774. Understanding and Exploiting Endogenous microRNA Regulation for Controlling Gene Expression

Both PB and BM was analyzed for vector expression in the various lineages defined by antibody positivity to the antigens CD3, CD4, CD8, CDllb, CDI4, CDI6, CD20, nucleated RBC. We found GFP expression in these lineages ranging from 6-11% in PB and from 7-10% in BM in animal Cl5B. Gf'P expression ranged from 2-4% in the PB and BM in another animal, RQ2617, and from 3-4% in the PB and 3-5% in BM ofanimal RQ3356. qPCR analysis for average vector copy number in PB and BM FACS sorted cells of different hematopoietic lineages number is in progress to evaluate the degree, if any, of vector silencing. Vector insertion site analysis by the linear amplification-mediated PCR method showed, as previously, highly polyclonal reconstitution. We found no evidence for common integration sites and no SIV vector integrants were detected in the MDS Il EV 1\ region, in contrast to previous results with the MLV gamrnaretroviral vector, supporting the possibility that lentiviral vectors may have enhanced biosafety relative to gammaretroviral vectors. This study is the first to report these levels of gene transfer this distant in time from the transplantation and indicate that the SIV vector system can achieve efficient gene transfer into the HSC of rhesus macaques with little gene silencing. Importantly, these levels of HSC gene transfer would likely be of clinical benefit in hematopoietic disorders in which genetically modified stem cells or early progenitors lack an in vivo advantage. GENE MODIFICATION

774. Understanding and Exploiting Endogenous micro RNA Regulation for Controlling Gene Expression Brian D. Brown ,' Alessio Cantore, I Bernhard Gentner,' Anna Zingale,' Luigi Naldini.' 'Telethon Institute for Gene Therapy, San Raffaele Institute, Milan. Italy. Recently, a complex network of gene regulation has been uncovered, which is mediated by small non-coding RNAs, known as microRNAs (miRNA). miRNAs act as a guide for the RNA Induced Silencing Complex (RISC) to control expression of a target gene. Molecular analysis has shown that miRNAs have distinct expression profiles in different tissues, and, together with functional studies, indieatc important roles for miRNAs in cstablishing ecll identity. We recently reported that endogenous miRNA regulation could be exploited for controlling transgene expression from a lentiviral vector (LV). By inserting a sequence complementary to a hematopoietic-specific miRNA, mir-142-3p, into our vector, transgene expression was completely prevented in all hematopoietic cells , while expression in non-hematopoietic cells remained unaffected (Brown et al. Nat Med 2006). This work presented a powerful demonstration that endogenous miRNA regulation could be used to effectively de-target expression of a transgene from a particular cellular lineage, and thus provided the basis for a new means of controlling vector expression. To develop a better framework for designing miRNA-regulated vector systems , we set out to determine the factors that influence the effectiveness of endogenous miRNA regulation. To monitor miRNA activity, we developed a novel LV reporter, which utilizes the bidirectional activity ofa single promoter to co-ordinately express two transgenes in opposite orientation. A panel ofmiRNA target sequences were cloned into one of the two transgenes within the vector, and miRNA activity was evaluated by monitoring changes in transgene expression. Using this approach, we provide the first evidence that endogenous miRNA activity is dependent on miRNA concentration within the cell, and that there is a direct relationship between target copy and miRNA activity. Since miRNA profiles are closely associated with cell identity, we next set out to determine whether miRNA regulation could be exploited to differentially regulate transgene expression between two S298

cellular states. As a model system, we used human dendritic cells (DCs). DCs can influence an immune response depending on their maturation status. Immature DCs (iDCs) present antigens to T cells, and induce antigen-specific tolerance, while mature DCs (mDCs) present antigens to T cells , and induce antigen-specific immunity. Initially, we evaluated the miRNA profile of iDC and mDCs, and found that there was a 50-fold elevation of a single miRNA in mDCs. Based on these findings, we modified an LV to contain target sequences for this miRNA. Amazingly, using this vector, we were able to achieve high levels of transgene expression in iDCs, while in mDCs, transgene expression was abolished. This expression pattern, which could not be obtained until now, provides a promising new means for achieving antigen-specific tolerance through genetic modification ofDCs. Overall, our findings provide new insights into the factors that govern miRNA activity, and have major implications for understanding endogenous miRNA regulation . They also provide a critical framework for developing new miRNA-regulated vector systems with highly specific cellular expression profiles.

775. The Woodchuck Hepatitis Virus PostTranscriptional Regulatory Element Reduces Readthrough Transcription from Retroviral Vectors To Improve Vector Titers and Expression Tomoyasu Higashimoto,' Fabrizia Urbinati,' Anil Pcrurnbcti.' Gang Jiang ," Alexander Zarzucla," Lung-Ji Chang,' Donald B. Kohn,' Punam Malik .' 'Division ofExperimental Hematology. Cincinnati Children Hospital Medical Center; Cincinnati. OH; "Dtvision ofHematology-Oncology, Childrens Hospital Los Angeles. Los Angeles. CA; JDepartmenl ofMolecular Genetics and Microbiology. University ofFlorida, Gainesville, FL.

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The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression from variety of viral vectors, although the precise mechanism is unknown. Since WPRE is most effective when placed downstream of the transgene, but proximal to the polyadenylation signal we hypothesized that WPRE likely reduces viral mRNA readthrough by improving transcript termination; which in turn would increase viral titers and expression. First, a series of plasmid constructs with the wild type (wt) or self-inactivating (SIN) , retroviral (RV) or lentiviral (LV) LTRs were made carrying the EFl-a promoter upstream ofthe LTRs. A promoter-less IRES-ere fragment was placed downstream of the LTRs, so that ere expression would only occur from transcriptional readthrough from the LTRs. WPRE was placed upstream of the LTRs. RV and LV LTR plasmid constructs, with and without the WPRE were transfeeted into the reporter cell line, TE26, which (~-gal) proportional to ere activity. Sigexpresses ~-galaetosidase activity was observed from constructs containing wt nificant ~-gal RV-LTR or the SIN RV-LTR. When WPRE was placed upstream of the wt RV-LTR or the SIN RV-LTR, it reduced transcriptional readthrough by 2-fold from the wt and SIN RV-LTR, respectively, to levels similar to controls (p