RU2773358C2 - Compositions and methods for enhancing pklr gene expression - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to a lentiviral gene delivery vector containing an expression cassette containing a promotor sequence, a sequence encoding polypeptide of human pyruvate kinase of liver and red blood cells (PKLR) and a signal of ribonucleic acid (RNA) export, as well as to a composition containing it. A method for the expression of transgene in erythroid cells in vitro is also disclosed, providing for bringing into contact of one or more erythroid cells with the above-mentioned vector.

EFFECT: invention is effective for the treatment or prevention of pyruvate kinase deficiency.

17 cl, 22 dwg, 4 tbl, 11 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority under U.S. Provisional Application No. 62/325,397, filed April 20, 2016, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[001] The present invention relates to gene therapy for pyruvate kinase deficiency.

BACKGROUND OF THE INVENTION

[002] Pyruvate kinase deficiency (PKD) is a monogenic metabolic disease caused by mutations in the PKLR gene, which leads to hemolytic anemia of various symptoms and can be fatal during the neonatal period. The recessive nature of the inheritance of the trait that causes PKD and the radical treatment of the disease with allogeneic bone marrow transplantation provide the most appropriate situation for the development of gene therapy approaches.

[003] Among many other congenital enzymatic defects affecting erythrocytes, pyruvate kinase deficiency (PKD) is the most common defect causing chronic nonspherocytic hemolytic anemia (CNSHA) (Zanella et al. 2007). The presentation and severity of PKD is highly variable and ranges from moderate to severe neonatal anemia, which in the most severe cases is fatal in childhood (Pissard et al 2006). Growth retardation, fetal hydrops and death during the neonatal period have also been reported with low frequency (Gilsanz et al. 1993). The incidence of PKD in the general Caucasoid population has been estimated at 1:20,000 (Beutler et al 2000), and more than 195 different mutations in the PKLR gene have been identified to date (http://www.lovd.nl/pklr). Allogeneic bone marrow transplantation (BMT) has been used successfully in patients with severe PKD (Tanphaichitr et al 2000), but the low availability of histocompatible donors and serious complications associated with BMT in these patients (i.e., graft versus host disease) , opportunistic infections, etc.) have made intermittent blood transfusions and splenectomy the main treatment options for most severe forms of PKD (Zanella et al 2005), dramatically increasing morbidity and mortality among patients (Hilgard et al 2005). The limited efficacy and side effects of therapy options for patients with severe PKD and its recessive inheritance pattern make PKD a disease for which gene therapy treatment is suitable.

[004] PKDs cause defects in the enzyme pyruvate kinase (PK) (Zanella 2005), which catalyzes the last ATP-generating reaction of the glycolysis pathway in all cells. In mature erythrocytes, PK becomes very important because only a specific R-type (RPK) isoform (RPK) is expressed in RBC (Kanno et al 1992) due to regulation of the erythroid cell-specific alternative promoter of the PKLR locus (Noguchi et al 1987). Thus, any loss of RPK activity impairs RBC metabolism and longevity (Zanella 2005), leading to CNSHA.

[005] A promising approach to the treatment and prevention of genetic and other diseases and disorders is the delivery of therapeutic agents using a gene therapy vector. Currently, viral vectors show the greatest efficiency in gene transfer and in the correction of genetic diseases that require stable gene expression, while vectors based on herpesviruses, retroviruses, lentiviruses, adenoviruses or AAVs are optimal due to the integrating nature of the life cycle of these viruses.

[006] Gene therapy for monogenic diseases, in particular those that affect the hematopoietic system, has provided compelling evidence that genetic repair of autologous hematopoietic stem cells (HSCs) is an alternative to the allogeneic HSCT treatment option that avoids its 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 diseases affecting the erythrocyte, such as β-thalassemia and sickle cell disease, has been considered in animal models (Pestina et al 20091, Breda et al 2012) as well as in humans (Cavazzana-Calvo et al 2010). However, gene therapy approaches for inherited erythroid metabolic deficiencies such as PKD are still limited. The suitability of HSC gene therapy for PKD has been demonstrated in both murine (Tani et al 1994; Meza et al 2009) and canine models of RPK deficiency (Trobridge et al 2012), showing that donor chimerism and transduction levels are key parameters for achieving effective correction of the hemolytic phenotype (Richard et al., 2004), taking into account the lack of selective advantage in HSCs corrected with the donor gene. Previous work in a mouse model of PKD showed that human RPK expression by retrovirus was able to completely correct the PKD phenotype when more than 25% of genetically corrected cells were transplanted (Meza et al 2009). A similar therapeutic threshold for corrected cells was recently reported in one Basenji dog with PKD infused with HSC in vivo expanded and corrected with a spumavirus vector (Trobridge et al 2012).

[007] A number of problems remain regarding the development of polynucleotide cassettes and expression vectors for use in gene therapy. One of the significant problems is to ensure sufficient expression of the transgene in target cells. There has been a longstanding unmet need in the art for sufficiently reliable expression of transgenes after gene transfer. In some cases, more efficient expression is required for the effectiveness of some vectors, such as plasmid DNA vectors. In other cases, more efficient gene expression cassettes are needed to provide a lower therapeutic dose that has a more favorable safety profile or a less invasive route of administration.

[008] High levels of transgene expression can be achieved using gamma-retroviral (gamma-RV) vectors due to the fact that therapeutic transgene expression is regulated by their LTR sequences. However, the first clinical trials based on this type of vector raised safety concerns as several patients unexpectedly developed leukemia (Hacein-Bey-Abina et al 2008). Strong promoter activity of LTR sequences could negatively impact the regulation of neighboring genes either by activating proto-oncogenic promoters or by inhibiting tumor suppressor genes, leading to insertional mutagenesis (Ott et al 2006, Howe et al 2008; Stein et al 2010; Braun et al 2014). These data highlight the need for safer and more effective vectors for PKD gene therapy than gamma-retroviral vectors.

BRIEF DESCRIPTION OF THE INVENTION

[009] In one embodiment, the present invention provides an expression cassette containing a polynucleotide sequence containing: a) a promoter sequence; b) the sequence encoding the gene product; and c) a ribonucleic acid (RNA) export signal, wherein the promoter sequence is operably linked to a sequence encoding a pyruvate kinase polypeptide and optionally a) to c) are present in the expression cassette in the 5'-3' direction. In certain embodiments, the promoter is a phosphoglycerate kinase (PGK) gene 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 erythrocyte pyruvate kinase (PKLR) polypeptide. In certain embodiments, the sequence encoding the gene product is codon-optimized. In specific embodiments, the RNA export signal is a mutant woodchuck hepatitis virus post-transcriptional 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 polypurine tract (PPT) or polyadenylation signal (polyA) sequence. In some embodiments, the expression cassette further comprises one or more of the following sequences: i) a packaging signal sequence; ii) a truncated Gag sequence; iii) Rev-sensing element (RRE); iv) central polypurine tract (cPPT); v) a central terminal sequence (CTS); and vi) an upstream sequence element (USE), optionally from simian virus 40 (SV40-USE). In some embodiments, the expression cassette further comprises 5' and 3' long terminal repeat sequences.

[0010] In a related embodiment, the present invention provides a recombinant gene delivery vector containing the expression cassette disclosed herein. In certain embodiments, the recombinant gene delivery vector is a virus or viral vector. In certain embodiments, the virus or viral vector is a lentivirus (LV).

[0011] In another related embodiment, the present invention relates to a cell containing the expression cassette or gene delivery vector disclosed herein. In some embodiments, the cell is a blood cell. In some embodiments, the cell is an erythroid cell. In some embodiments, the cell is a bone marrow cell, eg, a lineage-depleted bone marrow cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a CD34+ hematopoietic stem cell. In some embodiments, the cell is a committed hematopoietic erythroid progenitor cell.

[0012] 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 disclosed herein.

[0013] In another embodiment, the present invention provides a method of treating or preventing a disease or disorder in a subject in need thereof, comprising providing the subject with an expression cassette, gene delivery vector, or pharmaceutical composition disclosed herein. In one embodiment, the disease or disorder is pyruvate kinase deficiency (PKD) and the gene product is a pyruvate kinase (PK) polypeptide, optionally a liver and erythrocyte pyruvate kinase (PKLR) polypeptide. In certain embodiments, the pharmaceutical composition comprises a recombinant gene delivery vector. In other embodiments, the implementation of the pharmaceutical composition contains a cell. In one embodiment, the cell is autologous to the subject. In another embodiment, the cell is allogeneic to the subject.

[0014] In a related embodiment, the present invention provides a method for expressing a transgene in erythroid cells, comprising bringing one or more erythroid cells into contact with an effective amount of a recombinant viral vector, where the vector contains a human phosphoglycerate kinase gene promoter, a codon-optimized variant of the transgene based on pyruvate kinase cDNA human liver and erythrocyte (PKLR) and a mutant post-transcriptional woodchuck hepatitis virus post-transcriptional regulatory element, wherein after said contacting, PKLR is expressed at detectable levels in one or more erythroid cells.

BRIEF DESCRIPTION OF GRAPHICS

[0015] New features of the present invention are detailed in the appended claims. A better understanding of the features and advantages of the present invention will be assisted by reference to the following detailed description, set forth in the form of illustrative embodiments, in which the principles of the present invention are used, as well as accompanying drawings.

[0016] FIG. 1 is a diagram showing the positions of the various described elements present in the backbone of a lentiviral vector.

[0017] FIG. 2a is a schematic representation of self-inactivating lentiviral vectors used throughout gene therapy experiments that carry the human PGK gene promoter that regulates the expression of the EGFP transgene in the control vector (upper diagram) or the expression of the codon-optimized PKLR cDNA sequence (coRPK) in the therapeutic vector ( bottom chart).

[0018] FIG. 2b is a diagram of a gene therapy protocol performed to test the functionality of the designed lentiviral vector with PGK-coRPK.

[0019] FIG. 3a-d depict data showing PKD phenotype correction in peripheral blood in primary recipients after gene correction. In FIG. 3a shows the RBC level and FIG. 3b shows the level of reticulocytes in healthy (black bar, n=5) and PKD anemic mice (gray bar, n=6), as well as PKD anemic mice transplanted with cells transduced with EGFP (white bar, n =9) or coRPK (shaded bar, n=17). Data are presented as mean±SEM and analyzed using the non-parametric Kruskal-Wallis test. In FIG. 3c shows a flow cytometry technique used to detect biotin labeled RBCs over time, and FIG. 3d RBC survival kinetics in healthy mice (black line, n=2), anemic mice (grey line, n=2) and genetically corrected mice (dashed line, n=4). Data are presented as mean±SEM and analyzed by a two-way ANOVA test. Healthy, control mice, not transplanted; PKD, non-transplanted PKD mice; coRPK, PKD mice expressing the therapeutic transgene.

[0020] FIG. 4a-c show restoration of hematopoiesis of multiple lineages in secondary transplanted mice. In FIG. 4a is a diagram of a flow cytometry technique used to identify different hematopoietic lineages by labeling with PE-conjugated anti-CD3 antibody, PE-conjugated anti-B220 antibody, PECy5-conjugated anti-B220 antibody, biotin-conjugated anti-Gr1 antibody, and antibody to Mac1 conjugated with biotin, with the addition of SAV-PE-Cy5. In FIG. 4b shows typical scatter plots and FIG. 4c shows the percentages of each lineage in PB 140 days after transplantation. Bars represent the mean percentage ± SEM in healthy mice (n=2, black bar), PKD control mice (n=2, gray bar), and secondary transplanted mice expressing the therapeutic coRPK transgene (n=4, shaded bar). column).

[0021] In FIG. 5a-d depicts the correction of the PKD phenotype in secondary transplanted mice. In FIG. 5a shows brilliant cresyl blue staining of blood smears from non-transplanted mice and secondary recipients to identify the reticulocyte population (blue). In FIG. 5b shows flow cytometry analysis of reticulocyte levels in peripheral blood. In FIG. 5c shows the percentage of RBC and FIG. 5d shows the percentage of reticulocytes in secondary transplanted mice expressing the coRPK transgene (solid bar, n=4), healthy mice (black bar, n=3), and anemic control mice (gray bar, n=3). Data are presented as mean ± SEM and analyzed using a non-parametric two-tailed Mann-Whitney test.

[0022] FIG. 6a-c show the quantification of provirus integrations. In FIG. 6a shows the number of copies of the vector per cell in CFU from BM from individual transplanted mice 120-170 days after transplantation. The percentage of transduction and chimerism is also shown. In FIG. 6b shows the number of provirus copies in cells from various hematopoietic compartments. The columns represent the mean±SEM of different groups of transplanted mice. In FIG. 6c shows the kinetics of provirus integrations in BM cells in selected transplanted mice expressing EGFP (gray lines) and mice carrying the coRPK transgene (black lines).

[0023] FIG. 7a-c depict the normalization of the erythroid differentiation pattern in genetically corrected mice. In FIG. 7a shows the percentages of different erythroid subpopulations in the bone marrow and spleen 140 days after transplantation. In FIG. 7b depicts typical scatter plots obtained using flow cytometry techniques. The intensity of expression of the CD71 and Ter119 markers makes it possible to identify four erythroid subpopulations, population I:

early proerythroblasts (Ter119 ^med CD71 ^high ), population II: basophilic erythroblasts (Ter119 ^high CD71 ^high ), population III: late basophilic and polychromatophilic erythroblasts (Ter119 ^high CD71 ^med ) and population IV: orthochromatophilic erythroblasts, reticulocytes and mature erythroid cells (Ter119 ^high CD71 ^low ). In FIG. 7c shows plasma levels of Epo measured by ELISA in non-transplanted and transplanted mice. The dots represent the values of individual mice. Lines represent mean±SEM and were analyzed using the non-parametric Kruskal-Wallis test. Healthy, control mice, not transplanted; PKD, non-transplanted PKD mice; EGFP, PKD mice expressing the EGFP transgene; coRPK, PKD mice expressing the therapeutic transgene.

[0024] FIG. 8a-b show analyzes of hematopoietic progenitors in control mice and mice transplanted with transduced cells. The data show the total number of CFU from the spleen (Fig. 8a) and bone marrow (Fig. 8b) 140 days after transplantation. The dots represent the number of colonies per mouse analyzed, and the lines represent the mean ± SEM in each group. Statistical analysis of the data was performed using the nonparametric Kruskal-Wallis test.

[0025] FIG. 9a-c demonstrate reversal of splenomegaly and organ pathology in genetically corrected mice 140 days after transplantation. In FIG. 9a shows images of typical spleens, and FIG. 9b shows the ratio of spleen weight to total body weight in primary and secondary transplanted PKD mice. The dots represent the values of individual mice. The lines represent the group mean ± SEM. Data were analyzed using the non-parametric Kruskal-Wallis test. In FIG. 9c shows histological examination of the spleen and liver from primary transplanted PKD mice. The first and second columns show typical histological sections of the spleen and liver, stained with hematoxylin-eosin and photographed using a 4x and 10x objective lens, respectively, on a light microscope. Arrows indicate clusters of erythroid cells, which are a sign of extramedullary erythropoiesis. The third column shows Prussian blue (Fe) staining of liver sections to detect iron deposits, indicated by pointer arrows. Photographs were taken using a 20x objective. Group designations as in Fig. 7. 2nd ^coRPKs , secondary recipients.

[0026] FIG. 10a-g show metabolic profiling in RBC samples from mice transplanted with genetically modified cells. Analysis of significant changes in the metabolic profile in healthy and transplanted mice compared to animals with PKD in two independent experiments. In FIG. 10a shows the complete heat map of RBC obtained by non-targeted profiling, with higher and lower levels of metabolites shown in red and blue, respectively. The listed metabolites are characterized by at least one comparison that is significant when applying the following criteria: absolute fold change >1.5; minimum signal >2000; adjusted p-value <0.01. Black frames highlight a cluster of metabolite changes with a profile that differs in different groups. In FIG. 10b, 10c, and 10d depict, respectively, the levels of ATP, ADP, and pyruvate in RBC measured by non-targeted profiling in comparison with PKD mice 140 days after transplantation. Analysis 1: healthy mice (black bars), n=1, PKD (grey bars), n=1, hPGK-EGFP (white bars), n=2, hPGK-coRPK (shaded bars), n=3. Analysis 2: healthy mice, n=2, PKD, n=2, hPGK-EGFP, n=6, hPGK-coRPK, n=10. In FIG. 10e, 10f and 10g depict RBC-targeted metabolic profiling for a selected number of metabolites involved in the glycolysis pathway (PEP, 3-phosphoglyceric acid, and D-lactic acid, respectively) 280 days after transplantation. The dots represent the values of individual mice. Lines represent mean±SEM and were analyzed using the non-parametric Kruskal-Wallis test. Analysis 2: healthy mice, n=7, PKD, n=5, hPGK-EGFP, n=3, hPGK-coRPK, n=5.

[0027] FIG. 11a-c depict pyruvate kinase activity, FIG. 11b shows hexokinase activity and FIG. 11c - ratio of pyruvate kinase and hexokinase enzymatic activity in RBC from control mice and mice transplanted with transduced cells. RBC was isolated from blood samples by passing through a cellulose column to get rid of extraneous leukocyte PK activity and subjected to enzymatic activity evaluation. Black bars - healthy mice (n=2); white bars - mice transplanted with cells transduced with an EGFP-expressing vector (n=3); shaded bars - mice transplanted with cells transduced with coRPK-expressing vector (n=3). Bars filled with cells represent healthy volunteer values (n=1). Data are presented as mean ± SEM of each group.

[0028] FIG. 12a-d show off-target metabolic profiling in WBC samples from mice transplanted with genetically modified cells. In FIG. 12a shows a principal component analysis of the off-target metabolite profile in RBC (red dots; left and center) and WBC (blue dots; right cluster) in control and transplant mice. In FIG. 12b, 12c, and 12d depict the levels of ATP, ADP, and pyruvate, respectively, in WBC when compared to PKD mice. Analysis 1: healthy mice (black bars), n=1, PKD (grey bars), n=1, hPGK-EGFP (white bars), n=2, hPGK-coRPK (shaded bars), n=3. Analysis 2: healthy mice, n=2, PKD, n=2, hPGK-EGFP, n=6, hPGK-coRPK, n=10. Data are presented as mean ± SEM and analyzed using the non-parametric Kruskal-Wallis test.

[0029] FIG. 13 shows gels of Tsp509I LAM-PCR products from samples collected from all mice at different time points and tissues. Vector integration sites were identified by LAM-PCR amplification of the vector-genome 3'-LTR junctions. An automated MultiNA system was used to obtain a pattern characterized by several stripes. An internal control (IC) strip derived from the Tsp509I vector backbone is indicated by an arrow.

