Gene Delivery Using Viral Vectors - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 231

5.3.4.2.4 Assay
AAV vectors are usually characterized in terms of total number of particles and the
infectivity titer as determined in a replication assay. This principle allows the deter-
mination of the ratio of the particle to the infectivity titer of a vector preparation
and could allow for universal comparison of vectors. Some universal assays may be
adopted but this will still not provide a measurement of the particular biological effi-
ciency or transducing titer of individual vector preparations, because this will depend
on the particular gene that is expressed from the vector. Further, measurement of
transducing titers on cell lines in vitro will not necessarily be predictive for biologi-
cal activity in a particular target cell in vivo .
Slot-blot DNA hybridization assays or, more recently, TaqMan real-time DNA PCR
are used for determining the number of DNA genomes [132] . The number of DNase-
resistant particles (DRP) is calculated by assuming that each particle contains one
vector genome. An infectious center assay or endpoint assays are used for measuring
infectivity in cells that are also infected with adenovirus and wtAAV [154] . This three-
hit assay is difficult to perform reproducibly and robustly. Recently, the availability
of cells expressing rep and/or cap has also allowed the determination of infectivity in
a two-hit system in such cells by infecting with the vector and the helper adenovirus
[69,70] . Recently, HPLC can also be used for the rapid assay, which can give particle
assay readout on a sample in about 20 min [132] .
5.3.5 Application of Adeno-Associated Viral Vectors
5.3.5.1 Cancer Gene Therapy
Although recombinant adenoviral vectors have been utilized for both preclinical and
clinical trials in cancer gene therapy, they provide wide benefits for the treatment of
cancer. No T-cell-mediated cytoxicity to AAV vectors has been observed, even if AAV
vectors can induce strong humoral immune response. AAV vectors would appear less
mutagenic because they can initiate long-term transgene expression, and this transduc-
tion is attributed to episomal concatamer formation without integration into the host
chromosome. The AAV package capacity is reserved to less than 5 kb; most therapeutic
genes for cancer treatment fall into this range. Greater reduction of AAV packaging
size (2.5 kb) will still accommodate most anticancer genes (e.g., cytokines, RNAi, anti-
angiogenesis genes). AAV holds great promise as a viral vector delivering therapeutic
genes, such as those for immune regulation (e.g., cytokines) and antiangiogenesis
genes (e.g., endostatin, angiostatin, pigment epithelium-derived factor [PEDF]) for can-
cer gene therapy, with the new serotypes and potential developed targeting vectors. The
approaches for cancer gene therapy with rAAV vector can be divided into four major
categories: antiangiogenesis, immunomodulation, suicide gene therapy, and repair of
damaged tumor cells.
5.3.5.2 Antiangiogenesis
Targeting of the vasculature has proved to be the most attractive strategy in the treatment
of cancer, because solid tumor growth and metastasis depend on angiogenesis [135] .