Adenovirus - A Virus Heterozygous Carrier Construction And Virus Preparation

Various gene transfer vectors have been applied in gene therapy. Among them, adenovirus vector (Ad) is the most widely used one in cancer gene therapy because of its several merits including the easy production to high titers, efficient infection of both dividing and non-dividing cells, non integration into host genome, stable physical and chemical properties and so on. However, the application of adenovirus type 5 (Ad5) vector for hematopoietic cell transfer is limited by low gene transfer efficiency.Adenovirus enters cells mainly through binding of the coxsackie virus and adenovirus receptor (CAR) by its fiber. There are different cell targeting characters for adenovirus because of its variety in serotypes., The changes of adenovirus serotype by modification of fiber will cause cell retargeting for infection. To enhance the infection efficiency of Ad5 for hematopoietic cells, we replaced the Ad5 with B group of adenovirus type 11p (Ad11p) fiber head ball leading to cell retargeting of Ad5F11p in our previous work. This modification greatly enhanced the infection efficiency of adenovirus for hematopoietic cells (approximately 10 times higher than Ad5), but still very low in primary leukemia cells (particularly lymphoid leukemia cells).Recent studies show that alphavirus-based viral vector or its replicon could drive exogenous gene expression efficiently. And it has been widely used in vaccine preparation. The high efficiency of alphavirus in expressing exogenous genes dues to its regulation and production of a large number of mRNA on transcriptional level. However, alphavirus infects hematopoietic cells less efficiently.Based on these reasons, this issue was proposed to construct hybrid vectors with properties of high transcription efficiency of alphavirus and the hematopoietic cells targeting property of Ad5Fllp vector, and to evaluate its infection ablility to hematopoietic cells. The basic scheme of the experiment is as follows:In order to obtain hybrid adenovirus vectors, alphavirus replicon vector sequences,including the non-structural protein sequences of N-terminal, subgenomic replication origin and target gene, were inserted into the multiple clone site of Ad5Fllp vector. And the hybrid adenovirus was packaged, prepared and purified according to the manual of Ad-Easy system. This hybrid adenovirus with adenovirus framework can infect hematopoietic cells through the attachment of its fiber to CD46. After the virus enters the cells, its heterozygous genome will go into the nucleus. Driven by promoter (CMV), the alphavirus vector sequence is transcribed into RNA, which is transported to the cytoplasm.Then, non-structural proteins are expressed. And a large number of subgenomic mRNA synthesize followed by target genes expression.Firstly, we constructed the plasmid containing heterotic viral genome, including the construction of the framework plasmid, shuttle plasmid and the production of heterotic viral genome by homologous recombination.The concrete steps are as follows:(1) construct the framework plasmid pAdEasy-1/F11p-E4orf3. The elements of Alphavirus and target gene sequence exceed the carrying capacity of the adenovirus vector, so we deleted some regions of the framework plasmid of adenovirus (pAdEasy-1/Fllp). We isolated a small intermediate plasmid (pFiber5/11p,8.9 kb) from the framework plasmid pAdEasy-1/f11p(32.7 kb), and deleted parts of E4 region of pFiber5/llp through overlap extension PCR. Then reset the reformed intermediate plasmid to the corresponding location of the framework plasmid and obtained the new frameork plasmid, called pAdEasy-1/F11p-E4orf3. And the carrying capacity of the recombinant adenovirus will be up to nearly 10 kb. (2) Construct the heterotic shuttle plasmid. The hematopoietic cell specific promoter (CD11a promoter, CD11Ap), the element of alphavirus vector(nsP1～4),target gene (green fluorescent protein,GFP) and the tailed signal PolyA