Vector development for Gene Therapy

The main focus of our laboratory is the study and development of viral vectors and new molecular strategies with potential interest in gene therapy applications. The vector development laboratory is integrated by two groups:

• RNA and SV40 GROUP (PI: Puri Fortes)
• Semliki Forest virus (SFV) group (PI: Cristian Smerdou)

RNA and SV40 group

The main core of the lab studies gene expression inhibition based on RNA molecules. We work with RNA interference (RNAi) and inhibition with 5´-end modified U1 snRNPs (RNAu). RNAu is a novel technology developed in the lab based in the fact that 5´-end modified U1 snRNA (U1i) interaction with the terminal exon of target mRNAs blocks polyadenylation and, therefore, gene expression. Using this technique we have been able to block expression of cellular genes upon transient or stable transfection of plasmids expressing U1is (Fortes et al., 2003; Abad et al; submitted, in preparation). We still need to address the specificity of this technique. We are also combining RNAu and RNAi to see whether both techniques synergize to yield stronger inhibitions. RNAi has proven to be an efficient way to inhibit gene expression. However, there are several concerns about toxicity of RNAi in treated cells. We have analyzed if cellular miRNAs are altered in mouse liver infected with adenovirus expressing an exogenous RNAi targeted to the MRP2 transporter mRNA (MRP2RNAi). The results showed that endogenous miRNA levels are not altered by the expression of functional exogenous siRNAs (Narvaiza et al., 2006). During the course of these experiments we have detected a novel small RNA in adenovirus infected cells. This small RNA is generated by the processing of adenovirus VA RNA and has been named svaRNA. Surprisingly, svaRNA is able to inhibit the expression of target genes and is important for adenovirus viability (Aparicio et al., 2006). It would be interesting to identify svaRNA target genes.

Our model gene therapy vector is SV40, poorly used in gene therapy in spite of its advantages. In fact, intratumoral injection of dendritic cells infected with rSVIL15, which express interleukin 15 from a recombinant SV40 vector, showed up to 73% of tumor reversions (Vera et al, 2005). Also, a recombinant SV40 vector expressing IGF-I has showed therapeutic effects in the prevention of liver cirrhosis in rats (Vera et al., 2007). We want to test whether the same vector or a similar vector could function in the treatment of liver cirrhosis and address the molecular mechanisms that drive the therapeutic effects of IGF-I in liver cirrhosis.