Gene Therapy in Bone Regeneration: A Summary of Delivery Approaches for Effective Therapies - Intracellular Delivery: Fundamentals and Applications - page 827

remain cytoplasmic until the nuclear membrane is disrupted in mitosis (De Rijck
et al. 2007 ; Roe et al. 1993 ; Lewis and Emerman 1994 ). With the exception of
lentiviruses, this inability to pass through the nuclear envelope is a major bottle-
neck to most retroviral vectors and hence generally limits their use to cells with
high mitotic activity (Miller et al. 1990 ).

3.2.2 Adenovirus

Cellular internalization is initiated by most adenoviral serotypes through binding of
their fibre knobs to the Coxsackie-adenovirus receptor (CAR) (Bergelson et al.
1997 ). Subsequent binding of their penton base proteins to alpha-V integrins pre-
dominantly results in clathrin-mediated endocytosis (Wickham et al. 1993 ; Meier
and Greber 2004 ). Expanding the tropism of non-enveloped viruses through
pseudotyping is slightly more challenging than their enveloped counterparts due to
the fact that foreign proteins engineered into the virus must be structurally compat-
ible with the native capsid proteins and cannot simply be inserted into a lipid
bilayer (Waehler et al. 2007 ). That said, the tropism of individual adenoviral strains
have been successfully expanded by replacing fibre proteins with homologous pro-
teins from related serotypes, unrelated viruses and even with single-chain variable
fragment antibodies to confer specific targeting (Kanerva et al. 2002 ; Mercier and
Campbell 2004 ; Hedley et al. 2005 ). Once endocytosed, it is generally accepted that
the decrease in pH along the endocytic pathway induces a conformational change
of the penton base proteins causing membrane disruption and permitting escape to
the cytosol (Blumenthal et al. 1986 ; Wang et al. 2000 ). The virion is then trans-
ported to the nucleus in a dynein/dynactin-dependant manner where the dsDNA
genome is actively imported through the nuclear pore complex (NPC) (Kelkar et al.
2004 ; Saphire et al. 2000 ). Nuclear uptake is an elaborate process whereby NPC
docking is mediated by viral hexamer proteins followed by the entry of the genome
and the DNA-condensing proteins mu and VII through the NPC (Trotman et al.
2001 ; Wodrich et al. 2006 ). The host proteins Nup214, histone H1, and hsc70 have
also been implicated in adenovirus docking, capsid disintegration and DNA uptake
(Trotman et al. 2001 ; Saphire et al. 2000 ). This nuclear targeting is a considerable
feat considering that the commonly used serotypes Ad2 and Ad5 have a ~36 Kb
genome (virion size, 70-100 nm), which is much larger than the genome of most
non-viral plasmid-based systems (Davidson et al. 2003 ; McConnell and Imperiale
2004 ; Kennedy and Parks 2009 ). Generally, the adenoviral genome then remains
episomal in the nucleus (not integrating into the host genome) (Harui et al. 1999 ).
The episomal preference of adenoviral vectors, and their ability to actively import
into the nucleus, make them both safer and more efficient compared to most retro-
viral strains. That said, adenoviral vectors carry the disadvantages of occasional
integration, transient transgene expression, and immunological reactivity resulting
from prior exposure (Bangari and Mittal 2006 ). Some studies have focused on
using less common serotypes, than the typically used Ad2 and Ad5, in order to
decrease the possibility of an immune reaction (Loser et al. 2000 ).