Inhibition and promotion of tumor growth with adeno-associated virus carcinoembryonic antigen vaccine and Toll-like receptor agonists.

Carcinoembryonic antigen (CEA) is a cancer vaccines' target. Several features of recombinant adeno-associated virus (rAAV) are attractive for vaccine applications. Combining other viral vector vaccines with Toll-like receptor (TLR) agonists enhances antitumor immunity. Wild-type and CEA transgenic (Tg) mice were immunized with rAAV-expressing CEA, the TLR9 agonist, oligodinucleotide (ODN)1826 and the TLR7 agonist, imiquimod. Mice were challenged with MC38 colon tumor cells and MC38 cells expressing CEA. rAAV-CEA immunization combined with ODN1826 or imiquimod enhanced CEA-specific T-helper 1 immunity and protected against tumor challenge in wild-type but not in CEA-Tg mice. In contrast, immunization with rAAV-CEA in CEA-Tg mice could abrogate the antitumor effects of ODN1826 and promote tumor growth. Compared to wild-type, CEA-Tg mice were characterized by a greater myeloid suppressor cell and T-helper 2 response to TLR agonists and to syngeneic tumors. Depleting PDCA1(+) plasmacytoid dendritic cells and Gr1(+) myeloid cells increased anti-CEA immune responses in CEA-Tg mice to rAAV-CEA-ODN1826 immunization, whereas depleting CD25(+) T cells did not. There are differences in the response of wild-type and CEA-Tg mice to rAAV-CEA, TLR agonists and syngeneic tumor. In CEA-Tg mice, tumor growth can be promoted with rAAV-CEA and TLR agonists. Dendritic and myeloid cells play a regulatory role.

Therapeutic potential of genetically modified adult stem cells for osteopenia.

Adult stem cells have therapeutic potential because of their intrinsic capacity for self-renewal, especially for bone regeneration. The present study shows the utility of ex vivo modified mesenchymal stem cells (MSC) to enhance bone density in an immunocompetent mouse model of osteopenia. MSC were transduced ex vivo with a recombinant adeno-associated virus 2 (rAAV2) expressing bone morphogenetic protein 2 (BMP2) under the transcriptional control of collagen type-1alpha promoter. To enrich bone homing in vivo, we further modified the cells to transiently express the mouse alpha4 integrin. The modified MSC were systemically administered to ovariectomized, female C57BL/6 mice. Effects of the therapy were determined by dual-energy X-ray absorptiometry, 3D micro-CT, histology and immunohistochemistry for up to 6 months. Results indicated that mice transplanted with MSC expressing BMP2 showed significant increase in bone mineral density and bone mineral content (P < 0.001) with relatively better proliferative capabilities of bone marrow stromal cells and higher osteocompetent pool of cells compared to control animals. Micro-CT analysis of femora and other bone histomorphometric analyses indicated more trabecular bone following MSC-BMP2 therapy. Results obtained by transplanting genetically modified MSC from green fluorescent protein transgenic mouse suggested that production of BMP2 from transplanted MSC also influenced the mobilization of endogenous progenitors for new bone formation.

Cancer gene therapy using mesenchymal stem cells expressing interferon-beta in a mouse prostate cancer lung metastasis model.

Cell-based therapy for cancer is a promising new field. Among cell types that can be used for this purpose, mesenchymal stem cells (MSCs) appear to hold great advantage for reasons including easier propagation in culture, possible genetic modification to express therapeutic proteins and preferential homing to sites of cancer growth upon in vivo transfer. The present study evaluated the potential of genetically modified MSC, constitutively expressing interferon (IFN)-beta, in an immunocompetent mouse model of prostate cancer lung metastasis. A recombinant adeno-associated virus (rAAV) encoding mouse IFN-beta was constructed and initially tested in vitro for high-level expression and bioactivity of the transgenic protein. MSCs were transduced by the rAAV-IFN-beta or green fluorescent protein ex vivo and used as cellular vehicles to target lung metastasis of TRAMP-C2 prostate cancer cells in a therapy model. Cohorts of mice were killed on days 30 and 75 to determine the effect of therapy by measurement of tumor volume, histology, immunohistochemistry, enzyme-linked immunosorbent assay and flow cytometry. Results indicated a significant reduction in tumor volume in lungs following IFN-beta-expressing MSC therapy. Immunohistochemistry of the lung demonstrated increased tumor cell apoptosis and decreased tumor cell proliferation and blood vessel counts. A significant increase in the natural kill cell activity was observed following IFN-beta therapy correlating the antitumor effect. Systemic level of IFN-beta was not significantly elevated from this targeted cell therapy. These data demonstrate the potential of MSC-based IFN-beta therapy for prostate cancer lung metastasis.

