Transduction and Utility of the Granulocyte-Macrophage Colony-Stimulating Factor Gene into Monocytes and Dendritic Cells by Adeno-Associated Virus

Abstract

The genetic manipulation of antigen-presenting dendritic cells (DC) offers promise for stimulating the immune response, in particular for anticancer and antiviral protocols. As adeno-associated virus (AAV) has shown promise as a gene delivery vector for transducing a variety of hematopoietic cell types, we have investigated AAV's ability to genetically alter DC. In this analysis, we modified the standard granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) treatment of adherent monocytes to generate DC. In our protocol, adherent monocytes were first infected with an AAV/GM-CSF/Neo vector, and the addition of IL-4 was delayed for 2 days to allow for a brief period of monocyte proliferation. AAV-mediated transduction of the GM-CSF and Neo genes into monocytes/DC precursors was demonstrated by G418 selection, GM-CSF secretion, GM-CSF RNA expression (reverse transcriptase-polymerase chain reaction amplification [RT-PCR]), and cell proliferation. Cells resulting from infection with AAV/GM-CSF/Neo virus, and subsequent IL-4 and tumor necrosis factor-alpha (TNF-alpha) treatment, displayed multiple classic markers consistent with mature DC. Finally, chromosomal integration of the AAV vector was also demonstrated in sorted CD83+ DC. These data strongly suggest that AAV vectors will be useful for the genetic manipulation of DC and suggest that the transduction of the GM-CSF gene was able to fully replace the need for exogenous GM-CSF in the production of mature DC.

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

“…Partly because of this, there is a growing interest in the development of efficient methods for gene transfer into primary human monocyte-derived macrophages (MDM) and for manipulation of gene expression in these cells. Several groups have attempted to transduce MDM recently and different viral vectors have been tested for their effectiveness at transducing monocytes and macrophages [8,[26][27][28][29]. However, only limited success has been obtained to date.…”

“…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].…”

Overexpression of TGFβ1 by adeno-associated virus type-2 vector protects myocardium from ischemia–reperfusion injury

“…The latent or mutant rat TGFb 1 cDNA was inserted into AAV type 2 vector, dl6-95, in a manner described for other AAV vectors. 31 Hereafter, the recombinant AAV vector was referred to as AAV/TGFb 1 Latent or AAV/ TGFb 1 ACT , respectively. The AAV/Neo was generated as described earlier.…”

“…The AAV/Neo was generated as described earlier. 31 The virus stocks were generated and titered by dot blot hybridization as described previously. 31 The titers were calculated to be about 1 Â 10 11 encapsidated genomes per ml (10 10 infectious unit).…”

Adeno-associated virus vectors: potential applications for cancer gene therapy

“…Even though AAV vectors do not appear to infect DCs following intramuscular injection, several recent studies have indicated that immature DCs were transduced by AAV. [144][145][146][147] DCs infected with an AAV vector containing the HPV-16 E6 or E7 gene induced a strong CTL response against primary cervical cancer cell lines after 7 days of priming. Tumor cell killing was significantly blocked by the addition of anti-MHC class I antibodies, indicating CTL function via the MHC class I-restricted killing.…”