Molecular Therapies for Neuromuscular Disorders

Introduction

Most of the introduction basically went over the basics of gene expression and types of mutations, which I've also written about here. Also alternative splicing is important because the exons that get included in the final product can determine the function of the protein made. This post will focus mainly on interventions that target splicing.

Here are some examples of diseases caused by mutations:

• Huntington's disease is caused by "unstable repeats" of CAG. CAG codes for glutamine, so this mutation is also known as poly-Q. It is normal to have fewer than 36 CAG repeats. People with more than 40 are affected, and those in between might have issues and are more likely to pass Huntington's on to their children. Because these repeats are unstable, disease onset is earlier and more severe with each generation.
• Fragile X mental retardation is caused by having too many CGG repeats in a noncoding region. The normal amount of CGG repeats is 26-50. Fewer repeats may cause intellectual disability, but this is relatively stable. 50-58 is listed as "intermediate" in my notes (no idea what this means), 59-200 is unstable Fragile X pre-mutation syndrome, and more than 200 repeats results in Fragile X mental retardation syndrome.
• Fascioscapulohumeral muscular dystrophy (FSHD1A) is caused by the opposite problem: too few repeats. Fewer than 10 repeats in the relevant gene (the D4Z4 repeat array in the 4q35 subtelomeric region... whatever all that means) can cause the disorder.

Overview of genetic therapy strategies

See earlier post: Introduction to Gene Therapy

Generally "gene therapy" is used to describe replacing and repairing genes, whereas "molecular therapy" is the term given to altering gene expression. For example, inducing fetal gamma-globulin with butyrate or valproate may be used to treat thalassemia. Also, nonsense mutations (mutations that result in a premature stop codon) can be bypassed by using aminoglycoside antibiotics, though these can have unwanted side-effects.

The CRISPR-Cas system, which is basically the acquired immune system of prokaryotes, can be used to help with gene editing. CRISPRs consist of DNA loci with short repeats. Each repetition is followed by "spacer DNA," which recognises and silences exogenous genetic elements. Cas genes encode proteins related to CRISPRs.

To use CRISPRs, the plasmid or virus containing cas genes and specifically designed guide RNAs (single guide RNAs, or sgRNAs) are inserted. Cas9 endonuclease forms a complex with double-stranded DNA and sgRNA. Cas9:sgRNA binds the DNA to generate an R-loop, resulting in a double-stranded break where genes can be inserted.

Another strategy that is used, and that I'll talk about a lot in this post, is using antisense oligonucleotides to alter splicing and translation. This may be done to remove selected exons to bypass a mutation, or to degrade unwanted transcripts. Two RNA-like oligonucleotides commonly used in altering splicing are 2' O methyl phosphorothioate and phosphorodiamidate morpholino oligomer (PMO). These oligonucleotides are also known as "new-generation antivirals" as they have also been found to be effective against 75% of all human viruses. Transcript degradation can be induced by inducing RNase H (which can be induced by a DNA antisense oligonucleotide binding to an RNA strand) or by using siRNA (silencing RNA). Transcript stability can also be modified with the help of micro RNA (mir).

Duchenne Muscular Dystrophy (DMD)

See previous post: Introduction to Gene Therapy. Note that carriers of DMD are not entirely unaffected: they begin life with dystrophin in only ~50% of their muscle fibres.

Mice models of DMD (mdx mice) have an early stop codon in exon 23. Antisense oligonucleotides can be used to bypass this exon and allow production of a shorter yet functional (possibly BMD-like) form of dystrophin. Unfortunately, humans are a bit more complicated, and there are many different mutations that DMD patients may have.

Some patients with DMD are missing exon 50, resulting in a premature stop codon in exon 51. To circumvent this, Exondys51 (eteplirsen) can be used to splice out exon 51. Since many BMD patients are missing exons 50 and 51, eteplirsen can be used to create a BMD-like dystrophin. Clinical trials have found that eteplirsen can help in ambulation and also increases dystrophin expression (though not to normal levels).

As I just said, humans are complicated. Not every patient with DMD is missing exon 50 and therefore not all can be treated with the same drug. In fact, it is thought that only around 13% of patients will be able to benefit from this drug, and around 20% of patients are thought to have mutations that are not amenable to exon skipping. Oh well, baby steps I guess!

Spinal Muscular Atrophy (SMA)

Spinal muscular atrophy (SMA) is caused by a loss of the SMN1 gene on chromosome 5. SMN1 is important in all cells. It is made up of 8 exons, though around 10% of the time exon 7 is spliced out to create a non-functional protein (even in healthy individuals). Humans also have a similar gene called SMN2, which only differs from SMN1 by five bases. SMN2 can also produce a functional protein, though it is far more likely to skip exon 7 and produce a non-functional protein as compared to SMN1. Treatment strategies involve using antisense oligonucleotides to turn off silencing elements around exon 7 in order to hopefully upregulate SMN2 production. The idea is that if we upregulate SMN2 enough, it might be able to produce enough protein to overcome the effects of missing SMN1. One such treatment is Nusinersan, which is a 2' O methoxyethyl oligonucleotide that induces SMN2 exon 7 inclusion.