In vivo lentiviral gene therapy for the treatment of primary hyperoxaluria type 1

Abstract

The present invention relates to a lentiviral vector (LV) comprising a nucleic acid, the nucleic acid comprising a transcription unit comprising from 5' to 3' a hepatocyte-specific promoter, preferably an ET promoter, a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase protein (AGXT-RHEAM), a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), preferably a mutated WPRE, and at least one copy of at least one miRNA target sequence, preferably miRNA-142. The lentiviral vector of the present invention further comprises backbone elements necessary for the LV to perform its function, the backbone elements being at least one of the following: a 5' long terminal repeat (5'LTR), a primer binding site (PBS), a psi packaging signal, a stem loop 4 (SL4) region of wild-type HIV virus, a Rev response element (RRE), and a DNA flap central polypurine tract (cPPT), and a 3' long terminal repeat (3'LTR), or any combination thereof.

Description

[Technical field]

[0001] The present invention can be included in the field of gene therapy, in particular in the treatment of primary hyperoxaluria type 1. In particular, the present invention relates to a lentiviral vector for gene therapy that can treat primary hyperoxaluria type 1. [Background technology]

[0002] Primary hyperoxaluria type 1 (PH1) (OMIM number 259900) is a rare inherited autosomal recessive metabolic disorder. PH1 is caused by a deficiency of the enzyme alanine:glyoxylate aminotransferase (AGT, EC 2.6.1.44), encoded by the AGXT gene. In humans, AGT activity is restricted to hepatic peroxisomes. The enzyme is active only intracellularly and is not secreted into the extracellular space. PH1 is characterized by an overproduction of oxalate in the liver. Oxalate is the end product of metabolism in mammals and is normally excreted in the urine. When produced in large quantities, the kidneys are the first organs affected by calcium oxalate (CaOx) aggregates that form in the urinary cavity (urolithiasis) and in renal tissue (nephrocalcinosis), causing interstitial fibrosis and renal failure. As a consequence of renal damage, the glomerular filtration rate (GFR) decreases and chronic kidney disease increases. Eventually, about 60% of PH1 patients progress to end-stage renal disease by the age of 40. Subsequent systemic lesions with widespread CaOx deposition, called oxalosis, endanger the lives of PH1 patients (Non-Patent Document 1, Non-Patent Document 2). Current guidelines for PH1 patients provided by the European Consortium of Hyperoxaluria (http: / / www.oxaleurope.com / ) recommend prophylactic organ transplantation before systemic oxalosis appears if renal function is already damaged. Therefore, novel therapeutic approaches are needed that can be applied as soon as possible, ideally immediately after the disease is diagnosed. Since there is no accurate test to predict the decline of renal function and no available genetic diagnosis, novel treatments should be introduced at the early stage of the disease, aiming to reduce oxalate levels and preserve renal function.

[0003] Gene therapy has the potential to provide a one-time cure by restoring AGT activity through delivery of a functional copy of the AGXT gene. Currently, gene therapy for inherited metabolic liver diseases is mostly based on in vivo approaches. Liver sinusoidal endothelial cells have a fenestrated structure, facilitating access to and gene transfer into hepatocytes (Non-Patent Document 3). AAV vectors have been widely adopted for in vivo liver-directed gene therapy, given the remarkable safety and efficacy profile shown in preclinical models and clinical trials. AAV vector-based gene transfer of coagulation factors to the liver for the treatment of the coagulation disorder hemophilia is one of the most successful applications of gene therapy (Non-Patent Document 4, Non-Patent Document 5). In the specific case of PH1, since it is an autonomous cell disease in which functional cells cannot compensate for oxalate overproduction from defective cells, the percentage of corrected cells to be achieved after treatment is considered to be high in order to revert the hyperoxaluric phenotype and correct the disease (Non-Patent Document 6). Preclinical proof of concept of gene therapy efficacy has been provided using adenovirus vectors or adeno-associated virus vectors in PH1 mice (Non-Patent Document 7, Non-Patent Document 8). Despite this success, the non-integrative nature of AAV vectors poses a challenge for application to pediatric patients, as the transgene is diluted during liver development and immune responses currently prevent effective re-administration. Furthermore, widespread pre-existing immunity to AAV capsids reduces the proportion of eligible patients. As approximately 30% of PH1 patients have infantile onset (Non-Patent Document 9, Non-Patent Document 10), it is of great importance to develop gene therapy strategies that have the potential to persist for life after a single administration to pediatric patients.

[0004] Lentiviral vectors (LVs) are attractive vehicles for liver gene therapy due to their ability to stably integrate into the genome of target cells and the low prevalence of pre-existing immunity to vector components in humans. Recently, the use of LV-carrying FIX for the treatment of hemophilia B has shown promising results in mice, dogs and non-human primates (Non-Patent Document 11, Non-Patent Document 12, Non-Patent Document 13). In the present invention, LVs based on vectors used for the treatment of hemophilia B are used. These LVs are designed to target transgene expression to hepatocytes by a combination of transcriptional (synthetic liver-specific promoter) and post-transcriptional control (hematopoietic cell-specific microRNA 142 target sequence, involved in the degradation of any mRNA from the vector ectopically expressed in antigen-presenting cells of the liver and spleen) (Non-Patent Document 11). In addition, the proposed LVs contain an enhanced version of AGXT that confers higher stability to the protein (Non-Patent Document 14). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Cochat et al., 2012 [Non-Patent Document 2] Sas et al., 2020 [Non-Patent Document 3] Piccolo & Brunetti-Pierri, 2015 [Non-Patent Document 4] George et al., 2017 [Non-Patent Document 5] Pasi et al., 2020 [Non-Patent Document 6] Danpure, 2009 [Non-Patent Document 7] Salido et al., 2006 [Non-Patent Document 8] Salido et al., 2011 [Non-Patent Document 9] Sas et al., 2019 [Non-Patent Document 10] Mandrile et al., 2014 [Non-Patent Document 11] Annoni et al., 2013 [Non-Patent Document 12] Cantore et al., 2015 [Non-Patent Document 13] Milani et al., 2019 [Non-Patent Document 14] Mesa-Torres et al., 2014 [Brief description of the drawings]

[0006] [Figure 1] Illustrates lentiviral vector constructs. A. Reporter lentiviral vector: LV.ET.eGFP.142-3pT. The reporter lentiviral vector developed expresses the eGFP gene, which codifies green fluorescent protein (GFP). Hepatocyte-specific expression of this transgene is achieved by the presence of two distinct transcriptional control elements: a synthetic enhancer, a specific liver promoter (TTR) consisting of a part of the mouse TTR enhancer and mouse TTR promoter, and four target sequences of hematopoietic-specific miRNA-142-3 to block transgene expression in antigen-presenting cells (APCs) and thus the development of an immune response. B. Therapeutic lentiviral vector: LV.ET.AGXT-RHEAM.142-3pT. Developed from the same lentiviral vector structure as the reporter lentiviral vector, it expresses AGXT-RHEAM, an improved version of the AGXT gene with five amino acid changes that codify a more active form of the AGT protein, similar to the reporter LV, and hepatocyte-specific expression of the therapeutic transgene is achieved by the presence of two distinct transcriptional control elements, a specific liver promoter (TTR) and four repeats of the target sequence of the hematopoietic-specific miRNA-142-3. [Diagram 2]Figure 1 shows the in vivo test protocol for the gene therapy approach efficacy study. Two types of experiments were performed to study the efficacy of the proposed strategy. A. C57BL / 6 adult male mice were intravenously injected with a single dose of reporter lentiviral vector and sacrificed after 4 weeks. The transduction efficacy was then determined by analyzing the genome copy number of the integrated lentiviral vector and the percentage of transduced cells in the target tissue, the liver. In addition, biodistribution studies were performed to determine the security of the procedure by analyzing the vector copy number (VCN) in the spleen, lung, bone marrow, lymph node, thymus, brain, testis, pancreas, and kidney. B. For the analysis of the AGXT-expressing lentiviral vector therapeutic efficacy in a mouse model of PH1, the following protocol was performed. Adult Agxt1- / - male mice were intravenously injected with different doses of therapeutic lentiviral vector. In addition, three different control groups were included, two of which consisted of Agxt1- / - mice that were not injected or injected with the reporter lentiviral vector, and the other group consisted of non-injected C57BL / 6 wild-type mice. After 3 weeks of treatment, all mouse groups were subjected to an ethylene glycol challenge test, supplemented with 0.5% ethylene glycol in the drinking water to induce an overload in oxalate production. During the challenge test, two different PH1 endpoints, urinary oxalate concentration and body weight, were measured. After treatment, the mice were sacrificed to identify kidney damage. After sacrifice, the transduction efficacy of the therapeutic lentiviral vector was analyzed by VCN and AGXT expression in the liver. The percentage of transduced cells was also analyzed in the reporter lentiviral vector-injected Agxt1- / - mice to complete the results obtained with the therapeutic lentiviral vector. [Diagram 3]Figure 1 shows the number of viral genomes integrated into target tissues of lentiviral vector-injected mice. Lentiviral vector integration in the target tissue, liver, was analyzed by quantification of VCN by qPCR 4 weeks after lentiviral vector injection. Data from experiments performed in C57BL / 6 mice and Agxt1- / - are shown. Non-injected control mice (n=3) did not show lentiviral vector integration in any mouse strain. For experiments performed in C57BL / 6 mice, two different reporter lentiviral vector doses (1x108TU / mouse and 2.5x108TU / mouse) (n=3) were tested. At the low dose, 0.55 (0.34-0.56) lentiviral vector copies per diploid genome were observed, whereas at the high dose, 1.03 (0.94-1.56) lentiviral vector copies per diploid genome were obtained. Furthermore, intravenous injection of two different doses of LV.ET.eGFP.142-3pT into Agxt1 mice resulted in a VCN of 0.11 (0.09-0.49) in the liver of mice injected with 1.4 × 108 TU / mouse (n = 4), a VCN of 0.84 (0.73-1.58) in mice injected with 5 × 108 TU / mouse (n = 5), and a VCN of 0.615 (0.39-1.46), 0.765 (0.45-1.19), and 1.165 (0.85-0.32) in the liver of mice injected with 1.4 × 108 TU / mouse (n = 6), 5 × 108 TU / mouse (n = 10), and 1 × 109 TU / mouse (n = 6), respectively. [Figure 4]Figure 1: Analysis of AGXT expression in livers of Agxt1- / - injected mice after in vivo lentiviral vector therapy. Comparison with physiological mouse Agxt expression in C57BL / 6 wild type mice. AGXT expression in therapeutic lentiviral vector injected Agxt1- / - mice was analyzed by RT-qPCR. AGXT expression expression was referenced to housekeeping genes. Two different housekeeping genes with different expression levels were used in this analysis (msTbp, low level expression, and msActb, high level expression) to determine the difference between physiological Agxt expression in wild type mice (higher expression level) compared to AGXT expression induced by lentiviral vector integration after in vivo gene therapy strategy (lower expression level). A. AGXT expression referenced to msTbp expression in livers of LV.ET.AGXT-RHEAM.142-3pT injected Agxt1- / - mice and non-injected wild type mice. Therapeutic lentivirus-injected mice receiving doses of 2.5 × 108 TU / mouse (n = 6), 5 × 108 TU / mouse (n = 10) and 1 × 109 TU / mouse (n = 6) resulted in AGXT expression, expressed as % of msTbp expression, of 10.88 (5.62-16.79), 12.11 (4.38-14.48) and 19.88 (18.52-28.55), respectively, which correlates with the dose administered. Non-injected Agxt1- / - mice (n = 8) showed no AGXT expression. Furthermore, wild-type C57BL / 6 mice (n = 4) showed Agxt expression of 8563% of msTbp expression (6996%-8939%), significantly higher than that achieved in Agxt1- / - treated mice. B. AGXT expression relative to msActb expression in the liver of LV.ET.AGXT-RHEAM.142-3pT-injected Agxt1− / − mice and non-injected wild-type mice.Agxt expression expressed as % of msActb housekeeping gene expression was 0.1041 (0.06007-0.2452) (n=6) in Agxt1- / - mice administered a therapeutic lentiviral vector dose of 2.5 × 108 TU / mouse, 0.1857 (0.08878-0.2966) (n=10) in mice injected with 5 × 108 TU / mouse, and 0.3088 (0.1707-0.4418) (n=6) in mice injected with 1 × 109 TU / mouse. Physiological expression of Agxt referenced to msActb was 340.5% (141%-374.5%). Although the difference in Agxt expression levels was housekeeping gene dependent, the difference between groups was maintained. [Diagram 5] Figure 1 shows the percentage of transduced cells in reporter lentiviral vector C57BL / 6 and Agxt1- / - injected mice. The percentage of eGFP expressing hepatocytes in liver sections of reporter lentiviral vector injected wild type C57BL / 6 and Agxt1- / - mice was determined by immunostaining and subsequent image analysis with QuPath™ software. A-E. Representative images of immunostaining for eGFP in liver sections of all mouse groups. Green: eGFP, blue: nuclei stained with DAPI. F-H. Representative images of double immunostaining for eGFP and mouse albumin, a hepatocyte specific marker, in liver sections of C57BL / 6 mice. Red: eGFP, green: mouse albumin, blue: nuclei stained with DAPI. I. Percentage of transduced hepatocytes. For experiments performed in C57BL / 6 mice, two different reporter lentiviral vector doses were tested (1 × 108 TU / mouse (n = 3) and 2.5 × 108 TU / mouse (n = 3)). This corresponds to 1.751% (1.132% to 5.556%) and 12.92% (10.72% to 13.81%) positive hepatocytes, respectively. Furthermore, in Agxt1- / -, two different doses were tested (1.4 × 108 TU / mouse (n = 4) and 5 × 108 TU / mouse (n = 5)), resulting in 1.777% (0.399% to 4.572%) eGFP-positive hepatocytes and 5.5059% (2.109% to 8.754%) eGFP-positive hepatocytes, respectively. [Figure 6] Figure 1 shows biodistribution analysis. To determine the safety of the proposed therapeutic strategy, the number of lentiviral vector integrated genomes was analyzed in C57BL / 6 wild-type mice injected with two different doses of reporter lentiviral vector. The number of LV genome copies integrated in different tissues of injected wild-type C57BL / 6 mice was analyzed by qPCR. The integration of reporter lentiviral vector genome was observed only in the target tissue liver, as well as in lung, spleen, and bone marrow. C57BL / 6 mice injected with 1 × 108 TU / mouse (n = 3) showed off-target transduction with VCN values ​​of 2.72 (2.33-6.2) in the lung, 0.21 (0.14-0.32) in the spleen, and 0.05 (0.04-0.09) in the bone marrow, whereas mice injected with the higher dose of 2.5 × 108 TU / mouse (n = 3) showed VCN values ​​of 4.94 (0.185-7.79) in the lung, 0.35 (0.25-0.55) in the spleen, and 0.13 (0.09-0.18) in the bone marrow. [Figure 7]PH1 endpoint. Urinary oxalate concentrations during the ethylene glycol challenge test. Quantification of urinary oxalate (μmol / 24 h) at baseline and on days 3 and 7 of the ethylene glycol challenge test. All mice shown underwent a 7-day ethylene glycol challenge test with 0.5% ethylene glycol added to the drinking water. Two different negative control groups are shown, one consisting of Agxt1− / − uninjected mice (n=8) and the other consisting of Agxt1− / − mice (n=9) injected with two different doses of LV.ET.GFP.142-3pT (1.4×108 TU / mouse and 5×108 TU / mouse). A positive control group consisting of wild-type C57BL / 6 uninjected mice (n=5) is also included. Therapeutic lentiviral vector-injected Agxt1- / - mice were divided into three different groups according to the administered dose (2.5x108TU / mouse (n=6), 5x108TU / mouse (n=10) or 10x108TU / mouse (n=6)). Basal data show higher oxalate concentrations in treated and untreated Agxt1- / - mice compared to wild-type mice. During the ethylene glycol challenge, an increase in urinary oxalate levels was observed in all groups, but this increase was greater in untreated Agxt1 mice (uninjected and LV.ET.eGFP.142-3pT-injected Agxt1 mice) compared to Agxt1 mice treated with different doses of LV.ET.AGXT-RHEAM.142-3pT (2.5×108TU / mouse, 5×108TU / mouse, or 10×108TU / mouse) and wild-type C57BL / 6 mice. This difference was much greater in Agxt1 mice that received 5×108TU / mouse of LV.ET.AGXT-RHEAM.142-3pT on day 7 of the ethylene glycol challenge compared to the untreated group. Furthermore, no significant differences were observed between Agxt1- / - and wild-type C57BL / 6 mice treated with therapeutic LV at 5 × 108 TU / mouse on the 7th day of ethylene glycol treatment. [Figure 8]PH1 endpoint: Body weight change during ethylene glycol challenge. Body weight progression during ethylene glycol challenge was analyzed. Animal body weight was measured at baseline before the challenge and on days 3 and 7 of the challenge. The figure shows the weight gain or loss relative to the baseline body weight of each animal. Two different negative control groups are shown, one composed of Agxt1− / − non-injected mice and the other composed of Agxt1− / − mice injected with two different doses of LV.ET.GFP.142-3pT (1.4×108TU / mouse and 5×108TU / mouse). A positive control group composed of wild-type C57BL / 6 non-injected mice is also included. Therapeutic lentiviral vector-injected Agxt1− / − mice are divided into three different groups according to the administered dose (2.5×108TU / mouse, 5×108TU / mouse or 10×108TU / mouse). In the case of therapeutic lentiviral vector-treated mice, no weight loss was observed on day 7 of the challenge test, but an increase was observed, whereas in the case of untreated mice (non-injected or reporter lentiviral vector-injected Agxt1- / - mice), weight loss was observed during the challenge test. Furthermore, no significant difference was observed between wild-type C57BL / 6 mice and Agxt1- / - mice injected with therapeutic lentiviral vectors at doses of 5 x 108 TU / mouse or 10 x 108 TU / mouse, whereas a significant difference was observed between these groups and non-treated mice. [Figure 9]PH1 endpoint. Kidney damage score. Kidney damage development after ethylene glycol challenge. To determine kidney damage in treated and untreated mice subjected to ethylene glycol challenge, kidney tissue samples were classified according to the "kidney damage score" as follows: 0: no damage, 1: normal structure but some signs of damage such as dilated ducts, 2: partially affected structure with increasing presence of dilated ducts, 3: tissue structure is heavily affected and the presence of calcium oxalate crystals can be observed, indicating a stage of nephrocalcinosis. A. Representative images of hematoxylin-eosin kidney sections from each mouse group. Representative images of samples with kidney damage scores of 0-1 from each group (indicating no kidney damage or mild kidney damage) and samples with kidney damage scores of 2-3 (indicating moderate or severe kidney damage). The number of mice that developed that level of kidney damage relative to the total number of mice in each group is specified on each image. According to the different mouse groups, two different negative control groups are shown, one composed of Agxt1- / - non-injected mice (n=8) and the other composed of Agxt1- / - mice (n=9) injected with two different doses of LV.ET.GFP.142-3pT (1.4x108TU / mouse and 5x108TU / mouse). A positive control group composed of wild-type C57BL / 6 non-injected mice (n=5) is also included. Therapeutic lentiviral vector-injected Agxt1- / - mice are divided into three different groups according to the administered dose: 2.5x108TU / mouse (n=6), 5x108TU / mouse (n=10) or 10x108TU / mouse (n=6). B. The kidney damage stage of each animal is represented. The grey bands represent the development of nephrocalcinosis. Among the differently treated Agxt1− / − mice, none of the mice injected with intermediate doses of therapeutic lentiviral vectors developed nephrocalcinosis. [Figure 10]Figure 2: Transduction enhancer: Cyclosporine H increases reporter lentiviral vector transduction in HepG2 cell line. HepG2 cells were transduced with a combination of LV.ET.eGFP.142-3pT and Cyclosporine H (CsH) at different dosing regimens. The fold increase in the percentage of eGFP positive cells between different CsH treatment conditions compared to reporter lentiviral vector transduced cells without CsH is presented. In the first transduction protocol, HepG2 cells were exposed to different concentrations of CsH (4 μM, 8 μM or 16 μM) during lentiviral vector transduction. Cells were transduced with a lentiviral vector MOI of 0.3. Three technical replicates of each condition were performed. The basal transduction level was 36.67%, and the different CsH-treated samples showed transduction percentages of 37.67%, 44.3% and 51.5% in the 4 μM, 8 μM and 16 μM CsH conditions, which represents an increase of 1.03-fold, 1.2-fold and 1.4-fold, respectively. Additionally, another CsH dosing regimen was tested. HepG2 cells were pretreated with CsH for 16 h before transduction, and CsH was added together with LV.ET.eGFP.142-3pT during lentiviral transduction. The only CsH concentration tested was 16 μM. A 2.35-fold increase in eGFP-positive cells was observed with this CsH dosing regimen versus the control group. When cells were transduced with an MOI of 0.3 with LV.ET.eGFP.142-3pT, the percentage of transduced cells achieved was 13.2% in the control group and 31.03% in the CsH-treated cells. The observed difference in transduction levels at the same MOI is likely due to the difference in the virus stocks used. [Figure 11] FIG. 1 shows the LV.ET.eGFP.142-3pT construct. [Figure 12] FIG. 1 shows the LV.ET.AGXT-RHEAM.142-3pT construct. [Figure 13]FIG. 1 shows a comparison of LV.ET.eGFP.142-3pT transduction efficacy in vitro and in vivo. To determine in vitro transduction efficacy, HepG2 cells were transduced with LV.ET.eGFP.142-3pT at different multiplicities of infection (MOI). Three days after transduction, the percentage of eGFP positive cells was determined by flow cytometry. Using an MOI of approximately 1, a 50% transduction percentage was achieved, whereas at higher MOIs, 100% transduction was achieved. To determine in vivo transduction efficacy, adult C57BL / 6 (closed circles) or Agxt1− / − mice (open circles) were intravenously injected with LV.ET.eGFP.142-3pT at different doses (1×108 TU / mouse, 1.4×108 TU / mouse, 2×108 TU / mouse and 5×108 TU / mouse). The corresponding MOI was calculated solely according to the estimated number of hepatocytes in the livers of these mice as previously described (Park et al., 2000; Schmitt et al., 2010). Therefore, mice were injected with the following MOIs: 0.8, 1.1, 2, and 4. The percentage of transduced hepatocytes was determined in liver tissue samples after 4 weeks. [Figure 14]Figure 2 shows that dexamethasone treatment improves transduction efficacy in the liver of in vivo reporter lentiviral vector-treated C57BL / 6 mice. In vivo experiments were performed to test the use of dexamethasone as a transduction enhancer in the context of the proposed strategy. A. C57BL / 6 adult male mice were intravenously injected with a single dose of LV.ET.eGFP.142-3pT (5x108TU / mouse) by intravenous injection, either in combination with or without dexamethasone (Fortecortin™). Dexamethasone was administered by intraperitoneal injection at a dose of 5mg / kg body weight in three sequential doses, 12 hours and 2 hours before and 4 hours after lentiviral vector injection. Mice were sacrificed after 4 weeks. The transduction efficacy was then determined by analyzing the genome copy number of the integrated lentiviral vector and the percentage of transduced cells in the target tissue, the liver. B. Lentiviral vector integration in the liver was analyzed by quantification of VCN by qPCR. Two different mouse treatment groups are shown, one treated with lentiviral vector only (n=5) and the other with combination treatment (n=5). In mice treated with reporter lentiviral vector only, 1.48 (1.2-1.76) copies per diploid genome were found, whereas in mice treated with dexamethasone as well, 0.92 (0.38-1.4) copies per diploid genome were detected, a significant decrease compared to mice treated with lentiviral vector only (p<0.05). C. The percentage of eGFP-positive hepatocytes in liver sections was identified by immunostaining and subsequent image analysis with QuPath™ software. In mice treated with the reporter lentiviral vector alone, 4.434% (2.167%-7.55%) eGFP-positive hepatocytes were observed, whereas in mice treated with the combination of lentiviral vector and dexamethasone, 18.11% (6.381%-19.79%) eGFP-positive hepatocytes were obtained, a significant increase over mice treated with lentiviral vector alone (p<0.05). D and E. Representative images of immunostaining for eGFP in liver sections from all mouse groups. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] In a first aspect, the present invention provides a lentiviral vector (LV) comprising a nucleic acid, the nucleic acid comprising a transcription unit, the transcription unit being defined herein as comprising from 5' to 3' a hepatocyte-specific promoter, preferably an ET promoter, a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (hereinafter referred to as AGXT-RHEAM) protein, a Woodchuck Hepatitis Virus post-transcriptional regulatory element (hereinafter referred to as WPRE), preferably a mutated WPRE, and at least one copy of at least one miRNA target sequence, wherein the hepatocyte-specific promoter, the WPRE and the at least one copy of the at least one miRNA target sequence are operably linked to the AGXT-RHEAM and regulate expression of the AGXT-RHEAM.

