title: Verification of functional AAV-mediated neurotrophic and anti-apoptotic factor expression.
abstract: The use of viral vectors for gene delivery offer many advantages for both basic research and therapeutic application through the continuous expression of a gene product within a target region. It is vital however that any gene product is correctly expressed in a biologically active form, and this should be confirmed prior to large scale in vivo studies. Using adeno-associated viral (AAV) vectors to direct the expression of either a neurotrophic factor or an anti-apoptotic protein, we have developed a range of in vitro assays to verify functional transgenic protein expression. Brain-derived neurotropic factor (BDNF) activity was confirmed by demonstrating enhanced generation of GABAergic neurons in embryonic (E15) striatal cultures and AAV-mediated glial-derived neurotrophic factor (GDNF) function using an assay for dopaminergic differentiation of embryonic (E14) ventral mesencephalic cultures. To assess functional anti-apoptotic factor expression we designed cell-survival assays, using embryonic cortical cultures to confirm Bcl-x(L) activity and the HT1080 cell-line for X-linked inhibitor of apoptosis protein (XIAP) activity following AAV-mediated expression. This study demonstrates that the use of functional assays provides valuable confirmation of desired biotherapeutic expression prior to extensive investigation with new gene delivery vectors.
authors.affiliation: Neural Repair and Neurogenesis Laboratory, Department of Pharmacology and Clinical Pharmacology, The University of Auckland, Auckland, New Zealand.
authors.forename: Adrian P
authors.initials: AP
authors.lastname: Kells
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Henry
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Hughes
authors.forename: Bronwen
authors.initials: B
authors.lastname: Connor
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D017209:Apoptosis / Q000502:physiology*; D001681:Biological Assay / Q000379:methods*; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000235:genetics / Q000378:metabolism; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics / Q000378:metabolism*; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051034:Inhibitor of Apoptosis Proteins; D009414:Nerve Growth Factors