San Raffaele Telethon Institute for Gene Therapy

Safety of gene therapy and insertional mutagenesis

Group Leader

Eugenio Montini

Gene therapy (GT) applications in the recent years have advanced close to realizing their full potential. GT however is still in its infancy and a significantly uncharted territory and unique challenges remain to be addressed. Notably, the lack of entirely neutral gene delivery or editing platforms and consistent readouts for safety monitoring predictive for the long-term impact of these therapies represent an outstanding issue for the whole GT field.

The ‘Safety of gene therapy and insertional mutagenesis’ research unit is committed to overcome these challenges by unraveling the basic mechanisms governing genotoxicity in gene therapy and improve the safety of GT applications by modifying the current vector designs to deliver transgenes efficiently without impacting the expression or mRNA structure of genes nearby vector integration sites and to discover novel biomarkers predictive of the safety of GT treatments, outcome of the therapy and disease burden.

To reach these goals we have developed sensitive genetic mouse models for testing the genotoxic potential of different vector types and backbone designs, state of the art high throughput retrieval and next generation sequencing of vector insertion sites, methods to study the long-range interactions between vector and cellular genome and a solid bioinformatics and statistical framework to analyze and integrate genomic and phenotypic data.

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Research activity

Our activity is organized in three main areas:

Improving the safety of gene transfer. We use mouse models and powerful molecular technologies (4C, HiC, ChIP-seq, RNA-seq) to unravel the basic mechanisms governing genotoxicity and to compare the impact of integration of vectors with different designs and/or containing genetic elements to potentially improve their safety. We aim to capitalize on the gained knowledge to devise and validate novel vector designs with improved safety profile to be adopted as “gold standard” in future ex vivo and in vivo GT applications.
Characterization of the safety and dynamics of clonal reconstitution in ex vivo GT. We aim to understand several basic aspects of human hematopoiesis by dissecting the clonal dynamics of hematopoietic reconstitution in hematopoietic stem cell-GT patients and unveil factors that impact on the outcomes of the therapy in terms safety and efficacy under different genetic, clinical, and environmental conditions.
Uncovering the effects of insertional mutagenesis beyond cancer. We aim to identify markers for the detection of cells with risky cancer promoting insertions (or mutations), understand their impact on the surrounding microenvironment and at the whole organismal level. We also aim to devise methods that will allow to eliminate specifically cell harboring genotoxic insertions in vivo.

Selected publications

Calabria A, Cipriani C, Spinozzi G, Rudilosso L, Esposito S, Benedicenti F, Albertini A, Pouzolles M, Luoni M, Giannelli S, Broccoli V, Guilbaud M, Adjali O, Taylor N, Zimmermann VS, Montini E, Cesana D Intrathymic AAV delivery results in threapeutic site-specific integration at TCR loci Blood. 2023 Feb 15:blood.2022017378. doi: 10.1182/blood.2022017378. Online ahead of print.

Pais G, Spinozzi G, Cesana D, Benedicenti F, Albertini A, Bernardo ME, Gentner B, Montini E, Calabria A ISAnalytics enables longitudinal and high-throughput clonal tracking studies in hematopoietic stem cell gene therapy applications Brief Bioinform. 2022 Dec 21:bbac551. doi: 10.1093/bib/bbac551. Online ahead of print.

Del Core L, Cesana D, Gallina P, Serina Secanechia YN, Rudilosso L, Montini E, Wit EC, Calabria A and Grzegorczyk MA Normalization of clonal diversity in gene therapy studies using shape constrained splines. Sci Rep. 2022 Mar 9;12(1):3836. doi: 10.1038/s41598-022-05837-0.

