David A. Einfeld

Reducing the Native Tropism of Adenovirus Vectors Requires Removal of both CAR and Integrin Interactions

ABSTRACT The development of tissue-selective virus-based vectors requires a better understanding of the role of receptors in gene transfer in vivo, both to rid the vectors of their native tropism and to introduce new specificity. CAR and αv integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. We have constructed a set of four vectors, which individually retain the wild-type cell interactions, lack CAR binding, lack αv integrin binding, or lack both CAR and αv integrin binding. These vectors have been used to examine the roles of CAR and αv integrin in determining the tropism of Ad vectors in a mouse model following intrajugular or intramuscular injection. CAR was found to play a significant role in liver transduction. The absence of CAR binding alone, however, had little effect on the low level of expression from Ad in other tissues. Binding of αv integrins appeared to have more influence than did binding of CAR in promoting the expression in these tissues and was also found to be important in liver transduction by Ad vectors. An effect of the penton base modification was a reduction in the number of vector genomes that could be detected in several tissues. In the liver, where CAR binding is important, combining defects in CAR and αv integrin binding was essential to effectively reduce the high level of expression from Ad vectors. While there may be differences in Ad vector tropism among species, our results indicate that both CAR and αv integrins can impact vector distribution in vivo. Disruption of both CAR and αv integrin interactions may be critical for effectively reducing native tropism and enhancing the efficacy of specific targeting ligands in redirecting Ad vectors to target tissues.

Delivery of Human Immunodeficiency Virus Vaccine Vectors to the Intestine Induces Enhanced Mucosal Cellular Immunity

ABSTRACT Effective vaccines for human immunodeficiency virus type 1 (HIV-1) will likely need to stimulate protective immunity in the intestinal mucosa, where HIV-1 infection causes severe CD4+ T-cell depletion. While replication-competent recombinant adenovirus (rAd) vectors can stimulate adenovirus-specific mucosal immunity after replication, oral delivery of replication-defective rAd vectors encoding specific immunogens has proven challenging. In this study, we have systematically identified barriers to effective gut delivery of rAd vectors and identified sites and strategies to induce potent cellular and humoral immunity. Vector-mediated gene transfer by rAd5 was susceptible to low-pH buffer, gastric and pancreatic proteases, and extracellular mucins. Using ex vivo organ explants, we found that transduction with rAd5 was highest in the ileum and colon among all intestinal segments. Transgene expression was 100-fold higher after direct surgical introduction into the ileum than after oral gavage, with rAd5 showing greater potency than the rAd35 or the rAd41 vector. A single immunization of rAd5 encoding HIV-1 gp140B to the ileum stimulated potent CD8+ T-cell responses in the intestinal and systemic compartments, and these responses were further enhanced by intramuscular rAd5 boosting. These studies suggest that induction of primary immune responses by rAd5 gut immunization and subsequent systemic boosting elicits potent antigen-specific gut mucosal responses.

Binding of Adenoviral Fiber Knob to the Coxsackievirus- Adenovirus Receptor Is Crucial for Transduction of Fetal Muscle

Adenoviral (Ad) infection involves attachment mediated by the Ad fiber protein binding to the coxsackievirus-adenovirus receptor (CAR) of a target cell and internalization facilitated by the interaction of the Ad penton base protein with alpha(v) integrins. To understand the relative importance of the Ad binding and internalization steps for the transduction of fetal skeletal muscle, we used a panel of genetically modified vectors that specifically ablate the fiber-CAR interaction (AdL.F*), the penton base-alpha(v) integrin interaction (AdL.PB*), or both (AdL.PB*F*) to transduce embryonic day 16 (E-16) mouse muscle in vivo and primary E-16 muscle cells in vitro. Quantification of transgene expression and vector genome copies revealed a striking absence of E-16 muscle transduction by AdL.F* and AdL.PB*F*. In contrast, fetal muscle transduction with AdL.PB* was not significantly different than with the unmodified vector. Similar results were observed with in vitro Ad infection studies in primary E-16 muscle cells. From these data we conclude that the fiber-CAR interaction is important for the transduction of fetal muscle by Ad vectors. The high dependence on fiber-CAR binding will impact the development of strategies for Ad vector retargeting to achieve muscle-specific transduction in utero.

875. Adenoviral Vector Targeting |[ndash]| New Targets for Old Capsids

Top of pageAbstract The goal of our adenoviral vector-targeting program is to identify strategies that enhance delivery of therapeutic proteins to the site of disease as well as reduce the impact of the therapy on nontarget cells and tissue. Therapeutic areas under consideration are cancer, ocular disease, vaccine, hearing loss and balance disorders. To accomplish this we have created vectors that are ablated for binding to native CAR and integrin receptors, have introduced specific binding ligands for selective entry into the cells of choice, and have evaluated vector performance in these target therapeutic areas. We have evaluated the pharmacokinetics and biodistribution of tropism-modified vectors in regional (intra-peritoneal) and disseminated cancer disease models and have shown the importance of selective delivery to tumors after regional and systemic administration. Delivery to the eye and ear of tropism-modified vectors results in transduction of new target cells and the modifications may also impact the kinetics of transgene expression. We have demonstrated low-level expression of the normal entry receptors for adenovirus in the inner ear of mice, which is inconsistent with the amount of gene delivery observed by native tropism vectors. Adenovirus capsids ablated of normal receptor interactions (CAR and integrin) are still capable of gene delivery, suggesting that vector entry may occur through alternative pathways. The efficiency of gene delivery in the inner ear by capsid-modified vectors is dependent on target volume and local vector concentration. While efficient delivery within the inner ear is retained by the ablated vector, it has a restricted tropism for gene delivery to tissue outside of the inner ear. These results collectively lend support to the design of targeted adenovirus vectors that could enhance the delivery of the therapeutic protein and thereby be used for multiple clinical applications.

1020. Development and Evaluation of a Genetically-Modified Adenovirus Vector That Specifically Targets Tumors Via the Bloodstream Following Systemic Administration

The development of genetically modified adenovirus vectors capable of targeting tumors following systemic administration is an important goal for the treatment of disseminated cancer. Achieving this goal requires that the native adenovirus coat protein/receptor interactions are removed and replaced with new tumor-selective ligand/receptor interactions. Towards this goal, we have created vectors that are ablated for native receptor binding and that additionally contain a pseudo-receptor binding ligand or a tumor-selective peptide motif inserted into the HI loop of fiber. In vivo experiments showed that ablating both CAR and penton base binding is critical to reduce non-targeted tissue transduction. In addition, pharmacokinetic studies indicated that these tropism modified vectors efficiently enter and persist in the bloodstream following intraperitoneal (i.p.) administration. Interestingly, this sustained circulation was only observed after i.p. administration and not after intravenous administration. Furthermore, the combination of capsid modification and extended blood circulation could achieve receptor-mediated transduction on pseudo-receptor expressing subcutaneous tumor.