Adeno-Associated Virus Type 2-Mediated Transduction of Human Monocyte-Derived Dendritic Cells: Implications for Ex Vivo Immunotherapy

Abstract

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...

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

“…[4][5][6] In addition, potential applications of AAV as a vector for immunotherapy of infectious diseases and cancer are being developed. 2,7,8,10,12 Although most of the preclinical studies and clinical trials so far have utilized serotype 2 AAV vectors, recent studies suggest that alternate serotype vectors with different host cell receptor specificity could have better utility for gene transfer to rAAV2 refractory target cells. [16][17][18][19] One of the cell types that shows poor transduction efficiency for rAAV2 is DC, mouse DC in particular.…”

“…Our previous studies of human peripheral blood monocyte-derived DC indicated varying efficiency of AAV2 transduction. 12 Other preclinical studies of the application of rAAV to DC-based immunotherapies have also been limited by poor transduction efficiency of DC by serotype 2 vectors. [13][14][15] Alternate serotype vectors with different host cell receptor specificity have increased the repertoire of AAV available for gene therapy applications.…”

Immunity to adenovirus and adeno-associated viral vectors: implications for gene therapy

“…59 Whether immature DCs take up rAAV vectors via a receptor-mediated uptake remains to be determined considering the high phagocytic activity of this cell population. In summary, the current working hypothesis is that rAAV is incapable of recruiting immature APC in high enough numbers to the site of vector administration resulting in lack of T-cell activation and long-term gene transfer as described in many studies utilizing this vector platform.…”

HIV-1-based defective lentiviral vectors efficiently transduce human monocytes-derived macrophages and suppress replication of wild-type HIV-1

“…However, only limited success has been obtained to date. One of the major obstacles in delivering therapeutic genes into human MDM is the poor transduction efficiency under most currently available gene-transfer conditions [8,26,[28][29][30][31].…”