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Safe and efficient cancer therapies using adoptive transfer of engineered cells are very promising but very challenging approaches. Opportunities to improve gene transfer into primary cells involve a better design of the vectors used. Such improvements must lead to an increase of the efficiency of gene expression, the cell phenotype preservation and the increase of the gene number delivered. The use of lentiviral vectors has largely increased in clinical protocols over the past few years but safety concerns have slowed down this progression. Actually, the permanent genetic modification remains a focus of significant regulatory oversight. On another side, mRNA delivery is a versatile, flexible, and safe mean for protein therapies, but existing techniques, such as chemical or electroporation-based transfection protocols, are known to induce cell toxicity and phenotype modifications of the target cells. Here, we present an innovative tool, named LentiFlash, allowing RNA delivery into target cells without any genomic scar. This tool combines RNA delivery, providing the best expression system to get efficient and transient expression, and lentiviral delivery which exhibits the best efficiency of entry into primary and stem cells. The RNA encapsidation is mediated via an RNA/protein interaction using properties of the MS2 bacteriophage. This new vector breaks with all existing systems. The resulting lentiviral particle is able to efficiently deliver non-viral coding or non-coding RNA into the cytoplasm of any cell type. These LentiFlash particles, are able to transduce delicate primary cells, such as dendritic cells, T cells and hematopoietic stem cells. They show an efficient, fast and transient expression of protein and RNA, such as antigenic peptides or genome editing tools, with no cell phenotype modification. Here we show a proof of concept on primary murine dendritic cells transduction with LentiFlash particles, successfully used to deliver several tumoral antigens. As a result, these dendritic cells are able to present the antigens to the immune system which in turn can efficiently fight a tumor development into wild type mice. This new delivery system gives the opportunity for multiple antigens co-expression to enhance immune responses, avoiding tumor relapse. Such multiple co-expressions into immune cells are expected to mimic the innate and adaptive immune responses. This transient RNA delivery mediated by LentiFlash is a powerful tool to induce an efficient gene delivery. The possibility to express multiple genes at once in the target cells is an attractive therapeutic perspective. The absence of any viral sequence avoids any integration event, an important safety consideration for human use. Finally, another advantage of the LentiFlash system is its ability to utilize lentiviral production platforms already validated in clinical settings. Citation Format: Pascale Bouillé, Christine Duthoit, Nicolas Martin, Lucille Lamouroux, Jean-Christophe Pages. Non-integrative lentiviral particles for immunotherapy: RNA delivery to drive tumoral antigen presentation in a safe and efficient way [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4561. doi:10.1158/1538-7445.AM2017-4561

Gene editing by the CRISPR system shows great promises for gene therapy. Nevertheless, it must now face a number of challenges especially for the development of safe and efficient delivery tools for in vivo, as well as ex vivo gene editing. Cas9 and sgRNA delivery, mediated either by viral vectors (AAV- or Lentivirus-derived) or transfection protocols (chemical or by electroporation) have been largely and efficiently used but they bring major drawbacks incompatible with clinical applications. Indeed, viral vectors can display uncontrolled chromosomal integrations and transfection protocols are known to induce cell toxicity and/or phenotype modifications of the target cells.. Moreover, the CRISPR technology entails a “hit-and-run” mechanism that only requires a transient expression of the nuclease complex. Therefore, achieving an efficient delivery into hard-to-transfect cells, such as T cells, remains challenging and the need for delivery tools that would allow efficient transfer on most cell types without causing any cell damages is essential for downstream therapeutic applications. Here, we present an innovative tool, named LentiFlash, allowing RNA delivery into target cells without any genomic scar. The RNA encapsidation is mediated via an RNA/protein interaction: 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 protein is inserted into the Nucleocapsid. This new vector breaks with all existing systems, as the resulting lentiparticle is able to deliver non-viral coding or non-coding RNA, at high efficiency, into the cytoplasm of any cell type. Transduction of a large range of cells, from immortalized cells to delicate primary cells, such as T cells and hematopoietic stem cells, with LentiFlash shows an efficient, fast and transient expression of proteins and RNA, with no cell phenotype modification. In particular, LentiFlash particles were successfully used to deliver Cas9, alone or in association with an sgRNA not only targeting a reporter gene into immortalized cells, but outstandingly knocking-out the PD-1 gene into primary human T lymphocytes. This new RNA delivery system is an efficient and safe tool for the delivery of CRISPR editing machinery in most cell types without affecting cell viability and phenotype. The transient, RNA-based mechanism of LentiFlash vector, preventing the risk of integration, associated with its ability to utilize lentiviral production platforms already validated in clinical settings, make it a promising tool for CRISPR therapeutic applications. As a matter of fact, beyond gene editing efficiency, safety of delivery tools is a major concern that should be addressed to move forward with CRISPR clinical development. Citation Format: Pascale Bouillé, Régis Gayon, Lucille Lamouroux, Alexandra Iche, Christine Duthoit, Jean-Christophe Pages. Efficient KO of PD1 into primary T cells using a new non-integrative lentiviral particle expressing CRISPR/Cas9 system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5090. doi:10.1158/1538-7445.AM2017-5090

