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.
Highlights
The possibility to regulate transgene expression has been a discussed in the gene therapy field for a long time
We show that the system has the potential to be applied to biologically active proteins since a regulated fusion protein of destabilizing domain (DD) and glial cell derived neurotrophic factor (GDNF), a very relevant molecule for gene therapy in Parkinson’s disease [15] can be constructed using this system and the resulting fusion protein is functional
In the present study we evaluated the feasibility of using destabilizing domains fused to transgenes to regulate levels of transgene product
Summary
The possibility to regulate transgene expression has been a discussed in the gene therapy field for a long time (see e.g. [1,2]). The possibility to regulate transgene expression has been a discussed in the gene therapy field for a long time In clinical settings, regulated transgene expression would allow for increased or decreased transgene levels in response to clinical need. Regulating transgene expression would ideally provide a means to avoid adverse effects due to continuous overexpression of therapeutic genes. The ability to turn transgene expression off and on offers experimental advantages when studying causal effects of gene transfer in disease models. Many different regulated gene expression systems have been developed [3] and most operate at transcriptional levels. There is a regulated transcriptional system based on FRAP and rapamycin that has been developed for use in gene transfer paradigms [5]
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