[0008] Preferably, the nucleic acid is one of the following: an engineered hepatocyte-specific promoter having at least 98% sequence identity over the entire length of SEQ ID NO: 16; A nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM), wherein the AGXT-RHEAM protein has at least 98% amino acid sequence identity over the entire length of SEQ ID NO:52; A mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 98% sequence identity over the entire length of SEQ ID NO:21; At least a copy of at least one miRNA target sequence, wherein the miRNA target sequence is selected from the group consisting of miRNA-142-3, miR-181, miR-223, or miR-30b; Including, The engineered hepatocyte-specific promoter, the WPRE and at least one copy of the miRNA target sequence are operably linked to the AGXT-RHEAM protein to regulate the expression of the AGXT-RHEAM protein. Preferably, the amino acid sequence of the alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) consists only of SEQ ID NO:52. Preferably, the nucleotide sequence of the engineered hepatocyte-specific promoter consists only of SEQ ID NO:16. Preferably, the nucleotide sequence of the mutated optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) consists only of SEQ ID NO:21. Preferably, the nucleotide sequence coding for the optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein consists only of SEQ ID NO:29. Preferably, the at least one copy of the miRNA target sequence consists only of four copies of the target sequence of miRNA-142-3 having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO:22.

[0009] In a preferred embodiment, the nucleic acid contained in the lentiviral vector preferably comprises, 5' to 3', the following: a) a 5' long terminal repeat (LTR); b) a primer binding site (PBS); c) a psi packaging signal; d) the stem loop 4 (SL4) region of the wild-type HIV virus; and e) a Rev response element (RRE); f) DNA flap central polypurine tract (cPPT); g) a 3' long terminal repeat (LTR); Further comprising: Preferably, the nucleotide further comprises a DenvRF1 region located between d) and e) and a DenvRF2 region located between e) and f).

[0010] In a preferred embodiment of the first aspect or any of its embodiments, the lentiviral vector comprises a nucleic acid, the nucleic acid comprising from 5' to 3' the following nucleotides: a) a 5' long terminal repeat (LTR); b) a primer binding site (PBS); c) a psi packaging signal; d) the stem loop 4 (SL4) region of the wild-type HIV virus; and e) a Rev response element (RRE); f) DNA flap central polypurine tract (cPPT); g) an engineered hepatocyte-specific promoter having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO: 16; h) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO: 29; i) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO:21; j) at least one copy of at least one miRNA target sequence; k) a 3' long terminal repeat (LTR); Including, The long terminal repeat regions of a) and k) are substantially transcriptionally inactivated by a full or partial deletion in the U3 region of the LTR, and g), i) and j) are operably linked to h) and regulate the expression of h). In a preferred embodiment of the first aspect or any of its embodiments, the sequence identity of g), h), i) to SEQ ID NO: 16, SEQ ID NO: 29, and SEQ ID NO: 21, respectively, is 100%.

[0011] In a preferred embodiment of the first aspect or any of its embodiments, the at least copy of the at least one miRNA target sequence of j) consists of only four copies of the target sequence of miRNA-142-3 having at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO: 22.

[0012] In a preferred embodiment of the first aspect or any of its embodiments, the nucleotides comprised in the lentiviral vector are l) the DenvRF1 region located between d) and e); m) the DenvRF2 region located between e) and f); Further includes:

[0013] In a preferred embodiment of the first aspect or any of its embodiments, the 5' long terminal repeat comprised in the nucleotide sequence of said lentiviral vector has at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO:4.

[0014] In a preferred embodiment of the first aspect or any of its embodiments, the primer binding site contained in the nucleotide sequence of said lentiviral vector has at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO:9.

[0015] In a preferred embodiment of the first aspect or any of its embodiments, the psi packaging signal contained in the nucleotide sequence of the lentiviral vector has at least 95%, preferably 98%, and most preferably 100% sequence identity over the entire length of SEQ ID NO:10.

[0016] In a preferred embodiment of the first aspect or any of its embodiments, stem loop 4 (SL4) comprised in the nucleotide sequence of said lentiviral vector has at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO:11.

[0017] In a preferred embodiment of the first aspect or any of its embodiments, the Rev response element (RRE) comprised in the nucleotide sequence of said lentiviral vector has at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO: 13.

[0018] In a preferred embodiment of the first aspect or any of its embodiments, the DNA flap central polypurine tract (cPPT) comprised in the nucleotide sequence of said lentiviral vector has at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO: 15.

[0019] In a preferred embodiment of the first aspect or any of its embodiments, the 3' long terminal repeat comprised in the nucleotide sequence of said lentiviral vector has at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO:23.

[0020] In a preferred embodiment of the first aspect or any of its embodiments, the DenvRF1 comprised in the nucleotide sequence of said lentiviral vector has at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO: 12.

[0021] In a preferred embodiment of the first aspect or any of its embodiments, the DenvRF2 comprised in the nucleotide sequence of said lentiviral vector has at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO:14.

[0022] In a preferred embodiment of the first aspect or any of its embodiments, the full length nucleotide sequence of said lentivirus has at least 95%, preferably 98%, most preferably 100% sequence identity to SEQ ID NO: 30 (LV.ET.AGXT-RHEAM.142-3pT).

[0023] In a second aspect, the present invention relates to a host cell or a substantially pure population of cells comprising (e.g. transduced) a lentiviral vector as defined in the first aspect or any of its embodiments. In one embodiment of the second aspect, the host cell or substantially pure population of cells is a hepatocyte.

[0024] In a third aspect, the present invention relates to an isolated nucleic acid comprising a nucleotide sequence comprised in a lentiviral vector as defined in the first aspect or any of its embodiments.

[0025] In a fourth aspect, the present invention provides a pharmaceutical composition comprising a lentivirus as defined in the first aspect or any of its embodiments, a host cell or a substantially pure population of cells according to the second aspect or any of its embodiments, and / or a nucleic acid as defined in the third aspect or any of its embodiments, and a pharma- ceutically acceptable carrier or diluent.

[0026] In a fifth aspect, the present invention provides a lentiviral vector according to the first aspect or any of its embodiments, a host cell or a substantially pure population of cells according to the second aspect or any of its embodiments, an isolated nucleic acid according to the third aspect or any of its embodiments, or a pharmaceutical composition according to the fourth aspect or any of its embodiments, for use as a medicament.

[0027] In a sixth aspect, the present invention provides a lentiviral vector according to the first aspect or any of its embodiments, a host cell or substantially pure cell population according to the second aspect or any of its embodiments, an isolated nucleic acid according to the third aspect or any of its embodiments, or a pharmaceutical composition according to the fourth aspect or any of its embodiments, for use in the treatment of primary hyperoxaluria, preferably primary hyperoxaluria type 1, comprising administering said lentivirus, said host cell or cell population, or said composition to a subject.

[0028] Preferably, the use according to the fifth and sixth aspects is combined with an immune response suppressant, preferably dexamethasone, cyclosporin A, cyclosporin H and / or rapamycin, or any combination thereof.

[0029] General definition It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Further, unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0030] The term "about" is used herein to mean approximately, roughly, around, or approximately. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 10% above and below (high and low).

[0031] As used herein, the conjunction "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the first element being applicable except for the second element. The second option refers to the second element being applicable except for the first element. The third option refers to the first and second elements being applicable together. Any one of these options is understood to be within the meaning and thus meet the requirements of the term "and / or" as used herein. It is also understood that two or more options may be simultaneously applicable and thus meet the requirements of the term "and / or."

[0032] Throughout this specification and the appended claims, unless the context otherwise requires, the word "comprise", as well as variants such as "comprises" and "comprising", will be understood to mean the inclusion of the specified integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" may be replaced with the term "containing" or "including", or, as used herein, sometimes with the term "having". Any of the above terms (comprising, containing, including, having) whenever used herein in the context of an aspect or embodiment of the invention may be replaced with the term "consisting of", although this is less preferred.

[0033] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element.

[0034] A protein or nucleotide "consisting essentially of" a protein or nucleotide is one that has an amino acid or nucleotide sequence substantially identical to a particular protein or nucleotide.

[0035] A protein or nucleotide that has "essentially the same amino acid or nucleotide sequence" as a protein or nucleotide, respectively, typically has greater than 90% amino acid or nucleotide identity with that protein or nucleotide. This definition includes conservative amino acid substitutions.

[0036] The term "isolated" for purposes of the present invention refers to biological material (cells, polypeptides, polynucleotides, or fragments, variants, or derivatives thereof) that has been removed from its original environment (the environment in which it naturally occurs). For example, a polynucleotide occurring in the natural state in a plant or animal is not isolated, but the same polynucleotide separated from the adjacent nucleic acids in which it naturally occurs is considered "isolated."

[0037] "Nucleic acid", "nucleic acid molecule", "oligonucleotide", and "polynucleotide" are used interchangeably and refer to the polymeric form of phosphate ester of ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule"), or any of their phosphate analogs, such as phosphorothioates and thioesters, in single-stranded or double-stranded helical form. The term nucleic acid molecule, and in particular DNA molecule or RNA molecule, refers only to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. Thus, the term includes linear or circular DNA molecules (e.g., restriction fragments), double-stranded DNA found in plasmids, supercoiled DNA, and chromosomes, among others.

[0038] A "coding region" or "coding sequence" is a portion of a polynucleotide that consists of codons translatable into amino acids.

[0039] The term "downstream" refers to a nucleotide sequence located 3' to a reference nucleotide sequence. In certain embodiments, a downstream nucleotide sequence relates to a sequence that follows the start of transcription.

[0040] The term "upstream" refers to a nucleotide sequence located 5' to a reference nucleotide sequence. In certain embodiments, an upstream nucleotide sequence relates to a coding region or a sequence located 5' to the start of transcription.

[0041] As used herein, the term "gene regulatory region" or "regulatory region" refers to a nucleotide sequence located upstream (5' sequences), within, or downstream (3' sequences) of a coding region that influences the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. If the coding region is intended for expression in a eukaryotic cell, polyadenylation signals and transcription termination sequences are usually located 3' to the coding sequence.

[0042] The term "post-transcriptional regulatory element" refers to a DNA sequence that, when transcribed, produces a tertiary structure that enhances or represses the expression of a protein.

[0043] "Transcriptional control sequence" refers to a DNA regulatory sequence, such as promoters, enhancers, terminators, and the like, that provides for the expression of a coding sequence in a host cell.

[0044] The term "promoter" refers to a DNA sequence to which RNA polymerase can bind to initiate transcription. This sequence may further contain binding sites for various proteins that regulate transcription, such as transcription factors. A promoter sequence may be composed of different promoter fragments (either different fragments or the same fragment) that are closely located in the DNA sequence and may be separated by linkers or spacers. Such promoters are called chimeric promoters.

[0045] The terms "treatment" and "therapy" as used in this application refer to a set of hygienic, pharmacological, surgical and / or physical measures used with the goal of improving a health problem with the objective of curing and / or alleviating a disease and / or symptom. The terms "treatment" and "therapy" include preventative and curative methods, since both are directed to maintaining and / or restoring the health of an individual or animal. The administration of suitable medicines to alleviate and / or cure a health problem, regardless of the cause of the symptoms, disease and disability, should be interpreted as a form of treatment or therapy within the context of this application.

[0046] The term "therapeutically effective amount" refers to an amount of a substance that has a therapeutic effect and is capable of treating PH1.

[0047] The terms "individual", "patient" or "subject" are used interchangeably in this application and are not intended to be limiting in any way. An "individual", "patient" or "subject" may be of any age, sex and physical condition.

[0048] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable diluent" refers to any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to, additional buffers, preservatives, cosolvents, antioxidants including ascorbic acid and methionine, chelating agents such as EDTA, metal complexes (e.g., Zn-protein complexes), biodegradable polymers such as polyesters, salt-forming counterions such as sodium, polyhydric sugar alcohols, amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid and threonine, lactitol, stachyose, mannose, These include organic sugars or sugar alcohols such as sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol, sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate, low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins, and hydrophilic polymers such as polyvinylpyrrolidone.

[0049] As used herein, the phrase "subject in need thereof" includes subjects, such as mammalian subjects, who would benefit from the administration of a nucleic acid molecule, polypeptide, or vector provided herein, e.g., to improve hemostasis.

[0050] As used herein, the term "optimized" with respect to a nucleotide sequence refers to a polynucleotide sequence that encodes a polypeptide, where the polynucleotide sequence has been mutated to enhance a property of the polynucleotide sequence.

[0051] The term "lentivirus" (LV) refers to a genus of retroviruses that cause chronic and fatal diseases in humans and other mammalian species that are characterized by long latency periods. A "vector" is any vehicle that can be used to artificially deliver foreign genetic material into cells. Thus, a "lentiviral vector" refers to a recombinant lentivirus that delivers nucleic acid into cells.

[0052] As used herein, "transcription unit" refers to a nucleotide sequence contained in the LV vector of the present invention, comprising from 5' to 3' a hepatocyte-specific promoter, preferably an ET promoter, a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein, a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE), preferably a mutated WPRE, and at least one copy of at least one miRNA target sequence, wherein the hepatocyte-specific promoter, the WPRE, and at least one copy of at least one miRNA target sequence are operably linked to AGXT-RHEAM and regulate expression of AGXT-RHEAM.

[0053] explanation Although lentiviral vectors are an attractive tool for gene delivery, the efficiency of transduction is key to ensure that sufficient cells are genetically modified to obtain the desired therapeutic effect. Unfortunately, it is commonly found that the rate of transduction observed in vitro does not correspond to the transduction efficiency achieved in vivo, and this is particularly emphasized when the vector is not or cannot be delivered directly to the tissue where gene correction is desired (e.g., in the case of liver-specific diseases) but is delivered by other routes, such as the parenteral route, where the vector is taken up into the bloodstream and targeted by the host's immune response. The authors of the present invention show in Figure 13 the transduction efficiency of the same lentiviral vector used in vitro (in HepG2 cells) and in vivo (in C57BL / 6 mice), showing that there is a significant reduction in the percentage of transduced cells when the virus is in the in vivo context. Moreover, the transduction efficiency not only differs in vitro / in vivo, or between local and systemic delivery, but also depends on the type of lentivirus used and its ability to transduce cells and integrate into the host cell genome. In particular, the ability to transduce target cells is clearly influenced by the lentiviral backbone (Johnson et al., 2021, Mol Therapy). Often, small changes in the lentiviral backbone translate into large changes in its transduction efficiency. Taking all this into account, it can be asserted that it is difficult to determine whether a particular lentiviral vector containing a gene of interest (transgene) will work efficiently in vivo against a particular disease.

