Abstract 5090: Efficient KO of PD1 into primary T cells using a new non-integrative lentiviral particle expressing CRISPR/Cas9 system

Abstract

Abstract 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