RRE Dependent Packaging System for MLV Based Vectors Raises Biosafety Concerns

RNA VIRUS VECTORS: SAFETY, THERAPEUTICS RNA VIRUS VECTORS: SAFETY, THERAPEUTICS 822. A Rev/RRE Dependent Packaging System for MLV Based Vectors Raises Biosafety Concerns Hong Ma,1 Adam Cockrell,1 Ryan Bash,2 Teryy VanDyke,2 Tal Kafri.1 1 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; 2Linberger comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC. The HIV-1 Rev gene product is essential for nuclear export of unspliced viral RNAs as well as for efficient metabolism of Rev Responsive Elements (RRE)-containing mRNAs including translation, RNA splicing, and RNA stability. Not surprisingly, in most HIV-1 vector systems, both the vector, and the packaging expression cassettes contain the HIV-1 RRE sequence. Here we report on the ability of the HIV-1 Rev/RRE system to function as a secondary packaging system that mediates promiscuous packaging of RRE containing mRNA into HIV-1 particles. To characterize this ability of the HIV-1 Rev/RRE system, we incorporated a DNA fragment containing the HIV-1 cPPT and RRE sequences into traditional MLV based vectors. Using transient threeplasmid transfection, VSV-G pseudotyped chimeric vectors were packaged with either the MLV or the traditional HIV-1 packaging system. The titers of the chimeric MLV/RRE vectors expressing either the GFP or the GFP-Cre fusion protein (1-5x10e5 IU/ml) were determined on 293T and 293T-Loxp-stop-LoxP-LacZ cells, respectively. The fact that GFP expression levels from HIV-1 packaged chimera vectors were relatively low, and could not be detected following several cell divisions, indicates that the HIV-1 packaged chimera vectors lacked the ability to integrate into the target cells’ genome. Using deletion analysis of the chimeric MLV/RRE vectors, we showed that the Rev/RRE mediated packaging is independent of the parental MLV and HIV-1 packaging signals. Interestingly, HIV-1 packaged chimeric MLV/RRE vectors exhibited characteristics of traditional HIV-1 vectors, including the ability to transduce non-dividing cells and sensitivity to HIV-1 specific reverese-transcription inhibitors. Importantly, we showed that gag/pol encoding mRNAs containing the HIV-1 RRE sequence were packaged efficiently into HIV-1 particles in the process of vector production by transient threeplasmid transfection, and thus can be inadvertently transferred to potential target cells. In conclusion, the findings presented here demonstrate the ability of the HIV-1 packaging system to package RRE containing mRNAs. This opens new avenues in the development of novel vectors and yet raises biosafety concerns, which can be addressed by using Rev independent packaging systems.

823. Dissecting Overlapping Functions of Transcriptional Enhancer and Polyadenylation in the Lentiviral U3 Qing Yang,1 Wayne Chou,1 Lung-Ji Chang.1 1 Molecular Genetics & Microbiology, University of Florida, Gainesville, FL. The transcriptional readthrough activity of the 3’ long terminal repeat (LTR) of lentiviral vectors is critical to the safety profile of this vector system in future clinical applications. We have previously reported that the 3’ polyadenylation (polyA) readthrough activity of the murine leukemia virus long terminal repeat (LTR) is much higher than that of the human immunodeficiency virus type 1 (HIV1) LTR; however, deletion of the U3 or HIV-1 LTR to generate a self-inactivating (SIN) lentiviral vector results in a high transcriptional readthrough activity (J. Virol. 76: 7209-7219, 2002). This strongly S320

suggests that other transcriptional termination signals (TS) besides the polyA signal (AAUAAA) in the R region are present in the U3 region. By serially inserting back different lengths of the U3 sequence, we demonstrated that the readthrough activity of the HIV-1 3’ LTR was inversely correlated with the length of the U3, suggesting that the termination signals span across the entire U3 region. However, the transcriptional control region, containing 124 nt of the U3 sequence, preserves ∼ 70-80% of the wild type TS function. Furthermore, deletion of the Sp1 and NF-kB binding sites abolished most of the TS function. Knocking down NF-kB expression by siRNA down-regulated the TS function. The introduction of foreign TS elements including human T cell leukemia virus type I (HTLV-I) polyA signal, human growth hormone gene intron, a tRNA tertiary structure, or a tetracycline responsive element (TRE) with a TRE-binding factor did not restore the TS function. Therefore, the lentiviral U3 contains an overlapping transcriptional enhancer and termination function which is difficult to be separated. Further improving the transcriptional termination but not the enhancer/promoter function of the 3’ LTR will be critical to safety improvement of this vector system.

824. Oncoretroviral Vector Integration Biases in Mouse Hematopoietic Progenitors Mari Aker, Julie Tubb, George Stamatoyannopoulos, David W. Emery. 1 Dept. of Medicine, University of Washington, Seattle, WA. Although gene transfer vectors based on recombinant retroviruses have been known to have the potential to lead to insertional mutagenesis, that integrations can lead to oncogenic transformation of transduced target cells in a gene therapy setting has led to intensive research on integration site choice. Several groups have reported that oncoretroviral vectors have a slight preference for landing within genes, a strong preference for integration within the region immediately around transcriptional start sites, and a preference for actively transcribed genes. Using primary mouse bone marrow progenitors as target cells for an oncoretroviral vector, we addressed three questions: 1) Are genes preferentially the targets; 2) Are regions surrounding transcriptional starts favored; and 3) Does selection for the vector bias the repertoire of integration sites? We obtained bone marrow from 5-FU treated mice, prestimulated the cells for two days, transduced by co-cultivation on irradiated ecotropic producer lines at a low multiplicity of infection. The retrovirus has a bacterial drug resistance cassette for Zeocin in the long terminal repeat, an MSCV LTR transcribing a GFP reporter, and a separate internal Pgk-Neo cassette (used to select for expression). Progenitor cell colonies were recovered in methylcellulose +/- G418 seven days after transduction. Integration junctions were cloned directly from genomic DNA with the Zeocin cassette present in the LTRs, rather than by a PCR based strategy, enabling us to obtain larger genomic segments. Junction sequences were mapped by BLAST to build 33 of the mouse genome in the ENSEMBL database. We identified 130 integration sites from unselected pools and 130 integration sites from selected pools of progenitor cells. We also mapped an equivalent number of simulated integrations; this dataset was treated as the experimental dataset was, with unmappable sites (such as those that landed entirely in gaps or repetitive elements) removed. We have found that MSCV integrations do not occur more frequently into genes (defined as within known transcripts introns and exons, 42%) than random (41%), differing from what was previously reported for MLV integration into HeLa cells. Comparing the integrations that are within 5 kb upstream of a transcription start site, there is a highly significant difference between our experimental dataset (18%) and the simulated dataset (4%), P = < .0001. With the progenitor colonies having undergone selection, the frequency of landing within introns has increased from 35% to 43%. When the Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy