Gene therapies for CMT neuropathies: from the bench to the clinic.

PURPOSE OF REVIEW: Charcot-Marie-Tooth (CMT) neuropathies are rare, genetically heterogeneous and progressive diseases for which there are no approved treatments and their management remains mostly supportive and symptomatic. This review is intended to provide an update on recent developments in gene therapies for different CMT neuropathies. RECENT FINDINGS: Increasing knowledge of disease pathomechanisms underlying several CMT types has facilitated the development of promising viral and nonviral gene therapy approaches. Some of these therapies are currently approaching the crucial step of moving from the bench to the clinic, having passed the proof-of-concept stage in rodent models and some also in larger animals. However, questions of optimal delivery route and dose, off-target effects, and possible payload toxicity remain to be clarified for several of these approaches. Furthermore, limited resources, the rarity of most CMT subtypes, and issues of safety and regulatory requirements, create the need for consensus guidelines and optimal clinical trial design. SUMMARY: Promising gene therapies have been developed for several CMT neuropathies, with proof-of-principle demonstrated in relevant disease models. Advantages and drawbacks of each approach are discussed and remaining challenges are highlighted. Furthermore, we suggest important parameters that should be considered in order to successfully translate them into the clinic.

AAV9-mediated SH3TC2 gene replacement therapy targeted to Schwann cells for the treatment of CMT4C.

Type 4C Charcot-Marie-Tooth (CMT4C) demyelinating neuropathy is caused by autosomal recessive SH3TC2 gene mutations. SH3TC2 is highly expressed in myelinating Schwann cells. CMT4C is a childhood-onset progressive disease without effective treatment. Here, we generated a gene therapy for CMT4C mediated by an adeno-associated viral 9 vector (AAV9) to deliver the human SH3TC2 gene in the Sh3tc2-/- mouse model of CMT4C. We used a minimal fragment of the myelin protein zero (Mpz) promoter (miniMpz), which was cloned and validated to achieve Schwann cell-targeted expression of SH3TC2. Following the demonstration of AAV9-miniMpz.SH3TC2myc vector efficacy to re-establish SH3TC2 expression in the peripheral nervous system, we performed an early as well as a delayed treatment trial in Sh3tc2-/- mice. We demonstrate both after early as well as following late treatment improvements in multiple motor performance tests and nerve conduction velocities. Moreover, treatment led to normalization of the organization of the nodes of Ranvier, which is typically deficient in CMT4C patients and Sh3tc2-/- mice, along with reduced ratios of demyelinated fibers, increased myelin thickness and reduced g-ratios at both time points of intervention. Taken together, our results provide a proof of concept for an effective and potentially translatable gene replacement therapy for CMT4C treatment.

Charcot-Marie-Tooth neuropathies: Current gene therapy advances and the route toward translation.

Charcot-Marie-Tooth (CMT) neuropathies are a group of genetically and phenotypically heterogeneous disorders that predominantly affect the peripheral nervous system. Unraveling the genetic and molecular mechanisms, as well as the cellular effects of CMT mutations, has facilitated the development of promising gene therapy approaches. Proposed gene therapy treatments for CMTs include virally or non-virally mediated gene replacement, addition, silencing, modification, and editing of genetic material. For most CMT neuropathies, gene- and disease- and even mutation-specific therapy approaches targeting the neuronal axon or myelinating Schwann cells may be needed, due to the diversity of underlying cellular and molecular-genetic mechanisms. The efficiency of gene therapies to improve the disease phenotype has been tested mostly in vitro and in vivo rodent models that reproduce different molecular and pathological aspects of CMT neuropathies. In the next stage, bigger animal models, in particular non-human primates, provide important insights into the translatability of the proposed administration and dosing, demonstrating scale-up potential and safety. The path toward clinical trials is faced with further challenges but is becoming increasingly feasible owing to the progress and knowledge gained from clinical applications of gene therapies for other neurological disorders, as well as the emergence of sensitive outcome measures and biomarkers in patients with CMT neuropathies.

NCAM1 and GDF15 are biomarkers of Charcot-Marie-Tooth disease in patients and mice.

