Gerard Wagemaker

The potential for chemical mixtures from the environment to enable the cancer hallmark of sustained proliferative signalling.

The aim of this work is to review current knowledge relating the established cancer hallmark, sustained cell proliferation to the existence of chemicals present as low dose mixtures in the environment. Normal cell proliferation is under tight control, i.e. cells respond to a signal to proliferate, and although most cells continue to proliferate into adult life, the multiplication ceases once the stimulatory signal disappears or if the cells are exposed to growth inhibitory signals. Under such circumstances, normal cells remain quiescent until they are stimulated to resume further proliferation. In contrast, tumour cells are unable to halt proliferation, either when subjected to growth inhibitory signals or in the absence of growth stimulatory signals. Environmental chemicals with carcinogenic potential may cause sustained cell proliferation by interfering with some cell proliferation control mechanisms committing cells to an indefinite proliferative span.

Preclinical Efficacy and Safety Evaluation of Hematopoietic Stem Cell Gene Therapy in a Mouse Model of MNGIE

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early-onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is not available. Allogeneic bone marrow transplantation may improve clinical manifestations but carries disease and transplant-related risks. In this study, lentiviral vector-based hematopoietic stem cell gene therapy (HSCGT) was performed in Tymp−/−Upp1−/− mice with the human phosphoglycerate kinase (PGK) promoter driving TYMP. Supranormal blood TP activity reduced intestinal nucleoside levels significantly at low vector copy number (median, 1.3; range, 0.2–3.6). Furthermore, we covered two major issues not addressed before. First, we demonstrate aberrant morphology of brain astrocytes in areas of spongy degeneration, which was reversed by HSCGT. Second, long-term follow-up and vector integration site analysis were performed to assess safety of the therapeutic LV vectors in depth. This report confirms and supplements previous work on the efficacy of HSCGT in reducing the toxic metabolites in Tymp−/−Upp1−/− mice, using a clinically applicable gene transfer vector and a highly efficient gene transfer method, and importantly demonstrates phenotypic correction with a favorable risk profile, warranting further development toward clinical implementation.

OP45 – 3024: Efficient lentiviral vector-mediated hematopoietic stem cell gene therapy in MNGIE mice

Objective Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients are deficient in thymidine phosphorylase (TP) resulting in systemic thymidine (Thd) and deoxyuridine (dUrd) accumulation affecting mtDNA replication and causing mitochondrial dysfunction. Common symptoms are gastrointestinal dysmotility, progressive ophthalmoplegia and leukoencephalopathy. Allogeneic hematopoietic stem cell (HSC) transplantation has been shown to reduce disease symptoms, but is not well tolerated due to the inherent toxicity of the procedure. Application of lentiviral vector therapy. Methods Therefore, syngeneic ex vivo lentiviral vector HSC gene therapy overexpressing the native cDNA or the codon optimized (TPco) sequence driven by the phosphoglycerate kinase (PGK) or spleen focus forming virus (SFFV) promoters in Tp-/-Upp-/- double knockout mice, a model for MNGIE disease, was investigated. Results At 1 month post transplantation after sublethal total body irradiation, very low TP activity was detected in blood of control wild type mice (0.07±0.03nmoles/h/mg), but enzyme activities in PGK treated mice were at least 90-fold higher (PGK-TP = 150±4 and PGK-TPco=96±4 nmoles/h/mg), compared to a 400-fold increase observed in SFFV recipient mice (450±5) and consequently, a significant reduction of plasma and urine Thd and dUrd levels was observed. Long-term follow up (14 months post treatment) showed on average 1.2-fold TP wild type activity levels in LV-PGK-TP and LV-PGK-TPco and 36-fold in SFFV-TPco treated mice, sufficient for sustained reduction of plasma and urine nucleoside levels, which was achieved at 76.5±8.2% donor chimerism levels with low LV vector copy numbers (1.0±1.1VCN/donor cell). Conclusion Overall, stem cell gene therapy provided stable TP expression and long-term biochemical correction in MNGIE mice without genotoxicity or apparent phenotoxicity, which will be further evaluated for somatic and neurological phenotype correction and optimized to develop a clinical protocol to treat MNGIE patients.

Enhancement of mouse hematopoietic stem/progenitor cell function via transient gene delivery using integration-deficient lentiviral vectors

Highlights • Integration-deficient vectors (IdLVs) express genes transiently in dividing stem cells.• Hematopoietic stem/progenitor cells (HSPCs) can be programmed using IdLVs.• HOXB4 or Angptl3 expression from IdLVs improves engraftment of transplanted HSPCs.• Short-term gene delivery avoids the side effects associated with constitutive expression.

