Uncovering and dissecting the genotoxicity of self-inactivating lentiviral vectors in vivo.
Self-inactivating (SIN) lentiviral vectors (LV) have an excellent therapeutic potential as demonstrated in preclinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could still pose a significant risk in clinical applications. Taking advantage of novel in vivo genotoxicity assays, we tested a battery of LV constructs, including some with clinically relevant designs, and found that oncogene activation by promoter insertion is the most powerful mechanism of early vector-induced oncogenesis. SIN LVs disabled in their capacity to activate oncogenes by promoter insertion were less genotoxic and induced tumors by enhancer-mediated activation of oncogenes with efficiency that was proportional to the strength of the promoter used. On the other hand, when enhancer activity was reduced by using moderate promoters, oncogenesis by inactivation of tumor suppressor gene was revealed. This mechanism becomes predominant when the enhancer activity of the internal promoter is shielded by the presence of a synthetic chromatin insulator cassette. Our data provide both mechanistic insights and quantitative readouts of vector-mediated genotoxicity, allowing a relative ranking of different vectors according to these features, and inform current and future choices of vector design with increasing biosafety.
San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
Daniela
D
Cesana
San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
Marco
M
Ranzani
1] San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy [2] Vita Salute San Raffaele University, Milan, Italy.
Monica
M
Volpin
National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany.
Cynthia
C
Bartholomae
Laboratory of Biotechnology and Applied Pharmacogenetics, Ecole Normale Supérieure de Cachan, Cachan, France.
Caroline
C
Duros
Laboratory of Biotechnology and Applied Pharmacogenetics, Ecole Normale Supérieure de Cachan, Cachan, France.
Alexandre
A
Artus
1] San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy [2] Current address: Center for Translational Genomics and Bioinformatics, San Raffaele Scientific Institute, Milan, Italy.
Stefania
S
Merella
San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
Fabrizio
F
Benedicenti
San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
Lucia
L
Sergi Sergi
Department of Pathology, San Raffaele Scientific Institute, Milan, Italy.
Francesca
F
Sanvito
University Centre of Statistics for Biomedical Sciences, Vita Salute San Raffaele University, Milan, Italy.
Chiara
C
Brombin
University Centre of Statistics for Biomedical Sciences, Vita Salute San Raffaele University, Milan, Italy.
Alessandro
A
Nonis
University Centre of Statistics for Biomedical Sciences, Vita Salute San Raffaele University, Milan, Italy.
Clelia Di
CD
Serio
Department of Pathology, San Raffaele Scientific Institute, Milan, Italy.
Claudio
C
Doglioni
National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany.
Christof
C
von Kalle
National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany.
Manfred
M
Schmidt
1] Laboratory of Biotechnology and Applied Pharmacogenetics, Ecole Normale Supérieure de Cachan, Cachan, France [2] Department of Medical Oncology, Hospital Saint-Louis, Paris, France.
Odile
O
Cohen-Haguenauer
1] San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy [2] Vita Salute San Raffaele University, Milan, Italy.
Luigi
L
Naldini
1] San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy [2] Vita Salute San Raffaele University, Milan, Italy.
Eugenio
E
Montini
D063646:Carcinogenesis / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000009:adverse effects* / Q000627:therapeutic use; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000472:pathogenicity; D016254:Mutagenesis, Insertional / Q000235:genetics; D011401:Promoter Regions, Genetic