OBJECTIVE: To explore the effects of the rat FVIII light chain (rLC) on the activity of human FVIII heavy chain hHC745 and hHC1690. METHODS: hHC745, hHC1690, human FVIII light chain (hLC) and rLC were cloned into adeno-associated virus serotype 8 (AAV8) expression vectors with CB promoter (ubiquitous expression), respectively, and transfected into 293T cells using a dual-chain strategy of co-expression of HC and LC. The cultured cell supernatant was collected at 48 hours after transfection. The plasmids (pAAV8-CB-hHC745, pAAV8-CB-hHC1690, pAAV8-CB-hLC and pAAV8-CB-rLC) were hydrodynamically injected into hemophilia A (HA) mice via lateral tail vein. Forty-eight hours after injection, the peripheral blood of the mice was collected through retroorbital venous plexus and the plasma was obtained by centrifugation. The activity of FVIII was detected by activated partial thromboplastin time (aPTT) assay, and the antigen expression level of FVIII was determined by enzyme-linked immunosorbent assay (ELISA). The specific activity of FVIII was calculated based on the activity and the antigen expression level. RESULTS: In 293T cells, the coagulation activity of FVIII was significantly enhanced when hHC745 and hHC1690 combined with rLC compared with them combined with hLC. The FVIII activity of hHC745+rLC was about 4.6 times higher than that of hHC745+hLC, while hHC1690+rLC was about 2.9 times higher than that of hHC1690+hLC. The data from ELISA showed that there was no significant difference in FVIII antigen expression when hHC745 and hHC1690 combined with rLC and hLC. The specific activity of hHC745+rLC increased to about 4.1 times compared with hHC745+hLC, while that of hHC1690+rLC increased to about 2.8 times compared with hHC1690+hLC. In HA mice administrated with hydrodynamic injection, the FVIII activity of hHC745+rLC and hHC1960+rLC was significantly higher than that of hHC745+hLC and hHC1690+hLC at comparable expression level. The FVIII activity of hHC745+rLC was significantly higher than that of hHC745+hLC, increasing to about 5.1 times, while, that of hHC1690+rLC increasing to about 2.1 times than hHC1690+hLC. ELISA results also showed that there was no significant difference in FVIII antigen expression when hHC745 and hHC1690 combined with rLC and hLC. The specific activity of hHC745+rLC increased to about 4.2 times compared with hHC745+hLC, while that of hHC1690+rLC increased to about 1.8 times compared with hHC1690+hLC. In addition, the activity of hHC1690 combined with rLC was significantly higher than that of hHC745 combined with rLC. CONCLUSION rLC can significantly enhance the coagulation activity of FVIII when co-expressed with hHC of different length including hHC745 and hHC1690.
Rats ; Humans ; Mice ; Animals ; Dependovirus/genetics* ; Transfection

Dependovirus/genetics* ; Microglia ; Cells, Cultured ; Transduction, Genetic

Dependovirus ; genetics ; Genetic Therapy ; Genetic Vectors ; Humans ; Liver Diseases ; therapy

