Gene Therapy Strategy Curbed Disease Features in CMT1A Rat Model

A form of gene therapy that reduces levels of the PMP22 protein prevented the development of Charcot-Marie-Tooth disease 1A (CMT1A) features in a rat model, a study has found. 

The study, “AAV2/9-mediated silencing of PMP22 prevents the development of pathological features in a rat model of Charcot-Marie-Tooth disease 1 A,” was published in the journal Nature Communications. 

CMT1A is caused by an extra copy of the PMP22 gene, which encodes the PMP22 protein, a critical component of the myelin sheath produced by Schwann cells. The myelin sheath is a fatty coating surrounding nerve fibers (axons) that enhances electrical signals between nerve cells. 

Excess PMP22 protein leads to defects in the myelin sheath, myelin degeneration (demyelination), and loss of axons. 

Available treatments help manage the condition, but no therapy addresses the underlying cause. 

Gene therapy to reduce the expression (production) of PMP22 protein has been proposed as a potential strategy to provide CMT1A patients with long-term benefits. 

This study, led by a team of researchers based in Montpellier, France, used animal models to examine the impact of delivering so-called small hairpin inhibitory RNAs (shRNAs) that target the PMP22 messenger RNA (mRNA) — the molecule that carries the instructions from the gene to make the PMP22 protein.  

By blocking, or silencing, PMP22, the mRNA may reduce the levels of PMP22 protein, thereby preventing demyelination and nerve fiber loss. 

The first series of experiments demonstrated that the modified adeno-associated viral vector — AAV2/9 — delivered a fluorescent marker directly to Schwann cells following injection into the sciatic nerves of mice and rats. These results were confirmed in a non-human primate model. 

Based on these findings, the team designed two shRNAs (sh1 and sh2) as well as a non-specific control shRNA packaged in AAV2/9, and injected them into the sciatic nerves of control rats and a CMT1A rat model bred to overexpress a mouse version of PMP22. 

This model mimics the clinical aspects of human disease closely, with myelin sheath defects and demyelination. Treated and control animals were followed for up to 12 months (one year) after injection.

Although neither AAV2/9-sh1 nor AAV2/9-sh2 treatment reduced Pmp22 mRNAs, further analysis showed both shRNAs decreased PMP22 protein levels back to control levels in Schwann cells isolated from the sciatic nerves. 

“Taken together these data showed that AAV2/9 vectors carrying shRNAs targeting Pmp22 were able to prevent PMP22 overexpression in [Schwann cells] of CMT1A rats through a mechanism independent of the mRNA stability,” the team wrote. 

Compared to healthy rats, the myelin sheath of animals with CMT1A-like disease showed defects including thin myelin or demyelinated areas. While these defects were still present in treated rats, they were less abundant. Also, the number of gaps between nerve fibers was increased in untreated CMT1A rats. These gaps decreased significantly in shRNA-treated animals. 

A microscopic analysis of nerve fibers found that treated animals had a significant increase in myelinated fibers density and number of large myelinated axons. At the same time, treatment reduced the number of small myelinated axons, which indicated that, overall, “the treatment with AAV2/9-sh1 or -sh2 prevents myelin loss and the occurrence of myelinated fiber defects in CMT1A rats,” the team added. 

As CMT1A is a demyelinating disease, one of the first symptoms is a decrease in electrical signals between nerve cells, referred to as nerve conduction velocity (NCV). This parameter was significantly reduced in the rat model of CMT1A, compared to controls, as soon as one month after birth. However, when CMT1A animals were treated with either shRNA, the NCV remained similar to normal values for at least 12 months. 

Motor behavior tests also showed reduced performances of CMT1A animals compared to controls, starting two months after birth. These deficits were largely prevented by treatment, with no significant evidence of reduced effectiveness after 12 months. In addition, increased pain sensitivity of lower limbs at six and 12 months post-injection was prevented by treatment. 

“Consequently, treatment with AAV2/9 vectors that reduces PMP22 levels in [Schwann cells] constitutes an efficient and long-term preventive treatment for CMT1A symptoms in rats,” the scientists wrote.

The team confirmed that AAV2/9 vector treatment primarily targeted Schwann cells, and immune responses to the viral vectors were limited. 

Finally, previously characterized human skin biomarkers distinguished between treated and untreated animals, which indicated their potential as an outcome measure for this treatment strategy. 

“Taken together, this work suggests that an intra-nerve AAV2/9-mediated gene therapy represents an effective and attractive therapy for myelin-related CMT diseases,” the scientists concluded.