Therapeutic potential of genetically modified mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (MSC) are multipotent adult stem cells of mesodermal origin localized within the bone marrow compartment. MSC possess multilineage property making them useful for a number of potential therapeutic applications. MSC can be isolated from the bone marrow, expanded in culture and genetically modified to serve as cell carriers for local or systemic therapy. Despite their ability to differentiate into osteoblasts, chondrocytes, adipocytes, myocytes and neuronal cells under appropriate stimuli, distinct molecular signals that guide migration of MSC to specific targets largely remain unknown. The pluripotent nature of MSC makes them ideal resources for regenerative medicine, graft-versus-host disease and autoimmune diseases. Despite their therapeutic potential in a variety of diseases, certain issues need to be critically addressed both in in vitro expansion of these cells without losing their stem cell properties, and the long-term fate of the transplanted MSC in vivo following ex vivo modifications. Finally, understanding of complex, multistep and multifactorial differentiation pathways from pluripotent stem cells to functional tissues will allow us to manipulate MSC for the formation of competent composite tissues in situ. The present article will provide comprehensive account of the characteristics of MSC, their isolation and culturing, multilineage properties and potential therapeutic applications.

Targeting EGFR with metabolically biotinylated fiber-mosaic adenovirus.

Adenovirus (Ad)-based vectors are useful gene delivery vehicles for a variety of applications. Despite their attractive properties, many in vivo applications require modulation of the viral tropism. Targeting approaches applied to adenoviral vectors included genetic modification of the viral capsid, controlled expression of the transgene and combinatorial approaches that combine two or more targeting elements in single vectors. Most of these studies confirmed successful retargeting in cell cultures, however, in vivo gains of targeted adenoviral vectors have not been widely demonstrated. We have developed a combinatorial retargeting approach utilizing metabolically biotinylated Ad, where the biotin acceptor peptide was incorporated in one of the fibers in a dual fiber viral particle resulting in metabolically biotinylated fiber-mosaic Ad (mBfMAd). We have utilized this vector in complex with epidermal growth factor (EGF)-Streptavidin to retarget fiber-mosaic virus to EGF receptor (EGFR) expressing cells in vitro and confirmed an increased infectivity of the retargeting complex. Most importantly, the utility of this strategy was demonstrated in vivo in two distinct animal models. In both models tested, retargeted mBfMAd demonstrated an increased ratio of gene expression in target tissues compared to the liver expression profile. Thus, metabolically biotinylated fiber-mosaic virus in combination with appropriate adapters can be successfully exploited for adenoviral retargeting strategies.

Intraperitoneal gene therapy by rAAV provides long-term survival against epithelial ovarian cancer independently of survivin pathway.

Epithelial ovarian carcinoma is the leading cause of death from gynecological malignancies. Owing to the lack of an effective screening method, insidious onset, and non-specific symptoms, a majority of women present with advanced stage disease. Despite improvements from cytoreductive surgery and chemotherapy, recurrent disease remains a formidable challenge. In the present study, we demonstrate for the first time that stable intra-abdominal genetic transfer of endostatin and angiostatin (E+A) by recombinant adeno-associated virus (rAAV) provides sustained antitumor effects on the growth and dissemination of epithelial ovarian cancer in a mouse model. Further, when combined with paclitaxel (taxol), the effect of this therapy was dramatically increased and resulted in long-term tumor-free survival overcoming prior limitations of chemotherapy and gene therapy. The combined effects of angiosuppressive therapy and chemotherapy were found to be independently of survivin pathway. Evidence for the superior effects of the combination therapy was indicated by significantly lower ascites volume with less hemorrhage and tumor conglomerates, lower ascites vascular endothelial growth factor, higher tumor cell apoptosis and decreased blood vasculature, and long-term disease-free survival. Histopathology of visceral organs and liver enzyme assays indicated no toxicity or pathology.

Enhanced transduction of mouse bone marrow-derived dendritic cells by repetitive infection with self-complementary adeno-associated virus 6 combined with immunostimulatory ligands.

