363 Mouse Models To Assess the Risk of Vector Insertional Mutagenesis upon Systemic Delivery Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S141 R[.]
RNA VIRUS VECTORS II: VECTOR DESIGN AND INTEGRATION PROPERTIES complement fragments to AAV vector particles Nevertheless, AAV particles did not activate the alternative pathway of the complement cascade as suggested by this interaction Furthermore, AAV lacked cofactor activity for factor I-mediated degradation of C3b to iC3b Instead, our results suggest that the AAV capsid binds the complement regulatory protein factor H (fH) and that AAV-bound fH mediates cleavage and inactivation of C3b to iC3b Thus, AAV capsids bind and recruit the complement regulatory protein fH, resulting in decreased complement activation in solution and inactivation of particle bound C3 fragments Furthermore, generation of iC3b-modified AAV vector particles define host interactions based on the distribution of cellular iC3b receptors Interestingly, these receptors mediate the phagocytic clearance of iC3b opsinized AAV vector particles in the absence of inflammatory signaling and together with inhibition of complement activation by surface-bound fH account for the mild inflammatory responses observed in vivo with AAV vectors While we have previously shown that the complement system is an essential component of the host adaptive immune response to AAV Here we define an interaction between AAV and the complement system that attenuates host innate responses We show that AAV binds to serum fH and promotes the fH-mediated cleavage of C3b Because the AAV capsid lacks any sequence homology to known complement regulatory proteins, AAV appears to have evolved a novel viral immune evasion activity akin to that utilized by West Nile virus NS1, whereby the inflammatory and effector functions of complement are inhibited by recruiting and binding serum fH RNA Virus Vectors II: Vector Design and Integration Properties 362 Cell-Specific Transcriptional Regulatory Domains Attract γ-Retroviral Integration in the Human Genome Claudia Cattoglio,1 Barbara Felice,2 Davide Cittaro,2 Danilo Pellin,3 Alessandro Ambrosi,3 Ermanno Rizzi,4 Gianluca De Bellis,4 Chiara Bonini,1 Giulietta Maruggi,5 Francesca Miselli,5 Manfred Schmidt,6 Lucilla Luzi,2 Alessandra Recchia,5 Fulvio Mavilio.1,5 H San Raffaele Scientific Institute, Milan, Italy; 2FIRC Institute of Molecular Oncology, Milan, Italy; 3CUSSB, Vita-Salute San Raffaele University, Milan, Italy; 4CNR, Milan, Italy; 5University of Modena and Reggio Emilia, Modena, Italy; 6National Center for Tumor Diseases, Heidelberg, Germany Gene transfer vectors derived from γ-retroviruses target at high frequency genes involved in the control of growth and differentiation of the target cell, and may induce insertional tumors or pre-neoplastic clonal expansions in patients treated by gene therapy The gene expression program of the host cell appears instrumental in directing γ-retroviral integration, but the molecular basis of this phenomenon is poorly understood We recently reported a bioinformatics analysis of the distribution of transcription factor binding sites (TFBSs) flanking ∼4,000 proviruses in human hematopoietic and non-hematopoietic cells We showed that γ-retroviral, but not lentiviral, vectors integrate in genomic regions enriched in cell-type specific subsets of TFBSs Analysis of sequences flanking the integration sites of Moloney leukemia virus (MLV)-derived vectors with modified long terminal repeats (LTRs), and of a human immunodeficiency virus (HIV)-based vector packaged with the MLV integrase, showed that the MLV integrase and LTR enhancer are the viral determinants of the selection of TFBS-rich regions The study indicated that γ-retroviruses have evolved a peculiar strategy to interact with the host cell chromatin, based on a cooperation between the integrase and TFs bound to the LTR enhancers before integration to tether pre-integration complexes (PICs) to transcriptionally active regulatory regions To further explore the link between cell-specific transcription and Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy MLV integration, we performed deep sequencing of MLV insertions in human CD34+ hematopoietic cells (n=33,162), T lymphocytes (n=7,996), and skin keratinocytes (n=3,020) Compared to matched random distributions, MLV integrations are highly clustered along the chromosomes, with peaks of frequency around transcriptionally active loci Genes within or near the insertion clusters are shared in part among the three cell types, but are mostly involved in tissuespecific development, differentiation and functions, and differentially expressed, as evaluated by gene expression arrays Integration peaks overlap with non-coding elements highly conserved among mammals, again meaning that genomic regions presumably involved in transcriptional regulation are attractive for MLV PICs The status of chromatin around retroviral and random sites was checked by ChIP-on-chip technology, revealing marks of active transcription (H3K9ac, H3K4me2, H3K4me3) on regions flanking the sole MLV insertions Taken together, our observations state an overt preference of γ-retroviral vectors for genomic regions actively engaged in transcriptional regulation We speculate that MLV PICs are directed to these regions by interaction with general components of the enhancerbinding complexes, rather than with specific TFs or TF families 363 Mouse Models To Assess the Risk of Vector Insertional Mutagenesis upon Systemic Delivery Marco Ranzani,1,2 Daniela Cesana,1,2 Manfred Schmidt,3 Francesca Sanvito,4 Fabrizio Benedicenti,1 Cynthia Bartholomä,3 Maurilio Ponzoni,4 Alessio Cantore,1,2 Lucia Sergi Sergi,1 Claudio Doglioni,4 Christof VonKalle,3 Luigi Naldini,1,2 Eugenio Montini.1 HSR-TIGET, Milan, Italy; 2San Raffaele University, Milan, Italy; NCT, Heidelberg, Germany; 4HSR, Milan, Italy Lentiviral vectors (LV) can transduce a broad spectrum of nondividing cells in vivo including hepatocytes Efficient liver gene transfer and long term transgene expression may allow the treatment of several hepatic and systemic diseases However, vector integration may occasionally lead to transformation of hepatocytes, as it has been reported for AAV and non-primate LV Therefore, sensitive preclinical models to assess the genotoxic potential of vector integration in liver tissue are necessary to validate the safety of liver gene transfer We previously developed a sensitive in vivo genotoxicity assay based on transduction/transplantation of hematopoietic stem/progenitor cells (HSPC) from tumor prone Cdkn2a-/- mice By this approach we demonstrated that LV with self-inactivating (SIN) long terminal repeats (LTR) are less genotoxic than γ-retroviral vectors and that genotoxicity is strongly modulated by the vector design and the integration site selection of each vector type However, the predicted safety of an LV design validated in HSPC may not necessarily apply to the liver tissue To setup sensitive assays for liver cell genotoxicity in vivo, we exploited two tumor-prone mouse models and a tumorpromoting regimen applied to wild type mice after vector injection We validated these models by challenge with LV engineered with powerful enhancers/promoters in the LTR previously shown to be genotoxic in HSPC Temporal vein injection of this vector in newborn Cdkn2a-/- mice induced early onset of histiocytic lymphomas infiltrating the liver and spleen with respect to uninjected mice (p