Improved Method for Adenoviral Vectors Construction
DESCRIPTION (provided by applicant): Gutless (helper-dependent) adenovirus vectors constitute a promising tool for gene transfer, with potentially a wide variety of applications in research and medicine. Although high-titer gutless adenovirus preparations have recently been obtained, the generation of gutless adenovirus vectors remains a time-consuming and difficult process. It is therefore not surprising that no successful clinical trial using those vectors has been conducted so far. A very critical step in the construction of gutless adenovirus vectors is the rescue of the gutless virus from plasmid DNA. This step needs improvement, since the higher the number of gutless viral particles produced in rescue, the lower the number of passages required before purification. We have taken advantage of the adenovirus terminal protein, which can enhance recovery of the virus upon DNA transfection by more than two orders of magnitude. We are developing a technique for purifying the terminal protein in a form that can be easily linked to the ends of linear plasmid DNA used to generate adenoviruses. In Phase I, we successfully purified the terminal protein, and linked it to the ends of first-generation adenoviral DNAs derived from plasmids with almost 100% efficiency. We showed that transfection of such modified DNAs enables recovering the virus only two days after transfection and increases the initial virus titer by two to four orders of magnitude. In Phase II, we plan (1) to optimize the purification of the terminal protein by chromatography and scale up its production in bioreactors and, (2) test the product for the construction of gutless vectors. We expect that our technique will make the production of gutless adenovirus vectors faster, cheaper, and better. It is clear that the TP-ITR method will contribute to generating the high titer gutless virus preparations which are badly needed for animal and clinical experiments. Our system is very simple to use and easily adaptable to the systems for gutless vectors currently in development and also to the most popular systems for the production of first-generation adenoviral vectors.
Small Business Information at Submission:
O.D. 260, INC.
624 N. HILLVIEW DR BOISE, ID 83712
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