Protection of Human Pancreatic Islets Using a Lentiviral Vector Expressing Two Genes: cFLIP and GFP

Abstract

Pancreatic islet transplantation can provide insulin independence to diabetic patients. However, apoptosis of islets often leads to early graft failure. Genetic engineering with protective gene(s) can improve the viability of these cells. Here we show successful transduction of human islets with a feline immunodeficiency virus (FIV) vector expressing both a cytoprotective (cFLIP) gene and the green fluorescent protein (GFP). Despite using low virus titers to maximize safety, transduced islets expressed both genes, resulting in improved β-cell metabolic activity and viability. Although only ϳ10% of total islet cells were transduced, the significant viability advantages suggest a "barrier" effect in which protecting the periphery of the islet shields the core. These results provide the first demonstration that a lentiviral vector can express two genes in islets. Furthermore, the engineered islets are resistant to a variety of apoptotic stimuli, suggesting the potential of this approach in enhancing the viability of transplanted cells.

Genetically Engineered Human Islets Protected From CD8-mediated Autoimmune Destruction In Vivo

“…[37][38][39][40] However, studies illustrating the feasibility in human islets are rare but efficient thereby lentivirus-mediated expression of cFLIP in as little as 10% of the human islet cells was sufficient to protect these cells from IL-1β, TNF-α, and IFN-γ proinflammatory cytokines treatment. 41 Herpesviruses have the capacity to establish a lifelong infection of their host, a phenomenon associated with the expression of proteins with potent immunosuppressive properties, eluding CD8 + and CD4 + T cells, natural killer cells and innate immunity. 42,43 Among the different immunevasins, US2 is extremely effective in targeting MHC-I molecules for destruction.…”

Synthetic small interfering RNA down‐regulates caspase‐3 and affects apoptosis, IL‐1β, and viability of porcine proximal tubular cells

“…No single genetic modification is likely to be optimal for all gene therapeutic applications. “Perfect” regulators of gene expression will be administered by a non‐invasive delivery route, will target specifically to the desired cells within specific tissues, and will cause the overexpression or silencing of the target gene/s [Kay et al, 2001; Fenjves et al, 2008]. There are two fundamentally different gene delivery systems, viral and non‐viral.…”

RNA interference for improving the outcome of islet transplantation

“…Lymphocytic choriomeningitis virus (LCMV)-pseudotyped is more efficient in transducing human islet insulin-secreting beta cells and shows less toxicity compared with VSV-G pseudotyped lentivirus [252]. Lentivirus were successfully used for genetic modification of islets with various genes, including reporter genes (GFP, Luciferase) [253, 254], antioxidative genes [255], antiapoptotic genes (cFLIP) [253], anti-inflammatory and immunoregulatory genes (CTLA4, TGF-β, interleukin-1 receptor antagonist, and IL-4) [256–258]. Unlike Adv vectors which give high level transient gene expression, a prolonged gene expression is usually achieved after transduction islets of lentiviral vectors.…”

“…In addition, lentiviral vectors induced immune system activation was also minimal [258]. Under optimized conditions, lentivirus could transduce around 10% to 30% of islet cells in an intact islet, which are mainly the cells at the periphery [253, 259, 260]. This is still enough to have significant effect on the viability, probably due to the “barrier” effect, that is shield the core of islets by protecting the periphery of islets [253].…”