Natalya Belousova

Genetic Targeting of an Adenovirus Vector via Replacement of the Fiber Protein with the Phage T4 Fibritin

ABSTRACT The utility of adenovirus (Ad) vectors for gene therapy is restricted by their inability to selectively transduce disease-affected tissues. This limitation may be overcome by the derivation of vectors capable of interacting with receptors specifically expressed in the target tissue. Previous attempts to alter Ad tropism by genetic modification of the Ad fiber have had limited success due to structural conflicts between the fiber and the targeting ligand. Here we present a strategy to derive an Ad vector with enhanced targeting potential by a radical replacement of the fiber protein in the Ad capsid with a chimeric molecule containing a heterologous trimerization motif and a receptor-binding ligand. Our approach, which capitalized upon the overall structural similarity between the human Ad type 5 (Ad5) fiber and bacteriophage T4 fibritin proteins, has resulted in the generation of a genetically modified Ad5 incorporating chimeric fiber-fibritin proteins targeted to artificial receptor molecules. Gene transfer studies employing this novel viral vector have demonstrated its capacity to efficiently deliver a transgene payload to the target cells in a receptor-specific manner.

Modulation of Adenovirus Vector Tropism via Incorporation of Polypeptide Ligands into the Fiber Protein

ABSTRACT The efficacy of adenovirus (Ad)-based gene therapy might be significantly improved if viral vectors capable of tissue-specific gene delivery could be developed. Previous attempts to genetically modify the tropism of Ad vectors have been only partially successful, largely due to the limited repertoire of ligands that can be incorporated into the Ad capsid. Early studies identified stringent size limitations imposed by the structure of the Ad fiber protein on ligands incorporated into its carboxy terminus and thus limited the range of potential ligand candidates to short peptides. We have previously identified the HI loop of the fiber knob domain as a preferred site for the incorporation of targeting ligands and hypothesized that the structural properties of this loop would allow for the insertion of a wide variety of ligands, including large polypeptide molecules. In the present study we have tested this hypothesis by deriving a family of Ad vectors whose fibers contain polypeptide inserts of incrementally increasing lengths. By assessing the levels of productivity and infectivity and the receptor specificities of the resultant viruses, we show that polypeptide sequences exceeding by 50% the size of the knob domain can be incorporated into the fiber with only marginal negative consequences on these key properties of the vectors. Our study has also revealed a negative correlation between the size of the ligand used for vector modification and the infectivity and yield of the resultant virus, thereby predicting the limits beyond which further enlargement of the fiber knob would not be compatible with the virions integrity.

Genetically Targeted Adenovirus Vector Directed to CD40-Expressing Cells

ABSTRACT The success of gene therapy depends on the specificity of transgene delivery by therapeutic vectors. The present study describes the use of an adenovirus (Ad) fiber replacement strategy for genetic targeting of the virus to human CD40, which is expressed by a variety of diseased tissues. The tropism of the virus was modified by the incorporation into its capsid of a protein chimera comprising structural domains of three different proteins: the Ad serotype 5 fiber, phage T4 fibritin, and the human CD40 ligand (CD40L). The tumor necrosis factor-like domain of CD40L retains its functional tertiary structure upon incorporation into this chimera and allows the virus to use CD40 as a surrogate receptor for cell entry. The ability of the modified Ad vector to infect CD40-positive dendritic cells and tumor cells with a high efficiency makes this virus a prototype of choice for the derivation of therapeutic vectors for the genetic immunization and targeted destruction of tumors.

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.

Productive Replication of Human Adenovirus Type 5 in Canine Cells

ABSTRACT Development of immunocompetent patient-like models that allow direct analysis of human adenovirus-based conditionally replicative adenoviruses (CRAds) would be beneficial for the advancement of these oncolytic agents. To this end, we explored the possibility of cross-species replication of human adenovirus type 5 (Ad5) in canine cells. With a panel of canine tumor cell lines of both epithelial and mesenchymal derivations, we demonstrate that human Ad5 can productively infect canine cells. Since the biological behavior and clinical presentation of certain dog tumors closely resemble those of their human counterparts, our results raise the possibility of exploiting canine models for preclinical analysis of candidate CRAd agents designed for human virotherapy.

