1062. Validating Assays for Vector-Mediated Genotoxicity

thus showed that retroviral vectors with a self-inactivating (SIN) design , which are devoid of promoter/enhancer elements in the U3 region, are weaker insertional mutagens compared to their LTRdriven counterparts. However, SIN vector with strong internal enhancer-promoters (derived from murine leukemia viruses) were still able to transform cells by insertional transaetivation of protooncogenes such as Evi I. Interestingly, we found that SIN vectors with cellular promoters such as elongation factor I alpha (EF Ia) were sufficiently potent to express potentially therapeutic levels of methylguanine-DNA-methyltransferase (MGMT) or interleukin 2 receptor gamma chain (IL2RG) , two cDNAs which are of major interest for ongoing and future elinical trials. To assess whether the use of cellular promoters in SIN vectors increases biosafety, we evaluated their transactivating and transforming potent ial in 3 different assays : (I) In a plasmid-based assay we analyzed the enhancer interactions ofdifferent promoter/enhancer configurations on a minimal promoter driving a lueiferase expression cassette. (2) To test the effects in transduced cells, we constructed a SIN vector harbouring a minigene cassette in the 3'U3 deletion that can be transactivated by the internal promoter. (3) The same retroviral SIN configurations ineluding insulator sequences were compared in the IVIM assay. In all experimental settings, cellular promoters (PGK, EFla) were weaker transactivators than viral promoters (SFFV, MPSV, CMV, RSV). Interestingly, insertional transformation by the SFFV promoter could be decreased by introducing the cI-IS4 insulator (256 bp core element) into the SIN U3 region (P=0.025, n=4, IVIM assay), but this was not sufficient to prevent immortalisation of bone marrow cells. In contrast, SIN vectors using the EFla promoter were unable to immortalize primary bone marrow cells in the IVIM assay (despite dose escalation to as much as 40 copies per cell). Validation ofthese findings in murine bone marrow transplant studies is ongoing. In summary, we observed a correlation of transactivation potential in reporter assays and transformation potential in primal)' hematopoietic cells as defined by replating assays. Based on these results, we conclude that it is possible to design and evaluate integrating vectors with increased biosafety even for those gene therapy settings which require relatively high expression levels of therapeutic genes.

1062. Validating Assays for Vector-Mediated Genotoxicity

David W. Emery, I Chang Long Li,' George Stamatoyannopoulos.' {Department ofMedicine, University 0/ Washington, Seattle, Wi/.

Interest in the use of recombinant retroviral vectors for clinical gene therapy has been tempered by growing evidence ofveetor-mediatcd genotoxieity involving the activation of cellular oncogenes flanking sites ofvector integration. Studies into methods for reducing vector-mediated genotoxicity would be greatly facilitated by the establishment of validated genotoxicity assays that are robust and quantitative. We have been investigating two different approaches for assessing vector-mediated genotoxicity, as well as the ability of the cI-IS4 chromatin insulator to reduce this genotoxicity. The first approach involves directly measuring the rate with which vector provirus alters expression of cellular genes using panels of transduced I-IT I080 cell clones and RNA expression arrays. In these studies, flanking a gammaretroviral vector with the cI-IS4 insulator reduced the frequency of disregulated cellular genes located in cis with vector provirus 6-fold, from 13 per 161 provirus to 2 per 151 provirus (P=.006). Of the 15 disregulated genes, 8 contained provirus located within the primary transcript. Molecular analysis demonstrated several examples of'altcrnativc splicing or other effects on transcript processing, providing direct evidence for a causative role ofvector integration in cellular gene disregulation. We arc currently investigating the remaining cases, where the nearest provirus are located at Mb distances, for functional links between specific Molecular Therapy Volume 15 ~ Supplement I. May 2007 Copyright © Th c American Society of G ene Th erapy

provirus and disregulated genes . The second approach involves a functional assay based on vector-mediated transfonnation of the IL-3 dependent cell line 32D. In these studies, the cHS4 insulator reduced the frequency of vector-mediated transformation to IL-3 independence 8-fold, from 53 to 6.3 colonies per 10\ transduced cells (P=.005). Inclusion ofthe insulator also reduced the frequency of32D cell tumor formation in mice 6-fold, from 61 to 10 per 10\ transduced cells (P<.OOI). Integration site analysis revealed the presence of genes involved in cell cycle regulation, apoptosis, cytokine signaling, or oncogenic transformation within a 150 kb window ofvector provirus in 20 of21 transformed clones analyzed. This included 12 examples of oncogenes present in the Retroviral Tagged Cancer Gene Database. Such associations were significantly more frequent than for a simulated random integration site database (P=O.O I). Of the 45 integration sites analyzed , two common integration sites were noted. The first involves the gene Aatk that has previously been shown to playa role in the response of 32D cells to IL-3. The second involves the gene Gata2that has recently been implicated in oncogenic transformation mediated through Evi I, another gene commonly associated with gammaretroviral vector genotoxicity. These results provide evidence for a causative role of vector integration in the transformed 32D cell phenotype. In summary, we describe two independent approaches for assessing vector-mediated genotoxicity, and provide evidence validating both approaches as robust and quantitative.

1063. Modeling the Genotoxicity of Viral Vector Integration in a Tumor Prone Hematopoietic Stem Cell Transplantation Model Eugenio Montini,' Daniela Cesana,'> Manfred Schmidt,' Francesca Sanvito,' Maurilio Ponzoni,' Lucia Sergi Sergi,' Fabrizio Benedicenti,' Cynthia Bartholomae,' Clelia Di Serio,' Claudio Doglioni.s' Christof von Kalle,' Luigi Naldini.l-' {Sail Raffaele-Telethon Institute for Gene Therapy. Sail RaffaeleTelethon, Milan, Italy; 2 Vita Salute Sail Raffaele University, San Raffaele, Milan, Italy; JNatiollal Center for Tumor Diseases, DKFZ National Center for Tumor Diseases, Heidelberg, Germany; 'Department ofPathology; Sail Raffaele Hospital, Milan,

italy:

Insertional mutagenesis represents a major hurdle to successful gene therapy and mandates for sensitive pre-clinical assays of genotoxicity. We developed an in-vivo genotoxicity assay, based on transplantation of'Cdknza-thematopoietic stem cells, treated or not with prototypical retroviral (RV) and lentiviral (LV) vectors. Tumor prone Cdkn2a'{'mice are defective for p53 and pRb pathways and arc highly susceptible to a wide rangc if cancer-triggering genetic lesions. Therefore ifvector treatment is genotoxic, mice transplanted with vector-treated cells will show a significantly earlier onset of hematopoietic tumors with respect mice transplanted with untreated cells. Using this approach , we were able to detect a dose-dependent acceleration in tumor onset in the mice transplanted with cells treated with a conventional MLV-based RV. RV insertions targeting Common Integration Site genes (CIS; putative cancer genes previously identified by retroviral insertional mutagenesis) and cell cycle genes were enriched in tumors and significantly associated to early oncogenesis. On the other hand, LV treatment did not cause any tumor acceleration, nor selected integrations at any specific gene class . These data showed that LV have low oncogenic potential and provides a major rationale for their application in gene therapy. To dissect the role in oncogenesis of the strong enhancers in the RV LTRs and the different integration site selection of each vector, we tested RVs with self-inactivating LTR (SIN RVs) and a moderate promoter in interna l position, and LVs with strong RV enhancerpromoters into the LTRs or in internal position. Remarkably , SIN RVs showed near-abrogation of tumor acceleration respect to the S405