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.
Aldrich
W A
WA
Department of Medicine, The University of Alabama at Birmingham, Birmingham, AL, USA.
Ren
C
C
White
A F
AF
Zhou
S-Z
SZ
Kumar
S
S
Jenkins
C B
CB
Shaw
D R
DR
Strong
T V
TV
Triozzi
P L
PL
Ponnazhagan
S
S
D019264:Adoptive Transfer / Q000379:methods*; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D000918:Antibody Specificity; D017951:Antigen Presentation; D001854:Bone Marrow Cells / Q000276:immunology; D019013:CD40 Antigens / Q000235:genetics; D002272:Carcinoembryonic Antigen / Q000235:genetics; D002460:Cell Line; D018899:CpG Islands; D003713:Dendritic Cells / Q000276:immunology*; D000229:Dependovirus / Q000235:genetics* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry; D015870:Gene Expression; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007371:Interferon-gamma / Q000276:immunology; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes
D000914:Antibodies, Viral; D019013:CD40 Antigens; D002272:Carcinoembryonic Antigen; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma