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A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

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Destabilizing domains mediate reversible transgene expression in the brain.

Khalid Tai¹, Luis Quintino, Christina Isaksson

¹CNS Gene Therapy Unit, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, Lund, Sweden.

|October 3, 2012

Summary

A novel gene therapy system uses a destabilizing domain (DD) to control transgene expression in the brain with oral drugs. This system allows reversible, dose-dependent control of gene products, showing promise for treating neurological disorders.

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Area of Science:

Gene therapy
Molecular biology
Neuroscience

Background:

Controlling transgene expression in vivo is crucial for gene therapy.
Targeted proteasomal degradation offers a novel regulatory mechanism.

Purpose of the Study:

To develop and validate a novel, orally-regulatable gene expression system for the brain.
To assess the efficacy and safety of this system for therapeutic applications.

Main Methods:

A destabilizing domain (DD) from Escherichia coli dihydrofolate reductase (DHFR) was fused to reporter proteins (YFP) and a neurotrophic factor (GDNF).
The system's regulation was tested in vivo using trimethoprim (TMP) administration.
Dose-dependent and reversible expression was evaluated, along with potential adverse effects.

Main Results:

TMP administration induced YFP expression in the brain, which was dose-dependent and reversible upon TMP withdrawal.
DD-fusion proteins and TMP treatment showed no observable adverse effects.
N-terminal DD fusion enabled TMP-regulated release of biologically active GDNF.

Conclusions:

The DD system provides effective, reversible, and safe transgene regulation in the brain.
This technology holds significant potential for developing advanced gene therapies, particularly for neurodegenerative diseases like Parkinson's.