DMD, Going one level deeper – a personal problem…

I’ll keep these notes updated as a learn more about Duchenne Muscular Dystrophy (DMD) and the progress toward a cure.

I love Wired.  They have incredible content for people interested in STEM but after I read an article I’m often left with a feeling that I grasped the basics but I really didn’t understand the details–and I think it may be because I didn’t listen as well as I should have during high school biology.  For example, this article from August 2018 on DMD was very interesting to me because I have young relatives with the disease.  

Basically the article says the following:

  • Some King Charles Spaniels have a mutation on their X chromosomes, in a gene that codes for a muscle protein called dystrophin much like a human suffering from DMD.
  • Eric Olson from the University of Texas Southwestern Medical Center has successfully halted the progression of the disease in some of the dogs using a gene editing tool known as CRISPR but there is still a lot of work to be done (additional longer-term canine studies to test for safety) before human trials would be safe.
  • “Olson found a way to target an error-prone hot spot on exon 51, which he figured could, with a single slice, benefit approximately 13 percent of DMD patients.”
  • Olson licensed the technology and founded a startup called Exonics Therapeutics along with the CureDuchenne group (who invested $2M) and The Column Group (who invested $40M).
  • One of the challenges is figuring out how to manufacture enough viral delivery vehicles to inject CRISPR into all the muscles in the human body.

I get the basics and I should just move on but I can’t... I need to know more.  The new technology fascinates me: What is CRISPR and how does it work?  What is gene editing? What is a viral delivery vehicle?  What is dystrophin? ...but then there are also items I should understand but I don’t (items that I know I learned in high school but I’ve forgotten or never really grasped at the time): What’s a chromosome? What’s a gene?  What’s an Exon? What’s a protein and why is it important? …and how do dogs relate to humans? 

So the journey begins and it shows how I think and my limitations :-).  I know I won’t understand what Exonics does without understanding CRISPR/Cas9.  I won’t understand CRISPR/Cas9 without understanding ‘gene editing’.  I won’t understand ‘gene editing’ without understanding chromosomes & genes.  I won’t understand chromosomes & genes without understanding DNA.  I won’t understand DNA without understanding cells.   I won’t understand cells without understanding proteins. I won’t understand proteins without understanding molecules and atoms.  Hopefully, you get the point.  Most people know when to stop… me… unfortunately I need to go one step further and I constantly find myself realizing I didn’t retain much of what I learned in high school. …and then it becomes a bit of a puzzle. Some people like Sudoku… I like science. …but unfortunately, I’m not a scientist however I do have the passion (and motivation) to learn about this subject. 

Let’s start with the basic definitions (YES, high school biology)–humans only:

What’s a cell

The cell is the smallest unit of life.  The human body has >10Trillion cells.  A Cell has a membrane that contains receptors (proteins) that detect external signaling (ex. Hormones) and cytoplasm (all the stuff inside the cell like amino acids that perform functions and the nucleus).  

We have to take a detour to high school chemistry for a second: What are molecules and atoms?

An atom is the smallest unit of matter containing a nucleus (Protons, Neutrons) and electrons. The number of atoms in the human body–it’s staggering (here).

A molecule is 2 or more atoms held together by chemical bonds.  Much of the research references molecular formulas so you need to understand them. 

A molecular formula (example ‘a’) is a representation of a molecule that uses chemical symbols to indicate the types of atoms followed by subscripts to show the number of atoms of each type in the molecule. (A subscript is used only when more than one atom of a given type is present.)

The structural formula (example ‘b’) for a compound gives the same information as its molecular formula (the types and numbers of atoms in the molecule) but also shows how the atoms are connected in the molecule. The lines represent bonds that hold the atoms together. A chemical bond is an attraction between atoms or ions that holds them together in a molecule.

Example A and B are the formulae for methane as it contains one Carbon atom and four Hydrogen atoms.  Here are other examples for your reference:

A typical human cell has somewhere around 42 million protein moleculesYou can also find he number of molecules in the human body (here).

What is DNA (Deoxyribonucleic acid)?

DNA (and RNA) are nucleic acids, and, along with lipids, proteins and carbohydrates, constitute the four major macromolecules essential for all known forms of life.

