【Abstract】 Objective To construct a recombinant adeno-associated virus (AAV) shuttle vector expressing nervegrowth factor β (NGF-β) gene. Methods By PCR amplification, the structural element of pAAV-multi ple cloning site(MCS) and the functional element of pGenesil-1.1 were obtained and cloned into T-easy vector, respectively; the recombinant T-easy vectors were digested by restriction enzyme, then the target fragments were reclaimed and connected by DNA l igase, so the recombinant AAV shuttle vector pAAV-U6/CMV-enhanced green fluorescent protein (EGFP) containing U6 promoter and CMV promoter was obtained. The vector was transfected into 293 cells. The human Miapaca-2 cell l ine was cultured, and total RNA was extracted, then human NGF-β gene was obtained by RT-PCR. T-easy-NGF-β vector was constructed by cloning human NGF-β gene into T-easy vector and identified by RT-PCR, digestion, and DNA sequencing. As NGF-β gene was cloned into pAAV-U6/CMV-EGFP vector, the recombinant AAV shuttle vector expressing NGF-β gene was obtained and identified by RT-PCR, digestion, and DNA sequencing. Results The bands of 800 bp and 4 250 bp were detected when pAAV-U6/CMVEGFP was digested. The GFP was detected when pAAV-U6/CMV-EGFP was transfected into 293 cells. The bands of 736 bp and 3 015 bp were detected when T-easy-NGF-β was digested; DNA sequencing result of T-easy-NGF-β was fully consistent. The bands of 736 bp and 4 250 bp were detected when pAAV-U6/CMV-NGF-β was digested. DNA sequencing result of pAAV-U6/ CMV-NGF-β showed that sequences were completely correct. Conclusion The AAV shuttle vector pAAV-U6 /CMV-NGF-β is successfully constructed, providing experimental basis for investigation of the repair of spinal cord injury.
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.
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.
Objective To study the time effect of the gene expression 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 so as to lay a theoretical foundation for gene therapy of osteonecrosis. Methods The best multipl icity of infection (MOI) of BMSCs transfected with rAAV was detected by fluorescent cell counting. The 3rd generation rabbit bone mesenchymal stem cells (BMSCs) were transfected with rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (experimental group) and green fluorescent protein (GFP) labeled rAAV-IRES-GFP (control group), respectively. The expression of GFP was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by RT-PCR assay and Western blot assay in vitro. The transfected cells in 2 groups were prepared into suspension with 5 × 106 cells/mL, and injected into the rabbit thigh muscles of experimental group 1 (n=9) and control group 1 (n=9), respectively. The muscle injected with rAAV-IRES-GFP was sl iced by frozen section method and the expression of GFP protein was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by Western blot assay and ELISA assay in vivo. Results The best MOI of BMSCs transfected with rAAV was 5 × 104 v.g/cell. In vitro, the expressions of GFP, hVEGF165, and hBMP-7 genes started at 1 day after transfection, the expressions obviously increased at 14 days after transfection, and the expression maintained the b level at 28 days after transfection. In vivo, the expressions of GFP, hVEGF165, and hBMP-7 genes could be detected at 2 weeks after injection, and b expressions were shown at 6 to 8 weeks after injection. The values of hVEGF165 and hBMP-7 were (248.67 ± 75.58) pg/mL and (4.80 ± 0.61) ng/mL respectively in experimental group 1, and were (32.28 ± 8.42) pg/mL and (0.64 ± 0.42) ng/mL respectively in control group 1; showing significant differences between 2 groups (P lt; 0.05). Conclusion The rAAV-hVEGF165-IRES-hBMP-7 has efficient gene expression ability.
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.
