Singh, Ravi Nandan; (2008) Addressing the Limitations of Adenoviral-Mediated Gene Delivery through Coating the Viral Surface with Lipid Bilayers. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Recombinant adenovirus (Ad) is a powerful tool in gene therapy. However, the ability to deliver Ad to target tissues and cells following systemic administration is limited due to rapid clearance from blood circulation by the mononuclear phagocyte system (MPS), leading to transfection of non-target tissues (primarily liver), vector related toxicity and immunogenicity. The aim of this work was to engineer synthetic lipid envelopes around adenovirus in order to develop a novel gene therapy vector based on a hybrid viral/non-viral vector platform that would address some of the limitations of Ad. The interaction between the virions and phospholipid bilayers was systematically studied in order to develop a protocol for constructing this vector by using self-assembly principles without any alteration of the Ad genome or application of surface conjugation chemistry The hypothesis that envelopment of Ad using different lipid bilayers would produce nanoparticles with characteristics determined by the type of lipid used was tested using a variety of structural, physicochemical, and biological assays. Vector physicochemical characteristics such as size, degree of aggregation, stability in suspension, and surface charge were linked to biological function in vitro and in vivo. The results indicated several key parameters toward the development of stable, non-flocculated suspensions of lipid enveloped Ad, and how such characteristics affected the gene transfer efficiency, organ distribution, and pharmacokinetics of the resulting vectors. Further, these results demonstrated that it was possible to overcome problems of inherent viral tissue affinities and to reduce immunogenicity by introduction of the synthetic envelope. Next, the lipid components of the envelope were altered to optimize the vector for cancer therapy. Inclusion of stealth components like poly(ethylene glycol) (PEG)-lipids in the envelope extended blood circulation time. It was also found that enveloped Ad vectors could be tailored to preferentially accumulate in tumor tissue with little interaction with non-target tissue in vivo. Further, these vectors were demonstrated to possess enhanced tumor-penetration capability in vitro. Finally, the possibility of enhancing the therapeutic effect and reducing the toxicity of conditionally replication competent adenovirus (CRAd) used for cancer therapy was evaluated using both in vitro and in vivo models of human tumors. Although no improvements were observed in cancer therapy, the enveloped CRAd was far less toxic than the unmodified virus. This may lead to the development of cancer targeting vectors with low systemic toxicity, which will greatly broaden the horizon for oncolytic virotherapy of cancer. In summary, the studies in this thesis provide a new paradigm for modification of non-enveloped virions (such as Ad) by application of self-assembly principles and have contributed to a greater understanding of the importance of characterizing and manipulating Ad's physicochemical characteristics to enhance its biological activity. As such, this thesis provides the basis for development of a novel vector platform that may be used for the systemic treatment of primary or broadly disseminated cancer.

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