The potential of adeno-associated virus (AAV)-based vectors in human gene therapy is being explored for several diseases. Although sustained transgene expression and low vector-associated cellular immunity are attractive features of recombinant (r) AAV, the wider application of rAAV vectors encapsidated in serotype 2 capsid is hampered by poor transduction efficiency in many target tissues. These include ex vivo-generated dendritic cells (DC), which have demonstrated promising immunotherapeutic activity. We report here that efficient transduction of mouse bone marrow-derived DC can be achieved with self-complementary (sc) rAAV encapsidated in serotype 6 capsid. Sequential exposure of DC precursor cultures to IL-4 and GM-CSF with sc rAAV6 encoding the human tumor antigen, carcinoembryonic antigen (CEA), for 7 days followed by activation with CpG oligodeoxynucleotides (ODN) and anti-mouse CD40 antibody resulted in highly efficient transduction of DC. DC surface markers as determined by flow cytometry analysis of sc rAAV6-transduced DC were comparable to nontransduced DC. Efficiency of vector transduction and transgene expression were confirmed by immunostaining and real-time PCR. Microarray analysis of RNA from CpG ODN and CD40 antibody stimulated sc AAV6-transduced DC revealed upregulation of transcription factors and cytokines involved in immune activation and downregulation of inhibitory factors, suggesting a possible role of transcriptional activation in the observed effect. The adoptive transfer into syngeneic mice of the ex vivo-transduced and activated DC resulted in the development of CEA-specific antibody and T-helper 1-associated immune responses. Immunized mice also developed antibody to AAV6 capsid protein, which did not crossreact with AAV2 capsid protein. These studies demonstrate the potential utility of sc rAAV serotype 6-based vectors in transduction of DC for genetic vaccination approaches.

Activators of viral gene expression in polarized epithelial monolayers identified by rapid-throughput drug screening.

Epithelial polarity and tight junction formation limit the ability of adenovirus, retrovirus and adeno-associated virus (AAV) to deliver and express virally encoded genes. Using an extended half-life luciferase assay and high-throughput luminometry, we screened 23 000 compounds and natural product extracts as potentiators to overcome this barrier. Seven strong activators were discovered (up to several hundred fold above control) and two of these exhibited spectrum of activity in multiple cell types (HeLa (human cervical carcinoma), cystic fibrosis bronchial epithelial (human bronchial), HT29 (human colonic carcinoma), Calu3 (airway serous glandular)). Enhanced transduction by unrelated gene transfer vectors (adenovirus, lentivirus, AAV, liposomal) was also observed. These results establish a strategy for identifying compounds that improve viral gene transfer to resistant cell types, and provide new tools for examining epithelial defense against viral infection. The compounds should have broad usefulness in experimental therapies for cancer and genetic diseases.

Adeno-associated virus type 2-mediated transduction of human monocyte-derived dendritic cells: implications for ex vivo immunotherapy.

Dendritic cells (DCs) are pivotal antigen-presenting cells for regulating immune responses. A major focus of contemporary vaccine research is the genetic modification of DCs to express antigens or immunomodulatory molecules, utilizing a variety of viral and nonviral vectors, to induce antigen-specific immune responses that ameliorate disease states as diverse as malignancy, infection, autoimmunity, and allergy. The present study has evaluated adeno-associated virus (AAV) type 2 as a vector for ex vivo gene transfer to human peripheral blood monocyte (MO)-derived DCs. AAV is a nonpathogenic parvovirus that infects a wide variety of human cell lineages in vivo and in vitro, for long-term transgene expression without requirements for cell proliferation. The presented data demonstrate that recombinant AAV (rAAV) can efficiently transduce MOs as well as DCs generated by MO culture with granulocyte-macrophage colony-stimulating factor plus interleukin in vitro. rAAV transgene expression in MO-derived DCs could be enhanced by etoposide, previously reported to enhance AAV gene expression. rAAV transduction of freshly purified MO followed by 7 days of culture with cytokines to generate DCs, and subsequent sorting for coexpression of DC markers CD1a and CD40, showed robust transgene expression as well as evidence of nuclear localization of the rAAV genome in the DC population. Phenotypic analyses using multiple markers and functional assays of one-way allogeneic mixed leukocyte reactions indicated that rAAV-transduced MO-derived DCs were as equivalent to nontransduced DCs. These results support the utility of rAAV vectors for future human DC vaccine studies.