were cloned into the MSC of the pShuttle plasmid, called pSh-SFV-GFP.pSFV-cdlla was obtained by adding the promoter CD11Ap to the upstream of the nsP1-4 through the molecular biological methods. Then cloned the SV40 polyA into the MCS of the pSFV-cd11a and obtained plasmid pSFV-cdlla-polyA.And target gene GFP wasSubcloned into the suitable enzyme cutting site (BamHI). There is no suitable enzyme cutting site for the insertion of CD11Ap-nsP-GFP-polyA in the MCS of pShuttle plasmid. So we deleted the original MCS of the pShuttle plasmid, and introduced 8 nucleotide sequences (GGCGCGCC),which is the restriction enzyme site of AscI.After catalysed by AscI,the pSFV-cd11a-polyA was inserted into the AscI site of pShuttle-AscI, and obtained pSh-SFV-GFP.(3) The linearized pSh-SFV-GFP and the pAdEasy-1/F11p-E4orf3 cotransform the BJ5183 competent cell and obtained pAdE4orf3-SFV-GFP heterotic virus framewoek plasmid by homologous recombination.(4) In addition, the recombinant adenovirus plasmid pAdE4orf3-GFP without Alphavirus element was constructed to compare with the pAdE4orf3-SFV-GFP.Secondly, the rescue and amplification of heterotic virus requires helper-virus in packing cells. We constructed a hematopoietic cell targeted helper virus Ad5/f11p-HV. The processes of preparing helper virus include the reformation of the shuttle plasmid, the preparation of the helper virus genome, the rescue and purification of the helper virus. In order to attenuate the packaging efficiency of the helper virus and elevate the production of targeted virus in 293Cre4 by efficiently cutting the packing signals, we redesigned the incomplete packaging signal including loxP sequence.The helper virus pAd5/f11p-HV based on the hematopoietic cell targeted framework plasmid AdEasy-1/f11p. (1) Designed eight primers and synthetize 241bp packaging signal DNA fragment by overlap extension PCR, containing the enzyme cutting site and the loxP sequence. And then, substituted the original signal of the pShuttle, and obtained the shuttle plasmid pShuttle-SynES. (2) Obtained the recombination adenovirus plasmid pAd5/fllp-HV by homologous recombination of the linearized shuttle plasmid pShuttle-SynES and the framework plasmid AdEasy-1/fllp in BJ5183 competent cell.(3) The linearized Ad5/f11p-HV plasmid transfected 293 cells to pack the helper virus Ad5/f11p-HV.And then the virus were amplified in 293 cells and were purified by the CsCl density gradient centrifugation.Finally, hybrid virus was rescued by Ad5/f11p-HV, amplified in 293 cells, purified by CsCl density gradient centrifugation, evaluated the gene transfer efficiency in hematopoietic cells. Ad5/f11p-HV and AdE4orf3-SFV-GFP were linearized, and then were cotransfected into 293Cre4 cells to rescue the AdE4orf3-SFV-GFP virus. The amplified hybrid virus was purified through CsCl density gradient centrifugation, and the infection titer was 1.5×1010IU/ml measured by TCID50. Leukemia cell lines K562, U937, and myeloma cell lines U266, SKO-007 were infected by hybrid virus AdE4orf3-SFV-GFP, type 5 adenovirus Ad5-GFP and reconstructed 5 adenovirus Ad5/F11P-GFP at 10MOI(IU/cell),50MOI, 100MOI, and 200MOI respectively. The infection efficiency was evaluated by fluorescence microscope and flow cytometry. The results showed that infection efficiency of AdE4orf3-SFV-GFP in hematopoietic cells was similar to that of Ad5/F11P-GFP, but was significantly improved compare to that of the Ad5-GFP. For example, the GFP expression rates of U937 cells were 92.73%,94.73% and 2.95% respectively infected by AdE4orf3-SFV-GFP, Ad5/F11P-GFP, and Ad5GFP at 100MOI,.In summary, we successfully developed an adenovirus-alphavirus hybrid vector AdE4orf3-SFV-GFP which could be rescured by helper virus Ad5/f11p-HV. The infection efficiency of this hybrid virus for hematopoietic cells was obviously higher than that of Ad5-GFP, and similar to that of Ad5/F11P-GFP. Our works provide a novel adenovirus-alphavirus hybrid virus system for gene transfer of hematopoietic cells.