Xu-Shan Wang

Adeno-Associated Virus 2-Mediated Transduction and Erythroid Lineage-Restricted Expression from Parvovirus B19p6 Promoter in Primary Human Hematopoietic Progenitor Cells

Human parvovirus B19 gene expression from the viral p6 promoter (B19p6) is restricted to primary human hematopoietic cells undergoing erythroid differentiation. We have demonstrated that expression from this promoter does not occur in established human erythroid cell lines in the context of a recombinant parvovirus genome (Ponnazhagan et al. J Virol 69:8096-8101, 1995). However, abundant expression from this promoter can be readily detected in primary human bone marrow cells (Wang et al. Proc Natl Acad Sci USA 92:12416-12420, 1995; Ponnazhagan et al. J Gen Virol 77:1111-1122, 1996). In the present studies, we investigated the pattern of expression from the B19p6 promoter in primary human bone marrow-derived CD34+ HPC undergoing differentiation into myeloid and erythroid lineages. CD34+ cells were transduced with recombinant adeno-associated virus 2 (AAV) vectors containing the beta-galactosidase (lacZ) gene under the control of the following promoters/enhancers: the cytomegalovirus promoter (vCMVp-lacZ), B19p6 promoter (vB19p6-lacZ), B19p6 promoter with an upstream erythroid cell-specific enhancer element (HS-2) from the locus control region (LCR) from the human beta-globin gene cluster (vHS2-B19p6-lacZ), and the human beta-globin gene promoter with the HS-2 enhancer (vHS2-beta p-lacZ). Transgene expression was evaluated either 48 h after infection or following erythroid differentiation in vitro for 3 weeks. Whereas high-level expression from the CMV promoter 48 h after infection diminished with time, low-level expression from the B19p6 and the beta-globin promoters increased significantly following erythroid differentiation. Furthermore, in HPC assays, there was no significant difference in the level of expression from the CMV promoter in myeloid or erythroid cell-derived colonies. Expression from the B19p6 and the beta-globin promoters, on the other hand, was restricted to erythroid cell colonies. These data further corroborate that the B19p6 promoter is erythroid cell-specific and suggest that the recombinant AAV-B19 hybrid vectors may prove useful in gene therapy of human hemoglobinopathies in general and sickle cell anemia and beta-thalassemia in particular.

Cord blood transplantation and the potential for gene therapy. Gene transduction using a recombinant adeno-associated viral vector.

Cord blood, which contains a high frequency of immature stem/progenitor cells with extensive proliferative and replating capacity in vitro was used as a clinical source of transplantable stem and progenitor cells. These cells can be efficiently transduced with new genetic material by using AAV or retroviral vectors. Using a recombinant AAV vector, high level expression of the lacZ gene under a CMV promoter was demonstrated in immature subsets of cord blood progenitor cells.

Adeno-associated Virus 2-Mediated Transduction and Erythroid Lineage-Specific Expression in Human Hematopoietic Progenitor Cells

Parvoviruses are among the smallest of the DNA-containing viruses that infect a wide variety of vertebrates (Siegl et al. 1985). Two parvoviruses of human origin, the nonpathogenic adeno-associated virus 2 (AAV) and the parvovirus B19, a common human pathogen, have been studied extensively (Berns and Bohenzky 1987; Brown et al. 1994). AAV requires coinfection with a helper virus, such as adenovirus or herpesvirus, for its optimal replication (Berns 1990), but in the absence of a helper virus, the AAV genome establishes a latent infection in a site-specific manner (Kotin and Berns 1989; Kotin et al. 1990, 1991, 1992; Samulski et al. 1991). B19, by contrast, is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells (Ozawa et al. 1986, 1987; Yaegashi et al. 1989; Srivastava and Lu 1988; Takahashi et al. 1990). We have described the construction of a recombinant AAV-B19 hybrid genome, in which we combined the remarkable features of these two parvoviruses, and speculated that such a hybrid vector may prove useful for high efficiency transduction of primary human hematopoietic progenitor cells (Srivastava et al. 1989). Indeed, it has become increasingly clear that the AAV-based vector system may prove to be a useful alternative to the more commonly used retroviral and adenoviral vectors for its potential use in human gene therapy (Muzyczka 1992; Carter 1993; Srivastava 1994). Despite these advances, a number of fundamental questions related to AAV remain unanswered. For example, the molecular details of viral assembly and the mechanism of viral entry into the host cell have not been rigorously analyzed. Furthermore, the feasibility of obtaining tissue-specific expression of an AAV-transduced gene has not been adequately addressed. Here, we provide experimental evidence to suggest that the vector assembly requires a precise signaling mechanism and that AAV infection of human cells is receptor-mediated. We also document erythroid lineage restricted expression following AAV-B19 hybrid vector-mediated transduction of primary human hematopoietic progenitor cells. Elucidation of the molecular details of these aspects of AAV biology will have important implications in the potential use of AAV as a vector in human gene therapy.