Biomedical Engineering Reference
In-Depth Information
The immune response against AAV type 2-based vectors is an important consideration for
use in gene therapy. Determining the preexisting immunity due to naturally acquired infec-
tions is essential. Studies using seropositive antibodies against AAV-2A were determined to be
50-96%, with 18-67.5% being neutralized. 49-51 Also, in most therapeutic applications it is
necessary to readminister the vector. Chirmule et al 52 reported the efficacy of vector
readministration in skeletal muscle of mice and rhesus monkeys, even though neutralizing
antibodies were produced. 52 Other papers described animal experiments where neutralizing
antibodies were shown to eliminate 46,53 or greatly reduce 52,54 the levels of transgene expression
of the readministered vector. The variability of the outcome underscores the need to better
understand the immune response against AAV-2.
A major disadvantage of the AAV type 2 as a gene therapy vector is its large-scale produc-
tion. Recently, methods for producing a high titer 55 and purification 56 were published. These
advances will allow further studies using AAV vectors and make it available for clinical trials.
Another important characteristic and consideration for the use of AAV as a vector system, is its
natural tropism, which is predominantly skeletal muscle, neurons, and hepatocytes. 57 The pri-
mary AAV type 2 (AAV-2) receptor was recently shown to be membrane-associated heparan
sulphate proteoglycan (HSPG), mediating both AAV attachment to and transduction of target
cells. 58 The receptor characterization explains the broad host attachment and transduction. It
also should be noted that the transduction efficiency of AAV vectors varies greatly among
different cells and between tissues in vitro and in vivo, suggesting coreceptor involvement.
Human fibroblast growth factor receptor 1 was identified as a coreceptor for successful viral
entry into the host cell. 59 Also, α v β 5 integrin was also identified as a coreceptor for virus
purification and this provides critical information for in vivo distribution of AAV vectors. 60
The tissue tropism of AAV-2 could be viewed as a liability and many researchers are investigat-
ing approaches for targeting the vector to a specific cell type. As of yet, two approaches have
been used to modify the AAV's virion: chemically cross-linking bifunctional antibodies and
genetically manipulating the capsid gene.
Bartlett et al 1999, demonstrated that chemically cross-linked monoclonal antibodies such
as a bispecific F(ab' γ ) 2 that mediate an interaction between the AAV vector and a specific cell
surface receptor expressed on human megakaryocytes could target the AAV vectors to trans-
duce megakaryocyte cell lines. The data indicated that AAV vectors can be targeted to a specific
cell population and that transduction can be achieved by circumventing the normal virus re-
ceptor.
The second approach of altering the AAV-2 tropism using genetic manipulation of the
capsid gene is more challenging; a requirement for this to be successful is to determine the
surface amino acids and epitopes for manipulation. Moskalenko et al 49 defined peptide pools
that represent immunogenic regions on the virion surface. Rabinowitz et al 61 used random
linker insertion mutagenesis to also define the surface epitopes. In two other studies, using the
amino acid sequence alignment in conjunction with the crystal structure, both groups were
able to predict similar regions of the virion capsid that would be surface-localized. 62,63 These
various approaches will eliminate the AAV-2 tropism and once this is achieved, the focus will
turn to targeting the virion to specific tissues.
AAV Vectors in Gene Therapy
The adeno-associated virus (AAV) therapeutic applications are widening. Enthusiasm for
AAV is due not only to the relative safety of these vectors, but also to advances in the under-
standing of the unique biology of this virus. Compared to retrovirus, AAV vectors are able to
transduce nondividing cells in vivo including muscle, liver, and brain. 64-66 The broad host
range, low level of immune response, and longevity of gene expression observed with these
vectors in numerous disease paradigms has enabled the initiation of a number of clinical trials
using this gene delivery system. So far however, reports have been published only on cystic
fibrosis and hemophilia B.
 
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