Genetic incorporation of HSV-1 thymidine kinase into the adenovirus protein IX for functional display on the virion

Abstract

Adenoviral vectors have been exploited for a wide range of gene therapy applications. Direct genetic modification of the adenovirus capsid proteins has been employed to achieve alteration of vector tropism. We have defined the carboxy-terminus of the minor capsid protein pIX as a locus capable of presenting incorporated ligands on the virus capsid surface. Thus, we sought to exploit the possibility of incorporating functional proteins at pIX. In our current study, we incorporated the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) within pIX to determine if a larger protein of this type could retain functionality in this context. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional HSV-1 TK as a component of their capsid surface. DNA packaging and cytopathic effect were not affected by this genetic modification to the virus, while CAR-dependent binding was only marginally affected. Using an in vitro [3H]-thymidine phosphorylation assay, we demonstrated that the kinase activity of the protein IX-TK fusion protein incorporated into adenoviral virions is functional. Analysis of cell killing after adenovirus infection showed that the protein IX-TK fusion protein could also serve as a therapeutic gene by rendering transduced cells sensitive to gancyclovir. Using 9-[4-[18F]-fluoro-3-(hydroxymethyl)butyl]guanine ([18F]-FHBG; a positron-emitting TK substrate), we demonstrated that we could detect specific cell binding and uptake of adenoviral virions containing the protein IX-TK fusion protein at 1 h post-infection. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional HSV-1 TK as a component of their capsid surface. The alternative display of HSV-1 TK on the capsid may offer advantages with respect to direct functional applications of this gene product. In addition, the determination of an expanded upper limit of incorporable proteins on pIX highlights its unique utility as a locus for placement of functional vector constructs.

Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping

“…Besides its role as a structural protein, it is a transcriptional activator (Rosa-Calatrava et al, 2001;Sargent et al, 2004a) and reorganizes promyelocytic leukaemia (PML) nuclear bodies (Rosa-Calatrava et al, 2003) (reviewed by Parks, 2005). Protein IX has been extensively studied as a platform to express foreign peptides on the surface of the capsid (Dmitriev et al, 2002;Le et al, 2004;Li et al, 2005;Meulenbroek et al, 2004;Vellinga et al, 2004Vellinga et al, , 2007. Since protein IX has been so well characterized, we selected it as a fusion partner to test whether 2A sequences could be used to express foreign genes in adenoviruses.…”

Transductional targeting of adenovirus vectors for gene therapy

“…132,133 Further, other Ad vectors harboring complex ligands at the pIX locale have been reported. [134][135][136] As a whole, these studies have established pIX as a highly relevant capsid locale marked by the highest structural compatibility, with diverse targeting and imaging ligands observed to date.…”

“…For example, incorporation of genetically modified fibers, combined with imaging motifs on the pIX protein, could be used to simultaneously target tumor cells while monitoring viral replication and spread. In this regard, in addition to optical imaging-based ligands that have been incorporated at pIX as mentioned above, recently Li et al 136 have been successful in incorporating the enzyme Herpes Simplex Virus thymidine kinase (HSV-tk) at this capsid locale. This enzyme is compatible with available PET imaging ligands such as 18 F-penciclovir, providing an imaging system for viral replication that can directly be translated for clinical applications.…”

Assessment of Genetic Shielding for Adenovirus Vectors

“…To prove this concept, we determined that the minor capsid protein, pIX, would be a suitable anchorage position for the shielding molecule. This is due to the large body of work indicating that carboxy terminus of pIX can be fused to an array of molecules including polylysine [29], the biotin acceptor peptide (BAP) [30] and larger complex proteins including various fluorescent proteins, HSV thymidine kinase (TK) and TK fusions [31][32][33][34][35][36]. In these studies it was demonstrated that virus viability was not significantly affected and that the pIX-fused molecules retained functionality.…”

“…In addition to using hALB directly fused to pIX we decided to utilize another self-protein, alpha-1-antitrypsin (A1A), in parallel for direct incorporation. Although it is not as large as hALB, A1A is of a comparable size to HSV-TK, which has been successfully fused to pIX in replicating Ad vectors [31]. A1A is a serine proteinase inhibtitor with a primary function to inhibit the enzyme neutrophil elastase.…”