538. Transient Non-Viral RNA Delivery Mediated by a Lentiviral Particle

Safe and efficient gene therapies including gene-targeting technologies are very challenging but very promising approaches nowadays. The scientific and clinical communities have been working for a long time together to encounter substantial clinical advances they have made possible thanks to numerous improvements in cell culture and gene transfer methods. Opportunities to improve gene transfer into primary or stem cells involve a better design of the vectors used. Such improvements must lead to an increase of the transduction efficiency including the percentage of positive cells, as well as a better level and duration of expression, cell phenotype preservation and the number of genes delivered.. Lentiviral vectors have seen their use largely increased in clinical protocols over the past few years but safety concerns have been highlighted. First, the permanent genetic modification remains a focus of significant regulatory oversight and even integrase- or reverse transcriptase-deficient lentiviral vectors leads to residual integration events. Moreover, all the gene-editing technologies entail a “hit-and-run” mechanism that requires only a transient expression of the nuclease complex. In parallel, mRNA delivery is a versatile, flexible, and safe mean for protein therapies but chemical or electroporation-based transfection protocols are known to induce cell toxicity and phenotype modifications of the target cells. Here, we describe a new chimeric lentiviral platform that allows mRNA delivery into the target cells without any genomic signature. The respective properties of the MS2 bacteriophage and the lentiviral vectors have been combined to build a non-integrative packaging system in which the wild type HIV packaging sequence is replaced by the MS2 stem-looprepeats and the MS2 Coat sequence is inserted into the NucleoCapsid sequence. The resulting lentiviral particle is able to deliver a non-viral RNA into the cytoplasm of target cells, directly available for protein translation. Transduction of immortalized cells but also of T cells and HSC with these RNA lentiviral particles (RLP) shows an efficient, fast and transient expression of both reporters and functional proteins such as genome editingenzymes. Particles structure and functionality, cell transduction and characterization of such engineered cells have been compared with those obtained with an integrative lentiviral vector. Particularly by recruiting the RNA independently of dimerization with more than four molecules per particles, RLPs allow the cotransfer of different species of RNA into target cells. This new delivery system is a great candidate handle the safe and clinically suitable delivery of the editing machinery, which can transiently act without inducing cellular toxicity or immunogenicity.