Gene Therapy in Bone Regeneration: A Summary of Delivery Approaches for Effective Therapies - Intracellular Delivery: Fundamentals and Applications - page 828

3.2.3 Adeno-Associated Virus

Unlike adenovirus, AAV carries the distinct advantage of not being associated with
any pathogenic conditions and is generally considered to produce a less severe
immune response (Kay et al. 2001 ; Chirmule et al. 1999 ). Unlike adenoviruses,
AAV serotypes used in gene delivery studies bind to a diverse array of cellular
receptors naturally resulting in a variety of preferential cell-types. For example, the
commonly used serotype AAV2 utilizes heparan sulfate proteoglycan and the core-
ceptors FGFR1 and integrin a V b 5 (Summerford and Samulski 1998 ; Summerford
et al. 1999 ; Qing et al. 1999 ). The AAV4 uses sialic acid, as does AAV5, though
with a different carbohydrate linkage and an interaction with PDGF receptors
(Kaludov et al. 2001 ; Di Pasquale et al. 2003 ). The precise mechanism by which
AAV escapes the endocytic pathway after clathrin-mediated endocytosis has not
been fully elucidated. Endosomal acidification is required, but unlike adenovirus,
the intact virion appears to enter the nucleus without the loss its capsid coat; this is
debateable, however, and may be a serotype specific phenomenon (Bartlett et al.
2000 ; Wu et al. 2006 ). The ability of the entire capsid to pass through the NPC is
likely a result of its small size of ~26 nm, as molecules <40-60 kD may passively
diffuse through the NPC (Harbison et al. 2008 ; Weis 2003 ). Following viral uncoat-
ing in the nucleus, the ssDNA load may be converted to dsDNA and then remain
episomal like adenovirus, or integrate into the host's genome if the viral rep gene
is present (McCarty et al. 2004 ; Dyall et al. 1999 ). Much safer compared to retro-
viruses, AAV usually integrates at a highly specific location (AAVS1) in the human
genome on chromosome 19, hence having a lower probability of oncogenic muta-
tion (Giraud et al. 1994 ). AAV does carry disadvantages, however, most notably its
limited payload of ~5 Kb and the necessity of infecting packaging cells with helper
viruses (such as adenovirus or herpesvirus) due to its inability to replicate itself
(Grieger and Samulski 2005 ; Wu et al. 2006 ).

3.2.4 Herpesvirus

In stark contrast to AAV, Herpes simplex virus type I can hold over 30 Kb of
foreign sequence (Pechan et al. 1999 ). HSV infection is more complicated than the
fore-mentioned vectors in that cellular entry requires many protein interactions.
Cell binding is mediated by viral proteins gB and gC interacting with heparan sul-
fate, while gD binds to herpesvirus entry mediators HveA and HveC (Laquerre
et al. 1998 ). Membrane fusion between the cell and the enveloped virion seems to
require the presence of gB, gD, and gH/gL (Fink et al. 2000 ). The tropism of HSV
may be expanded by genetically altering the glycoproteins gB, gD, and more suc-
cessfully gC (Laquerre et al. 1998 ; Anderson et al. 2000 ; Grandi et al. 2004, 2010 ).
Following active uptake into the nucleus, wild-type HSV may either follow a lytic
cycle initiating transcription or (as in the case of engineered constructs) exist in a
latent episomal state (Jackson and DeLuca 2003 ; Fink et al. 2000 ). The transcrip-
tional persistence of the foreign sequences largely depends on what specific class