Gene therapies approaches for Charcot-Marie-Tooth diseases: targeting the right cell type and compartment – GENERATE

This project is organized into two tasks:

Task 1 consists of developing tools and therapeutic approaches for CMT2A.

Task 2 consists of developing tools and therapeutic approaches for CMT4H.

For both tasks, the goal is to deliver the repair gene into the relevant cell type. To achieve this, we combine our therapeutic gene with a regulatory sequence called a promoter, which will allow either targeting of the affected neurons (motor neurons and sensory neurons for CMT2A) or Schwann cells for CMT4H.

We have also optimized the serotype of the viral vector (called AAV) as well as the method of administration (lumbar intrathecal injection) to increase targeting of the peripheral nervous system while reducing spread to peripheral organs and thereby minimizing potential side effects.

For Task 1, the therapeutic genes will aim to counterbalance the effects of the mutated MFN2 gene responsible for CMT2A, notably by restoring communication between neuronal organelles.

For Task 2, the therapeutic genes will counterbalance the effects of the mutated FGD4 gene, which encodes the protein Frabin and is responsible for CMT4H, by producing either a normal (wild-type) protein or a miniaturized version that restores Schwann cell function.

During the course of this project, we obtained promising results:

In the context of CMT2A, we demonstrated that our gene therapy approaches significantly improved CMT2A pathology. In particular, treated CMT2A mouse models showed an improvement in motor function. Interestingly, this therapy was beneficial both when delivered before and after disease onset in mice. One of the proposed approaches has been subject to an invention disclosure and a project submission currently underway.

In the context of CMT4H, we first optimized the viral serotype, notably by selecting one known to promote long-distance transport, which is a considerable advantage for a disease like CMT that primarily affects distal nerves.

We then tested the therapeutic effect of our gene replacement strategy (replacing the mutated gene with the wild-type gene) in our CMT4H mouse model.

We showed that this strategy significantly reduced the proportion of abnormal myelin produced by the affected Schwann cells. However, at present, this treatment works mainly on proximal nerve segments (those closer to the body’s midline) and less effectively on distal nerves, which are predominantly affected.

This project opens many perspectives. First, the valorization of therapeutic approaches targeting CMT2A through a patent constitutes an initial step toward collaborating with potential investors and raising funds to test the safety of the therapy in larger animals, an essential stage before considering a clinical transfer, which I hope will ultimately benefit patients.

In parallel, we continue to optimize available viral tools to create the safest possible therapeutic approach. Recently, new tools capable of very specifically targeting motor neurons have been developed and would be of great interest to test in the context of CMT2A.

In the case of CMT4H, the results obtained thanks to this project’s funding are very encouraging. This represents the first gene therapy approach for CMT4H. Moving forward, we aim to improve our tools and dosing to target a greater number of Schwann cells, particularly those located in the distal parts of the nerves, which are primarily affected in CMT patients. Furthermore, our approaches could subsequently be applied to other subtypes of demyelinating CMT

Charcot-Marie-Tooth disease (CMT) is a rare and incurable disease of the peripheral nervous system. CMTs are defined according to the cell type affected: either the Schwann Cell (SC) in demyelinating CMT, or motor (MNs) and sensory neurons (SNs) in axonal CMT. More than 100 causative CMT-related genes have been identified. This genetic and cell-type diversity limits the prospect of a broadly applicable therapeutic approach. Given that most CMT subtypes are monogenic and caused by the dysfunction of a well-defined population of cells (SC or MNs and SNs), we believe that gene therapy strategies aimed at restoring the altered function of the mutated protein in the appropriate cell type or even compartment could be a promising approach for CMT.
We will focus on two different CMT forms which mechanisms are currently studied by the GENERATE project members: the axonal form CMT2A and the demyelinating type CMT4H.
CMT2A is linked to heterozygous mutations in the MFN2 gene. MFN2 protein is localized at the mitochondrial membrane, where it regulates mitochondria fusion with its homolog, MFN1. In contrast to this latter, MFN2 has been also observed at the surface of the endoplasmic reticulum (ER) where it controls ER-mitochondria contacts, called MAM. Previous work suggest that MAM dysregulation contributes to CMT2A pathology via a loss-of-function mechanism, which argues for an approach aimed at rescuing MAM function. On this premise, we aim at counteracting the effects of CMT2A mutation by overexpressing wild-type MFN2 in MNs and SNs. As an alternative approach, we will engineer a construct enabling the ectopic expression of the MFN2 protein-homolog MFN1 in the ER. Those constructs will be selectively expressed in MNs and SNs using the appropriate AAVs vectors and neuronal-specific promoter (human Synapsin).
CMT4H is linked to homozygous mutations in the FGD4 gene encoding FRABIN. FRABIN is a guanine exchange factor that regulates the activity of the small RhoGTPase cdc42. Nerves from CMT4H patients display myelin abnormalities. Similar alterations were observed in the sciatic nerves from mice in which Fgd4 or cdc42 is specifically deleted in SCs. That suggests that the pathological effect molecular basis of CMT4H relies on the loss of function of FGD4/FRABIN and cdc42 in SCs. Therefore, we aim at developing a gene-replacement therapy for CMT4H based on the selective expression of either the wildtype full-length FRABIN or a shorten version of FRABIN comprising only the functional domains required for the activity of cdc42 in SCs. Those constructs will be selectively expressed in SCs using the appropriate AAVs and Lvs vectors and SC-specific promoter (Mpz/P0).
We will test the effect of the aforementioned therapeutical strategies on the molecular, cellular and functional alterations associated with CMT2A or CMT4H (MAM and axonal degeneration in CMT2A, myelination abnormalities in CMT4H, motor and sensory functions for both CMT forms). To that end we will use a combination of molecular biology and imaging strategies as well as behavioral testing, in vitro preparations (cellular models originating from rodents and patients with CMT) and in vivo transgenic mouse models.
We believe the GENERATE project is achievable in three years since: 1) most of the tools and models are available 2) most of the tasks described above rely on solid preliminary data 3) members of the project display all the necessary conceptual and technical expertise to perform each proposed experiment. This project will benefit from the close collaboration of our team with clinicians and patients, an important asset given the anticipated translation of the project towards clinical application. Therefore, the GENERATE project fully complies with the ANR’s and the French State’s 2021 strategic priority regarding translational research on rare diseases.