To construct the recombinant adeno-associated virus (rAAV) vector co-expressinghVEGF165 and hBMP-7 depending on internal ribosome entry site (IRES) sequence, to measure the virus titer and to ver ify the correct recombination. Methods The AAV helper-free system was used to generate the rAAV co-expressing hVEGF165 and hBMP-7 genes. The IRES sequence from the bicistronic eukaryotic expression plasmid pIRES was cut down and subcloned into the ITR/MCS containing vector pAAV-MCS to get pAAV-MCS A-IRES-MCS B, in which upstream MCS A and downstream MCS B was constructed. The hVEGF165 and hBMP-7 genes were ampl ified by PCR and inserted into MCS A and MCS B respectively. The recombinant expression plasmid pAAV-hVEGF165-IRES-hBMP-7 was co-transfected into AAV-293 cells with pHelper and pAAV-RC for packaging of recombinant AAV. The green fluorescent protein (GFP) labeled rAAVIRES GFP was simultaneously packaged by using the parallel plasmid pAAV-IRES-GFP. The efficiency of rAAV packagingwas monitored under fluorescent microscope and recombinant viral particles were harvested from infected AAV-293 cells.The virus titer was measured through infecting AAV-HT1080 cells, and the recombinant rAAV-hVEGF165-IRES-hBMP-7was verified by PCR of the exogenous interest genes of hVEGF165 and hBMP-7. Results The recombinant plasmid pAAVhVEGF165- IRES-hBMP-7 was verified by double digestion. Using the AAV helper-free system, GFP expression could be observed under fluorescent microscope 72 hours after triple plasmid co-transfection and the system provided a high packing ratio of 95%-100%. The rAAV has a high purity and high titer of 5.5 × 1011vp/mL, and AAV-HT1080 cell could be infected at a ratio of 90%. The recombinant virus was confirmed by PCR of exogenous hBMP-7 and hVEGF165 genes. Conclusion Re combinant rAAV-hVEGF165-IRES-hBMP-7 was successfully constructed with a high virus titer, which may offer the basement
【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.
Adeno-associated viral vector (AAV) is the most important viral tool and has been widely used in gene therapy. Because of its small size, non-enveloped, non-pathogenic and other characteristics, so it is one of the main means to treat hereditary retinal diseases. Aiming at MERTK for retinitis pigmentosa, ND4 for Leber hereditary optic neuropathy or RPE1 for choroideremia, AAV gene therapy improved half patients’ visual acuity in clinic tests. Besides, there are some clinic tests in progress for Leber’s congenital amaurosis, X-linked retinoschisis, Achromatopsia, age-related macular degeneration. But more researches need to be found before clinic test for Stargardt disease, Usher syndrome and nanophthalmos. At present, AAV gene therapy is mainly used for recessive hereditary retinal diseases, and technology is needed to intervene for dominant retinal diseases. For the treatment of hereditary retinal diseases, this will be an important and complex systematic project, which requires more human and material resources to participate in and study together, and we expect to have a great breakthrough in the near future.
Leber hereditary optic neuropathy (LHON) is a matrilineal hereditary optic neuropathy in which mitochondrial DNA mutations lead to retinal ganglion cell degeneration. At present, the treatment for LHON is limited. Early symptomatic treatment and medical treatment may improve the vision of patients. In recent years, rapid progress has been made in gene therapy. Many clinical studies have confirmed its safety and efficacy. Monocular gene therapy is helpful to improve the visual function of LHON patients, and it can also improve the visual acuity of uninjected eyes. Patients do not have serious eye or systemic adverse events during the treatment period, showing good safety and tolerance. Studies with larger sample size and longer follow-up time are needed to further verify the efficacy and safety of gene therapy in the future. Gene therapy is expected to become a safe and effective treatment, bringing hope to LHON patients.
At present, 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. There are currently three ways to deliver AAV vector-mediated target genes to the retina. 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. Moreover, studies on X-linked retinoschisis have been undergoing clinical trials, but intravitreal injection has poor transduction in retinal pigment epithelium (RPE) cell layer and photoreceptor cell (PR) layer. Subretinal injection of AAV vector can deliver the target gene to the local retina, resulting in stronger efficiency of transfection and gene expression in the RPE and PR layers, which has been undergoing in animal and clinical trials, 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, which is less invasive than intravitreal and subretinal injection, Suprachorioidal injection is an important delivery route for future exploration of AAV vector mediated gene therapy for retinal diseases.