Challenges and Opportunities in Gene Delivery - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 69

To further understand the consequences of cotrafficking the cationic lipids and DNA to
the nucleus, microinjection of DNA complexes with cationic lipid or polymer directly
into the nucleus has demonstrated the accessibility of the complexed transgene for
transcription. When the cationic lipids dimyristyloxypropyl-3-dimethyl-hydroxyethyl
ammonium (DMRIE)-DOPE, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),
or dioleoylglycerylspermine (DOGS) were used, no transgene expression was detected
after nuclear microinjection of complexes containing an excess of positive charge
[149,150] . Decreasing the charge ratio of DMRIE-DOPE complexes to less than 0.5
(cationic lipid)-1 (anionic DNA) demonstrated similar levels of transgene expression
such as were observed for plasmid alone [150] . These studies indicated the absence
of free DNA for transcription in the nucleus if complexed with an excess of positive
charge, and thus the inability of the DNA to be released from the cationic lipid com-
plex inside the nucleus. In the case of complexes of DNA with an excess of the cat-
ionic polymers, such as poly-L-lysine and PEI, both the polymers showed a significant
extent of transgene expression, demonstrating the accessibility of DNA for transcrip-
tion and transgene expression [149] . Such observations are consistent with an ability of
these cationic polymers to remain bound to the plasmid after endosomal escape while
facilitating nuclear translocation and subsequent transcription.
2.4.4 Transgene Expression and Post-translational Changes
in Proteins
Even after successfully entering the nucleus, the transcription and translation of the
DNA must efficiently happen for the formation of desired therapeutic protein at the
desired rate and quantity. The time period for which transgene is expressed depends
on the origin of the DNA and the vector used. The disposition of DNA is an impor-
tant factor in the expression period of the transgene in cells. Although viral vectors
like retroviruses, adenoviruses, and adeno-associated viruses have a tendency to
integrate the host genome, thus providing long-term transgene expression, they may
prove to be carcinogenic in nature if improperly integrated or mutated during cell
division; hence, they are not safe to use [186] . When the plasmid DNA by the non-
viral vector is inserted into the nucleus, the transgene on a plasmid is not integrated
into the host genome, thus providing transient gene expression. The plasmid DNA
will be reduced in copy number according to cell division (in dividing cells), in addi-
tion to sustaining loss by degradation by cytoplasmic nuclease. However, if a pro-
moter providing site-specific integration is added to the plasmid, then it may be used
to provide transgene expression for a long period. However, as compared to viral
vectors, these DNAs existing as extrachromosomal DNAs are likely to be safer from
the viewpoint of cancer promotion.
After entering the cell nucleus, with or without integration into the host's genome,
the desired DNA transcription is a vital necessity during transgene expression. The
transcription of the gene on DNA occurs to form the mRNA. However, proper tran-
scription control of the transgene is an important issue in forming the therapeutically
effective protein. The transcription of the DNA is dependent on the promoter region
of DNA. The promoter contains a binding site for the RNA polymerase and the