An inducible CAG repeat selective small molecule screening cell line

Background:

Neurodegenerative disorders caused by CAG repeat expansions, such as Huntington’s disease and various spinocerebellar ataxias (SCAs), represent a significant challenge in biomedical research and clinical practice. These diseases are characterized by the abnormal expansion of CAG trinucleotide repeats within specific genes, leading to the production of toxic RNA and proteins that disrupt normal cellular function and ultimately cause progressive neurological decline. Despite advances in understanding the genetic basis of these disorders, there remains a critical unmet need for disease-modifying therapies that can specifically target the underlying molecular pathology. The identification of small molecules capable of selectively reducing the levels of toxic expanded CAG repeat RNAs is a promising therapeutic strategy, as it has the potential to mitigate both RNA-mediated toxicity and the downstream production of harmful polyglutamine proteins. Current approaches to discovering such selective small molecules are hampered by several limitations. Many existing cell-based screening systems lack the specificity to distinguish between expanded and non-expanded CAG repeat transcripts, making it difficult to identify compounds that act selectively on the disease-causing RNA. Additionally, constitutive expression of expanded CAG repeats in cell lines often leads to significant toxicity, resulting in selective pressure against high repeat length and robust expression, which compromises the stability and longevity of the screening platform. This toxicity also limits the ability to conduct long-term studies or to assess the effects of potential therapeutics across a range of expression levels, which is crucial for understanding dose-response relationships and therapeutic windows. Furthermore, the absence of robust, multiplexed readouts for both RNA and protein levels in many systems reduces the sensitivity and throughput of screening efforts, slowing the pace of therapeutic discovery.

Technology Overview:

This technology is an advanced inducible cell line system designed for the high-throughput screening of small molecules that selectively reduce expanded CAG repeat RNA levels, a hallmark of neurodegenerative disorders such as Huntington’s disease and spinocerebellar ataxias. The system utilizes engineered HEK293-based cells containing two distinct reporter constructs. Each construct includes unique qPCR probe sites, enabling precise multiplex RT-qPCR quantification of RNA levels, while dual luciferase assays provide ratiometric protein measurements. Critically, both constructs are controlled by an antibiotic-inducible promoter, allowing researchers to temporally and quantitatively regulate expression, thereby reducing toxicity and enabling long-term studies. The system’s design is intentionally gene-agnostic, ensuring broad applicability across various CAG repeat expansion disorders. What differentiates this technology is its combination of inducibility, dual-reporter specificity, and robust validation, which together overcome major limitations of previous constitutive systems. The inducible promoter extends cell line viability by mitigating the toxicity associated with continuous expression of expanded CAG repeats, allowing for sustainable long-term research and screening. The dual-reporter setup enables highly sensitive and specific detection of selective effects on expanded versus non-expanded repeats, facilitating the identification of compounds that target disease-causing transcripts without affecting normal cellular function. Additionally, the unique qPCR probe sites and ratiometric luciferase assays provide orthogonal, multiplexed readouts at both RNA and protein levels, enhancing assay reliability and throughput. This platform not only accelerates the discovery of disease-modifying therapeutics for CAG repeat disorders but also serves as a versatile tool for mechanistic studies and preclinical validation, setting a new standard for translational research in the field of repeat expansion diseases. Lead compounds for SCAs have been identified with this cell line and are included in this technology.

Advantages:

•    Enables selective identification of small molecules that reduce toxic expanded CAG repeat RNA levels.
•    Provides precise temporal and quantitative control of CAG repeat expression via a doxycycline-inducible promoter, reducing toxicity and extending cell line viability.
•    Utilizes dual reporter constructs and multiplex RT-qPCR for sensitive and specific differentiation between expanded and non-expanded CAG transcripts.
•    Supports high-throughput screening for potential therapeutics targeting multiple CAG repeat expansion disorders, including Huntington’s disease and various spinocerebellar ataxias.
•    Facilitates detailed mechanistic studies on CAG repeat expression and its modulation by candidate drugs.
•    Offers a broadly generalizable platform applicable to diverse microsatellite expansion diseases beyond CAG repeats.
•    Improves preclinical validation of disease-modifying therapies by enabling assessment of minimum therapeutic reductions in toxic polyglutamine protein levels.

Applications:

•    High-throughput drug screening assays
•    Preclinical validation of therapeutics
•    Mechanistic studies of CAG toxicity
•    Screening for RNA-targeting compounds

Intellectual Property Summary:

Patent Pending 19/196,499

Stage of Development:

TRL 4

Licensing Status:

This technology is available for licensing.