A METHOD OF MAKING ADENOVIRUS AND CORRESPONDING PLASMIDS

摘要

The present disclosure relates to a method of making an adenovirus plasmid comprising a part or all of an adenovirus genome and one or more original restriction sites allowing rapid and flexible manipulation of the adenovirus genome, and methods of preparing adenovirus constructs, for example comprising a transgene. The disclosure also extends to novel intermediates employed in and generated by the method, to plasmids and shuttle vectors of the method and to adenoviruses or adenoviral vectors obtainable from the plasmid and/or method. The disclosure further relates to use of the viruses or vectors, for example obtained from a method disclosed herein, in therapy, such as use in the treatment of cancer.

主权项

1. A method of preparing a shuttle vector comprising a selection marker gene and a low copy bacterial replication of origin, and an adenovirus genome comprising a 5′ ITR, a 3′ ITR, an L5 gene said method comprising the steps:
a) preparing an adenovirus shuttle vector comprising ligating equal proportions the following three fragments:
i) a vector fragment comprising a selection marker gene and a low copy bacterial replication of origin, wherein the 5′ end of the vector fragment starts with a first restriction enzyme site and terminates at the 3′ end of the vector fragment in a second restriction enzyme site,ii) a 5′-arm comprising the 5′ end of the adenovirus genome including the 5′ ITR, wherein the 5′ end of the 5′ arm starts with a second restriction enzyme site and terminates at the 3′ end of the 5′ arm with a third restriction enzyme site,iii) a 3′-arm comprising the 3′ end of the adenovirus genome including the 3′ ITR and the LS gene, wherein the 5′ end of the 3′ arm starts with a third restriction enzyme site andterminates at the 3′ end of the 3′ arm with a first restriction enzyme site, and performing a one-step three-way ligation to join: the 3′ end of the 3′ arm (fragment iii) to the 5′ end of the vector fragment (fragment i) at the first restriction enzyme site, the 3′ end of the vector fragment (fragment i) to the 5′ end of the 5′ arm (fragment ii) at the second restriction enzyme site, and the 3′ end of the 5′ arm (fragment ii) to the 5′ end of the 3′ arm (fragment iii) at the third restriction enzyme site at least the L5 gene, to form a circularised shuttle vector arranged as a first restriction enzyme site followed by a vector fragment followed by a second restriction enzyme site followed by a 5′ arm, followed by a third restriction enzyme site followed by a 3′ arm, b) introducing at least one original restriction site and/or transgene into the shuttle vector in a location between the L5 gene and a site selected from the group comprising an E3 site, an E4 site or both said sites.