Replication-defective recombinant adenovirus (Ad) vectors are under development for a wide variety of gene therapy indications. A potential limiting factor associated with virus gene therapy requiring repeated treatment is the development of a humoral immune response to the vector by the host. In animal models, there is a dose-dependent rise in neutralizing antibodies after primary vector administration, which can preclude effective repeat administration. The strategy we have developed to circumvent the neutralization of adenovirus vectors by antibodies is to mask their surface by covalent attachment of the polymer polyethylene glycol (PEG). Covalent attachment of PEG to the surface of the adenovirus was achieved primarily by using activated PEG tresylmonomethoxypolyethylene glycol (TMPEG), which reacts preferentially with the epsilon-amino terminal of lysine residues. We show that the components of the capsid that elicit a neutralizing immune response, i.e., hexon, fiber, and penton base, are also the main targets for PEGylation. Several protocols for PEGylation of an adenovirus vector were evaluated with respect to retention of virus infectivity and masking from antibody neutralization. We show that covalent attachment of polymer to the surface of the adenovirus can be achieved with retention of infectivity. We show further that PEG-modified adenovirus can be protected from antibody neutralization in the lungs of mice with high antibody titers to adenovirus, suggesting that PEGylation will improve the ability to administer Ad vectors on a repeated basis.

To help clarify the role of DBF2, a previously described cell cycle protein kinase, high copy number suppressors of the dbf2 mutation were isolated. Three open reading frames (ORF) have been identified. One ORF encodes a protein which has homology to a human small nuclear riboprotein, while the remaining two are genes which have been identified previously, SIT4 and SPO12. SIT4 is known to have a role in the cell cycle but the nature of the interaction between SIT4 and dbf2 is unclear. SPO12 has until now been implicated exclusively in meiosis. However, we show that SPO12 is expressed during vegetative growth, moreover it is expressed under cell cycle control coordinately with DBF2. SPO12 is a nonessential gene, but it becomes essential in a DBF2 delete genetic background. Furthermore, detailed analysis of the cell cycle of SPO12 delete cells revealed a small but significant delay in mitosis. Therefore, SPO12 does have a role during vegetative growth and it probably functions in mitosis in association with DBF2.