Objective To study the effect of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothel ial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes on bone regeneration and angiopoiesis in vivo so as to provide a theoretical basis for the gene therapy of avascular necrosis of thefemoral head (ANFH). Methods Twenty-four male adult New Zealand rabbits were made the ischemic hind l imb model and divided into 4 groups (n=6). The 3rd generation rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with the following 4 virus and were administered intramuscularly into the ischemic thigh muscle of 4 groups, respectively: rAAVhVEGF165- internal ribosome entry site (IRES)-hBMP-7 (group A), rAAV-hVEGF165-green fluorescent protein (GFP) (group B), rAAV-hBMP-7-GFP (group C), and rAAV-IRES-GFP (group D). At 8 weeks after injection, the blood flow of anterior tibial artery in the rabbit hind l imb was detected by ultrasonographic image. Immunohistochemical staining for CD34 was performed to identify the prol iferation of capillary. Another 24 male adult New Zealand rabbits were made the femur muscle pouch model and divided into 4 groups (n=6). The above 4 BMSCs transfected with rAAV were administered intramuscularly into the muscle pouch. At 8 weeks after injection, X-ray radiography was used to assess orthotopic bone formation, and von Kossa staining to show mineral ization. Results No symptoms of local or systemic toxicity were observed after rAAV injection. At 8 weeks after injection, the ratio of ischemic to normal blood flow and the number of capillaries in group A were the highest among 4 groups (P lt; 0.05). The ratio of ischemic to normal blood flow and the number of capillaries in group B were significantly higher than those in group C and group D (P lt; 0.05). However, there was no significant difference between group C and group D (P gt; 0.05). At 8 weeks after injection, orthotopic ossification and mineral ization were evidently detected in group A and group C, and group A was ber than group C. No obvious evidence of orthotopic ossification and mineral ization were observed in group B and group D. Conclusion rAAV-hVEGF165-IRES-hBMP-7 vector has the biological activities of inductive bone regeneration and angiopoiesis in vivo.
Age-related macular degeneration (AMD) represents a significant cause of visual impairment and blindness in individuals over 65 years old. In recent years, gene therapy has emerged as a research hotspot for wet AMD, with adeno-associated virus (AAV) vectors being widely utilized due to their non-pathogenic nature, low immunogenicity, broad tissue tropism, and capacity for sustained transgene expression. Several related studies have progressed to clinical trial stages. Although challenges persist, including immunogenicity concerns, limited vector capacity, and potential long-term adverse effects, the continuous advancement of research strategies and technologies holds promise. Future developments may employ AAV delivery systems to achieve gene supplementation, gene editing, or gene silencing of angiogenesis-related signaling molecules, thereby providing novel therapeutic approaches for wet AMD.
Objective To investigate the neuroprotective effects of recombinant adeno-associated virus (rAAV) expressing vascular endothel ial growth factor (VEGF) on traumatic spinal cord injury (SCI) of rat and its mechanisms. Methods The 144 male Sprague Dawley rats were randomly divided into 4 groups, and each group contained 36 rats. The rats in sham group (group A) received dorsal laminectomy without SCI and microinjection, the rats in model control group (group B), rAAV-green fluorescent protein (GFP) group (group C), and rAAV-hVEGF165-GFP group (group D) received dorsallaminectomy with SCI and injection of 20 μL sal ine, rAAV-GFP viruses, or rAAV-hVEGF165-GFP viruses, respectively. At 3 and 7 days after operation, Basso-Beattie-Bresnahan (BBB) score was used to evaluate the neurologic function. At 7 days after operation, Nissl’s body staining was used to evaluate the histopathological changes; apoptosis was confirmed by transmission electron microscope examination and TUNEL staining; the expression of aquaporin 4 (AQP-4) was detected by Western blot assay. At 1, 3, 5, and 7 days, ELISA assay was used to detect the VEGF165 protein expression. Results According to BBB scores, the neurologic function in group D was significantly better than those in groups B and C at 3 and 7 days after operation (P lt; 0.05). Nissl’s body staining showed that tissue damage in group D was significantly milder than those in groups B and C at 7 days after operation (P lt; 0.05). ELISA results showed that VEGF165 protein expression was slowly-released in low dose in group D, and the expression in group D was significantly higher than that in groups A, B, and C at 3, 5, and 7 days after operation (P lt; 0.05). The results of transmission electron microscope and TUNEL staining showed that apoptosis rate of spinal cord neurons in group D was significantly lower than that in groups B and C at 7 days after operation (P lt; 0.05). The results of Western blot showed that AQP-4 expression in group D was significantly decreased when compared with that in groups B and C at 7 days after operation (P lt; 0.05). Conclusion TherAAV expressing VEGF has neuroprotective effects by inhibiting apoptosis of spinal cord neurons and relieving spinal cord edema.