[0030] FIG. 14 shows gels of HpyCH4IV5 LAM-PCR products from samples collected from all mice at different time points and tissues. Vector integration sites were identified by LAM-PCR amplification of the vector-genome 3'-LTR junctions. An automated MultiNA system was used to obtain a pattern characterized by several stripes. An internal control (IC) strip derived from the HpyCH4IV5 vector backbone is indicated by an arrow.

[0031] In FIG. 15 is a schematic diagram of an integration site mapping assay performed on mice transplanted with genetically modified hematopoietic progenitor cells. Bone marrow and white blood cell samples from transplanted mice were analyzed, referring to two independent experiments (Table 3) and collected at different time points after transplantation, as described in the auxiliary methods following the shown workflow.

[0032] FIG. 16a-b show the distribution of LV integrations along the genome of transplanted mice. In FIG. 16a depicts the distribution density of integration sites (IS) near the transcription start site (TSS) of the nearest RefSeq gene covering 500 kb. upstream and downstream of the TSS. The numbers at the top represent the number of ISs found for all samples and time points. In FIG. 16b depicts the chromosome distribution of LV integration sites in transplanted mice expressing the EGFP transgene (black bars) or the therapeutic coRPK transgene (gray bars), showing no bias towards any particular chromosome.

[0033] FIG. 17a-c show an analysis of the abundance of clones in cells transduced with coRPK-LV. The scatter plot represents the abundance of pooled integration clones for each mouse from analyzes 1 (FIG. 17a) and 2 (FIGS. 17b and 17c). The relative percentage (y-axis) of each integration site is calculated relative to the total number of sequence reads obtained in each dataset. IS - integration site; BM - bone marrow; PB - peripheral blood; coRPK1-14 mice transplanted with hematopoietic cells transduced with a therapeutic vector.

[0034] FIG. 18 shows traced integrations that were common between primary and secondary recipient mice carrying the therapeutic LV vector with PGK-coRPK. The integrations identified in each mouse in any organ and at any point in time are combined. Secondary recipients received pooled BM from mice transplanted with coRPK11-14. The rest of the identified IS were detected either in primary or secondary recipients. The numbers in the cells show the representation of the corresponding integration as a percentage in the mentioned mouse. In addition to the >5% filter applied in the integration assay, all integrations with <3 sequences were excluded.

[0035] FIG. 19 shows an abundance analysis of EGFP-LV transduced cell clones. The dot plot shows the abundance of pooled integration clones in each mouse in the bone marrow. The relative percentage (y-axis) of each integration site is calculated relative to the total number of sequence reads obtained in each dataset. Similar to cells transduced with co-RPK (FIG. 17), the diagram shows that the vast majority of transplanted mice show a polyclonal hematopoietic cell repopulation pattern. IS - integration site.

[0036] FIG. 20 depicts the genomic integration profile of LV. Gene ontology analysis (GO) was performed using GREAT software on transplanted mouse samples. All data on integrations obtained in this study (N=2220) showed the prevalence of gene functions indicated on the left side of the figure. To examine whether the most common integrations were enriched in terms of certain gene classes, all integration sites with relative sequence numbers >5% of the total dataset were selected (shown in Fig. 17), showing no predominance of GO gene classes.

[0037] FIG. 21 is a schematic diagram of a drug product (LV with PGK-coRPK).

[0038] FIG. 22a-b depict the mechanism of action of the drug product. In FIG. 22a shows that ectopic expression of an LV-based drug product with PGK-coRPK will restore the wild-type phenotype in PKD erythrocytes that are otherwise unable to produce functional RPK protein to produce enough energy to carry out their functions. In FIG. 22b shows a gene therapy strategy for PKD patients based on ex vivo transduction of RPK-deficient CD34 ⁺ hematopoietic progenitor cells with a drug product and subsequent transplantation into the patient. The developed drug product, carrying the therapeutic cDNA of the human PKLR gene, will be integrated into the genome of the patient's CD34 ⁺ cells in the field of ex vivo transduction. Such genetically corrected cells will then be infused back into the patient where they will produce RBCs expressing the therapeutic transgene and hence will produce functional RPK proteins that will correct the pathological PKD phenotype. Figure from the Boston Children's Clinic blog with modifications.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0039] As used herein, a "vector" refers to a macromolecule or association of macromolecules that contains or is associated with a polynucleotide and that can be used to mediate delivery of the polynucleotide into a cell. Illustrative vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles.

[0040] The term "LV" is an abbreviation for lentivirus and may be used to refer to the virus itself or derivatives thereof. The term includes all subtypes, as well as both naturally occurring and recombinant forms, unless otherwise indicated.

[0041] As used herein, the term "gene" or "coding sequence" refers to an in vitro or in vivo nucleotide sequence that encodes a gene product. In some cases, the gene consists or actually consists of a coding sequence, that is, a sequence that encodes a gene product. In other cases, the gene contains an additional non-coding sequence. For example, a gene may or may not include regions before and after the coding region, such as 5'-untranslated (5'-UTR) or "leader" sequences and 3'-UTR or "trailer" sequences, as well as intermediate sequences. (introns) between individual coding segments (exons).

[0042] As used herein, a "therapeutic gene" refers to a gene that, when expressed, provides a beneficial effect in the cell or tissue in which it resides, or in the mammal in which the gene is expressed. Examples of beneficial effects include alleviating a sign or symptom of a condition or disease, preventing or suppressing a condition or disease, or providing a desirable characteristic. Therapeutic genes include genes that correct for a genetic deficiency in a cell or in a mammal.

[0043] As used herein, a "transgene" is a gene that is delivered into a cell by a vector.

[0044] As used herein, the term "gene product" refers to the desired expression product of a polynucleotide sequence, such as a polypeptide, peptide, protein, or interfering RNA, including short interfering RNA (siRNA), miRNA, or small hairpin RNA (shRNA).

[0045] As used herein, the terms "polypeptide", "peptide", and "protein" refer to polymers of amino acids of any length. The terms also cover an amino acid polymer that has been modified; for example, by disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0046] By "comprising" it is meant that said elements are necessary, for example, in a composition, method, kit, etc., but other elements may be included within the scope of the claimed claim to form, for example, a composition, method, kit, and etc. For example, an expression cassette "containing" a gene encoding a therapeutic polypeptide operably linked to a promoter is an expression cassette that may include other elements in addition to the gene and promoter, such as a polyadenylation sequence, enhancer elements, other genes , linker domains, etc.

[0047] By "consisting essentially of" is meant limiting the scope of, e.g., a described composition, method, kit, etc., to specified materials or steps that do not materially affect the essential and novel feature(s), e.g., composition, method , set, etc. For example, an expression cassette "consisting essentially of" a gene encoding a therapeutic polypeptide operably linked to a promoter and a polyadenylation sequence may include additional sequences, such as linker sequences, provided that they do not significantly affect transcription or translation of a gene. As another example, a variant or mutant polypeptide fragment "consisting essentially of" said sequence has the amino acid sequence of said sequence plus or minus approximately 10 amino acid residues at the sequence boundaries, based on the full-length unexposed polypeptide from which it was derived, e.g. , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue less than said boundary amino acid residue, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues more than the boundary amino acid residue mentioned.

[0048] By "consisting of" is meant the exclusion from a composition, method, or kit of any element, step, or ingredient not listed in a claim. For example, an expression cassette "consisting of" a gene encoding a therapeutic polypeptide operably linked to a promoter and a post-transcriptional regulatory element consists only of a promoter, a polynucleotide sequence encoding a therapeutic polypeptide, and a post-transcriptional regulatory element. As another example, a polypeptide "consisting of" said sequence contains only said sequence.

[0049] As used herein, "expression vector" encompasses a vector, such as a plasmid, minicircle, viral vector, liposome, and the like, discussed above or known in the art, containing a polynucleotide that encodes a gene product of interest, and it used to effect expression of the gene product in the intended target cell. The expression vector also contains control elements operably linked to the coding region to facilitate expression of the gene product in the target. The combination of control elements, eg, promoters, enhancers, UTRs, miRNA targeting sequences, etc., and the gene or genes to which they are operably linked for expression, is sometimes referred to as an "expression cassette". Many such control elements are known and available in the art, or can be easily constructed from components that are available in the art.

[0050] As used herein, a "promoter" encompasses a DNA sequence that directs the binding of an RNA polymerase and thereby enables RNA synthesis, i.e., the minimum sequence sufficient to direct transcription. Promoters and the corresponding expression of a protein or polypeptide can be ubiquitous, meaning that they exhibit strong activity in a wide range of cells, tissues and species, or cell type specific, tissue specific or species specific. Promoters can be "constitutive", meaning permanently active, or "inducible", meaning that the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors. Also included in the nucleic acid construct or vectors of the present invention are enhancer sequences, which may or may not be adjacent to a promoter sequence. Enhancer sequences affect promoter-dependent gene expression and can be located in the 5' or 3' regions of the native gene.

[0051] As used herein, "enhancer" encompasses a cis-acting element that stimulates or represses the transcription of adjacent genes. An enhancer that suppresses transcription is also called a "silencer". Enhancers can function (i.e., can be associated with a coding sequence) in any orientation, up to several thousand base pairs (kb) away from the coding sequence, and from a position upstream of the transcribed region.

[0052] As used herein, a "termination signal sequence" encompasses any genetic element that causes RNA polymerase to terminate transcription, such as, for example, a polyadenylation signal sequence.

[0053] As used herein, the terms "operably linked" or "operably linked" refer to the contiguous position of genetic elements, e.g., promoter, enhancer, termination signal sequence, polyadenylation sequence, etc., where the elements are in a relationship that allows them function as expected. For example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intermediate residues between the promoter and the coding region, provided that a functional relationship is maintained.

[0054] As used herein, the term "heterologous" means derived from an entity that is genotypically different from the rest of the entity to which it is being compared. For example, a polynucleotide introduced using genetic engineering techniques into a plasmid or vector derived from another species is a heterologous polynucleotide. As another example, a promoter removed from its native coding sequence and operably linked to a coding sequence to which it is not naturally linked is a heterologous promoter. Thus, for example, an LV vector that includes a heterologous nucleic acid encoding a heterologous gene product is an LV vector that includes a nucleic acid that is not normally included in naturally occurring wild-type LV, and the encoded heterologous gene product is is a gene product not normally encoded by naturally occurring wild-type LV.

[0055] The term "endogenous" as used herein in relation to a nucleotide molecule or gene product refers to a nucleic acid sequence, e.g., a gene or genetic element, or a gene product, e.g., RNA, a protein, that occurs naturally in a virus. host or cell or associated with them.

[0056] As used herein, the term "native" refers to a nucleotide sequence, such as a gene, or a gene product, such as RNA, protein, that is present in a wild-type virus or cell.

[0057] As used herein, the term "variant" refers to a mutant of a reference polynucleotide or polypeptide sequence, e.g., a native polynucleotide or polypeptide sequence, i.e., it has less than 100% sequence identity with the reference polynucleotide or polypeptide sequence. In other words, the variant contains at least one amino acid difference (eg, amino acid substitution, amino acid insertion, amino acid deletion) relative to a reference polynucleotide sequence, eg, a native polynucleotide or 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% or 80% or more identity, 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 a full length native polypeptide sequence, e.g., 75% or 80% or more identity, e.g. 85%, 90% or 95% or more than, for example, 98% or 99% identity with the full-length native polypeptide sequence. Variants may also include variant fragments of a reference, e.g. native, sequence that share a sequence identity of 70% or more with a fragment of the reference, e.g., native sequence, e.g., 75% or 80% or more identity, e.g., 85%, 90% or 95% or more, such as 98% or 99% identity with the native sequence.

[0058] As used herein, the terms "biological activity" and "biologically active" refer to the activity inherent in a particular biological element in a cell. For example, the "biological activity" of an immunoglobulin, antibody, or fragment or variant thereof refers to the ability to bind an antigenic determinant and thereby facilitate immunological function. As another example, the biological activity of a polypeptide or functional fragment or variant thereof refers to the ability of the polypeptide or functional fragment or variant thereof to perform its native functions, e.g., binding, enzymatic activity, etc. As a third example, the biological activity of a gene regulatory element , e.g., promoter, enhancer, kozak sequence, etc., refers to the ability of a regulatory element or functional fragment or variant thereof to regulate, i.e., provide, enhance or activate, translation, and accordingly, the expression of the gene to which it is operatively linked .

[0059] As used herein, the terms "use" or "administration" refer to the delivery of a vector for expression of a recombinant protein into a cell, cells and/or organs of a subject or subject. Such use or administration may take place in vivo, in vitro or ex vivo. The gene product expression vector can be introduced into a cell by transfection, which generally means introducing the heterologous DNA into the cell by physical means (eg, calcium phosphate transfection, electroporation, microinjection, or lipofection); infection, which generally refers to administration by an infectious agent, i.e., a virus; or transduction, which generally means the stable infection of a cell with a virus or the transfer of genetic material from one microorganism to another by a viral agent (eg, a bacteriophage).

[0060] "Transformation" is generally used in relation to bacteria containing heterologous DNA, or cells that express an oncogene and are therefore put into a continuous growth mode, such as tumor cells. The vector used to "transform" the cell may be a plasmid, virus, or other carrier.

[0061] Typically, a cell is referred to as "transduced," "infected," "transfected," or "transformed," depending on the means used to apply, introduce, or introduce the heterologous DNA (ie, vector) into the cell. The terms "transduced", "transfected" and "transformed" can be used interchangeably herein, regardless of the method of introduction of heterologous DNA.

[0062] As used herein, the term "host cell" refers to a cell that has been transduced, infected, transfected, or transformed with a vector. The vector may be a plasmid, a virus particle, a phage, etc. Culture conditions such as temperature, pH, etc. are those previously applied to the host cell chosen for expression and will be apparent to those skilled in the art. areas. It will be appreciated that the term "host cell" refers to the original transduced, infected, transfected or transformed cell and its progeny.

[0063] The terms "treatment", "treatment", etc. are used in this document mainly to refer to obtaining the desired pharmacological and/or physiological effect. The effect may be prophylactic in the sense of completely or partially preventing the disease or symptom thereof, e.g., reducing the likelihood that the disease or symptom thereof will occur in the subject, and/or may be therapeutic in the sense of partially or completely curing the disease and/or adverse effects inherent in this disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal and includes: (a) preventing the onset of the disease in a subject who may be predisposed to the disease but not yet diagnosed as having the disease; (b) disease suppression, i.e. cessation of its development; or (c) amelioration of the disease, i.e. providing regression of the disease. The therapeutic agent can be used before, during or after the onset of a disease or injury. Of particular interest is the treatment of an ongoing disease, wherein the treatment stabilizes or reduces unwanted clinical symptoms in the patient. Such treatment is preferably performed before the complete loss of function in the affected tissues. Said therapy is desirably applied during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

[0064] The terms "individual", "host", "subject", and "patient" are used interchangeably herein and refer to a mammal, including, without limitation, humans and non-human primates, including monkeys and humans; mammals used in sports (for example, horses); agricultural mammals (eg sheep, goats, etc.); domestic mammals (dogs, cats, etc.) and rodents (eg mice, rats, etc.).

[0065] The terminology used herein is intended to describe specific embodiments and is not intended to limit the present invention. As used herein, the singular forms are also intended to include plural forms, unless the context clearly indicates otherwise. In addition, the terms "comprising", "includes", "having", "has", "with", or variations thereof, to the extent they are used or in the detailed description and/or claims, are intended to include analogy with the term "containing".

[0066] The term "approximately" or "approximately" means within an acceptable range of error for a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, according to the established practice in this field. Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of this value. Alternatively, in particular in relation to biological systems or processes, the term may mean within an order of magnitude, preferably within 5 times and more preferably within 2 times the value. Where specific values are described in the present application and the claims, unless otherwise indicated, the term "about" should be taken to mean within the margin of error for a particular value.

[0067] Unless otherwise indicated, all terms used herein have the same meaning as those of skill in the art, and the practice of the present invention will utilize conventional microbiological techniques and recombinant DNA technologies that are known experts in this field.

[0068] Unless otherwise indicated, conventional cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology techniques known to those skilled in the art are used to practice the present invention. Such techniques are fully described in the literature, for example, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D. M. Weir & C. C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J. M. Miller & M. P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J. E. Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0069] In some embodiments, the present invention 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, for example, in an individual, for example, to treat or prevent a disorder. These and other objects, advantages, and features of the present invention will become apparent to those skilled in the art upon reading the detailed description of the compositions and methods, which are described more fully below.

[0070] The present invention generally relates to the fields of molecular biology and virology and, in particular, to genetic expression cassettes and vectors containing them, applicable for the delivery of nucleic acid segments encoding selected therapeutic constructs (including, for example, peptides, polypeptides, ribozymes and catalytic RNA molecules), into selected cells and tissues of vertebrates. In particular, such genetic constructs are useful in the development of gene therapy vectors, including, for example, lentiviral vectors, for the treatment of diseases, disorders, and dysfunctions in mammals, and in particular humans.

[0071] The disclosed compositions may be used in a variety of research, diagnostic, and therapeutic regimens, including the prevention and treatment of a variety of human diseases. Various compositions and methods of the present invention are described below.

[0072] While specific compositions and methods are exemplified herein, it should be appreciated that any of a variety of alternative compositions and methods are applicable and suitable for use in the practice of the present invention. It should also be appreciated that the evaluation of the expression constructs and methods of the present invention can be performed using standard procedures in the prior art.

[0073] In certain embodiments, methods and compositions are provided for preparing compositions based on gene therapy vectors, e.g., viral vectors containing such genetic expression cassettes, for use in the preparation of drugs useful in basic and targeted gene therapy for diseases, disorders, and dysfunctions in animals and, in particular, in humans.

[0074] In some embodiments, the present invention provides a PKD gene therapy based on a lentiviral vector carrying the eukaryotic hPGK promoter that drives expression of the PKLR cDNA. This therapeutic vector can be used for transduction of PKD mouse hematopoietic stem cells (HSC) and subsequent transplantation into myeloablated PKD mice. Ectopic expression of RPK normalizes the erythroid compartment, correcting the hematological phenotype and reversing the pathology of the organ. Metabolic studies demonstrate a functional correction of the glycolysis pathway in RBCs derived from genetically corrected HSCs with PKD in the absence of metabolic disorders in leukocytes. Analysis of lentivirus insertion sites in the genome of transplanted hematopoietic cells demonstrates no evidence of genotoxicity in any of the transplanted animals. Overall, the results highlight the therapeutic potential of the hPGK-coRPK lentiviral vector and hold great promise for gene therapy for PKD and other metabolic genetic disorders in erythroid cells.

