Discipline: Biological Sciences
Ambermarie Dawdy - University of South Florida
Co-Author(s): Christian Schuetz, Dylan Finneran, Joaquin Torello, Amanda Pitre, and Kevin Nash, USF Health Byrd Alzheimer's Institute, FL Joaquin Torello, USF Health Byrd Alzheimer's Institute, FL. Amanda Pitre, USF Health Byrd Alzheimer's Institute, FL. Kevin Nash, USF Health Byrd Alzheimer's Institute, FL.
Angelman syndrome (AS) is caused by a disruption to the Ube3a gene and is a complex genetic disorder that primarily affects the central nervous system (CNS). Since AS is a single gene disorder it could potentially be corrected using gene therapy. However, in order for a gene therapy to be effective we would need to target the majority, if not all, neurons with in the CNS. To this end we are examining viral gene delivery into the CNS of rodents. We have chosen to use recombinant adeno-associated virus (rAAV) because it offers a number of advantages to CNS delivery; including long-term expression, low immunogenicity, infects neurons and has shown safety in a large number of clinical trails. Using various rAAV serotypes and green fluorescent protein (GFP) as a reported gene we will examine different routes of administration, which will vary from invasive (intracranial delivery) to less invasive (intravenous delivery). Level of GFP expression will be measured to determine which route of administration and which serotype offers the best global distribution in the CNS. One to two months after injection the rodents will be euthanized and the brain collected for analysis. In our intracranial injections we examined a number of different sites of administration; Lateral ventricle (virus could potentially cross from CSF into parenchyma), Raphe nuclei and Thalamus which both have significant neuronal connections to many different brain regions. The lateral ventricle injections show transgene expression in a number of brain structures including the hippocampus and cortex but it is rather restricted. Raphe and thalamus injections show significant expression at site of injection but also significant projections into other brain regions including hippocampus, cortex, striatum and cerebellum. The raphe injection seemed to be better than the thalamus and injection at this site could be ideal for a secreted protein molecule to be distributed throughout the brain. Examination of serotypes would indicate that rAAV9 would offer the most robust expression with this administration. With intravenous injections we examined novel rAAV constructs. Immunohistochemical examination of the intravenous injections demonstrated significant neuronal transduction with the published PHP.B vector. This vector shown significant GFP expression with both a ubiquitous promoter and a neuronal specific promoter, thus offering the most potential for a global gene delivery to the CNS. Future research includes other various therapeutic protein injections in order to deliver better distribution throughout the brain to give a better neurocognitive response in rodent models.
Funder Acknowledgement(s): Florida Georgia Louis Strokes Alliance for Minority Participation
Faculty Advisor: Kevin Nash, firstname.lastname@example.org
Role: I did the slicing, staining, mounting and analysis of the brains. Also I did cell culture, virus creation and injection along with the immunohistochemistry tests and data analysis that went along with the study.