Specifically, DNA is a molecule composed of two chains that coil around each other to form a double helix carrying the genetic instructions used in the growth, development, functioning, and reproduction of all known living organisms.  All the cells in a person’s body have the same DNA and the same genes. However, the difference between cells in different tissues and organs is that the “expression” of the genes differs between cells. Expression generally means that the message from the DNA is being copied and made into protein. For example, liver cells have different proteins than skin cells, even though their DNA is the same.

DNA is made up of Nucleotides (sugar, phosphates and nitrogenbases).  There are 4 types of nitrogen bases: Thymine (T), Adenine(A), Guanine (G), Cytosine(C)

“A” bonds only with “T” and “C” only bonds with “G”

What is RNA (Ribonucleic acid)? 

RNA is a molecule essential in various biological roles in coding, decoding, regulation, and expression of genes. Like DNA, RNA is assembled as a chain of nucleotides, but unlike DNA it is more often found in nature as a single-strand folded onto itself. Cellular organisms use messenger RNA (mRNA) to convey genetic information (using the nitrogenous bases of guanine, uracil, adenine, and cytosine, denoted by the letters (G, U, A, and C) that directs the synthesis of specific proteins. Many viruses encode their genetic information using an RNA genome.

What is a chromosome?

A chromosome is a DNA molecule that contains part of a human’s genetic material.  A  human cell nucleus contains 23 pairs (46 total) of chromosomes (DNA molecules) which are long strands of DNA tightly wound into coils (note that sperm and egg cells contain only 23 total chromosomes). If you unwound each cells DNA it would be about 6 foot long. 

What is a gene?

gene is a sequence (section) of DNA or RNA that uses a set of rules to translate information encoded within the DNA or mRNA sequences into proteins for a molecule that has a function.

Genes are either turned ‘on’ or ‘off’ mixed among other non-coded ‘junk DNA’.

Human beings have roughly 20,500 genes, all coiled up in DNA, housed in each cell. That’s 20,500 places where the machinery of human life can be altered.

Genes are divided into sections called exons and introns (junk DNA). Exons are the sections of DNA that code for the protein and they are interspersed with introns.

The HUGO Gene Nomenclature Committee (HGNC) designates an official name and symbol (an abbreviation of the name) for each known human gene. The Committee has named more than 13,000 of the estimated 20,000 to 25,000 genes in the human genome.

Genes can also mutate…  Although the human genome consists of 3 billion nucleotides, changes in even a single base pair can result in dramatic physiological malfunctions.  For example, sickle-cell anemia is a disease caused by the alteration of a single nucleotide in the gene for the beta chain of the hemoglobin protein (the oxygen-carrying protein that makes blood red) and that is all it takes to turn a normal hemoglobin gene into a sickle-cell hemoglobin gene. This single nucleotide change alters only one amino acid in the protein chain– the results are devastating! Beta hemoglobin is a single chain of 147 amino acids, but because of the single-base mutation, the sixth amino acid in the chain is valine, rather than glutamic acid. Note below that ‘Wild-Type’ is the normal hemoglobin. 

To understand amino acids like valine and glutamic acid you need to understand the codon table found here:

Gene Sequencing

DNA sequencing is the process of determining the order of nucleotides in DNA.  DNA molecules are incredibly long and consist of billions of nitrogen bases. In fact, if all the DNA bases of the human genome were typed as A, C, T, and G, the 3 billion letters would fill 4,000 books of 500 pages each.  The Human Genome Project was the effort to map all the human nucleotides and genes. 

The sickle-cell gene mentioned above is CLLU1 and if you were to compare the human gene sequence to that of a chimp or a macaque it would look like the following:


There are 2 common Genome Browsers (and several others).  One from Ensembl and another from the University of California Santa Cruz Genomics Institute browser.

Let’s look at an Ensembl example:

Within the chromosome you can view the detail of a region (1) and inspect the genes (2).  For example, here (3) you can see the sickle-cell anemia gene CLLU1

The sequence will provide the order of nucleotides in the gene and you can begin to see the sequence from the chimp / macaque example from above (1). 

Now, with that backdrop, we can now begin to understand the content in the Wired article.

What is CRISPR/Cas9 and gene editing? 

