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17.4 Cationic polymer method of transgenesis

• a.  Lipofection : This method involves the use of cationic lipids mixed with DNA in aqueous solution forming liposomes that encapsulate DNA. Such DNA preparations are taken in either by pinocytosis or phagocytosis, depending on the cell type. Use of liposomes in vivo is limited by its rapid plasma clearance and high toxicity.

b.  Cationic peptides : These are chain of basic amino acids, which compact DNA into spherical complexes, or chromatin components such as histones or protamine, which compact DNA in a structured manner allowing it to enter cell by their interaction with sulfated membrane-bound proteoglycans.

c.  Polyethylenimine : Polyethylenimine is a linear or branched polymer with many protonable amino nitrogen atoms that allows efficient DNA condensation and cell entry. The polymer behaves as an effective ‘proton sponge' that causes the rupture of endosome by osmotic swelling and releases the polyethylenimine–DNA complexes into the cytoplasm.

d.  Receptor-mediated delivery : In this method polycations conjugated DNA bound to a cell-specific ligand is targeted to cells via receptor-mediated internalization.


All these non viral vectors have proven well in in vitro transfection but lacks in in vivo transfection efficiency and only allow transient transgene expression. This inefficiency of non viral vectors can be attributed to the following:

•  Interaction of the non viral–DNA complex with blood plasma proteins, undesirable cells and extracellular matrix.

•  Inability to escape from liposome or endosome enclosed moiety.

•  Vulnerability to cytoplasmic degradative enzymes.

•  Inability to pass through the double membrane nuclear envelope and subsequent degradation during breakdown of nuclear membrane at mitosis.

Thus to increase the in vivo stability of non viral vectors novel modular vectors have been developed. These vectors have reduced affinity for intracellular proteins and cell surfaces and posses ligands for receptor-mediated endocytosis, peptide sequences that assist DNA compaction, endosomal disruption sequences and nuclear-import signals. Modular vectors can be easily modulated and they also mimic the ability of viruses to overcome the cellular barriers to DNA delivery. GD5 is one such modular vectors, which forms complex with DNA using DNA binding domain (DBD) of GAL4 transcription factor, attaches to tumor cells via a single chain antibody against ERBB2 antigen leading to receptor mediated endocytosis and facilitates endosomal escape using translocation domain of diphtheria toxin in acidic pH.

Modular vectors can provide efficient delivery of therapeutic gene complexed to non viral vectors but stable nuclear maintenance and replication of such vectors can be achieved by either site specific integration into safe genomic regions or maintenance of extra chromosomal segments.