Gene Therapy in Bone Regeneration: A Summary of Delivery Approaches for Effective Therapies - Intracellular Delivery: Fundamentals and Applications - page 832

(Brunner et al. 2000 ). This 'passive' uptake seems to be operational with polymeric
carriers as well, since our recent studies with lipid-substituted polymers also indi-
cate a strong correlation between the localization of the particles in the nuclear
periphery and the ability of a cell to express a transgene (Hsu et al. 2010 ).
Most viral vectors are far more effective at nuclear delivery and hence integrating
their targeting peptides into non-viral systems has become a common approach to
overcoming this bottleneck. Studies using nuclear import signals have overwhelm-
ingly utilized the Large T-antigen NLS (nuclear localization sequence) of SV40
(Simian Virus 40). This monopartite NLS binds importin-a, which itself has a
bipartite NLS that can bind to importin-b resulting in the import of the entire com-
plex through the nuclear pore (Fontes et al. 2000 ). A number of approaches have
been undertaken to facilitate nuclear DNA delivery using the SV40 NLS. Plasmid
DNA has been modified with the peptide by simple electrostatic interactions, pep-
tide nucleic acid coupling, streptavidin-biotin systems, and covalent linking
(Aronsohn and Hughes 1998 ; Branden et al. 1999 ; Ludtke et al. 1999 ; Sebestyen
et al. 1998 ). The peptide can also be associated with polymer or lipid-based carriers
instead of being linked to the DNA directly (Talsma et al. 2006 ; Chan et al. 2000 ;
Kurihara et al. 2009 ). While eclipsed by the use of SV40 NLS, other viral peptides
can facilitate nuclear targeting of non-viral carriers as well. The HIV-derived TAT
peptide, mentioned above for its membrane-destabilizing properties, can also serve
as a nuclear-localizing agent when complexed with DNA (Rudolph et al. 2003 ).
Similarly, the mu peptide from the core complex of adenovirus was initially used
to condense DNA but was later found to have nuclear localizing properties when
used in liposomal formulations (Murray et al. 2001 ; Tagawa et al. 2002 ; Keller
et al. 2003 ).
Although nuclear transcription of exogenous DNA is not generally thought to be
rate-limiting, the choice of sequences surrounding the transgene can significantly
influence expression. Plasmids with viral sequences have had poorer expression
compared to non-viral promoters; following robust expression, a steady decline in
transgene expression was observed in vitro and in vivo (Aviles et al. 2010 ). In addi-
tion to this steady decline, the immune response to foreign material must be con-
sidered. Unmethylated CpG plasmid DNA is recognized by Toll-like Receptor 9
(Hemmi et al. 2000 ), and can lead to cytokine production, inflammation and other
undesirable immune responses.

4 Current State of Affairs on Non-viral Gene Delivery

for Bone Regeneration

Local injection of plasmid, rather than systemic injection, is favoured for bone
regeneration because of undesired effects should the recombinant protein be
expressed ectopically. Injections of the therapeutic agents are advantageous with
respect to their minimum invasiveness compared to surgical implantation. The lat-
ter approach, however, enables one to employ biomaterial scaffolds whose