Biotechnology harnesses cellular and biomolecular processes to develop technologies and products that help improve our lives and the health of our planet. Some examples include:

• combat rare diseases
• reduce environmental footprint
• use less and cleaner energy
• have safer, cleaner, and more efficient inducstrial manufacturing processes
• agricultural biotechnology to increase yields and prevent damage from insects

About DMD

Dystrophin is a protein found in muscle cells. It's essentially a rope-like material used in muscles to pull in actin filament in muscle fiber to the cytoskeleton of a muscle cell. It is essential for muscle contractions as a result.

DMD is a condition almost exclusively to boys, due to the relationship of it cutting the Dystrophin production in DNA via the X chromosome. Boys with DMD experience muscle weakness when they are 3 and cannot walk by the time they're 10. See this link for more information.

Diagnosing DMD

There's two ways to test for DMD

1. Genetic Test: via blood samples (much preferred)
   1. Genetic material is copies
   2. Identify what genetic material was copied in the first stage
2. Biopsy: via sample from a muscle tissue and testing for the existence of the dystrophin protein

Genetic Testing - PCR and Gel Electrophoresis

Polymerase chain reaction (PCR) is a process where a target stretch of DNA is selected and copied over and over. The starts and ends are identified and called primers. We may choose a pair of primers that mark the beginning and the end of the dystrophin gene on the X chromosome.

PCR can take a really small amount of DNA and create billions of copies of the target sequence. One simple test that could be run using PCR and gel electrophoresis:

1. Run PCR with a perimer set that reproduces normal dystrophin and another PCR with a primer set that reproduces the most common form of DMD-mutation
2. Compare the lengths of the genetic material produces to see what type of gene is in the sample

But before we can do this, we need to know the exact sequence of the dystrophin gene, so we know where to look in the DNA.

Sanger Sequencing

Frederick Snager made such a method.

TL;DR

• You add dexoy-deoxyribose versions of the ribse in DNA to the sequence
• These new versions will STOP the sequence at those points
• You do this for each nucleotide: A,C,T,G
• You then use electrophorisis to find the amounts of each subchain, and create the sequence going from most probably amount to least probable amount.

Treating the Symptoms

A combination of methods are used to treat DMD:

Muscle Health

DMD creates issues with muscle strength, so some solutions are to:

• Use pharmacology, ie: steroids, but can have serious side effects
• Therapy - doing stretching and other exercises made for people with DMD can help them prolong certain desired activities
• Assistive Devices - braces and splints, wheelchairs, etc.

Bone Health

Usually prescriptions are offered to help give essential vitamins and minerals so the body can build bone when weakened by the condition.

Heart Health

The heart can become weak and not pump blood properly. Cardiologists may prescribe medications to make the heart contract more forcefully or to control arrhythmia.

Other concerns

• Nutrition - having a healthy diet is important when mental anguish comes from having DMD. Further, over time people may not be able to swallow, so a feeding tube may be prescribed.
• Emotional and Mental Health - DMD is a stressful condition to have so physicians may encourage independence an dinvolvement in decision making; social and learning skills; etc.

Gene Editing

(1979+) Before, scientists had no way to edit genes, other than to "knock out" certain genes. And this was done only amongst yeast and mice. Removing a gene then replicating yeast or a mouse could show what the removed gene did. But this had limited clinical relevance and was only limited to these species.

(1995) Sloan-Kettering finds that cutting a strand of DNA at a certain location and trying to introduce a new strand of genetic material into the genome before repair would edit the gene.

(2012) Jennifer Dougna (UC Berkeley) and Emmanuelle Charpentier proposed a power method of targeted gene editing. This uses CRSPR and Cas9.

Gene Therapy

Gene Therapy has the promise to cure DMD. But it's really only accomplishable in experimental models and not yet humans. The problem is finding a way to introduce the functional dystrophin gene into the skeletal and cardiac muscles of the person with DMD.

Delivering Gene Therapy

There are three ways to deliver genes to a patient:

1. Naked DNA: Inserting the fixed DNA gene into cells. This doesn't work though since the DNA wouldn't survive. But it doesn't cause an immune response
2. Viral Vectors: Using viruses to inject DNA; however, the immune system tries to kill the virus. Further, viruses can only carry a small amount of DNA, not enough for the lengthy dystrophin gene
3. Plasmids packed in liposomes: take the naked DNA and package it into liposomes. This helps the DNA survive in the body is a very inefficient way to transfer genes into cells compared to viruses

Protein Therapy

Manufacturing protiens into the body could work. It won't cure DMD, but it can limit the damage of the DMD mutation and imporve muscle function. There are two main targets for protein therapy in DMD.

Myostatin Antibodies

Myostatin is a protein in muscle cells that effectively limits muscle growth. Species of cattle called Belgain Blue were found to be missing the protein, and thus had weird muscly tendons.

For DMD, a compound was tested in clinical trials but didn't really improve strength in DMD patients. Other trials have been done but results seem underwhelming.

Exon Skipping

Exon skipping attempts to identify the location of the mutation and skip over the entire exon that includes the mutation. This cuts out a section of the gene, but leaves the remained of the gene intact including the binding regions on either end of the protein. These treatments were approved by the FDA beginning in 2021.