9. Mobilization of Sendai Virus Vectors (SeVV) by Phylogenetic Related Human Pathogens: Implications for Future Vector Generations

Abstract

In an attempt to make HIV-1 based vectors safer for use in the research and clinical setting, a significant modification to the HIV-1 genome has been the deletion of the TATA box and enhancer elements from the U3 region of the long terminal repeat (LTR). Vectors containing this 400bp U3 deletion are thought to have no LTR-directed transcription, and are thus called self-inactivating (SIN) lentivectors. As with wild-type LTR vectors, any transcripts initiated at the U3-R junction in the 5' LTR of SIN lentivectors contain the packaging signal found in the psi region and 5' portion of gag. Transcripts containing the packaging signal have the potential to be encapsidated and mobilized by wild-type HIV-1. Using four distinct approaches, we show that SIN lentivectors continue to have promoter activity near the 5' LTR which is responsible for the production of full-length vector genome transcripts. To verify that transcripts derived from the LTR in SIN lentivectors are competent for encapsidation and integration, we transduced a lentiviral packaging cell line with a standard SIN lentivector and then observed production of viable vector particles containing full-length SIN lentivector genomes. We also demonstrate that a Herpes Simplex Virus thymidine kinase gene placed downstream of the SIN LTR, but upstream of the vector's internal promoter, is produced in quantities high enough to render cells containing this vector partially succeptible to gancyclovir-induced cell death. Additionally, we show that SIN lentivectors containing no internal promoter express an eGFP transgene. Using RT-PCR we demonstrate that transcripts initiated at the 5' LTR in SIN lentivectors can be readily detected, and using this assay, we conclude that the SIN LTR retains approximately 15% of the transcriptional activity of the wild-type LTR (in the absence of Tat). We have also identified sequences in the SIN lentivector which are responsible for transcriptional activation at the 5' LTR. With a promoter assay using different segments of the LTR and leader region of the SIN lentivector, we have determined that the residual promoter activity does not depend on any LTR sequence, including the HIV-1 start-site initiator (Inr) element. Rather, the promoter activity is contained entirely within the leader region, and although this element is downstream of the transcription initiation site, it is capable of initiating transcription from the 5' end of R in the LTR. Mutation of leader region binding sites for the transcriptional activators DBF1 and SP1 reduces transcription from the SIN LTR by up to 80%; however, a significant decrease in vector titer results from this mutation, perhaps owing to disruption of the folding of psi, which overlaps the SP1 binding site. In conclusion, knowledge of the potential for mobilization of SIN lentivectors by HIV-1 will be important for the design of future gene therapy trials using such vectors. It is also possible that low level expression from the SIN LTR may, in some instances, be adequate for the production of small amounts of transgene in a non-cell-type specific manner, which may be relevant to studies using SIN lentivectors to introduce cell-specific expression cassettes.