Defective RNA packaging is responsible for low transduction efficiency of CAEV-based vectors}

Abstract

Replication defective retroviral vectors are regularly used for transfer and expression of exogenous genes into dividing cells and in animals. Since lentiviruses are able to infect terminally differentiated and non-dividing cells, their use to produce replication defective vectors may overcome this limitation. We developed two replication-defective lentiviral vectors based on the genome of Caprine Arthritis Encephalitis Virus (CAEV). The first vector (pBNL2) carries the neo and lacZ marker genes. Neo gene is expressed from a genomic RNA and lacZ gene from a subgenomic RNA. The second vector (pCSHL) carries a single fusion gene encoding both phleomycin resistance and beta-galactosidase activity. Replication-competent CAEV was used as helper virus to provide the viral proteins for transcomplementation of these vectors. Our data demonstrated that the genomes of both vectors were packaged into CAEV virions and transduced into goat synovial membrane cells following infection. However, the vector titers remained 3 to 4 logs lower than those of CAEV. Further analysis showed a lack of accumulation of unspliced pBNL2 RNA into the cytoplasm of producer cells resulting in the packaging of pBNL2 sub-genomic RNA only. In contrast, RNA produced from pCSHL vector was correctly transported to the cytoplasm and more efficiently packaged than the pBNL2 sub-genomic RNA as revealed by slot-blot and quantitative RT/PCR analyses. However this higher packaging efficiency of pCSHL genome did not result in a higher transduction efficiency of lacZ gene.

Comparison of Gene Transfer Efficiencies and Gene Expression Levels Achieved with Equine Infectious Anemia Virus- and Human Immunodeficiency Virus Type 1-Derived Lentivirus Vectors

“…Our data demonstrate that EIAV-based vectors are effective gene transfer vehicles in dividing and nondividing human cells, a finding in direct contrast to those for two other nonprimate lentivirus vectors based on caprine arthritis encephalitis virus and visna virus (3,12). However, other nonprimate lentiviral vectors, such as those based on feline immunodeficiency virus, can also effectively transduce human cells.…”

Lack of Functional Receptors Is the Only Barrier That Prevents Caprine Arthritis-Encephalitis Virus from Infecting Human Cells

“…When the monolayer reached 50% CPE, virus stock was harvested 8 to 16 h after replacement with fresh culture medium. Titer of the virus stock was determined by infection of GSM cells with serial dilutions and scoring CPE development as described previously (23,29).…”

“…RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase and random hexamer primers as reported previously (23). PCR analysis was performed using the Gag primers GEX5 and GEX3 previously described (34).…”

Lentiviral vectors: optimization of packaging, transduction and gene expression

“…These include the immunodeficiency viruses derived from human (HIV-1 [1] and HIV-2 [2]), simian (SIV [3]), feline (FIV [4]) or bovine (BIV [5]) origins, the caprine arthritis encephalopathy virus (CAEV [6]), the equine infectious anemia virus (EIAV [7,8]) and the Jembrana disease virus (JDV [9]) of bovine origin. The minimal packaging activities for these different LV vectors will be described and the newly optimized LV genetic systems will be discussed in term of production yield, transduction efficiency and biosafety parameters.…”