RESUMO
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.
Assuntos
Técnicas de Transferência de Genes , Vírus Sendai/metabolismo , Proteínas do Envelope Viral/química , Animais , Cátions/química , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Dicroísmo Circular , DNA/química , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Endossomos , Feminino , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Histidina/química , Humanos , Imuno-Histoquímica , Cinética , Lipídeos/química , Fígado/citologia , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Modelos Químicos , Polímeros/química , Conformação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , TransfecçãoRESUMO
Herein, employing a previously reported disulfide-linker strategy, we have designed and synthesized a novel cationic lipid 2 with a disulfide-linker and its non-disulfide control analog lipid 1. The relative efficacies of lipids 1 and 2 in transfecting CHO, COS-1 and MCF-7 cells were measured using both reporter gene and whole cell histochemical staining assays. In stark contrast to the expectation based on the disulfide-linker strategy, the control non-disulfide cationic lipid 1 showed phenomenally superior in vitro transfection efficacies to its essentially transfection incompetent disulfide counterpart lipid 2. Results in DNase I protection experiments and the electrophoretic gel patterns in the presence of glutathione, taken together, are consistent with the notion that the success of the disulfide-linker strategy may depend more critically on the DNase I sensitivity of the lipoplexes than on the efficient DNA release induced by intracellular glutathione pool.
Assuntos
Dissulfetos/síntese química , Transfecção/métodos , Animais , Neoplasias da Mama , Células CHO , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetinae , Desoxirribonuclease I/metabolismo , Desenho de Fármacos , Humanos , Indicadores e ReagentesRESUMO
The clinical success of gene therapy is critically dependent on the development of efficient and safe gene delivery reagents, popularly known as "Transfection Vectors". The transfection vectors commonly used in gene therapy are mainly of two types: viral and non-viral. The efficiencies of viral transfection vectors are, in general, superior to their non-viral counterparts. However, the myriads of potentially adverse immunogenic aftermaths associated with the use of viral vectors are increasingly making the non-viral gene delivery reagents as the vectors of choice. Among the existing arsenal of non-viral gene delivery reagents, the distinct advantages associated with the use of cationic transfection lipids include their: (a) robust manufacture; (b) ease in handling & preparation techniques; (c) ability to inject large lipid:DNA complexes and (d) low immunogenic response. The present review will highlight the successes, set-backs, challenges and future promises of cationic transfection lipids in non-viral gene therapy.