[0054] Furthermore, some diseases such as hemophilia (e.g., hemophilia A) can be treated with lentiviral vectors, even though the transduction efficiency of lentiviral vectors reaches less than 10% of cells in vivo. This is because in these cases, a low in vivo transduction efficiency of 5% can be sufficient to obtain a sufficient therapeutic effect to reverse the disease phenotype. However, in the specific case of PH1, it is widely accepted that at least 40% of the hepatocytes need to be corrected, i.e., efficiently produce AGXT protein, to at least partially reverse the PH1 phenotype. Castello et al. have previously demonstrated that hepatocyte transplantation strategies are unlikely to be successful because correction of PH1 requires a large number of engrafted hepatocytes, which is likely to exceed the capacity of this procedure (Castello et al. 2015. doi: 10.1038 / gt.2015.107). Moreover, Jiang and colleagues reveal that some experts estimate that at least 75% of the overproducing mutant hepatocyte mass must be replaced by wild-type hepatocytes to achieve a clinical cure (Jiang et al. 2008. DOI: 10.1097 / TP.0b013e31816de49e). However, the present authors have developed a lentiviral vector that is able to correct the defect in the expression of AGXT protein in hepatocytes, causing a reduction in oxalate accumulation and thus partially reverting the PH1 phenotype, as shown in Figures 7-9. Surprisingly, this effect provided by the lentiviral vector described herein was achieved despite the fact that said lentiviral vector was only able to transduce about 5% of cells in vivo, as shown in Figure 5.

[0055] Therefore, the present invention fulfills an important need in the art by providing a lentiviral vector comprising a codon-optimized AGXT sequence, which increases the intracellular level of AGXT protein upon transduction into hepatocytes, resulting in superior therapeutic efficacy for treating PH1. A schematic diagram of the nucleic acid sequence contained in the lentiviral vector provided herein is shown in Figure 12.

[0056] Thus, in a first aspect, the present invention provides a lentiviral vector (LV) comprising a nucleic acid, the nucleic acid comprising a transcription unit, the transcription unit being defined herein as comprising from 5' to 3' a hepatocyte-specific promoter, preferably an ET promoter, a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (hereinafter referred to as AGXT-RHEAM) protein, a Woodchuck Hepatitis Virus post-transcriptional regulatory element (hereinafter referred to as WPRE), preferably a mutated WPRE, and at least one copy of at least one miRNA target sequence, the hepatocyte-specific promoter, the WPRE and the at least one copy of at least one miRNA target sequence being operably linked to AGXT-RHEAM and regulating the expression of AGXT-RHEAM. A coding sequence and a gene expression control sequence are said to be operably linked when they are linked in such a way that the expression or transcription and / or translation of the coding sequence is under the influence or control of the gene expression control sequence. For example, a hepatocyte-specific promoter, a WPRE and at least one copy of at least one miRNA target sequence are operably linked to a nucleotide sequence encoding an optimized AGXT-RHEAM protein such that the expression level of AGXT is regulated by the promoter and the WPRE.

[0057] In one embodiment, the nucleic acid contained in the LV vector according to the first aspect further comprises one or more lentiviral backbone elements, defined as nucleic acid encoding proteins necessary for LV to carry out its functions (infecting cells and persisting within the cell via integration into the viral genome) and nucleic acid encoding regulatory sequences necessary for LV to carry out its functions.

[0058] Thus, the LV vector of the first embodiment comprises a nucleic acid, which comprises 1) a transcription unit and 2) one or more LV vector backbone elements. Note that all of the nucleic acids contained in the transcription unit and the LV vector backbone can be combined. Each of these two components is described in detail below.

[0059] 1) Transcription unit As defined above, in this specification, a transcription unit refers to a nucleic acid sequence contained in the LV vector of the present invention, which comprises from 5' to 3' a hepatocyte-specific promoter, a nucleotide sequence encoding an optimized AGXT-RHEAM protein, a WPRE element, and at least one copy of at least one miRNA target sequence, wherein the hepatocyte-specific promoter, the WPRE, and at least one copy of at least one miRNA target sequence are operably linked to AGXT-RHEAM and regulate the expression of AGXT-RHEAM.

[0060] Hepatocyte-specific promoter The hepatocyte-specific promoter directs AGXT-RHEAM expression following lentiviral vector genome integration into target cells.

[0061] In some embodiments, the nucleic acid sequence contained in the lentiviral vector comprises at least one tissue-specific promoter, i.e., a promoter that will regulate the expression of the optimized AGXT protein in a specific tissue or cell type. In some embodiments, the tissue-specific promoter in the lentiviral vector selectively enhances the expression of the optimized AGXT protein in target liver cells. In some embodiments, the target liver cells are hepatocytes or their precursor cells. In some embodiments, the isolated nucleic acid molecule contained in the lentiviral vector is stably integrated into the genome of the target cell or tissue, for example, the genome of a hepatocyte.

[0062] In some embodiments, the lentiviral vector comprises a hepatocyte-specific promoter for transgene expression. In one embodiment, the nucleotide comprising the hepatocyte-specific promoter is a genetically engineered hepatocyte-specific promoter. In some embodiments, the tissue-specific promoter that selectively enhances the expression of the optimized AGXT protein in the target liver cells comprises a transthyretin promoter (TTRp). In one embodiment, the hepatocyte-specific promoter is a chimeric promoter. In some embodiments, the chimeric hepatocyte-specific promoter is an ET promoter consisting of only a synthetic enhancer, a transthyretin enhancer (hereinafter referred to as TTR enhancer), a transthyretin promoter (hereinafter referred to as TTRp), and a transthyretin 5'UT (hereinafter referred to as mTTR 5'UT).

[0063] In a preferred embodiment, the synthetic enhancer of the ET promoter comprises SEQ ID NO: 17 or a sequence that is at least 98% identical over the entire length of SEQ ID NO: 17. Preferably, the synthetic enhancer of the ET promoter comprises SEQ ID NO: 17 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 17. More preferably, the synthetic enhancer of the ET promoter is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 17 or SEQ ID NO: 17.

[0064] In some embodiments, the nucleotide sequence of the synthetic enhancer of the ET promoter contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:17.

[0065] In a preferred embodiment, the TTR enhancer of the ET promoter is a mouse TTR enhancer (mTTR enhancer) and comprises SEQ ID NO: 18 or a sequence that is at least 98% identical over the entire length of SEQ ID NO: 18. Preferably, the mTTR enhancer of the ET promoter comprises a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 18. More preferably, the mTTR enhancer of the ET promoter is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 18 or SEQ ID NO: 18.

[0066] In some embodiments, the nucleotide sequence of the mTTR enhancer of the ET promoter contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:18.

[0067] In a preferred embodiment, the transthyretin promoter (TTRp) of the ET promoter is mouse TTRp (mTTRp) and comprises SEQ ID NO: 19 or a sequence that is at least 98% identical over the entire length of SEQ ID NO: 19. Preferably, the mTTRp of the ET promoter comprises a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 19. More preferably, the mTTRp of the ET promoter is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 19 or SEQ ID NO: 19.

[0068] In some embodiments, the nucleotide sequence of the ET promoter mTTRp contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:19.

[0069] In a preferred embodiment, the transthyretin 5'UT (TTR 5'UT) of the ET promoter is the mouse TTR 5'UT (mTTR 5'UT) and comprises SEQ ID NO:20 or a sequence that is at least 98% identical over the entire length of SEQ ID NO:20. Preferably, the mTTR 5'UT of the ET promoter comprises a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:20 or SEQ ID NO:20. More preferably, the mTTR 5'UT of the ET promoter is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:20 or SEQ ID NO:20.

[0070] In some embodiments, the nucleotide sequence of the mTTR 5'UT of the ET promoter contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:20.

[0071] The sequence identity between two sequences can be determined by conventional methods, for example, by using standard alignment algorithms known in the art, such as BLAST (Altschul et al., 1990. J Mol Biol. 215(3): 403-10). In a preferred embodiment, the sequence identity between two sequences is determined using BLAST.

[0072] In a preferred embodiment, the hepatocyte-specific promoter is an ET promoter consisting only of the synthetic enhancer of SEQ ID NO: 17, the mTTR enhancer of SEQ ID NO: 18, the mTTRp of SEQ ID NO: 19, and the mTTR 5'UT of SEQ ID NO: 20, or sequences that are at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 17, 18, 19, or 20, respectively.

[0073] In a preferred embodiment, the nucleotide sequence of the hepatocyte-specific ET promoter comprises SEQ ID NO: 16 or a sequence that is at least 98% identical over the entire length of SEQ ID NO: 16. Preferably, the nucleotide sequence of the hepatocyte-specific ET promoter comprises SEQ ID NO: 16 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 16. More preferably, the nucleotide sequence of the hepatocyte-specific ET promoter is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 16 or SEQ ID NO: 16.

[0074] In some embodiments, the nucleotide sequence, which is a liver-specific promoter contained in the lentiviral vector and which selectively enhances expression of the optimized AGXT protein in target liver cells, comprises, consists of, or essentially consists of the ET promoter of SEQ ID NO:16.

[0075] Nucleotide sequence encoding optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein The lentiviral vector provided herein comprises a codon-optimized polynucleotide encoding an optimized AGXT protein. In one embodiment, the codon-optimized polynucleotide AGXT is operably linked to at least one, preferably two, expression control sequences comprising a promoter and a post-transcriptional element. In some embodiments, the present invention provides a lentiviral vector comprising an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having an activity similar to AGXT. In one embodiment, the nucleotide sequence contained in the lentiviral vector and coding for AGXT-RHEAM encodes a signal peptide that places newly synthesized proteins in the peroxisomes of cells.

[0076] In a preferred embodiment, the nucleotide sequence encoding for AGXT-RHEAM comprises SEQ ID NO:29 or a sequence that is at least 98% identical over the entire length of SEQ ID NO:29. Preferably, the nucleotide sequence encoding for AGXT-RHEAM comprises a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:29 or SEQ ID NO:29. More preferably, the nucleotide sequence encoding for AGXT-RHEAM is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:29 or SEQ ID NO:29.

[0077] In some embodiments, the nucleotide sequence contained in the lentiviral vector encoding AGXT-RHEAM comprises, consists of, or consists essentially of SEQ ID NO:29.

[0078] In a preferred embodiment, the amino acid sequence of AGXT-RHEAM comprises SEQ ID NO:52 or a sequence that is at least 98% identical over the entire length of SEQ ID NO:52. Preferably, the amino acid sequence of AGXT-RHEAM comprises SEQ ID NO:52 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:52. More preferably, the amino acid sequence of AGXT-RHEAM is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:52 or SEQ ID NO:52.

[0079] In some embodiments, the AGXT-RHEAM protein encoded by the nucleotide sequence contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:52.

[0080] WHV (Woodchuck Hepatitis Virus) Posttranscriptional Regulatory Element (WPRE) The lentiviral vector provided herein also comprises at least one post-transcriptional regulatory element operably linked to the nucleotide coding for optimized AGXT.In one embodiment, the post-transcriptional regulatory element is WHV (Woodchuck Hepatitis Virus) post-transcriptional regulatory element (WPRE).In one embodiment, the post-transcriptional regulatory element is mutated to enhance the safety of the vector.In a preferred embodiment, the mutated WPRE comprises a mutation in gene X frame.

[0081] In a preferred embodiment, the nucleotide sequence comprising the mutated WPRE comprises SEQ ID NO:21 or a sequence that is at least 98% identical over the entire length of SEQ ID NO:21. Preferably, the nucleotide sequence comprising the mutated WPRE comprises SEQ ID NO:21 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:21. More preferably, the nucleotide sequence comprising the mutated WPRE is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:21 or SEQ ID NO:21.

[0082] In some embodiments, the nucleotide sequence WPRE contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:21.

[0083] At least one copy of at least one miRNA target sequence The present invention also provides at least one copy of at least one miRNA target sequence operably linked to the optimized AGXT-RHEAM nucleotide sequence.To increase the efficiency of the system, two or more copies of the miRNA target sequence can be included in the transcription unit.The one or more miRNA target sequences can be the same or different.In one embodiment, the transcription unit included in the lentiviral vector comprises one, two, three, four, five, six, seven or eight copies of the same or different miRNA target sequence.

[0084] In one embodiment, the target sequence is a miRNA-142-3 target. In some embodiments, the lentiviral vector comprises one or more target sequences for miRNA-142-3 to reduce immune response to the transgene product. In one embodiment, the lentiviral vector comprises one, two, three, preferably four, five, six, seven or eight repeats or copies of the miRNA-142-3 target sequence. In some embodiments, the incorporation of one or more target sequences for miRNA-142-3 into the transcription unit of the lentivirus of the present invention allows for a desired transgene expression profile and / or reduced immunogenicity. In particular, the incorporation of one or more target sequences for miRNA-142-3 can suppress transgene expression in intravascular and extravascular hematopoietic lineages, while transgene expression is maintained in non-hematopoietic cells. In certain embodiments, the complementary sequence of a hematopoietic-specific microRNA, such as miRNA-142-3, is incorporated into the 3' untranslated region of the lentiviral vector, rendering the transgene-encoded transcript susceptible to miRNA-mediated downregulation.

[0085] The target sequence may be fully or partially complementary to the miRNA. The term "fully complementary" means that the target sequence has a nucleic acid sequence that is 100% complementary to the sequence of the miRNA that recognizes it. In one embodiment, the nucleic acid sequence contained in the transcription unit includes four repeats of the target sequence of miRNA-142-3.

[0086] In a preferred embodiment, the nucleotide sequence comprising miRNA-142-3 comprises SEQ ID NO:22 or a sequence that is at least 98% identical over the entire length of SEQ ID NO:22. Preferably, the nucleotide sequence comprising miRNA-142-3 comprises SEQ ID NO:22 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:22. More preferably, the nucleotide sequence comprising miRNA-142-3 is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:22 or SEQ ID NO:22.

[0087] In some embodiments, the nucleotide sequence of miRNA-142-3 contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:22.

[0088] In some embodiments, the target sequence is a miR-181 target. In some embodiments, the target sequence is a miR-223 target. In some embodiments, the target sequence is a miR-122 target. In some embodiments, the target sequence is a miR-30b target. In preferred embodiments, the miRNA target sequence is selected from the group consisting of miRNA-142-3, miR-181, miR-223, or miR-30b.

[0089] It should be understood that all the above embodiments of the different elements of the transcription unit (hepatocyte-specific promoter, the nucleotide sequence encoding the optimized AGXT-RHEAM protein, the WPRE element and at least one copy of at least one miRNA target sequence) can be combined with each other. In a preferred embodiment, the transcription unit contained in the nucleic acid of the lentiviral vector comprises: a) an engineered hepatocyte-specific promoter having at least 95%, preferably 98%, and most preferably 100% sequence identity over the entire length of SEQ ID NO: 16; b) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein having at least 95%, preferably 98%, and most preferably 100% sequence identity over the entire length of SEQ ID NO: 29; c) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 95%, preferably 98%, and most preferably 100% sequence identity over the entire length of SEQ ID NO:21; d) at least one copy of at least one miRNA target sequence; comprising or consisting of a), c) and d) are operably linked to b) and regulate the expression of b). In a further preferred embodiment, d) at least one copy of at least one miRNA target sequence preferably consists of only four copies of the target sequence of miRNA-142-3 having at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO:22.

[0090] In a preferred embodiment, the transcription unit contained in the nucleic acid of the lentiviral vector comprises a) an engineered hepatocyte-specific promoter having at least 95%, preferably 98%, and most preferably 100% sequence identity over the entire length of SEQ ID NO: 16; b) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein, the AGXT-RHEAM protein having at least 95%, preferably 98%, and most preferably 100% amino acid sequence identity over the entire length of SEQ ID NO: 52; c) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 95%, preferably 98%, and most preferably 100% sequence identity over the entire length of SEQ ID NO:21; d) at least a copy of at least one miRNA target sequence, wherein the miRNA target sequence is selected from the group consisting of miRNA-142-3, miR-181, miR-223, or miR-30b; comprising or consisting of a), c) and d) are operably linked to b) and regulate the expression of b). In a further preferred embodiment, d) at least one copy of at least one miRNA target sequence preferably consists of only four copies of the target sequence of miRNA-142-3 having at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO:22.

[0091] In a preferred embodiment, the transcription unit contained in the nucleic acid of the lentiviral vector comprises a) a genetically engineered hepatocyte-specific promoter consisting solely of SEQ ID NO: 16; b) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein, the AGXT-RHEAM protein consisting solely of SEQ ID NO: 52; c) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) consisting of only SEQ ID NO: 21; d) at least a copy of at least one miRNA target sequence, wherein the miRNA target sequence is selected from the group consisting of miRNA-142-3, miR-181, miR-223, or miR-30b; comprising or consisting of a), c) and d) are operably linked to b) and regulate the expression of b). In a further preferred embodiment, d) at least one copy of at least one miRNA target sequence preferably consists of only four copies of the target sequence of miRNA-142-3 having at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO:22.

[0092] In a preferred embodiment, the transcription unit contained in the nucleic acid of the lentiviral vector comprises a) an engineered hepatocyte-specific promoter having at least 95%, preferably 98%, and most preferably 100% sequence identity over the entire length of SEQ ID NO: 16; b) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein having at least 95%, preferably 98%, and most preferably 100% sequence identity over the entire length of SEQ ID NO: 29; c) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 95%, preferably 98%, and most preferably 100% sequence identity over the entire length of SEQ ID NO:21; d) at least a copy of at least one miRNA target sequence, wherein the miRNA target sequence is selected from the group consisting of miRNA-142-3, miR-181, miR-223, or miR-30b; comprising or consisting of a), c) and d) are operably linked to b) and regulate the expression of b). In a further preferred embodiment, d) at least one copy of at least one miRNA target sequence preferably consists of only four copies of the target sequence of miRNA-142-3 having at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO:22.

[0093] In a preferred embodiment, the transcription unit contained in the nucleic acid of the lentiviral vector comprises a) a genetically engineered hepatocyte-specific promoter consisting solely of SEQ ID NO: 16; b) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein consisting of SEQ ID NO: 29 alone; c) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) consisting of only SEQ ID NO: 21; d) at least a copy of at least one miRNA target sequence, wherein the miRNA target sequence is selected from the group consisting of miRNA-142-3, miR-181, miR-223, or miR-30b; comprising or consisting of a), c) and d) are operably linked to b) and regulate the expression of b). In a further preferred embodiment, d) at least one copy of at least one miRNA target sequence preferably consists of only four copies of the target sequence of miRNA-142-3 having at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO:22.

[0094] 2) Lentiviral vector backbone elements As defined above, lentiviral vector backbone elements are defined as nucleic acid sequences necessary for LV to carry out its functions (infecting cells and persisting within the cells by integrating into the viral genome).

[0095] Lentiviruses include those belonging to the bovine lentivirus group, the equine lentivirus group, the feline lentivirus group, the ovine and caprine lentivirus group, and the primate lentivirus group.