Molecular markers scalable for clinical use are critical for the development of effective treatments and the design of clinical trials. Here, we identify proteins in sera of patients and mouse models with Charcot-Marie-Tooth disease (CMT) with characteristics that make them suitable as biomarkers in clinical practice and therapeutic trials. We collected serum from mouse models of CMT1A (C61 het), CMT2D (GarsC201R, GarsP278KY), CMT1X (Gjb1-null), CMT2L (Hspb8K141N) and from CMT patients with genotypes including CMT1A (PMP22d), CMT2D (GARS), CMT2N (AARS) and other rare genetic forms of CMT. The severity of neuropathy in the patients was assessed by the CMT Neuropathy Examination Score (CMTES). We performed multitargeted proteomics on both sample sets to identify proteins elevated across multiple mouse models and CMT patients. Selected proteins and additional potential biomarkers, such as growth differentiation factor 15 (GDF15) and cell free mitochondrial DNA, were validated by ELISA and quantitative PCR, respectively. We propose that neural cell adhesion molecule 1 (NCAM1) is a candidate biomarker for CMT, as it was elevated in Gjb1-null, Hspb8K141N, GarsC201R and GarsP278KY mice as well as in patients with both demyelinating (CMT1A) and axonal (CMT2D, CMT2N) forms of CMT. We show that NCAM1 may reflect disease severity, demonstrated by a progressive increase in mouse models with time and a significant positive correlation with CMTES neuropathy severity in patients. The increase in NCAM1 may reflect muscle regeneration triggered by denervation, which could potentially track disease progression or the effect of treatments. We found that member proteins of the complement system were elevated in Gjb1-null and Hspb8K141N mouse models as well as in patients with both demyelinating and axonal CMT, indicating possible complement activation at the impaired nerve terminals. However, complement proteins did not correlate with the severity of neuropathy measured on the CMTES scale. Although the complement system does not seem to be a prognostic biomarker, we do show complement elevation to be a common disease feature of CMT, which may be of interest as a therapeutic target. We also identify serum GDF15 as a highly sensitive diagnostic biomarker, which was elevated in all CMT genotypes as well as in Hspb8K141N, Gjb1-null, GarsC201R and GarsP278KY mouse models. Although we cannot fully explain its origin, it may reflect increased stress response or metabolic disturbances in CMT. Further large and longitudinal patient studies should be performed to establish the value of these proteins as diagnostic and prognostic molecular biomarkers for CMT.

AAV9-mediated Schwann cell-targeted gene therapy rescues a model of demyelinating neuropathy.

Mutations in the GJB1 gene, encoding the gap junction (GJ) protein connexin32 (Cx32), cause X-linked Charcot-Marie-Tooth disease (CMT1X), an inherited demyelinating neuropathy. We developed a gene therapy approach for CMT1X using an AAV9 vector to deliver the GJB1/Cx32 gene under the myelin protein zero (Mpz) promoter for targeted expression in Schwann cells. Lumbar intrathecal injection of the AAV9-Mpz.GJB1 resulted in widespread biodistribution in the peripheral nervous system including lumbar roots, sciatic and femoral nerves, as well as in Cx32 expression in the paranodal non-compact myelin areas of myelinated fibers. A pre-, as well as post-onset treatment trial in Gjb1-null mice, demonstrated improved motor performance and sciatic nerve conduction velocities along with improved myelination and reduced inflammation in peripheral nerve tissues. Blood biomarker levels were also significantly ameliorated in treated mice. This study provides evidence that a clinically translatable AAV9-mediated gene therapy approach targeting Schwann cells could potentially treat CMT1X.

Emerging Therapies for Charcot-Marie-Tooth Inherited Neuropathies.

Inherited neuropathies known as Charcot-Marie-Tooth (CMT) disease are genetically heterogeneous disorders affecting the peripheral nerves, causing significant and slowly progressive disability over the lifespan. The discovery of their diverse molecular genetic mechanisms over the past three decades has provided the basis for developing a wide range of therapeutics, leading to an exciting era of finding treatments for this, until now, incurable group of diseases. Many treatment approaches, including gene silencing and gene replacement therapies, as well as small molecule treatments are currently in preclinical testing while several have also reached clinical trial stage. Some of the treatment approaches are disease-specific targeted to the unique disease mechanism of each CMT form, while other therapeutics target common pathways shared by several or all CMT types. As promising treatments reach the stage of clinical translation, optimal outcome measures, novel biomarkers and appropriate trial designs are crucial in order to facilitate successful testing and validation of novel treatments for CMT patients.

Lumbar Intrathecal Injection in Adult and Neonatal Mice.

This article describes a step-by-step process of lumbar intrathecal injection of Evans blue dye and AAV9-EGFP in adult (2-month-old) and neonatal (postnatal day 10) mice. Intrathecal injection is a clinically translatable technique that has already been extensively applied in humans. In mice, intrathecal injection is considered a challenging procedure that requires a trained and experienced researcher. For both adult and neonatal mice, lumbar intrathecal injection is directed into the L5-L6 intervertebral space. Intrathecally injected material enters the cerebrospinal fluid (CSF) within the intrathecal space from where it can directly access the central nervous system (CNS) parenchyma. Simultaneously, intrathecally injected material exits the CSF with pressure gradient and enters the endoneurial fluid and ultimately the peripheral nerves. While in the CSF, the injectable material also enters the bloodstream and systemic circulation through the arachnoid villi. A successful lumbar intrathecal injection results in adequate biodistribution of the injectable material in the CNS, PNS, and peripheral organs. When correctly applied, this technique is considered as minimally invasive and non-disruptive and can be used for the lumbar delivery of any solute. © 2024 Wiley Periodicals LLC. Basic Protocol 1: C57BL/6 adult and P10 mice lumbar intrathecal injection Basic Protocol 2: Tissue collection and preparation for evaluating Evans blue dye diffusion Basic Protocol 3: Tissue collection and preparation for immunohistochemistry staining Basic Protocol 4: Tissue collection and vector genome copy number analysis.

