Optimization of Feline Immunodeficiency Virus Vectors for RNA Interference

Abstract

RNA interference (RNAi) occurs naturally in plant and animal cells as a means for modulating gene expression. This process has been experimentally manipulated to achieve targeted gene silencing in cells, tissues, and animals, using a variety of vector systems. Here, we tested the hypothesis that vectors based on feline immunodeficiency virus (FIV) could be used for coexpression of reporter constructs and RNAi expression cassettes. We found, unexpectedly, in our

Human gene therapy vectors derived from feline lentiviruses

“…These results have been confirmed with this system and with independently-derived similar systems in other laboratories (Johnston et al, 1999;Curran et al, 2000;Song et al, 2003). FIV vectors have now been applied with favorable results in the brain, eye, ear, airway, liver, muscle, pancreas and hematopoietic system Johnston et al, 1999;Wang et al, 1999;Alisky et al, 2000;Curran et al, 2000;Loewen et al, 2001;Stein et al, 2001;Brooks et al, 2002;Curran et al, 2002;Curran and Nolan, 2002a, b;Derksen et al, 2002;Djalilian et al, 2002;Hughes et al, 2002;Kang et al, 2002;Loewen et al, 2002;Lotery et al, 2002;Price et al, 2002;Stein and Davidson, 2002;Loewen et al, 2003a;Loewen et al, 2003b;Haskell et al, 2003;Sinn et al, 2003;Loewen et al, 2004;Kang et al, 2005;Harper et al, 2006;Saenz et al, 2006;Khare et al, 2007). In addition to animal models, success has been evident in explanted human organs (Wang et al, 1999;Loewen et al, 2001;Loewen et al, 2002).…”

Connecdenn, A Novel DENN Domain-Containing Protein of Neuronal Clathrin-Coated Vesicles Functioning in Synaptic Vesicle Endocytosis

“…siRNA#2 (GGCCCAGGCTGCTTTCTTT) and siRNA#3 (GAGCTGCTTCTGTATCTTA) were effective in knocking down transfected mouse connecdenn, with siRNA#3 the most effective in knocking down endogenous connecdenn in COS-7 cells (data not shown). This sequence was thus selected for generation of short hairpin RNA (shRNA) packaged in a lentivirus delivery system, which was prepared as described previously (Harper et al, 2006). Briefly, PCR was used to amplify a mouse U6 promoter followed by a stem-loop-stem structure encoding the sequence of siRNA#3.…”

“…The reverse primer, matching the 3Ј end of the U6 promoter followed by the siRNA antisense strand, a loop, the siRNA sense strand, and an RNA Pol III termination sequence, had the sequence GCGCAATTGAAAAAAAGAGCTGCTTCTGTATCTTAT-GACAGGAAGTAAGATACAGAAGCAGCTCTTCAAAACAAGGCTT-TTCTCCAAGG. The resulting PCR fragment was cloned into a shuttle vector that was used to generate feline immunodeficiency virus (FIV) expressing the connecdenn shRNA and GFP as described previously (Harper et al, 2006). Control virus expressing GFP alone was described previously (Harper et al, 2006).…”

Molecular Characterization of Feline Immunodeficiency Virus Budding

“…Thus, FIV in domestic cats represents an important smallanimal model for HIV vaccine research, for testing potential antiretroviral agents, and for understanding the cell biology of lentiviral replication (10,29,32). FIV has also been developed as an ideal lentiviral vector system for the delivery of genes and small interfering RNAs (siRNAs), since it has the capacity to transduce nondividing cells for long-term gene expression but is not infectious or pathogenic in humans (24,28,36,52,58,60). Despite the importance of the FIV system, little is known, relative to the primate lentivirus systems, about the molecular mechanisms of FIV Gag protein trafficking, virus assembly, release, and replication (29).…”