PERSIST: Persisting Transgenesis.

The overall objective of the first WP was to develop tools for effective gene delivery to cells without toxicity arising as a result of semi-random genome integration. Considering that the high efficiency of lentivirus-mediated gene transfer, coupled with specific modifications to limit or re-direct integration may have widespread utility, the consortium planned to explore the mechanisms of action, the effectiveness of gene transfer and expression, and the potential for toxicity of integrase-defective lentiviral vectors. Then the reagents would be utilised for correction of models of disease outlined in WP10-12 according to technologies developed in WP3-4.

New methods to deliver DNA-modifying enzymes, mainly via transfer of mRNA or proteins, as required for the target cell populations of PERSIST’s disease-related work program, were explored in WP2. Partners used episomal and non-integrating lentivirus-based modes of DNA delivery to express the enzymes of interest. Efficiency and biosafety of mRNA and protein delivery could be determined using novel technologies and experimental systems introduced by the investigators of PERSIST, including both physicochemical and viral methods. The goal was to define novel and potentially clinically useful means of transient delivery of DNA-modifying enzymes into somatic (stem) cells, and to explore basic mechanisms of mRNA and protein delivery into somatic cells.

The objective of WP3 was the development of integrase- and transposase-based technology for efficient and safe site-specific integration of therapeutic transgenes into the human genome. This included the design and testing of Rep and phiC31 integrases, Sleeping Beauty-derived transposases, and group-II introns with increased targeting efficiency and reduced genotoxicity. New hybrid vector platforms for the combined delivery of site-specific recombinase/transposase and donor DNA for site-specific integration were developed and tested in relevant target cells. Genome-wide analysis of the full spectrum of integration properties of integrases and transposases was crucial to assess their potential genotoxicity for clinical applications. In addition, the WP aimed at developing a platform based on large-capacity HD-Ad vectors to deliver integrase- and transposase-based integration systems to specific cell and tissues in vitro and in vivo, and at validating the new site-specific integration technology in cells (hematopoietic stem cells, epidermal stem cells) and organs (liver, muscle) of clinical relevance.

The main activities of the WP4 “Homologous recombination-based gene targeting” aimed to (i) improve ZFN performance in terms of on target efficacy, specificity and limit their toxicity, (ii) develop effective delivery strategies for ZFN and donor DNA templates into target cell types relevant for gene therapy application, such as lymphocytes and hematopoietic stem cells (HSC), (iii) identify genomic target sites tolerant to insertion and allowing robust expression (safe genomic harbours”); (iv) generate ZFN-based vector systems for efficient gene addition to a selected “safe genomic harbour” and validate them in the above mentioned relevant cell types. A new design for autonomous Zinc Finger Nucleases was developed and allowed simultaneous delivery of multiple ZFN pairs targeting different loci. Moreover, a safe genomic acceptor site for transgenes was validated by showing robust transgene expression upon targeted integration without any detectable impact on endogenous transcription at the targeted and flanking loci. This strategy allowed defining a novel modality of “sustainable” gene transfer with minimal to undetectable impact on the acceptor genome. In addition, a new strategy to assess ZFN specificity genome wide in vivo was developed and used to validate the amazing specificity of late-generation designer ZFN action on the human genome.

The work plan described in the WP5 related to “Surface targeting” aimed at i) exploring the resources of envelope glycoproteins and cellular molecules that can naturally interact with specific target receptors ii) engineering these components to optimise their incorporation on LV surface iii) developing methodologies to display on the LV surface ligands that specifically bind receptors expressed on target cells iv) evaluating these strategies to target cells of interest such as HSC and hepatocytes v) optimizing and testing the developed LV to target these cells in vivo. During the last months of the project, the activities mainly focused on testing the novel BAEVgp pseudotyped lentivectors for transduction of human and macaque HSCs and comparison to RDTR and VSVG pseudotyped vectors. Partners involved in this WP also investigated whether HSCs ex vivo transduced by CD133-LV could repopulate in immunodeficient mice and whether CD133-LV was superior to commonly used VSV-G LV in transducing long-term repopulating CD133+/CD34+ HSC.

The activities carried out in WP6 involved (i) the definition of microRNA activity in different hematopoietic lineages, (ii) the identification of hematopoietic stem cell (HSC) and progenitor cell-specific microRNAs, (iii) the combination of epigenetics and RNA interference to control transgene expression, (iv) the design of novel synthetic transcription factors which control therapeutic transgenes in response to physiologic/pathologic metabolites, (v) the in vivo validation of hepatocyte-specific promoters developed using novel bio-informatics algorithms and (vi) the evaluation of immune responses to microRNA-regulated gene transfer vectors.