Study On Methods And Mechanism To Augment Gene Expression Mediated By Adenovirus And Adeno-Associated Virus

Objective: To investiage the efficiency and cell tropism after in vivo and in vitro transduction of retina cells by various viral vectors rAAV1/2, rAAV2, Ad5, Ad5/F35 and Lentivirus. Methods: Human Retina Pigment Epithelium (hRPE) cell line, primary human RPE cells and primary retinal cells of SD rat were transduced by rAAV2-EGFP, rAAV2/1-EGFP, Ad5-GFP, Ad5/F35-GFP or Lentivirus-GFP respectively. The onset of GFP fluorescence and fluorescence intensity were observed by an inverted fluorescence microscopy. hRPE cells were harvested at 7 and 14 day after transduction for flow cytometry analysis. The onset, distribution and the duration of GFP fluorescence in rat retina were monitored in vivo and photographed by fluorescence stereoscopy after subretinal injection of each viral vector. Morever, cell tropism of each viral vector in rat retina was detected by confocal microscopy. The distribution of cell surface receptors necessary for viral entry including CAR,αⅤβⅤ-integrin, HSPG, CD4 and CD46 were measured by use of immunohistochemistry staining. Results Efficient transduction was observed in cultured hRPE cell lines, primary cultured adult RPE cells and primary cultured retinal cells mediated by rAAV2-EGFP, Ad5-EGFP, Ad5/F35-EGFP and Lentivirous-EGFP but not rAAV2/1-EGFP. The percentage of GFP positive cells in hRPE cells at 7 and 14d after transduction by rAAV2-EGFP, rAAV2/1-EGFP, Ad5-EGFP, Ad5/F35-EGFP or Lentivirous-EGFP were the following: 13.5±1.7% and 15.6±0.82%, 1.09±0.5% and 1.98±0.45%, 99.1±1.7% and 74.6±3.7%, 84.3±2.6% and 70.2±5.6%, 85.7±1.7% and 90.4±1.7%. Mean intensity of GFP fluorescence were 2.75±1.7% and 3.80±0.72 , 1.12±0.09 and 1.75±0.2 ,173.4±11.7and 143.6±15.2, 154.8±16.3 and 123.2±13.6, 89.5±10.1 and 85.2±12.4. All vrial vectors achieved efficient transduction in rat retina in vivo. Earlier onset and shorter duration of GFP expression in retina mediated by Ad5-GFP or Ad5/F35-GFP were observed comparable with the transduction mediated by rAAV2-EGFP, rAAV2/1-EGFP or Lentivirous-GFP. These vectors displayed different cell tropism in rat retina among which LV-GFP selectively transduced RPE cells, Ad5/F35-GFP tansduced RPE cells, photoreceptors and their inner segment. Ad5-GFP tansduced RPE cells, some of photoreceptors, ganglion cells and cells in inner nuclear lays. rAAV2-EGFP transduced almost all kinds of cells of retina comparable with AAV2/1-GFP which transduced especially in RPE cells and photoreceptors. Another important observation was that there was no severe immune response and inflammation in retina after subretinal injection of rAAV1/2, rAAV2, Ad5, Ad5/F35 and LV. Cell tropism of rAAV2 was in accordance with the distribution of HSPG, whereas the distribution of CD4-positive cells or CD46-positive cells did not coincide with the cell tropism of Lentivirus or Ad5/F35. Conclusions rAAV1/2, rAAV2, Ad5, Ad5/F35 and Lentivirous display different efficiency and cell tropism after delivery into the retinal cells in vivo and in vitro. These results provided an insight into selection of an appropriate vector when a specific retinal disease was considered for gene therapy. Objective To search and verify an adjuvant which can enhance the transduction efficiency and gene expression mediated by Adeno-Associated Virus type-2 in retinal cells. Lower usage of adenovirus and anticancer chemotherapeutant were evaluated in vitro and in vivo.Methods The established hRPE cell line,primary human RPE,IPE cells and rat retinal cells were infected by rAAV2-GFP alone or with lower dosage of adenovirus and anticancer chemotherapeutant. GFP fluorescence were observed by inverted fluorescent microscopy and quantified by Fluorescence Activated Cell Sorting (FACS). GFP protein was detected by Western blotting. DNA copies and mRNA expression of GFP as well as mRNA expression of HSPG,FGFR-1 andαvintegrin in the infected tumor cells were detected by real-time PCR and RT-PCR. The rAAV2-GFP/rAAV2-Luc alone or with lower dosage of adenovirus and chemotherapeutant was injected into subretinal space of SD rats and Balb/c mice. GFP expression in the living retina was monitored using fluorescent stereoscope. Bioluminescene imaging was performed to evaluate dynamics of transgene expression in living retina. The rAAV2-CNTF alone or with adenovirus