The development of targeted vectors, capable of tissue-specific transduction, remains one of the important aspects of vector modification for gene therapy applications. Recombinant adeno-associated virus type 2 (rAAV-2)-based vectors are nonpathogenic, have relatively low immunogenicity, and are capable of long-term transgene expression. AAV-2 vectors bind primarily to heparan sulfate proteoglycan (HSPG), a receptor that is present in many tissues and cell types. Because of the widespread expression of HSPG on many tissues, targeted transduction in vivo appears to be limited with AAV-2 vectors. Thus, development of strategies to achieve transductional targeting will have a profound benefit in the future application of these vectors. We report here a novel conjugate-based targeting method to enhance tissue-specific transduction of AAV-2-based vectors. The present report utilized a high-affinity biotin-avidin interaction as a molecular bridge to cross-link purified targeting ligands, produced genetically as fusion proteins to core-streptavidin, in a prokaryotic expression system. Conjugation of the bispecific targeting protein to the vector was achieved by biotinylating purified rAAV-2 without abolishing the capsid structure, internalization, and subsequent transgene expression. The tropism-modified vectors, targeted via epidermal growth factor receptor (EGFR) or fibroblast growth factor 1␣ receptor (FGFR1␣), resulted in a significant increase in transduction efficiency of EGFR-positive SKOV3.ip1 cells and FGFR1␣-positive M07e cells, respectively. Further optimization of this method of targeting should enhance the potential of AAV-2 vectors in ex vivo and in vivo gene therapy and may form the basis for developing targeting methods for other AAV serotype capsids.Adeno-associated virus (AAV)-based vectors are becoming increasingly popular for gene therapy of human diseases (3,16,22,33,34). Despite the potential for long-term expression, genomic integration, and low immunogenicity, the transduction efficiency of AAV type 2 (AAV-2) vectors varies significantly among cell types. For example, the cells of muscle and brain are highly permissive for AAV-2 infection. However, certain human megakaryocytic cell lines and primary cells of hematopoietic origin show inconsistencies in infection (8,15,19,20,23), which may be due to either insufficient receptor numbers, an absence of optimal amounts of intracellular factors necessary for AAV second-strand synthesis, or a combination of both (2, 6, 7, 10, 30). Thus, improvements to overcome these limitations will have a positive impact on the application of AAV vectors in gene therapy. Whereas recent studies on intracellular events conducive for AAV transgene expression have identified the roles of several host cell factors (10,14,30), in order to overcome the limitations of receptormediated AAV infection, both genetic and nongenetic approaches are currently being developed (19,24,26,37,38). Although genetic modification of AAV capsid may be a preferred means of achieving vector r...

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.Dendritic cells (DCs) are potent antigen-presenting cells (APC) for initiating T-cell immunity, due to their ability to take up and process antigens for presentation by major histocompatibility complex (MHC) class I and class II molecules, migrate to T-cell areas of lymphoid tissues, and present antigen in conjunction with the appropriate T-cell costimulatory molecules and cytokines (reviewed in references 3, 4, 44, and 51). DCs can be manipulated ex vivo to express antigens in order to generate effective vaccines for a variety of immunotherapy applications. In the simpler approaches, DCs are pulsed by incubation with purified proteins, microbial or tumor cell lysates, synthetic MHC-binding peptides, or crude peptides eluted from tumor cells, all of which have shown promising results, although the persistence of antigens on pulsed DCs is of relatively short duration (4, 44, 51). Alternatively, the transfer of genes encoding antigens into DCs offers the advantages of sustained antigen expression and a broader spectrum of MHC peptide epitopes presented by DCs and allows modulation of DC receptors and cytokine secretion to further finetune the immune response (3,4,44,51). Both nonviral and viralvector-mediated gene transfer have been used for DC-based immunotherapy in animal models and human clinical trials, with the majority of viral-mediated DC transductions employing r...

