The San Raffaele Telethon Institute for Gene Therapy (SR-Tiget)
Vector integration core
Lentiviral vectors are very proficient tools for gene therapy, due to their ability to integrate in the host genome. This makes them also useful to genetically mark and follow cells in vivo, but poses safety concerns. Thus, the analysis of vector integration sites in target cells is extremely important to address both biological and safety issues.
The SR-Tiget vector integration core has set up several state of the art and new techniques to study vector integration and its impact on gene transcript structure and expression levels. The setup of a core activity with validated Standard Operating Procedures, dedicated computational infrastructures and data analysis pipelines has enabled the high-throughput integration site monitoring in different clinical trials and several other scientific projects at SR-Tiget in a fast and cost-effective fashion.
New wet-lab protocols for integration site retrieval and sequencing and bioinformatics research projects are being developed to approach the massive amount of samples and data generated, and to enable the correct interpretation of the physiological significance of differences detected in integration profiles.
Quantitative retrieval of vector integration junctions
We have set up an automated protocol of Linear Amplification Mediated (LAM) PCR for the retrieval of vector/genome junctions. Moreover, novel methods based on DNA sonication and sequencing of vector-genome junctions using the Illumina sequencing technology are being setup to accurately determine the relative abundance of cell clones harboring specific integration sites in a given biological sample. Moreover, the Illumina sequencing deepness grants the retrieval of a higher number of integration sites compared to previous sequencing methods, strengthening the power of the safety studies on one hand and allowing to picture the overall stem cell derived clone dynamics on the other.
Schematic outline of LAM-PCR for LTR – genomic junction amplification.
Development of automated pipelines for ISs analysis on distributed computational environments
Analysis of chromosomal vector integration sites in vector marked cells from gene therapy patients and preclinical models has enabled to detect in vivo selection of gain-of-function insertional mutants even before they progress to overt malignancy. For this reason, over the last years there has been a constant increase in the amount of sequencing and mapping of vector/genomic DNA junctions and statistical tools to improve biological investigations and their interpretation. Recently, Next Generation Sequencing (NGS) approaches have been exploited in Gene Therapy to greatly enhance the analytical power of integration site analysis. However, NGS analytical advances are balanced by computational drawbacks such as the flooding of data that needs to be carefully managed and processed with smart pipelines on distributed high-performances infrastructures. Thus NGS-structured and standardized pipelines for biologists are strongly required.
We developed a NGS bioinformatics pipeline, both for Illumina and Roche platforms, for integration sites analysis, enabling quality controls and high-performance properties. We designed data processing activities grouped in: (a) reads quality control, avoiding unreliable data analysis, (b) raw data cleaning and vector sequences trimming, (c) reads grouping and clustering, to estimate the number of integration sites before mapping sequences, (d) reads mapping to reference genome (e) post-processing data filtering and refinement of integration sites for subsequent analyses (i.e. integration sites association to annotated genomic features, common integration site identification and complexity estimation as surrogate of clonal diversity of vector marked cells).
Comprehensive Data Mining Approach applied to Integration Sites Analysis
Analysis of chromosomal vector integration sites (ISs) in vector marked cells from gene therapy patients is a key point for unraveling gene therapy progresses, like safety and efficacy, and getting new biological insights into stem cell biology. By Next-Generation Sequencing (NGS) technologies we sequenced >10x10e6 proviral/host genomic junctions from cells of 6 patients from 2 ongoing hematopoietic stem cell (HSC)-based clinical trials with self-inactivating lentiviral vectors in Milan (metachromatic leukodystrophy and Wiskott–Aldrich syndrome).
Overall we retrieved millions of univocally mapped ISs resulting in may thousands of unique ISs distributed into cell lineages and tissues during time. Given the large amount of IS data to be analyzed and the diversification of the variables associated to it, we designed a new comprehensive data-mining framework, divided into three main blocks: (1) NGS data processing, to handle high-throughput IS mapping; (2) data quality, to filter ISs by quality parameters and new methods for collisions detection and data normalization; (3) ISs biological analysis. The third block addresses different biological problems: (a) common insertion sites identification, using a gene-centric method; (b) clonal abundance studies; (c) stem cell activity during time by analyzing shared ISs in CD34+, myeloid and lymphoid lineages; (d) lineage-based clonality and diversity analyses.
Ranzani M, Cesana D, Bartholomae CC, Sanvito F, Pala M, Benedicenti F, Gallina P, Sergi LS, Merella S, Bulfone A, Doglioni C, von Kalle C, Kim YJ, Schmidt M, Tonon G, Naldini L, Montini E. Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer. Nature Methods. 2013 Feb;10(2):155-61 IF: 23.565, Cited 1 time
Cesana D*, Sgualdino J*, Rudilosso L, Merella S, Naldini L, Montini E. Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations. Journal of Clinical Investigation. 2012. 122(5):1667-76; IF: 14.152; Cited 3 times. (Commentary on JCI same issue from D.Trono: Gene therapy: too much splice can spoil the dish; Highlight in Nature Biotechnology from J. Kreisberg, Safer lentiviral vectors, 30,508, 07 June 2012)
Montini E, Cesana D. Genotoxicity assay for gene therapy vectors in tumor prone Cdkn2a-/- mice. Methods Enzymology. 2012. 507:171-85. IF: 1.626
Biffi A, Bartolomae CC, Cesana D, Cartier N, Aubourg P, Ranzani M, Cesani M, Benedicenti F, Plati T, Rubagotti E, Merella S, Capotondo A, Sgualdino J, Zanetti G, von Kalle C, Schmidt M, Naldini L*, Montini E*. Lentiviral vector common integration sites in Preclinical Models and a Clinical Trial Reflect a Benign Integration Bias and not Oncogenic Selection. Blood. 2011. 117(20):5332-9. IF: 10.558. Cited 21 times.
Cassani B*, Montini E*, Maruggi G, Ambrosi A, Mirolo M, Selleri S, Biral E, Frugnoli I, Hernandez-Trujillo V, Di Serio C, Roncarolo MG, Naldini L, Mavilio F, Aiuti A. Integration of retroviral vectors induces minor changes in the transcriptional activity of T cells from ADA-SCID patients treated with gene therapy. Blood. 2009. 114(17):3546-56. IF: 10.558. Cited 27 times. (*equal contribution)
Montini E*§, Cesana D*, Schmidt M, Sanvito F, Bartholomae C, Ranzani M, Benedicenti F, Sergi Sergi L, Ambrosi A, Ponzoni M, Doglioni C, Di Serio C, von Kalle C, Naldini L§. The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of hematopoietic stem cell gene therapy. Journal of Clinical Investigation. 2009. 119(4):964-75. IF: 14.152; Cited 153 times. (Commentary from C. Baum on JCI same issue: Preventing and exploiting the oncogenic potential of integrating gene vectors). (*equal contribution; §co-corresponding authors)