The San Raffaele Telethon Institute for Gene Therapy (SR-Tiget)

New Gene Transfer Technologies

As we test late generation lentiviral vectors in the clinic, we are busy at the bench to create the next generation vector, in order to overcome the known risks and limitations of the platforms currently being tested, as well as to be ready to address unexpected hurdles that may surface in the path to clinical translation for other diseases. Thus, we continue to devote a large effort to further advance gene transfer technology. We are stringently assessing the biosafety of lentiviral vectors and further improving their current design, notably by avoiding gene disruption at the integration site by reducing the likelihood of aberrant splicing and transcriptional termination of endogenous transcription within the vector sequence. These improved vector designs are then validated taking advantage of new sensitive in vivo models to score genotoxicity and of high-throughput genomic analyses and deep sequencing to monitor the impact of vector integration both in experimental models and in our clinical trials.

At the same time, we are continuing to develop gene targeting approaches based on engineered Zinc Finger Nucleases (ZFNs) that bring the possibility of targeted integration and gene correction within the reach of gene therapy. These advances may offer radical new solutions to overcome some of the major hurdles that have long hindered progress of the gene therapy field. Gene correction, as opposed to gene replacement, not only restores the function of a diseased gene but also its physiological expression control, while avoiding the risks of insertional mutagenesis. We have optimized the application of ZFN-mediated gene editing to several cell types, including human primary lymphocytes, fibroblasts, neural stem cells, hematopoietic progenitors and demonstrated the feasibility, efficiency and specificity of gene disruption and site-specific integration. We have studied the transcriptional and epigenetic impact of different transgene expression cassettes targeted into different genomic loci and tailored locus choice and cassette design to achieve robust and uniform transgene expression without inducing detectable transcriptional perturbation of the targeted locus and its flanking genes. These studies provide a framework for “sustainable” gene transfer that can be exploited in novel experimental paradigms and safer therapeutic applications. Indeed, we are now developing a protocol for the correction of SCID-X1 (X-linked Severe Combined ImmunoDeficiency) and IPEX in HSPC, which may become the first clinical application of ZFN-induced homologous recombination mediated gene repair.

We have combined gene correction with strategies for safe genetic reprogramming of patient-derived fibroblasts to induced Pluripotent Stem Cells (iPSC), in order to provide an alternative and potentially unlimited source of patient-derived, gene-corrected, reprogramming factor free stem cells. These iPSC represent invaluable tools to explore methods for inducing their differentiation into hematopoietic cell types and design new cell replacement approaches.

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