Elena Kashentseva

Ectodomain of Coxsackievirus and Adenovirus Receptor Genetically Fused to Epidermal Growth Factor Mediates Adenovirus Targeting to Epidermal Growth Factor Receptor-Positive Cells

ABSTRACT Human adenovirus (Ad) is extensively used for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to target cells expressing marginal levels of the Ad fiber receptor. Therefore, the present generation of Ad vectors could potentially be improved by modification of Ad tropism to target the virus to specific organs and tissues. The fact that coxsackievirus and adenovirus receptor (CAR) does not play any role in virus internalization, but functions merely as the virus attachment site, suggests that the extracellular part of CAR might be utilized to block the receptor recognition site on the Ad fiber knob domain. We proposed to design bispecific fusion proteins formed by a recombinant soluble form of truncated CAR (sCAR) and a targeting ligand. In this study, we derived sCAR genetically fused with human epidermal growth factor (EGF) and investigated its ability to target Ad infection to the EGF receptor (EGFR) overexpressed on cancer cell lines. We have demonstrated that sCAR-EGF protein is capable of binding to Ad virions and directing them to EGFR, thereby achieving targeted delivery of reporter gene. These results show that sCAR-EGF protein possesses the ability to effectively retarget Ad via a non-CAR pathway, with enhancement of gene transfer efficiency.

Engineering of Adenovirus Vectors Containing Heterologous Peptide Sequences in the C Terminus of Capsid Protein IX

ABSTRACT The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.

Double modification of adenovirus fiber with RGD and polylysine motifs improves coxsackievirus-adenovirus receptor-independent gene transfer efficiency.

Adenoviral vectors based on serotype 5 (Ad5) have been widely used to deliver therapeutic genes to different organs and tissues. However, many tissues are poorly infected with Ad5 because of low-level expression of its primary receptor, coxsackievirus-adenovirus receptor (CAR). Two motifs, RGD and polylysine (pK7), have been shown to enhance Ad5 infection via CAR-independent pathways when incorporated into fiber separately. Because the two motifs bind to different cell surface proteins (RGD motif binds to integrins, and pK7 binds to heparan sulfate-containing receptors), we hypothesized that the two motifs function additively to improve gene transfer efficiency. In this study, we sought to improve infectivity of Ad5 by incorporating both RGD and pK7 motifs into fiber. We created an Ad5 vector containing an RGD motif in the HI loop and a pK7 motif at the C terminus of fiber (Ad5.RGD.pK7). Compared with unmodified and singly modified Ad5 vectors Ad5.RGD and Ad5.pK7, the doubly modified Ad5 demonstrated the highest infectivity in both CAR-positive and CAR-negative cells. The enhanced infectivity appeared to be mediated by additive effects of the two motifs. More importantly, Ad5.RGD.pK7 lost the natural CAR-dependent pathway while employing novel targeting mechanisms. This strategy thus may be used to overcome CAR deficiency and to achieve vector retargeting.

Improved gene transfer efficiency to primary and established human pancreatic carcinoma target cells via epidermal growth factor receptor and integrin-targeted adenoviral vectors.

In this study we analyzed two ways of retargeting of Ad-vectors to human pancreatic carcinoma with the aim of enhancing the gene transfer efficiency. First, we analyzed the expression of the epidermal growth factor receptor (EGFR) on primary, as well as established pancreatic carcinoma cells by flow cytometry which revealed high expression levels of EGFR on the surface of these cells. We showed that EGFR-retargeted entry pathway using a bispecific fusion protein formed by a recombinant soluble form of truncated Coxsackie and Adenovirus Receptor (sCAR) genetically fused with human EGF (sCAR-EGF) redirects them to the EGFR leading to an enhanced gene transfer efficiency to pancreatic carcinoma cells. Since flow cytometry revealed absence of CAR expression, but the presence of at least one of both αv integrins on the pancreatic carcinoma cells, a second way of targeting was investigated using a genetically modified Ad vector which has an RGD (Arg-Gly-Asp)-containing peptide inserted into the HI-loop of the fiber knob. This RGD targeted Ad (AdlucRGD) revealed efficient CAR-independent infection by allowing binding to cellular integrins resulting in a dramatic enhancement of gene transfer. These findings have direct relevance for Ad-vector based gene therapy strategies for pancreatic carcinoma.

Construction and Characterization of Adenovirus Serotype 5 Packaged by Serotype 3 Hexon

ABSTRACT Adenovirus serotype 5 (Ad5) has great potential for gene therapy applications. A major limitation, however, is the host immune response against Ad5 infection that often prevents the readministration of Ad5 vectors. In this regard, the most abundant capsid protein, hexon, has been implicated as the major target for neutralizing antibodies. In this study, we sought to escape the host neutralization response against Ad5 via hexon replacement. We constructed a chimeric adenovirus vector, Ad5/H3, by replacing the Ad5 hexon gene with the hexon gene of Ad3. The chimeric viruses were successfully rescued in 293 cells. Compared to that for the control Ad5/H5, the growth rate of Ad5/H3 was significantly slower and the final yield was about 1 log order less. These data indicate that the Ad3 hexon can encapsidate the Ad5 genome, but with less efficiency than the Ad5 hexon. The gene transfer efficacy of Ad5/H3 in HeLa cells was also lower than that of Ad5/H5. Furthermore, we tested the host neutralization responses against the two viruses by using C57BL/6 mice. The neutralizing antibodies against Ad5/H3 and Ad5/H5 generated by the immunized mice did not cross-neutralize each other in the context of in vitro infection of HeLa cells. Preimmunization of C57BL/6 mice with one of the two types of viruses also did not prevent subsequent infection of the other type. These data suggest that replacing the Ad5 hexon with the Ad3 hexon can circumvent the host neutralization response to Ad5. This strategy may therefore be used to achieve the repeated administration of Ad5 in gene therapy applications.

