Pascale Bouillé

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-loop repeats 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 editing enzymes. 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.

Abstract 3743: New non-integrative MS2-based lentiviral particles for mRNA delivery: A safe and efficient opportunity for gene editing and immunotherapy applications

Safe and efficient cancer therapies using adoptive transfer of engineered T cells or gene editing 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 T cells or hematopoietic stem cells involve a better design of the vectors used. Such improvements must lead to an increase of the efficiency in 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 it has been shown that some resulting CAR T cells can exhibit toxicity due to high and persistent expression. If mRNA delivery is a versatile, flexible, and safe mean for protein therapies, 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-loop repeats 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 target cell, directly available for protein translation. Transduction 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 editing enzymes. Cell viability of such engineered cells and multiple genes expression analyses will be presented. This transient mRNA delivery mediated by a lentiviral particle is a powerful tool, useful to induce an efficient CAR delivery and ensure a complete loss of the CAR-driven T cells activity. The possibility to express multiple genes at once in the target cells is an attractive therapeutic perspective. One of the advantages of the MS2RLP system is its ability to utilize lentiviral production platforms already validated in clinical settings. The RNAs transferred by the MS2RLPs are directly expressed into the cytoplasm, which completely removes the risk of integration, an important safety consideration for human use. Citation Format: Pascale Bouille, Cedric Auffray, Jean-Christophe Pages, Christine Duthoit, Regis Gayon. New non-integrative MS2-based lentiviral particles for mRNA delivery: A safe and efficient opportunity for gene editing and immunotherapy applications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3743.

Abstract 353: How highly purified lentiviral vectors matter when it comes to genetically modifying your cells for the generation of in vitro or in vivo predictive cancer models

The development of relevant predictive cancer models including genetic modification is crucial for gene target validation and the assessment of molecules efficacy. Such models must be conceived through the use of a tool ensuring both the original phenotype of the cells to be strictly maintained and a stable expression of the sequence of interest in all target cells including primary cells. In these delicate cells as hematopoietic lineages, classical transfection protocols are not only transient and inefficient but they also induce cytotoxic effects and/or proliferative arrests. Gene transfer using lentiviral vectors is safer for such cells and the expression of the sequence of interest (cDNA, shRNA, miRNA) is stable thanks to the vector DNA integration into genomic DNA, bringing stable cell line at once. Using highly purified and concentrated lentivectors, we show that not only is it possible to transduce 100% of T lymphocytes, monocytes, M1/M2 Macrophages, and dendritic cells, but we also manage the integrated copy number and level of expression. With such transduction efficiency, it doesn’t worth to perform any antibiotic selection, allowing to save time and ensuring to keep the cells phenotype. In vivo, we show that lentiviral vectors expressing luciferase or fluorescent reporters are a good tool for noninvasive analyses of tumor animal models. They can be efficiently used through direct injections into adult animals and also through in vitro transductions of any tumoral cells subsequently reimplantated in the context of cancer models. The transplantation of genetically modified HSC offers an attractive alternative to transgenic animal models, allowing a more rapid and cost-effective in vivo validation of target genes for models of hematopoietic malignancies. Here, we present data obtained using concentrated and purified lentiviral suspension for the transduction of Lin- bone marrow cells before their transfer into recipient irradiated mice. These data bring out that lentiviral vectors have to be highly concentrated and purified in order to maintain the original cell phenotype, proliferation and viability in the goal to achieve an effective transgene expression. These two additional criterias combined with the lentiviral vector properties are required to ensure the development of trustworthy predictive cancer models, and thus build the link between in vitro assays and in vivo results to finally translate to therapeutic applications. Citation Format: Adriana Georges, Regis Gayon, Christine Duthoit, Yohann Moal, Raphael Sevrain, Nicolas Martin, Pascale Bouille. How highly purified lentiviral vectors matter when it comes to genetically modifying your cells for the generation of in vitro or in vivo predictive cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 353. doi:10.1158/1538-7445.AM2014-353