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 type 2-mediated gene transfer: role of epidermal growth factor receptor protein tyrosine kinase in transgene expression.

Adeno-associated virus type 2 (AAV), a single-stranded, DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have recently documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays an important role in AAV-mediated transgene expression (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997) and that a strong correlation exists between the phosphorylation state of the ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing et al., J. Virol. 72:1593-1599, 1998). In this report, we document that treatment of cells with specific inhibitors of the epidermal growth factor receptor protein tyrosine kinase (EGF-R PTK) activity, such as tyrphostin, leads to significant augmentation of AAV transduction efficiency, and phosphorylation of the ssD-BP is mediated by the EGF-R PTK. Treatment of cells with EGF results in phosphorylation of the ssD-BP, whereas treatment with tyrphostin causes dephosphorylation of the ssD-BP and consequently leads to increased expression of the transgene. Furthermore, AAV transduction efficiency inversely correlates with expression of the EGF-R in different cell types, and stable transfection of the EGF-R cDNA causes phosphorylation of the ssD-BP, leading to significant inhibition in AAV-mediated transgene expression which can be overcome by the tyrphostin treatment. These data suggest that the PTK activity of the EGF-R is a crucial determinant in the life cycle of AAV and that further studies on the interaction between the EGF-R and the ssD-BP may yield new insights not only into its role in the host cell but also in the successful use of AAV vectors in human gene therapy.

Characterization of wild-type adeno-associated virus type 2-like particles generated during recombinant viral vector production and strategies for their elimination.

The pSub201-pAAV/Ad plasmid cotransfection system was developed to eliminate homologous recombination which leads to generation of the wild-type (wt) adeno-associated virus type 2 (AAV) during recombinant vector production. The extent of contamination with wt AAV has been documented to range between 0.01 and 10%. However, the precise mechanism of generation of the contaminating wt AAV remains unclear. To characterize the wt AAV genomes, recombinant viral stocks were used to infect human 293 cells in the presence of adenovirus. Southern blot analyses of viral replicative DNA intermediates revealed that the contaminating AAV genomes were not authentic wt but rather wt AAV-like sequences derived from recombination between (i) AAV inverted terminal repeats (ITRs) in the recombinant plasmid and (ii) AAV sequences in the helper plasmid. Replicative AAV DNA fragments, isolated following amplification through four successive rounds of amplification in adenovirus-infected 293 cells, were molecularly cloned and subjected to nucleotide sequencing to identify the recombinant junctions. Following sequence analyses of 31 different ends of AAV-like genomes derived from two different recombinant vector stocks, we observed that all recombination events involved 10 nucleotides in the AAV D sequence distal to viral hairpin structures. We have recently documented that the first 10 nucleotides in the D sequence proximal to the AAV hairpin structures are essential for successful replication and encapsidation of the viral genome (X.-S. Wang et al., J. Virol. 71:3077-3082, 1997), and it was noteworthy that in each recombinant junction sequenced, the same 10 nucleotides were retained. We also observed that adenovirus ITRs in the helper plasmid were involved in illegitimate recombination with AAV ITRs, deletions of which significantly reduced the extent of wt AAV-like particles. Furthermore, the combined use of recombinant AAV plasmids lacking the distal 10 nucleotides in the D sequence and helper plasmids lacking the adenovirus ITRs led to complete elimination of replication-competent wt AAV-like particles in recombinant vector stocks. These strategies should be useful in producing clinical-grade AAV vectors suitable for human gene therapy.

Recombinant human parvovirus B19 vectors: erythroid cell-specific delivery and expression of transduced genes.

A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562-566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111-1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and acquired human diseases affecting cells of erythroid lineage.

Adeno-associated virus type 2-mediated gene transfer: correlation of tyrosine phosphorylation of the cellular single-stranded D sequence-binding protein with transgene expression in human cells in vitro and murine tissues in vivo.

