title: Destabilizing domains mediate reversible transgene expression in the brain.
abstract: Regulating transgene expression in vivo by delivering oral drugs has been a long-time goal for the gene therapy field. A novel gene regulating system based on targeted proteasomal degradation has been recently developed. The system is based on a destabilizing domain (DD) of the Escherichia coli dihydrofolate reductase (DHFR) that directs fused proteins to proteasomal destruction. Creating YFP proteins fused to destabilizing domains enabled TMP based induction of YFP expression in the brain, whereas omission of TMP resulted in loss of YFP expression. Moreover, induction of YFP expression was dose dependent and at higher TMP dosages, induced YFP reached levels comparable to expression of unregulated transgene., Transgene expression could be reversibly regulated using the DD system. Importantly, no adverse effects of TMP treatment or expression of DD-fusion proteins in the brain were observed. To show proof of concept that destabilizing domains derived from DHFR could be used with a biologically active molecule, DD were fused to GDNF, which is a potent neurotrophic factor of dopamine neurons. N-terminal placement of the DD resulted in TMP-regulated release of biologically active GDNF. Our findings suggest that TMP-regulated destabilizing domains can afford transgene regulation in the brain. The fact that GDNF could be regulated is very promising for developing future gene therapies (e.g. for Parkinson's disease) and should be further investigated.
authors.affiliation: CNS Gene Therapy Unit, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, Lund, Sweden.
authors.forename: Khalid
authors.initials: K
authors.lastname: Tai
authors.forename: Luis
authors.initials: L
authors.lastname: Quintino
authors.forename: Christina
authors.initials: C
authors.lastname: Isaksson
authors.forename: Fredrik
authors.initials: F
authors.lastname: Gussing
authors.forename: Cecilia
authors.initials: C
authors.lastname: Lundberg
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007276:Injections, Intraventricular; D016086:Lentivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D023421:Models, Animal; D058990:Molecular Targeted Therapy; D010300:Parkinson Disease / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes; D014295:Trimethoprim / Q000494:pharmacology
chemical_list: D000890:Anti-Infective Agents; D001426:Bacterial Proteins; C495039:GDNF protein, human; D051100:Glial Cell Line-Derived Neurotrophic Factor; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase