Shariat-Panahi, Ali Reza; (2004) Genetically engineered hepatitis B surface antigen: A potential vector for liver-directed gene therapy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The liver is the body's main synthetic and metabolic organ and one of the main targets for gene therapy. Conventional viral and synthetic gene delivery vectors have so far resulted in limited clinical application due to concerns over viral re-activation, immune-toxic responses and lack of tissue specificity. Recently, novel composite vectors have emerged which incorporate a synthetic or viral base with a cell-targeting moiety. Hepatitis B surface antigen (HBsAg) has been suggested as a potential liver-specific targeting protein, due to its highly hepato-tropic properties. This thesis describes work earned out to clone, express, characterise recombinant HBsAg in vitro and attempt to utilise it as part of a liver-targeting vector. The full-length large (L) surface antigen gene was cloned into a baculovirus expression system and expressed in insect cells. Secretion of recombinant HBsAg into culture medium was severely retarded due to intracellular retention. A variant HBsAg construct was engineered by fusing the hepatotropic preS1 domain (amino acids 21-47) onto the carboxyl terminal domain of the S gene, in order to eliminate the retention sequences in preS1. The engineered constructs (HBS-TS and HBS-TS-[His]) were expressed and purified from mammalian COS7 cells. The recombinant HBsAg products were spherical particles of ~20nm in diameter with buoyant densities of 1.21g/ml, and thus very similar to wild type HBsAg particles found in infected plasma. Western blot analysis revealed a principle protein of 27 kDa, reactive to anti-S and anti-preS1 antibodies. HBS-TS particles, radio-labelled with 35S Methionine, demonstrated specific binding to hepatic cells, while HBS-TS probed with fluorescent antibodies showed extensive binding to primary human hepatocytes. Two composite, lipid-based vectors were constructed using HBS-TS as the targeting ligand within a LipofectAMINE-DNA complex and second a DOPE-DOGS-DNA composite. Both resulted in limited gene transfer and conferred no improvement on gene transfer efficiency. Reconstituted HBS-TS micelles were produced, which demonstrated high hepato-tropicity and a potential for use as liver-targeting vectors.

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