Establishment of a Personalized Antisense Oligonucleotide (ASO) Screening Platform Based on Patient-Derived Organoids

Academic Background

In recent years, with the rapid development of genome sequencing technologies, an increasing number of rare genetic diseases have been found to be associated with specific gene mutations. Antisense oligonucleotides (ASOs), as a therapeutic approach capable of targeting specific RNA sequences, have shown potential in the treatment of various genetic disorders. ASOs bind to target mRNAs, regulating RNA processing or influencing protein expression levels, thereby correcting pathological phenotypes caused by gene mutations. However, despite significant achievements in both laboratory and clinical settings, the personalized design and preclinical evaluation of ASOs still face substantial challenges in terms of time and cost. Particularly for patients with rare diseases, developing ASOs tailored to their specific gene mutations requires efficient model systems to validate their efficacy.

To address this issue, researchers have developed a rapid and scalable ASO screening platform based on patient-derived organoids. This platform utilizes patient-derived induced pluripotent stem cells (iPSCs) to generate organoid models and evaluates ASOs through these models for preclinical assessment. This approach not only enables the rapid generation of patient-specific disease models but also validates the therapeutic effects of ASOs in a short time frame, offering new possibilities for personalized treatment of rare diseases.

Source of the Paper

This paper was co-authored by John C. Means, Anabel L. Martinez-Bengochea, Daniel A. Louiselle, and other researchers from the Genomic Medicine Center and Children’s Research Institute at Children’s Mercy Kansas City, as well as the University of Missouri-Kansas City School of Medicine and the University of Kansas Medical Center. The paper was accepted on November 27, 2024, and published in the journal Nature.

Research Process and Results

1. Rapid Generation of Patient-Derived iPSCs

The research team first developed an efficient iPSC generation platform. Using peripheral blood mononuclear cells (PBMCs) from patients as starting material, they generated iPSCs in just 3 weeks through an optimized reprogramming method. This method combined various reprogramming factors (such as the GSK inhibitor CHIR99021 and the MEK inhibitor PD0325901) and utilized a centrifugation step to promote cell attachment to Matrigel. The team successfully generated nearly 300 patient-derived iPSC lines with a success rate exceeding 93%. These iPSCs maintained stable pluripotency and genomic integrity even after multiple passages.

2. Generation and Validation of Organoid Models

The researchers differentiated patient-derived iPSCs into three-dimensional organoid models, including cardiac and brain organoids. These organoid models were able to recapitulate disease-related phenotypes. For example, cardiac organoids derived from patients with Duchenne muscular dystrophy (DMD) exhibited a loss of dystrophin expression. Through genome sequencing and RNA sequencing, the team confirmed the presence of gene mutations in these organoid models and validated their consistency with the pathological phenotypes observed in patients.

3. Design and Delivery of ASOs

The research team designed various ASOs targeting different RNA regions, including translation initiation sites, splice donor sites, and splice acceptor sites. They delivered these ASOs into cardiac organoids and evaluated their effects using immunofluorescence, Western blot, and RNA sequencing. The results showed that ASOs effectively inhibited the expression of target genes or modulated RNA splicing. For instance, ASOs targeting cardiac troponin T (TNNT2) significantly reduced the expression of this protein and led to the loss of contractile function in cardiac organoids.

4. Evaluation of Existing ASO Therapeutics

The team also evaluated the efficacy of FDA-approved ASOs in patient-derived organoids. They selected DMD patients with gene mutations causing the deletion of exons 46-53. By designing ASOs matching the exon 45 skipping therapy, the researchers successfully restored dystrophin expression and improved the contractile function of cardiac organoids. These results demonstrated that patient-derived organoid models can be used to assess the efficacy of existing ASOs.

5. Development and Validation of Personalized ASOs

For two DMD patients carrying deep intronic mutations, the research team designed two personalized ASOs. These ASOs targeted the mutation sites and successfully restored normal RNA splicing, leading to the recovery of dystrophin expression. Using calcium imaging, the researchers observed that ASO-treated cardiac organoids regained normal contractile rhythms. These findings indicated that personalized ASOs exhibited significant therapeutic effects in patient-derived organoid models.

Conclusions and Significance

This study developed a rapid and scalable ASO screening platform that utilizes patient-derived organoid models for the preclinical evaluation of personalized ASOs. This platform not only accelerates the development of ASOs but also provides new tools for the personalized treatment of rare diseases. Through this platform, researchers can generate patient-specific disease models in a short time and validate the therapeutic effects of ASOs, thereby offering more precise treatment options for patients with rare diseases.

Research Highlights

1. Efficient Patient-Derived iPSC Generation Platform: The research team developed a method to generate iPSCs in just 3 weeks, significantly shortening the time required to establish disease models.
2. Broad Application of Organoid Models: Using patient-derived organoid models, researchers were able to recapitulate disease phenotypes and validate the therapeutic effects of ASOs.
3. Development of Personalized ASOs: The team successfully designed personalized ASOs targeting specific gene mutations and validated their efficacy in organoid models.
4. Rapid Preclinical Evaluation: This platform enables the preclinical evaluation of ASOs in a short time frame, offering new possibilities for the treatment of rare diseases.

Additional Valuable Information

The research team also demonstrated the potential of this platform in other disease models, such as brain organoids and skeletal muscle models. These results indicate that patient-derived organoid models can be used not only for ASO evaluation but also for other drug development and high-throughput screening applications. Additionally, the team provided detailed experimental methods and data analysis workflows, offering valuable references for other researchers.

This study provides new tools and methods for the personalized treatment of rare diseases, holding significant scientific and practical value.