Lentiviral Vector Production Titer Is Not Limited in HEK293T by Induced Intracellular Innate Immunity.

Most gene therapy lentiviral vector (LV) production platforms employ HEK293T cells expressing the oncogenic SV40 large T-antigen (TAg) that is thought to promote plasmid-mediated gene expression. Studies on other viral oncogenes suggest that TAg may also inhibit the intracellular autonomous innate immune system that triggers defensive antiviral responses upon detection of viral components by cytosolic sensors. Here we show that an innate response can be generated after HIV-1-derived LV transfection in HEK293T cells, particularly by the transgene, yet, remarkably, this had no effect on LV titer. Further, overexpression of DNA sensing pathway components led to expression of inflammatory cytokine and interferon (IFN) stimulated genes but did not result in detectable IFN or CXCL10 and had no impact on LV titer. Exogenous IFN-ß also did not affect LV production or transduction efficiency in primary T cells. Additionally, manipulation of TAg did not affect innate antiviral responses, but stable expression of TAg boosted vector production in HEK293 cells. Our findings demonstrate a measure of innate immune competence in HEK293T cells but, crucially, show that activation of inflammatory signaling is uncoupled from cytokine secretion in these cells. This provides new mechanistic insight into the unique suitability of HEK293T cells for LV manufacture.

Enhancing Lentiviral and Alpharetroviral Transduction of Human Hematopoietic Stem Cells for Clinical Application.

Ex vivo retroviral gene transfer into CD34+ hematopoietic stem and progenitor cells (HSPCs) has demonstrated remarkable clinical success in gene therapy for monogenic hematopoietic disorders. However, little attention has been paid to enhancement of culture and transduction conditions to achieve reliable effects across patient and disease contexts and to maximize potential vector usage and reduce treatment cost. We systematically tested three HSPC culture media manufactured to cGMP and eight previously described transduction enhancers (TEs) to develop a state-of-the-art clinically applicable protocol. Six TEs enhanced lentiviral (LV) and five TEs facilitated alpharetroviral (ARV) CD34+ HSPC transduction when used alone. Combinatorial TE application tested with LV vectors yielded more potent effects, with up to a 5.6-fold increase in total expression of a reporter gene and up to a 3.8-fold increase in VCN. Application of one of the most promising combinations, the poloxamer LentiBOOST and protamine sulfate, for GMP-compliant manufacturing of a clinical-grade advanced therapy medicinal product (ATMP) increased total VCN by over 6-fold, with no major changes in global gene expression profiles or inadvertent loss of CD34+CD90+ HSPC populations. Application of these defined culture and transduction conditions is likely to significantly improve ex vivo gene therapy manufacturing protocols for HSPCs and downstream clinical efficacy.

Impaired EIF2S3 function associated with a novel phenotype of X-linked hypopituitarism with glucose dysregulation.

BACKGROUND: The heterotrimeric GTP-binding protein eIF2 forms a ternary complex with initiator methionyl-tRNA and recruits it to the 40S ribosomal subunit for start codon selection and thereby initiates protein synthesis. Mutations in EIF2S3, encoding the eIF2γ subunit, are associated with severe intellectual disability and microcephaly, usually as part of MEHMO syndrome. METHODS: Exome sequencing of the X chromosome was performed on three related males with normal head circumferences and mild learning difficulties, hypopituitarism (GH and TSH deficiencies), and an unusual form of glucose dysregulation. In situ hybridisation on human embryonic tissue, EIF2S3-knockdown studies in a human pancreatic cell line, and yeast assays on the mutated corresponding eIF2γ protein, were performed in this study. FINDINGS: We report a novel hemizygous EIF2S3 variant, p.Pro432Ser, in the three boys (heterozygous in their mothers). EIF2S3 expression was detectable in the developing pituitary gland and pancreatic islets of Langerhans. Cells lacking EIF2S3 had increased caspase activity/cell death. Impaired protein synthesis and relaxed start codon selection stringency was observed in mutated yeast. INTERPRETATION: Our data suggest that the p.Pro432Ser mutation impairs eIF2γ function leading to a relatively mild novel phenotype compared with previous EIF2S3 mutations. Our studies support a critical role for EIF2S3 in human hypothalamo-pituitary development and function, and glucose regulation, expanding the range of phenotypes associated with EIF2S3 mutations beyond classical MEHMO syndrome. Untreated hypoglycaemia in previous cases may have contributed to their more severe neurological impairment and seizures in association with impaired EIF2S3. FUND: GOSH, MRF, BRC, MRC/Wellcome Trust and NIGMS funded this study.

Efeitos da administração de metformina sobre a pressão arterial e o metabolismo glicídico de ratos espontaneamente hipertensos tornados obesos pela injeção neonatal de glutamato monossódico / Metformin effects upon blood pressure and glucose metabolism of monossodium glutamate induced-obese spontaneously hypertensive rats

OBJETIVOS: Produzir um modelo experimental de síndrome metabólica (SM) e analisar efeitos da metformina sobre pressão arterial (PA), peso corporal (PC), metabolismo glicídico e conteúdo de gordura epididimal (GE). MÉTODO: Os machos SHR receberam 2 mg/kg/dia de glutamato monossódico (MSG) até o 11º dia de vida. Os controles receberam salina. Após 12 semanas, foram separados em dois grupos e tratados com 500 mg/kg/dia de metformina ou veículo. Foram acompanhados a PA e o PC dos dois grupos. Ao final do seguimento, realizou-se o teste de tolerância à glicose oral (TTGO) e mediu-se o índice de sensibilidade à insulina. Após sacrifício dos animais, a GE foi pesada. RESULTADOS: A administração de MSG intensificou a resistência insulínica e aumentou o conteúdo de GE, sem, no entanto, alterar a PA. O tratamento com metformina promoveu melhora da sensibilidade insulínica e redução da GE e PA. CONCLUSÕES: Observou-se importante papel da resistência hepática à insulina na SM e efeitos cardiovasculares benéficos da melhora na sensibilidade insulínica.

OBJECTIVES: To make available experimental model for the metabolic syndrome (MS) and verify effects of chronic oral treatment with metformin upon blood pressure (BP), body weight (BW), glucose metabolism, epididimal fat content (EF). METHOD: Males SHR received monossodium glutamate (MSG, 2 mg/kg/day/sc) during first 11 days of life. Control animals received saline. After 12 weeks, animals were separated in two groups, treated either with metformin 500 mg/ kg/day or vehicle during 12 weeks. PA and BW were determined. At the end of the follow-up, animals underwent an oral glucose tolerance test (OGTT) and insulin sensitivity index was determined. Upon sacrifice EF was measured. RESULTS: MSG worsened insulin resistance and induced visceral obesity in SHR, without change BP. Treatment with metformin improved glucose metabolism and reduces EF and BP. CONCLUSIONS: These observations emphasize the role of hepatic insulin resistance on MS and point out for beneficial cardiovascular effects with improvement in the insulin sensitivity.