Cesana D, Calabria A, Rudilosso L, Gallina P, Benedicenti F, Spinozzi G, Schiroli G. Magnani A, Acquati S, Fumagalli F, Calbi V, Witzel M, Bushman FD, Cantore A, Genovese P, Klein C, Fischer A, Cavazzana M, Six E, Aiuti A, Naldini L, Montini E. Retrieval of vector integrations sites from cell-free DNA (2021) Nature Medicine. Aug;27(8):1458-1470

Biavasco R*, Lettera M*, Giannetti K, Gilioli D, Beretta S, Conti A, Scala S, Cesana D, Gallina P, Norelli M, Basso-Ricci L, Bondanza A, Cavalli G, Ponzoni M,, Dagna L, Doglioni C, Alessandro A, Merelli I, Di Micco R§, Montini E§. (*equal contribution, §co-corresponding) Oncogene-induced senescence in hematopoietic progenitors features myeloid restricted hematopoiesis, chronic inflammation and histiocytosis. (2021) Nature Communications. Jul 27; (12): 4559

Cesana D*, Santoni de Sio FR, Rudilosso L, Gallina P, Calabria A, Beretta S, Merelli I, Bruzzesi E, Passerini L, Nozza S, Vicenzi E, Poli G, Gregori S, Tambussi G, Montini E*. *co-corresponding. HIV-1-mediated insertional activation of STAT5B and BACH2 trigger viral reservoir in T regulatory cells. Nat Commun. 2017 Sep 8;8(1):498.

Calabria A, Leo S, Benedicenti F, Cesana D, Spinozzi G, Orsini M, Merella S, Stupka E, Zanetti G, Montini E. VISPA: a computational pipeline for the identification and analysis of genomic vector integration sites. Genome Med. 2014 Sep 3;6(9):67.

Ranzani M, Annunziato S, Calabria A, Brasca S, Benedicenti F, Gallina P, Naldini L, Montini E. Lentiviral vector-based insertional mutagenesis identifies genes involved in the resistance to targeted anticancer therapies. Mol Ther. 2014 Dec;22(12):2056-68.

Cesana D, Ranzani M, Volpin M, Bartholomae C, Duros C, Artus A, Merella S, Benedicenti F, Sergi Sergi L, Sanvito F, Brombin C, Nonis A, Serio CD, Doglioni C, von Kalle C, Schmidt M, Cohen-Haguenauer O, Naldini L, Montini E. Uncovering and dissecting the genotoxicity of self-inactivating lentiviral vectors in vivo. Mol Ther. 2014 Apr;22(4):774-85.

Biffi A*, Montini E*, Lorioli L, Cesani M, Fumagalli F, Plati T, Baldoli C, Martino S, Calabria A, Canale S, Benedicenti F, Vallanti G, Biasco L, Leo S, Kabbara N, Zanetti G, Rizzo WB, Mehta NA, Cicalese MP, Casiraghi M, Boelens JJ, Del Carro U, Dow DJ, Schmidt M, Assanelli A, Neduva V, Di Serio C, Stupka E, Gardner J, von Kalle C, Bordignon C, Ciceri F, Rovelli A, Roncarolo MG, Aiuti A, Sessa M, Naldini L. *equal contribution. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science. 2013 Aug 23;341(6148):1233158.

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. Nat Methods. 2013 Feb;10(2):155-61.

Cesana D, Sgualdino J, Rudilosso L, Merella S, Naldini L, Montini E. Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations. J Clin Invest. 2012 May;122(5):1667-76.

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 May 19;117(20):5332-9.

Montini E*, Cesana D*, Schmidt M, Sanvito F, Bartholomae CC, Ranzani M, Benedicenti F, Sergi LS, Ambrosi A, Ponzoni M, Doglioni C, Di Serio C, von Kalle C, Naldini L. *equal contribution. The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy. J Clin Invest. 2009 Apr;119(4):964-75.

Montini E, Cesana D, Schmidt M, Sanvito F, Ponzoni M, Bartholomae C, Sergi Sergi L, Benedicenti F, Ambrosi A, Di Serio C, Doglioni C, von Kalle C, Naldini L. Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration. Nat Biotechnol. 2006 Jun;24(6):687-96.