DEFECTIVE RNA PACKAGING IS RESPONSIBLE FOR LOW TRANSDUCTION EFFICIENCY OF CAEV-BASED VECTORS

Replication defective retroviral vectors are regularly used for transf er and expression of exogenous genes into dividing cells and in animal s. Since lentiviruses are able to infect terminally differentiated and non-dividing cells, their use to produce replication defective vector s may overcome this limitation. We developed two replication-defective lentiviral vectors based on the genome of Caprine Arthritis Encephali tis Virus (CAEV). The first vector (pBNL2) carries the neo and lacZ ma rker genes. Neo gene is expressed from a genomic RNA and lacZ gene fro m a subgenomic RNA. The second vector (pCSHL) carries a single fusion gene encoding both phleomycin resistance and beta-galactosidase activi ty. Replication-competent CAEV was used as helper virus to provide the viral proteins for transcomplementation of these vectors. Our data de monstrated that the genomes of both vectors were packaged into CAEV vi rions and transduced into goat synovial membrane cells following infec tion. However, the vector titers remained 3 to 4 logs lower than those of CAEV Further analysis showed a lack of accumulation of unspliced p BNL2 RNA into the cytoplasm of producer cells resulting in the packagi ng of pBNL2 sub-genomic RNA only. In contrast, RNA produced from pCSHL vector was correctly transported to the cytoplasm and more efficientl y packaged than the pBNL2 sub-genomic RNA as revealed by slot-blot and quantitative RT/PCR analyses. However this higher packaging efficienc y of pCSHL genome did not result in a higher transduction efficiency o f lacZ gene.