Many researchers are using viruses to deliver genes of interest into the brains of laboratory animals. However, certain target brain cells are not easily infected by viruses. Moreover, the differential tropism of different viruses in monkey brain is not well established. We investigated the cellular tropism of lentivirus and adeno-associated virus (AAV) toward neuron and glia in the brain of cynomolgus monkeys (Macaca fascularis). Lentivirus and AAV were injected into putamen of the monkey brain. One month after injection, monkeys were sacrificed, and then the presence of viral infection by expression of reporter fluorescence proteins was examined. Tissues were sectioned and stained with NeuN and GFAP antibodies for identifying neuronal cells or astrocytes, respectively, and viral reporter GFP-expressing cells were counted. We found that while lentivirus infected mostly astrocytes, AAV infected neurons at a higher rate than astrocytes. Moreover, astrocytes showed reactiveness when cells were infected by virus, likely due to virus-mediated neuroinflammation. The Sholl analysis was done to compare the hypertrophy of infected and uninfected astrocytes by virus. The lentivirus infected astrocytes showed negligible hypertrophy whereas AAV infected astrocytes showed significant changes in morphology, compared to uninfected astrocytes. In the brain of cynomolgus monkey, lentivirus shows tropism for astrocytes over neurons without much reactivity in astrocytes, whereas AAV shows tropism for neurons over glial cells with a significant reactivity in astrocytes. We conclude that AAV is best-suited for gene delivery to neurons, whereas lentivirus is the best choice for gene delivery to astrocytes in the brain of cynomolgus monkeys.
Animals, Laboratory ; Antibodies ; Astrocytes ; Brain* ; Dependovirus* ; Fluorescence ; Haplorhini ; Hypertrophy ; Lentivirus* ; Macaca fascicularis* ; Neuroglia ; Neurons ; Putamen ; Tropism*

We have developed the recombinant adeno-associated virus (AAV) carrying the EGFP gene under the control of the microphtalmia-associated transcription factor-M (MITF-M) promoter region for melanoma-specific expression. MITF-M distal enhancer (MDE) region enhances the specific expression of the reporter gene specifically in cultured melanoma cells. Expression of EGFP protein was very high in AAV-CMV-EGFP infected cells but relatively low in cells infected with AAV-Mitf(Enh/Pro)-EGFP. After an in vitro infection by a recombinant AAV carrying the EGFP gene under the control of human MITF-M promoter, the reporter gene was expressed in MITF-M producing melanoma cell lines (SK-28 and G361), but not in MITF-M non-producing cell lines (HaCat). These results suggest that the utilization of the MITF-M promoter in a recombinant AAV vector could provide benefits in gene therapy applications.
Cell Line ; Dependovirus ; Genes, Reporter ; Genetic Therapy ; Green Fluorescent Proteins ; Humans ; Lifting ; Melanoma ; Promoter Regions, Genetic

Coagulation factor VIII (fVIII) is a secretion protein and plays a crucial role in the coagulation cascade. Hemophilia A resulted from deficiency of fVIII is the most common X-linked recessive bleeding disorder. Gene therapy is recognized as an attractive strategy for the eventual cure of this disease. However, the gene therapy is hampered by the big size of fVIII gene when using the most promising gene vectors, adeno-associated virus (AAV) vectors. In this study we explored the intein-mediated protein trans-splicing to deliver a Phe(309)-->Ser mutant full-length fVIII (F309SfVIII) gene by using a dual-vector system. An intein is a protein sequence embedded within a precursor protein and can excise itself through protein splicing. The F309SfVIII is proven to be beneficial to its secretion. The F309SfVIII gene was broken into heavy and light chains before Ser(1239) in B domain and fused with the coding sequences of Ssp DnaB intein respectively to construct a pair of plasmid vectors by inserting them into the pcDNA3.1 vectors. Forty-eight hours after co- or separate transfection of 293 cells, the co-transfected cell lysate showed an obvious ligated F309SfVIII protein band by Western blot with a polyclonal antibody against fVIII. The amounts of secreted F309SfVIII protein in culture supernatants and their bioactivities were (71+/-9) ng/mL and (0.38+/-0.09) IU/mL determined by ELISA and Coatest assay respectively. The supernatant from combined cells with separate transfections also displayed lower levels of F309SfVIII antigen and fVIII activity [(25+/-6) ng/mL and (0.12+/-0.05) IU/mL], indicating the F309SfVIII could be formed by splicing both before and after secretion. The content of F309SfVIII heavy chain protein from co-transfected cell supernatant was higher than that of intein-fused heavy chain transfection alone [(135+/-10) ng/mL vs (37+/-7) ng/mL, P<0.01)]. These data demonstrated that intein could be used as a technical strategy in a dual-vector system delivering F309SfVIII gene with improved secretion of fVIII providing an alternative approach to circumvent the packaging limitation of AAV for F309SfVIII gene transfer, which encourages our continuing study in hemophilia A gene therapy in vivo.
Cell Line ; Dependovirus ; Factor VIII ; metabolism ; Genetic Vectors ; Humans ; Inteins ; Protein Splicing

