Retargeting Adenovirus Serotype 48 Fiber Knob Domain by Peptide Incorporation

Abstract

Adenovirus type 5 (Ad5) is a commonly used vector for gene therapy, but its efficacy is limited by high seroprevalence and off-target hepatic and splenic sequestration. In order to circumvent these limitations, the use of vectors derived from rare species adenoviruses is appealing. The opportunity to retarget rare species vectors to defined cell types through the incorporation of peptide ligands would be advantageous, particularly in targeting tumors and disseminated metastases. We used predictive structural modeling to assess the CD, DG, HI, and IJ loops of the Ad48 fiber knob and identify optimal incorporation locales for the 20-mer peptide, A20FMDV2 (A20). A20FMDV2 targets ανβ6 integrin, which is overexpressed in human carcinomas. Recombinant Ad48 fiber knob proteins Knob48, Knob48-CD-A20, Knob48-DG-A20, Knob48-HI-A20, and Knob48-IJ-A20 were engineered and purified after expression in Escherichia coli. We confirmed that Knob48, Knob48-CD-A20, and Knob48-IJ-A20 formed stable homotrimers. However, Knob48-DG-A20 and Knob-HI-A20 failed to form a trimer. All A20-modified knob proteins blocked the transduction of Ad5-EGFPA20 via ανβ6, demonstrating that the inserted A20 peptide was functional. In conclusion, we show that the CD and IJ loops of Ad48 represent suitable sites for targeting peptide incorporation. Interestingly, in vitro gene transfer mediated by the non-factor-X-binding Ad48 vector was not sensitive to immunoglobulins and complement when incubated in the presence of mouse serum, unlike Ad5. These data support the future generation of the corresponding Ad48 viral vectors, Ad48-CD-A20 and Ad48-IJ-A20, which may offer favorable characteristics for targeted delivery in vivo.

Pseudotyped αvβ6 integrin-targeted adenovirus vectors for ovarian cancer therapies

“…Predictive 3D models [ 39 ] showed A20 in Ad48 DG and HI loops in a favorably-exposed conformation for receptor binding ( Figure S1 ). A20 insertion into DG and HI loops of recombinant kn48 protein has previously been shown to impair fiber trimerization, suggesting these sites to be suboptimal [ 34 ]. Conversely, in this study DG was the only loop that tolerated A20 insertion in combination with a 13-aa deletion, allowing functional Ad5/kn48 virus production (Figure 1A-1B ).…”

“…Viral genomes were based on luciferase (Luc)-expressing replication-deficient (ΔE1/ΔE3) (Ad5.Luc) and introduced modifications were verified by sequencing [ 20 ]. A20 insertion sites in Ad5 [ 28 ] and Ad48 knob loops [ 34 ] were selected based on conformational compatibility for receptor binding ( Figure S1 ). Ad48 knob sequences were PCR-amplified from previously generated pQE30knob48 plasmids [ 34 ] (primers, Table S1 ) and inserted into Ad5.Luc fiber knob (Ad5 fiber sequence before TLW hinge region (aa 400–402), Ad48 fiber afterwards).…”

Adenovirus serotype 26 utilises sialic acid bearing glycans as a primary cell entry receptor

“…SG13009 E.coli harbouring pREP-4 plasmid and pQE-30 expression vector containing the fiber-knob DNA sequence were cultured in 20ml LB broth with 100μg/ml ampicillin and 50μg/ml kanamycin overnight from glycerol stocks made in previous studies( 76–78 ). 1L of TB (Terrific Broth, modified, Sigma-Aldrich) containing 100μg/ml ampicillin and 50μg/ml were inoculated with the overnight E.coli culture and incubated at 37°C until they reached OD0.6.…”

Designer Oncolytic Adenovirus: Coming of Age

“…The A20 peptide was incorporated into the FIber-knob loops (CD and IJ) of the rare species D adenoviral serotype Ad48, replicating the success of the Ad5.A20 vector. Ad48, in contrast to Ad5, was shown to be insensitive to neutralisation by serum, raising interest in improved systemic delivery 279 .…”