[0096] In some embodiments, the lentiviral vector provided herein is a "third generation" lentiviral vector. As used herein, the term "third generation" lentiviral vector refers to a lentiviral packaging system that has the characteristics of a second generation vector system and further lacks a functional tat gene, for example, the tat gene is deleted or inactivated. Typically, the gene encoding rev is provided on a separate expression construct. As used herein, the "second generation" lentiviral vector system refers to a lentiviral packaging system that lacks a functional accessory gene, for example, the accessory genes vif, vpr, vpu, and nef are deleted or inactivated. As used herein, "packaging system" refers to a set of viral constructs that include genes encoding viral proteins involved in packaging recombinant virus. Typically, the construct of the packaging system is ultimately integrated into a packaging cell.

[0097] In some embodiments, the third generation lentiviral vector provided herein is a self-inactivating lentiviral vector. In some embodiments, the lentiviral vector is a VSV.G pseudotyped lentiviral vector.

[0098] In certain embodiments, the lentiviral vector is a recombinant lentiviral vector capable of infecting non-dividing cells. In certain embodiments, the lentiviral vector is a recombinant lentiviral vector capable of infecting liver cells (e.g., hepatocytes). Lentiviral genomes and proviral DNA typically have three genes found in retroviruses: gag, pol, and env, which are flanked by two long terminal repeat (LTR) sequences. The gag gene encodes internal structural (matrix, capsid, and nucleocapsid) proteins, the pol gene encodes RNA-dependent DNA polymerase (reverse transcriptase), protease, and integrase, and the env gene encodes viral envelope glycoproteins. The 5'LTR and 3'LTR serve to facilitate transcription and polyadenylation of virion RNA. The LTR contains all other cis-acting sequences required for viral replication. In some embodiments, the lentivirus has additional genes including vif, vpr, tat, rev, vpu, net and vpx (in HIV-I, HIV-2 and / or SIV).

[0099] In certain embodiments, lentiviral vectors are deleted for the HIV virulence genes env, vif, vpr, vpu, and nef without compromising the ability of the vector to transduce non-dividing cells.

[0100] Adjacent to the 5'LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and efficient encapsidation of viral RNA into particles (Psi site). If sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) can be missing from the viral genome, the cis defect will prevent encapsidation of genomic RNA. However, the resulting mutant remains capable of directing the synthesis of all virion proteins.

[0101] Thus, as defined above, the lentiviral vector provided herein comprises a nucleic acid, and the nucleic acid comprises a transcription unit. Furthermore, in another embodiment, the nucleic acid contained in the lentiviral vector comprises, upstream of the transcription unit defined above, i.e., in the 5' region, in the order of 5' to 3', at least one of the following, or any combination thereof: a) 5' long terminal repeat (5'LTR), b) primer binding site (PBS), c) psi packaging signal, d) wild-type HIV virus stem loop 4 (SL4) region, e) Rev response element (RRE), and f) DNA flap central polypurine tract (cPPT). In a preferred embodiment, the nucleic acid contained in the lentiviral vector further comprises, upstream of the transcription unit defined above, i.e., in the 5' region, l) a DenvRF1 region located between d) and e), and m) a DenvRF2 region located between e) and f), or any combination thereof. Each of these nucleotides is further defined below.

[0102] In another embodiment, the nucleic acid contained in the lentiviral vector further comprises a 3' long terminal repeat (LTR) downstream of the transcription unit defined above, i.e., in the 3' region. In some embodiments, the lentiviral vector comprises a deletion of the U3 region of the 5'LTR. The deletion of the U3 region of the 5'LTR can be a complete deletion or a partial deletion.

[0103] It is to be understood that any combination possible between nucleotides a) to f) and j) to k) is encompassed in the present invention and in combination with the transcription units described in the sections above. Each of the nucleotides a) to f) and j) to k) and their preferred embodiments are further defined below.

[0104] Furthermore, in another embodiment, the nucleic acid contained in the lentiviral vector preferably further comprises, upstream (i.e., in the 5' region) of the transcription unit defined above, preferably in the order of 5' to 3', at least one of the following: a) 5' long terminal repeat (5'LTR), b) primer binding site (PBS), c) psi packaging signal, d) wild-type HIV virus stem loop 4 (SL4) region, e) Rev response element (RRE), and f) DNA flap central polypurine tract (cPPT), or any combination thereof. In a preferred embodiment, the nucleic acid contained in the lentiviral vector preferably further comprises, upstream (i.e., in the 5' region) of the transcription unit defined above, h) DenvRF1 region located between d) and e), and i) DenvRF2 region located between e) and f), or any combination thereof. In a further preferred embodiment, the nucleic acid contained in the lentiviral vector preferably further comprises downstream (i.e. in the 3' region) of the transcription unit defined above: g) a 3' long terminal repeat (LTR). As above, it is to be understood that any possible combination between nucleotides a), b), c), d), e), f), h) and i) and g) is included in the present invention and in combination with the transcription units described in the above sections.

[0105] In a preferred embodiment, the nucleotide sequence comprising the 5'LTR comprises SEQ ID NO:4 or a sequence that is at least 98% identical over the entire length of SEQ ID NO:4. Preferably, the nucleotide sequence comprising the 5'LTR comprises SEQ ID NO:4 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:4. More preferably, the nucleotide sequence comprising the 5'LTR is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:4.

[0106] In some embodiments, the nucleotide sequence of the 5'LTR comprised in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:4.

[0107] In one embodiment, the 5'LTR region comprises a CMV promoter, preferably a chimeric CMV promoter, and / or a RU5 element. In one embodiment, the chimeric CMV promoter comprises or consists of a CMV enhancer and a CMV promoter. In a preferred embodiment, the 5'LTR region comprises or consists of a CMV enhancer, a CMV promoter, a RU5 element, and / or any combination thereof.

[0108] In a preferred embodiment, the nucleotide sequence comprising the chimeric CMV promoter comprises SEQ ID NO:5 or a sequence that is at least 98% identical over the entire length of SEQ ID NO:5. Preferably, the nucleotide sequence comprising the chimeric CMV promoter comprises SEQ ID NO:5 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:5. More preferably, the nucleotide sequence comprising the chimeric CMV promoter is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:5 or SEQ ID NO:5.

[0109] In some embodiments, the nucleotide sequence of the chimeric CMV promoter contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:5.

[0110] In a preferred embodiment, the nucleotide sequence comprising the CMV enhancer comprises SEQ ID NO:6 or a sequence that is at least 98% identical over the entire length of SEQ ID NO:6. Preferably, the nucleotide sequence comprising the CMV enhancer comprises SEQ ID NO:6 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:6. More preferably, the nucleotide sequence comprising the CMV enhancer is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:6.

[0111] In some embodiments, the nucleotide sequence of the CMV enhancer contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:6.

[0112] In a preferred embodiment, the nucleotide sequence comprising the CMV promoter comprises SEQ ID NO:7 or a sequence that is at least 98% identical over the entire length of SEQ ID NO:7. Preferably, the nucleotide sequence comprising the CMV promoter comprises SEQ ID NO:7 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:7. More preferably, the nucleotide sequence comprising the CMV promoter is SEQ ID NO:7 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:7.

[0113] In some embodiments, the nucleotide sequence of the CMV promoter contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:7.

[0114] In a preferred embodiment, the nucleotide sequence comprising an RU5 element comprises SEQ ID NO:8 or a sequence that is at least 98% identical over the entire length of SEQ ID NO:8. Preferably, the nucleotide sequence comprising an RU5 element comprises SEQ ID NO:8 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:8. More preferably, the nucleotide sequence comprising an RU5 element is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:8.

[0115] In some embodiments, the nucleotide sequence of the RU5 element contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:8.

[0116] In one embodiment, adjacent to the 5'LTR is a sequence necessary for reverse transcription of the genome, which is a tRNA primer binding site (hereinafter referred to as PBS or PBS SL23).

[0117] In a preferred embodiment, the nucleotide sequence comprising PBS SL23 comprises SEQ ID NO:9 or a sequence that is at least 98% identical over the entire length of SEQ ID NO:9. Preferably, the nucleotide sequence comprising PBS SL23 comprises SEQ ID NO:9 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:9. More preferably, the nucleotide sequence comprising PBS SL23 is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:9.

[0118] In some embodiments, the nucleotide sequence of PBS SL23 contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:9.

[0119] In some embodiments, the lentiviral vectors provided herein contain at least a psi (Ψ) packaging signal (also called a psi site) for efficient encapsulation of viral RNA into particles.

[0120] In a preferred embodiment, the nucleotide sequence comprising the psi packaging signal comprises SEQ ID NO: 10 or a sequence that is at least 98% identical over the entire length of SEQ ID NO: 10. Preferably, the nucleotide sequence comprising the psi packaging signal comprises a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 10 or SEQ ID NO: 10. More preferably, the nucleotide sequence comprising the psi packaging signal is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 10 or SEQ ID NO: 10.

[0121] In some embodiments, the nucleotide sequence of the psi packaging signal contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO: 10. In one embodiment, the nucleotides of the PSI packaging signal overlap with the stem loop 4 (SL4) region of PBS SL23 and wild-type HIV virus, as defined below.

[0122] In certain embodiments, the lentiviral vectors provided herein further comprise one or more nucleotide sequences encoding a gag protein, DenvRF1, a Rev response element (hereinafter referred to as RRE), DenvRF2, a central polypurine track (hereinafter referred to as cPPT), and / or any combination thereof.

[0123] In some embodiments, the lentiviral vectors provided herein contain at least the stem loop 4 (SL4) region of the wild-type HIV virus (hereinafter referred to as SL4mgag).

[0124] In a preferred embodiment, the nucleotide sequence comprising SL4mgag comprises SEQ ID NO: 11 or a sequence that is at least 98% identical over the entire length of SEQ ID NO: 11. Preferably, the nucleotide sequence comprising SL4mgag comprises SEQ ID NO: 11 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 11. More preferably, the nucleotide sequence comprising SL4mgag is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 11 or SEQ ID NO: 11.

[0125] In some embodiments, the nucleotide sequence of SL4mgag contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:11.

[0126] In some embodiments, the lentiviral vectors provided herein contain at least a DenvRF1 element.

[0127] In a preferred embodiment, the nucleotide sequence comprising DenvRF1 comprises SEQ ID NO: 12 or a sequence that is at least 98% identical over the entire length of SEQ ID NO: 12. Preferably, the nucleotide sequence comprising DenvRF1 comprises SEQ ID NO: 12 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 12. More preferably, the nucleotide sequence comprising DenvRF1 is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 12 or SEQ ID NO: 12.

[0128] In some embodiments, the nucleotide sequence of DenvRF1 contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO: 12. In one embodiment, the nucleotides of DenvRF1 overlap with the nucleotides of SL4mgag defined above and the nucleotides of RRE defined below.

[0129] In a preferred embodiment, the nucleotide sequence comprising an RRE comprises SEQ ID NO: 13 or a sequence that is at least 98% identical over the entire length of SEQ ID NO: 13. Preferably, the nucleotide sequence comprising an RRE comprises SEQ ID NO: 13 or a sequence that is at least 75%, 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 13. More preferably, the nucleotide sequence comprising an RRE is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 13 or SEQ ID NO: 13.

[0130] In some embodiments, the nucleotide sequence of the RRE contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:13.

[0131] In some embodiments, the lentiviral vectors provided herein contain at least a DenvRF2 element.

[0132] In a preferred embodiment, the nucleotide sequence comprising DenvRF2 comprises SEQ ID NO: 14 or a sequence that is at least 98% identical over the entire length of SEQ ID NO: 14. Preferably, the nucleotide sequence comprising DenvRF2 comprises SEQ ID NO: 14 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 14. More preferably, the nucleotide sequence comprising DenvRF2 is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 14 or SEQ ID NO: 14.

[0133] In some embodiments, the nucleotide sequence of DenvRF2 contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:14.

[0134] In a preferred embodiment, the nucleotide sequence comprising cPPT comprises SEQ ID NO: 15 or a sequence that is at least 98% identical over the entire length of SEQ ID NO: 15. Preferably, the nucleotide sequence comprising cPPT comprises SEQ ID NO: 15 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 15. More preferably, the nucleotide sequence comprising cPPT is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO: 15 or SEQ ID NO: 15.

[0135] In some embodiments, the nucleotide sequence of the cPPT contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:15.

[0136] In some embodiments, the lentiviral vector comprises a nucleic acid, the nucleic acid comprising a 3' long terminal repeat (hereinafter referred to as 3'LTR), which is involved in mRNA polyadenylation during lentiviral vector production.

[0137] In a preferred embodiment, the 3'LTR comprises a deletion of the U3 region of the 3'LTR. The deletion of the U3 region of the 3'LTR can be a complete deletion or a partial deletion.

[0138] In a preferred embodiment, the nucleotide sequence comprising the 3'LTR comprises SEQ ID NO:23 or a sequence that is at least 98% identical over the entire length of SEQ ID NO:23. Preferably, the nucleotide sequence comprising the 3'LTR comprises SEQ ID NO:23 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:23. More preferably, the nucleotide sequence comprising the 3'LTR is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:23 or SEQ ID NO:23.

[0139] In some embodiments, the nucleotide sequence of the 3'LTR comprised in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:23.

[0140] In a preferred embodiment, the 3'LTR is composed of a truncated U3 element (hereinafter referred to as ΔU3) derived from wild-type HIV-1, which is a self-inactivating lentiviral vector, and elements R and U5 (hereinafter referred to as RU5).

[0141] In a preferred embodiment, the nucleotide sequence comprising ΔU3 comprises SEQ ID NO:24 or a sequence that is at least 98% identical over the entire length of SEQ ID NO:24. Preferably, the nucleotide sequence comprising ΔU3 comprises SEQ ID NO:24 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:24. More preferably, the nucleotide sequence comprising ΔU3 is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:24 or SEQ ID NO:24.

[0142] In some embodiments, the nucleotide sequence of ΔU3 contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:24.

[0143] In a preferred embodiment, a nucleotide sequence comprising RU5 comprises SEQ ID NO:25 or a sequence that is at least 98% identical over the entire length of SEQ ID NO:25. Preferably, a nucleotide sequence comprising RU5 comprises SEQ ID NO:25 or a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:25. More preferably, a nucleotide sequence comprising RU5 is a sequence that is at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% identical over the entire length of SEQ ID NO:25 or SEQ ID NO:25.

[0144] In some embodiments, the nucleotide sequence of RU5 contained in the lentiviral vector comprises, consists of, or consists essentially of SEQ ID NO:25.

[0145] In one embodiment, the RU5 nucleotide is located between the chimeric CMV promoter and the PBS SL123 nucleotide. In one embodiment, the PBS SL123 nucleotide is located between the 5'LTR nucleotide and the Psi packaging signal. In one embodiment, the Psi packaging signal nucleotide is located between the PBS SL123 nucleotide and the nucleotide of the stem loop 4 (SL4) region of the wild-type HIV virus. In one embodiment, the DenvRF1 nucleotide is located between the SL4mgag nucleotide and the RRE nucleotide. In one embodiment, the DenvRF2 nucleotide is located between the RRE nucleotide and the cPPT nucleotide. In one embodiment, the cPPT nucleotide is located between the RRE nucleotide and the ET promoter nucleotide. In one embodiment, at least one copy of at least one miRNA target sequence is located between the mutated WPRE nucleotide and the 3'LTR nucleotide.

[0146] In a further preferred embodiment of the first aspect or any of its embodiments, the lentiviral vector comprises a nucleic acid, the nucleic acid comprising from 5' to 3' the following nucleotides: a) a 5' long terminal repeat (LTR); b) a primer binding site (PBS); c) a psi packaging signal; d) the stem loop 4 (SL4) region of the wild-type HIV virus; and e) a Rev response element (RRE); f) DNA flap central polypurine tract (cPPT); g) an engineered hepatocyte-specific promoter having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO: 16; h) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO: 29; i) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO:21; j) at least one copy of at least one miRNA target sequence; k) a 3' long terminal repeat (LTR); Including, The long terminal repeat regions of a) and k) are substantially transcriptionally inactivated by a full or partial deletion in the U3 region of the LTR, and g), i) and j) are operably linked to and regulate the expression of h). In a preferred embodiment, at least one copy of at least one miRNA target sequence consists of only four repeats of the target sequence of miRNA-142-3.

[0147] In some embodiments, the nucleotide sequence contained in the lentiviral vector is l) d) the DenvRF1 region located between the stem loop 4 (SL4) region of the wild-type HIV virus and e) the Rev response element (RRE); m) the DenvRF2 region located between e) the Rev response element (RRE) and f) the DNA flap central polypurine tract (cPPT); Further includes:

[0148] In a most preferred embodiment of the first aspect or any of its embodiments, the lentiviral vector comprises a nucleic acid, the nucleic acid comprising from 5' to 3' the following nucleotides: a) a 5' long terminal repeat (LTR) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:5; b) a primer binding site (PBS) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:9; c) a psi packaging signal having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 10; d) a wild-type HIV virus stem loop 4 (SL4) region having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 11; e) a Rev response element (RRE) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 13; f) a DNA flap central polypurine tract (cPPT) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 15; g) an engineered hepatocyte-specific promoter having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 16; h) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 29; i) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:21; j) at least one copy of at least one miRNA target sequence; k) a 3' long terminal repeat (LTR) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:3; Including, The long terminal repeat regions of a) and k) are substantially transcriptionally inactivated by a full or partial deletion in the U3 region of the LTR, and g), i) and j) are operably linked to and regulate the expression of h). In a preferred embodiment, at least one copy of the at least one miRNA target sequence consists of only four repeats of the target sequence of miRNA-142-3 having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:22.

[0149] In some embodiments, the nucleotide sequence contained in the lentiviral vector is l) a DenvRF1 region having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 12, d) located between the stem loop 4 (SL4) region of a wild-type HIV virus and e) the Rev response element (RRE); m) a DenvRF2 region having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 14, and located between e) a Rev response element (RRE) and f) a DNA flap central polypurine tract (cPPT); Further includes:

[0150] In a further preferred embodiment of the first aspect, the lentiviral vector comprises a nucleic acid, the nucleic acid comprising from 5' to 3' the following nucleotides: a) a 5' long terminal repeat (LTR); b) a primer binding site (PBS); c) a psi packaging signal; d) the stem loop 4 (SL4) region of the wild-type HIV virus; and e) a Rev response element (RRE); f) DNA flap central polypurine tract (cPPT); g) an engineered hepatocyte-specific promoter having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 16; h) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 29; i) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:21; j) at least one copy of at least one miRNA target sequence; k) a 3' long terminal repeat (LTR); l) the DenvRF1 region located between d) and e); m) the DenvRF2 region located between e) and f); Including, The long terminal repeat regions of a) and k) are substantially transcriptionally inactivated by a full or partial deletion in the U3 region of the LTR, and g), i) and j) are operably linked to and regulate the expression of h). In a preferred embodiment, at least one copy of at least one miRNA target sequence consists of only four repeats of the target sequence of miRNA-142-3.

[0151] In a further preferred embodiment of the first aspect, the lentiviral vector comprises a nucleic acid, the nucleic acid comprising from 5' to 3' the following nucleotides: a) a 5' long terminal repeat (LTR) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:5; b) a primer binding site (PBS) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:9; c) a psi packaging signal having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 10; d) a wild-type HIV virus stem loop 4 (SL4) region having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 11; e) a Rev response element (RRE) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 13; f) a DNA flap central polypurine tract (cPPT) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 15; g) an engineered hepatocyte-specific promoter having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 16; h) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 29; i) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:21; j) at least one copy of at least one miRNA target sequence; k) a 3' long terminal repeat (LTR) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:3; l) a DenvRF1 region having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 12 and located between d) and e); m) a DenvRF2 region having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 14 and located between e) and f); Including, The long terminal repeat regions of a) and k) are substantially transcriptionally inactivated by a full or partial deletion in the U3 region of the LTR, and g), i) and j) are operably linked to and regulate the expression of h). In a preferred embodiment, at least one copy of the at least one miRNA target sequence consists of only four repeats of the target sequence of miRNA-142-3 having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:22.