Angiogenesis is characteristic of solid tumor growth and a surrogate marker for metastasis in many human cancers. Inhibition of tumor angiogenesis using antiangiogenic drugs and gene transfer approaches has suggested the potential of this form of therapy in controlling tumor growth. However, for long-term tumor-free survival by antiangiogenic therapy, the factors controlling tumor neovasculature need to be systemically maintained at stable therapeutic levels. Here we show sustained expression of the antiangiogenic factors angiostatin and endostatin as secretory proteins by recombinant adeno-associated virus 2 (rAAV)-mediated gene transfer. Both vectors provided significant protective efficacy in a mouse tumor xenograft model. Stable transgene persistence and systemic levels of both angiostatin and endostatin were confirmed by in situ hybridization of the vector-injected tissues and by serum ELISA measurements, respectively. Whereas treatment with rAAV containing either endostatin or angiostatin alone resulted in moderate to significant protection, the combination of endostatin and angiostatin gene transfer from a single vector resulted in a complete protection. These data suggest that AAV-mediated long-term expression of both endostatin and angiostatin may have clinical utility against recurrence of cancers after primary therapies and may represent rational adjuvant therapies in combination with radiation or chemotherapy.

Gene therapy for osteopenic conditions including osteoporosis is a potential alternative to pharmacotherapy for cost effectiveness, long-term viability, and the ability to enhance bone mass by anabolic approaches. Increased understanding of mesenchymal stem cell (MSC) lineage differentiation during osteogenesis, and of the molecular pathways involved in bone cell production, provides an opportunity for the advancement of gene therapy approaches for osteopenic conditions. The potential of MSCs in osteoblast differentiation and the relative ease of MSC isolation and culturing offer a promising resource for the development of ex vivo gene therapy for bone defects. In an effort to develop ex vivo gene therapy for osteoporosis, we used gene-modified MSCs in a preclinical mouse model to determine the efficiency of transduction of murine MSCs by recombinant adeno-associated virus 2 (AAV) vectors carrying reporter genes and determined their osteogenic potential after recombinant AAV-mediated expression of bone morphogenic protein 2, known to induce osteoblast differentiation. Although surgical ovariectomy is believed to induce progressive bone loss in mouse models, similar to an osteoporosis-like phenotype in humans, several factors, including hormonal alteration and dietary habits, significantly affect both the onset and progression of the disease. Thus, in the present study, we determined the influence of these factors and developed an immunocompetent mouse model of osteoporosis with degenerative bone loss as in the human pathology.

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Antiangiogenic gene transfer has the potential to be more efficacious than protein-based therapies or pharmacotherapies for the control of solid tumor growth, invasion and metastasis. For a sustained antiangiogenic effect, a vector capable of long-term expression without vector-associated immunity or toxicity is advantageous. The present study evaluated the potential of a recombinant adeno-associated virus-2 (rAAV) encoding the human soluble FMS-like tyrosine kinase receptor 1 (sFlt-1), which functions by both sequestering vascular endothelial growth factor (VEGF) and forming inactive heterodimers with other membranespanning VEGF receptors, in vitro and in vivo. Results indicated significant growth inhibitory activity of the transgenic factor in a human umbilical vein endothelial cell proliferation assay in vitro and protection against the growth of an angiogenesis-dependent human ovarian cancer cell line, SKOV3.ip1, xenograft in vivo with increased disease-free survival. Stable expression of the secretory factor and transgene persistence were confirmed by immunohistochemistry and in situ hybridization analyses, respectively. Increased therapeutic effects on both the growth index of the implanted tumor cells and tumor-free survival also correlated with an increasing dose of the vector used. These studies indicate that rAAV-mediated sFlt-1 gene therapy may be a feasible approach for inhibiting tumor angiogenesis, particularly as an adjuvant/therapy.

Recombinant adeno-associated virus 2 (rAAV) vectors have been successfully used for sustained expression of therapeutic genes. The potential of using rAAV as a cancer vaccine vector and the impact of a bacterial plasmid adjuvant on this activity were investigated. C57BL/6 mice received a single intramuscular injection of rAAV expressing the human tumor-associated antigen, carcinoembryonic antigen (CEA). Three weeks later, when CEA expression was optimal, a bacterial plasmid containing methylated DNA motifs was injected into the same muscle. Mice were challenged 1 week later with syngeneic MC38 tumor cells stably expressing CEA. Immunization with rAAV-CEA alone resulted in sustained transgene expression and the elicitation of a humoral immune response to CEA. Cellular immune response, however, was weak, and tumor protection was not significant. In contrast, immunization with rAAV-CEA and the plasmid adjuvant resulted in stronger cellular immune response to CEA and tumor protection. The addition of plasmid adjuvant increased both myeloid dendritic cell recruitment in situ and CEA-specific T-helper-1-associated immune response. These data indicate that robust rAAV transgene expression of a tumor antigen followed by transient plasmid delivery to recruit and activate dendritic cells is an effective method of eliciting antitumor cellular immune responses.

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