The advent of human induced pluripotent stem cells (hiPSC) is revolutionizing many research fields including cell-replacement therapy, drug screening, physiopathology of specific diseases and more basic research such as embryonic development or diseases modeling. Despite the large number of reports on reprogramming methods, techniques in use remain globally inefficient. We present here a new optimized approach to improve this efficiency. After having tested different monocistronic vectors with poor results, we adopted a polycistronic cassette encoding Thomson's cocktail OCT4, NANOG, SOX2 and LIN28 (ONSL) separated by 2A peptides. This cassette was tested in various vector backbones, based on lentivirus or retrovirus under a LTR or EF1 alpha promoter. This allowed us to show that ONSL-carrier retrovectors re- programmed adult fibroblast cells with a much higher efficiency (up to 0.6%) than any other tested. We then compared the reprogramming ef- ficiencies of two different polycistronic genes, ONSL and OCT4, SOX2, KLF4 and cMYC (OSKM) placed in the same retrovector backbone. Interestingly, in this context ONSL gene reprograms more efficiently than OSKM but OSKM reprograms faster suggesting that the two cocktails may reprogram through distinct pathways. By equally mixing RV-LTR-ONSL and RV-LTR-OSKM, we indeed observed a remarkable synergy, yielding a reprogramming efficiency of .2%. We present here a drastic improvement of the reprogramming efficiency, which opens doors to the development of automated and high throughput strategies of hiPSC production. Furthermore, non-integrative reprogramming protocols (i.e. mRNA) may take advantage of this synergy to boost their efficiency.

BackgroundHuman pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods.ResultsWe used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration.ConclusionsWe have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also forin vitroapplications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

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 Bouillé, Cédric Auffray, Jean-Christophe Pagès, Christine Duthoit, Régis 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.

The initiation of an appropriate T cell response is a key event in the establishment of efficient anti-tumoral response. In a context of tumor development, different T cell populations are mobilized and understanding the pathways involved in their functions and in their interactions is a major field of research in cancer immunotherapy. Thus, while the activation of antigen-specific naive CD4+ T cells that can differentiate into appropriate effector T cells and home to the tumor might be highly beneficial, regulatory CD4+ T cells are considered as deleterious. Many efforts are thus made to understand the behavior of both CD4+ T cells subsets and to decipher their functions within the tumor micro-environment. A key step to reach this goal is the development of relevant predictive cancer models including genetic modifications of T cell populations in vitro and in vivo, either for gene target studies, or to create new tools for sensitive readouts. While human and activated mouse T cells are easily transduced with lentiviral vectors, transduction of naive and regulatory murine T cells is a technical challenge since these target cells are poorly permissive to transfection or viral transduction. Here, extensive in vitro transduction data show that the use of highly concentrated (1E9 TU/mL) and pure (1E8 TU/mg of proteins) lentiviral vectors increases significantly the transduction level of naive and regulatory CD4+ T cells. Furthermore, transduced T cells exhibit an original cell phenotype without any changes of T cells specific markers expression (CD44, CD69, CD25, Ly-6C) attesting that the composition of this pure batch of lentiviral vector does not have any impact on T cells phenotype. Transduced T cells were then assessed for their properties once adoptively transferred into recipient mice. Analysis of these mice show that not only the genetically modified T cells/regulatory cells can be easily followed thanks to the fluorescent reporter but also their phenotype is not modified by the transduction, as shown in vitro. Our data bring out that the composition of lentiviral vectors in terms of titer, specific activities and purity are the success keys to ensure transduction of murine T cells. This major technological leap will allow to tightly control expression of various modifiers of these cells and thus paves the way to design original tools and develop cell-based cancer. Citation Format: Pascale Bouillé, Christine Duthoit, Régis Gayon, Vincent Feuillet, Cédric Auffray. Predictive Immunotherapy Models: Overcoming the challenge of T cells gene transfer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3161. doi:10.1158/1538-7445.AM2015-3161

