This proposed R21 application for the RFA entitled """"""""Therapeutics Delivery for Neurodegenerative Diseases"""""""" directly addresses the need for a novel gene delivery technology that is capable of crossing the Blood Brain Barrier and delivering DNA to neurons. The proposed project will utilize a novel gene delivery vector, termed Protofection (Protein Mediated Transfection), which is comprised of a Protein Transduction Domain and a recombinant protein able to bind DNA. This goal of the project is to provide the proof of principle for the delivery and stability of full-length mtDNA to cells. The project will address (i) delivery and long-term stability of the transfected mtDNA, (ii) any acute or long-term effects mitochondrial transfection may have on the health of cells, and (iii) the mechanism by which the vector delivers DNA into cells and mitochondria.
In Aim I, full- length mtDNA engineered to have a new RFLP or to produce a marker protein (Green Fluorescent Protein, GFP) will be complexed with the protofection vector protein and delivered to the neuroblastoma cell line, SH-SY5Y. The delivery of mtDNA to cells will be quantified by following GFP fluorescence and by PCR for the introduced RFLP.
Aim II will address mitochondrial function and cell viability utilizing spectrophotometry for electron transport chain function, oxygen respiration, and cell death. Finally, Aim III will address the mechanism of uptake of the vector protein into mitochondria by identifying what role mitochondrial fusion plays in lipid raft targeting to mitochondria. Successful achievement of the aims will form the basis for an extensive investigation into the efficacy and safety of protofection as a treatment for the mtDNA disease MELAS (Myopathy, Encephalopathy, Lactic Acidosis, Stroke-like episodes) in a planned R01 grant submission. Protofection will enable novel investigations into mitochondrial genetics and determination of the potential roles of mitochondrial genomes in aging, neurodegenerative diseases, cancer, diabetes, and the metabolic syndrome as well as provide a unique therapeutic option where none has existed.
The aims carried out under this proposal will show feasibility for an extensive investigation into the efficacy and safety of protofection as a treatment for mtDNA disease. MELAS, which will be pursued in a planned RO1 submission, has no effective treatment or cure. The age of onset ranges from neonatal to young adult, with the average survival of 6.5 years from the onset. The broader impact on public health for this proposal is also significant. Successful validation of the protofection delivery system can enable novel investigations into mitochondrial genetics and determination of the potential roles of mitochondrial genomes in aging, neurodegenerative diseases, cancer, diabetes, and the metabolic syndrome as well as provide a unique therapeutic option where none has existed. ? ? ? ? ?
| Swerdlow, Russell H; Burns, Jeffrey M; Khan, Shaharyar M (2010) The Alzheimer's disease mitochondrial cascade hypothesis. J Alzheimers Dis 20 Suppl 2:S265-79 |
