Semliki forest virus vectors engineered to express higher IL-12 levels induce efficient elimination of murine colon adenocarcinomas

To evaluate the use of alphavirus vectors for tumor treatment we have constructed and compared two Semliki Forest virus (SFV) vectors expressing different levels of IL-12. SFV-IL-12 expresses both IL-12 subunits from a single subgenomic promoter, while in SFV-enhIL-12 each IL-12 subunit is expressed from an independent subgenomic promoter fused to the SFV capsid translation enhancer. This latter strategy provided an eightfold increase of IL-12 expression. We chose the poorly immunogenic MC38 colon adenocarcinoma model to evaluate the therapeutic potential of SFV vectors. A single intratumoral injection of 10(8) viral particles of SFV-IL-12 or SFV-enh-IL-12 induced>or=80% complete tumor regressions with long-term tumor-free survival. However, lower doses of SFV-enhIL-12 were more efficient than SFV-IL-12 in inducing antitumoral responses, indicating a positive correlation between the IL-12 expression level and the therapeutic effect. Moreover, repeated intratumoral injections of suboptimal doses of SFV-enhIL-12 increased the antitumoral response. In all cases SFV vectors were more efficient at eliminating tumors than a first-generation adenovirus vector expressing IL-12. In addition, the antitumoral effect of SFV vectors was only moderately affected by preimmunization of animals with high doses of SFV vectors. This antitumoral effect was produced, at least partially, by a potent CTL-mediated immune response

Increased efficacy and safety in the treatment of experimental liver cancer with a novel adenovirus-alphavirus hybrid vector

An improved viral vector for cancer gene therapy should be capable of infecting tumors with high efficiency, inducing specific and high-level expression of transgene in the tumor and selectively destroying tumor cells. In the design of such a vector to treat hepatocellular carcinoma, we took advantage of (a) the high infectivity of adenoviruses for hepatic cells, (b) the high level of protein expression and proapoptotic properties that characterize Semliki Forest virus (SFV) replicon, and (c) tumor selectivity provided by alpha-fetoprotein (AFP) promoter. We constructed a hybrid viral vector composed of a helper-dependent adenovirus containing an SFV replicon under the transcriptional control of AFP promoter and a transgene driven by SFV subgenomic promoter. Hybrid vectors containing murine interleukin-12 (mIL-12) genes or reporter gene LacZ showed very specific and high-level expression of transgenes in AFP-expressing hepatocellular carcinoma cells, both in vitro and in an in vivo hepatocellular carcinoma animal model. Infected hepatocellular carcinoma cells were selectively eliminated due to the induction of apoptosis by SFV replication. In a rat orthotopic liver tumor model, treatment of established tumors with a hybrid vector carrying mIL-12 gene resulted in strong antitumoral activity without accompanying toxicity. This new type of hybrid vectors may provide a potent and safe tool for cancer gene therapy

Generation and characterization of human iPSC line generated from mesenchymal stem cells derived from adipose tissue

Abstract In this work, mesenchymal stem cells derived from adipose tissue (ADSCs) were used for the generation of the human-induced pluripotent stem cell line G15.AO. Cell reprogramming was performed using retroviral vectors containing the Yamanaka factors, and the generated G15.AO hiPSC line showed normal karyotype, silencing of the exogenous reprogramming factors, induction of the typical pluripotency-associated markers, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation ability to the three germ layers

Treatment of chronic viral hepatitis in woodchucks by prolonged intrahepatic expression of interleukin-12

Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 10(10) viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-gamma) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-gamma in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 10(10) vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers

Generation of NKX2.5(GFP) Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts

Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5(GFP) reporter cardiac fibroblasts. We first established a new NKX2.5(GFP) reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells. The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5(GFP) hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5(GFP) cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.This work was supported by PID 2019-107150RB-I00/AEI/ 10.13039/501100011033 to XC-V; by the “Ramón y Cajal” State Program, Ministry of Economy and Competitivenes

Discovery of first-in-class reversible dual small molecule inhibitors against G9a and DNMTs in hematological malignancies

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.We particularly acknowledge the Biobank of the University of Navarra for its collaboration. We thank Dr Edorta Martínez de Marigorta and Dr Francisco Palacios from Departamento de Química Orgánica I, Facultad de Farmacia, Universidad del Pais Vasco for 13C NMR determination and Angel Irigoyen Barrio and Dr Ana Romo Hualde, from University of Navarra, for HRMS determination. Dr. Irene de Miguel Turrullols from Small Molecule Discovery Platform, CIMA, University of Navarra is acknowledged for NMR data interpretation. This work was funded by grants from Instituto de Salud Carlos III (ISCIII) PI10/01691, PI13/01469, PI14/01867, PI10/2983, TRASCAN (EPICA), CIBERONC, cofinanciacion FEDER, RTICC RD12/0036/0068, Fundació La Marató de TV3 (20132130-31-32) and ‘Fundación Fuentes Dutor’. B.P. is supported by a Sara Borrell fellowship CD13/00340 and X.A. is a Marie Curie researcher under contract ‘LincMHeM-330598’.S

CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I

CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1(-/-) mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites

Development of the advanced genetic therapies for Primary Hyperoxaluria type I

Primary Hyperoxaluria type I (PH1) is an inherited inborn error of the glyoxylate metabolism in the liver. It is caused by mutations in the AGXT gene, a gene that codes the peroxisomal enzyme alanine-glyoxylate aminotransferase (AGT). As a result of AGT deficiency oxalate, which is an end-product of glyoxylate metabolism, is overproduced in the liver. In healthy individuals, oxalate is excreted into urine, but when it is produced at high concentration there is a tendency for calcium oxalate (CaOx) crystals to be generated and deposited in the renal parenchyma, where kidney stones can form. As a consequence, PH1 patients present with severe kidney damage and poor survival of kidneys, developing end-stage renal disease (ESRD) in most of the cases. The only curative treatment is liver transplantation, which is usually combined with kidney transplantation because of the loss of renal function. The main goal of this study was to develop new therapeutic alternatives for PH1 based on advanced genetic treatment. In the initial part of this thesis we tried to improve PH1 gene therapy using adeno-associated viral (AAV) vectors. First, human AGXT was codon optimized in order to improve the expression levels of the protein. In this case, the optimization of the sequence of the AGXT gene resulted in no therapeutic advantage in comparison to the WT version of the gene. Second, we worked on the optimization of AAV gene delivery to the liver in non-human primates (NHP) changing the route of administration. It was demonstrated that the direct administration of AAV vectors into the hepatic blood flow resulted in a higher transduction of the liver in comparison to the systemic intravenous route. In addition, a completely novel approach based on gene editing using the recently discovered clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system was designed and characterized. This treatment was focused on a substrate reduction therapy (SRT) strategy, i.e. the reduction of glyoxylate production (the precursor of oxalate). Glycolate oxidase (GO) enzyme is a liver peroxisomal enzyme in charge of the production of glyoxylate. The inhibition of GO synthesis is known to reduce oxalate production. Therefore, a specific CRISPR/Cas9 system was designed to target and disrupt the Hao1 gene (the gene that codes GO) in hepatocytes. Using this strategy we were able to efficiently reduce GO protein levels. Moreover, the treatment resulted in a significant reduction of oxalate production and of renal damage in PH1 mice challenge with oxalate precursors, in absence of toxicity. In conclusion, several strategies to treat PH1 were developed and optimized during this project, which were able to reduce oxalate excretion in the urine of the PH1 mouse model

Development of the advanced genetic therapies for Primary Hyperoxaluria type I

Primary Hyperoxaluria type I (PH1) is an inherited inborn error of the glyoxylate metabolism in the liver. It is caused by mutations in the AGXT gene, a gene that codes the peroxisomal enzyme alanine-glyoxylate aminotransferase (AGT). As a result of AGT deficiency oxalate, which is an end-product of glyoxylate metabolism, is overproduced in the liver. In healthy individuals, oxalate is excreted into urine, but when it is produced at high concentration there is a tendency for calcium oxalate (CaOx) crystals to be generated and deposited in the renal parenchyma, where kidney stones can form. As a consequence, PH1 patients present with severe kidney damage and poor survival of kidneys, developing end-stage renal disease (ESRD) in most of the cases. The only curative treatment is liver transplantation, which is usually combined with kidney transplantation because of the loss of renal function. The main goal of this study was to develop new therapeutic alternatives for PH1 based on advanced genetic treatment. In the initial part of this thesis we tried to improve PH1 gene therapy using adeno-associated viral (AAV) vectors. First, human AGXT was codon optimized in order to improve the expression levels of the protein. In this case, the optimization of the sequence of the AGXT gene resulted in no therapeutic advantage in comparison to the WT version of the gene. Second, we worked on the optimization of AAV gene delivery to the liver in non-human primates (NHP) changing the route of administration. It was demonstrated that the direct administration of AAV vectors into the hepatic blood flow resulted in a higher transduction of the liver in comparison to the systemic intravenous route. In addition, a completely novel approach based on gene editing using the recently discovered clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system was designed and characterized. This treatment was focused on a substrate reduction therapy (SRT) strategy, i.e. the reduction of glyoxylate production (the precursor of oxalate). Glycolate oxidase (GO) enzyme is a liver peroxisomal enzyme in charge of the production of glyoxylate. The inhibition of GO synthesis is known to reduce oxalate production. Therefore, a specific CRISPR/Cas9 system was designed to target and disrupt the Hao1 gene (the gene that codes GO) in hepatocytes. Using this strategy we were able to efficiently reduce GO protein levels. Moreover, the treatment resulted in a significant reduction of oxalate production and of renal damage in PH1 mice challenge with oxalate precursors, in absence of toxicity. In conclusion, several strategies to treat PH1 were developed and optimized during this project, which were able to reduce oxalate excretion in the urine of the PH1 mouse model

Generation of an induced pluripotent stem cell line (CIMAi001-A) from a compound heterozygous Primary Hyperoxaluria Type I (PH1) patient carrying p.G170R and p.R122* mutations in the AGXT gene.

Primary Hyperoxaluria Type I (PH1) is a rare autosomal recessive metabolic disorder characterized by defects in enzymes involved in glyoxylate metabolism. PH1 is a life-threatening disease caused by the absence, deficiency or mistargeting of the hepatic alanine-glyoxylate aminotransferase (AGT) enzyme. A human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a PH1 patient being compound heterozygous for the most common mutation c.508G>A (G170R), a mistargeting mutation, and c.364C>T (R122*), a previously reported nonsense mutation in AGTX. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for drug development