[0075] In certain embodiments, the present invention provides a lentiviral vector (LV) with RPK for genetic correction of PKD. Genetic modification of mouse PKD-HSC with this vector can effectively correct the hemolytic phenotype and RBC metabolite profile in transplanted PKD mice. Of note, no evidence of leukocyte metabolic disturbances and genotoxicity resulting from vector integration was observed, confirming the therapeutic potential of the LV vector with PGK-coRPK. Overall, the results provide encouraging evidence for the suitability of PKD gene therapy with an LV designed for clinical use.

[0076] In some embodiments, the present invention provides a self-inactivating lentiviral vector expressing a codon-optimized variant of the human PKLR gene. The expression vector contains a promoter region, a coding sequence, and a post-transcriptional regulatory element.

[0077] In some embodiments, the implementation of the polynucleotide cassettes of the present invention contain a promoter region containing a promoter sequence or a functional fragment. In one embodiment, the promoter is the human phosphoglycerate kinase (PGK) gene promoter.

[0078] In some embodiments, implementation of the present invention provides polynucleotide cassettes for enhanced expression of pyruvate kinase. In some embodiments, the polynucleotide cassette contains a codon-optimized human PKLR (coRPK) cDNA variant to enhance transcriptional stability of the mRNA. For optimization, GeneArt® software can be used to increase GC content and remove cryptic splicing sites to eliminate transcriptional silencing, thereby increasing transgene expression. The optimized coRPK sequence showed 80.4% homology with the human PKLR gene, with no changes in the amino acid sequence of the protein. Alternatively, any optimization method known in the art can be used.

[0079] In some embodiments, the polynucleotide cassette contains an RNA export signal downstream from a second enhancer. The RNA export signal may contain the woodchuck hepatitis virus post-transcriptional element (WPRE) sequence. In some embodiments, a mutant woodchuck hepatitis virus (Wpre) post-transcriptional regulatory element, without any residual open reading frame, is also included (Schambach, Bohne et al. 2006) to improve the level of expression and stability of the therapeutic gene.

[0080] In some aspects of the present invention, gene delivery vectors are provided that comprise the polynucleotide cassette of the present invention. In some embodiments, the gene delivery vector is a lentivirus.

[0081] In some aspects of the present invention, pharmaceutical compositions are provided comprising a polynucleotide cassette of the present invention and a pharmaceutical excipient. In some embodiments, the pharmaceutical composition comprises a gene delivery vector of the present invention and a pharmaceutical excipient.

[0082] In some aspects of the present invention, methods for expressing a transgene in mammalian cells are provided. In some embodiments, the method comprises contacting one or more mammalian cells with an effective amount of a polynucleotide cassette of the present invention or a gene delivery vector of the present invention, wherein the transgene is expressed at detectable levels in one or more mammalian cells. In some embodiments, the method comprises contacting one or more mammalian cells with an effective amount of a polynucleotide cassette of the present invention or a gene delivery vector of the present invention, wherein the transgene is expressed at therapeutic levels in one or more mammalian cells. In some embodiments, the method is carried out in vitro. In other embodiments, the method is carried out in vivo.

[0083] In some aspects, the present invention provides methods for treating or preventing a disease or disorder in a mammal in need of treating or preventing a disease or disorder. In some embodiments, the method comprises administering to a mammal an effective amount of a pharmaceutical composition of the present invention, wherein the coding sequence encodes a therapeutic gene product.

Compositions

[0084] In some aspects of the present invention, compositions are provided for expressing a transgene in eukaryotic cell(s). In some aspects, the eukaryotic cell is a mammalian cell. In some aspects, the mammalian cell is a hematopoietic stem cell. In some embodiments, the cell is a bone marrow cell, eg, a lineage-depleted bone marrow cell. In some aspects, the mammalian cell is a committed hematopoietic erythroid progenitor cell.

[0085] In some embodiments, the implementation of the present invention, the composition is a polynucleotide cassette. By "polynucleotide cassette" is meant a polynucleotide sequence containing two or more functional polynucleotide sequences, e.g., regulatory elements, translation initiation sequences, coding sequences and/or termination sequences, etc., typically in operative relationship with each other. Similarly, by "polynucleotide cassette for expression of a transgene in a mammalian cell" is meant a combination of two or more functional polynucleotide sequences, e.g., a promoter, an enhancer, a 5'-UTR, a translation initiation sequence, a coding sequence and/or termination sequences, etc. , which ensures the expression of the transgene in the cell.

[0086] For example, in some embodiments, the polynucleotide cassette comprises a human phosphoglycerate kinase (PGK) gene promoter, a codon-optimized human PKLR (coRPK) cDNA variant, and a woodchuck hepatitis virus (Wpre) mutant post-transcriptional regulatory element.

[0087] In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the human PKLR gene promoter comprises or consists of the following sequence, a functional fragment thereof, or a sequence characterized by at least 80%, at least 85% , at least 90%, at least 95%, at least 98%, or at least 99% identity with the following sequence:

ATTATGGTAAATCCACTTACTGTCTGCCCTCGTAGCCATCGAGATAAACCCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCCC (SEQ ID NО: 4).

[0088] In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the human PKLR gene promoter comprises or consists of the following sequence, a functional fragment thereof, or a sequence characterized by at least 80%, at least 85% , at least 90%, at least 95%, at least 98%, or at least 99% identity with the following sequence:

TCCACGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTCGTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGGCGCCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAG (SEQ ID NО: 8).

[0089] In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the RPE sequence contains or consists of the following sequence, or a sequence characterized by at least 80%, at least 85%, at least 90% , at least 95%, at least 98%, or at least 99% identity with the following sequence:

TCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGTGGAAAGATACCT (SEQ ID NO: 3).

[0090] In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the psi sequence is an HIV-1 psi sequence, or the psi sequence contains or consists of the following sequence or sequence characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the following sequence:

TCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAG (SEQ ID NO: 5).

[0091] In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the 5'-LTR contains or consists of the following sequence, or a sequence characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the following sequence:

(SEQ ID 6)

[0092] In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the 3'-LTR contains or consists of the following sequence, or a sequence characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the following sequence:

SEQ ID: 7 ID

[0093] In some embodiments, the implementation of the polynucleotide cassettes of the present invention provide enhanced expression of the transgene in mammalian cells. As demonstrated in the working examples of the present invention, the inventors of the present invention have found a number of polynucleotide elements, i.e., improved elements compared to elements known in the art, which individually and synergistically provide enhanced expression of transgenes in mammalian cells. In certain embodiments, the location of two or more functional polynucleotide sequences within the polynucleotide cassettes of the present invention provides enhanced expression of the transgene in mammalian cells. By "enhanced" is meant that the expression of the transgene is increased, increased, or become more potent in cells carrying the polynucleotide cassettes of the present invention compared to cells carrying a transgene operably linked to comparable regulatory elements, such as those known in the art. In other words, the expression of the transgene is upregulated, increased, or potentiated by the polynucleotide cassettes of the present invention compared to expression by a polynucleotide cassette lacking one or more of the optimized elements of the present invention, i.e., a reference control. In a certain embodiment, the enhanced expression is specific to or limited to one or more desired cell types.

[0094] For example, expression of a transgene may increase, or increase, or become more potent in cells containing a polynucleotide cassette containing the promoter disclosed herein compared to expression in cells that carry a transgene operably linked to a different promoter, e.g. known from the prior art. As another example, the expression of a transgene can be increased or increased, increased or become stronger in cells containing a polynucleotide cassette containing an enhancer sequence disclosed herein, compared to expression in cells that carry a transgene operably linked to a different enhancer sequence.

[0095] Promoter and enhancer elements can be tissue specific or stage specific. For example, a tissue-specific promoter or enhancer preferentially directs expression (or higher levels of expression) in one or more specific cell types. Examples of cell types include, without limitation, hematopoietic stem cells, long-lived hematopoietic stem cells, short-lived hematopoietic stem cells, multipotent progenitors, hematopoietic CD34+ cells, and cells with any subset of the differentiation cluster within the CD34+ population. A stage-specific promoter or enhancer advantageously directs expression (or higher levels of expression) during one or more specific stages of the cell cycle or development. These include, but are not limited to, the beta-globin gene locus control region, the spectrin gene promoter, and the erythroid-specific promoter.

[0096] While not wishing to be bound by any particular theory, it is believed that increased expression of the transgene in cells is due to more rapid buildup of the gene product in cells or a more stable gene product in cells. As a consequence, enhanced transgene expression by the polynucleotide cassettes of the disclosed invention can be observed using a variety of methods. For example, enhanced expression can be observed by detecting transgene expression soon after contact of the polynucleotide cassette with cells earlier, for example, 2 days earlier, 7 days earlier, 2 weeks earlier, 3 weeks earlier, 4 weeks earlier, 8 weeks earlier , 12 weeks earlier or more than the expression that would be detected if the transgene were operably linked to comparable regulatory elements, such as those known in the art. Increased expression can also be observed as an increase in the amount of gene product per cell. For example, there may be a 2-fold increase or more, such as a 3-fold increase or more, a 4-fold increase or more, a 5-fold increase or more, or a 10-fold increase or more in the amount of gene product per mammalian cell. Increased expression can also be observed as an increase in the number of mammalian cells that express the transgene carried by the polynucleotide cassette at detectable levels. For example, there may be a 2-fold increase or more, such as a 3-fold increase or more, a 4-fold increase or more, a 5-fold increase or more, or a 10-fold increase or more in the number of mammalian cells that express the transgene at detectable levels. . As another example, a polynucleotide of the present invention can provide detectable levels of a transgene in a higher percentage of cells compared to a conventional polynucleotide cassette; for example, if a traditional cassette can provide detectable levels of expression of a transgene, for example, in less than 5% of cells in a certain area, then the polynucleotide of the present invention provides detectable levels of expression in 5% or more of the cells in that area; e.g. 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, in some cases 50% or more , 55% or more; 60% or more, 65% or more, 70% or more, or 75% or more, such as 80% or more, 85% or more, 90% or more, or 95% or more of the cells that were brought into contact will express the gene product at detectable levels. Increased expression can also be observed as a change in cell viability and/or function.

[0097] The polynucleotide cassettes of the present invention typically contain a promoter region. Any suitable promoter region or promoter sequence therein can be used in the inventive polynucleotide cassettes, provided that the promoter region allows expression of the coding sequence in eukaryotic cells. In certain embodiments, the promoter region allows expression of a coding sequence in mammalian cells. In some cases, the promoter is a ubiquitous promoter, ie a promoter that is active in a wide range of cells, tissues and species. In other instances, the promoter is the human phosphoglycerate kinase (PGK) gene promoter.

[0098] In some embodiments, the implementation of the polynucleotide contains one or more enhancers. Enhancers are nucleic acid elements known in the art for enhancing transcription, and they can be located anywhere in association with the gene they regulate, for example, upstream, downstream, in an intron, etc. e. Any enhancer element can be used in the polynucleotide cassettes and gene therapy vectors of the present invention, provided that it enhances gene expression when used in combination with a promoter.

[0099] The coding sequence to be expressed in cells can be any polynucleotide sequence, e.g., a gene or cDNA, that encodes a gene product, e.g., an RNA-based polypeptide or therapeutic agent (siRNA, antisense sequence, ribozyme, shRNA, etc. .). A coding sequence may be heterologous with respect to the promoter sequence to which it is operably linked, i.e., not operably associated with it in nature. Alternatively, the coding sequence may be endogenous to the promoter sequence with which it is operably linked, i.e., associated with the promoter in nature. The gene product may act inside the mammalian cell, or it may act outside, for example, it may be secreted. For example, if the transgene is a therapeutic gene, then the coding sequence can be any gene that encodes the desired gene product, or a functional fragment or variant thereof, that can be used as a therapeutic agent for the treatment of a disease or disorder. In various preferred embodiments, the transgene encodes for human PKLR.

[00100] In one embodiment of the present invention, the coding sequence of the transgene is modified or "codon-optimized" to enhance expression by replacing infrequently present codons with more frequently present codons. A coding sequence is a portion of an mRNA sequence that codes for amino acids for translation. During translation, each of the 61 trinucleotide codons is translated into one of 20 amino acids, resulting in degeneracy or redundancy in the genetic code. However, different cell types and different animal species use tRNAs (each with an anticodon) that code for the same amino acids at different frequencies. If the gene sequence contains codons that are rarely present in the corresponding tRNA, then the work of the ribosomal translation apparatus may slow down, which prevents efficient translation. Expression can be improved by species-specific "codon optimization" in which the coding sequence is changed to code for the same protein sequence but with codons that are highly represented and/or used in highly expressed human proteins (Cid- Arregui et al., 2003; J. Virol. 77: 4928). In one aspect of the present invention, the coding sequence of a transgene is modified to replace codons that are rarely expressed in mammals or primates with codons that are often expressed in primates. For example, in some embodiments, the coding sequence encoded by the transgene encodes a polypeptide having at least 85% sequence identity with a polypeptide encoded by a sequence disclosed above or herein, such as at least 90% sequence identity, such as at least at least 95% sequence identity, at least 98% identity, at least 99% identity, wherein at least one codon of the coding sequence has a higher frequency of tRNA in humans than the corresponding codon in the sequence disclosed above or herein.

[00101] In a further embodiment of the present invention, the coding sequence of the transgene is modified to enhance expression by terminating or removing open reading frames (ORFs) that do not code for the required 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 in forward or reverse orientation and may be "in-frame" or "out-of-frame" relative to the gene of interest. Such open reading frames could potentially be expressed in the expression cassette at the same time as the gene of interest, and this could lead to undesirable side effects. In one aspect of the present invention, the coding sequence of the transgene has been modified to remove open reading frames by further changing the frequency of codon usage. This was done by deleting start codons (ATG) and introducing stop codons (TAG, TAA, or TGA) into the reverse orientation or out of frame ORF while maintaining the amino acid sequence and maintaining highly used codons in the gene of interest (i.e. avoiding codons with a frequency of <20%. In the present invention, the coding sequence of a transgene can be optimized either by codon optimization or by removing ORFs not belonging to the transgene, or by using both techniques. As will be understood by one of ordinary skill in the art, it is preferable to remove or minimize non-transgene ORFs after codon optimization in order to remove ORFs introduced during codon optimization.

[00102] In some embodiments, the polynucleotide cassette of the present invention further comprises an RNA export signal. Exemplary RNA export sequences include, but are not limited to, sequences from Woodchuck Hepatitis Virus Post-Transcriptional Element (WPRE). Woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (Wpre) significantly upregulates transgene expression in target cells by increasing transgene RNA stability in a promoter- and vector-independent manner (Zuffrey et al, 1999). However, it can result in the expression of a 60 amino acid truncated protein derived from the WHV X gene involved in liver cancer (Kingsman et al, 2005). Therefore, most preclinical protocols and clinical trials include a mutant version of the Wpre element (Zanta-Boussif et al, 2009). On the other hand, the use of two SV40-USE elements in SIN-LV vectors was found to be more effective in suppressing transcriptional readout than the WPRE sequence (Schambach et al, 2007). More precisely, the WPRE disclosed herein is a chimeric WPRE that carries 589 nucleotides from the modified WPRE prepared by Axel Schambach (nucleotides 1-589) (WO 2008136670 A2; [5]) and 88 from the former WPRE ( nucleotides 590-677) (Zuffrey et al, 1999). The data disclosed in this document shows that this chimeric wpre performs better than the old WPRE. The sequence of the chimeric WPRE provides for the sequence shown in the table below.

[00103] Table 1. Modified WPRE sequence

[00104] The present invention also includes a nucleic acid, for example, a polynucleotide sequence, containing a sequence characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least at least 99% identity with the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 9. In specific embodiments, the polynucleotide sequence contains the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 9.

[00105] In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the Wpre sequence contains or consists of a sequence under SEQ ID NO: 1 or a sequence characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the sequence under SEQ ID NO: 1.

[00106] In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the Wpre sequence contains or consists of the following sequence, or a sequence characterized by at least 80%, at least 85%, at least 90% , at least 95%, at least 98%, or at least 99% identity with the following sequence:

CGAGCATCTTACCGCCATTTATTCCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTCGGGGAAGGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG (SEQ ID NО: 9).

[00107] In certain embodiments, an expression cassette or gene delivery vector, such as a lentivirus, contains a polynucleotide sequence containing the following sequences in the 5'-3' direction:

a) a PGK promoter sequence, optionally a human PGK promoter sequence;

b) a sequence encoding a pyruvate kinase polypeptide, optionally a codon-optimized coding sequence or an RPK cDNA sequence; and

c) a mutant Wpre sequence optionally containing or consisting of the sequence under SEQ ID NO: 1.

[00108] In certain embodiments, an expression cassette or gene delivery vector, such as a lentivirus, contains a polynucleotide sequence containing the following sequences in the 5'-3' direction:

a) cPPT sequence;

b) a PGK promoter sequence, optionally a human PGK promoter sequence;

c) a sequence encoding a pyruvate kinase polypeptide, optionally a codon-optimized coding sequence or an RPK cDNA sequence; and

d) a mutant Wpre sequence optionally containing or consisting of the sequence under SEQ ID NO: 1.

[00109] In certain embodiments, an expression cassette or gene delivery vector, such as a lentivirus, contains a polynucleotide sequence containing the following sequences in the 5'-3' direction:

a) 5'-LTR, optionally modified with 5'-LTR;

b) cPPT sequence;

c) a PGK promoter sequence, optionally a human PGK promoter sequence;

d) a sequence encoding a pyruvate kinase polypeptide, optionally a codon-optimized RPK coding sequence or a cDNA sequence;

e) mutant sequence Wpre, optionally containing or consisting of the sequence under SEQ ID NO: 1; and

f) 3'-LTR, optionally modified with 3'-LTR.

[00110] In certain embodiments, the gene delivery vector is a LV with PGK-coRPK or contains the elements depicted in FIG. 21.

[00111] In specific embodiments of any of the expression cassettes and gene delivery vectors described herein, the codon-optimized cDNA or RPK coding sequence encodes a PKLR polypeptide that contains or consists of a sequence disclosed under any of the GenBank accession numbers XP 016856982 1, XP 011507942.1, XP 006711449.1, NP 870986 1 or NP 000289.1, or a functional fragment of any of these sequences, or a sequence characterized by 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.

[00112] Other combinations of elements, both disclosed herein and known in the art, will be apparent to those of ordinary skill in the art.

[00113] In addition, one of ordinary skill in the art will appreciate that the polynucleotide cassettes may optionally contain other elements, including, without limitation, restriction sites to facilitate cloning and regulatory elements for a particular gene expression vector.

[00114] In some aspects of the present invention, the claimed polynucleotide cassettes are used to deliver a gene to animal cells, for example, to determine the effect that this gene has on the viability and / or functioning of the cell, to treat a cellular disorder, etc. Therefore, in some aspects of the present invention, a composition that provides expression of a transgene in mammalian cells is a gene delivery vector, wherein the gene delivery vector contains the polynucleotide cassettes of the present invention.