In vivo analysis of a genetically modified adenoviral vector targeted to human CD40 using a novel transient transgenic model.

Retargeting is necessary to overcome the limitations of adenovirus (Ad)‐based gene therapy vectors. To this end, we previously constructed an adenovirus with the fiber knob domain replaced by a fibritin trimerization motif fused to the CD40 ligand (Ad5Luc.FF/CD40L). We demonstrated the utility of this fiber replacement strategy for targeting CD40 (hCD40) on human dendritic cells in vitro. The in vivo targeting capacity of this virus, however, is unknown, and there is a limited repertoire of animal models that present hCD40 at an accessible site. Therefore, a new animal model for evaluating CD40‐targeted vectors is required.

122. Expression of hCD40 in the Pulmonary Endothelium Targeted Via a Fiber-Modified Adenovirus Improves Gene Delivery to the Lung

The lung is an attractive target for gene therapy since many diseases such as cystic fibrosis, lung cancer, primary pulmonary hypertension and acute respiratory distress syndrome require novel therapeutic approaches. However, pulmonary gene transfer by adenovirus (Ad) vectors via the vascular route has proven to be limited with respect to efficiency due to low expression of human coxsackie adenovirus receptor (hCAR) in the lung. Conversely, the liver expresses high levels of hCAR leading to ectopic transduction and the risk of liver damage. Therefore, development of CAR-independent gene transfer strategy to the lung is needed. We recently constructed an Ad in which the fiber was replaced by human CD40 ligand (Ad5 Luc FF / CD40L) and showed the utility of this fiber replacement strategy for genetic targeting of the virus to human CD40 (Belousova et al. J. Virol 77:11367–77, 2003). In addition, we recently showed the promoter for vascular endothelial growth factor receptor 1 (fms-like tyrosine kinase-1; flt-1) has high activity in pulmonary endothelial cells, low activity in hepatocytes (Nicklin et al. Hypertension 38:65–70, 2001), and also provided transcriptional targeting to the lung vasculature (Reynolds et al. Nat. Biotechnol.19:838–42, 2001). We hypothesized that hCD40 expression, transcriptionally targeted to the vascular endothelium via the flt-1 promoter, could provide CD40-targeted gene delivery to the lung in a CAR-independent manner. We constructed an E1-deleted recombinant adenovirus expressing hCD40 driven by the flt-1 promoter (Ad5flt1-hCD40). We then analyzed hCD40 expression and flt-1 activity in several cell lines by luciferase assay and fluorescence-activated cell sorting (FACS) analysis. We selected NTERA-2 cells as low hCD40 and flt-1 positive cell line, and HeLa cells as low hCD40 and flt-1 negative cell line as control for further experiments. Forty-eight hours after the first infection with Adflt1-hCD40 and the control virus, we quantitated hCD40 expression on the cell surface by FACS analysis. Adflt1-hCD40 infected NTERA-2 cells had markedly increased hCD40 expression compared to either control virus infected cells or flt-1 negative HeLa cells. Further, we evaluated infectivity enhancement gained by increased hCD40 in the flt-1 positive cell line. Forty-eight hours after the first infection, cells were infected with Ad5 Luc FF / CD40L vectors at an MOI of 1000 viral particles / cell to determine infectivity enhancement accrued via CD40 targeting. Luciferase assays were performed 24 hours post second infection. Ad5 Luc FF / CD40L vector infectivity in Ad flt-1 hCD40 infected NTERA-2 cells showed 13-fold greater luciferase reporter gene activities versus negative control virus infected cells. These data suggest that expression of hCD40 in pulmonary endothelial cells using recombinant Ad vectors containing hCD40 gene under the control of the flt-1 promoter and targeted with a novel fiber modified Ad vector can increase the efficacy and specificity of adenoviral gene therapy for lung disease.