[0152] In another embodiment, the lentiviral vector comprises a nucleic acid, the nucleic acid comprising from 5' to 3' the following nucleotides: a) a 5' long terminal repeat (LTR); b) a primer binding site (PBS); c) a psi packaging signal; d) the stem loop 4 (SL4) region of the wild-type HIV virus; and e) a Rev response element (RRE); f) DNA flap central polypurine tract (cPPT); g) an engineered hepatocyte-specific promoter having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO: 16; h) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO: 29; i) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO:21; j) 4 copies of a target sequence of miRNA-142-3 having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO: 22; k) a 3' long terminal repeat (LTR); Including, The long terminal repeat regions of a) and k) are substantially transcriptionally inactivated by a total or partial deletion in the U3 region of the LTR, and g), i) and j) are operably linked to h) and regulate the expression of h).

[0153] In some embodiments, the nucleotide sequence contained in the lentiviral vector is l) d) the DenvRF1 region located between the stem loop 4 (SL4) region of the wild-type HIV virus and e) the Rev response element (RRE); m) the DenvRF2 region located between e) the Rev response element (RRE) and f) the DNA flap central polypurine tract (cPPT); Further includes:

[0154] In a preferred embodiment, the lentiviral vector comprises a nucleic acid, which preferably comprises, 5' to 3', the following: a) a 5' long terminal repeat (LTR); b) a primer binding site (PBS); c) a psi packaging signal; d) the stem loop 4 (SL4) region of the wild-type HIV virus; and e) a Rev response element (RRE); f) DNA flap central polypurine tract (cPPT); g) an engineered hepatocyte-specific promoter having at least 95%, preferably 98%, and most preferably 100% sequence identity over the entire length of SEQ ID NO: 16; h) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein, the AGXT-RHEAM protein having at least 95%, preferably 98%, and most preferably 100% amino acid sequence identity over the entire length of SEQ ID NO: 52; i) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 95%, preferably 98%, and most preferably 100% sequence identity over the entire length of SEQ ID NO:21; j) at least a copy of at least one miRNA target sequence, the miRNA target sequence being selected from the group consisting of miRNA-142-3, miR-181, miR-223, or miR-30b; k) a 3' long terminal repeat (LTR); Including, The long terminal repeat regions of a) and k) are substantially transcriptionally inactivated by a full or partial deletion in the U3 region of the LTR, and g), i) and j) are operably linked to h) and regulate the expression of h). In a further preferred embodiment, the at least one copy of the at least one miRNA target sequence of j) preferably consists of only four copies of the target sequence of miRNA-142-3 having at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO:22.

[0155] In some embodiments, the nucleotide sequence contained in the lentiviral vector is l) d) the DenvRF1 region located between the stem loop 4 (SL4) region of the wild-type HIV virus and e) the Rev response element (RRE); m) the DenvRF2 region located between e) the Rev response element (RRE) and f) the DNA flap central polypurine tract (cPPT); Further includes:

[0156] In a preferred embodiment, the lentiviral vector comprises a nucleic acid, which preferably comprises, 5' to 3', the following: a) a 5' long terminal repeat (LTR); b) a primer binding site (PBS); c) a psi packaging signal; d) the stem loop 4 (SL4) region of the wild-type HIV virus; and e) a Rev response element (RRE); f) DNA flap central polypurine tract (cPPT); g) a genetically engineered hepatocyte-specific promoter consisting solely of SEQ ID NO: 16; h) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein, the protein consisting of SEQ ID NO: 52; i) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) consisting of only SEQ ID NO: 21; j) at least a copy of at least one miRNA target sequence, the miRNA target sequence being selected from the group consisting of miRNA-142-3, miR-181, miR-223, or miR-30b; k) a 3' long terminal repeat (LTR); Including, The long terminal repeat regions of a) and k) are substantially transcriptionally inactivated by a full or partial deletion in the U3 region of the LTR, and g), i) and j) are operably linked to h) and regulate the expression of h). In a further preferred embodiment, the at least one copy of the at least one miRNA target sequence of j) preferably consists of only four copies of the target sequence of miRNA-142-3 having at least 95%, preferably 98%, most preferably 100% sequence identity over the entire length of SEQ ID NO:22.

[0157] In some embodiments, the nucleotide sequence contained in the lentiviral vector is l) d) the DenvRF1 region located between the stem loop 4 (SL4) region of the wild-type HIV virus and e) the Rev response element (RRE); m) the DenvRF2 region located between e) the Rev response element (RRE) and f) the DNA flap central polypurine tract (cPPT); Further includes:

[0158] In a further preferred embodiment of the first aspect, the lentiviral vector comprises a nucleic acid, the nucleic acid preferably comprising from 5' to 3' the following nucleotides: a) a 5' long terminal repeat (LTR) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:5; b) a primer binding site (PBS) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:9; c) a psi packaging signal having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 10; d) a wild-type HIV virus stem loop 4 (SL4) region having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 11; e) a Rev response element (RRE) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 13; f) a DNA flap central polypurine tract (cPPT) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 15; g) an engineered hepatocyte-specific promoter having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 16; h) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein, the AGXT-RHEAM protein having at least 95%, preferably 98%, and most preferably 100% amino acid sequence identity over the entire length of SEQ ID NO: 52; i) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:21; j) at least one copy of at least one miRNA target sequence, wherein the miRNA target sequence is selected from the group consisting of miRNA-142-3, miR-181, miR-223, or miR-30b; k) a 3' long terminal repeat (LTR) having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:3; l) a DenvRF1 region having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 12 and located between d) and e); m) a DenvRF2 region having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO: 14 and located between e) and f); Including, The long terminal repeat regions of a) and k) are substantially transcriptionally inactivated by a full or partial deletion in the U3 region of the LTR, and g), i) and j) are operably linked to and regulate the expression of h). In a preferred embodiment, at least one copy of the at least one miRNA target sequence consists of only four repeats of the target sequence of miRNA-142-3 having at least 95%, preferably 98%, more preferably 100% sequence identity over the entire length of SEQ ID NO:22.

[0159] In a preferred embodiment, the full length nucleotide sequence contained in the lentiviruses provided herein has at least 75%, 80%, 85%, 89%, 90%, 91%, 92%, 95%, 97%, 98%, 99%, or 100% sequence identity over the entire length of SEQ ID NO:30 (LV.ET.AGXT-RHEAM.142-3pT). In another preferred embodiment, the full length nucleotide sequence contained in the lentiviruses provided comprises, consists of, or consists essentially of SEQ ID NO:30.

[0160] In one embodiment, the lipid coat of a viral particle can comprise membrane-associated polypeptides that were previously present on the surface of a host cell.

[0161] In some embodiments, the lentiviral vector has on its surface modified expression of one or more polypeptides that suppress an immune response to the lentiviral vector after administration to a human subject. In some embodiments, the surface of the lentiviral vector comprises one or more CD47 molecules. CD47 is a marker of "self proteins" and is ubiquitously expressed on human cells. Surface expression of CD47 suppresses macrophage-induced phagocytosis of endogenous cells through the interaction of CD47 and macrophage-expressed SIRPa. Cells expressing high levels of CD47 are less likely to be targeted and destroyed by human macrophages in vivo. In some embodiments, the lentiviral vector comprises a high concentration of CD47 polypeptide molecules on its surface. In some embodiments, the lentiviral vector is produced in a cell line with a high expression level of CD47. In certain embodiments, the lentiviral vector is produced in a cell line, the cell line with a high expression of CD47 on the cell membrane. In certain embodiments, the lentiviral vector is produced in a cell line with a high expression of CD47 on the cell membrane. In certain embodiments, the lentiviral vector is produced in a CD47high The lentiviral vector is produced in HEK293T cells, which have high expression of CD47 on the cell membrane. In some embodiments, the HEK293T cells are modified to have increased expression of CD47 compared to unmodified HEK293T cells. In certain embodiments, the CD47 is human CD47. In one embodiment, the surface of the lentiviral vector lacks or has reduced expression of one or more class I major histocompatibility complexes (MHCI). In certain embodiments, the MHCI is human MHCI.

[0162] In a second aspect, the invention relates to a host cell (e.g. a hepatocyte) or a substantially pure population of cells comprising (e.g. transduced) a lentiviral vector as defined in the first aspect or any of its embodiments. In one embodiment of the second aspect, the host cell or substantially pure population of cells is a hepatocyte.

[0163] In a third aspect, the present invention relates to an isolated nucleic acid comprising a nucleotide sequence comprised in a lentiviral vector as defined in the first aspect or any of its embodiments.

[0164] Pharmaceutical Compositions In a fourth aspect, the present invention provides a pharmaceutical composition comprising a lentivirus as defined in the first aspect or any of its embodiments, a host cell or a substantially pure population of cells according to the second aspect or any of its embodiments, and / or a nucleic acid as defined in the third aspect or any of its embodiments, and a pharma- ceutically acceptable carrier or diluent.

[0165] In some embodiments, the substantially pure cell population according to the second aspect or any of its embodiments, or the composition for administration of the pharmaceutical composition of the fourth aspect or any of its embodiments, is contacted or transduced, either in vivo, in vitro, or ex vivo, with a lentiviral vector according to the first aspect or any of its embodiments.

[0166] The pharmaceutical compositions described herein may also contain other substances, including, but not limited to, cryoprotectants, lyoprotectants, surfactants, bulking agents, antioxidants, and stabilizers. In some embodiments, the pharmaceutical compositions may be lyophilized.

[0167] The term "cryoprotectant" as used herein includes agents that provide lentiviral vectors with stability against freezing-induced stress by being preferentially excluded from the surface of the lentiviral vector. Cryoprotectants can also provide protection during primary and secondary drying and during long-term storage of the product. Non-limiting examples of cryoprotectants include sugars such as sucrose, glucose, trehalose, mannitol, mannose, and lactose, polymers such as dextran, hydroxyethyl starch, and polyethylene glycol, surfactants such as polysorbates (e.g., PS-20 or PS-80), and amino acids such as glycine, arginine, leucine, and serine. Cryoprotectants that exhibit low toxicity in biological systems are commonly used.

[0168] In one embodiment, a lyoprotectant is added to the pharmaceutical composition described herein. The term "lyoprotectant" as used herein includes agents that provide stability to lentiviral vectors during the lyophilization or dehydration process (primary and secondary lyophilization cycles) by providing an amorphous glassy matrix, binding to the surface of the lentiviral vector through hydrogen bonds, and replacing water molecules that are removed during the drying process. This minimizes product degradation during the lyophilization cycle and improves the long-term stability of the product. Non-limiting examples of lyoprotectants include sugars such as sucrose or trehalose, amino acids such as sodium glutamate, amorphous glycine or histidine, methylamines such as betaine, lyotropic salts such as magnesium sulfate, polyols such as trihydric or higher sugar alcohols (e.g., glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol), propylene glycol, polyethylene glycol, Pluronic®, and combinations thereof. The amount of lyoprotectant added to a pharmaceutical composition is generally an amount that does not cause an unacceptable amount of degradation of the strain when the pharmaceutical composition is lyophilized.

[0169] In some embodiments, a bulking agent is included in the pharmaceutical composition. The term "bulking agent" as used herein includes agents that provide structure to the lyophilized product without directly interacting with the pharmaceutical product. In addition to providing a pharma-ceutical elegant cake, bulking agents can also impart useful properties with respect to modifying collapse temperature, providing freeze-thaw protection, and enhancing strain stability over long-term storage. Non-limiting examples of bulking agents include mannitol, glycine, lactose, and sucrose. Bulking agents may be crystalline (e.g., glycine, mannitol, or sodium chloride) or amorphous (e.g., dextran, hydroxyethyl starch) and are generally used in formulations in amounts of 0.5% to 10%.

[0170] Other pharma- ceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), may also be included in the pharmaceutical compositions described herein, so long as they do not adversely affect the desired properties of the pharmaceutical composition. As used herein, "pharma-ceutically acceptable carriers" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are compatible with pharmaceutical administration. The use of such media and agents for pharma-ceutically active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and may include additional buffering agents, preservatives, cosolvents, antioxidants including ascorbic acid and methionine, chelating agents such as EDTA, metal complexes (e.g., Zn-protein complexes), biodegradable polymers such as polyesters, salt-forming counterions such as sodium, polyhydric sugar alcohols, amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine, lactitol, stachyose, mannose, sucrose, sorbitol ... Examples of suitable sugars or sugar alcohols include organic sugars or sugar alcohols such as sugar, sorbose, xylose, ribose, ribitol, myo-initose, myo-inititol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), and polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [α]-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers such as polyvinylpyrrolidone.

[0171] The pharmaceutical composition can be prepared for oral, sublingual, buccal, intravenous, intramuscular, subcutaneous, intraperitoneal, conjunctival, rectal, transdermal, intrathecal, topical and / or inhalation-mediated administration. In a preferred embodiment, the pharmaceutical composition can be a solution suitable for intravenous, intramuscular, conjunctival, transdermal, intraperitoneal and / or subcutaneous administration. In another embodiment, the pharmaceutical composition can be a solution suitable for sublingual, buccal and / or inhalation-mediated administration routes. In an alternative embodiment, the pharmaceutical composition can be a gel or solution suitable for intrathecal administration. In an alternative embodiment, the pharmaceutical composition can be an aerosol suitable for inhalation-mediated administration. In a preferred embodiment, the pharmaceutical composition can be prepared for intrathecal administration.

[0172] The pharmaceutical composition may further include common excipients and carriers known in the art. For solid pharmaceutical compositions, conventional non-toxic solid carriers may be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For injectable solutions, the pharmaceutical composition may further include cryoprotectants, lyoprotectants, surfactants, bulking agents, antioxidants, stabilizers, and pharma- ceutical acceptable carriers. For aerosol administration, the pharmaceutical composition is generally supplied in finely divided form together with a surfactant and a propellant. The surfactant must, of course, be non-toxic and generally soluble in the propellant. Representative of such agents are esters or partial esters of fatty acids containing 6 to 22 carbon atoms, such as caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid, and oleic acid, with aliphatic polyhydric alcohols or their cyclic anhydrides. Mixed esters, such as mixed or natural glycerides, may also be used. A carrier can also be included as desired, such as, for example, lecithin for intranasal delivery. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides.

[0173] Thus, suitable pharmaceutical compositions for injection may include buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), optionally stabilizers (e.g., human albumin), etc. However, in other embodiments consistent with the teachings herein, lentiviral vectors may be delivered directly to the site of the harmful cell population, i.e., hepatocytes, thereby increasing exposure of the diseased tissue to the therapeutic agent.

[0174] The lentiviral vectors provided herein can optionally be administered in combination with other agents that are effective in treating the disorder or condition requiring treatment (e.g., prophylactically or therapeutically). Administration of the lentiviral vectors provided herein in conjunction with or in combination with adjunctive therapy refers to sequential, simultaneous, coextensive, simultaneous, concurrent, or simultaneous administration or application of the therapy and the disclosed polypeptide. In one embodiment, the lentiviral vector is administered in combination with other agents that can induce hepatocyte proliferation, such as, but not limited to, triiodothyronine hormone, human interleukin 6, and / or derivatives thereof. In another embodiment, the lentiviral vector is administered in combination with other agents that can suppress immune responses to the lentiviral vector, such as, but not limited to, dexamethasone, cyclosporine A, cyclosporine H, rapamycin, and / or derivatives thereof. In another embodiment, the lentiviral vector is administered in combination with other agents that can increase the number of specific receptors in target cells for VSV-G pseudotyped lentivirus, such as, but not limited to, statins, iron chelators, and / or derivatives thereof. In another embodiment, the lentiviral vector is administered in combination with other agents capable of blocking apoptosis in transduced hepatocytes, such as, but not limited to, antioxidants, e.g., N-acetylcysteine, and / or derivatives thereof.

[0175] In certain embodiments, the lentiviral vector according to the first aspect or any of its embodiments is administered in combination with cyclosporine H (CsH), which acts as a transduction facilitator. In preferred embodiments, CsH is administered at a dose ranging from 20 mg / kg to 1 mg / kg, 15 mg / kg to 1 mg / kg, 10 mg / kg to 1 mg / kg, 5 mg / kg to 1 mg / kg, or 5 mg / kg to 2.5 mg / kg. In one embodiment, CsH is administered before, simultaneously, or after administration of the lentivirus. Intermediate doses in the above ranges are also intended to be within the scope of the invention. In some embodiments, CsH is administered once. In other embodiments, CsH is administered two or more times, at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times. The repeated administrations of CsH may be administered on the same day or at separate times. In one embodiment, the interval between single doses of CsH can be daily, weekly, monthly, or yearly.

[0176] Medical Use and Schedule, Route and Dosage of Administration In a fifth aspect, the present invention provides a lentiviral vector according to the first aspect or any of its embodiments, a host cell or substantially pure cell population according to the second aspect or any of its embodiments, an isolated nucleic acid according to the third aspect or any of its embodiments, or a pharmaceutical composition according to the fourth aspect or any of its embodiments, for use as a medicament. In a sixth aspect, the present invention provides a lentiviral vector according to the first aspect or any of its embodiments, a host cell or substantially pure cell population according to the second aspect or any of its embodiments, an isolated nucleic acid according to the third aspect or any of its embodiments, or a pharmaceutical composition according to the fourth aspect or any of its embodiments, for use in the treatment of primary hyperoxaluria, preferably primary hyperoxaluria type 1, comprising administering said lentivirus, said host cell or cell population, or said composition to a subject.

[0177] Preferably, the use of the fifth and sixth aspects is combined with an immune response suppressant, preferably dexamethasone, cyclosporin A, cyclosporin H and / or rapamycin, or any combination thereof.

[0178] The present invention also provides a method of treating, preventing or ameliorating a PH1 disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a lentiviral vector according to the first aspect or any of its embodiments, a host cell or a substantially pure population of cells according to the second aspect or any of its embodiments, an isolated nucleic acid of the third aspect or any of its embodiments, or a pharmaceutical composition of the fourth aspect or any of its embodiments.

[0179] In another embodiment, administration of a lentiviral vector, host cell or substantially pure cell population, or composition provided herein does not induce an immune response in a subject, or induces a very low immune response in the subject.

[0180] In some embodiments, the lentiviral vector, host cell or substantially pure cell population, or composition of the present invention is administered as a single dose or multiple doses. In some embodiments, the dose of the lentiviral vector, host cell or substantially pure cell population, or composition of the present invention is administered once or divided into multiple sub-doses, for example, two sub-doses, three sub-doses, four sub-doses, five sub-doses, six sub-doses, or more than six sub-doses. In some embodiments, two or more lentiviral vectors are administered. Most preferably, the lentiviral vector, host cell or substantially pure cell population, or composition of the present invention is administered as a single dose.

[0181] In some embodiments, a dose of a lentiviral vector of the invention, a host cell or substantially pure cell population of the invention, or a pharmaceutical composition is administered repeatedly at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times.

[0182] The lentiviral vector of the present invention, the host cell or substantially pure cell population of the present invention, or the pharmaceutical composition can be administered locally or systemically. In one embodiment, the route of administration of the lentiviral vector is parenteral. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. In one embodiment, the lentiviral vector can be administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, for example, orally, sublingually, buccal, sublingually, nasally, rectally, vaginally, or via pulmonary route. Intravenous forms of parenteral administration are preferred. In one embodiment, the administration form will be a solution for injection, particularly a solution for intravenous or intraarterial injection or infusion.

