Selvarangan Ponnazhagan

Adeno-associated virus 2-mediated gene transfer in vivo: organ-tropism and expression of transduced sequences in mice☆

Adeno-associated virus 2 (AAV), a non-pathogenic human parvovirus, is gaining attention as a vector for its potential use in human gene therapy. However, few studies have examined the safety and the efficacy of this vector system in vivo. We report here that recombinant AAV vectors, when directly injected intravenously in mice, accumulated predominantly in liver cells, suggesting that AAV may possess in vivo organ-tropism for liver. The transduced lacZ reporter gene was expressed in hepatocytes in the liver and, at the level examined, did not appear to induce any detectable cytotoxic T lymphocyte response against beta Gal. AAV-mediated transduction of murine hematopoietic progenitor cells ex vivo followed by transplantation into lethally irradiated syngeneic mice also revealed high-efficiency gene transfer into progeny cells without any observable cytotoxicity or deleterious effect. The transduced reporter gene sequences were also expressed in mice in vivo. The AAV-based vectors may thus prove useful as a potentially safe alternative to the more commonly used retrovirus- and adenovirus-based vector systems.

Mouse silver. mutation is caused by a single base insertion in the putative cytoplasmic domain of Pmel 17

This laboratory has established in previous studies that Pmel 17, a gene expressed specifically in melanocytes, maps near the silver coat color locus (si/si) on mouse chromosome 10. In the current study, we have focused on determining whether or not the si allele carries a mutation in Pmel 17. Pmel 17 cDNA clones, isolated from wild-type and si/si murine melanocyte cDNA libraries, were sequenced and compared. A single nucleotide (A) insertion was found in the putative cytoplasmic tail of the si/si Pmel 17 cDNA clone. This insertion is predicted to alter the last 24 amino acids at the C-terminus. Also predicted is the extension of the Pmel 17 protein by 12 residues because a new termination signal created downstream from the wild-type reading frame. The mutation was confirmed by the sequence of the PCR-amplified genomic region flanking and including the mutation site. The fact that si/si Pmel 17 was not recognized by antibodies directed toward the C-terminal 15 amino acids of wild-type Pmel 17, indicated a defect in this region. We conclude from these results that silver pmel 17 protein has a major defect at the carboxyl terminus. The chromosomal location and the identification of a potentially pathologic mutation in si-Pmel 17 support our conclusion that Pmel 17 is encoded at the silver locus.

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.

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.