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, Régis Gayon, Christine Duthoit, Yohann Moal, Raphael Sevrain, Nicolas Martin, Pascale Bouillé. 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

Previously, we have observed a strong restriction of the Moloney murine leukemia virus (MoMLV) replicative cycle in a cell line displaying resistance to topoisomerase II (topo II)-interactive drugs. Resistance towards these antitumoral inhibitors was associated with decreased expression and activity of topo II, suggesting that such a decrease may be responsible for MoMLV restriction. To more specifically assess the role of topo II during the retroviral cycle, we have used the antisense strategy to obtain a selective decrease of cellular topo II expression. The RNA antisense was isolated from a retroviral library expressing random fragments of human topo II (α form). This system allowed us to investigate the HIV-1 replicative cycle in two related human CEM cell lines expressing different levels of topo II. Expression of the enzyme is decreased four- to sixfold following formation of a sense-antisense RNA hybrid. Repression of the topo II enzyme results in an impairment of the HIV-1 replicative cycle. Using the polymerase chain reaction, we showed that the number of integration events was decreased in cells repressing the enzyme, although viral DNA synthesis and circularization were equivalent to those in the parent cells.

We have assessed the suitability of retroviral vectors for gene therapy of recessive dystrophic epidermolysis bullosa (RDEB) in dogs expressing a mutated collagen type VII. Isolation and analysis of the 9 kb dog collagen type VII cDNA identified the causative genetic mutation G1906S and disclosed the interspecies conservation of collagen type VII. Highly efficient transfer of the wild-type collagen type VII cDNA to both dog RDEB and human primary RDEB collagen type VII-null keratinocytes using recombinant vectors derived from LZRS-Ires-zeo and MSCV retroviruses achieved sustained and permanent expression of the transgene product. The expression and post-translational modification profile of the recombinant collagen type VII was comparable to that of the wild-type counterpart. The recombinant canine collagen type VII in human RDEB keratinocytes and dog cells corrected the observable defects caused by RDEB keratinocytes in cell cultures and in vitro reconstructed skin. Hypermotility was fully reverted in human RDEB keratinocytes, and strongly reduced in the dog RDEB cells. This observation suggests that not only infection efficiency but also high expression levels are required to ensure therapeutic efficacy in the presence of mutated gene products. Our results set the basis for preclinical gene therapy assays in the first immune-competent large animal model for an inherited skin disease and broaden the spectrum of preclinical and clinical applications of retroviral vectors in the transfer of large recombinant genes in epithelial cells.

Outgrowth of the dendrites and the axon is the basis of the establishment of the neuronal shape, and it requires addition of new membrane to both growing processes. It is not yet clear whether one or two exocytotic pathways are responsible for the respective outgrowth of axons and dendrites. We have previously shown that tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) defines a novel network of tubulovesicular structures present both at the leading edge of elongating dendrites and axons of immature hippocampal neurons developing in primary culture and that TI-VAMP is an essential protein for neurite outgrowth in PC12 cells. Here we show that the expression of the N-terminal domain of TI-VAMP inhibits the outgrowth of both dendrites and axons in neurons in primary culture. This effect is more prominent at the earliest stages of the development of neuronsin vitro. Expression of the N-terminal domain deleted form of TI-VAMP has the opposite effect. This constitutively active form of TI-VAMP localizes as the endogenous protein, particularly concentrating at the leading edge of growing axons. Our results suggest that a common exocytotic mechanism that relies on TI-VAMP mediates both axonal and dendritic outgrowth in developing neurons.