Long-term expression of a transferred gene in Epstein-Barr virus transformed human B cells

Delivering a gene into the Epstein-Barr virus (EBV)-transformed B cells is useful in studying effects of the gene on B-cell functions. However, although people have been able to efficiently transfer genes into and get them expressed in B-lympho blastoid cells for a time probably long enough to kill the cells using vectors harbouring oriP, the expression time of the delivered gene is not long enough in order to study the gene function in B cells. To solve this problem, we constructed an adeno-associated virus (AAV) plasmid pAGX(+) based on plasmids pSub201 and pRc/CMV. We developed and packaged recombinant AAV (rAAV) expression vectors containing an antisense or a sense DNA fragment of 6A8 cDNA encoding a human alpha-mannosidase, or an antisense fragment of 5D4 cDNA encoding a human cell membrane protein, or EYFP DNA. EBV-transformed B cell SKW6 and 3D5 were transduced with those rAAV or the mock. Transduction with the rAAV-EYFP showed an infection frequency of 64 +/- 3.5% and 58 +/- 6.2% for SKW6 and 3D5 cell, respectively. Genomic polymerase chain reaction (PCR) for neoR gene indicated an integration of the transferred gene into the host DNA. After being cultured and propagated for over 12 months, the cells were detected for the expression of the transferred gene. The RT-PCR, enzymatic assay and Con A binding test demonstrated an inhibition of 6A8 alpha-mannosidase in both SKW6 and 3D5 cells transduced with the antisense 6A8 DNA. Immunofluorescence staining with monoclonal antibodies (MoAb) 5D4 showed a reduction of the 5D4 protein expression on both the cells transduced with the antisense 5D4 DNA. The DNA fragmentation assay showed a resistance of the cells with 6A8 alpha-mannosidase inhibition to apoptosis induction by anti-Fas antibody. The data indicate that the AAV vector pAGX(+) can efficiently introduce genes into EBV-transformed B cells and the delivered gene can be expressed in the cells for more than 12 months which may be long enough for the study of gene functions in B cells.