Tunable Gene Therapy Expression System Regulated by Acyclovir

Background & Unmet Need

• Existing gene therapy systems rely on constitutive protein expression
• However, not all genetic diseases require continuous replacement of the defective gene product
• Prolonged or excessive expression of therapeutic proteins can also lead to toxicity
• Tailored therapy is essential as patients differ in required gene product levels, balancing therapeutic efficacy and toxicity
• Gene expression can be regulated by controlling the inclusion or exclusion of exons, a process known as alternative splicing
• Challenges related to off-target effects, efficiency, immunogenicity, and safety have arisen in gene expression systems employing alternative splicing
• Unmet Need: Simplified and safer method for regulating gene expression

Technology Overview

• The Technology: A system that utilizes acyclovir to regulate gene expression via alternative splicing of a poison exon
• A novel splicing cassette which incorporates exon 7 of survival motor neuron 1 (SMN1) as a poison exon
• The poison exon contains an in-frame stop codon, and a TSL2 stem loop modified with an acyclovir binding aptamer that can control inclusion of the exon
• In the presence of acyclovir, the poison exon is removed from mature mRNA allowing functional protein expression
• Acyclovir is an FDA-approved antiviral that is known to be well tolerated during chronic dosage
• PoC Data: Insertion of the splicing cassette into a luciferase reporter gene led to significant repression of reporter expression, with dose-dependent induction up to 300-fold upon acyclovir addition

Technology Applications

• Incorporated into current gene therapy systems for better-controlled gene expression in terms of both levels and timing
• Adaptable tool for cellular biology research, where controlling protein expression is essential

Technology Advantages

• Utilizes FDA-approved acyclovir for regulation, offering a safer and more specific approach
• Avoids the addition of exogenous amino acids, reducing the risk of protein misfolding and immune responses

Figure 1: Schematic showing how splicing cassette regulates gene expression (top). Acyclovir-induced activation of the luciferase reporter gene correlated with dosage (bottom).