60 tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc ctgggtctcg cacattcttc 120 acgtccgttc gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc cccggcgacg 180 cttcctcgtc cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga 240 caaacggaag ccgcacgtct cactagtacc ctcgcagacg gacagcgcca gggagcaatg 300 gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct gctcagcagg ggcgcccgag 360 agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg 420 ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc 480 tccctcgttg accgaatcac cgacctctct ccccag 516 <210> 9 <211> 677 <212> DNA <213> Woodchuck hepatitis virus <400> 9 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

Claims

1. (a) Promoter sequence; (b) A sequence encoding a therapeutic gene product which is a human pyruvate kinase polypeptide; and (c) A variant woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) containing a sequence having at least 90% identity with SEQ ID NO:

1. An expression cassette comprising a polynucleotide sequence, wherein the promoter sequence is functionally linked to a sequence encoding a pyruvate kinase polypeptide.

2. The expression cassette according to claim 1, wherein WPRE contains the sequence of SEQ ID NO:

1.

3. The expression cassette according to claim 1, wherein the promoter is a phosphoglycerate kinase (PGK) promoter.

4. The expression cassette according to claim 1, wherein the pyruvate kinase polypeptide is an R isoform pyruvate kinase (RPK) polypeptide.

5. The expression cassette according to claim 1, wherein the pyruvate kinase polypeptide is a liver and red blood cell type pyruvate kinase (PKLR) polypeptide.

6. The expression cassette according to claim 1, wherein the sequence encoding the pyruvate kinase polypeptide is codon-optimized.

7. A recombinant gene delivery vector comprising an expression cassette according to any one of claims 1 to 6.

8. The recombinant gene delivery vector according to claim 7, further comprising one or more enhancer sequences.

9. The recombinant gene delivery vector according to claim 7, further comprising a polyprint lactate (PPT) or polyadenylation (PolyA) signal sequence.

10. The recombinant gene delivery vector according to claim 7, further comprising one or more of the following sequences: (i) Packing signal sequence; (ii) Shortened Gag sequences; (iii) Rev response element (RRE); (iv) Central polyprint lactate (cPPT); and (v) Central Terminal Array (CTS).

11. The recombinant gene delivery vector according to claim 7, further comprising 5' and 3' long terminal repeat sequences.

12. A recombinant gene delivery vector according to claim 7, which is a virus or a viral vector.

13. A cell comprising an expression cassette according to any one of claims 1 to 6.

14. Cells transduced by a recombinant gene delivery vector according to any one of claims 7 to 12.

15. The cell according to claim 13 or 14, which is a hematopoietic stem cell.

16. The cell according to claim 13 or 14, which is a committed hematopoietic erythrocyte progenitor cell.

17. The cell according to claim 13 or 14, which is a CD34+ cell.

18. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a recombinant gene delivery vector according to any one of claims 7 to 12.

19. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and cells according to any one of claims 13 to 17.

20. At least 2 x 10 per kg of body weight 6 The pharmaceutical composition according to claim 19, comprising individual living CD34+ cells.

21. The pharmaceutical composition according to claim 19 or 20, wherein the vector copy number per cell (VCN / cell) is at least 0.

5.

22. The pharmaceutical composition according to claim 21, wherein the VCN / cell ratio is 5 or less.

23. A pharmaceutical composition according to any one of claims 19 to 22, wherein cells are cryopreserved.

24. A pharmaceutical composition comprising a recombinant gene delivery vector according to any one of claims 7 to 12, for use in treating or preventing a disease or disorder in a subject requiring such treatment, wherein the disease or disorder is pyruvate kinase deficiency (PKD).

25. A pharmaceutical composition comprising cells according to any one of claims 13 to 17, for use in treating or preventing a disease or disorder in a subject requiring such treatment, wherein the disease or disorder is pyruvate kinase deficiency (PKD).

26. The pharmaceutical composition according to any one of claims 24 or 25, wherein the subject is a patient with pyruvate kinase deficiency who has a history of severe transfusion-dependent anemia resistant to splenectomy.

27. The pharmaceutical composition according to claim 25, wherein the cells are self to the target.

28. The pharmaceutical composition according to claim 25, wherein the cells are homogeneous with respect to the target.

29. The pharmaceutical composition according to any one of claims 25 to 28, wherein the cells are transduced CD34+ cells.

30. At least 2 x 10 per kg of body weight 6 A pharmaceutical composition according to any one of claims 25 to 29, comprising individual living CD34+ cells.

31. A pharmaceutical composition according to any one of claims 25 to 30, wherein the vector copy number per cell (VCN / cell) is at least 0.

5.

32. A pharmaceutical composition according to any one of claims 25 to 31, wherein the VCN / cell ratio is 5 or less.

33. A pharmaceutical composition according to any one of claims 25 to 32, wherein cells are cryopreserved.

34. CD34+ cells: at least 2 × 10⁶ cells per kg of weight of the subject. 6 A pharmaceutical composition according to any one of claims 25 to 33, administered by infusion with individual CD34+ cells.

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