Abstract
Recent studies have demonstrated that covalent grafting of a single histidine residue into a twin-chain aliphatic hydrocarbon compound enhances its endosome-disrupting properties and thereby generates an excellent DNA transfection system. Significant increase in gene delivery efficiencies has thus been obtained by using endosome-disrupting multiple histidine functionalities in the molecular architecture of various cationic polymers. To take advantage of this unique feature, we have incorporated L-histidine (N,N-di-n-hexadecylamine) ethylamide (L(H)) in the membrane of hepatocyte-specific Sendai virosomes containing only the fusion protein (F-virosomes (Process for Producing a Targeted Gene (Sarkar, D. P., Ramani, K., Bora, R. S., Kumar, M., and Tyagi, S. K. (November 4, 1997) U. S. Patent 5,683,866))). Such L(H)-modified virosomal envelopes were four times more (p < 0.001) active in terms of fusion with its target cell membrane. On the other hand, the presence of L(H) in reconstituted influenza and vesicular stomatitis virus envelopes failed to enhance spike glycoprotein-induced membrane fusion with host cell membrane. Circular dichroism and limited proteolysis experiments with F-virosomes indicated that the presence of L(H) leads to conformational changes in the F protein. The molecular mechanism associated with the increased membrane fusion induced by L(H) has been addressed in the light of fusion-competent conformational change in F protein. Such enhancement of fusion resulted in a highly efficient gene delivery system specific for liver cells in culture and in whole animals.
Highlights
Completion and annotation of the human genome sequence has significantly enhanced the possibility of using designer DNA for gene therapy
We report for the first time that histidine functionality of a cationic amphiphile enhances F glycoprotein-induced membrane fusion and
An enveloped animal virus belonging to paramyxovirus family, contains two glycoproteins (hemagglutinin neuraminidase (HN) and fusion factor (F)) in the outer leaflet of its lipid bilayer [19]
Summary
Completion and annotation of the human genome sequence has significantly enhanced the possibility of using designer DNA for gene therapy. It turns out that the most up-hill task toward ensuring clinical success of gene therapy is to design safe, efficacious and target-specific transfection vectors Toward designing such gene carriers, significant progress has been made on gene transfection by novel non-glycerol-based histidylated cationic amphiphiles, presumably via the endosome-disrupting properties of the histidine functionalities [2]. The beneficial role of such complexes was clearly envisaged by the amplification of binding and entry of adenovirus particles into host cells Despite all these encouraging results, its failure in targeted gene transfer in whole animal and inherent cytopathic/cytotoxic side effects limit its applications in liver gene therapy [5]. We observed that fusion efficiency of Sendai virus F protein is much reduced in the absence of its native attachment glycoprotein, hemagglutinin neuraminidase (HN) [7] To overcome such impediments, we tested the efficacy of histidine functionality in disrupting endosomes. Modified Sendai Viral Envelopes for Targeted Gene Delivery results in efficient gene delivery. The possible mechanisms of LH-induced activation of F-virosome-target membrane fusion have been investigated
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