[00115] Any suitable gene therapy vector that finds use in delivering polynucleotide sequences to mammalian cells is included in the gene delivery vectors of the present invention. For example, the vector may contain single or double stranded nucleic acid, such as single or double stranded DNA. For example, the gene delivery vector may be DNA, eg, deproteinized DNA, eg, plasmid, minicircle, etc. The vector may contain single or double stranded RNA, including modified forms of RNA. In another example, the gene delivery vector may be RNA, such as mRNA or modified mRNA.

[00116] As another example, the gene delivery vector can be a viral vector derived from a virus, e.g., adenovirus, adeno-associated virus, lentivirus (LV), herpesvirus, alphavirus, or retrovirus, e.g., Moloney murine leukemia virus (M-MuLV), Moloney Mouse Sarcoma Virus (MoMSV), Harvey Mouse Sarcoma Virus (HaMuSV), Mouse Mammary Tumor 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 covering the use of lentivirus are described in more detail below, it is expected that one of ordinary skill in the art will appreciate that similar knowledge and skill in the art can also be applied to non-LV gene therapy vectors. In some embodiments, the gene delivery vector is a self-limiting lentivirus.

[00117] In such embodiments, the inventive polynucleotide cassette is flanked at the 5' and 3' ends by functional long terminal repeat (LTR) sequences. In one embodiment, the positions of the various elements present in the lentiviral vector backbone are depicted in Figure 1. Both LTR sequences were modified to produce self-inactivating (SIN) LV vectors. SIN vectors have a 400 bp deletion. into a 3'-LTR spanning promoter/enhancer elements from the U3 region. In this regard, transgene expression depends on internal promoters, which reduces the risk of RCL and reduces promoter interference (Ginn et al, 2003). Such a deletion in the 3'-LTR removes the TATA box, preventing transcription initiation (Miyoshi et al. 1998; Zuffrey et al 1998) and hence inactivating the vector. The U3 region in the 5'-LTR was replaced with other heterologous promoter sequences (i.e. CMV or RSV) to allow for Tat-independent transcription and to enhance genomic RNA synthesis resulting in an increase in viral titer. Since the 5'-U3 region controls the expression of primary transcripts, its modifications will 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 element (USE) from simian virus 40 (SV40-USE) (Schambach et al. 2007) have also been included into the R region of the viral 3'-LTR to reduce transcriptional read-through from internal promoters (Zaiss et al, 2002) or residues from the remote U3 region of SIN-LV vectors (Almarza et al. 2011), which prevents potential transcriptional activation of genes, located along the transcription. The leader region contains a packing signal (ᴪ), and it was believed that approximately 300 bp was required for LV vectors. the Gag gene in this area. This Gag sequence has now been reduced to only 40 bp. (figure 1). A Rev responsive element (RRE) has also been included to improve the efficiency of gene transfer, although it contains Env residues adjacent to it. The central polypurine tract (cPPT), which facilitates nuclear translocation of pre-integration complexes, together with the central terminal sequence (CTS) involved in reverse transcriptase compartment, has been found to increase viral titer (Zennou, et al. 2000; Follenzi et al. 2000) . In specific embodiments, cPPT present in any of the expression cassettes or gene delivery vectors described herein contains or consists of the following sequence: TTTAAAAGAAAAGGGGGGATTGGGGGGT (SEQ ID NO: 2) or a sequence characterized by at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 2.

[00118] The dNEF/PPT signal is required for reverse transcription and its inclusion significantly improves LV vector production.

[00119] Gene therapy vectors encapsulating the polynucleotide cassettes of the present invention can be prepared using standard techniques. For example, in the case of LV virions, an LV expression vector of the present invention may be introduced into the producer cell, followed by the introduction of an LV helper construct, where the helper construct includes LV coding regions that are capable of being expressed in the producer cell and that complement the functions of the LV helper. , which are absent from the LV-vector. This is followed by the introduction of the helper virus and/or additional vectors into the producer cell, wherein the helper virus and/or additional vectors provide helper functions capable of supporting efficient production of the LV virus. The producer cells are then cultured to produce LV. These steps are performed using standard methods.

[00120] Any suitable method for the production of viral particles can be used to deliver the claimed polynucleotide cassettes, including, without limitation, those described in the examples below. For contact with mammalian cells in vitro or in vivo, any concentration of viral particles suitable for efficient transduction of mammalian cells can be prepared. For example, viral particles can be formulated at a concentration of 10 ⁸ vector genomes per ml or more, eg 5 x 10 ⁸ vector genomes per ml; 10 ⁹ vector genomes per ml; 5 x 10 ⁹ vector genomes per ml, 10 ¹⁰ vector genomes per ml, 5 x 10 ¹⁰ vector genomes per ml; 10 ¹¹ vector genomes per ml; 5 x 10 ¹¹ vector genomes per ml; 10 ¹² vector genomes per ml; 5 x 10 ¹² vector genomes per ml; 10 ¹³ vector genomes per ml; 1.5 x 10 ¹³ vector genomes per ml; 3 x 10 ¹³ vector genomes per ml; 5 x 10 ¹³ vector genomes per ml; 7.5 x 10 ¹³ vector genomes per ml; 9 x 10 ¹³ vector genomes per ml; 1 x 10 ¹⁴ vector genomes per ml, 5 x 10 ¹⁴ vector genomes per ml or more, but generally not more than 1 x 10 ¹⁵ vector genomes per ml.

[00121] In preparing the claimed LV compositions, any host cell can be used to produce LV virions, including, for example, mammalian cells (eg, 293 cells), insect cells (eg, SF9 cells), microorganisms, and yeast. The host cells can also be packaging cells, in which the rep and cap LV genes are stably maintained in the host cell, or producer cells, in which the LV vector genome is stably maintained and packaged. The exemplary packaging and producer cells are derived from SF-9, 293, A549 or HeLa cells. LV vectors are purified and compiled using standard techniques known in the art.

[00122] In certain embodiments, the present invention includes a cell containing the expression cassette or gene delivery vector disclosed herein. In related embodiments, the cell is transduced with a viral vector containing an expression cassette as disclosed herein or has an expression cassette as disclosed herein integrated into the cell's genome. In certain embodiments, the cell is a cell used to produce a viral gene delivery vector. In other embodiments, the cell is a cell to be delivered to a subject to provide the subject with the gene product encoded by the expression cassette. Thus, in certain embodiments, the implementation of the cell is autologous in relation to the subject to be treated, or was obtained from the subject to be treated. In other embodiments, the implementation of the cell is allogeneic in relation to the subject to be treated, or was obtained from a donor other than the subject to be treated. In specific embodiments, the cell is a mammalian cell, such as a human cell. In certain embodiments, the cell is a blood cell, an erythrocyte, a hematopoietic progenitor cell, a bone marrow cell, e.g., a lineage-depleted bone marrow cell, a hematopoietic stem cell (e.g., CD34+), or a committed hematopoietic erythroid progenitor cell.

[00123] The present invention includes pharmaceutical compositions comprising the polynucleotide cassette, gene delivery vector, or cell described herein, and a pharmaceutically acceptable carrier, diluent, or excipient. The inventive polynucleotide cassette, gene delivery vector, or cell may be combined with pharmaceutically acceptable carriers, diluents, and reagents useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for use in primates. Such excipients may be solid, liquid, semi-liquid or, in the case of an aerosol composition, gaseous. Examples of such excipients, carriers, or diluents include, without limitation, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Additional active compounds may also be included in the formulations. Solutions or suspensions used for formulations may include a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; surfactants such as Tween 20 to prevent aggregation and compounds to control tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. In specific embodiments, the pharmaceutical compositions are sterile.

[00124] Pharmaceutical compositions suitable for use in the present invention further include sterile aqueous solutions or dispersions, as well as sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.

[00125] Sterile solutions can be prepared by incorporating the required amount of the active compound in an appropriate solvent with one or a combination of the ingredients listed above, if necessary, followed by filtered sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains the basic dispersion medium and the other necessary ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze drying, which yield a powder of the active ingredient with any additional required ingredient from a previously filter-sterilized solution thereof.

[00126] In one embodiment, the compositions are prepared with carriers that will protect the gene cassette or expression vector from rapid excretion from the body, such as in the case of a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials can also be obtained commercially.

[00127] It is especially convenient to formulate compositions for oral, ophthalmic or parenteral administration in unit dosage form for ease of administration and dosage uniformity. As used herein, "unit dosage form" refers to physically discrete units suitable as single dosages for a subject to be treated; wherein each unit contains a predetermined amount of active compound, calculated to obtain the desired therapeutic effect, in combination with the required pharmaceutical carrier. The specification of the unit dosage forms of the present invention is subject to and depends directly on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as on the limitations in formulating such an active compound for the treatment of individuals.

[00128] Pharmaceutical compositions may be included in a container, sachet, or dispenser, such as a syringe, such as a pre-filled syringe, along with instructions for use.

[00129] The pharmaceutical compositions of the present invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound that, upon administration to an animal, including a human, is capable of providing (directly or indirectly) its biologically active metabolite or residue.

[00130] The term "pharmaceutically acceptable salt" refers to physiologically and pharmaceutically acceptable salts of the compounds of the present invention, i.e., salts that retain the desired biological activity of the parent compound and do not cause undesirable toxicological effects inherent in it. 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 later editions), in “Encyclopaedia of Pharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. sci. 66:2 (1977). See also a review of suitable salts in Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[00131] Pharmaceutically acceptable base addition salts are formed with metals or amines such as alkali and alkaline earth metals or organic amines. Metals useful as cations include sodium, potassium, magnesium, calcium, and the like. Amines include N-N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., "Pharmaceutical Salts," J. Pharma Sci., 1977, 66, 119). The base addition salts of these acidic compounds are prepared by contacting the free acid form with a sufficient amount of the required base to form the salt in the conventional manner. The free acid form can be recovered by contacting the salt form with the acid and isolating the free acid in the conventional manner. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but the salts are otherwise equivalent to their corresponding free acid for purposes of the present invention.

[00132] The inventive polynucleotide cassette, gene delivery vector, e.g., recombinant virus (virions), or cell (e.g., transduced with the gene delivery vector disclosed herein) can be included in pharmaceutical compositions for use in mammalian patients, in particular in primates and more specifically in humans. The inventive polynucleotide cassette, gene delivery vector, e.g. virions, or cell can be formulated in non-toxic, inert, pharmaceutically acceptable aqueous vehicles, preferably at a pH ranging from 3 to 8, more preferably ranging from 6 to 8. Such sterile compositions will contain a vector or a virion containing a nucleic acid encoding a therapeutic molecule, dissolved in an aqueous buffer with an acceptable pH after dissolution.

[00133] In some embodiments, a pharmaceutical composition provided herein comprises a therapeutically effective amount of a cell, vector, or virion disclosed herein, in admixture with a pharmaceutically acceptable carrier and/or excipient, e.g., saline, phosphate buffered saline , phosphate and amino acids, polymers, polyhydric alcohols, sugar, buffers, preservatives and other proteins. Exemplary amino acids, polymers, sugars, and the like are octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose , trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hanks' solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycol. Preferably, this composition remains stable for at least six months at 4°C.

[00134] In some embodiments, the pharmaceutical composition provided herein contains a buffer, such as phosphate buffered saline (PBS) or sodium phosphate/sodium sulfate, tris buffer, glycine buffer, sterile water, and other buffers known to those of ordinary skill in the art. field, such as those described in Good et al. (1966) Biochemistry 5:467. The pH of the buffer containing the pharmaceutical composition containing the tumor-suppressing gene contained in the adenoviral vector-based delivery system may be in the range of 6.5 to 7.75, preferably 7 to 7.5 and most preferably 7.2 up to 7.4.

[00135] In certain embodiments, viral vectors can be formulated in any suitable single dosage including, without limitation, 1 x 10 ^s vector genomes or more, e.g., 1 x 10 ⁹ , 1 x 10 ¹⁰ , 1 x 10 ¹¹ , 1 x 10 ¹² or 1 x 10 ¹³ vector genomes or more, in certain cases 1 x 10 ¹⁴ vector genomes, but usually not more than 4 x 10 ¹⁵ vector genomes. In some cases, a single dosage is not more than about 5 x 10 ¹⁵ vector genomes, for example, 1 x 10 ¹⁴ vector genomes or less, such as 1 x 10 ¹³ , 1 x 10 ¹² , 1 x 10 ¹¹ , 1 x 10 ¹⁰ or 1 x 10 ⁹ vector genomes or less, in certain cases 1 x 10 ⁸ vector genomes or less, and generally not less than 1 x 10 ⁸ vector genomes. In some cases, a single dosage is from 1 x 10 ¹⁰ to 1 x 10 ¹¹ vector genomes. In some cases, a single dosage is from 1 x 10 ¹⁰ to 3 x 10 ¹² vector genomes. In some cases, a single dosage is from 1 x 9 ¹⁰ to 3 x 10 ¹³ vector genomes. In some cases, a single dosage is from 1 x 8 ¹⁰ to 3 x 10 ¹⁴ vector genomes. In one embodiment, the range is from about 5 x 10 ¹⁰ to about 1 x 10 ¹¹ vector genomes. In some embodiments, the range is from about 1 x 10 ⁹ to about 1 x 10 ¹⁰ vector genomes.

[00136] In some cases, a single dosage of a pharmaceutical composition can be measured using multiplicity of infection (MOI). By MOI is meant the ratio or multiple of vector or viral genomes to cells into which the nucleic acid can be delivered. In some cases, the MOI may be 1 x 10 ⁶ . In some cases, the MOI may be 1 x 10 ⁵ - 1 x 10 ⁷ . In some cases, the MOI may be 1 x 10 ⁴ - 1 x 10 ⁸ . In some cases, the recombinant viruses of the present invention are characterized by at least about 1 x 10 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 x 10 ¹⁸ MOI. In some cases, the recombinant viruses of the present invention are characterized by 1 x 10 ⁸ - 3 x 10 ¹⁴ MOI. In some cases, the recombinant viruses of the present invention are characterized by no more than about 1 x 10 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 x 10 ¹⁸ moi. In some embodiments, the range is from about 20 to about 400 MOI.

[00137] In some aspects, a certain amount of the pharmaceutical composition contains from about 1 x 10 ⁸ to about 1 x 10 ¹⁵ recombinant viruses, from about 1 x 10 ⁹ to about 1 x 10 ¹⁴ recombinant viruses, from about 1 x 10 ¹⁰ to about 1 x 10 ¹³ recombinant viruses or from about 1 x 10 ¹¹ to about 3 x 10 ¹² recombinant viruses.

Ways

[00138] As disclosed herein, the claimed polynucleotide cassettes and gene delivery vectors, collectively referred to herein as the "claimed compositions", find use in transgene expression in animal cells. For example, the claimed compositions can be used in research, for example, to determine the effect that a given gene has on the viability and/or functioning of a cell. As another example, the claimed compositions can be used in medicine, for example, to treat or prevent a disease or disorder. Thus, in some aspects of the present invention, methods are provided for expressing a gene in cells, wherein the method comprises contacting the cells with a composition of the present invention. In some embodiments, the implementation of bringing into contact is carried out in vitro or ex vivo. In some embodiments, bringing into contact is carried out in vivo, i.e. the claimed composition is applied to the subject.

[00139] In cases where mammalian cells are to be brought into contact in vitro or ex vivo with the inventive polynucleotide cassette or gene delivery vector containing the inventive polynucleotide cassette, the cells can be obtained from any species of mammals, for example, a rodent (for example, mice, rats, gerbils, squirrels), rabbit, feline, canine, goat, ovine, pig, horse, bovine, primate, human. Cells may be derived from stable cell lines, or they may be primary cells, with "primary cells", "primary cell lines", and "primary cultures" being used interchangeably herein to refer to cells and cell cultures that were obtained from a subject. and grew in vitro over a limited number of passages, i.e. culture divisions. For example, primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times to pass the crisis stage. Typically, primary cell lines of the present invention are maintained for less than 10 in vitro passages.

[00140] Embodiments of the present invention provide mammalian cells (eg, CD34+ cells) transduced with a viral delivery vector, eg, a lentiviral vector containing the human hepatic and erythroid pyruvate kinase (PKLR) gene. Therefore, the present invention includes a method for transducing a mammalian cell, e.g., a human hematopoietic stem cell or other cell as described herein, comprising contacting the cell with a viral delivery vector, e.g., a lentiviral vector containing an expression cassette as described herein. In certain embodiments, the implementation of the cell previously received from the subject to be treated, or from another donor. In specific embodiments, the subject has been diagnosed with PKD and the cell has been transduced with an LV containing an expression cassette encoding pyruvate kinase, such as a codon-optimized region or cDNA encoding RPKs. It should be understood that the disclosed methods, for example, used to deliver a pyruvate kinase gene product to a subject, for example, using the coPRK cDNA sequence, can also be used to treat hemolytic anemia and/or normalize erythroid cell differentiation, increase the number of functional mature erythrocytes, reduce extramedullary erythropoiesis, reduction of splenomegaly and other secondary effects of hemolytic anemia or PKD.

[00141] To allow expression of the transgene, the inventive polynucleotide cassette or gene delivery vector containing the inventive polynucleotide cassette will be brought into contact with cells for from about 30 minutes to 24 hours or more, 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.

[00142] The claimed polynucleotide cassette or gene delivery vector containing the claimed polynucleotide cassette can be applied to the claimed cells once or more, for example, once, twice, three times or more than three times, and the cells are left to incubate the cells with the agent ( ami) for a certain amount of time after each contact event, for example, within 16-24 hours, after which the medium is replaced with fresh medium and the cells are cultured further. Bringing cells into contact can occur in any culture medium and under any culture conditions that ensure the survival of the cells. The culture may contain growth factors to which the cells are sensitive. Growth factors, as defined herein, are molecules capable of promoting cell survival, growth and/or differentiation, either in culture or in intact tissue, through specific effects on a transmembrane receptor. Growth factors include polypeptides and factors other than polypeptides.

[00143] Typically, an effective amount of the inventive polynucleotide cassette or gene delivery vector containing the inventive polynucleotide cassette is used to effect transgene expression in cells. As discussed elsewhere herein, an effective amount can readily be determined empirically, for example, by detecting the presence or levels of a transgenic gene product, by detecting an effect on cell viability or function, etc. Generally, an effective amount of the claimed polynucleotide cassette or vector delivery of a gene containing the claimed polynucleotide cassette will provide higher expression of the transgene in cells than the same amount of polynucleotide cassette known from the prior art. Typically, expression will be 2-fold or more enhanced compared to expression by a reference or control polynucleotide cassette, such as known in the art, such as 3-fold, 4-fold, or 5-fold or more, in some cases 10 -fold, 20-fold or 50-fold or more, for example 100-fold.