Although the adeno-associated virus type 2 (AAV)-based vector system has gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy, the single-stranded nature of the viral genome, and consequently the rate-limiting second-strand viral DNA synthesis, significantly affect its transduction efficiency. We have identified a cellular tyrosine phosphoprotein, designated the single-stranded D sequence-binding protein (ssD-BP), which interacts specifically with the D sequence at the 3' end of the AAV genome and may prevent viral second-strand DNA synthesis in HeLa cells (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997). In the present studies, we examined whether the phosphorylation state of the ssD-BP correlates with the ability of AAV to transduce various established and primary cells in vitro and murine tissues in vivo. The efficiencies of transduction of established human cells by a recombinant AAV vector containing the beta-galactosidase reporter gene were 293 > KB > HeLa, which did not correlate with the levels of AAV infectivity. However, the amounts of dephosphorylated ssD-BP which interacted with the minus-strand D probe were also as follows: 293 > KB > HeLa. Predominantly the phosphorylated form of the ssD-BP was detected in cells of the K562 line, a human erythroleukemia cell line, and in CD34+ primary human hematopoietic progenitor cells; consequently, the efficiencies of AAV-mediated transgene expression were significantly lower in these cells. Murine Sca-1+ lin- primary hematopoietic stem/progenitor cells contained predominantly the dephosphorylated form of the ssD-BP, and these cells could be efficiently transduced by AAV vectors. Dephosphorylation of the ssD-BP also correlated with expression of the adenovirus E4orf6 protein, known to induce AAV gene expression. A deletion mutation in the E4orf6 gene resulted in a failure to catalyze dephosphorylation of the ssD-BP. Extracts prepared from mouse brain, heart, liver, lung, and skeletal-muscle tissues, all of which are known to be highly permissive for AAV-mediated transgene expression, contained predominantly the dephosphorylated form of the ssD-BP. Thus, the efficiency of transduction by AAV vectors correlates well with the extent of the dephosphorylation state of the ssD-BP in vitro as well as in vivo. These data suggest that further studies on the cellular gene that encodes the ssD-BP may promote the successful use of AAV vectors in human gene therapy.

Adeno-associated virus type 2-mediated transduction in primary human bone marrow-derived CD34+ hematopoietic progenitor cells: donor variation and correlation of transgene expression with cellular differentiation.

Although the adeno-associated virus type 2 (AAV) is known to possess a broad host range that transcends the species barrier, we suggested in an earlier study that AAV infection of human cells is receptor mediated (S. Ponnazhagan et al., J. Gen. Virol. 77:1111-1122, 1996). In the present studies, we investigated the ability of AAV to infect primary human hematopoietic progenitor cells capable of multilineage differentiation. Bone marrow-derived CD34+ cells from 12 hematologically normal volunteer donors were infected with a recombinant AAV containing the beta-galactosidase gene under the control of the cytomegalovirus immediate-early promoter (vCMVp-lacZ). Whereas 15 to 80% of the cells from approximately 50% of the donors showed various levels of lacZ gene expression, the expression was undetectable in cells from the remaining donors. However, if cells from both sets of donors were stimulated with various combinations of cytokines to induce differentiation into myeloid and lymphoid lineages following AAV infection, then the level of expression of the transduced gene increased up to 20-fold over a period of 14 days. The results of virus-binding assays suggested that the observed difference between the two groups was due to the differential susceptibility of CD34+ cells to AAV infection rather than to differences in transcription and translation of the transduced gene. To corroborate these results, CD34+ cells from the two donor groups, KB (human nasopharyngeal carcinoma) cells, and M07e (human megakaryocytic leukemia) cells were infected with vCMVp-lacZ. KB cells served as a positive control for AAV infection, and M07e cells served as a negative control. Whereas abundant hybridization to the single-stranded viral DNA on Southern blots was detected in KB and CD34+ cells that were positive for lacZ gene expression, little activity was detected in M07e and CD34+ cells that did not show expression of the lacZ gene. These results suggest that the levels of expression of the putative cellular receptor for AAV vary widely in CD34+ cells from different donors. These studies have implications for the potential use of AAV vectors in human gene therapy involving primary human primitive hematopoietic stem and progenitor cells.

Encapsidation of adeno-associated virus type 2 Rep proteins in wild-type and recombinant progeny virions: Rep-mediated growth inhibition of primary human cells.

