#AANAM — Combined Gene Therapy/RNA Strategy May Correct Cellular Changes in CMT2A, Study Suggests

A combination of gene therapy and a strategy called RNA interference (RNAi) successfully repaired disease-related alterations in nerve cells derived from patients with Charcot-Marie-Tooth disease type 2A (CMT2A), according to a study.

The study, “RNAi/gene therapy combined approach as therapeutic strategy for Charcot-Marie-Tooth 2A,” was presented by Valentina Melzi at the 2019 American Academy of Neurology (AAN) annual meeting, recently held in Philadelphia.

CMT2A is caused by mutations in MFN2, a gene that provides instructions for a protein called mitofusin 2, which is involved in the fusion of mitochondria (which provide energy to cells).

Although MFN2 mutations have been characterized and several disease-related processes have been proposed, CMT2A still lacks effective treatments.

To address this, a team from Italy and the U.K. investigated a new therapeutic approach that combines gene therapy with RNAi — a naturally occurring mechanism that uses tiny RNA molecules to suppress gene expression.

In induced pluripotent stem cells (iPSCs) — developed by reprogramming cells so that they revert to an embryonic-like state able to grow into all cell types — from CMT2A patients, the scientists targeted the mutant MFN2 gene copy with specific short hairpin RNAs (shRNAs) — artificial RNA molecules that block gene expression.

Simultaneously, they administered an altered MFN2 gene able to resist shRNAs and generate the normal mitofusin 2 protein. In this way, the combination therapy can silence the disease-causing protein and restore the normal, correct version.

The researchers then differentiated iPSCs into spinal motor neurons — specialized cells that control muscle contraction — to analyze the impact of their strategy on CMT2A-associated changes they had previously reported. These alterations included a reduced amount and altered location of mitochondria, particularly in motor nerve cells.

Results revealed that shRNAs successfully suppressed the expression of the cells’ own MFN2 gene. In turn, the delivered gene enabled the production of functional protein without altering the cells’ ability to develop into all types of adult cells.

Findings also revealed that motor neurons from both treated and untreated CMT2A stem cells had a typical shape and appearance, and produced their main markers.

Importantly, the combination therapy effectively addressed CMT2A-related changes in motor nerve cells, as reflected by an increased number of mitochondria, improved transport of these organelles along nerve fibers, restored sensitivity to apoptosis — which refers to “programmed” cell death, as opposed to death caused by injury — and reduced degradation of mitochondria by a cellular process called autophagy.

“Overall, our results led to a significant level of rescue in CMT2A [motor neurons], suggesting RNAi/gene therapy combined approach as a promising therapeutic strategy for CMT2A,” the scientists wrote.