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B - Gene Targeting and Gene Correction -> B1 – Gene Targeting and Gene Correction – In Vivo Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

Advancing Epigenetic Editing with CRISPR-GNDM: Novel Muscle-Tropic AAV Vectors Deliver Promising Single-Dose Treatment for LAMA2-CMD

Type: Oral Abstract Session

Presentation Details
Session Title: Late-breaking Abstracts 2
Location: Concourse Hall 152 & 153
Start Time: 5/19/2023 10:45
End Time: 5/19/2023 11:00

Epigenetic editing holds great promise as a next-generation gene therapy platform. While animal proof-of-concept (POC) for the concept has been demonstrated in the literature, there has been no report demonstrating the mechanism of action (MOA) in larger animals. This is due to the challenge in allometric scaling associated with gene therapy, and particularly with epigenetic editing, where one more step is needed beyond expressing the transgene to act on the gene’s endogenous locus. Our epigenetic editors are designed to efficiently target genes and control chromatin conformation by coupling an engineered Cas9 variant with epigenetic effector domains; referred to as GNDM (Guide Nucleotide Directed Modulation). Here, we present promising POC data for our CRISPR-GNDM® platform, advancing its pipeline of single-vector therapies for LAMA2-CMD and other disorders.
LAMA2-CMD is a severe, early onset form of congenital muscular dystrophy characterized by muscle weakness, loss of ambulation, and respiratory failure caused by LAMA2 mutations. Unfortunately, there is no cure or effective treatment available. In mouse models, LAMA1 protein expression is able to compensate the loss of LAMA2. Since the LAMA1 gene is too large for AAV packaging, and not actively transcribed in muscle tissues, it serves as an ideal target for CRISPR-GNDM® technology.
Most AAV capsids used for systemic treatments of muscle diseases require high doses, leading to safety concerns. To overcome such obstacles, we assessed two novel, muscle-tropic AAV capsids, allowing for reduced doses, increased safety margins, and higher therapeutic efficacy. Moreover, we codon-optimized the GNDM transgene and paired it with two different compact poly-A signals to further enhance transgene expression.
To validate the efficacy of our therapeutic candidates, we evaluated them in DyW mice, a well-characterized mouse model for LAMA2-CMD. Mice were dosed intravenously at 2E13 vg/kg. After six weeks, biochemical analyses revealed significantly higher muscle tissue transduction levels for all three lead candidates than mice dosed with AAV9 at a 10-times higher dose (2E14 vg/kg). Immunofluorescent staining showed robust and evenly distributed LAMA1 expression in the basement membranes of skeletal muscle fibers. More importantly, treated animals exhibited significantly improved grip strength and body weights as compared to untreated controls, confirming therapeutic benefit.
Translating high levels of transduction observed in mouse models to large animals is often challenging and can compromise predicted clinical efficacy. We evaluated our lead candidates in juvenile NHPs, which were systemically dosed via IV infusion at 1E14 vg/kg, and sacrificed six weeks post-administration. We observed no obvious toxicity apart from transient elevation of liver enzymes. DNA and RNA analysis showed robust distribution of the vector to muscle tissue with enhanced expression of GNDM and significantly induced LAMA1 gene expression. Compared to our historical datasets, our novel constructs showed up to 60-fold enhanced GNDM expression in muscle tissues compared to AAV9 while maintaining low liver transduction. The confirmation of high levels of induced gene expression in a large animal model using a transcriptional activator such as GNDM is a novel finding and indicates the suitability of this technology for clinical application.
In summary, our proprietary CRISPR-GNDM® technology, packaged in novel, muscle-tropic AAV vectors offers a safe and effective single-dose treatment for LAMA2-CMD, and demonstrates the leading approach for epigenetic editing.

Yuanbo Qin, Talha Akbulut, Alison Shottek, Seth Levy, Rajakumar Mandraju, Keith Connolly, John Bechill, Farzaneh Assadian, Payal Pranami, Tanuj Thakkar, Julia Paquette, Chanikarn Power, Claudia Foster, Rebekah Garfolo, Jamie Benoit, Tetsuya Yamagata

Modalis Therapeutics Inc., Waltham, MA
 Y. Qin: None.

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