Biomedical Engineering Reference
In-Depth Information
Similarly, HSV/AAV hybrid virus with an AAV-ITR vector cassette inserted in the
HSV genome was infectious for neural cells, and gene expression from the AAV vec-
tor was maintained over 2 weeks, even though the HSV genome sequences were lost
from the vector [126,127] .
5.3.4.2.2 Pseudo-Wild-Type or Replication-Competent AAV
Initially AAV vectors were produced by cotransfection with helper plasmids that had
overlapping homology with the vector, and they generated vector particles that contam-
inated with wild-type AAV due to homologous recombination. Reduction of the over-
lapping AAV sequence homology between the vector and helper plasmids, such as in
the widely used pAAV/Ad packaging system, reduced but did not eliminate the genera-
tion of wild-type AAV [115,116] . Combining vector plasmid and packaging plasmid,
in which the AAV region containing the P5 promoter was not present in either plasmid
[116] , prevented generation of wtAAV, but some pseudo-wild-type AAV was generated
at lower frequency by nonhomologous recombination. This nonhomologous recom-
bination may be decreased in a packaging system like split-gene packaging, carrying
rep and cap genes in separate cassettes, which would require three or four recombina-
tion events to generate rcAAV. An alternate method to decreasing pseudo-wild-type or
recombinant AAV is to insert a large intron within the rep gene in the helper plasmid
so that any recombinants would tend to be too large to package in AAV particles [128] .
All vector production systems may have a tendency to generate pseudo-wild-type AAV
or other recombinants of AAV at low frequency, because it is not possible to elimi-
nate nonhomologous recombination in DNA. Packaging systems in which transfection
is avoided may help to reduce the frequency of such recombination. For detection of
pseudo-wild-type or recombinant species in AAV vector, rather than rep or cap immu-
noassays, an assay that employs two cycles of amplification by replication and then a
sensitive readout such as hybridization or polymerase chain reaction (PCR) is likely
to be preferred. Use of cell lines that provide rep- and cap-complementing functions
allows evaluation of such recombinant species without wtAAV, and this will facilitate
detection of species that are very poor at replication [129,130] .
5.3.4.2.3 Purification
In the past, purification of AAV was done by proteolytic digestion of cell lysates in
the presence of detergents, followed by banding in CsCl gradients to concentrate and
purify the particles and separate adenoviral particles. Considerable progress has been
made in the downstream processing of AAV vectors, leading to much higher quality
and purity. This is significantly important for preclinical and clinical trials.
The original CsCl centrifugation technique is being abandoned because it is a
cumbersome procedure and does not provide high purity; it may inactivate some
AAV vector; and it is difficult to envision its use for commercial production. A num-
ber of groups have employed nonionic iodixonal gradients as an initial bulk-recovery
method. Various chromatographic methods, including ion exchange, antibody, and
heparin affinity resins have been used in both conventional and high-performance
liquid chromatography (HPLC) formats [131-134] . Chromatographic procedures are
more acceptable for AAV vectors therapeutic applications.
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