One of the long-term interests of our laboratory has been to develop parvovirus-based vectors for the potential treatment of hemoglobinopathies in general and sickle cell anemia and β-thalassemia in particular (34
). We have previously reported stable transduction and expression of human globin gene sequences mediated by recombinant AAV vectors both in vitro and in vivo (22
). However, AAV vectors may not always be desirable, considering the need to specifically target a selected type of cells in a heterogeneous population, given the broad host range of AAV (14
). We sought to exploit the salient feature of parvovirus B19: it possesses a remarkable tropism for erythroid cells in human bone marrow because infection by B19 virus is mediated by the erythrocyte P antigen (4
). These studies were prompted by our previously published reports that the B19 viral genome encapsidated within the AAV capsid structure is both infectious and replication competent in cells of erythroid lineage in human bone marrow (35
). In addition, we have reported that the B19p6 promoter, the only authentic promoter in the B19 viral genome, is capable of conferring autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells (37
). Previous studies utilizing a baculovirus system have indicated that empty capsids of B19 virus could be assembled with VP2 protein alone, since VP2 comprises the majority of the capsid protein (38
). In the present studies, although we were able to obtain recombinant B19 encapsidated virions with sequences of VP2 alone using the helper plasmid pSP-42, the addition of VP1 sequences in the helper plasmid pSP-46 increased the packaging efficiency approximately 10-fold. Still, the packaging efficiency of the B19 virus helper plasmid pSP-46 was significantly lower than that of the AAV helper plasmid pAAV/Ad, which is most probably due to the efficiency of different promoters regulating capsid gene expression. Western blot analyses of capsid gene expression corroborated this possibility (data not shown). Replacing the CMV promoter by the authentic AAVp40 promoter or the B19p6 promoter driving expression of the B19 virus capsid gene did not lead to successful production of recombinant B19 virus vectors.
The results of several different experiments with both established and primary human cells clearly document that the recombinant B19 virus vectors can indeed specifically target cells of erythroid lineage in human bone marrow cells. It is perhaps not surprising, therefore, that despite low viral titers, the recombinant B19 virus vector is able to infect cells in the erythroid lineage at a significantly higher efficiency than the recombinant AAV vector. It is intriguing to note, however, that the B19 virus vector is able to transduce CD34+ cells that are positive for glycophorin A on the day of analysis. It is likely that erythroid-differentiated cells which express both glycophorin A and the CD34 marker on their cell surfaces are infected by B19 more readily than are more-undifferentiated CD34+ cells. This is corroborated by the fact that the nonerythroid population of cells that was initially positive for the CD34 marker on the day of sorting did not show any expression of the transduced lacZ gene. However, if multipotential CD34+ cells were transduced by the B19 vector, there should have been transgene expression following differentiation into erythroid, myeloid, and lymphoid lineages; this was not detected. These results indicate that the recombinant B19 virus vector can selectively mediate transduction even in less-differentiated erythroid cells.
The observation that erythroid-differentiated, but not undifferentiated, MB-02 cells showed expression of the transduced lacZ
gene suggests that expression of the P antigen receptor correlates with erythroid differentiation of these cells. In this context, it is noteworthy that previous studies from our laboratory have documented that wild-type B19 virus DNA replication and gene expression occur only in erythroid-differentiated MB-02 cells (13
). It may now be of interest to investigate whether nonerythroid cells, such as myocardial cells and endothelial cells, that express the P antigen receptor but are nonpermissive for B19 replication can be successfully transduced by the recombinant B19 virus vector. This may help resolve the issue of whether a putative intracellular factor that is present only in cells of erythroid lineage is crucial for successful replication of B19 virus and whether an additional coreceptor(s) is required for successful infection by B19 virus.
The recombinant B19 virus vector system described here offers several potential advantages over the currently used AAV vector system. First, since it remains to be unequivocally established whether stable integration of a recombinant AAV genome in primary cells leads to insertional mutagenesis, it may be desirable to transduce committed erythroid progenitor cells rather than pluripotent hematopoietic stem cells. Second, it may also be desirable to obtain tissue-specific delivery of the therapeutic gene so as not to affect the normal functions of other cell lineages. Third, since approximately 90% of the human population is seropositive for AAV capsid proteins (2
), the potential use of AAV vectors in in vivo gene therapy protocols may be limited. The use of B19 capsids composed of VP1 plus VP2 proteins to encapsidate a potentially therapeutic gene can at least partially overcome the problem of neutralizing antibodies against AAV, since only approximately 60% of the human population is seropositive for the B19 capsid proteins (4
). It is also tempting to speculate that the use of B19 capsids composed entirely of VP2 proteins may be especially advantageous, since all antigenic epitopes to date have been mapped within the VP1 region (38
). Further development of this novel vector system may prove useful in its application for gene therapy of human diseases involving cells of erythroid lineage in general and sickle cell anemia and β-thalassemia in particular.