[00144] In cases where cells are to be brought into contact in vivo with the claimed polynucleotide cassette or gene delivery vector containing the claimed polynucleotide cassette, the subject can be any mammal, for example, a rodent (for example, mice, rats, gerbils), a rabbit, a feline, a canine, a goat, an ovine, a pig, a horse, a bovine, or a primate. In a further preferred embodiment, the primate is a human. In an additional embodiment, the cells are CD34+ cells.

[00145] The methods and compositions of the present invention find use, for example, in the treatment of pyruvate kinase deficiency.

[00146] In another embodiment, the present invention includes a method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of cells transduced with a gene delivery vector, e.g., a viral vector that expresses the therapeutic gene product in the cells. In specific embodiments, the cells are autologous to the subject. In certain embodiments, the cells are erythroid cells, such as hematopoietic stem cells or committed hematopoietic erythroid progenitor cells. In some embodiments, the cell is a bone marrow cell, eg, a lineage-depleted bone marrow cell. In specific embodiments, the method is used to treat PKD and the viral vector is an LV containing an expression construct disclosed herein, comprising a human PGK gene promoter operably linked to a codon-optimized cDNA or human PKLR gene coding sequence, and a mutant Wpre, disclosed in this document. In specific embodiments, cells are administered to a subject parenterally, such as by intravenous injection.

[00147] In another embodiment, the present invention includes a method of treating PKD in a subject in need thereof, comprising administering to the subject an effective amount of autologous C34+ stem cells transduced with a lentiviral vector that expresses a codon-optimized PKLR cDNA in cells wherein the lentiviral vector contains a gene promoter A human PGK operably linked to a codon-optimized cDNA or human PKLR coding sequence, and a mutant Wpre sequence disclosed herein. In specific embodiments, the cells are hematopoietic stem cells or committed hematopoietic erythroid progenitor cells, such as bone marrow cells. In specific embodiments, cells are administered to a subject parenterally, such as by intravenous injection.

[00148] In another embodiment, the present invention provides treatment for a disease in a subject in need thereof, comprising administering to the subject an effective amount of a gene delivery vector, e.g., a viral vector that expresses a therapeutic gene product in the subject. In specific embodiments, the method is used to treat PKD and the viral vector is an LV containing an expression construct disclosed herein, comprising a human PGK gene promoter operably linked to a codon-optimized cDNA or human PKLR gene coding sequence, and a mutant Wpre, disclosed in this document. In specific embodiments, the gene delivery vector is administered parenterally to a subject, such as by intravenous injection.

[00149] In specific embodiments, gene delivery cells or vectors are administered to a subject in pharmaceutical compositions.

[00150] In some embodiments, the claimed methods result in a therapeutic benefit, such as preventing the development of a disorder, halting the progression of a disorder, reversing the progression of a disorder, etc. In some embodiments, the claimed method includes the step of detecting that a therapeutic benefit has been achieved. One of ordinary skill in the art will appreciate that such measurements of therapeutic efficacy will be appropriate for the particular disease to be modified and will be aware of the appropriate detection methods to use in measuring therapeutic efficacy.

[00151] It is expected that the expression of the transgene using the claimed transgene is reliable. Therefore, in some cases, transgene expression, for example, as detected by measuring gene product levels, by measuring therapeutic efficacy, etc., can be observed two months or less after administration, for example, 4, 3, or 2 weeks or less after administration. , for example, 1 week after the introduction of the claimed composition. It is also expected that the expression of the transgene is maintained over time. Therefore, in some cases, transgene expression, e.g., as detected by measuring levels of the gene product, by measuring therapeutic efficacy, etc., can be observed for 2 months or more after the administration of the inventive composition, for example, within 4, 6, 8 or 10 months or more, in some cases for 1 year or more, for example, for 2, 3, 4 or 5 years, in certain cases for more than 5 years.

[00152] In certain embodiments, the method comprises the step of detecting expression of a transgene in cells or in a subject, where expression is enhanced relative to expression from a polynucleotide cassette lacking one or more of the improved elements of the present invention. Typically, expression will be 2-fold or more enhanced compared to expression by a reference, i.e. control, polynucleotide cassette, such as known in the art, such as 3-fold, 4-fold, or 5-fold or more , in some cases 10-fold, 20-fold or 50-fold or more, for example, 100-fold, as evidenced by, for example, earlier detection, higher levels of the gene product, stronger functional effect on cells, etc. .

[00153] In general, if the inventive composition is an LV containing the inventive polynucleotide cassette of the present invention, then the effective amount to achieve the change will be approximately 1 x 10 ⁸ vector genomes or more, in some cases 1 x 10 ⁹ , 1 x 10 ¹⁰ , 1 x 10 ¹¹ , 1 x 10 ¹² or 1 x 10 ¹³ vector genomes or more, in certain cases 1 x 10 ¹⁴ vector genomes or more, and usually not more than 1 x 10 ¹⁵ vector genomes. In some cases, the number of vector genomes that is delivered is no more than about 1 x 10 ¹⁵ vector genomes, for example, 1 x 10 ¹⁴ vector genomes or less, for example, 1 x 10 ¹³ , 1 x 10 ¹² , 1 x 10 ¹¹ , 1 x 10 ¹⁰ or 1 x 10 ⁹ vector genomes or less, in certain cases 1 x 10 ⁸ vector genomes and, as a rule, not less than 1 x 10 ⁸ vector genomes. In some cases, the number of vector genomes that is delivered is from 1 x 10 ¹⁰ to 1 x 10 ¹¹ vector genomes. In some cases, the number of vector genomes that is delivered is from 1 x 10 ¹⁰ to 3 x 10 ¹² vector genomes. In some cases, the number of vector genomes that is delivered is from 1 x 10 ⁹ to 3 x 10 ¹³ vector genomes. In some cases, the number of vector genomes that is delivered is from 1 x 10 ⁸ to 3 x 10 ¹⁴ vector genomes.

[00154] In some cases, the amount of a pharmaceutical composition to be administered can be measured using multiplicity of infection (MOI). In some cases, MOI may mean the ratio or multiple of vector or viral genomes to cells into which the nucleic acid can be delivered. In some cases, the MOI may be 1 x 10 ⁶ . In some cases, the MOI may be 1 x 10 ⁵ - 1 x 10 ⁷ . In some cases, the MOI may be 1 x 10 ⁴ - 1 x 10 ⁸ . In some cases, the recombinant viruses of the present invention are characterized by at least about 1 x 10 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 x 10 ¹⁸ MOI. In some cases, the recombinant viruses of the present invention are characterized by 1 x 10 ⁸ - 3 x 10 ¹⁴ MOI. In some cases, the recombinant viruses of the present invention are characterized by no more than about 1 x 10 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 x 10 ¹⁸ moi.

[00155] In some aspects, a certain amount of the pharmaceutical composition contains from about 1 x 10 ⁸ to about 1 x 10 ¹⁵ particles of recombinant viruses, from about 1 x 10 ⁹ to about 1 x 10 ¹⁴ particles of recombinant viruses, from about 1 x 10 ¹⁰ to about 1 x 10 ¹³ recombinant virus particles, or about 1 x 10 ¹¹ to about 3 x 10 ¹² recombinant virus particles.

[00156] In a mammal, any total number of viral particles suitable to achieve appropriate cell transduction may be used to provide the desired effect or treatment of the disease. In various preferred embodiments, at least 10 ⁸ are injected; 5 x 10 ⁸ ; ¹⁰⁹ ; 5 x 10 ⁹ , 10 ¹⁰ , 5 x 10 ¹⁰ ; 10 ¹¹ ; 5 x 10 ¹¹ ; 10 ¹² ; 5 x 10 ¹² ; 10 ¹³ ; 1.5 x 10 ¹³ ; 3 x 10 ¹³ ; 5 x 10 ¹³ ; 7.5 x 10 ¹³ ; 9 x 10 ¹³ , 1 x 10 ¹⁴ virus particles or 5 x 10 ¹⁴ virus particles or more, but generally not more than 1 x 10 ¹⁵ virus particles. Any suitable number of applications of the vector to a mammal or primate eye may be made. In one embodiment, the methods include a single application; in other embodiments, multiple applications are carried out over a period of time that the attending physician considers appropriate. In some embodiments, a single administration (24 hour transduction) requires at least 2 x 10 ⁸ VG/ml in 5 x 10 ⁵ cells/ml to obtain high transduction efficiencies.

[00157] As a rule, the composition of individual doses includes an amount that is not less than the amount necessary to produce a measurable effect on the subject, and they can be determined on the basis of pharmacokinetics and pharmacology in terms of absorption, distribution, metabolism and excretion (“ADME” ) of the claimed composition or its by-products and thus based on the pharmacokinetic steps in the subject. This includes consideration of the route of administration as well as the amount of dosage. Effective dosage amounts and/or dosing schedules can readily be determined empirically from preclinical analyses, from safety and dose escalation and range testing, individual physician-patient relationships, and in vitro and in vivo assays such as those described herein. document and illustrated in the examples.

[00158] Some aspects of the present invention are described herein with reference to typical uses for illustration. It should be appreciated that numerous specific details, relationships and methods are set forth to provide a thorough understanding of the present invention. However, one of ordinary skill in the art will readily appreciate that the present invention may be practiced without one or more specific details, or by other means. The present invention is not limited to the illustrated order of actions or events, as some actions may be performed in a different order and/or simultaneously with other actions or events. In addition, not all illustrated actions or events are necessary to implement methods in accordance with the present invention.

[00159] In addition, it should be noted that claims may be drafted with the exclusion of any optional element. In this regard, this statement is intended to be used as the previously mentioned ground for the use of such exclusive terminology as "exclusively", "only", etc., in relation to the presentation of the claimed elements or for the use of a "negative" limitation.

[00160] The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the present invention is not entitled to backdate such publication in view of prior invention. In addition, the publication dates shown may differ from the actual publication dates, which may need to be confirmed independently.

[00161] 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 mentioned in this specification and/or listed in the Application Information Sheet are incorporated herein by reference in in its entirety, for example, to disclose and describe the methods and/or materials in connection with which these publications are cited. It should be appreciated that this disclosure supersedes any disclosure of an incorporated publication where there is a conflict.

[00162] It will be understood from the foregoing that while specific embodiments of the present invention are described herein for illustrative purposes, various modifications can be made without departing from the spirit and scope of the present invention. Accordingly, the invention is not limited except by the appended claims.

EXAMPLES

[00163] The following examples are provided to provide those of ordinary skill in the art with a complete disclosure and description of the embodiments and uses of the present invention, and are not intended to limit the scope of what the present inventors consider their invention, and it is not intended that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy in terms of the numbers used (eg amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric or near atmospheric.

Experimental methods

[00164] Obtaining vectors and supernatant containing lentivirus. LV was obtained as described in this document. The coRPK sequence was constructed using GeneArt® software to increase the GC content of the sequence and avoid cryptic splicing sites. Vectors were designed using the pCCL.sin.ppt.hPGK-EGFP-Wpre* construct as a backbone, generously provided by Dr. Naldini (HSR-TIGET, San Raffaele Telethon Institute, Milan, Italy). VSV-G pseudotyped LV vector material was obtained by calcium phosphate-mediated transfection with 3-plasmids of 293T cells (ATCC: CRL-1573, Rockville, MD, USA) as previously described [Follenzi A, et al. (2000). Nat Genet 25: 217-222]. Infectious LV titers were determined in HT1080 cells (ATCC: CCL-121) by qPCR as described elsewhere [Charrier S, et al. (2005). Gene Ther 12: 597-606]. The original lentiviral material with titers of 10 ⁷ -10 ⁸ virus particles (vp)/ml was obtained in the traditional way.

[00165] Purification and transduction of mouse HSCs. BM from 8-14 week old male PKD mice were harvested from leg bones and lineage-negative (Lin ^- ) cells were purified using the Lin ^- Cell Depletion Kit (Miltenyi Biotec, Gladbach, Germany) to give 70-90% purity. Lin ^- cells were prestimulated 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, Minnesota) 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, Massachusetts) for 24 hours, and then transduced with LVs carrying EGFP or coRPK in two transduction cycles at an MOI of 1-10 vp/cell Each transduction was performed for 24 hours in the presence of the aforementioned cytokines on plates pre-coated with a CH-296 fibronectin fragment (2 μg/cm ² ; Retronectin, Takara Shuzo, Otsu, Japan) overnight at 4°C.

[00166] In vivo survival of RBC. Transplanted mice carrying the coRPK transgene were injected with three consecutive intravenous injections (12 hours apart) of biotin-3-sulfo-N-hydroxysuccinimide ester sodium (50 mg/kg) (Sigma Aldrich, St. Louis, MO) . Twelve hours after the last injection, blood was collected from the tail vein and labeled with 2 µg/mL PE-conjugated anti-mouse Ter119 antibody (BD Bioscience, San Jose, CA) and FITC-conjugated streptavidin (50 µg/mL , BD Biosciences, San Jose, CA) for 30 minutes at 4°C. Samples were analyzed on an EPICS XL flow cytometer (Beckman Coulter, Brea, CA) every 2-4 days for 40 days post-injection. RBC survival kinetics were measured as the percentage of biotinylated cells within the total RBC population.

[00167] CFC analysis. CFC analysis was performed on BM and spleen from control and transplant mice according to the procedure of the media manufacturer Methocult GF M3434 (Stem Cell Technologies, Vancouver, Canada). BM cells were harvested at different time points post-transplant from all groups of mice and CFUs (clusters of 30 or more cells) were assessed 7 days post-seeding using a Nikon Diaphot-TMD microscope.

[00168] Identification of hematopoietic lineages. PBMCs were obtained from the tail vein of transplanted animals and labeled with an antibody panel to detect various hematopoietic cells. Myeloid cells were detected with biotinylated anti-GR-1 and Mac-1 antibodies (BD Bioscience, San Jose, CA, 5 μg/mL), while lymphoid cells were detected with anti-CD3 antibody conjugated to PE in the case of T- cells and anti-B220 antibody conjugated to PE and anti-B220 antibody conjugated to PECy5 in the case of B cells (BD Bioscience, San Jose, CA, 10 μg/ml) together with a SAV-TRC secondary antibody (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts). Samples were analyzed on a BD LSR Fortessa cytometer (BD Bioscience, San Jose, CA, CIF) supplemented with DAPI (Boehringer, Ingelheim, Germany, 2 μg/mL) to exclude dead cells.

[00169] Structural and histological studies. The spleens were collected, photographed and weighed on a precision balance to determine the presence of splenomegaly. Histological studies were performed on sections of the spleen and liver, obtained by performing traditional histological methods and stained with hematoxylin (Gill-2 hematoxylin, Thermo, Pittsburgh, USA) and eosin (eosin alcohol solution, Thermo Fisher Scientific, Waltham, Massachusetts). Iron deposits in the spleen were also examined by Prussian blue or Perls stain (Sigma Aldrich, St. Louis, MO) according to the manufacturer's instructions. All sections were viewed using an Olympus BX40 light microscope and photographed with an Olympus DP21 camera at a final magnification of 100x or 200x.

[00170] Differentiation of erythroid cells. Flow cytometric analysis of the intensity of the Ter119 and CD71 markers in BM and spleen was used to identify different subpopulations of erythroid cells, as described elsewhere [Socolovsky M, et al. (2001). Blood 98: 3261-3273], using 4 μg/mL anti-mouse Ter119 PE-conjugated antibody (BD Bioscience, San Jose, CA), 10 μg/mL biotinylated anti-CD71 antibody (BD Bioscience, San Jose, CA ) and streptavidin conjugated to Tricolor (Invitrogen, Thermo Fisher Scientific, Waltham, MA). Cells were then analyzed on an EPICS XL flow cytometer (Beckman Coulter, Brea, CA) using propidium iodide (IP, 2 μg/mL) to detect live cells.

[00171] Provirus quantification. Detection and quantification of integrated provirus per cell was performed using primers complementary to the proviral packaging sequence (ψ) and the mouse Titin housekeeping gene. Total 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). Twenty to 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 the primers and probes previously described [Charrier S, et al. (2011). Gene Ther 18: 479-487].

[00172] Chimerism. The presence of donor cells was quantified by qPCR detection of the SRY gene on the Y chromosome and the housekeeping gene of mouse β-actin. The primers and probes described previously [Navarro S et al (2006)] were used. Mol Ther 14: 525-535], and genomic DNA from transplanted PB mice was amplified using the 7500 Fast real-time PCR system (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA). Standard curves were generated using gDNA extracts from samples containing 0% to 100% BM cells from a mixture of cells from male/female mice, and chimerism was calculated as: % engraftment of donor cells = 100 x 2 ( ^{β-actin number - SRY number} ) .

[00173] LAM-PCR procedure. To identify vector integration sites, the 3'-LTR junctions of the vector-genome were 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. Linear amplification products were purified using streptavidin-coated magnetic beads, followed by complementary strand synthesis, parallel digestion with 2 different restriction enzymes (Tsp509I and HpyCH4IV), and two ligation reactions using linker cassettes complementary to the ends left after enzyme cutting. The resulting fragments were amplified using two additional steps of exponential PCR. LAM-PCR products were separated and quantified by gel electrophoresis on an automated MultiNA system (Shimadzu).

[00174] Preparing LAM-PCR products for sequencing with Illumina MiSeq. According to the method published by Parazynski et al [Paruzynski A, et al. (2010). Nat Protoc 5: 1379-1395], 40 ng of second exponential PCR products derived from Tsp509I and HpyCH4IV enzymes were re-amplified using overlapping primers containing specific sequences that allow matched-end sequencing on an Illumina MiSeq sequencer. LAM-PCR samples were adapted for 454-pyrosequencing by overlapping PCR by adding Roche 454 GS-FLX adapters: Adapter A, along with an 8-nt barcode, was added to the LTR end of the LAM-PCR generated amplicon; adapter B was added from the side of the linker cassette. The final amplicon in 5'-3' orientation was as follows: adapter A, barcode, LTR sequence, unknown genomic sequence, linker cassette sequence, and primer B. Purified PCR products with overlapping primers were run and quantified on the MultiNA automated electrophoretic system and pooled to give a final equimolar library of 10 nM. The final library was then re-quantitated using the Illumina Sequencing Platform KAPA Library Quantification Kit (Kara Biosystems, Wilmington, MA) on a Viia7 real-time PCR system (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA) to obtain a calculated concentration , which is 16.35 nM. Finally, the libraries were sequenced using the Illumina MiSeq Reagent Kit.

[00175] Bioinformatics analysis. To isolate vector integration sites (IS) from a high-throughput sequencing platform, both Roche 454 and Illumina MiSeq/HiSeq developed a workflow that collects raw data as input (typically in the FastQ file format), producing a list of trusted ISs and the nearest gene. Higher level analyzes regarding quantification of clone abundance and gene ontology enrichment were performed using Excel, GraphPad Prism(TM) and tools available online.