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) method is based on a natural system used by bacteria to protect themselves from infection by viruses.  When a bacterium detects the presence of virus DNA it produces 2 types of short RNA one of which contains a sequence that matches that of the invading virus.  These 2 RNAs form a complex with a protein enzyme called Cas9. Cas9 can cut DNA (think of Cas9 as a set of molecular scissors). When the matching sequence known as a “guide” RNA finds it matching target within the viral genome the Cas9 cuts the target DNA disabling the virus.

Cas9 can be engineered to cut any DNA sequence (not just viral DNA) at a precise location by changing the guide RNA to match the target DNA. Once inside the nucleus of the cell, the RNA-Cas9 complex will locate and lock on to a short target sequence known as the PAM (Protospacer Adjacent Motif). The Cas9 will then unzip the DNA and match it to its target RNA and if the match is complete the Cas9 will use its tiny molecular scissors to cut the DNA.  Once the CRISPR system has made the cut this new DNA can pair up with the cut ends recombining and replacing the original sequence with the new version.

Here is the basic process:

  1. Build the guide RNA (gRNA).  This guide RNA will direct the protein (Cas9) to its target DNA sequence. The guide RNA consists of a tracrRNA (a scaffold sequence necessary for Cas-binding) and a crRNA sequence (a user-defined ∼20 nucleotide spacer) that is identical to the target. The crRNA can be any ∼20 nucleotide DNA sequence, provided it meets two conditions:
    • The sequence is unique compared to the rest of the genome.
    • The target is present immediately adjacent to the Protospacer Adjacent Motif (PAM). The PAM sequence is essential for target binding, but the exact sequence depends on which Cas protein you use (check out the list of additional Cas proteins and PAM sequences).
  2. Guide RNA + CAS9. Once expressed, the Cas9 protein and the gRNA form a complex through interactions between the gRNA scaffold and surface-exposed positively-charged grooves on Cas9. Cas9 undergoes a conformational change upon gRNA binding that shifts the molecule from an inactive, non-DNA binding entity into an active DNA-binding entity. Importantly, the spacer region of the gRNA remains free to interact with target DNA.
  3. Bind. Once the Cas9-gRNA complex finds a DNA target, the seed sequence (8-10 bases at the 3′ end of the gRNA targeting sequence) will begin to bind to the target DNA. If the seed and target DNA sequences match, the gRNA will continue to bind to the target DNA in a 3′ to 5′ direction.
  4. Cut. Once Cas9 binds to the target DNA it cuts the target DNA ∼3-4 nucleotides upstream of the PAM sequence.
  5. REPAIR: (NHEJ or HDR) Once the CRISPR system has made the cut this new DNA can pair up with the cut ends recombining and replacing the original sequence with the new version.
    • The efficient but error-prone non-homologous end joining (NHEJ) pathway
    • The less efficient but high-fidelity homology-directed repair (HDR) pathway

CRISPR can also be used to target many genes at once which is helpful for complex diseases that are caused not by one single mutation but by many genes acting together.

If you want to geek out you can try CRISPR yourself by ordering a kit here.  … here is a YouTube video that shows the basics.  If you want to go very very deep on CRISPR read this PMC article.

What is Dystrophin and how is it important to Duchenne Muscular Dystrophy (DMD)?

In the study published in Science, a team led by Eric Olson at the University of Texas Southwestern Medical Center used CRISPR to successfully modify the DNA of four young dogs, reversing the molecular defect responsible for the canine version of DMD

The dystrophin gene (view it in Ensembl) is the largest in the human genome, and there are thousands of different mutations that can all result in the disease. Olson found a way to target an error-prone hot spot on exon 51 (Ensembl), which he figured could, with a single slice, benefit approximately 13 percent of DMD patients

However, a challenge is manufacturing enough viral delivery vehicles to inject CRISPR into all the muscles in the human body and it is expensive.

What is Exonics doing?

From PMC Oct 2018 Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy

From Oct 2018 “We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression…” “dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. ” You can purchase the full report for $30 here.

From PMC Nov 2017 Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy

From the funding PR release Nov 2017: “Exonics has used SingleCut CRISPR to genetically repair and restore dystrophin, the key protein missing in children with Duchenne.”

From April 2017 CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice “pathophysiological hallmarks of muscular dystrophy were corrected in mdx mice following Cpf1-mediated germline editing”

These folks at Exonics are heros!

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