The treatment of hereditary retinopathy depends on gene replacement or editing therapy, and adeno-associated virus (AAV) vector is one of the most widely used gene transfer vectors. The delivery methods of AAV vector-mediated target genes to the retina inlucde intravitreal injection, subretinal injection, and suprachorioidal injection. Intravitreal injection of AAV vector is currently the most commonly used delivery route, which can effectively improve the functions of retina disorders such as blinding retinal dystrophy in mice. Subretinal injection of AAV vector can deliver the target gene to the local retina, resulting in stronger efficiency of transfection and gene expressio, however, the high technical operations are required. In recent years, as a new high-profile delivery route suprachorioidal injection of AAV vector can achieve more extensive transfection of target genes in the retina of rabbits and rats. At present, the efficiency of AAV vector transduction in the retina is affected by the delivery mode. In the future, it is necessary to further explore the effect of AAV vector delivery mode on the transduction efficiency in order to find an important delivery route for mediating gene therapy for retinal diseases.
Objective To use an improved technique to construct the recombinant adeno-associated virus 2 (rAAV2) mediated gene which can transfer human tissue inhibitor of metalloproteinase-1 (TIMP1). Methods Human TIMP1 gene was amplified from pDNR-LIB plasmid by PCR and cloned into the rAAV2 vector pSNAV to recombinant pSNAV-TIMP1, then was transferred into BHK-21 cells by means of lipofectamine. Using G418 selection, a mixed cell named BHK-21/rAAV2-TIMP1 was isolated, which was capable to express TIMP1. The cell was subsequently infected with recombinant herpes simplex virus 1 (rHSV1-rc/△UL2) that was able to package the rAAV2-TIMP1. After purification, rAAV2-TIMP1 was obtained. Results The rAAV2 carrying human TIMP1 gene was constructed successfully. The viral titer of the rAAV2-TIMP1 was 1×1012 v.g./ml. Conclusion rAAV2-TIMP1 was constructed successfully, which would provide experimental basis for carrying the TIMP1 into hepatocellular carcinoma effectively and inhibiting the invasiveness and migratory capacity of hepatocellular carcinoma in vitro and in vivo models.
Objective The human amniotic epithel ial cells (hAECs) are a recently identified new type of stem cells.It has previously been shown that hAECs express hepatocyte-related gene and possess intracellular features and functional properties of hepatocytes. The hAECs may be a candidate seed cell for l iver regeneration. To research the survival and migrationin vivo of hAECs via adeno-associated virus-mediated the green fluorescent protein gene (AAV-GFP) transfection, and toexplore the expression of hepatocyte-l ike function. Methods Thirty nude mice (aging 6-8 weeks, half males and females, and weighing 20-22 g) were randomly divided into 3 groups (groups A, B, and C, n=10). The mice of groups A and C were made the 2/3 partial hepatectomy model, and the mice of group B underwent open abdominal operation without hepatectomy. The hAECs transfected by AAV-GFP were transplanted into the inferior end of the spleen in groups A and B with a cell density of 5 × 106/mL and a volume of 0.2 mL; the same volume of normal sal ine was injected in group C. At 4 hours, the nude mice were sacrificed and the samples of l iver, spleen, heart, lung, brain, and kidney were harvested and the general observation, histological observation, and immunofluorescence detection were performed for the hAECs survival, migration, and the functional properties of hepatocytes. Results No tumor tissue was found in l iver and spleen of 3 groups, and HE staining showed no tumor cells. There were a lot of roundl ike and deeply-stained cells with less cytoplasm and large nucleus in the spleen and the l iver of group A; no abnormal cells were found in l iver and spleen of groups B and C and in kidney, heart, bung, and brain of groups A, B, and C. The GFP+ cells were detected in the spleen and l iver of group A with expressing human albumin, but no GFP+ cells was found in l iver and spleen of groups B and C and in heart, kidney, lung, and brain of groups A, B, and C. Conclusion AAV-GFP infected hAECs transplanted into SCID nude mice with hepatectomy can keep the hepatocyte-l ike function. It will be beneficial to further identify their biological characteristics.