Identification of Sites in Adenovirus Hexon for Foreign Peptide Incorporation

ABSTRACT Adenovirus type 5 (Ad5) is one of the most promising vectors for gene therapy applications. Genetic engineering of Ad5 capsid proteins has been employed to redirect vector tropism, to enhance infectivity, or to circumvent preexisting host immunity. As the most abundant capsid protein, hexon modification is particularly attractive. However, genetic modification of hexon often results in failure of rescuing viable viruses. Since hypervariable regions (HVRs) are nonconserved among hexons of different serotypes, we investigated whether the HVRs could be used for genetic modification of hexon by incorporating oligonucleotides encoding six histidine residues (His6) into different HVRs in the Ad5 genome. The modified viruses were successfully rescued, and the yields of viral production were similar to that of unmodified Ad5. A thermostability assay suggested the modified viruses were stable. The His6 epitopes were expressed in all modified hexon proteins as assessed by Western blotting assay, although the intensity of the reactive bands varied. In addition, we examined the binding activity of anti-His tag antibody to the intact virions with the enzyme-linked immunosorbent assay and found the His6 epitopes incorporated in HVR2 and HVR5 could bind to anti-His tag antibody. This suggested the His6 epitopes in HVR2 and HVR5 were exposed on virion surfaces. Finally, we examined the infectivities of the modified Ad vectors. The His6 epitopes did not affect the native infectivity of Ad5 vectors. In addition, the His6 epitopes did not appear to mediate His6-dependent viral infection, as assessed in two His6 artificial receptor systems. Our study provided valuable information for studies involving hexon modification.

Generation of an adenoviral vector containing an addition of a heterologous ligand to the serotype 3 fiber knob

As an initial assessment of the feasibility of employing the adenovirus serotype 3 (Ad3) fiber knob as a locale for introducing a tropism-modifying motif, we generated an adenoviral vector containing a six-histidine tag genetically fused to the carboxy-terminus of the Ad3 fiber knob. The heterologous tag proved to be accessible for binding in the context of the virion and, moreover, had rendered the modified vector capable of mediating gene transfer through an artificial, non-Ad3 receptor.

Artificial Extension of the Adenovirus Fiber Shaft Inhibits Infectivity in Coxsackievirus and Adenovirus Receptor-Positive Cell Lines

ABSTRACT Recent studies demonstrate that virus-cellular receptor interactions are not the sole determinants of adenovirus (Ad) tropism. It has been shown that the fiber shaft length, which ranges from 6 to 23 β-repeats in human Ads, also influences viral tropism. However, there is no report that investigates whether artificial extension of the shaft alters the infectivity profile of Ad. Therefore, we constructed Ad serotype 5 (Ad5) capsid-based longer-shafted Ad vectors by incorporating Ad2 shaft fragments of different lengths into the Ad5 shaft. We show that “longer-shafted” Ad vectors (up to 32 β-repeats) could be rescued. We also show that longer-shafted Ad vectors had no impact on knob-CAR (coxsackievirus and Ad receptor) interaction compared to wild-type Ad. Nevertheless, gene transfer efficiencies of longer-shafted Ad vectors were lower in CAR-positive cell lines compared to wild-type Ad. We suggest that artificial extension of the shaft can inhibit infectivity in the context of CAR-positive cell lines without modification of knob-CAR interaction.

Localization of the N-terminus of minor coat protein IIIa in the adenovirus capsid

Minor coat protein IIIa is conserved in all adenoviruses (Ads) and is required for correct viral assembly, but its precise function in capsid organization is unknown. The latest Ad capsid model proposes that IIIa is located underneath the vertex region. To obtain experimental evidence on the location of IIIa and to further define its role, we engineered the IIIa gene to encode heterologous N-terminal peptide extensions. Recombinant Ad variants with IIIa encoding six-histidine (6His) tag, 6His, and FLAG peptides, or with 6His linked to FLAG with a (Gly(4)Ser)(3) linker were rescued and analyzed for virus yield, capsid incorporation of heterologous peptides, and capsid stability. Longer extensions could not be rescued. Western blot analysis confirmed that the modified IIIa proteins were expressed in infected cells and incorporated into virions. In the Ad encoding the 6His-linker-FLAG-IIIa gene, the 6His tag was present in light particles, but not in mature virions. Immunoelectron microscopy of this virus showed that the FLAG epitope is not accessible to antibodies on the viral particles. Three-dimensional electron microscopy and difference mapping located the IIIa N-terminal extension beneath the vertex complex, wedged at the interface between the penton base and peripentonal hexons, therefore supporting the latest proposed model. The position of the IIIa N-terminus and its low tolerance for modification provide new clues for understanding the role of this minor coat protein in Ad capsid assembly and disassembly.

Addition of Six-His-Tagged Peptide to the C Terminus of Adeno-Associated Virus VP3 Does Not Affect Viral Tropism or Production

ABSTRACT Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6×His tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6×His)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6×His-tagged VP3mutant recombinant AAV. The 6×His-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6×His-modified AAV were equivalent to those of wild-type particles. The 6×His-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6×His tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6×His-tagged AAV VP3 capsid protein and to utilize the 6×His-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.