Biomedical Engineering Reference
In-Depth Information
event of unintended recombination between constructs (Kay et al.
2001
). In this
way, the engineered viral particles cannot replicate post-infection due to the
absence of the packaging construct and cause the detrimental effects of viral repli-
cation, cell lysis, immunoreaction, and rampant infection.
Viral vectors often utilized include members of the retrovirus family (lentivirus,
oncoretroviruses, spumaviruses), adenovirus, adeno-associated virus, and herpesvi-
rus (Kay et al.
2001
; Verma and Weitzman
2005
). While retroviral vectors are
particularly easy to engineer, more complicated vectors such as adenovirus pose
more of a challenge. The retroviral genome can essentially be swapped out of its
flanking LTRs (long terminal repeats) in exchange for foreign sequence to make the
vector construct and retroviral particles can easily be manufactured in packaging
cell lines (Sinn et al.
2005
). Viruses such as adenovirus, however, require elaborate
recombination strategies and helper-dependent systems to produce functional par-
ticles free of pathogenic and replication-associated genes (Segura et al.
2008
;
Howarth et al.
2010
; Alba et al.
2005
). The complex genetic engineering and opti-
mization of viral gene combinations to create safe and functional constructs are two
hurdles associated with viruses that non-viral gene delivery largely circumvents.
That said, the necessary engineering of viral carriers is rewarded with exceptional
transduction efficiencies due to their ability to efficiently navigate their genome
past numerous cellular barriers. Cellular barriers for effective viral nucleic acid
delivery include passage through cell membrane (either by direct fusion or vesicu-
lar escape) and trafficking through the cytoplasm and the nuclear envelope. As
outlined below, each virus has acquired highly specialized features (due to evolu-
tion and human modifications) allowing them to overcome these barriers and suc-
cessfully express a gene sequence.
3.2.1
Retrovirus
Retroviral infection begins with nonspecific binding to cell surface followed by
specific attachment between the virus's envelope glycoproteins (Env proteins) and
their target receptors (Pizzato et al.
1999
; Haynes et al.
2003
). Following this
binding, the membranes of both the cell and enveloped-virus fuse mediating the
entry of the retroviral nucleocapsid into the cytoplasm (Harrison
2005
). The tro-
pism of retroviral vectors has been greatly expanded by exchanging the wild-type
attachment-protein genes with those from related viruses (in the packaging cell
line or packaging construct), a technique known as pseudotyping (Cronin et al.
2005
). Pseudotyping is most commonly performed with the Env glycoprotein of
vesicular stomatitis virus (VSV-G), which bestows an extremely broad tropism
(Burns et al.
1993
). Following the release of the nucleocapsid into the cytoplasm,
the viral RNA genome is reverse-transcribed into dsDNA (with the exception of
Foamy Virus particles which can reverse-transcribe their genome and deliver a
DNA genome) (Sinn et al.
2005
; Deyle et al.
2010
). Prior to inserting into the
host's genome, the preintegration complex can either be actively imported through
the nuclear envelope by cellular proteins (in the case of lentiviruses, like HIV) or
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