[00176] NGS Data Processing and Workflow Use. The NGS data processing step is associated with data management from the Illumina MiSeq high-throughput sequencing platform and aims to identify an IS where all valid sequence reads are aligned with the reference genome. Data processing involves two main processes. 1. Data quality check and analysis, in which lentiviral vector sequences and other contaminants are truncated. 2. Identification of the integration site, in which all valid sequence reads are aligned with the template and valid ISs are found.

[00177] Data quality analysis. To identify IS based on raw Illumina MiSeq data, a bioinformatics workflow was developed. Standard LAM-PCR products contain an LTR sequence, a flanking human genomic sequence, and a linker cassette (LC) sequence. 459 technology has made it possible to obtain LAM-PCR sequences with lengths ranging from 10 bp. up to 900 bp Similar results were obtained with Illumina MiSeq mated-end reads. These length limits are important parameters to consider in the quality analysis process, as they affect both the subsequent alignment procedure and the vector component identification algorithm. Sequences that were too short to align correctly with the reference gene and those that exceeded the maximum size achievable with NSG technology were discarded to avoid missing part or all of the LC sequence. Once the workflow for each pool was completed, all integration sites were collected both in files (archived in the TIGET -NAS- attached file storage) and in an internal database and stored on a storage server that keeps track of the modified copies.

[00178] Identification of integration sites. To identify unique integration sites and extract an Excel file with all ISs in rows and each sample in columns (IS matrix) with nearest gene annotations, the following steps were performed. 1. Creating an IS matrix using a program called create matrix, which allows you to identify conflicts between projects. This program will create a tab-delimited (TSV) file. 2. Annotate the IS matrix file, using the annotate bed program, into a file that will be named as follows for each pool using the input TSV file: awk '{print”chr”$1”\t”$2”\t”$2}' TSV_FILE \tail-n+2>TSV_FILE.bed; annotate bed-a/opt/genome/mouse/mm9/annotation/mm9.refGene. TIGET. gtf -b TSV_FILE.bed -o TSV_FILE.annotated.bed. 3. Import both the annotated file and the matrix file into a new Excel worksheet, called XLS in this case.

[00179] Collision detection. To obtain a robust IS data set from each transplanted mouse, potential contamination/conflict and false positive data were filtered based on the number of sequences. To combine the integration sites obtained as a result of various experiments, an additional stage of data normalization was required.

[00180] The term "conflict" is used to identify the presence of identical ISs in independent samples. Under the experimental conditions of the present invention, integration of a vector into the same genomic position in different cells is a very low probability event. Thus, detection of identical ISs in independent samples is likely due to contamination that can occur at various stages of wet laboratory procedures (sample purification, DNA extraction, LAM-PCR, and sequencing). Although the process of the present invention is designed to minimize the occurrence of contact between samples, high throughput IS analysis inherently carries a degree of background contamination. Identification of the degree of contamination between samples is also critical because obtaining the same IS in different samples obtained from the same mouse is used in the following steps to infer the biological properties of vector-labeled hematopoietic cells (i.e., the ability to differentiation into multiple lineages and sustained clonogenic activity). Thus, it should be possible to distinguish the actual occurrence of the same IS in different samples (from the same mouse) from contamination/conflict. To solve these problems, as a way to measure the degree of conflict in the assays of the present invention, among samples obtained from different test subjects and mice, the degree of common IS was assessed, and then rules were developed to discard those IS from each mouse data set that could be attributed to conflict. , and minimized the probability of counting false positives when looking for a common IS between samples from the same mouse. The present inventors have developed a conflict detection process that allows each integration locus to be validated. The overall result would be that if there is a set I of integration loci, if integration locus i in I is classified as a conflict, i is dropped from I. The present inventors used a conflict detection process between 3 independent transplant groups. 1. coPKR170: mice from analysis 1 sacrificed 170 days after transplantation of Lin.sup.- cells transduced with ^a coRPK-expressing LV vector (coRPK 1-3). 2. EGFP: Assay 2 mice transplanted with Lin.sup.- cells carrying ^an EGFP-expressing LV vector (EGFP 1-6). 3. coPKR-TC: Assay 2 mice transplanted with ^Lin.sup.- cells transduced with coPKR-expressing LV vector (coRPK 1-14), whose blood and BM were analyzed at different time points, including second recipients transplanted with pooled BM from a subgroup of mice subjected to primary transplantation (coRPK 11-14).

[00181] Each identical IS has a different sequence read (number of sequences) in different mice. The number of sequences can be used to determine if samples from one mouse are contaminated samples from another mouse based on an abundance criterion. According to this principle, an integration found in two mice will be attributed to the mouse showing the highest abundance, while in the other mouse it will be considered as a contaminant. Therefore, it was possible to identify a threshold for the number of different sequences that would allow a given conflict to be attributed to a mouse and removed from others. The authors derived a threshold value based on the data of the present invention with a value of 10, which means that for each IS among all TIs, if the IS has an abundance value (percentage of the ratio of the number of sequences) 10 times lower than the highest abundance value ( percentage of the number of sequences) of other TIs, then it is discarded from the current TI. The present inventors applied these rules to both TI and selected groups, and Excel files were used to calculate collision detection by applying the following rules (TI filtering is detailed here, but this also applies to group filtering). 1. Isolation of each TI, grouping all samples of the same TI together by summing the number of sequences. 2. For three received TIs, for each IS, calculate the percentage of the number of IS sequences as a function of the total amount of reads for the TI. 3. Then apply the following rule to calculate the decision stage with a threshold value of 10, which ensures that each IS is assigned to a reliable TI.

[00182] After identifying an IS to be deleted, the reads of this IS were removed from the group so that it would not also be attributed to this group. The filter described above was used in mice transplanted with different populations of ex-vivo transduced cells (one cohort of EGFP expressing mice from analysis 2 and two cohorts of coPKR mice belonging to two independent transplant experiments). Moreover, for the coPKR-TC group (assay 2), the filtering method described above was modified in two ways: one or more mice, then the integration will be maintained for all time points, even if the number of sequences for it is less than 10% of the maximum number of sequences among mice; b) for lineage tracing relationships, the present inventors applied a more stringent filter by eliminating IS with sequence count less than 3 and a 10% sequence count filter for co-presence at different time points. This means that an integration common to two points in time will only be kept or discarded if it is greater or less than the other, respectively.

[00183] Gene ontology analysis. All gene ontology analyzes were performed using the online GREAT software (http://bejerano.stanford.edu/great/public/html/). The web page allows downloading genomic coordinates of integrations from each dataset and calculates enrichment levels in the test dataset by matching position information (based on binomial distribution analysis for p-value calculations) and annotated function of genes closest to integration sites (based on analysis hypergeometric distribution for p-value calculations) [Groeschel S, et al. (2011). J Inherit Metab Dis 34: 1095-1102]. Biological processes and molecular functions were selected from the gene ontology database for enrichment analysis. For both statistical analyzes, only gene classes with a false positive rate of <0.05 were considered (FIG. 20).

[00184] Data storage. All data, both raw data and results, are stored in the TIGET Network Attached File Storage (NAS) in the root folder, where all alignments from the workflow, as well as the abundance matrix and plots, are available. The NAS is protected by authentication and authorization policies and has been built on a reliable and scalable infrastructure using a redundant RAID 5 disk storage array and is backed up on the TIGET registered CrashPlan software of the present invention.

Example 1

Therapeutic lentiviral vector with PGK-coRPK leads to stable and long-term correction of the anemia phenotype in genetically corrected PKD mice

[00185] In vivo efficacy of LV with PGK-coRPK (FIG. 2a) was analyzed by transduction and transplantation of lineage-depleted BM cells (Lin ⁻ cells) from PKD mice (FIG. 2b). In FIG. 2a is a schematic representation of the self-inactivating lentiviral vectors used throughout gene therapy experiments that contain the human PGK gene promoter that regulates the expression of the EGFP transgene in the control vector (upper diagram) or the expression of the codon-optimized PKLR cDNA sequence (coRPK) (coRPK) in the therapeutic vector (lower diagram). The coRPK sequence showed 80.4% homology with the human PKLR cDNA and 76.5% homology with the mouse Pklr cDNA, with no change in amino acid sequence. Figure 2b is a diagram of a gene therapy protocol performed to study the functionality of the designed lentiviral vector with PGK-coRPK. Correction of the PKD phenotype was investigated within 4-9 months after transplantation in PB and BM by hematological analysis and metabolic profiling. Integration analysis was performed in various tissues and at different time points in all mice to study the safety of the LV vector. 280 days after transplantation, total BM from primary transplanted mice carrying the coRPK transgene was transplanted back into lethally irradiated female PKD mice (secondary recipients) to test the stability and safety of engraftment. Lethally irradiated, cell-deficient PKD mice transduced with LV coRPK showed significant improvement in all blood parameters tested for erythroid cells compared to non-transplanted PKD littermates or cells-transplanted mice transduced LV with EGFP (Figure 3 and Table 2).

[00186] Table 2. Hematological parameters recorded 140 days after transplantation in peripheral blood

[00187] Data represent mean±SEM and were statistically analyzed by comparison with mice expressing EGFP using the non-parametric Kruskal-Wallis test. *p<0.05; **p<0.01.

[00188] The number of RBCs increased no later than 40 days after transplantation (Fig. 3a), and constitutive reticulocytosis, which is one of the most common signs of PKD, was largely reversed in mice carrying the PGK-coRPK transgene, reaching levels similar to those observed in healthy controls, for at least 9 months after transplantation (Fig. 3b). In contrast, PKD animals transplanted with EGFP-transduced LV cells showed anemia and marked reticulocytosis similar to PKD mice at all time points analyzed. Hemoglobin (HGB), hematocrit index (HTC), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) levels were also corrected in lentivirus-corrected cell transplant mice compared to untreated PKD littermates. transplantation (table 2). This hematological correction was achieved with 63.66 ± 4.45% donor chimerism and transduction efficiency ranging from 60% to 90% (Table 3).

[00189] Table 3. Relevant molecular parameters in mice transplanted with genetically modified cells

[00190] Data represent mean ± SEM, n. about. - not defined. ^a Estimated percent transduction obtained by interpolation in linear regression based on Experiment 1 (X-axis: VCN/WBC, Y-axis: % cfu provirus ⁺ ).

[00191] For transduced cells, an average of 1.65 ± 0.08 integrated vector copies per cell was shown, indicating that the LV vector with PGK-coRPK provided sufficient human RPK transgene expression to reverse hemolytic anemia. Of note, expression of the coRPK transgene resulted in an extended half-life of erythroid cells compared to non-transplanted PKD mice (Fig. 3c, d). On average, PKD mice showed an RBC half-life of 19 days, while in genetically corrected mice it was extended to 25 days (an extension of 6 days), reaching values close to the wild-type RBC half-life (Fig. 3d). Thus, RBCs from coRPK-expressing mice showed intermediate survival kinetics between cells from healthy and deficient control mice (Fig. 3c), most likely due to the fact that complete chimerism was not achieved in these animals (Table 3).

[00192] Nine months after transplantation, hematopoietic progenitor cells from primary recipients were transplanted into secondary recipients who maintained engraftment levels (62.89 ± 5.61%) and VCN (1.44 ± 0.08 copies) (Table 3) . Secondary transplant recipients showed recovery of multiple lineage hematopoiesis up to 5 months post-transplant (FIG. 4) and a significant improvement in all erythroid parameters in PB (FIG. 5 and Table 2). Figure 4a is a diagram of a flow cytometry technique used to identify different hematopoietic lineages by labeling with PE-conjugated anti-CD3 antibody, PE-conjugated anti-B220, PECy5-conjugated anti-B220, biotin-conjugated anti-Gr1, and an anti-Mac1 antibody conjugated to biotin supplemented with SAV-PE-Cy5. Figure 4b shows typical scatter plots and percentages (Figure 4c) of each lineage in PB 140 days after transplantation. Bars represent the mean percentage ± SEM in control, healthy (n=2, black bar), PKD mice (n=2, gray bar), and secondary transplant mice expressing the therapeutic coRPK transgene (n=4, shaded bar). In addition, provirus integration was detected in variously committed hematopoietic progenitor cells (Fig. 6a,b) and their number remained constant over time (Fig. 6c), demonstrating the stability of genetic modification and highlighting the safety of LV with PGK-coRPK. Figure 6a shows the number of copies of the vector per cell in CFU from BM from individual transplanted mice, 120-170 days after transplantation. The percentage of transduction and chimerism is also shown. Figure 6b shows the number of copies of the provirus in cells from different hematopoietic compartments. The columns represent the mean±SEM of different groups of transplanted mice. Figure 6c shows the kinetics of provirus integrations in BM cells in individual transplanted mice expressing EGFP (gray lines) and mice carrying the coRPK transgene (black lines).

Example 2

Expression of RPK by lentivirus normalizes erythroid differentiation and ensures the production of functional mature erythrocytes

PKD mice show a characteristic expansion of the erythroid compartment caused by a compensatory mechanism of erythropoiesis (Min-oo et al 2004). Examination of the pattern of erythroid differentiation in transplanted mice indicated that ectopic expression of RPK reversed this mechanism (Fig. 7a,b). For mice expressing PKD and EGFP, a predominance of immature erythroid progenitors (subpopulation I: proerythroblasts, and subpopulation II: basophilic erythroblasts) in the BM and spleen is shown, as well as a marked drop in the number of late erythroid cells (population IV: reticulocytes and mature erythrocytes), then both mice transplanted with cells transduced with LV with coRPK showed a significant decrease in the number of immature erythroid progenitors (subpopulations I and II) in the BM and spleen, as well as a significant increase in the latest erythroid compartment (subpopulation IV), equivalent to healthy mice (Fig. 7a, b). In addition, in contrast to mice expressing PKD and EGFP, mice carrying the coRPK transgene showed a significant decrease in plasma erythropoietin (Epo) levels (Fig. 7c). Figure 8a shows the total number of CFU for the spleen, and figure 8b shows it for the bone marrow 140 days after transplantation. The dots represent the number of colonies per mouse analyzed, and the lines represent the mean ± SEM in each group. Statistical analysis of the data was performed using the nonparametric Kruskal-Wallis test. Normalization of erythropoiesis in PKD mice treated with the therapeutic vector was accompanied by a decrease in the number of progenitors in the spleen to normal levels (Fig. 8a), although there was no change in the content of CFU in BM (Fig. 8b).

Example 3

Transplantation of cells transduced with coRPK lentiviral vector reverses extramedullary erythropoiesis and organ pathology

[00193] Due to the active destruction of RPK-deficient erythrocytes, mice expressing PKD and EGFP exhibit acute splenomegaly with an increase in spleen mass and size greater than 200% compared to healthy controls (Fig. 9a, b). Also in these animals, a disorganized structure of the splenic tissue and expansion of the red pulp of the spleen was observed, indicating intense extramedullary erythropoiesis, which is also confirmed by the presence of clusters of erythroid cells on liver sections in animals expressing PKD and EGFP (Fig. 9c). Of note, ectopic expression of the coRPK transgene completely reversed spleen and liver pathology in genetically corrected mice, with a reduction in RBC aggregations and normalization of the histological structure and size of the spleen (Fig. 9). In addition, histological studies showed a complete absence of iron deposits in the liver of genetically corrected mice, while PKD mice from both the non-transplanted group and the HSC transplanted group transduced with the EGFP-carrying vector showed intense supersaturation. iron due to the continuous process of hemolysis (Fig. 9c). Overall, transplantation of genetically corrected HSCs into PKD mice restored the normal status of erythropoiesis and all secondary effects caused by hemolytic anemia.

Example 4

Expression driven by LV with PGK-coRPK restores the glycolysis pathway to RBC without modifying WBC metabolic equilibrium

Next, an extended metabolomic analysis of all transplanted and control mice was performed to investigate the functional correction of RPK enzymatic activity. Following a non-targeted profiling strategy, significant changes in RBC glycolysis intermediates were observed across groups, with the identification of three broad clusters of metabolite patterns with distinct trends (Fig. 10a). RBCs from coRPK-expressing mice showed increased levels of metabolites from cluster 1, similar to healthy controls, but in contrast to transplanted mice that carried the EGFP transgene. Similarly, cluster 3 showed a trend towards reduced metabolites in genetically corrected mice, similar to wild-type mice, but in contrast to mice expressing EGFP. However, cluster 2 from analysis 1 showed no difference in metabolite profile between groups of transplanted mice (mice expressing EGFP and coRPK) (FIG. 10a). Non-targeted metabolic profiling also showed that genetic modification was able to modify several important glycolysis intermediates to achieve increased levels of ATP (Fig. 10b), ADP (Fig. 10c), and pyruvate (Fig. 10d) in erythrocytes isolated from transplanted mice. HSC transduced with LV with PGK-coRPK. Given the trends for these metabolites, other metabolites localized near the PK catalyzed reaction were then analyzed using a targeted profiling approach. Levels of the direct PK substrate, phosphoenolpyruvate (PEP) (FIG. 10e), and 3-phosphoglycerate (3-PG) (FIG. 10f), located in the opposite direction from the PK catalyzed reaction, approached levels in healthy control mice. Deficient erythrocytes expressing the coRPK transgene also produced increased amounts of D-lactate (Fig. 10g), an end product of anaerobic glycolysis, compared to mice expressing PKD and EGFP. To test whether the compensation of glycolysis metabolites was the result of normalization of PK activity in mature erythrocytes, the activity of this enzyme was measured and normalized to that of hexokinase to avoid the influence of high reticulocyte counts in deficient animals. RBCs were purified by passing through a cellulose column to prevent contamination with leukocyte PK activity. Complete compensation of PK activity was observed in coRPK expressing animals, which achieved ratios similar to those obtained in healthy wild-type animals and normal healthy voluntary blood donors (Fig. 11). In FIG. 11a shows pyruvate kinase activity, FIG. 11b shows hexokinase activity and FIG. 11c shows the ratio of pyruvate kinase and hexokinase enzyme activities in RBCs from control mice and mice transplanted with transduced cells. RBC was isolated from blood samples by passing through a cellulose column to get rid of extraneous leukocyte PK activity and subjected to enzymatic activity evaluation. Black bars - healthy mice (n=2); white bars - mice transplanted with cells transduced with an EGFP-expressing vector (n=3); shaded bars - mice transplanted with cells transduced with coRPK-expressing vector (n=3). Bars filled with cells represent healthy volunteer values (n=1). Data are presented as mean ± SEM of each group.

[00194] Principal component analysis showed that the pattern of metabolites in RBC differed depending on the group and differed markedly from the WBC profile (Fig. 12a). In contrast, WBC subgroups clustered together with very little difference between groups, indicating no change in leukocyte metabolic balance when ectopic coRPK was expressed (Fig. 12a). In addition, the specific metabolite changes seen with non-targeted RBC profiling were absent in WBC (Fig. 12b-d).

