Reconstitution of the Ataxia-Telangiectasia Cellular Phenotype With Lentiviral Vectors.

Ataxia-telangiectasia (A-T) is a complex disease arising from mutations in the ATM gene (Ataxia-Telangiectasia Mutated), which plays crucial roles in repairing double-strand DNA breaks (DSBs). Heterogeneous immunodeficiency, extreme radiosensitivity, frequent appearance of tumors and neurological degeneration are hallmarks of the disease, which carries high morbidity and mortality because only palliative treatments are currently available. Gene therapy was effective in animal models of the disease, but the large size of the ATM cDNA required the use of HSV-1 or HSV/AAV hybrid amplicon vectors, whose characteristics make them unlikely tools for treating A-T patients. Due to recent advances in vector packaging, production and biosafety, we developed a lentiviral vector containing the ATM cDNA and tested whether or not it could rescue cellular defects of A-T human mutant fibroblasts. Although the cargo capacity of lentiviral vectors is an inherent limitation in their use, and despite the large size of the transgene, we successfully transduced around 20% of ATM-mutant cells. ATM expression and phosphorylation assays indicated that the neoprotein was functional in transduced cells, further reinforced by their restored capacity to phosphorylate direct ATM substrates such as p53 and their capability to repair radiation-induced DSBs. In addition, transduced cells also restored cellular radiosensitivity and cell cycle abnormalities. Our results demonstrate that lentiviral vectors can be used to rescue the intrinsic cellular defects of ATM-mutant cells, which represent, in spite of their limitations, a proof-of-concept for A-T gene therapy.

Engineered LINE-1 retrotransposition in nondividing human neurons.

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80-100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought.

Retrotransposons in pluripotent cells: Impact and new roles in cellular plasticity.

Transposable Elements are pieces of DNA able to mobilize from one location to another within genomes. Although they constitute more than 50% of the human genome, they have been classified as selfish DNA, with the only mission to spread within genomes and generate more copies of themselves that will ensure their presence over generations. Despite their remarkable prevalence, only a minor group of transposable elements remain active in the human genome and can sporadically be associated with the generation of a genetic disorder due to their ongoing mobility. Most of the transposable elements identified in the human genome corresponded to fixed insertions that no longer move in genomes. As selfish DNA, transposable element insertions accumulate in cell types where genetic information can be passed to the next generation. Indeed, work from different laboratories has demonstrated that the main heritable load of TE accumulation in humans occurs during early embryogenesis. Thus, active transposable elements have a clear impact on our pluripotent genome. However, recent findings suggest that the main proportion of fixed non-mobile transposable elements might also have emerging roles in cellular plasticity. In this concise review, we provide an overview of the impact of currently active transposable elements in our pluripotent genome and further discuss new roles of transposable elements (active or not) in regulating pluripotency. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.