Therapeutic area

Hybrid adenovirus-alphavirus vector Semliki

Gene therapy represents a promising approach for the treatment of different diseases, including cancer. The aim of this new type of therapy is to transfer therapeutic genes to tumor cells in order to promote their destruction either through local toxicity or by estimulating antitumoral immune responses. In the latter case the transfer of interleukin-12 (IL-12) gene has shown great efficacy to eliminate different types of tumors in many animal models. Gene transfer requires the use of appropriate vectors able to carry the genetic material into target cells. The most commonly used vectors are based on genetically modified viruses and include adenoviruses, retroviruses, and alphaviruses, like Semliki Forest virus (SFV). Each type of vector has advantages and disadvantages for cancer gene therapy. Adenoviral vectors, which contain DNA as genome, have a large cloning capacity, and a natural tropism for the liver, including hepatocellular carcinoma (HCC). However, adenoviral vectors expressing IL-12 have only shown limited efficacy in patients with advanced liver, pancreas or colon cancer. Alphavirus vectors like SFV are based on a self-replicating RNA which leads to very high expression levels and induction of apoptosis in infected cells. This latter property is thought to potentiate the antitumoral effect of IL-12, since apoptotic infected cells can release tumor antigens that can be uptaken by antigen presenting cells, estimulating in this way the antitumoral immune response. SFV vectors have shown a high antitumoral efficacy in several animal models of liver and colon cancer. Vector-mediated expression of IL-12 can be very beneficial in the tumor environment, but when this cytokine is expressed outside the tumor it leads to toxicity mediated by the induction of g-interferon. For this reason an ideal vector would be one that could express IL-12 or other transgenes especifically in tumor cells.

For that purpose a new viral vector able to express IL-12 specifically in HCC has been developed. This vector combines the main advantages of adenovirus and alphavirus vectors, like high infectivity for liver cells, high levels of transgene expression and induction of apoptosis in infected cells. The new vector is based on a third generation adenovirus vector devoid of all adenoviral genes, which carries the sequence of an SFV replicon under the transcriptional control of the alpha-fetoprotein (AFP) promoter, a promoter specific for HCC cells. In this hybrid vector the transgene is inserted within the alphavirus replicon, requiring the transcription and replication of the SFV RNA in order to be expressed. The adenovirus-SFV vector can be packaged into viral particles by using a helper adenovirus, which endows these particles with an adenovirus-like tropism. However, transcription and replication of the SFV replicon included in this vector will only take place in cells where thet AFP promoter is active, limiting the expression of  the transgene and induction of apoptosis to HCC cells, and reducing in this way IL-12 toxicity. Hybrid adenovirus-SFV vectors expressing either IL-12 or reporter gene LacZ have been constructed and evaluated, showing a very high specificity of expression and apoptosis induction in HCC cells, both in vitro and in vivo. Furthermore, hybrid vector adenovirus-SFV-IL-12 has shown a very high antitumoral efficacy in an orthotopic model of rat HCC. This efficiency, which was similar to the one provided by conventional SFV-IL-12 particles, did not lead to any liver toxicity, probably due to the higher specificity of the hybrid vector for HCC cells.

This new type of hybrid vector could also be used for the treatment of other tumor types, by means of substituting the AFP promoter with another tumor specific promoter, representing a very powerful and safe tool for cancer therapy.

The figure shows the structure of the adenovirus-SFV hybrid vector (A), including the inverted terminal repeats (ITR), stuffer DNA (HPRT and C346), AFP promoter and an SFV vector containing IL-12 gene downstream of the viral subgenomic promoter (sg Pr). In part B two possible scenarios for infection with the hybrid vector are depicted. In the left part: non-HCC cells that can be infected by the vector but in which no transcription of the SFV replicon takes place. In the right part: HCC cells that can be infected by the vector leading to SFV RNA transcription and replication resulting in IL-12 production and apoptosis