371. Optimization of Feline Immunodeficiency Viral Vectors for RNA Interference

Scott Harper; Patrick Staber; Christine Rowley; Sarah Fineberg; Dalyz Ochoa; Colleen Stein; Beverly Davidson

doi:10.1016/j.ymthe.2006.08.430

RNA interference (RNAi) refers to post-transcriptional reduction of cellular gene expression mediated by sequence specific, 21 nucleotide double-stranded RNA molecules. RNAi occurs naturally in mammals (microRNAs), and appears to be particularly involved in controlling development of the nervous system. In addition, RNAi can be induced through exogenous expression of short hairpin RNAs (shRNA) using viral vectors. Feline immunodeficiency virus (FIV)- based lentiviral vectors are useful tools for neuronal gene delivery because they can stably infect difficult-to-transduce cells, are easy to produce, and, unlike the more broadly utilized HIV-based vectors, are non-human pathogens that pose a lower risk of generating replication-competent viruses in humans. Despite these advantages, compared to HIV vectors, less is known about the biology and construction of FIV and FIV-based vectors. Here, we describe thedevelopment of a second-generation FIV-based vector system that expresses U6 promoter-transcribed shRNAs (shLacZ) and a GFP reporter gene from the same vector. We found that the position of the U6 expression cassette with respect to the GFP reporter and viral rev-responsive element (RRE) was critical for reporter gene and shRNA expression. In particular, placement of the U6 promoter alone or an U6.shLacZ cassette near the 3' end of the vector genome between the GFP reporter cassette and the viral RRE caused 31 and 131-fold reductions, respectively, in transducing unit titer compared to an FIV vector expressing GFP alone. Reduced titer was also observed when a similarly-organized RNA pol III promoter (H1) was cloned into the same site. In contrast, TU titer was slightly increased (2.8-fold) when a similarly-sized, non-coding fragment of firefly luciferase was cloned into the same site downstream of GFP. Movement of U6.shLacZ to a site upstream of GFP resulted in complete TU titer restoration. Taken together, these data demonstrate that GFP expression is inhibited when a pol III-based promoter is located downstream of the reporter, suggesting that this inhibition may depend on recruitment of pol III transcription machinery.In a second set of studies we refined the FIV system to simultaneously express natural microRNAs and reporter genes. MicroRNAs, which arise from longer (several kbp; called pri- microRNA) RNA polymerase II (pol II) transcribed sequences may be better tolerated by cells. Also, others' and our data show that processing to release the active guide strand may be more efficient than shRNA-based vectors. To achieve this we generated FIV vectors that produce a translated reporter gene (GFP or Neo) and a microRNA from the same pol II transcript. These vectors produced mature, biologically active microRNAs and allowed for detectable reporter gene expression; inclusion of the microRNA sequence in the reporter gene 3' UTR caused only a modest decrease in GFP positive cells (2.5 fold) and mean GFP fluorescence/cell (2-fold).In summary, these studies helped to better define FIV vector development in general and resulted in development of optimized FIV vectors for RNAi delivery.

371. Optimization of Feline Immunodeficiency Viral Vectors for RNA Interference

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