[0183] The effective dose of the compositions provided herein for the treatment of PH1 depends on many different factors, including the means of administration, the target site, the physiological condition of the subject, whether the subject is human or animal, other drugs administered, and whether the treatment is preventive or therapeutic. Usually, the subject is a human, but non-human mammals, including transgenic mammals, can also be treated. To optimize safety and efficacy, routine techniques known to those skilled in the art can be used to set the treatment dosage. In one embodiment, the subject includes, but is not limited to, an individual with PH1. In some embodiments, the subject is a pediatric subject, while in other aspects, the subject is an adult subject.

[0184] The lentiviral vector of the present invention, the host cell or substantially pure cell population of the present invention, or the pharmaceutical composition can be administered as a single dose or multiple doses, and the multiple doses can be administered continuously or at specific time intervals. In vitro or in vivo assays can be used to determine the optimal dose range and / or schedule of administration. Furthermore, effective doses can be extrapolated from dose-response curves obtained from animal models.

[0185] In a preferred embodiment, the lentiviral vector of the invention, the host cell or substantially pure cell population of the invention, or the pharmaceutical composition comprises at least 1×10 9 Vector genomes / kg body weight, preferably 1 x 10 10 Vector genome / kg body weight, 1 × 10 11 Vector genome / kg body weight or 1×10 12 More preferably, the vector is administered at a dose of at least or about 4.6×10 12 Vector genomes / kg body weight are administered.

[0186] In one embodiment, the dose of lentivirus is at least 5×10 2 Transducing units / kg (TU / kg), 5 x 10 3 TU / kg, 5×10 4 TU / kg, 5×10 5 TU / kg, 5×10 6 TU / kg, 5×10 7 TU / kg, 5×10 8 TU / kg, 5×10 9 TU / kg, 5×10 10 TU / kg, 5×10 11 TU / kg, 5×10 12 TU / kg, 5×10 13 TU / kg, 5×10 14 TU / kg, 5×10 15 In one embodiment, the dose of lentivirus is 5×10 2 Transducing units / kg (TU / kg) ~ 5 x 10 3 TU / kg, 5×103 TU / kg to 5×10 4 TU / kg, 5×10 4 TU / kg to 5×10 5 TU / kg, 5×10 5 TU / kg to 5×10 6 TU / kg, 5×10 6 TU / kg to 5×10 7 TU / kg, 5×10 8 TU / kg to 5×10 9 TU / kg, 5×10 9 TU / kg to 5×10 10 TU / kg, 5×10 10 TU / kg to 5×10 11 TU / kg, 5×10 11 TU / kg to 5×10 12 It is TU / kg. In one embodiment, the dose of lentivirus is about 1×10 2 transducing units / kg (TU / kg), 1×10 3 TU / kg, 1×10 4 TU / kg, 1×10 5 TU / kg, 1×10 6 TU / kg, 1×10 7 TU / kg, 1×10 8 TU / kg, 1×10 9 TU / kg, 1×10 10 TU / kg, 1×10 11 TU / kg, 1×10 12 TU / kg, 1×10 13 TU / kg, 1×10 14 TU / kg, 1×10 15 TU / kg, or about 5×10 2 transducing units / kg (TU / kg), 5×10 3 TU / kg, 5×10 4 TU / kg, 5×10 5 TU / kg, 5×10 6 TU / kg, 5×10 7 TU / kg, 5×10 8 TU / kg, 5×10 9 TU / kg, 5×10 10 TU / kg, 5×10 11 TU / kg, 5×10 12 TU / kg, 5×1013 TU / kg, 5 x 10 14 TU / kg, 5 x 10 15 TU / kg, or approximately 7.5 x 10 2 Transducing units / kg (TU / kg), 7.5 x 10 3 TU / kg, 7.5 x 10 4 TU / kg, 7.5 x 10 5 TU / kg, 7.5 x 10 6 TU / kg, 7.5×10 7 TU / kg, 7.5×10 8 TU / kg, 7.5×10 9 TU / kg, 7.5×10 10 TU / kg, 7.5×10 11 TU / kg, 7.5×10 12 TU / kg, 7.5×10 13 TU / kg, 7.5×10 14 TU / kg, 7.5 x 10 15 It is TU / kg.

[0187] Doses intermediate in the above ranges are also intended to be within the scope of the invention.

[0188] The lentiviral vector of the present invention, the host cell or substantially pure cell population of the present invention, or the pharmaceutical composition provided herein can be administered on multiple occasions. The interval between single administrations can be daily, weekly, monthly, or yearly. The intervals can also be irregular, depending on the progression of the disease or the curative effect of the lentivirus.

[0189] The dosage and frequency of administration of the lentiviral vector of the present invention, the host cell or substantially pure cell population of the present invention, or the pharmaceutical composition may vary depending on whether the treatment is preventive or therapeutic.In preventive applications, the composition comprising the lentiviral vector provided herein is administered to a subject who is not yet in a disease state to enhance the subject's resistance or minimize the effect of the disease.Such an amount is defined as a "prophylactically effective dose".A relatively low dosage is administered at relatively infrequent intervals over a long period of time.Some subjects may continue to receive treatment for the rest of their lives.

[0190] item The present invention further provides the following items.

[0191] 1. A lentiviral vector, the lentiviral vector comprising a nucleic acid, the nucleic acid comprising from 5' to 3' the following nucleotides: a) a 5' long terminal repeat (LTR); b) a primer binding site (PBS); c) a psi packaging signal; d) the stem loop 4 (SL4) region of the wild-type HIV virus; and e) a Rev response element (RRE); f) DNA flap central polypurine tract (cPPT); g) an engineered hepatocyte-specific promoter having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO: 16; h) a nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO: 29; i) a mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO:21; j) at least one copy of at least one miRNA target sequence; k) a 3' long terminal repeat (LTR); Including, A lentiviral vector, wherein the long terminal repeat regions of a) and k) are substantially transcriptionally inactivated by a total or partial deletion in the U3 region of the LTR, and g), i) and j) are operably linked to h) and regulate the expression of h).

[0192] 2. The lentiviral vector according to item 1, wherein the sequence identity of g), h), and i) to SEQ ID NO: 16, SEQ ID NO: 29, and SEQ ID NO: 21 is 100%, respectively.

[0193] 3. The lentiviral vector according to any one of items 1 and 2, wherein the at least one copy of the at least one miRNA target sequence of j) consists of only four copies of the target sequence of miRNA-142-3 having at least 95%, preferably 98%, sequence identity over the entire length of SEQ ID NO: 22.

[0194] 4. The nucleotide contained in the lentiviral vector is l) the DenvRF1 region located between d) and e); m) the DenvRF2 region located between e) and f); 4. The lentiviral vector according to any one of items 1 to 3, further comprising:

[0195] 5. The lentiviral vector according to any one of items 1 to 4, wherein the full-length nucleotide sequence of the lentivirus has at least 95%, preferably 98%, sequence identity to SEQ ID NO: 30 (LV.ET.AGXT-RHEAM.142-3pT).

[0196] 6. The lentiviral vector of item 5, which has 100% sequence identity with SEQ ID NO: 30.

[0197] 7. A substantially pure cell population transduced with a lentiviral vector according to any one of items 1 to 6.

[0198] 8. A pharmaceutical composition comprising the lentiviral vector according to any one of items 1 to 6, or the substantially pure cell population according to item 7, and a pharma- ceutically acceptable carrier or diluent.

[0199] 9. The lentiviral vector according to any one of items 1 to 6, the substantially pure cell population according to item 7, or the pharmaceutical composition according to claim 8, for use as a medicament.

[0200] 10. The lentiviral vector according to any one of items 1 to 6, the substantially pure cell population according to item 7, or the pharmaceutical composition according to claim 8, for use in treating a subject in need of treatment of primary hyperoxaluria, the method comprising administering the lentiviral vector, the cell population, or the pharmaceutical composition to the subject.

[0201] 11. The lentiviral vector, substantially pure cell population, or pharmaceutical composition for use according to item 10, wherein the primary hyperoxaluria is type 1. EXAMPLES

[0202] Example 1: Materials, methods and sequences 1.1 Lentiviral vector construction A reporter lentiviral vector (LV) construct expressing an enhanced version of green fluorescent protein (eGFP) was developed from a LV construct (LV.ET.FIX.142-3pT) previously described by the group of Naldini and Cantore (Brown et al., 2007). The eGFP-expressing LV was developed by classical cloning techniques. LV.ET.FIX.142-3pT was digested with NheI and SalI restriction enzymes (New England Biolabs™) to remove the FIX sequence. The target sequences of NheI and SalI are at the 5' and 3' ends of the FIX gene, respectively. After digestion of the plasmid with both enzymes, the LV construct was isolated by electrophoresis and band purification.

[0203] Furthermore, we amplified the eGFP sequence from the #241.pCCL.sin.PPT.TTR.GFP.Wpre plasmid by PCR. Primers were designed to add NheI (SEQ ID NO: 1 Fw: 5'-ATTGGCTAGCATGGTGAGCAAGGGCGAGGA-3') and SalI (SEQ ID NO: 2 Rv: 5'-CATTGTCGACTTACTTGTACAGCTCGTCCA-3') restriction enzyme target sequences at the 5' and 3' ends, respectively. Finally, both sequences were ligated to obtain LV.ET.eGFP.142-3pT.

[0204] The expression cassette of LV.ET.eGFP.142-3pT contains an engineered hepatocyte-specific promoter (enhanced transthyretin, modified from Genbank accession number AY661265) and a target sequence for hematopoietic lineage-specific microRNA 142 (mature human microRNA 142 SEQ ID NO: 3 - miRNA-142-3p: UGUAGUGUUUCCUACUUUAUGGA) with the aim to avoid off-target transgene expression in antigen-presenting cells and thus the generation of an immune response against the transduced cells.

[0205] LV.ET.eGFP.142-3pT sequence: 5'LTR (SEQ ID NO: 4): The 5'LTR is involved in proviral transcription during lentiviral vector production. In this lentiviral vector construct, the 5'LTR is composed of a chimeric version of the CMV promoter and RU5, which is part of the wild-type HIV-1 5'LTR. The absence of the U3 sequence in the wild-type 5'LTR means that this lentiviral vector construct is Tat-independent and therefore it is produced in the third generation. tggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccggggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcag

[0206] Chimeric CMV promoter (SEQ ID NO: 5): This sequence directs proviral transcription during lentiviral vector production. The chimeric CMV promoter is composed of two different sequences, one with enhancer activity and the other with promoter activity. tggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacg gtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagt acgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgac tcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaacc

[0207] CMV enhancer (SEQ ID NO:6): gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattg acgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatg

[0208] CMV promoter (SEQ ID NO:7): gtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttgg caccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagct

[0209] RU5 (SEQ ID NO: 8): Truncated 5'LTR without RU3. This sequence is required for retrotranscription and integration of the proviral genome into transduced cells. gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcag

[0210] PBS SL23 SL123 (primer binding site) (SEQ ID NO: 9): tRNA is bound to the PBS SL23 element during reverse transcription of the proviral genome after target cell transduction and prior to integration into the cellular genome. tggcgcccgaacagggacTtgaaagcgaaagggaaaccagagGAGctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagag

[0211] Ψ (packaging signal) (SEQ ID NO: 10): This element is involved in dimerization and packaging of lentiviral vector particles. ctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtc

[0212] SL4mgag (SEQ ID NO: 11): This element is part of the wild-type mgag HIV-1 gene involved in proviral packaging. atgggtgcgagagcgtcagtattaagcgggggaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaata ctgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattataataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgat

[0213] DenvRF1 (SEQ ID NO: 12): tcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaata

[0214] RRE (Rev response element) (SEQ ID NO: 13): Rev binds to this sequence in the transcript of the lentiviral vector and aids in the nuclear export of the provirus during lentiviral vector production. aggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcGtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaa tttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcct

[0215] DenvRF2 (SEQ ID NO: 14): gggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaat taacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaa cataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattc accattatcgtttcagacccacctcccaacccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatc

[0216] cPPT (central polypurine tract) (SEQ ID NO: 15): This element acts as a primer for positive DNA strand transcription during reverse transcription of lentiviral vectors after transduction of target cells. It also increases proviral DNA nuclear import, thus increasing lentiviral vector transduction efficacy. It also increases the titer of lentiviral vectors. ttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaattcaaaatttt

[0217] ET promoter (SEQ ID NO: 16): The ET promoter is a genetically engineered hepatocyte-specific promoter. This sequence directs transgene expression after lentiviral vector genome integration into target cells. The ET promoter is a chimeric promoter composed of a synthetic enhancer, an mTTR enhancer, and an mTTR promoter (Enhanced Transthyretin, Genbank Accession No. AY661265). cgcgagttaataattaccagcgcgggccaaataaataatccgcgaggggcaggtgacgtttgcccagcgcgcgctggtaattattaacctcgcgaatattgattcgaggccgcgattgccgcaatcgcgaggggcagg tgacctttgcccagcgcgcgttcgccccgccccggacggtatcgataagcttaggagcttgggctgcaggtcgagggcactgggaggatgttgagtaagatggaaaactactgatgacccttgcagagacagagtatt aggacatgtttgaacaggggccgggcgatcagcaggtagctctagaggatccccgtctgtctgcacatttcgtagagcgagtgttccgatactctaatctccctaggcaaggttcatatttgtgtaggttacttattc tccttttgttgactaagtcaataatcagaatcagcaggtttggagtcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtcacacagatccacaagctcctg

[0218] Components of the ET promoter: Synthetic enhancer (SEQ ID NO: 17): cgcgagttaataattaccagcgcgggccaaataaataatccgcgaggggcaggtgacgtttgcccagcgcgcgctggtaattattaacctcgcgaatattgattcgaggccgcgattgccgcaatcgcgaggggcaggtgacctttgcccagcgcgcg

[0219] mTTR enhancer (SEQ ID NO: 18): cactgggaggatgttgagtaagatggaaaactactgatgacccttgcagagacagagtattaggacatgtttgaacaggggccgggcgatcagcaggtag

[0220] mTTR promoter (SEQ ID NO: 19): gtctgtctgcacatttcgtagagcgagtgttccgatactctaatctccctaggcaaggttcatatttgtgtaggttacttattctccttttgttgactaag tcaataatcagaatcagcaggtttggagtcagcttggcagggatcagcagcctgggttggaaggagggggtataaaagccccttcaccaggagaagccgtc

[0221] mTTR 5'UT (SEQ ID NO: 20): acacagatccacaagctcctg

[0222] eGFP sequence (SEQ ID NO:51): This sequence codifies an enhanced version of the green fluorescent protein gene. tggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgaggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccacc tcgtgaccaccctgacctacggcgtgcagtgcttcagccgctacccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctgg tgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccg accactaccagcagaacaccccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaa

[0223] Mutated WPRE (WHV (Woodchuck Hepatitis Virus) Post-transcriptional Regulatory Element) (SEQ ID NO: 21): The mutated WPRE increases transgene expression in transduced cells by improving polyadenylation, RNA export from the nucleus, and protein synthesis. The mutated WPRE contains a mutation in the gene X frame to increase security. caacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggcttt cattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattg ccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattcc gtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgc

[0224] 142-3pT (SEQ ID NO: 22): Target sequence of 142-3 microRNA specifically expressed in hematopoietic cells. This element is a transcriptional control element that enhances the expression specificity of the transgene in transduced cells. tccataaagtaggaaacactaca

[0225] 3'LTR: DR3RU5 (SEQ ID NO: 23): The 3'LTR is responsible for mRNA polyadenylation during lentiviral vector production. In this case, the 3'LTR is composed of a truncated U3 element from wild-type HIV-1, a self-inactivating lentiviral vector, and elements R and U5. The integrated viral genome contains a deletion of the viral promoter region (U3), resulting in transcriptional inactivation of the potentially packaging-competent viral genome in transduced cells. tggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagca

[0226] ΔU3 (SEQ ID NO: 24): tggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtact

[0227] RU5 (SEQ ID NO: 25): gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcag

[0228] LV.ET.eGFP.142-3pT sequence (SEQ ID NO: 26):

[0229] LV.ET.eGFP.142-3pT map: LV.ET.eGFP.142-3pT construct. The various elements mentioned above are shown in FIG.

[0230] At the same time, we developed a therapeutic LV expressing an improved version of the AGXT gene (AGXT-RHEAM). We used the isolated construct of LV.ET.FIX.142-3pT obtained as described above. We amplified by PCR the AGXT-RHEAM sequence (14), kindly provided by Eduardo Salido's group. Primers were designed to add NheI (SEQ ID NO: 27 Fw: 5' CTA GCT AGC ATG GCC TCT CAC AAG CTG CT 3') and SalI (SEQ ID NO: 28 Rv: 5' CAA GTC GAC TCA CAG CTT CTT CTT GGG GC 3') target sequences to the 5' and 3' ends, respectively, for use in cloning. Finally, the LV.ET.FIX.142-3pT construct and the AGXT-RHEAM sequence were ligated to yield the LV.ET.AGXT-RHEAM.142-3pT vector.

[0231] LV.ET.AGXT-RHEAM.142-3PT sequence: The LV.ET.AGXT-RHEAM.142-3pT sequence contains the same elements as LV.EG.eGFP.142-3pT, except eGFP has been replaced with the AGXT-RHEAM coding sequence.

[0232] AGXT-RHEAM (SEQ ID NO: 29): This sequence, developed by Eduardo Salido's group (Non-Patent Document 14), codifies an improved version of the AGXT gene with five amino acid changes to increase the stability and half-life of the protein. ATGGCCTCTCACAAGCTGCTGGTGACCCCCCCAAGGCCCTGCTCAAGCCCCTCTCCATCCCCAAC CGTCTCCTGCTGGGGCCTGGTCCTTCCAACCTGCCTCCTCGCATCATGGCAGCCGGGGGGCTGCAGATGATCGGG CAC ATGAGCAAG GAA ATGTACCAGATCATGGACGAGATCAAGGAAGGCATCCAGTACGTGTTCCAGACCAGGAACCCACTCACACTGGTCATCTCCGGCTCGGGACACTGTGCCCTGGAGGCCGCCCTGGTCAATGTGCTGGAGCCTGGGGACTCCTTCCTGGTTGGGGCCAATGGCATTTGGGGGCAGCGAGCC GCG GACATCGGGGAGCGCATAGGAGCCCGAGTGCACCCGATGACCAAGGACCCCGGAGGCCACTACACACTGCAGGAGGTGGAGGAGGGCCTGGCCCAGCACAAGCCAGTGCTGCTGTTCTTAACCCACGGGGAGTCGTCCACCGGCGTGCTGCAGCCCCTTGATGGCTTCGGGGAACTCTGCCACAGGTACAAGTGCCTGCTCCTGGTGGATTCGGTGGCATCCCTGGGCGGGACCCCCCTTTACATGGACCGGCAAGGCATCGACATCCTGTACTCGGGCTCCCAGAAGGCCCTGAACGCCCCTCCAGGGACCTCGCTCATCTCCTTCAGTGACAAGGCCAAAAAGAAGATGTACTCCCGCAAGACGAAGCCCTTCTCCTTCTACCTGGACATCAAGTGGCTGGCCAACTTCTGGGGCTGTGACGACCAGCCCAGGATGTACCATCACACAATCCCCGTCATCAGCCTGTACAGCCTGAGAGAGAGCCTGGCCCTCATTGCGGAACAGGGCCTGGAGAACAGCTGGCGCCAGCACCGCGAGGCCGCGGCGTATCTGCATGGGCGCCTGCAGGCACTGGGGCTGCAGCTCTTCGTGAAGGACCCGGCGCTCCGGCTTCCCACAGTCACCACTGTGGCTGTACCCGCTGGCTATGACTGGAGAGACATCGTCAGCTACGTC ATGGACCACTTCGACATTGAGATCATGGGTGGCCTTGGGCCCTCCACGGGGAAGGTGCTGCGGATCGGCCTGCTGGGCTGCAATGCCACCCGCGAGAATGTGGACCGCGTGACGGAGGCCCTGAGGGCGGCCCTGCAGCACTGCCCCAAGAAGAAGCTGTGA

[0233] LV.ET.AGXT-RHEAM.142-3pT(SEQ ID NO: 30):

[0234] LV.ET.AGXT-RHEAM.142-3pT map. LV.ET.AGXT-RHEAM.142-3pT construct. The various elements mentioned above are shown in FIG.