The adeno-associated virus type 2 (AAV) arrests the growth of primary human fibroblasts in vitro at high particle-to-cell ratios. To test the role of AAV gene expression in the observed growth inhibition, primary human cells were infected, under identical conditions, with wild-type (wt) AAV or with recombinant AAV that lacked all viral promoters and coding sequences. Significant, dose-dependent growth inhibition of primary human cells was observed with both wt and recombinant AAV at particle-to-cell ratios equal to or exceeding 10(4). In contrast, neither virus affected the growth of immortalized human cells even at a 10-fold-higher particle-to-cell ratio. AAV-induced growth arrest could be overcome by reculturing cells after treatment with trypsin. Even after reculturing, cells still harbored the proviral AAV genome. Thus, neither integration nor expression of the AAV genome appears to be required for the virus-induced growth-inhibitory effect on primary human cells. The growth-inhibitory effect of AAV was hypothesized to be mediated by virion-associated AAV Rep proteins, since these proteins have been reported to inhibit cellular DNA synthesis. Rep proteins tightly associated with wt as well as recombinant AAV could be detected on Western blots. Coinfection by adenovirus was necessary and sufficient for ample replication of recombinant AAV genomes lacking the rep gene. Although wt AAV-like particles arose during production of the recombinant AAV stocks, their low-titer levels were insufficient to cause the observed growth inhibition. AAV rep gene expression from these contaminating particles was not required for replication of the recombinant AAV genomes, which could be detected even in the absence of de novo Rep protein synthesis. Exposure of recombinant AAV to anti-AAV Rep protein antibodies did not abrogate viral infectivity. These results suggest that biologically active Rep proteins are encapsidated in mature progeny AAV particles. AAV Rep protein-mediated growth inhibition of primary human cells has implications in the use of AAV-based vectors in human gene therapy.

Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression.

The adeno-associated virus 2 (AAV), a single-stranded DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the single-stranded nature of the viral genome significantly impacts upon the transduction efficiency, because the second-strand viral DNA synthesis is the rate-limiting step. We hypothesized that a host-cell protein interacts with the single-stranded D sequence within the inverted terminal repeat structure of the AAV genome and prevents the viral second-strand DNA synthesis. Indeed, a cellular protein has been identified that interacts specifically and preferentially with the D sequence at the 3' end of the AAV genome. This protein, designated the single-stranded D-sequence-binding protein (ssD-BP), is phosphorylated at tyrosine residues and blocks AAV-mediated transgene expression in infected cells by inhibiting the leading strand viral DNA synthesis. Inhibition of cellular protein tyrosine kinases by genistein results in dephosphorylation of the ssD-BP, leading not only to significant augmentation of transgene expression from recombinant AAV but also to autonomous replication of the wild-type AAV genome. Dephosphorylation of the ssD-BP also correlates with adenovirus infection, or expression of the adenovirus E4orf6 protein, which is known to induce AAV DNA replication and gene expression. Thus, phosphorylation state of the ssD-BP appears to play a crucial role in the life cycle of AAV and may prove to be an important determinant in the successful use of AAV-based vectors in human gene therapy.

Evaluation of recombinant adeno-associated virus as a gene transfer vector for the retina.

PURPOSE: To evaluate recombinant adeno-associated virus (AAV) as an in vivo gene transfer vector for the retina. METHODS: A recombinant AAV, vCMVp-lacZ, in which the bacterial beta-galactosidase reporter gene (lacZ) was placed under the control of a cytomegalovirus (CMV) early promoter, was injected into the vitreous body or the subretinal space of mouse eyes. The reporter gene expression was followed by histochemical analyses from 10 to 100 days post-injection. The effect of several variables on the extent of AAV-mediated gene transfer was examined, including routes of delivery, presence of an underlying mutation that caused retinal degeneration, and prior treatment with hydroxyurea. RESULTS: As measured by reporter gene expression, the AAV vector mediated gene transfer to three major cell types in the retina: the retinal pigment epithelium (RPE), ganglion cells and photoreceptor cells. Following a single injection, more than half of the total retinal areas were typically positive for gene transfer. Reporter gene expression was stable for at least 3 months, the farthest time point examined. Gene transfer to photoreceptor cells was observed only following subretinal delivery, and was greatly enhanced in mice undergoing early retinal degeneration. Cells in the inner nuclear layer were rarely transduced. Systemic administration of a genotoxic drug, hydroxyurea, 2 days prior to AAV delivery did not affect the patterns and extent of reporter gene expression. There was minimal histopathology associated with AAV transduction in the retinas of recipient mice, as determined by light microscopy. CONCLUSION: Recombinant AAV mediates efficient gene transfer to RPE and ganglion cells, and to photoreceptor cells under certain conditions. Persistence of transgene expression is of long duration and without apparent histopathology. The greater stability, lower cytopathicity, and the ability to transduce retinal ganglion cells are three distinct features of the AAV vector compared to current adenovirus-based vectors.

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.