CMT1A current gene therapy approaches and promising biomarkers.

Charcot-Marie-Tooth neuropathies (CMT) constitute a group of common but highly heterogeneous, non-syndromic genetic disorders affecting predominantly the peripheral nervous system. CMT type 1A (CMT1A) is the most frequent type and accounts for almost ~50% of all diagnosed CMT cases. CMT1A results from the duplication of the peripheral myelin protein 22 (PMP22) gene. Overexpression of PMP22 protein overloads the protein folding apparatus in Schwann cells and activates the unfolded protein response. This leads to Schwann cell apoptosis, dys- and de- myelination and secondary axonal degeneration, ultimately causing neurological disabilities. During the last decades, several different gene therapies have been developed to treat CMT1A. Almost all of them remain at the pre-clinical stage using CMT1A animal models overexpressing PMP22. The therapeutic goal is to achieve gene silencing, directly or indirectly, thereby reversing the CMT1A genetic mechanism allowing the recovery of myelination and prevention of axonal loss. As promising treatments are rapidly emerging, treatment-responsive and clinically relevant biomarkers are becoming necessary. These biomarkers and sensitive clinical evaluation tools will facilitate the design and successful completion of future clinical trials for CMT1A.

A translatable RNAi-driven gene therapy silences PMP22/Pmp22 genes and improves neuropathy in CMT1A mice.

Charcot-Marie-Tooth disease type 1A (CMT1A), the most common inherited demyelinating peripheral neuropathy, is caused by PMP22 gene duplication. Overexpression of WT PMP22 in Schwann cells destabilizes the myelin sheath, leading to demyelination and ultimately to secondary axonal loss and disability. No treatments currently exist that modify the disease course. The most direct route to CMT1A therapy will involve reducing PMP22 to normal levels. To accomplish this, we developed a gene therapy strategy to reduce PMP22 using artificial miRNAs targeting human PMP22 and mouse Pmp22 mRNAs. Our lead therapeutic miRNA, miR871, was packaged into an adeno-associated virus 9 (AAV9) vector and delivered by lumbar intrathecal injection into C61-het mice, a model of CMT1A. AAV9-miR871 efficiently transduced Schwann cells in C61-het peripheral nerves and reduced human and mouse PMP22 mRNA and protein levels. Treatment at early and late stages of the disease significantly improved multiple functional outcome measures and nerve conduction velocities. Furthermore, myelin pathology in lumbar roots and femoral motor nerves was ameliorated. The treated mice also showed reductions in circulating biomarkers of CMT1A. Taken together, our data demonstrate that AAV9-miR871-driven silencing of PMP22 rescues a CMT1A model and provides proof of principle for treating CMT1A using a translatable gene therapy approach.

Genetic mechanisms of peripheral nerve disease.

Peripheral neuropathies of genetic etiology are a very diverse group of disorders manifesting either as non-syndromic inherited neuropathies without significant manifestations outside the peripheral nervous system, or as part of a systemic or syndromic genetic disorder. The former and most frequent group is collectively known as Charcot-Marie-Tooth disease (CMT), with prevalence as high as 1:2,500 world-wide, and has proven to be genetically highly heterogeneous. More than 100 different genes have been identified so far to cause various CMT forms, following all possible inheritance patterns. CMT causative genes belong to several common functional pathways that are essential for the integrity of the peripheral nerve. Their discovery has provided insights into the normal biology of axons and myelinating cells, and has highlighted the molecular mechanisms including both loss of function and gain of function effects, leading to peripheral nerve degeneration. Demyelinating neuropathies result from dysfunction of genes primarily affecting myelinating Schwann cells, while axonal neuropathies are caused by genes affecting mostly neurons and their long axons. Furthermore, mutation in genes expressed outside the nervous system, as in the case of inherited amyloid neuropathies, may cause peripheral neuropathy resulting from accumulation of ß-structured amyloid fibrils in peripheral nerves in addition to various organs. Increasing insights into the molecular-genetic mechanisms have revealed potential therapeutic targets. These will enable the development of novel therapeutics for genetic neuropathies that remain, in their majority, without effective treatment.