Biomedical Engineering Reference
In-Depth Information
2.5
Gene Expression
It is still unclear how and when DNA delivered by nonviral vectors is released. It is
commonly thought that genomic DNA displaces the DNA condensing agents in the
nucleus, thus releasing the therapeutic gene and enabling gene expression (Remy
et al. 1995 ). Upon arrival into the nucleus, exogenous genes must be expressed to
perform their therapeutic effects. Episomal transgene retention may result in short
term expression, while transgenic insertion into the host genome is always neces-
sary for a stable, long term expression. Several types of DNA elements have been
employed to direct genome insertion, including transposons, site-specific elements,
and transcription-regulating promoters among them (Yant et al. 2000 ; Held et al.
2005 ; Auricchio et al. 2002 ). Transposon-mediated genome integration has the
drawback of random insertion into the host genome, which may elicit unwanted
side effects. This can be overcome via site specific integration using site-specific
elements such as FC31 integrase or hybrid systems containing DNA binding
domain. However, the limitations of these strategies have spurred scientists to
explore more efficient approaches, such as transcription-regulating promoters, to
achieve desirable transgene expression.
3
Gene Vectors Based on Synthetic Polymers
3.1
Polycations with Peptide Backbone
3.1.1
Polylysine
Poly(L-lysine) (PLL, as shown in Table 1 ) is available in a large variety of
molecular weights, which are generally synthesized by the polymerization of
N-carboxyanhydride of e-(benzyoxycarbonyl)-L-lysine combined with a subse-
quent de-protection(Deming 2006 ). PLL is one of the first cationic polymers used
for gene delivery investigation (Zauner et al. 1998 ). The epsilon amino groups of
PLL are positively charged at physiological pH, and therefore they can ionically
interact with the negatively charged phosphate groups of DNA or RNA, condensing
the related gene species into polyplexes. Depending on preparation methods and
formulations, the structure of polyplexes could be toroid, rod-like, or spherical
particle (Wagner et al. 1991 ; Perales et al. 1994 ). PLL itself, however, is a poor
delivery vector for DNA. Even in the presence of chloroquine, only moderate trans-
fection was achieved (Pack et al. 2005 ). Various targeting ligands, on the other
hand, can greatly enhance in vitro and in vivo delivery efficiency of PLL-DNA
polyplexes. For examples, asialoorosomucoid glycoprotein(Wu and Wu 1987 ),
transferring (Wagner et al. 1990 ), sugars like lactose and mannose (Erbacher et al.
1995 ; Ferkol et al. 1996 ), folate (Mislick et al. 1995 ), RGD-containing peptides
(Hart et al. 1995 ) and antibodies (Suh et al. 2001 ) have been covalently conjugated
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