Example 5

Cells transduced with LV with PGK-coRPK provide polyclonal hematopoietic recovery without evidence of vector genotoxicity

[00195] The integration profile of LVs carrying one of the coRPK or EGFP transgenes was analyzed in transplanted mice. Based on the genome-wide integration profile of LV, each insert creates a unique genetic tag that can be used to track clonal behavior in individual transduced cells. Genomic DNA (gDNA) was obtained from WBC and BM cells from mice subjected to primary and secondary transplantation, as well as from pools of transduced cells before transplantation (cells Lin ^- ). Linear amplification-mediated PCR (LAM-PCR) (FIGS. 13 and 14) was used to amplify vector/genome junctions and to identify insertion sites (IS) of the vector. Figure 13 shows vector integration sites identified by LAM-PCR amplification of vector-genome 3'-LTR junctions. An automated MultiNA system was used to obtain a pattern characterized by several stripes. An internal control (IC) strip derived from the Tsp509I vector backbone is indicated by an arrow. Figure 14 shows vector integration sites identified by LAM-PCR amplification of vector-genome 3'-LTR junctions. An automated MultiNA system was used to obtain a pattern characterized by several stripes. An internal control (IC) strip derived from the HpyCH4IV5 vector backbone is indicated by an arrow.

[00196] The PCR products were sequenced using the MiSeq Illumina platform and the resulting sequences were mapped into the mouse genome using a bioinformatics workflow and filtered for conflicts as described above in Methods (FIG. 15). Figure 15 depicts a general outline of an integration site mapping assay performed on mice transplanted with genetically modified hematopoietic progenitor cells. Bone marrow and white blood cell samples from transplanted mice were analyzed, referring to two independent experiments (Table 3) and collected at different time points after transplantation, as described in the auxiliary methods following the shown workflow.

[00197] A total of 5,173,892 sequencing reads were mapped into the transplanted mouse genome, yielding 2220 unique vector integration sites. The genomic distribution of IS from two independent experiments was consistent with the previously reported preferential integration of LV within transcription units (especially within the first 50 kb downstream of the transcription start site -TSS-) (Fig. 16a), showing no bias towards any particular chromosome in the mouse genome (Fig. 16b). Figure 16a shows the distribution density of integration sites (IS) near the transcription start site (TSS) of the nearest RefSeq gene covering 500 kb. upstream and downstream of the TSS. The numbers at the top represent the number of ISs found for all samples and time points. Figure 16b shows the chromosomal distribution of LV integration sites in transplanted mice expressing the EGFP transgene (black bars) or the coRPK therapeutic transgene (gray bars), showing no bias towards any particular chromosome.

[00198] The safety of LV-based gene therapy with PGK-coRPK was investigated by assessing the abundance of clones, calculating the percentage of the number of sequences for each IS (clonal tag) relative to the total number of sequences in the data set. Dot plots and heat map representations of the relative abundance of each IS obtained for each mouse (FIGS. 17, 18 and 19) showed large fluctuations in clonal composition across mice and in in vitro cultured Lin ⁻ cells. Figure 17 is a graph of traced integrations that were common between primary and secondary recipient mice carrying the therapeutic LV vector with PGK-coRPK. The integrations identified in each mouse in any organ and at any point in time are combined. Secondary recipients received pooled BM from mice transplanted with coRPK11-14. The rest of the identified IS were detected either in primary or secondary recipients. The numbers in the cells show the representation of the corresponding integration as a percentage in the mentioned mouse. In addition to the >5% filter applied in the integration assay, all integrations with <3 sequences were excluded. The figure 19 presents in the form of a scatter plot the abundance of clones of pooled integrations in each mouse in the bone marrow. The relative percentage (y-axis) of each integration site is calculated relative to the total number of sequence reads obtained in each dataset. Similar to cells transduced with co-RPK (FIG. 17), the diagram shows that the vast majority of transplanted mice show a polyclonal hematopoietic cell repopulation pattern.

[00199] In addition, it could be understood that in the case of some samples, a small number of integrations led to a large number of sequence reads (Fig. 17 and 18), revealing a polyclonal repopulation pattern of transduced HSCs. In addition, tracked integration shared between primary transplanted mice bearing therapeutic LV with PGK-coRPK and post-secondary transplanted mice showed no large co-presence of integrations across groups, confirming the absence of clonal dominance (Fig. 18) .

[00200] To determine whether hallmarks of insertional mutagenesis were present in transplanted mice, the appearance of common insertion sites (CIS) was assessed, similar to ongoing clinical trials associated with LV. CISs are insertional hot spots that can result from integration bias during transduction or in vivo selection of clones containing vector integrations that confer a growth advantage. CIS was identified using the algorithm from Abel et al. and Grubbs' test for outliers, no CIS was detected and thus there were no warning signs of genotoxicity in this reading. Moreover, gene ontology (GO) analysis did not reveal bias towards gene classes involved in cancer, cell proliferation, or regulation of apoptosis in any of the integration datasets sorted by tissue distribution, time point, or abundance of repopulating hematopoietic cell clones. (Fig. 20). Figure 20 shows the genomic integration profile of LV. Gene ontology analysis (GO) was performed using GREAT software on transplanted mouse samples. All data on integrations obtained in this study (N=2220) showed the prevalence of gene functions indicated on the left side of the figure. To examine whether the most common integrations were enriched in terms of certain gene classes, all integration sites with relative sequence numbers >5% of the total dataset were selected (shown in Fig. 17), showing no predominance of GO gene classes.

[00201] These results indicate neutral vector integration and demonstrate the safety of LV with PGK-coRPK in preclinical studies.

Example 6

Human clinical trial

[00202] A clinical trial was conducted to evaluate the safety and preliminary efficacy of autologous hematopoietic stem cell transplantation (HSCT) using drug product EU/3/14/1130 (autologous CD34+ hematopoietic stem cells transduced with a lentiviral vector containing the RPK gene) in patients with deficiency pyruvate kinase with a history of severe and transfusion-dependent anemia refractory to treatment with splenectomy.

[00203] ODD EU/3/14/1130 contains a self-inactivating lentiviral vector expressing a codon-optimized variant of the therapeutic human PKLR gene (Figure 21).

[00204] Self-inactivating lentiviral vectors (SIN-LV) provide more robust expression (Ellis 2005) and are less susceptible to transcriptional silencing than gamma retroviral vectors (Pfeifer, Ikawa et al. 2002). They also show a much safer integration profile (Schroder, Shinn et al. 2002), (Mitchell, Beitzel et al. 2004), (Wu, Li et al. 2003), and due to a 400 bp deletion, which they carry in the 3'-LTR sequence (Miyoshi, Blomer et al. 1998) (Zufferey, Dull et al. 1998), transgene expression is regulated by internal promoters, which increases the safety of LV-based genetic modification.

[00205] The vector sequence of the acceptable lentiviral vector also includes several modifications to improve the expression and safety of the transgene in target cells.

[00206] One modification is the use of the human phosphoglycerate kinase (PGK) gene promoter, already characterized by its stable in vivo activity and improved safety properties 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 transgene expression and lower susceptibility to transcriptional silencing (Gerolami, Uch et al. 2000, Zychlinski, Schambach et al. 2008).

[00207] Another modification is a codon-optimized human PKLR (coRPK) cDNA variant to enhance mRNA stability after transcription. GeneArt® software was used for optimization, increasing the GC content and removing cryptic splicing sites to eliminate transcriptional silencing, thereby increasing transgene expression. The optimized coRPK sequence showed 80.4% homology with the human PKLR gene, with no changes in the amino acid sequence of the protein.

[00208] Another modification is also a mutant woodchuck hepatitis virus post-transcriptional regulatory element (Wpre), without any residual open reading frame (Schambach, Bohne et al. 2006) to improve the expression level and stability of the therapeutic gene. The backbone, promoter and Wpre* sequences of this lentiviral vector (LV with PGK-coRPK) are the same as the sequence corresponding to the drug product in the Lentiviral vector containing the Fanconi anemia A (FANCA) gene for the therapy of Fanconi anemia Type A patients trial » (No. 141/2000), as well as the vector backbone used in the ongoing clinical trial for metachromatic leukodystrophy (MLD) (Biffi, Montini et al. 2013).

[00209] Mechanism of action

[00210] Once CD34 ⁺ progenitor cells have been harvested, either from bone marrow (BM) or mobilized peripheral blood cells from PKD patients, they will be transduced ex vivo with the drug product and the therapeutic vector integrated into the cells' genome. Once integrated, the human therapeutic gene (coRPK) will be transcribed and translated in deficient cells to produce the therapeutic RPK protein, which is absent or reduced in mature erythrocytes in PKD. Transduced hematopoietic progenitor cells with PKD will then be genetically corrected and thus acquire the ability to produce RBCs with sufficient amounts of ATP to perform their functions (Figure 22). These genetically corrected hematopoietic progenitor cells (which will be part of the drug product) will then be transplanted back into the patient, and after engraftment, they will give normal red blood cells that cure the disease for life.

[00211] The active principle will consist of a cell suspension of corrected hematopoietic stem cells (CD34 ⁺ ) with the therapeutic lentiviral vector PGK.coRPK.wpre developed as a rare disease drug by the European Commission (ODD EU/3/14/1130 ) for the treatment of pyruvate kinase deficiency. Thus, this new drug should be included in the group of advanced therapeutic developments within the subclass of gene therapy.

[00212] The composition of the active principle will include a suspension of genetically modified cells at the rate of at least 2 x 10 ⁶ CD34 ⁺ cells/kg of body weight, containing at least 0.1 copies of the therapeutic vector per cell. Cells will be suspended in saline with 2% HSA.

[00213] The final therapeutic product will be produced according to GMP rules, so the product requirements for its release and infusion into the patient will be related to the quality of the product. Therefore, these specifications would be >30% cell viability, sterility (Gram test and pharmacopeia sterility), absence of mycoplasma, absence of replicative competing lentiviral particles, and demonstration of effective therapeutic potential by detecting the presence of at least 0.1 copy of the vector per cell with using quantitative PCR. In addition, studies and scientific studies of the content of hematopoietic progenitor cells and the number of copies of the vector in them will be carried out. Three independent validations will be performed with healthy control cells to ensure that the procedure achieves the requirements described above.

[00214] The final product will be finally packaged in a heat-sealed transfusion bag and, especially if frozen and stored prior to infusion into the patient, samples will be taken prior to this for precise quality control checks.

[00215] Mobilization

[00216] Patients will be mobilized at their respective hospitals, although the first two patients will be mobilized at Hospital del Niño Jesús, Madrid (Spain). The mobilization process will consist of administering recombinant granulocyte colony stimulating factor (G-CSF, Neupogen, Amgen, Thousand Oaks, CA, USA) at doses of 12 mg/kg twice daily up to eight days from birth, plerixaphora, 4 Day 2, 240 mg/kg/day (Mozobil®, Genzyme Europe BV, Naarden, The Netherlands), as four subcutaneous doses for 4 consecutive days. Hematopoietic progenitor cells from peripheral blood will be collected by high volume leukapheresis from day 5 of mobilization through a cell separator and according to standard protocols at the Hospital Niño Jesús in Madrid. All instruments and solutions are CE marked and comply with legal specifications for medical devices.

[00217] Purification of CD34 ⁺ cells

[00218] In accordance with the mobilization process, the apheresis will take place in a hospital where the patient is being mobilized. Apheresis will be processed immediately to select hematopoietic progenitor cells (CD34 ⁺ cells) using the MACS "magnetic cell sorting" technology (Miltenyi Biotec, Germany), which allows cells to be separated using a high magnetic field gradient through a powerful permanent magnet and a separation column with a ferromagnetic matrix. The CliniMACS system (Miltenyi Biotec, Bregisch Gladbach, Germany) consists of a computer (the CliniMACS®plus device), dedicated CD34 ⁺ cell sampling software, a set of sterile tubes (CliniMACS tube sets), a magnetically controlled reactive sterile device (reagent kit for isolation of CD34 CliniMACS cells) and sterile buffer (PBS/EDTA buffer CliniMACS). The device and reagents used will be CE marked and will comply with legal specifications for medical devices. During this phase and subsequent washes, non-specific immunoglobulin (phlebogamma 5% intravenous, 0.5 g, Grifols) and human albumin (Grifols® human albumin 20%, Grifols) are used, which are later removed by washing after centrifugation. The CD34+ cells will then be quantified. Microbiological controls of the obtained products will be carried out by taking standard samples for fungi, aerobic bacteria and anaerobic microorganisms for cultivation according to certain protocols.

[00219] Transduction of CD34 ⁺ cells

[00220] Transduction of purified CD34 ⁺ cells using ODD EU/3/14/1130 will be performed under GMP conditions within a time period of 48 hours from the time of extraction of cells from the patient (apheresis). Ex-vivo cell culture will last less than 48 hours and will be performed according to established standards, including the use of properly formulated X-vivo-20 medium (Lonza), the addition of hematopoietic growth factors (100 ng/ml hrSCF, 100 ng/ml hrFlt-3 , 100 ng/mL TPO and 20 ng/mL IL-3 (all from Prepotech), 1 µg/mL Pulmozyme and controlled concentration of 5% O _2. Transduction will be performed with a GMP batch of lentivirus ODD EU/3/14/1130 obtained from VIVEbiotech (San Sebastian, Spain) After transduction, cells will be washed in X-vivo-20 (Lonza) for final packaging in a transfusion bag suitable for cryopreservation Specific samples will be collected to determine if the final product meets all the specifications already mentioned for its final release.Three independent validations will be carried out to evaluate the stability of the product.All products and solutions, including the vector, will meet the specifications of the legislation for medical devices and clinical applications. Prior to receipt, all raw materials (including consumables, biological reagents, and chemical powders) will be inspected by CliniStem's Quality Control (QC) team according to Standard Operating Procedures (SOPs).

[00221] Conditioning

[00222] Patients will be conditioned according to standardized and specific protocols provided for testing. Considering the patient undergoing conditioning, an alternative reserve of 2 x 10 ⁶ untreated CD34 ⁺ cells/kg will be kept frozen for use in the event that the resulting product does not completely restore hematopoiesis in the treated patient.

[00223] Infusion

[00224] Prior to infusion, an eligible patient will be screened for study eligibility. The day of infusion, premedication and prophylactic medications used will be recorded.

Example 7

Preclinical development

[00225] Previous work has demonstrated the suitability of HSC gene therapy for the treatment of PKD in mice transplanted with over 25% gene-corrected cells. These results imply that a significant number of HSCs corrected with the donor gene (Zaucha, Yu et al. 2001) and high levels of transgene expression are required to achieve a therapeutic effect on PKD. The present inventors have developed a new therapeutic lentiviral vector proposed for this clinical trial, which contains the eukaryotic hPGK gene promoter driving the expression of the PKLR cDNA, which was developed as a drug for the treatment of rare diseases in August 2014 (EU/3/14/1130 ). This vector was used in a preclinical PKD gene therapy protocol in a mouse model of the disease. At lentivirus dosages based on clinical standards, ectopic RPK expression was able to normalize the erythroid compartment, correcting the hematologic phenotype and reversing organ pathology. Metabolic studies have demonstrated a functional correction of the glycolysis pathway in RBCs subjected to genetic correction, in the absence of metabolic disorders observed in leukocytes. It should be noted that in the case of WBCs analyzed in parallel, no changes in the metabolic equilibrium in leukocytes were shown during ectopic expression of RPK under the action of a ubiquitous promoter such as PGK, which excludes the metabolic advantage of leukocytes as a possible safety problem and enhances the therapeutic potential of the EU/3/ vector. 14/1130.

[00226] The recovery of multiple lineages and the absence of any leukemic complication or clonal expansion in secondary recipients after proliferative stress induced by BM retransplantation demonstrate the long-term stability and safety of the LV vector-based protocol with PGK-coRPK. The use of a eukaryotic human PGK gene promoter that i) appears to result in more physiological expression of the RPK transgene, ii) has been shown to be a weak transactivator, and iii) is currently being used in a clinical trial for metachromatic leukodystrophy (MLD), can also explain the safety of the procedure as a whole.

[00227] To assess the long-term safety of HSC gene therapy by analyzing vector integration sites, next generation sequencing was used to predict the risk of insertional oncogenesis in HSC. More than 5,173,892 sequence reads were mapped into the mouse genome, resulting in a total of 2220 unique IS vectors, with no evidence of in vivo expansion or selection of clones containing IS. In contrast, the data of the present invention indicate the clonal composition and dynamics of hematopoiesis after transplantation of transduced HSCs in mice, implying a true and stable in vivo genetic modification of HSCs over time. Overall, the analysis of the vector integration pattern highlights the safety properties of the LV vector with PGK-coRPK, which provides a genetic correction of PKD, in the absence of evidence of genotoxicity.

Example 8

Clinical Development

[00228] To date, no clinical studies of this medicinal product have been conducted. For the first time, an application for assistance in drafting study protocols is submitted to the regulatory authority. The purpose of the authors of the present invention is to perform a clinical trial sponsored by the European Commission. The ForGeTPKD consortium, made up of various physicians and fundamental researchers from Europe, was formed to focus on PKD research and the development of new therapeutic strategies. The ForGeTPKD clinical trial will be the first human use of this medicinal product. It is designed as an international, multicentre, open-label phase I/II trial to evaluate the safety and efficacy of transplantation of autologous CD34+ cells transduced ex vivo with a lentiviral vector containing the erythrocyte pyruvate kinase (RPK) gene (EU/3/14/1130) in patients with severe pyruvate kinase deficiency. .

[00229] Regulatory status

[00230] The medicinal product is not currently marketed. The aim of the PKD Consortium is to advance the clinical development of a drug product in order to eventually obtain marketing authorization.

[00231] This final product will be produced with a lentiviral vector that has received drug designation for the treatment of orphan diseases.

[00232] Indication. Treatment of pyruvate kinase deficiency.

[00233] Criteria. The only definitive treatment for PKD is allogeneic BMT, which has been used in patients with transfusion-dependent severe anemia refractory to other treatments. However, allogeneic BMT is not a widely accepted treatment for PKD (only one patient reported in the literature (Tanphaichitr, Suvatte et al. 2000)) because it is associated with severe complications associated with intensive conditioning before allogeneic BMT with chemotherapy or chemoradiotherapy; and acute and chronic graft-versus-host disease (GVHD). The hypothesis of the present inventors is that gene therapy using autologous hematopoietic stem cells transduced with viral vectors containing the wild-type gene variant presented as ODD EU/3/14/1130 may offer the potential to cure such patients, while avoiding the risks of GVHD, a major cause of failure in hematopoietic progenitor transplantation.

[00234] Active ingredient. Autologous CD34 ⁺ hematopoietic stem cells transduced with a lentiviral vector containing the erythrocyte pyruvate kinase (RPK) gene (ODD EU/3/14/1130) that express the wild-type protein variant.

