Biomedical Engineering Reference
In-Depth Information
of vector was used: a recombinant HSV-1 vector or a HSV-derived amplicon vector.
A rHSV-1 vector is replication deficient and can be produced in large quantities but
its transgene expression is transient; additionally, it still contains large regions of
the wild-type genome and carries the risk of expressing toxic and immunogenic
proteins (Verma and Weitzman
2005
). HSV-derived amplicon vectors only carry
essential
cis
-acting sequences along with a transgene that can be expressed long-
term, but their production requires a replicating helper virus or the use of cosmids
or artificial chromosomes expressing the essential structural proteins (Smith et al.
1995
; Stavropoulos and Strathdee
1998
; Kong et al.
1999
).
The appropriate choice of a viral vector for a clinical application will ultimately
depend on the nature of the condition being treated in terms of target cell-type,
time-frame of protein expression, size of the transgene needed, and the degree of
risk appropriate for the condition. Beyond their direct utility, viral vectors provide
useful models of nucleic acid delivery and their effective mechanisms may be
exploited to create safer non-viral vectors.
3.3
Mimicking Viral Mechanisms for the Purpose
of Non-viral Gene Delivery
Non-viral carriers share general properties with viral particles, such as the nano-
scale size and a capacity to penetrate the plasma membrane. With non-specific
carriers (i.e., in the absence of specific ligands), plasmid particle uptake appears to
mostly follow clathrin-mediated endocytosis, however some particles may enter the
cell via other forms of endocytosis. The physiochemical properties of particles
appear to influence whether clathrin- or caveolae-mediated endocytosis is the major
uptake method. For example, PLGA nanospheres alone were shown to be mainly
taken up via clathrin-mediated endocytosis, however, surface coating of such
nanospheres with polysorbate 80 led to equivalent uptake with clathrin- and
caveola-mediated endocytosis (Tahara et al.
2010
). In an effort to increase particle
uptake, many groups have focused on modifying gene carriers with specific
peptides that display cell affinity. The expectation is that increased cell membrane
binding will lead to increased complex uptake, ultimately improving the transfec-
tion efficiency. This is generally accomplished through incorporating short viral
peptides into the non-viral system permitting binding to and penetration through
the plasma membrane, and targeting to and through the nuclear envelope (Fig.
3
).
3.3.1
Cellular Uptake and Vesicular Escape
The internalization of carrier/DNA complexes by clathrin-mediated endocytosis or
other mechanisms (such as caveolae-mediated endocytosis, clathrin/caveole-
independent endocytosis and macropinocytosis) must be followed by vesicular
escape to allow nuclear uptake. The fate of an endosome is dependent on the
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