HTT Splicing Assay Service

Aberrant splicing of the huntingtin (HTT )gene plays a pivotal role in the pathogenesis of Huntington's Disease (HD). Understanding the precise mechanisms of pseudoexon inclusion and exon skipping is crucial for developing targeted therapeutic interventions. For further information regarding the products and services provided, project-specific consultation, and pricing, please submit an inquiry here.

Introduction

Splicing regulation of the HTT gene is a critical focus in HD research due to its direct impact on disease progression. Alternative splicing events within the HTT gene generate multiple transcript variants, each potentially contributing to disease pathogenesis. These variations influence protein levels and functionality. Notably, alternative polyadenylation (APA) of introns 9 and 10 is implicated in modulating HTT protein expression levels, offering a potential therapeutic target. Furthermore, aberrant splicing of exon 1 results in the production of atypical transcripts, such as mHTT1a. These truncated transcripts are particularly concerning, as they form intracellular aggregates, known as inclusion bodies, which induce significant neuronal dysfunction, contributing to the hallmark neurodegeneration observed in HD. Therefore, understanding and controlling HTT splicing is paramount.

HTT Splicing Assays

• High-Throughput Sequencing

To decipher the complex regulation of the HTT gene implicated in HD, we employ RNA-Seq technology. This high-throughput sequencing approach allows for the detailed analysis of the HTT transcriptome, uncovering alternative splicing events. A study utilizing a custom analysis pipeline detected 12 out of 25 potential splicing variations, highlighting the power of RNA-Seq to quantify diverse HTT gene isoforms.

• qPCR Validation

To validate the identified HTT gene splice variants, quantitative PCR (qPCR) offers a precise detection method using isoform-specific primers. For instance, a primer set can be designed with one primer amplifying both the full-length and short isoforms and the second primer specifically targeting only the short isoform. This approach allows for the accurate amplification and quantification of both the full-length and short HTT gene isoforms from cDNA templates.

• Splicing Regulator Intervention Assays

In order to validate the effectiveness of splicing regulators as potential HD therapy, we will conduct in vitro efficacy evaluations. These evaluations will focus on confirming the regulators' ability to enhance the degradation of HTT mRNA, a crucial step in reducing the production of the harmful huntingtin protein.

Case Study: Compounds affect HTT lowering through HTT pre-mRNA splicing.

Fig.1 Splicing of human HTT pre-mRNA resulting in lowering of HTT mRNA.^{2, 3}

Our company is committed to providing a full-chain solution from gene to protein for HD research. To that end, we rely on the world's leading splicing analysis platform and neurological disease model library. Please contact us to discuss the customized service plan that we can provide for your needs.

References

1. Jarosińska, Olga D., and Stefan GD Rüdiger. "Molecular strategies to target protein aggregation in Huntington's disease." Frontiers in molecular biosciences 8 (2021): 769184.
2. Bhattacharyya, Anuradha, et al. "Small molecule splicing modifiers with systemic HTT-lowering activity." Nature communications 12.1 (2021): 7299.
3. Distributed under Open Access license CC BY 4.0, without modification.