Advancements in AAV Gene Therapy for GNE Myopathy

Catherine Kozwara is developing AAV gene therapy to treat GNE myopathy through optimized gene delivery and expression studies.

### Developing AAV Gene Therapy for GNE Myopathy
Noah Wise Leader welcomes attendees to the GEG NEM Symposium and introduces Catherine Kozwara, a Ph.D. student at Yale, as the speaker. Kozwara's research focuses on using **CRISPR-Cas9 technology** to correct mutations in GNE myopathy and establishing a **biobank of GNE patient cells**. The project aims to develop an **AAV gene replacement therapy** for GNE myopathy, with a focus on **optimizing the transgene cassette in vitro** before moving to **in vivo mouse studies**.

### Developing Gene Therapy for GNE Myopathy
The treatment for GNE myopathy is being approached through **supplementation and gene therapy**. The gene therapy aims to **replace the mutant GNE gene with a healthy copy**, using AAV vectors to deliver the gene to affected cells. The research focuses on **optimizing the gene cassette design**, including the promoter, intron, and transgene, to achieve the **highest GNE protein expression**.

### Isoform Expression and Gene Therapy for Muscle Disease
The GNE1 isoform showed **high expression** in healthy muscle tissue, while the GNE2 isoform had **weak expression**. Two constructs, one from our lab and another from the Adriana Rosa lab, both using the GNE1 isoform, demonstrated **strong GNE protein and RNA expression** in various cell lines. AAV9 particles containing these constructs were used for a dose-dependent biodistribution study in mice, showing **promising GNE1 expression** in muscle tissues and **minimal expression** in the liver.

### Progress in AAV Gene Therapy for GNE Myopathy
The current research on AAV gene therapies for GNE myopathy has not shown a **strong pattern in protein expression** in muscle samples from treated mice. The team is working on **optimizing the detection of the GNE protein** and is planning to try an **even higher dose** to improve results. Future work includes characterizing a mouse model of GNE myopathy for potential **gene therapy efficacy experiments**.

### AAV Gene Therapy: Treatment Approach and Immune Response
AAV gene therapy is typically a **one-dose treatment**, but emerging technologies are being developed to address the need for **redosing**. Immune reactivity to AAV can vary among individuals, posing a risk in delivering the therapy, but the field is working to **identify those at risk**. The aim of gene therapy is to achieve **normal or slightly higher levels of GNE expression** in muscle cells, with studies showing that **overexpressing GNE does not have overtly toxic effects**.

### Ensuring Efficient AAV Distribution to Skeletal Muscles
The distribution of AAV to skeletal muscles is controlled by the **serotype used**, with ongoing efforts to **optimize these serotypes**. Future work aims to determine the **amount of gene protein needed for therapeutic purposes**, with current doses being **relatively low**. Different isoforms of the gene may have varying expression in different muscle types, and the aberrant gene's expression is not expected to cause **cell damage**, but further studies are needed to clarify the **pathogenic mechanisms involved**.

### Gene Therapy and Its Mechanisms
Mutations causing genomyopathy typically result in a **loss of gene function**, rather than a gain, which is why carriers of a single GNE mutation are usually healthy. AAV (Adeno-Associated Virus) is the **most efficient gene delivery vehicle**, particularly for the neuromuscular field, due to its natural evolution to **infect cells effectively**. The use of induced pluripotent stem cells from skin biopsies is being explored as a model to **test the safety and efficacy of gene therapy** before introducing the virus into patients.

### Differentiating Stem Cells for Research
The speaker is working on differentiating fibroblasts from skin cells into **muscle-like cells** to create a bank of various cell lines for AAV testing and studying **genomopathy**. They have shared patient-derived skin cells with other labs, including NIH, for **iPSC conversion**. The speaker discusses the challenges of using cell models to test the **systemic immune response** and emphasizes the importance of **multiple models for clinical confidence** in therapeutic development.

### Advancements in Gene Therapy for GNE Myopathy: Insights from the GEG NEM Symposium
* 👋 Welcome to the GEG NEM Symposium Speaker Series, hosted by the Nebraska Disease Foundation, with **Noah Wise Leader** introducing today's session on **GNE myopathy**.
* 🧬 Catherine Kozwara, a Ph.D. student from Yale School of Medicine, is presenting her work on developing an **AAV gene therapy** aimed at treating **GNE myopathy**, an autosomal recessive disorder affecting **approximately one in one million people** globally.
* 🔬 GNE myopathy is caused by mutations in the GNE gene, leading to **progressive muscle atrophy and weakness**, characterized by the presence of **rim vacuoles and inclusion bodies** in muscle tissues.
* 💡 The project focuses on optimizing the AAV transgene cassette for effective gene delivery, with significant emphasis on **selecting the right promoter, intron, and transgene** to ensure **high expression levels of the GNE protein** in muscle cells.
* 📊 Preliminary results from in vitro studies indicate that the CK80 promoter, along with the SV40 intron and GNE1 isoform, yields the **highest protein expression**, leading to further testing in mouse models for **biodistribution and efficacy**.
* 🐭 Initial biodistribution studies show promising **dose-dependent expression of GNE** in muscle tissues, although challenges remain in **detecting GNE protein levels effectively**, prompting ongoing optimization efforts.
* 🔄 Future work aims to **refine the detection of GNE protein expression** and explore the **safety and efficacy of the gene therapy** in disease models, with collaboration across various research labs enhancing the overall effort.
* 🤝 The session concluded with a robust Q&A, highlighting the **collaborative nature of the research** and the **importance of continued exploration in gene therapy for GNE myopathy**, with upcoming presentations scheduled for December.

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2022 NDF GNEM Symposium with Katherine Koczwara: Developing An AAV Gene Therapy For GNEM: Part ll

2022 NDF GNEM Symposium with Katherine Koczwara: Developing An AAV Gene Therapy For GNEM: Part ll