[00235] Finished product. Frozen sachet containing at least 2 x 10 ⁶ active ingredient/kg patient body weight, which is suspended in saline buffer with 2% HAS.

Example 9

Pharmacology

[00236] Completed studies. The drug product under development includes several modifications in its sequence that provide some of the advantages of PKD gene therapy: 1) the use of the SIN-LV vector design made it possible to relatively easily and safely obtain viral starting materials capable of efficiently transducing HSC; 2) the use of a weak and eukaryotic promoter such as hPGK, which is less susceptible to silencing by methylation (Gerolami, Uch et al. 2000), results in more physiological expression of the transgene, reaching therapeutic levels at viral dosing (1.65 VCN) within clinical standards (Matrai, Chuah et al. 2010); and 3) the codon-optimized sequence of the transgene and the presence of the mutant Wpre sequence increase the stability of the transgene mRNA: the reporter gene is not included in the therapeutic vector sequence, which prevents possible problems with immunogenicity (Morris, Conerly et al. 2004); (Stripecke, Carmen Villacres et al. 1999).

[00237] The developmental LV drug product with hPGK-coRPK effectively reversed PKD pathology in both primary and secondary progenitor transplant deficient mice transduced and corrected with ODD EU/3/14/1130. Correction was performed by cells carrying an average of 1.65 copies of the therapeutic transgene per cell.

[00238] The human PGK gene promoter was sufficiently efficient to express clinically appropriate levels of coRPK protein to compensate for the hemolytic phenotype in transplanted mice.

[00239] Genetic modification was able to extend the half-life of RBC; normalize hematological parameters and reticulocyte levels; reverse compensatory erythropoiesis constitutively activated in PKD mice; get rid of the pathology of the spleen and liver, significantly reduce iron overload, which is one of the life-threatening complications of PKD. In addition, human RPK ectopic expression corrected the energy defect in the RBC without altering the metabolic balance in the WBC, highlighting the efficacy and safety of the drug product.

Example 10

Ongoing research

[00240] Transduction of human hematopoietic progenitor cells from healthy donors and PKD patients to study: 1) transduction efficiency of ODD EU/3/14/1130 into human cells; 2) determining the optimal vector copy number/cell to obtain efficient and therapeutic expression of the therapeutic RPK protein; and 3) determining the optimal conditions to obtain therapeutic levels of transduction without loss of hematopoietic stem cell potential.

[00241] Planned studies include setting up large-scale transduction on GMP equipment, as well as pre-validation and 3 validation studies to establish optimal conditions to achieve the required specifications defined for the final therapeutic product.

[00242] Toxicology

[00243] Completed studies include the following: 1) human RPK ectopic expression corrected the energy defect in the RBC without altering the metabolic balance in the WBC; 2) analysis of genomic integration of the vector showed that (i) analysis of the relative abundance of specific cell clones revealed oligoclonal recovery of hematopoietic cells in some mice, indicating the absence of clonal dominance in any mice subjected to primary and secondary transplantation; (ii) common integration sites (CIS, dense clusters of vector integrations at specific intervals of the genome), considered a hallmark of insertional mutagenesis, did not show signs of genotoxicity, there was no abnormal enrichment of CIS over time, and CIS identified in two independent gene therapy experiments, performed on mice were not represented by a large number of sequences and were not targeted primarily at oncogenes; (iii) gene ontology (GO) analysis of genes targeted by lentivirus integration and examination of the position of vector integrations in specific regions of the genome demonstrated no bias towards the class of genes involved in cancer, cell proliferation, or regulation of apoptosis; and (iv) overall drug product integration analysis showed no evidence of genotoxicity.

[00244] Planned studies include the following: 1) Recombinant competent lentivirus (RCL) production assay: Human T-lymphocytes from healthy donors and from PKD patients will be transduced with ODD EU/3/14/1130 and cultured in vitro for long periods of time ; the presence of p24 viral protein will be analyzed in supernatants by ELISA to assess potential RCL production, and 2) drug product biodistribution: mouse hematopoietic progenitor cells will be transduced with ODD EU/3/14/1130 and transplanted into lethally irradiated recipients; Animals will be sacrificed one month after transplantation and various organs (gonads, liver, kidney, brain, bone marrow, spleen and peripheral blood) will be analyzed for the presence of vector DNA; and 3) vector integre in human cells: hematopoietic progenitors from healthy donors and from PKD patients will be transduced with ODD EU/3/14/1130 and transplanted into severely immunocompromised mice to allow engraftment and proliferation of human hematopoietic cells. At various time points (1, 2 and 3 months after transplantation) blood and BM samples will be taken, human cells sorted and analyzed for vector integrome, as already done with mouse cells.

Example 11

Human clinical trial

[00245] A ForgetPKD trial will be conducted to test clinical efficacy. The aim of the proposed clinical trial is to evaluate the safety and preliminary efficacy of autologous hematopoietic stem cell transplantation (HSCT) using the drug product EU/3/14/1130 (autologous CD34 ⁺ hematopoietic stem cells transduced with a lentiviral vector containing the erythrocyte pyruvate kinase (RPK) gene) in patients with pyruvate kinase deficiency with a history of severe and transfusion-dependent anemia that cannot be treated with splenectomy.

[00246] The main goal is to evaluate the safety and tolerability/suitability of the treatment. In this regard, the following endpoints will be measured: 1) occurrence and characterization of adverse events (AEs), including AEs associated with infusion of transduced cells, AEs resulting from conditioning prior to cell infusion, and AEs resulting from clonal evolution associated with transduced cells; and 2) the number of patients with stem cell engraftment 30 days after transplantation.

[00247] Secondary goals are a preliminary assessment of the effectiveness of treatment. In this regard, the following endpoints will be measured: the number of patients who became “transfusion independent” at the end of the study; in patients who still need transfusions after treatment, the ratio of the average number of transfusions needed during the study period (1 year) to the average number of transfusions in the last 1.5 years before baseline assessment; a clinically significant reduction in anemia, defined as the number of patients with an increase in hemoglobin levels of 2 g/dl from baseline at the end of the study; a clinically significant reduction in reticulocytosis, defined as the number of patients with a 50% reduction from baseline at the end of the study; and the number of patients with stem cell engraftment, with 1% of transduced cells detectable at 6 and 12 months after cell infusion and at the end of the study.

[00248] The search goal is to evaluate the effect of treatment on the patient's quality of life. In this regard, the following endpoints will be measured: improvement in quality of life from baseline at the end of the study using a quality of life questionnaire (SF-36 for adults or PEDSQL for children) and its validated versions translated into the language of the participating countries (Italian, Dutch and Spanish).

[00249] The ForGetPKD trial is a multicenter, international trial that will be performed in 3 EU member states: Spain, Italy, and the Netherlands. Participating centers include competent national researchers and institutes regarding the diagnosis and treatment of PKD.

[00250] The test will be the first human application of the described product. It is designed as a Phase I/II non-comparative, open-label study.

[00251] The duration of the global study will be 2 years from the first visit of the first patient to the last visit of the last patient. It includes 1 year recruitment period and 1 year treatment and early (immediate) follow-up period. After the end of the trial, the included subjects will be asked to participate in a follow-up study that will monitor safety and efficacy up to 5 years after transplantation.

[00252] In accordance with the incidence of the disease and the design of the study, the authors of the present invention plan to enroll 6 patients within one year. This assessment was made taking into account that 3 potential participants have already been identified.

[00253] The study procedures include a screening period, a treatment period, and a follow-up period. Details of visits in each phase and associated study procedures are detailed below and summarized in Table 4.

[00254] Table 4.

[00255] Screening period

[00256] Visit -1. Prescreening visit

[00257] Potential candidates will be informed of the purpose and characteristics of the trial, and will receive written informed consent completed and signed by the patient (or their legal representative, if the patient is underage) in duplicate. To be eligible for study participation, patients must fully meet all of the inclusion criteria and none of the exclusion criteria to be tested. It includes a pre-treatment procedure to mobilize and obtain viable CD34 ⁺ cells to carry out: A. storage of 2 x 10 ⁶ CD34+ cells/kg body weight as a reserve in case of lack of engraftment, B. transduction of at least 6 x 10 ⁶ CD34+ cells/ kg of body weight using the vector EU/3/14/1130 to obtain a medicinal product and to perform all quality controls necessary for the release of a medicinal product. Only patients with a sufficient number of transduced cells available after drug product release (2 x 10 ⁶ transduced CD34 ⁺ cells/kg body weight) will be included in the study.

[00258] This visit will also include the following procedures:

- registration of the relevant medical anamnesis or surgical operations in the anamnesis;

- registration of demographic data and clinically significant results of the physical examination;

- registration of relevant concomitant medicinal products;

- testing of peripheral blood with a standard clinical blood test (CBC), biochemical analysis, determination of coagulation and serology;

- performing an echocardiogram, a lung function test and a chest X-ray;

- filling in the quality of life questionnaire (SF-36 or PEDSQL);

- genetic diagnosis of PKD.

[00259] To be eligible for study participation, patients must fully meet all of the following inclusion criteria and none of the exclusion criteria.

[00260] Inclusion criteria are as follows: male or female patients, age >2 years at time of recruitment, willingness to give signed informed consent (to be signed by parents or legal guardian in the case of children under 18 years of age), previously diagnosed PKD, confirmed genetic testing, history of severe transfusion-dependent anemia unresponsive to splenectomy, candidate for autologous hematopoietic stem cell transplantation, availability of ≥ 2 x 10 ⁶ transduced CD34 ⁺ cells/kg body weight, and treatment and follow-up for at least last 2 years in a referral center with detailed medical records including history of transfusions.

[00261] Exclusion criteria are as follows: positive test results for human immunodeficiency virus type 1 or 2 (HIV 1 and HIV 2), uncorrected bleeding disorder, presence of other causes of hemolysis, any previous or current malignant, or myeloproliferative, or immunodeficiency disorder , a close relative with a known or suspected familial cancer syndrome (including, but not limited to, hereditary breast and ovarian cancer syndrome, hereditary non-polyposis colorectal cancer syndrome, and familial adenomatous polyposis), in the case of patients with a previous allogeneic transplant, the presence of residual cells of donor origin, patients with severe complications who, after medical evaluation, are considered to be suffering from pathologies of cardiac, pulmonary, hepatic or renal function III / IV degree, uncontrolled seizure disorder, diffusing capacity of the lungs for carbon monoxide (DLco) < 50% of pro predictable (hemoglobin-adjusted), any other evidence of severe iron overload that in the investigator's opinion warrants exclusion, participation in another clinical trial with an experimental drug within 30 days prior to screening, availability of an HLA-identical donor relative for allogeneic bone marrow transplantation , for women, pregnancy or lactation, patients who, according to the investigator's criteria, fail to understand the study objectives, benefits and risks, and/or fail to comply with study procedures, poor functional status as evidenced by a Kamofsky index <80 in adults or a Lansky score <80 in children.

[00262] The statistical analysis of the study will be descriptive. Qualitative endpoints will be described as frequencies and percentages. Qualitative endpoints include adverse events, number of patients with engraftment of stem cells, number of patients who become "transfusion independent", number of patients who have a clinically significant reduction in anemia, number of patients who have a clinically significant decrease in reticulocytosis, number of patients with engraftment of stem cells, which can be detected by the presence of transduced cells, and improvement in quality of life compared to baseline, measured using the SF-36 questionnaire or PEDSQL. Qualitative endpoints will be described using the mean and standard deviation, or using the median and quartiles. Qualitative endpoints include reduction in anemia and reticulocytosis from baseline, the number of transfusions needed during the study period relative to the number of transfusions in the last 1 year before baseline, and the number of copies of the vector in peripheral blood and bone marrow cells. All endpoints will be described at the end of the study.

[00263] Previous work has demonstrated the suitability of HSC gene therapy for the treatment of PKD in mice transplanted with over 25% gene-corrected cells. These results imply that a significant number of HSCs corrected with the donor gene (Zaucha, Yu et al. 2001) and high levels of transgene expression are required to achieve a therapeutic effect on PKD. The present inventors have developed a new therapeutic lentiviral vector proposed for this clinical trial, which contains the eukaryotic hPGK gene promoter driving the expression of the PKLR cDNA, which was developed as a drug for the treatment of rare diseases in August 2014 (EU/3/14/1130 ). This vector was used in a preclinical PKD gene therapy protocol in a mouse model of the disease. At lentivirus dosages based on clinical standards, ectopic RPK expression was able to normalize the erythroid compartment, correcting the hematologic phenotype and reversing organ pathology. Metabolic studies have demonstrated a functional correction of the glycolysis pathway in RBCs subjected to genetic correction, in the absence of metabolic disorders observed in leukocytes. It should be noted that in the case of WBCs analyzed in parallel, there is no change in the metabolic balance in leukocytes during ectopic expression of RPK under the action of a ubiquitous promoter such as PGK, which excludes the metabolic advantage of leukocytes as a possible safety problem and enhances the therapeutic potential of the EU/3/ vector. 14/1130.

[00264] The recovery of multiple lineages and the absence of any leukemic complication or clonal expansion in secondary recipients after proliferative stress induced by BM retransplantation demonstrate the long-term stability and safety of the LV vector-based protocol with PGK-coRPK. The use of the eukaryotic human PGK gene promoter, which appears to result in more physiological expression of the RPK transgene, which has been shown to be a weak transactivator and is currently being used in a metachromatic leukodystrophy (MLD) clinical trial, may also explain overall safety of the procedure.

[00265] To evaluate the long-term safety of HSC gene therapy by analyzing vector integration sites, next generation sequencing was used to predict the risk of insertional oncogenesis in HSC. More than 5,173,892 sequence reads were mapped into the mouse genome, resulting in a total of 2220 unique IS vectors, with no evidence of in vivo expansion or selection of clones containing IS. In contrast, the data of the present invention indicate the clonal composition and dynamics of hematopoiesis after transplantation of transduced HSCs in mice, implying a true and stable in vivo genetic modification of HSCs over time. Overall, the analysis of the vector integration pattern highlights the safety properties of the LV vector with PGK-coRPK, which provides a genetic correction of PKD, in the absence of evidence of genotoxicity.

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SEQUENCE LIST

<110> CENTRO DE INVESTIGACIONES ENERGETICAS, MEDIOAMBIENTALES AND TEKNOLOJICAS

FUNDACION INSTITUTO DE INVESTIGACION SANITARIA FUNDACION GIMENES DIAZ

CENTRO DE INVESTIGACION BIOMEDIC EN RED

SEGOVIA, Jose S.

GOMEZ, Mary G.

NAVARRO, Susana

MESA, Nestor

BUEREN, Juan

Bravo, Mary G.

<120> COMPOSITIONS AND METHODS FOR ENHANCED PKLR GENE EXPRESSION

<130> ROPA-001/01US 329592-2034

<140> PCT/US2017/028695

<141> 2017-04-20

<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 post-transcriptional regulatory element

woodchuck hepatitis virus (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 ttgtggccg 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 ccctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg 660

ggccgcctcc ccgcctg 677

<210> 2

<211> 28

<212> DNA

<213> Human Immunodeficiency Virus 1

<400> 2

tttaaaagaaaaggggggat 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

tccggggggcg 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

120

cagtattaag cgggggag 138

<210> 6

<211> 236

<212> DNA

<213> Artificial sequence

<220>

<223> Modified LTR nucleotide 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 nucleotide 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 promoter sequence

Human PKLR

<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 ttgtggccg 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 ccctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg 660

ggccgcctcc ccgcctg 677

<---

Claims (27)

1. Recombinant gene delivery vector containing an expression cassette containing a polynucleotide sequence containing the following in the 5'-3' direction: a) a promoter sequence; b) a sequence encoding the therapeutic gene product, wherein the therapeutic gene product is a human erythrocyte and liver pyruvate kinase (PKLR) polypeptide; and c) ribonucleic acid (RNA) export signal, wherein the promoter sequence is operably linked to a sequence encoding a PKLR polypeptide, and wherein the recombinant gene delivery vector is a lentivirus (LV). 2. The recombinant gene delivery vector of claim 1, wherein the promoter is a phosphoglycerate kinase (PGK) promoter. 3. The recombinant gene delivery vector according to claim 1 or 2, wherein the sequence encoding the gene product is codon-optimized. 4. Recombinant gene delivery vector according to any one of paragraphs. 1-3, where the RNA export signal is a mutant woodchuck hepatitis virus post-transcriptional regulatory element (Wpre). 5. The recombinant gene delivery vector of claim 4, wherein the mutant Wpre is a chimeric Wpre containing a sequence at least 80% identical to SEQ ID NO: 1. 6. Recombinant gene delivery vector according to any one of paragraphs. 1-5, additionally containing one or more enhancer sequences. 7. Recombinant gene delivery vector according to any one of paragraphs. 1-6 further comprising a polypurine tract (PPT) sequence or a polyadenylation signal (polyA) sequence. 8. Recombinant gene delivery vector according to any one of paragraphs. 1-7, additionally containing one or more of the following sequences: i) packing signal sequence; ii) a truncated Gag sequence; iii) Rev-sensing element (RRE); iv) central polypurine tract (cPPT); v) Central Terminal Sequence (CTS) and vi) a sequence element (USE) located upstream of transcription, optionally from simian virus 40 (SV40-USE). 9. Recombinant gene delivery vector according to any one of paragraphs. 1-8, additionally containing 5'- and 3'-terminal long terminal repeat sequences. 10. Pharmaceutical composition for the treatment or prevention of pyruvate kinase deficiency (PKD), containing an effective amount of cells containing a recombinant gene delivery vector according to any one of paragraphs. 1-9. 11. Pharmaceutical composition according to claim 10, wherein the cells are hematopoietic stem cells. 12. The pharmaceutical composition of claim 11 wherein the cells are committed hematopoietic erythroid progenitor cells. 13. Pharmaceutical composition according to any one of paragraphs. 10-12, where the cells are autologous to the subject. 14. Pharmaceutical composition according to any one of paragraphs. 10-12, where the cells are allogeneic with respect to the subject. 15. Pharmaceutical composition for the treatment or prevention of pyruvate kinase deficiency (PKD), containing a pharmaceutically acceptable excipient and an effective amount of a recombinant gene delivery vector according to any one of paragraphs. 1-9. 16. A method for expressing a transgene in erythroid cells in vitro , comprising bringing one or more erythroid cells into contact with an effective amount of a recombinant gene delivery vector according to any one of paragraphs. 1-9. 17. A method for expressing a transgene in erythroid cells in vitro , comprising bringing one or more erythroid cells into contact with an effective amount of a recombinant gene delivery vector according to any one of paragraphs. 1-9, wherein after said contacting, PKLR is expressed at detectable levels in one or more erythroid cells.

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