Differential expression in human cells from the p6 promoter of human parvovirus B19 following plasmid transfection and recombinant adeno-associated virus 2 (AAV) infection: human megakaryocytic leukaemia cells are non-permissive for AAV infection

Abstract

Expression from the human parvovirus B 19p6 promoter fused to the firefly luciferase (' Luc') reporter gene was evaluated in a non-erythroid human nasopharyngeal carcinoma cell line, KB, and a human megakaryocytic leukaemia cell line, MB-02, known to become permissive for B19 replication following erythroid-differentiation. The B19p6-Luc construct was introduced into KB and MB-02 cells, both in undifferentiated and differentiated states, either via DNA-mediated transfection, or via infection with recombinant adeno-associated virus 2 (AAV), a non-pathogenic human parvovirus known to possess a broad host-range. Although Luc activity was readily detected in KB cells following transfection of the B 19p6-Luc plasmid DNA, no expression from the B 19p6 promoter was observed following infection with recombinant virus. In addition, transfection of the reporter plasmid resulted in high-level expression of Luc in differentiated but not in undifferentiated MB-02 cells. However, no Luc activity was detected, even in differentiated MB-02 cells, following infection with recombinant virus. Further studies with an additional recombinant as well as wild-type (wt) AAV revealed that MB-02 cells were non-permissive for AAV infection. A second human megakaryocytic leukaemia cell line, M07e, was likewise resistant to infection by recombinant as well as wt AAV. Taken together, these studies identify the first human cell type that cannot be infected by AAV.They indicate that expression from the B 19p6 promoter, in the context of an AAV genome, is restricted to primary human haematopoietic cells, perhaps because parvoviral DNA replication and transcription are intrinsically coupled.

High-Efficiency Transduction of Fibroblasts and Mesenchymal Stem Cells by Tyrosine-Mutant AAV2 Vectors for Their Potential Use in Cellular Therapy

“…However, a number of nonpermissive cell types have also been identified (Ponnazhagan et al, 1996;Bartlett et al, 1999;Girod et al, 1999). Even in cells that allow infection, the level of infection or transgene expression is variable due to well-documented impediments in virus-host cell interactions during the life cycle of AAV2 (Qing et al, 1999(Qing et al, , 2001Hansen et al, 2000Hansen et al, , 2001aZhong et al, 2006).…”

Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors

“…However, the site specificity is lost in rAAV vectors and they have been shown to either persist as episomes or to integrate randomly into the host genome. [2][3][4][5] Therefore, the potential for rAAV to have adverse sideeffects due to insertional mutagenesis may exist.…”

Library selection and directed evolution approaches to engineering targeted viral vectors

“…Despite these advantageous properties, AAV suffers from several shortcomings, including the inability to target delivery to specific cell types (Muzyczka and Warrington, 2005), inefficient gene delivery to a number of ''nonpermissive'' cell types (Hughes et al, 2002;Ponnazhagan et al, 1996;Smith-Arica et al, 2003;Stacchini et al, 1999), a limited packaging insert size (Dong et al, 1996), and the prevalence of pre-existing immunity to human AAV serotypes in the human population (Moskalenko et al, 2000;Sun et al, 2003).…”

“…Perabo et al (2003) also produced AAV2 libraries carrying random seven amino acid insertions at position 587 (of the AAV2 capsid), and mutants were selected for the ability to infect cell lines ordinarily non-permissive to AAV infection. While the selected variants mediated enhanced transduction of target cells non-permissive to wild-type AAV2 (the human megakaryocytic cell line M-07e and B-cell chronic lymphocytic leukemia B-CLL cells) (Ponnazhagan et al, 1996;Stacchini et al, 1999), the ability of the selected mutants to transduce cells expressing heparan sulfate (human colon carcinoma CO-115 cells) was reduced approximately 12-50%, implying enhanced specificity toward the target cell types.…”