Efficacy of lentivirus-mediated gene therapy in an Omenn syndrome recombination-activating gene 2 mouse model is not hindered by inflammation and immune dysregulation

Background Omenn syndrome (OS) is a rare severe combined immunodeficiency associated with autoimmunity and caused by defects in lymphoid‐specific V(D)J recombination. Most patients carry hypomorphic mutations in recombination‐activating gene (RAG) 1 or 2. Hematopoietic stem cell transplantation is the standard treatment; however, gene therapy (GT) might represent a valid alternative, especially for patients lacking a matched donor. Objective We sought to determine the efficacy of lentiviral vector (LV)–mediated GT in the murine model of OS (Rag2R229Q/R229Q) in correcting immunodeficiency and autoimmunity. Methods Lineage‐negative cells from mice with OS were transduced with an LV encoding the human RAG2 gene and injected into irradiated recipients with OS. Control mice underwent transplantation with wild‐type or OS‐untransduced lineage‐negative cells. Immunophenotyping, T‐dependent and T‐independent antigen challenge, immune spectratyping, autoantibody detection, and detailed tissue immunohistochemical analyses were performed. Results LV‐mediated GT allowed immunologic reconstitution, although it was suboptimal compared with that seen in wild‐type bone marrow (BM)−transplanted OS mice in peripheral blood and hematopoietic organs, such as the BM, thymus, and spleen. We observed in vivo variability in the efficacy of GT correlating with the levels of transduction achieved. Immunoglobulin levels and T‐cell repertoire normalized, and gene‐corrected mice responded properly to challenges in vivo. Autoimmune manifestations, such as skin infiltration and autoantibodies, dramatically improved in GT mice with a vector copy number/genome higher than 1 in the BM and 2 in the thymus. Conclusions Our data show that LV‐mediated GT for patients with OS significantly ameliorates the immunodeficiency, even in an inflammatory environment.

279. Efficient and Safe Lentiviral Vector-Mediated Hematopoietic Stem Cell Gene Therapy in MNGIE Mice

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients are deficient in thymidine phosphorylase(TP) resulting in systemic thymidine(Thd) and deoxyuridine(dUrd) accumulation affecting mtDNA replication and causing mitochondrial dysfunction. Common symptoms are gastrointestinal dysmotility, progressive ophthalmoplegia and leukoencephalopathy. Allogenic hematopoietic stem cell (HSC) transplantation has been shown to reduce disease symptoms, but is not well tolerated due to the inherent toxicity of the procedure. Therefore, syngeneic ex vivo lentiviral vector HSC gene therapy overexpressing the native cDNA or the codon optimized (TPco) sequence driven by the phosphoglycerate kinase (PGK) or spleen focus forming virus (SFFV) promoters in Tp−/-Upp−/- double knockout mice, a model for MNGIE disease, was investigated. At 1 month post transplantation after sublethal total body irradiation, very low TP activity was detected in blood of control wild type mice (0.07±0.03nmoles/h/mg), but enzyme activities in PGK treated mice were at least 90-fold higher (PGK-TP = 150±4 and PGK-TPco = 96±4 nmoles/h/mg), and in SFFV recipient mice 400-fold higher (450±5 nmoles/h/mg). Consequently, a significant reduction of plasma and urine Thd and dUrd levels was observed. Long-term follow up (14 months) showed on average 1.2-fold wild type TP activity levels increase in LV-PGK-TP and LV-PGK-TPco and 36-fold in SFFV-TPco treated mice. This was sufficient for sustained reduction of plasma and urine nucleoside levels, which was achieved at 76.5±8.2% donor chimerism levels with low LV vector copy numbers (1.0±1.1VCN/donor cell). The LV integration profile in bone marrow cells of primary recipients was analyzed; LVs displayed the expected tendency to integrate within highly expressed genes and the integration pattern did not differ from that of other SIN-LV vectors in other disease models (primary immune deficiencies and lysosomal enzyme storage disorders). Overall, stem cell gene therapy provided stable TP expression and long-term biochemical correction in MNGIE mice without genotoxicity or apparent phenotoxicity, which will be further evaluated for somatic and neurological phenotype correction and optimized to develop a clinical protocol to treat MNGIE patients.