San Raffaele Telethon Institute for Gene Therapy
Vector Integration Core
Insertional mutagenesis is one of the major hurdles of gene therapy with integrating vectors. Analysis of genomic vector integration sites present in cells from gene therapy patients and preclinical models in vivo allows the detection of cells that have acquired a selective advantage because of oncogene activation caused by nearby vector insertions. Indeed, since vector integration sites are stable genetic marks, distinctive for each independently transduced cell and its progeny, their characterization allows to track thousands of transduced cell clones over time, in different tissues or cell lineages, evaluate the clonal composition of the engrafted population, identify the genes targeted by vector integrations and quantify the relative abundance of clones harboring a specific integration sites in order to detect or exclude clonal expansions. Therefore, the analyses of integration sites is crucial to monitor the biological unfolding and ultimate safety of the administered gene therapies.
The mission of the Vector Integration Core is to provide a comprehensive molecular monitoring of safety and effectiveness of Advanced Therapy Medicinal Products (ATMP) in the context of gene therapy and gene editing applications. The services apply to preclinical and clinical testing, commercialized products as well as to R&D and early discovery stages.
Activities
The Vector Integration Core has set up state of the art procedures, bioinformatics pipelines and rigorous statistical analyses to perform genome-wide profiling of vector integration sites and monitor the behavior of transduced cell clones in:
- pre-clinical safety studies performed under research grade or Good Laboratory Practice;
- clinical gene therapy pharmacovigilance studies;
- basic scientific projects.
The Vector Integration Core has been fundamental to address the safety and the clonal dynamics of hematopoietic reconstitution in several preclinical models and clinical trials such as for metachromatic leukodystrophy, Wiskott-Aldrich, beta-thalassemia and mucopolysaccharidosis type I, Hurler variant carried out at SR-Tiget.
The Core developed and consolidated the technologies and analytical pipelines to retrieve and analyze vector insertion sites for various vector platforms. Two of the most important techniques set in the facility are:
- Sonication Linker-mediated-PCR (SLiM-PCR), a highly efficient method for integration sites retrieval from genomic DNA, which amplifies virus or vector-genome junctions from cells and tissues in a genome-sequence unbiased and quantitative fashion;
- Liquid Biopsy Integration Site-Sequencing (LiBIS-Seq; UK Patent filed in April 2019) a novel method that allows retrieving integration sites from cell-free DNA from blood plasma or other body fluids to examine genetically modified cells in virtually any tissue/ organ of the body, overcoming the need of invasive tissue biopsies to retrieve test material
Both methods are tailored to different vector platforms including LV, RV, Transposons and AAV and were successfully adapted to other constructs such as plasmids or DNA cassettes for site-specific gene editing applications.
Laboratory Equipment: The main equipment consists in 4 certified PCR hoods, 2 liquid handlers for sample processing automation in 96- and 384-well plates, 6 thermal cyclers, 2 systems for real-time quantitative PCR, 2 systems for automated capillary electrophoresis, several refrigerators and laboratory equipment for molecular biology work. All experimental procedures are performed in dedicated rooms to avoid cross contaminations. DNA samples are processed automatically through robotized solutions Illumina platforms (available at the sequencing facility in the Ospedale San Raffaele Center for Omics Science Research [link]) are used to simultaneously sequence hundreds of samples, univocally tagged to allow correct association of the biological information to sequencing data.
Computational Resources: Next Generation Sequencing (NGS) data analyses are executed on a powerful computational infrastructure distributed on two computer clusters available through the collaboration with the National Research Council and CINECA interuniversity consortium, two workstations (Intel, dual node Xeon 8 cores, 256 GB of shared RAM DDR3) with an attached storage system of 16 TB (RAID 5) and a backup on a network attached storage (NAS) of 8 TB to store integration site results.
