Functional studies on factor VIII variants containing different lengths of the B-domain AND The construction of a new helper vector for the production of helper-dependent adenovectors

Abstract

<strong>Functional studies on factor VIII variants containing different lengths of the B-domain</strong><br /> To evaluate the role of the factor VIII (FVIII) B-domain on FVIII expression, (activity and activation), we constructed several FVIII expression constructs with various lengths of the B- domain and expressed them transiently as well as stably.<br /> Practically, B-domain deleted recombinant FVIII (BDD-rFVIII) was generated from the full- length FVIII-cDNA by ligating (on the protein level) Ser743 to Arg1634; the corresponding FVIII-cDNA was then cloned into the pMT2 expression vector. Then, cDNA-fragments of different length (on the protein level: 101, 202, 292, and 394 amino acid long fragments of the B-domain starting from its amino terminal) were cloned between Ser743 and Arg1634. Constructs were then transiently expressed in COS-7, HEK 293 and CHO cells and stably expressed in CHO-DHFR(-) cells by methotrexate selection and amplification. FVIII proteins were then assessed for activity in culture medium by the clotting assay and the chromogenic assay and for antigen in cellular lysates by ELISA. FVIII proteins were purified from stable cell lines media. <br /> Our transient expression studies showed that FVIII expression was the highest in COS-7 cells followed by HEK 293 cells. The CHO cells showed the lowest FVIII expression. Transient expression in COS-7 cells (for forty-eight hours) revealed that the constructs containing added lengths of the B-domain showed a higher FVIII: C (~1.1-2.5 folds) and a higher overall expressed protein (total antigen in medium and cellular lysates; ~1.1-3 folds) compared the BDD-rFVIII. The functionality of the expressed proteins in terms of FVIII specific activity was higher in all constructs in comparison to the BDD construct. Comparing the clotting FVIII activity by that of the chromogenic assay, we found that those constructs showed a slightly higher FVIII value within the clotting assay than within the chromogenic assay. In contrast, the BDD construct showed the opposite effect and also showed a significant discrepancy between both assay methods. Activation of purified FVIII proteins by thrombin revealed that the BDD-rFVIII showed a faster generation of the A2 domain than the constructs containing added lengths of the B-domain.<br /> In conclusion, our data showed that the inclusion of a 100 amino acids or more of the B- domain sequence eliminates the assay discrepancy and increases the overall expression and functionality of rFVIII protein.<br /><strong>The construction of a new helper vector for the production of helper-dependent adenovectors</strong><br /> Fully deleted Helper-Dependent Adenovectors (HD-AdV) are so far the ultimate form of Adenovirus vector modifications which lack all the adenovirus genes except the replication and packaging elements. They can accommodate an insert of up to 36 kb. HD-AdVs are produced in HEK 293 cells in the presence of a helper adenovirus that supplies the viral genes in trans. Using this system very high titers of HD-AdV can be prepared now, with small but still measurable helper adenovirus contamination.<br /> Within this study we have tried to design a novel hybrid helper virus that can support the production of a HD-AdV, carrying a B-domain-deleted factor VIII-cDNA, in high efficiency without any contamination. In order to avoid contamination of HD-AdV production, the adenoviral sequences in the novel hybrid helper virus was lacking the adenoviral ITRs and the packaging signal, but was flanked by adeno-associated virus (AAV) ITRs. This adenoviral helper sequences was then cloned into the baculovirus expression system. <br /> The lack of the Adenovirus packaging signal renders the hybrid helper virus packaging incompetent and thus eliminates the risk of contaminating the HD-AdV preparations. Since the helper virus also lacks the adenoviral ITRs, it will not compete with the HD-AdV for the Adenovirus 5 replication proteins. The lack of the capsid genes within the AAV-sequences should also prevent the generation of AAV particles. <br /> To drive the replication of the Ad5-genome in the hybrid helper virus theAAV2-Rep genes were cloned in a separate baculoviral construct. A GFP baculoviral construct was prepared to be used as positive control for transfecting insect cells and infecting HEK 293 cells. <br /> We successfully constructed the hybrid helper vector pBac-AAV2-Ad5 with a deletion in the 3’ITR region. The recombinant baculovirus particles (vBac-AAV2-Ad5, vBac-AAV2-Rep and vBac-EGFP) was then produced in insect cells, plaque purified and amplified to high titers. Since the deletion within the 3’-ITR region should have no effect on helper virus production, the produced baculoviruses were used to infect HEK 293 cells, together with the HD-AdV carrying the B-domain-deleted factor VIII-cDNA. Depending on the presence of AAV-ITRs, we expected that the genome of the helper-virus will be replicated, thus improving its efficiency in providing helper function. To verify our strategy in synthesizing the helper virus, we showed that the baculovirus is able to infect HEK 293 cells by eGFP expression and that the Rep gene was expressed in both SF9 cells and HEK 293 cells. <br /> After three rounds of infecting HEK293 cells with the helper hybrid virus, we couldn’t see any signs of HD-AdV production. Unexpectedly, the analysis of extracted extra chromosomal DNA showed that the material was the original transfected material and not synthesized within the HEK 293 cells. <br /> In conclusion, as the baculovirus was able to infect HEK 293 cells and the Rep genes were also expressed in those cells, we propose that the HD-AdV was not produced because of inefficient or non occurring replication of the Ad5 genes in the Bac/Ad5 hybrid. The presence of only one ITR may be a possible cause. The reconstruction of an helper hybrid with both ITRs would clarify the function of such an adenoviral helper virus system.