Gammaretroviral vector integration preference for DNAse hypersensitive sites

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ABSTRACTS / Blood Cells, Molecules, and Diseases 40 (2008) 248–294

Hct is eliminated upon removal of the ContigenTM-embedded Epo + MSCs implants (Ref. 2). Moreover, we discovered that allogeneic MSCs cannot be used for protein delivery in vivo, at least not in mice, through our observation that class I and II MHC-mismatched murine MSCs provoke a cellular immune response by allogeneic mice with normal immune systems, and that rejection is amplified by repeated challenge (Ref. 3). We thereafter tested our Epo + MSCs in an autologous mouse model of anemia induced by experimental chronic renal failure, and observed that these cells, when implanted subcutaneously embedded in Contigen™, lead to a Hct rise that is cell dose dependent, as well as to an exercise capacity amelioration as assessed by swimming duration (Ref. 4). In conclusion, our findings support the use of a neo-organoid consisting of autologous ex vivo gene-engineered MSCs as a biopharmaceutical platform for in vivo delivery of plasma soluble therapeutic proteins. References: 1. Eliopoulos, N., Al-Khaldi, A., Crosato, M., Lachapelle, K, Galipeau, J., Gene Therapy, 10(6): 478–489, 2003. 2. Eliopoulos, N., Lejeune, L., Martineau, D., Galipeau, J., Molecular Therapy, 10:741–748, 2004. 3. Eliopoulos, N., Stagg, J., Lejeune, L., Pommey, S., Galipeau, J., Blood, 106:4057–4065, 2005. 4. Eliopoulos, N., Gagnon, R., Francois, M., Galipeau, J., Journal of the American Society of Nephrology, 17:1576–1584, 2006. doi:10.1016/j.bcmd.2007.10.036

27 Gammaretroviral vector integration preference for DNAse hypersensitive sites Mingdong Liu, Peter J. Sabo, Michael S. Kuehn, John A. Stamatoyannopoulos, David W. Emery Department of Medicine, University of Washington, Seattle, WA, USA Concerns surrounding the oncogenic potential of recombinant gammaretroviral vectors have spurred a great deal of interest into the integration site preferences of such vectors. Early studies of murine leukemia virus-transformed cell clones, and more recent studies with recombinant gammaretroviral vectors in unselected cell pools, indicated a correlation between viral integration sites and DNase hypersensitive sites, which mark open chromatin regions and are often associated with cisregulatory elements such as enhancers and promoters. Although compelling, these previous studies are difficult to interpret: the older studies involved the use of wild-type virus and transformed clones, while the latter compared integration sites to DNase hypersensitive sites identified in a high throughput screen of an unrelated cell type. In order to study this issue more directly, we looked for correlations between gammaretroviral vector integration sites identified in the absence of selection,

and DNase hypersensitive sites mapped within the same cell line (the human fibrosarcoma HT1080) using robust conventional methods. To date we have analyzed 17 independent integration sites located on 12 different chromosomes. Of these sites, a total of 10 were found to be located directly within DNase hypersensitive sites, a frequency that is highly unlikely by chance. The remaining sites were distributed over a distance of 20 bp to 1115 bp from the border of the nearest DNase hypersensitive site. As a control, we used an extensive data set of validated DNase hypersensitive sites mapped in the ENCODE region using the DNase-array method (Sabo et al., Nature Methods 3:511, 2006). Specifically, we calculated the distances between 753 DNase hypersensitive sites from extended segments of most of the chromosomes used for the integration site analysis. For this data set, the distances between borders of adjacent DNase hypersensitive sites ranged from 16 bp to over 200 kb, with an mean of 9100 bp and a median of 619 bp. The notable discrepancy between the mean and median values reflects a fat-tailed Cauchy-like distribution. Although the median distances for both the experimental and control data sets were similar, comparison of these two data sets using the non-parametric, distribution free Kolmogorov–Smirnov test, showed a clear difference between the overall distributions (P b 0.001). Specifically, the integration site data set lacked examples of integrations located at distances greater than 1 kb, whereas over 40% of the distances in the control data set were greater than 1 kb. Taken together, these results provide direct evidence that gammaretrovirus vectors have a preference for integration within, or at least near, DNase hypersensitive sites. Current studies are focused on developing means of assessing this preference on a genome-wide scale in clinically relevant cell types, as well as assessing the impact of vector provirus on the pattern of DNase hypersensitive sites surrounding sites of integration. doi:10.1016/j.bcmd.2007.10.037

28 Reducing retroviral vector genotoxicity with chromatin insulators David W. Emery, Chang Long Li, George Stamatoyannopoulos Department of Medicine, University of Washington, Seattle, WA, USA The therapeutic application of gene transfer vectors based on recombinant retroviruses has been limited by problems of vector expression and, more recently, vector-mediated genotoxicity. These problems arise in large part from interactions between vector provirus and the genomic environment surrounding ectopic sites of integration. Advances have been made in overcoming both of these problems through the modification of deleterious vector sequences, the inclusion of better enhancers and promoters, and the use of alternative virus systems. However, these modifications often add additional restrictions on vector design, which in turn can further limit therapeutic