The Construction Of Double-stranded Adeno-associated Virus Vector And The Pilot Study Of Single-stranded Adeno-associated Virus Mediated Gene Overexpression Of Angiopoietin-1 In Rat Cerebral Ischemia Model

IntroductionGene therapy for ischemic stroke has a promising future. It is very important to select an appropriate gene delivery system. In recent years, adeno-associated virus (AAV) vertor has been widely used in the gene therapy for ischemic stroke.The single-stranded AAV has a property of non-neurotoxity, low immunogenicity and long-lasting gene expression. However, the efficiency of these vectors is hinderd by the need to convert the single-stranded DNA (ssDNA) genome into double-stranded DNA (dsDNA) prior to expression. This step can be entirely circumvented through the use of self-complementary vectors, which package an inverted repeat genome that can fold into dsDNA without the requirement for DNA synthesis or base-pairing between multiple vector genomes.Therefore, the application of dsDNA is beneficial to the faster onset of gene expression.Uncontroversially, the conversion process, which requires host-cell DNA synthesis machinery, is one of the major limiting steps for the efficiency of rAAV. Furthermore if the AAV genome is half wild-type size, viral particule can package either two copyies of a single-stranded AAV genome or one double stranded AAV genome. Thus double-stranded AAV (dsAAV) also called self-complementary AAV (scAAV) vectors have been developed to bypass the rate-limiting step of second strand synthesis. Deletion of the D-sequence or mutation of the terminal resolution site of one inverted terminal repeat (ITR) can yield high percentage of dsAAV vector.Undoubtfully, the application of dsDNA in the gene therapy for ischemic stroke provides a suitable strategy for the faster onset of the therapeutic gene expression. Meanwhile, the appropriate application of therapeutic genes is crucial to the ischemic stroke gene therapy.Vascular endothelial growth factor (VEGF), a secreted mitogen associated with angiogenesis, has been widely applied to the gene therapy of ischemic stroke for a long period of time. Unfortunately, recent studies clearly indicated that acute VEGF therapy during reperfusion significantly aggravated ischemic injury by increased leakage of blood-brain barrier (BBB) and intracerebral hematoma formation in experimental stroke. Fortunately, several new reports also indicated that the VEGF-induced permeability effect in the acute stage of stroke could be counteracted by angiopioetin-1 (Ang-1), which shows a synergic effect on inducing ischemic angiogenesis with VEGF. Ang-1, the ligand of the Tie-2 receptor, is an angiogenic factor that plays an important role in the stabilization and maturation of blood vessles in prenatal and postnatal angiogenesis. Ang-1 counteracts VEGF-induced inflammation in endothelial cells while having an addictive effect on vessel formation. Therefore, combined treatment with Ang-1 and VEGF may be better than treatment with either factor alone for enhancing therapeutic vascularization and angiogenesis.In the present study, we constructed dsAAV vector for the faster onset of enhanced green fluorescent protein (EGFP). Meanwhile, we injected combination of rAAV1-Angl and rAAV1-VEGF165 into the lateral ventricle of adult rats eight weeks before transient middle cerebral artery occlusion (tMCAO) for gene therapy and examined its possible effects on ischemic insults at the early stage of stroke.Materials and methodsPart 1 Construction of pSC-CMV-EGFPThe EGFP fragment was harvested from pSNAV2.0-EGFP using restriction enzymes SalI and KpnI. Then the harvested EGFP was connected to pSC-CMV-lacZ alpha, resulting in the plasmid pSC-CMV-EGFP. Part 2 Construction of dsAAV-EGFPThe dsAAV vectors were prepared by three-plasmid transfections in 293 cells, using pADHelper, pT-r2cl and pSC-CMV-EGFP. Southern blot was used to determine the dsAAV-EGFP genome size. And the purity of dsAAV-EGFP was evaluated by electrophosis in SDS-PAGE. Comparison of transduction efficiencies by dsAAV and ssAAV vectors was performed in BHK-21 and HEK-293 cell lines. Part 3 Constructiuon of pSC-CMV-VEGFThe VEGF gene was excised from pRRLSIN-VEGF165-EGFP by SaiI/KpnI, and inserted into the pSC-CMV-LacZ alpha, resulting in the plasmid pSC-CMV-VEGF.Part 4 rAAVl-mediated gene overexpression of Ang-1 and VEGF-165 in rat cerebral ischemia modelSixty-two adult Wistar rats were divided randomly into two equal groups to be injected with rAAV-Ang 1/rAAV-VEGF or rAAV-VEGF/rAAV-null, respectively. And tMCAO models were induced after 8 weeks. During the following 48 hours, Ang-1 and VEGF level, BBB permeability, microvessle density, AQP4 expression, c-fos expression, and occludin expression were determined respectively and compared statistically with that of the control group. ResultsPart 1 Construction of pSC-CMV-EGFPExamination of the plasmid pSC-CMV-EGFP using PCR and restriction analysis showed a correct product size. The identity of the amplified products was verified by sequencing.Part 2 Construction of dsAAV-EGFPThe construction of dsAAV-EGFP is successfully verified with molecular technology like Southern blot, SDS-PAGE, PCR, et al. Transduction of BHK-21 and HEK-293 cell is substantially higher using dsAAV-EGFP, when compared to ssAAV-EGFP.Part 3 Constructiuon of pSC-CMV-VEGFExamination of the plasmid pSC-CMV-VEGF using PCR and restriction analysis showed a product size of 580 bp. The identity of the amplified products was also verified by sequencing.Part 4 rAAV1-mediated gene overexpression of Ang-1 and VEGF in rat cerebral ischemia modelIntraventricular application of rAAV-Angl/rAAV-VEGF eight weeks before tMCAO resulted in Ang-1 and VEGF overexpression, and significantly decreased EB permeability and AQP4 expression following ischemia (P<0.05). In addition, the cerebral microvessle density was also significantly increased in peri-infarct zone in rAAV-VEGF/rAAV-Angl group compared to rAAV-VEGF/rAAV-null group(P< 0.05). Moreover, there were more positive cells expressing c-fos or occludin in the brain of rat coming from rAAV-VEGF/rAAV-Angl group than that from rAAV-VEGF/rAAV-null group(P< 0.05).Conclusions1 The plasmid pSC-CMV-EGFP was successfully constructed.2 The dsAAV-EGFP vector was smoothly constructed.3 The plasmid pSC-CMV-VEGF was also constructed uneventfully.4 In cerebral ischemia, the combination of Ang-1 and VEGF could be used early to promote the formation of mature neovessles and protect the injured cells without inducing side effects on BBB permeability. And the early intraventricualr injection of mixed rAAV-VEGF and rAAV-Ang1 may be a rational therapeutic strategy in the gene therapy for experimental stroke.