[0235] AGXT-RHEAM. Amino acid sequence. SEQ ID NO:52 MASHKLLVTPPKALLKPLSIPNRLLLGPGPSNLPPRIMAAGGLQMIGHMSKEMYQIMDEIKEGIQYVFQTRNPLTLVISGSGHCALEAALVNVLEPGDSFLVGANGIWGQRAADIGERIGARVHPMTKDPGGHYTLQEVEEGLAQHKPVLLFLTHGESSTGVLQPLDGFGELCHRYKCLLLVDSVASLGGTPLYMD RQGIDILYSGSQKALNAPPGTSLISFSDKAKKKMYSRKTKPFSFYLDIKWLANFWGCDDQPRMYHHTIPVISLYSLRESLALIAEQGLENSWRQHREAAAYLHGRLQALGLQLFVKDPALRLPTVTTVAVPAGYDWRDIVSYVMDHFDIEIMGGLGPSTGKVLRIGLLGCNATRENVDRVTEALRAALQHCPKKKL*

[0236] 1.2 LV Production Laboratory-grade VSV.G pseudotyped third generation SIN LVs were generated by calcium phosphate transient transfection into HEK293T cells. Ten million HEK293T cells were cultured at 150 cm 2per cell culture dish and 24 hours later, cells were transfected with a solution containing a mixture of LV genome transcription plasmids (LV.ET.AGXT-RHEAM.142-3pT or LV.ET.eGFP.142-3pT) and packaging plasmids pMDLg / pRRE, pRSV.Rev, and pMD2.VSV.G. The medium was changed 6 hours after transfection and the supernatant was harvested 2 days later. Usually, the second harvest was performed 24 hours after the first. The harvested supernatant was clarified by filtration (0.22 μm PES) and concentrated by centrifugation at 20000g for 120 min at 16°C. The LV pellet was resuspended in an appropriate volume of sodium chloride 0.9% and stored at -80°C.

[0237] 1.3 LV dose setting LV.ET.AGXT-RHEAM.142-3pT dose setting was performed in the hepatic cell line, HepG2, due to the target tissue specificity of this strategy. For LV dose setting, 2 × 10 5 Cells were seeded in 24-well plates. 24 hours after seeding, cells were transduced with serial dilutions of LV in culture medium and cell numbers were determined on day 0. Five days after transduction, cells were harvested and genomic DNA (gDNA) was extracted using the NucleoSpin™ Tissue Kit (Macherey Nagel™) according to the manufacturer's instructions.

[0238] The number of lentiviral vector genomes integrated into transduced cells (VCN) was determined by qPCR. To analyze copies of lentiviral vector genomes, we used primers (SEQ ID NO: 31 Fw: 5' CAGGACTCGGCTTGCTGAAG 3', SEQ ID NO: 32 Rv: 5' TCCCCCGCTTAATACTGACG 3') and probe (SEQ ID NO: 33 5'-CGCACGGCAAGAGGCGAGG-3') (Taqman™, Thermo Fisher Scientific) designed against the Ψ sequence of the lentiviral vector. To determine the amount of endogenous DNA, we used primers (SEQ ID NO: 34 Fw: 5' GCTGTCATCTCTTGTGGGCTG 3', SEQ ID NO: 35 Rv: 5' ACTCATGGGAGCTGCTGGTTC 3') and probe (SEQ ID NO: 36 5' CCTGTCATGCCCACACAAATCTCTCC 3') (Taqman™, Thermo Fisher Scientific) against the human albumin gene. We also use the standard curve to determine the number of copies of the ψLV-specific sequence and the number of diploid genomes per sample, which allows us to calculate the number of LV copies per cell (VCN = ψ copies / diploid genomes). Finally, based on this parameter, we calculate the LV title, defined as the number of transducing units per milliliter, according to the following formula: Title (TU / mL) = number of cells on day 0 × VCN / LV volume per diploid genome (mL) The calculation was carried out using

[0239] Standard curves for extrapolating qPCR data were generated using serial dilutions of a DNA fragment containing Ψ and human albumin sequences. All reactions were performed in duplicate on a 7500 Fast Real-Time PCR System (Applied Biosystems™).

[0240] For LV.ET.eGFP.142-3pT dose setting, we also transduced HepG2 as described above. However, 3 days after transduction, the percentage of GFP-expressing positive cells was analyzed by flow cytometry. This parameter was calculated using the following formula: Title (TU / mL) = (cell count on day 0 x fluorescence % / 100) x (dilution factor / LV volume (ml)) was used to calculate the LV title.

[0241] 1.4 Experimental animals Agxt1 - / - Mouse (B6.129SvAgxt tm1Ull ) was developed by targeted mutagenesis in embryonic stem cells (Non-Patent Document 7). Wild-type C57BL / 6 mice were purchased from Jackson Laboratory™.

[0242] 1.5 Animal Treatment LV.ET.AGXT-RHEAM.142-3pT or LV.ET.GFP.142-3pT was administered via tail vein injection to adult male mice (12-14 weeks of age). - / - Experiments performed in mice showed that mice were given a single dose of LV.ET.AGXT-RHEAM.142-3pT (2.5 × 10 8 TU / mouse, 5×10 8 TU / mouse, 1×10 9 TU / mouse) or LV.ET.eGFP.142-3pT (1.4 × 10 8 TU / mouse, 5×10 8 TU / mouse). LV was injected in a total volume of 200 μl / mouse. Wild-type C57BL / 6 mice were administered a single dose of LV.ET.eGFP.142-3pT (1 × 10 8 TU / mouse, 2.5×10 8 TU / mouse). LV was injected in a total volume of 200 μl / mouse.

[0243] Three weeks after LV injection, Agxt1 - / -Mice were subjected to an ethylene glycol challenge test. It has been reported that oxalate overload is necessary to develop clinical signs of primary hyperoxaluria type 1 in this mouse model. For this purpose, 0.5% ethylene glycol (Ethylene glycol Reagent Plus™, Sigma-Aldrich, REF 102466), a precursor of glyoxylic acid metabolism, was administered in drinking water for 7 days. During the ethylene glycol challenge test, mice were isolated in metabolic cages and urine was collected for 24 hours. Furthermore, the weight of the animals was monitored throughout the treatment.

[0244] Agxt1 - / - Mice were sacrificed 7 days after the start of the ethylene glycol challenge test in the case of mice, or 4 weeks after LV injection in the case of wild-type C57BL / 6 mice.

[0245] For autopsy, mice were anesthetized with ketamine (2.5 mg / 20 g mouse) + medetomidine (0.2 mg / 20 g mouse). Upon losing consciousness, mice were sacrificed by diaphragm destruction, and thus 20 ml of PBS (1x) was injected to remove blood from tissues to facilitate the following analysis. In sacrificed animals, various tissues were harvested (liver, spleen, lung, bone marrow, lymph nodes, thymus, brain, testis, pancreas, and kidney).

[0246] Agxt1 - / -All animal procedures involving mice were performed at the CIMA (Centro de Investigacion Medica Aplicada) in Pamplona. All experimental procedures were approved by the Ethics Committee of the University of Navarra and the Navarra Institute of Public Health, in accordance with European Council guidelines. All experiments using animal models complied with all relevant ethical regulations. All animal procedures involving C57BL / 6 mice were performed at the CIEMAT (Centro de Investigaciones Energeticas y Medioambientales y Tecnologicas) in Madrid. All experimental procedures were approved by the Direccion General de Medio Ambiente of Comunidad de Madrid under PROEX 165-18.

[0247] 1.6 Determination of VCN in LV-injected mice After sacrifice of the animals, different tissues were harvested (liver, spleen, lung, bone marrow, lymph nodes, thymus, brain, testis, pancreas, and kidney) and stored at −80° C. For the determination of the LV genome integrated in these tissues, genomic DNA was extracted with the NucleoSpin™ Tissue Kit (Macherey Nagel) according to the manufacturer's instructions.

[0248] The number of integrated lentiviral vector genomes (VCN) in injected mouse tissues was determined by qPCR. To analyze the number of lentiviral vector genomes, we used primers (SEQ ID NO: 37 Fw: 5' CAGGACTCGGCTTGCTGAAG 3', SEQ ID NO: 38 Rv: 5' TCCCCCGCTTAATACTGACG 3') and probe (SEQ ID NO: 39 5'-CGCACGGCAAGAGGCGAGG-3') (Taqman™, Thermo Fisher Scientific) designed against the Ψ sequence of the lentiviral vector. To determine the amount of endogenous DNA, we used primers (SEQ ID NO: 40 Fw: 5' AAAACGAGCAGTGACGTGAGC 3', SEQ ID NO: 41 Rv: 5' TTCAGTCATGCTGCTAGCGC 3') and probe (SEQ ID NO: 42 5'-TGCACGGAAGCGTCTCGTCTCAGTC-3') against the mouse titin gene. Once the LV copy number and diploid genome number per sample were determined, the VCN was calculated (VCN=Ψ copy number / diploid genome number).

[0249] Standard curves were generated using serial dilutions of DNA fragments containing the Ψ and titin sequences. All reactions were performed in duplicate on a 7500 Fast Real-Time PCR System (Applied Biosystems™).

[0250] 1.7 Determination of AGXT expression Injection Agxt1 - / -To analyze the functionality of therapeutic lentiviral vectors in mice, we determined the transgene expression in the liver of these mice. To identify this parameter, RNA was extracted from the liver by the Trizol protocol. In brief, liver fragments were homogenized in Trizol, followed by the addition of chloroform to separate the aqueous phase. RNA was precipitated with isopropanol and glycogen, washed with 70% ethanol, and finally resuspended in nuclease-free water. To ensure that the RNA samples were free of genomic DNA, the samples were digested with DNase using the DNase Max™ kit (Qiagen) according to the manufacturer's instructions. RNA samples were kept at -80°C.

[0251] Afterwards, the RT-PCR technique was carried out to synthesize cDNA from the RNA samples by the RETROscript™ kit (Ambion, USA) according to the manufacturer's instructions.

[0252] Transgene expression was determined by qPCR. We designed primers for AGXT-RHEAM (SEQ ID NO: 43 Rv: 5' GTCTTGCGGGAGTACATCTT 3', SEQ ID NO: 44 Fw: 5' CAAGGCATCGACATCCTGTA 3') and two housekeeping genes mouse Tbp (SEQ ID NO: 45 Rv: 5' GATGGGAATTCCAGGAGTCA 3', SEQ ID NO: 46 Fw: 5' GGGAGAATCATGGACCAGA 3') and mouse Actb (SEQ ID NO: 47 Rv: 5' CTA AGG CCA ACC GTG AAA AG 3', SEQ ID NO: 48 Fw: 5' ACC AGA GGC ATA CAG GGA CA 3'). All reactions were performed in duplicate in a Fast Real-Time PCR System (Applied Biosystems™). Quantification of relative expression of the transgenes was performed by ΔΔCt analysis (Schefe et al., 2006) referenced separately to two housekeeping genes, mouse Tbp and mouse β-actin.

[0253] We also analyzed the physiological expression of mouse Agxt in the liver of non-injected C57BL / 6 mice. RNA was extracted from liver samples of non-injected wild-type mice and RT-PCR was performed as described above. To analyze gene expression, primers were designed for the mouse Agxt gene (SEQ ID NO: 49 Rv: 5' GACAAAGCCAGTCTCCTTCTAC 3', SEQ ID NO: 50 Fw: 5' GTGACAGGTGTGGTATGATGAA 3'). For expression analysis, mouse Tbp and mouse Actb were also measured by RT-qPCR. All reactions were performed in duplicate in a Fast Real-Time PCR System (Applied Biosystems™). Quantification of the relative expression of the transgene was performed by ΔΔCt analysis (Schefe et al., 2006) referenced separately to two housekeeping genes, mouse Tbp and mouse Actb.

[0254] 1.8 Determining the percentage of transduced cells by immunostaining To analyze the percentage of transduced cells in target tissues, we performed immunostaining assays to detect eGFP-positive cells in the livers of LV.ET.eGFP.142-3pT-injected mice.

[0255] Samples from paraffin sections were deparaffinized by incubation in xylene and decreasing ethanol dilutions. Samples were then incubated in blocking solution (10% donkey serum in PBS (1x)) for 1 hour and overnight with goat-αeGFP (ab6673 abcam™) 1:100 and rabbit-αmsALB (AHP1478) 1:500 in blocking solution. The next day, samples were incubated with secondary antibodies (donkey-α goat AF™488 (A11055 invitrogen™), donkey-α rabbit AF™488 (A21206 invitrogen™), donkey-α goat AF™594 (A11058 invitrogen™)) 1:1000 and DAPI 1:1000 in blocking solution for 1 hour. The samples were then mounted with Mowiol™ and visualized with a Zeiss AxioImager microscope. Images were acquired for each sample.

[0256] Determination of eGFP positive cells in liver immunostained samples was performed by QuPath™ software. The percentage of eGFP expressing cells relative to the total number of liver cells in each image was determined. At least 10 different fields were analyzed per sample (400x magnification).

[0257] 1.9 Urinary oxalate determination A 24-hour urine was collected from the mice to measure the total amount of oxalate removed from the organism during this period. Each 24-hour urine was collected in 50 μl of 5N HCl and the amount was measured. The urine samples were clarified with activated charcoal and oxalate was measured by enzymatic quantification by oxalate oxidation using an oxalate kit (Trinity Biotech, REF 591-D) according to the manufacturer's instructions. To determine the concentration of oxalate (μmol) in each sample, a reference curve of known concentrations of oxalate was generated (oxalate standard 0.5 mmol / l REF 591-3 Trinity Biotech). Finally, the oxalate concentration and urine volume were used to calculate the oxalate quantification (μmol / 24 hours).

[0258] 1.10 Determination of Renal Injury: Renal Injury Score After the animals were sacrificed, the kidneys were fixed in formalin and embedded in paraffin. Then, 5 μm sections were obtained and stained with hematoxylin-eosin. To determine the damage that occurred, we developed a "kidney damage score" that classified the stage of each sample into four different stages according to the characteristics observed. 0: No damage 1: The structure is normal but there are some signs of damage such as dilated vessels. 2: The structure is partially affected, with an increased presence of dilated ducts. 3: The tissue structure is severely affected and the presence of calcium oxalate crystals can be observed, indicating a stage of nephrocalcinosis.

[0259] 1.11 Transduction enhancer: in vitro testing of cyclosporine H To increase the in vivo transduction efficacy after treatment with the developed lentiviral vector, we tested the effect of the transduction enhancer cyclosporine H (CsH) during the in vitro transduction protocol. Two different regimens of CsH were tested. HepG2 cells, a hepatic cell line, were used because of the important relevance of the target tissue origin.

[0260] For the first CsH regimen, 2 × 10 5 Cells were seeded in 24-well plates. One day later, cells were transduced with a culture medium solution containing LV.ET.eGFP.142-3pT and CsH at an MOI of 0.3. Different CsH concentrations were tested (4 μM, 8 μM, and 16 μM). Three days after transduction, the percentage of eGFP-positive cells was analyzed by flow cytometry. All conditions were performed as triplicates.

[0261] For the second CsH regimen, 2 × 10 5Cells were seeded in 24-well plates. After 1 day, the culture medium was changed and the cells were preincubated with culture medium supplemented with 16 μM CsH. After 16 h, cells were transduced with a solution containing LV.ET.eGFP.142-3pT at an MOI of 0.3 and 16 μM CsH. Three days after transduction, the percentage of eGFP-positive cells was analyzed by flow cytometry. All conditions were performed as triplicates.

[0262] 1.12 Statistical analysis Significant differences between groups were determined by the Mann-Whitney test, a non-parametric test used to compare two independent groups without using a Gaussian distribution. Analyses were performed with Graphpad Prism™ software. A value of p<0.05 was considered significant.

[0263] 1.13 Transduction enhancer: in vivo testing of dexamethasone To enhance the transduction efficacy of the lentiviral vectors of the present invention, we tested the combination of lentiviral vector administration with dexamethasone, a suppressor of the innate immune response. Analysis was performed in C57BL / 6 adult male mice.

[0264] C57BL / 6 mice were injected with a single dose of LV.ET.eGFP.142-3pT (5 × 10 8 TU / mouse) was administered via tail vein injection. Additionally, mice were treated with 5 mg / kg body weight dexamethasone (Fortecortin™) and injected intraperitoneally in three sequential doses, 12 hours and 2 hours before and 4 hours after lentiviral vector injection. A control group injected with lentiviral vector only was also included in the experiment.

[0265] Mice were sacrificed 4 weeks later and transduction efficacy analysis (lentiviral vector integration per diploid genome and percentage of eGFP positive cells in liver) was performed.

[0266] Example 2: Lentiviral vectors were administered intravenously to C57BL / 6 mice and Agxt1 mice - / - Efficiently transducing the liver in a mouse model First, as a proof of concept to study the feasibility of the developed gene therapy tool and to perform transduction percentage and biodistribution analysis, we performed an in vivo experiment in which adult C57BL / 6 male mice were injected with a single dose of LV.ET.GFP.142-3pT (Fig. 1a, Fig. 2a). We administered two different doses (1 × 10 8 TU / mouse, 2.5×10 8 TU / mouse). We tested 1×10 8 In C57BL / 6 mice injected with a dose of 1.0 × 10 TU / mouse, we observed 0.55 (0.34–0.56) lentiviral vector copies per diploid genome, compared with 2.5 × 10 8 In C57BL / 6 mice injected with a dose of 1.03 (0.94-1.56) lentiviral vector copies per diploid genome were obtained (Figure 3). Immunostaining analysis reveals that this corresponds to 1.751% (1.13%-5.55%) and 12.92% (10.72%-13.81%) eGFP-positive hepatocytes at the low and high doses, respectively (Figure 5). Representative images of these mice can be observed in Figure 5 (A-C, F-H).

[0267] Next, to further characterize the in vivo transduction efficiency of the lentiviral vector LV.ET.GFP.142-3pT and compare it with the in vitro transduction efficiency of the same lentiviral vector, HepG2 cells were transduced with LV.ET.eGFP.142-3pT at different multiplicities of infection (MOI). Three days after transduction, the percentage of eGFP-positive cells was determined by flow cytometry. Using an MOI of approximately 1, a 50% transduction percentage was achieved, whereas at higher MOIs 100% transduction was achieved in vitro. For the determination of in vivo transduction efficacy, adult C57BL / 6 (Figure 13, black circles) or Agxt1- / - Mice (Figure 13, open circles) were injected with different doses (1 × 10 8 TU / mouse, 1.4×10 8 TU / mouse, 2×10 8 TU / mouse and 5×10 8 LV.ET.eGFP.142-3pT was intravenously injected at 100 ng / mouse (100 ng / TU). The corresponding MOI was calculated only according to the estimated number of hepatocytes in the liver of these mice as previously described (Park et al., 2000; Schmitt et al., 2010). Mice were therefore injected with the following MOIs: 0.8, 1.1, 2, and 4. The percentage of transduced hepatocytes was determined in liver tissue samples after 4 weeks. The results shown in Figure 13 indicate that the transduction efficiency of this lentiviral vector is significantly reduced when used in vivo compared to in vitro experiments.

[0268] Therefore, to test the feasibility of in vivo administration of therapeutic lentiviral vectors as a gene therapy strategy to treat primary hyperoxaluria type 1, we used adult male Agxt1 - / - Mice were intravenously injected with a single dose of the LV.ET.AGXT-RHEAM.142-3pT lentiviral vector (Figure 1B). Three different lentiviral vector doses (2.5 × 10 8 TU / mouse, 5×10 8 TU / mouse, 1×10 9 Three weeks after injection, the mice were subjected to a 7-day ethylene glycol challenge test (FIG. 2B) and then sacrificed.

[0269] The copy number of genomic lentiviral vector integrated in the liver was determined at the time of sacrifice. Dose-related efficacy in liver transduction was observed. Liver VCN achieved in three different groups was 2.5 × 10 8 Agxt1 administered TU / mice - / - Mouse: 0.615 (0.39–1.46), 5 × 10 8 Agxt1 administered TU / mice - / -Mouse: 0.765 (0.45–1.19), 10 × 10 8 Agxt1 administered TU / mice - / - The mean p value for the lentiviral vector genome in the non-injected mice was 1.165 (range, 0.85-0.32) (Figure 3). We also analyzed this parameter in non-injected mice, and, as expected, we did not detect any copies of the lentiviral vector genome in these animals.

[0270] As a control, we also used Agxt1 - / - Mice were injected with the reporter lentiviral vector. Two different LV doses were tested (1.4 × 10 8 TU / mouse, 5×10 8 We also observed a dose-related liver transduction efficacy. In low-dose injected mice, a liver VCN of 0.11 (0.09-0.49) was observed, whereas at the high dose, a liver VCN of 0.84 (0.73-1.58) was observed (Figure 3). Furthermore, reporter LV transduction efficacy was significantly higher in mice treated with Agxt1 than in mice treated with Agxt1. - / - This was comparable to the efficacy achieved with therapeutic lentiviral vectors in mice.

[0271] With the intention of identifying the percentage of transduced cells achieved after lentiviral vector injection, we also performed a LV.ET.eGFP.142-3pT Agxt1 - / - The percentage of positive cells in liver sections of injected mice was analyzed (Figure 5). 8 Agxt1 administered TU / mice - / - In mice, 1.777% (0.399%–4.572%) of eGFP-positive hepatocytes were observed, whereas 5 × 10 8 Agxt1 administered TU / mice - / - In mice, 5.5059% (2.109%-8.754%) of eGFP-positive hepatocytes were observed. Representative images of these mice are shown in Figure 5(D,E). Because the reporter lentiviral vector and therapeutic lentiviral vector appear to function similarly, 5 × 10 8 TU / Agxt1 mice administered therapeutic lentiviral vectors- / - In mice, Agxt1 was administered the same dose of reporter lentiviral vector. - / - It was expected that similar transduction percentages to those observed in mice would be achieved.

[0272] Agxt1 - / - Differences in transduction efficacy between injected mice with and wild type C57BL / 6 may be due to variability in the genetic background of the mice or titering of LV lab grade batches.

[0273] Example 3: Therapeutic lentiviral vectors treat Agxt1 - / - Efficiently expressed in mouse liver The present inventors have developed a therapeutic lentiviral vector, Agxt1 - / - Having confirmed the presence of the therapeutic lentiviral vector in the target tissues of injected mice, we wanted to test whether this construct was efficiently expressed. - / - Transgene expression in mouse liver was analyzed by RT-qPCR. As a reference, two different housekeeping genes were used in this analysis (msTbp and msActb), and Agxt1 - / - The difference between physiological mouse Agxt1 expression in C57BL / 6 wild type mice compared to AGXT expression induced by lentiviral vector integration following an in vivo gene therapy strategy in mice was appropriately determined.

[0274] 2.5×10 8 Therapeutic lentivirus-injected mice receiving a dose of TU / mouse showed 10.88% (5.62%-16.79%) AGXT expression in reference to msTbp expression and 0.1041% (0.06007%-0.2452%) AGXT expression in reference to msActb. 5 × 10 8 Agxt1 administered at a dose of TU / mouse - / -Mice had 12.11% (4.38%-14.48%) AGXT expression in reference to msTbp and 0.1857% (0.08878%-0.2966%) AGXT expression in reference to msActb. 9 Agxt1 in TU / mouse - / - Therapeutic lentiviral vector-injected mice showed 19.88% (18.52%-28.55%) AGXT expression with reference to msTbp and 0.3088% (0.1707%-0.4418%) AGXT expression with reference to msActb (Figure 4). Thus, the different therapeutic lentiviral vectors Agxt1 - / - The difference in AGXT expression levels between injected mice correlated with the dose administered. - / - The mice showed no AGXT expression.

[0275] Furthermore, to investigate the difference between AGXT expression achieved after therapeutic lentiviral vector injection and physiological mouse Agxt1 expression, we analyzed this expression in wild-type C57BL / 6 mice. - / - The mouse Agxt1 expression of 8563 (6996-8939) referenced to msTbp and 340.5 (141-374.5) referenced to msActb was significantly higher than that achieved in treated mice.

[0276] Example 4: In vivo lentiviral vector injection into C57BL / 6 mice reduces off-target transduction To determine the security of the proposed therapeutic strategy, we analyzed the number of lentiviral vector integrated genomes in C57BL / 6 wild-type mice injected with two different doses of reporter lentiviral vector. The number of LV genome copies integrated in different tissues of injected wild-type C57BL / 6 mice was analyzed by qPCR. The integration of reporter lentiviral vector genome was observed only in the target tissue liver, as well as in the lung, spleen and bone marrow, which could occur due to the administration route used in this strategy. 1 × 10 8C57BL / 6 mice injected with TU / mouse showed VCN of 2.72 (2.33–6.2) in the lung, 0.21 (0.14–0.32) in the spleen, and 0.05 (0.04–0.09) in the bone marrow. 8 Mice injected with TU / mouse showed VCN values ​​of 4.94 (0.185-7.79) in the lungs, 0.35 (0.25-0.55) in the spleen, and 0.13 (0.09-0.18) in the bone marrow (Figure 6). Although the presence of integrated vector genomes in these tissues was observed, reporter gene expression was not observed by immunofluorescence analysis, indicating a reduced risk resulting from off-target transduction. Furthermore, no signs of tumor formation were observed.

[0277] Example 5: Therapeutic lentiviral vector therapy Agxt1 - / - Mice showed reduced urinary oxalate levels Agxt1 - / - Primary hyperoxaluria type 1 pathological phenotype analysis in mouse models requires an ethylene glycol challenge to obtain a phenotype closer to the human phenotype. This challenge produces an overload in oxalate production, which is necessary to analyze the pathological phenotype of these mice.

[0278] Over the course of a 7-day ethylene glycol challenge, mice were quarantined for 24 hours and urine was collected for 24 hours. Urinary oxalate concentrations were measured at three different time points: before the start of treatment (basal measurement) and on the 3rd and 7th days of treatment.

[0279] To analyze the pathological phenotype, 2.5 × 10 of three different therapeutic lentiviral vector doses were 8 TU / mouse, 5×10 8 TU / mouse and 10x10 8 In addition, three different control groups were used: PBS or Agxt1 mice injected with a reporter lentiviral vector (LV.ET.GFP.142-3pT). - / -Two negative control groups of mice and a positive control group of C57BL / 6 wild-type mice were included. All groups were subjected to ethylene glycol challenge.

[0280] Before treatment, urinary oxalate concentrations were low in all conditions. After the initiation of the ethylene glycol challenge, an increase in urinary oxalate concentrations was observed in all groups. However, the observed increase was greater in untreated Agxt1 mice than in those treated with the therapeutic lentiviral vector. - / - Moreover, the variance of the data related to this value, a specific feature of this mouse model, was significantly greater in the LV-treated Agxt1 group. - / - than untreated Agxt1 mice - / - It was big in the mouse. 5×10 8 TU / Agxt1 mice administered therapeutic lentiviral vectors - / - Urinary oxalate concentrations in mice were significantly higher in untreated Agxt1 mice than in control mice. - / - The urinary oxalate concentrations in the rats were significantly lower than those in the control group on days 3 and 7. Importantly, on day 7, the urinary oxalate concentrations were 5×10 8 Therapeutic LV injection in TU / Mice Agxt1 - / - There was no significant difference between the mice and wild-type C57BL / 6 groups (Figure 7).

[0281] Example 6: Treated mice gain weight during ethylene glycol challenge Agxt1 - / - Another important parameter for tracking the pathological phenotype of mice is the weight gain or loss of the animals during the ethylene glycol challenge test. Regarding this parameter, we analyzed the weight trends of all mice at the two reference time points and compared them with the weight measurements at the basal time point. We observed a decrease in the weight of the animals in the untreated control group. This decrease was not observed in the therapeutic LV treatment group. Furthermore, weight gain was achieved (Figure 8). Seven days after the ethylene glycol challenge test, 5 × 10 8 TU / mouse and 10x10 8 Therapeutic LV injection in TU / Mice Agxt1 - / - Weight gain in the mice was not significantly different from that in C57BL / 6 wild-type mice.

[0282] Examples 5 and 6 show that although the AGT expression observed in the corrected cells is much lower than that observed in normal hepatocytes, the phenotypic correction obtained in the treated animals is unexpectedly much higher. Thus, considering the hypothesis that "more than 40% of the cells need to be corrected to achieve a therapeutic effect", an unexpected degree of phenotypic conversion was found. In the most efficient groups, the percentage of transduction is below 20% (see FIG. 5 with reporter LV).

[0283] Example 7: Treated mice show reduced incidence of nephrocalcinosis One of the most important clinical signs of patients with primary hyperoxaluria type 1 is the development of nephrocalcinosis due to calcium oxalate crystal formation in the kidney parenchyma. To determine the damage in the kidneys of animals, we developed a kidney damage score to classify the damage caused by ethylene glycol treatment, where 0 represents no damage, 1 represents mild damage in the kidney parenchyma, and 2 represents moderate damage that may turn into stage 3 where obvious signs of nephrocalcinosis can be observed. Each animal was classified with respect to this score. With respect to this classification, a higher percentage of mice developed nephrocalcinosis in the non-treated group compared to the treated group. 5×10 8 Therapeutic LV injection in TU / Mice Agxt1 - / - In mice, no mice were observed to develop nephrocalcinosis (FIG. 9A).

[0284] This score can be simplified into mice with no or mild kidney damage (score 0–1), and moderate or severe kidney damage (score 2–3), which represents mice with progressive kidney damage as determined by the ethylene glycol tolerance test. - / - Compared to mice, progression of kidney damage occurred in half the cases, one-quarter, with a 2.5 × 10 8 TU / mouse or 10x10 8 Therapeutic lentiviral vector injection Agxt1 at a dose of TU / mouse - / - Progression of kidney damage was also observed in mice. 8Therapeutic lentiviral vector injection in TU / Mice Agxt1 - / - In mice, only 1 in 10 treated mice developed kidney damage progression, with the intermediate dose (5 × 10 8 ) did not develop nephrocalcinosis (Figure 9B).

[0285] [Table 1]

[0286] Example 8: Use of cyclosporine H in lentiviral transduction protocols increases transduction efficacy in hepatic cell lines in vitro To increase the amount of corrected hepatocytes without increasing the amount of ineffective viral dose as observed in previous experiments, we looked for potential transduction enhancers to optimize LV transduction. Cyclosporine H, among others, has been described to promote in vitro LV transduction in hematopoietic stem cells (HSCs), where IFITM3, the target protein of CsH, is highly expressed under basal conditions. However, no data on hepatocytes as target cells were suggested or tested. With the aim of testing this possibility, hepatic HepG2 cells were transduced with LV.ET.eGFP.142-3pT in two different dosing regimens and different CsH concentrations.

[0287] In the first transduction protocol, HepG2 cells were exposed to different concentrations of CsH (4 μM, 8 μM, or 16 μM) during the lentiviral vector transduction protocol. Cells were transduced with a lentiviral vector MOI of 0.3. Three technical replicates of each condition were performed. The basal transduction level was 36.67%, and in the different CsH-treated samples, transduction percentages of 37.67%, 44.3%, and 51.5% were observed in the 4 μM, 8 μM, and 16 μM CsH conditions, representing a 1.03-, 1.2-, and 1.4-fold increase over the control sample, respectively ( FIG. 10 ).

[0288] In the second CsH dosing regimen, HepG2 cells were subjected to a 16 h CsH pretreatment prior to transduction and CsH was also included. The only CsH concentration tested was 16 μM. In this CsH dosing regimen, a 2.35-fold increase in eGFP-positive cells was observed over the control sample (Figure 10). When cells were transduced with an MOI of 0.3 LV.ET.eGFP.142-3pT, the percentage of transduced cells achieved was 13.2% in control conditions and 31.03% in CsH-treated cells.

[0289] The observed difference in transduction levels between the control conditions at the same MOI is likely due to a decrease in LV titers due to the thaw-freeze cycles. The two different dosing regimens were independent experiments.

[0290] Thus, it is noteworthy that addition of CsH as a transduction enhancer increases the transduction efficiency (in vitro) of the developed lentiviral vector in hepatic cell lines such as HepG2, even though the CsH target gene IFITM3 is not thought to be expressed in the hepatic lineage.

[0291] Example 9: Dexamethasone treatment improves transduction efficacy in the liver of reporter lentiviral vector-treated C57BL / 6 mice in vivo The use of additional compounds may be a useful tool to increase the amount of transduced hepatocytes, thereby improving the therapeutic efficacy of the treatment with the lentivirus of the present invention. In addition, the immune response mediated by type 1 interferon (IFN-1) that lentiviral vectors may cause may also interfere with the transduction efficacy, and therefore the use of inhibitors of said immune response also represents a potential combination treatment after in vivo administration of lentiviral vectors. For this reason, the inventors performed experiments in which C57BL / 6 mice were treated with dexamethasone (following the protocol described in Agudo et al. 2012, Molecular Therapy vol. 20 no. 12, 2257-2267) in combination with lentiviral vectors.

[0292] C57BL / 6 mice were administered a single dose of LV.ET.eGFP.142-3pT (5 × 10 8 Mice were treated with 5 mg / kg body weight dexamethasone (Fortecortin™) intraperitoneally in three sequential doses, 12 hours and 2 hours before and 4 hours after lentiviral vector injection. Mice were sacrificed 4 weeks after the combined treatment ( FIG. 14A ).

[0293] In mice treated with a combination of dexamethasone and reporter lentiviral vector, 0.92 (0.38-1.4) integrated copies per diploid genome were found, whereas in mice treated with lentiviral vector alone, 1.48 (1.2-1.76) integrated copies per diploid genome were detected, a significant increase compared to mice treated with the combination treatment (Figure 14B). However, immunostaining analysis reveals a 4.08-fold increase in the percentage of eGFP-positive hepatocytes in mice treated with the combination treatment versus mice treated with lentiviral vector alone (18.11% (6.381%-19.79%) eGFP-positive hepatocytes and 4.434% (2.167%-7.55%) eGFP-positive hepatocytes, respectively). Representative images of these mice can be observed in Figure 14D and E.

[0294] These results suggest that Agxt1 - / - In the context of mice, our results suggest that the combination of dexamethasone and LV.ET.AGXT-RHEAM.142-3pT would improve transduction efficacy and, plausibly, therapeutic efficacy, supporting the feasibility of lentiviral vector-based in vivo strategies for the treatment of PH1 patients.

Claims

1. 1. A lentiviral vector comprising a nucleic acid, the nucleic acid comprising: an engineered hepatocyte-specific promoter having at least 98% sequence identity over the entire length of SEQ ID NO: 16; A nucleotide sequence encoding an optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM), wherein the AGXT-RHEAM protein has at least 98% amino acid sequence identity over the entire length of SEQ ID NO:52; A mutation-optimized woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 98% sequence identity over the entire length of SEQ ID NO:21; at least a copy of at least one miRNA target sequence, wherein the miRNA target sequence is selected from the group consisting of miRNA-142-3, miR-181, miR-223, or miR-30b; Including, A lentiviral vector, wherein the engineered hepatocyte-specific promoter, the WPRE and at least a copy of the at least one miRNA target sequence are operably linked to the AGXT-RHEAM protein and regulate expression of the AGXT-RHEAM protein.

2. The lentiviral vector of claim 1, wherein the amino acid sequence of the alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) consists solely of SEQ ID NO:

52.

3. The lentiviral vector according to claim 1 or 2, wherein the nucleotide sequence of the genetically engineered hepatocyte-specific promoter consists solely of SEQ ID NO:

16.

4. 3. The lentiviral vector of claim 1 or 2, wherein the nucleotide sequence of the mutation-optimized Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE) consists solely of SEQ ID NO:

21.

5. The lentiviral vector according to claim 1 or 2, wherein the nucleotide sequence coding for the optimized alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT-RHEAM) protein consists solely of SEQ ID NO:

29.

6. The lentiviral vector of claim 1 or 2, wherein the at least one copy of the at least one miRNA target sequence consists of only four copies of the target sequence of miRNA-142-3 having at least 95% sequence identity over the entire length of SEQ ID NO:

22.

7. The nucleic acid is selected from the group consisting of: a) a 5′ long terminal repeat (LTR); b) a primer binding site (PBS); c) a psi packaging signal; and d) the stem loop 4 (SL4) region of the wild-type HIV virus; and e) a Rev response element (RRE); and f) DNA flap central polypurine tract (cPPT); g) a 3′ long terminal repeat (LTR); The lentiviral vector of claim 1 or 2, further comprising:

8. The nucleotide contained in the lentiviral vector is h) a DenvRF1 region located between d) and e); i) the DenvRF2 region located between e) and f); The lentiviral vector of claim 1 or 2, further comprising:

9. The lentiviral vector of claim 1 or 2, wherein the full-length nucleotide sequence of the lentivirus has at least 95% sequence identity with SEQ ID NO: 30 (LV.ET.AGXT-RHEAM.142-3pT).

10. The lentiviral vector of claim 9, which has 100% sequence identity with SEQ ID NO:

30.

11. A substantially pure cell population transduced with the lentiviral vector of claim 1.

12. A pharmaceutical composition comprising the lentiviral vector of claim 1 and a pharma- ceutically acceptable carrier or diluent.

13. 13. A lentiviral vector as claimed in claim 1 or 2, a substantially pure cell population as claimed in claim 11, or a pharmaceutical composition as claimed in claim 12 for use as a medicament.

14. 13. The lentiviral vector of claim 1 or 2, the substantially pure cell population of claim 11, or the pharmaceutical composition of claim 12, for use in treating a subject in need of treatment for primary hyperoxaluria, the method comprising administering the lentiviral vector, the substantially pure cell population, or the pharmaceutical composition to the subject.

15. 15. The lentiviral vector, substantially pure cell population, or pharmaceutical composition for use according to claim 14, wherein the primary hyperoxaluria is type 1.

16. 14. The lentiviral vector, substantially pure cell population, or pharmaceutical composition for use according to claim 13, wherein said use is in combination with an immune response suppressor.

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