Animals ; Dependovirus ; genetics ; immunology ; Gene Transfer Techniques ; instrumentation ; Genetic Therapy ; instrumentation ; Genetic Vectors ; genetics ; immunology ; Humans

Recombinant adeno-associated virus (rAAV) has been widely used as vector for gene therapy. However, the effectiveness of gene therapy based on rAAV needs to be further improved. Enhancement of the transduction efficiency is one of the most important fields for rAAV-based gene therapy. Recent results have showed that the ubiquitin-proteasome system plays an important role in the trafficking of rAAV vector in cytoplasm, and regulation of its function may significantly improve the transduction efficiency of rAAV vector in various types of cells and tissues.
Animals ; Dependovirus ; genetics ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Humans ; Transduction, Genetic ; Ubiquitin ; metabolism

OBJECTIVE: To investigate the expression efficiency of exogenous gene mediated by different serotypes of adeno-associated virus (AAV) vectors in retina, and to compare the expression efficiency of AAV vector and two kinds of promoters commonly used in ophthalmology after transfection into mouse retina, so as to provide the basis for selecting appropriate AAV vector and promoter for gene therapy of retinitis pigmentosa. METHODS: AAV2/2, AAV2/5, AAV2/8 and AAV2/9 were prepared. The C57BL/6J mice were injected subretinally with 1 μL purified AAV vectors (1.00×10¹³ mg/L). Then the mice were killed 2 or 4 weeks after treatment, and the eyes were enucleated for frozen section. The expression of green fluorescent protein (GFP) was observed under the confocal microscope. Two kinds of promoters, CMV and CAG, were selectd, and the expression of AAV2/8-GFP-CMV and AAV2/8-GFP-CAG was observed under confocal microscope. RESULTS: No bacterial infection or immune response were seen in the injected mice. 2 weeks after injection, the GFP green fluorescence of AAV2/8 and AAV2/9 in the mouse retina was obvious, which indicated that the GFP green fluorescence of AAV2/8 and AAV2/9 was high after transfection into the mouse retina. In these two serotypes, GFP green fluorescence of AAV2/8 was mainly concentrated in photoreceptor cells while AAV2/8 was expressed in the whole retina, indicating that AAV2/8 was more specific to photoreceptors. Further experiments on AAV2/8 showed that the GFP green fluorescence of the mouse retina was obvious 4 weeks after injection, indicating that the exogenous gene mediated by AAV2/8 could be stably expressed in vivo. For CMV and CAG promoters, CMV promoter was expressed stronger in retinal pigment epithelium (RPE)cells, while CAG promoter was stronger in photorecepters. In photorecepters, CAG promoter was expressed almost the same as CMV promoter, while CMV promoter was stronger in RPE cells. CONCLUSION AAV vectors could express transgene robustly in retinal cells; Among several AAV serotypes, AAV2/2 and AAV2/5 showed weaker GFP fluorescence than AAV2/8 and AAV2/9. AAV2/9 showed expression in each layer of the retina including ganglion cells. AAV2/8 was more specific for photoreceptor; CAG promoters had higher specificity for photoreceptors than CMV promoters.
Animals ; Dependovirus/genetics* ; Genetic Vectors ; Mice ; Mice, Inbred C57BL ; Retina ; Serogroup ; Transduction, Genetic

OBJECTIVE: To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody. METHODS: The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay. RESULTS: The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10. CONCLUSION We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.
Animals ; Antibodies ; Capsid ; Capsid Proteins/genetics* ; Dependovirus/genetics* ; Prokaryotic Cells ; Rabbits ; Recombinant Proteins/genetics*