【Abstract】ObjectiveTo construct a recombinant adeno-associated virus(rAAV2) vectors carrying the combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter for the purpose of targeted gene therapy for hepatocellular carcinoma (HCC). MethodsThe fragment of combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter was amplified through polymerase chain reaction (PCR) and cloned into the promoter site of pAAV-IRES-hrGFP instead of the CMV promotor in AAV Helper-Free System to construct the rAAV2 expression plasmid pAAV-IRES-hrGFP-EP. Then the packaging cell lines (HEK 293 cell) was co-transfected with the pAAV-IRES-hrGFP-EP together with the control plasmid pAAV-RC and pHelper in AAV Helper-Free System by means of lipofectamine.The recombinant adenoassociated virus vector(rAAV2-EP) carrying the combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter was packaged and amplified in the HEK 293 cell. Then the viral titer was checked by GFP. ResultsThe recombinant adeno-associated virus vector(rAAV2-EP) carrying the combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter was constructed successfully, the b green fluorescence was observed in HEK 293 cells under fluorescence microscope. The viral titer was 1.2×105. ConclusionConstruction of the recombinant adeno-associated virus vector rAAV2-EP driven by the combined transcriptional regulatory sequences of α-fetoprotein enhancer and albumin promoter would provide a sound basis and improved vector for targeted gene therapy for HCC.
In the present study, packaging system composed of pAAV-CMV-GFP, pAAV-RC and pHelper were transfected into human embryonic kidney 293 cells (HEK293 cells) mediated by polyethyleneimine (PEI) to explore an optimal transfection condition. Different total plasmid DNA dosages (1, 2, 3, 4, 5, 6μg) and different PEI/Plasmid ratios (1:1, 3:1, 5:1, 7:1) were tested with detection of green fluorescence protein (GFP) with ImagePro Plus6.0 Software. Then transfection efficiency of the optimized transfection system was further observed for different time periods(12, 24, 36, 48, 60, 72 h). The results showed that total plasmid dosage of 4μg/well with PEI/plasmid ratio of 3:1~5:1 was an efficient transfection condition. Transfection efficiency-time curve was an S-shaped curve. Transfection efficiency reached a plateau at 60 h after transfection. The optimized conditions for PEI-mediated transfection at the optimal time result in enhanced transfection efficiency of triple plasmid into HEK293 cells.
Objective To construct the recombinant adeno-associated virus vector with human bone morphogenetic protein 4 gene（AAV-hBMP4）. Methods The hBMP-4 gene primer was designed basing on the corresponding gene sequence in GenBank. EcoR I site was introduced into the upstream of the primer and Sal Ⅰ site into downstream. The hBMP-4 gene was amplifiedwith the template of EX-A0242-M01-hBMP-4, then was cloned into pUC18 vectorto construct recombinant plasmid pUC18-hBMP-4. The plasmids pUC18-hBMP-4 and plasmid pSNAV cut by EcoR Ⅰ and Sal Ⅰenzyme, the fragments were collected and linked with T4 DNA ligase at 16℃ over night, recombinant plasmid pSNAVhBMP-4 was obtained. The recombinant plasmid was then transfected into BHK21 cells using Lipofectamine TM2000. The G418 resistant cells were obtained consequently. Thesecells were infected with HSV1-rc/△UL2 which has the function of packaging andcopying the recombinant AAV. After purification, the construction of recombinant AAV-hBMP-4 was completed. Results The construction of the recombinant pSNAV-hBMP-4 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequence in the recombinant pSNAV-hBMP-4 wascorrect. The virus titer was about 1.5×1012 μg/ml.The purity of the virus was more than 95% using the SDSPAGE method. Conclusion With this method, high virus titers and purity of AAV-hBMP-4 can be acquired successfully and it is useful to bone tissue engineering.