and chemotherapeutant was injected into subretinal space of RCS rats at the age of 3 weeks. Histologic examination was carried out 5 weeks later to observe the protection of photoreceptor lose. The eyes were also examined for apoptosis using TUNEL in situ assay in both normal and RCS rats.Results Combined lower dosage adenovirus enhances GFP protein and mRNA expression 1-5 or 3-5 folds respectively more than alone. And chemotherapeutant augment GFP protein and mRNA expression mediated by rAAV2-GFP 10-20 or 9-27 folds. However, the DNA copies of GFP had no significant change in the cells infected by rAAV2-GFP either with or without chemotherapeutants or adenovirus. mRNA expression of cellular surface receptors increased slightly. Earlier onset of gene expression, GFP and Luceferase, stronger and prolonged fluorescence was observed in the living retina received injection of AAV2 combined adenovirus or chemotherapeutant in comparing with that received injection of AAV2 alone. The retinal cells had no significant apoptosis with or without usage of chemotherapeutant and adenovirus. And the RCS rat, received injection of AAV2-CNTF combined adenovirus or chemotherapeutant, possessed three-four or four to five rows of photoreceptors respectively whereas the control, received injection of AAV2-CNTF alone, had only one to two rows of photoreceptors (p<0.01).Conclusions Lower dosage of could significantly enhance the transduction efficiency and transgene expression of rAAV2 in human, rat as well as mouse retinal cells. And the dosage of adenovirus or chemotherapeutant, used to enhance rAAV2 mediated gene expression in this study, showed no remarkable cytotoxity to eye both in vivo and in vitro. Objective To investigate the potential to use chemotherapeutants as an adjuvant to enhance transgene expression mediated by adenovirus or adeno-associated virus in tumour cells. Methods The tumour cells of NCIH446, NCI-H460,A549, SGC7901, SMMC7721, SK-BR-3, BTT, KB and KB/VCR were infected by rAAV2-CMV-GFP or Ad5-CMV-GFP alone or combined with different chemotherapeutant respectively. GFP fluorescence were observed by inverted fluorescent microscopy and quantified by Fluorescence Activated Cell Sorting (FACS).GFP protein were dectected by Western blotting. DNA copies and mRNA expression of GFP in the rAAV2-CMV-GFP or Ad5-CMV-GFP infected cells with or without chemotherapeutant were detected by real-time PCR. And also mRNA expression of cellular surface receptors HSPG,FGFR-1 andανintegrin in the tumor cells infected by rAAV2-CMV-GFP alone or combined with chemotherapeutant were detected by RT-PCR. The amount of GMCSF in the tumour cells of NCI-H460,A549,SMMC-7721,SGC7901,SKOV-3 and BTT infected by Ad-CMV-GMCSF alone or combined with chemotherapeutant was detected by ELISA. RFP Fluorescence of BTT tumor model of T739 mice was observed by in vivo imaging system after infected by Ad5-CMV-RFP alone or combined with chemotherapeutant.Results Advanced onset of GFP expression and improved transgene expression was observed in all the tumour cell lines when rAAV2-CMV-GFP or Ad5-CMV-GFP infection was associated with chemotherapeutants compared with rAAV2-CMV-GFP or Ad5-CMV-GFP infection alone. Chemotherapeutants could make an enhancement of protein and mRNA expression of exogene mediated by rAAV2-CMV-GFP approximately 1-5 fold and 15-25 fold respectively. And the enhancement of gene expression mediated by Ad5-CMV-GFP was 10-20 fold in protein and 8-10 fold in mRNA. However, the DNA copies of GFP had no significant change in the tumor cells infected by either vector with or without chemotherapeutants. mRNA Of HSPG,FGFR-1 andανintegrin in the tumor cells infected by rAAV2-CMV-GFP with slightly increased compared with rAAV2-CMV-GFP infection alone. The amount of GMCSF increased approximately 1-4 fold in the Ad-CMV-GMCSF with chemotherapeutant infected cells more than Ad-CMV-GMCSF infected alone. Earlier onset of RFP gene expression, stronger and prolonged fluorescene was observed in the living BTT tumor model of T739 mice received tumor-in-situ injection of Ad5-CMV-RFP combined chemotherapeutant compared with injection of Ad5-CMV-RFP alone.Conclusions Chemotherapeutants could be used as an adjuvant to improve adenovirus of adeno-associated virus mediated gene expression in tumour cells. This new approach might be useful for AdV or AAV-mediated cancer gene therapy.