Gene Therapy for Duchenne Muscular Dystrophy

Gene therapies aim to treat disease by correcting the underlying genetic problem. Duchenne Muscular Dystrophy (DMD) gene therapies, such as micro-dystrophin, supply a functional dystrophin gene to muscle. Exon skipping and nonsense mutation readthrough therapies allow the faulty dystrophin gene to gain partial function.

There are several gene therapy approaches to correcting the body’s ability to make functional dystrophin. One is to introduce a replacement dystrophin gene, which gives the muscle cells a new set of instructions for making dystrophin. Other approaches alter how the genetic instructions are followed, allowing the faulty dystrophin gene to gain partial function. How gene therapies might help people with DMD will be described in more detail after first establishing a bit of foundation on how gene mutations disrupt protein production.

Genes and Proteins

The long double-helix DNA molecules located in the nucleus of almost all our cells contain functional segments called genes. DNA and genes are comprised of four base molecules known as A, C, G, and T linked together in a particular sequence known as genetic code. For protein-coding genes, the sequence of these base molecules code for the sequence of amino acids in a protein.

Before protein is produced inside the cell, an RNA transcript or copy of the gene is made. RNA contains the same sequence of bases as DNA, except that in RNA the bases are A, C, G, and U instead of T. Genes have coding regions called exons and intervening non-coding regions called introns. Exons are joined together as introns are removed during the maturation of the RNA into messenger RNA (mRNA).

Protein production machinery in the cell translates the mRNA transcript into groups of three base molecules called codons. Each codon designates the addition of one amino acid. In this process, called translation, proteins are built by adding one amino acid at a time as specified by the genetic code. Once a stop codon is reached, the translation process is terminated.

Gene Mutations

DMD is caused by mistakes in the genetic code called mutations. Mutations include missing gene segments called deletions, duplicated regions, or point mutations. An example of a point mutation would be a change in a single base in the genetic sequence, like from ACCGGT to ACTGGT. If the point mutation changes a codon to designate a different amino acid this is called a missense mutation. A nonsense mutation is a point mutation that changes an amino acid codon into a stop codon causing the protein to be truncated. Since the translational machinery reads the bases in groups of three, deletions and insertions that are not a multiple of three can cause a frameshift, resulting in different amino acid codons.

Due to mutations in dystrophin, people with Duchenne produce no functional dystrophin. The normal function of dystrophin is to stabilize muscle cell membranes and provide support to the contractile apparatus of muscle. This is a very important job considering that all our movements from walking to breathing require the shortening and lengthening of muscle tissue via its contractile apparatus. Gene therapy hopes to give people with DMD some functional dystrophin to perform this job.

Gene Therapy Delivery

Many gene therapies aim to replace a defective gene with a functioning gene. Delivering a replacement gene requires specialized packages that transfer the gene to the inside of cells. These specialized packages, called gene therapy vectors, may be derived from viruses, with modifications so that instead of transferring viral genetic material into cells, they transfer the desired gene therapy. Adeno-associated virus (AAV) is commonly used in gene therapy and is efficient at transferring genetic material into muscle. Non-viral vectors are sort of like bubble-like structures made of lipids or polymers that can carry the replacement gene and release it inside cells.

The type of vector used has an impact on safety and ability to effectively transfer the therapeutic product into the target cells. Vectors that keep the gene separate from the patient’s DNA are considered safer than those that integrate the therapeutic gene into the patient’s DNA because the latter carries a higher risk of causing cancer. Adenovirus and AAV usually do not integrate genes into the patient’s DNA, but this can occur at very low frequency, which carries potential risk.

Many people have developed immunity to adenoviruses and AAV from common respiratory infections. These prior infections or previous gene therapy can lead to an immune response that renders the therapy ineffective. There is a risk for more serious inflammatory reactions that cause health complications. This risk is lower for AAV than adenoviral vectors.

Gene replacement therapy

A challenge for DMD is that the dystrophin gene is so large that it is difficult to fit inside gene therapy vectors. So rather than trying to replace the entire dystrophin gene, scientists have come up with other strategies. Mini-dystrophin and micro-dystrophin are DMD gene therapies that introduce a smaller version of the dystrophin gene as a replacement. While they don’t result in the production of full-length dystrophin, the shorter dystrophin protein produced can partially function.

Mini-dystrophin gene therapy introduces a shorter version of dystrophin. It is similar to the partially functional dystrophin produced in the milder disease, Becker muscular dystrophy. An AAV vector carries the shortened version of dystrophin with a gene element called a promotor that causes the gene to be active in muscle tissue. The micro-dystrophin gene therapy is trimmed down even further, to the most essential parts of the protein. A specialized type of AAV is used that is particularly efficient at targeting muscle tissue and the treatment. In clinical trials, recipients produced micro-dystrophin protein in their muscles and showed improvements in their motor function. Both mini-dystrophin and micro-dystrophin gene therapies are delivered by intravenous injection.

Skipping over mutations

Exon skipping and nonsense mutation readthrough therapies are different in that they do not deliver a replacement gene or set of instructions. Instead, these approaches trick the cellular machinery into skipping over the mutations on their dystrophin gene and producing a somewhat functional dystrophin protein. These therapies deliver types of molecules that target steps after RNA transcription and before protein translation.

Exon Skipping

Exon-skipping gene therapies induce the RNA transcript to skip over or exclude an exon with a deletion and produce a smaller but still functional dystrophin protein. The therapeutic molecule is an antisense oligonucleotide, which is a short molecule that is complementary to and sticks to the dystrophin RNA transcript. When the transcript is being processed, this prevents the targeted exon from being included when the exons are joined together. The resulting mRNA does not contain the mutation and codes for a dystrophin protein that can function, at least partially. Research is ongoing to target different exons and to improve the delivery methods of exon-skipping therapies.

Nonsense Mutation Readthrough

Nonsense mutation readthrough is a type of therapy that targets nonsense mutations, which account for 13% of Duchenne muscular dystrophy cases. Proteins produced by nonsense mutations stop translation prematurely so they can be missing essential parts. Some nonsense mutations produce partially functional dystrophin, such as seen in Becker muscular dystrophy. DMD nonsense mutations result in no dystrophin. This is because the defective protein and the defective mRNA transcript tend to be disposed of by cellular clean-up processes. Nonsense mutation readthrough therapy introduces a small molecule, that can be taken orally, that encourages the translation machinery to read through or ignore the stop signal and continue building full-length dystrophin.

DMD Clinical Trials

For DMD a handful of exon skipping gene therapies have completed clinical trials and are approved in various jurisdictions. There are other exon-skipping therapies currently in Duchenne clinical trials. Nonsense mutation readthrough, mini-dystrophin, and micro-dystrophin are in clinical trials for Duchenne. Nonsense mutation readthrough has approval in some areas.

In addition to gene therapies focused on correcting the ability to produce dystrophin, Duchenne therapies also target other parts of the disease such as chronic inflammation and muscle weakness. A steroid alternative is in clinical trials that has anti-inflammatory properties similar to corticosteroid but with less of the undesirable side effects.

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