I want to take a brief moment to introduce my guest blogger today, Jon Moulton. Jon works for Gene Tools. Below, he gives a brief biography but here is what he doesn't say is:
- he is a friend of duchenne parents
- actively involved in education scientists as well as amateurs like me! Wow! I understand so much more now!
- a scientists of the finest caliber
- a caring and compassionate human being.
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This is an introduction to Morpholino antisense oligos, with notes on their potential application in treatment of Duchenne muscular dystrophy (DMD). Toward the bottom I'll include some links to other places on the web which have more information about Morpholinos.
My name is Jon Moulton, I'm a Ph.D. biologist and a former adjunct professor, and about 10 years ago I leapt from the bottom rung of the professorial ladder to work for Dr. Jim Summerton at Gene Tools LLC. Gene Tools is an Oregon company that manufactures Morpholinos for the research reagent market. Most of our custom-sequence Morpholino oligos go to academic labs worldwide for research.
Morpholinos have genetic bases identical to those of DNA connected to a synthetic, non-natural backbone. The backbone contains morpholine rings, which is where the name Morpholino oligo comes from. Usually a Morpholino oligo is about 25 bases in length and can be described by a base sequence, like:
CCTCTTACCTCAGTTACAATTTATA.
Morpholinos are called antisense oligos because they are typically designed to be complementary to mRNA (an mRNA sequence is called a "sense" sequence) so that they stick to the complementary part of the mRNA, binding A to T and C to G just like in a DNA helix (the DNA strands are also called the sense and antisense strands).
DMD is caused by mutations in the dystrophin gene. Morpholino oligos appear promising as therapeutics for DMD. Morpholinos can be used to alter RNA and RNA determines the structure of proteins (more about that below). The amazing thing about using a Morpholino oligo as therapy for a genetic disease is that the Morpholino is treating the disease by altering the mutated form of the RNA so that it codes for a protein that functions better. That is, the Morpholino treats the disease instead of treating the symptoms. If Morpholinos are approved to treatment of DMD, many more human genetic diseases may be treatable by changing the sequence of the Morpholino oligo so it targets different locations on human RNA.
Morpholino antisense oligos from AVI BioPharma Inc. are currently in clinical trial for Duchenne muscular dystrophy in England. Prior to this trial, preclinical data was gathered from Morpholino experiments with cell cultures, mice, and dogs. The oligos used in DMD-targeted experiments change how pre-mRNA is spliced. The oligos for the clinical trials are used to excise exons from the RNA in order to change the reading frame of sequence downstream of the excision.
I suppose I'd better explain "reading frame". RNA bases are used to specify the order of amino acids in proteins. Proteins are basically strings of amino acids, and the order of the amino acids determines the shape of the protein and how it will function. Three bases of RNA are translated into one amino acid; the three-base groups are called codons. This means that a string of RNA bases can be interpreted several ways by the protein-making machinery of the cell, depending on which RNA base it starts reading from, because this leads to reading different codons.
The RNA sequence:
TTCGACCGTACCGGGTGTA,
can be divided up into codons three ways; each leads to a different amino acid sequence, depending which of the three reading frames is chosen. I ignored the partial codons at the ends.
[TTC] [GAC] [CGT] [ACC] [GGG] [TGT] [A
would make:
- Phenylalanine - Aspartic acid - Arginine - Threonine - Glycine - Cystine -
T] [TCG] [ACC] [GTA] [CCG] [GGT] [GTA]
would make:
- Serine - Threonine - Valine - Proline - Glycine - Valine -
TT] [CGA] [CCG] [TAC] [CGG] [GTG] [TA
would make:
- Arginine - Proline - Tyrosine - Arginine - Valine -
Consider what would happen if you inserted a base halfway along one of these sequences. That would change the reading frame of the sequence to the right (since codons are always three bases, the edges of the codons would move over by one base). This base insertion is one way a mutation can cause a frameshift. Bases can also be deleted and cause a frameshift. If the string of amino acids is part of a functional protein and a change in the RNA causes all the sequence to the right of the change to become different amino acids, there is a good chance that the protein would no longer function.
Many DMD-causing mutations result in altered reading frames and the exon-skipping oligos are intended to restore the "healthy" reading frame to the dystrophin mRNA by removing an exon which has a number of bases that is not evenly divisible by three, changing the reading frame "downstream" (the cellular machinery that translates RNA into protein starts at one end of the RNA, called the upstream (or 5') end, and moves toward the downstream (or 3') end). Treatment with an exon-skipping oligo produces a modified form of dystrophin which, it is hoped, will retain most of the function of the dystrophin of a healthy individual. So far no data have been published showing functional improvement as a result of administration of exon-skipping Morpholinos into humans. There are several kinds of oligos that work by a mechanism similar to Morpholinos. These are called "steric blocking" oligos, and include peptide nucleic acids (PNA), locked nucleic acids (LNA) and 2'-O-methyl phosphorothioate oligos. Another company, Prosensa B.V., has clinical trials ongoing for DMD using 2'-O-methyl phosphorothioate oligos and are using a similar exon-skipping approach to restore the healthy reading frame in the dystrophin protein.
Morpholinos can knockdown gene function more effectively and with less off-target gene modulation than many other methods, such as siRNA and the older phosphorothioate oligos. This has led to their use in developmental biology (the field that used to be called embryology), for which the oligos are injected into eggs or early zygotes to learn the function of genes in development of an organism. The combination of good efficacy and good specificity (less off-target gene modulation) have made Morpholinos the standard gene knockdown reagent injected into zebrafish or Xenopus (clawed frog) embryos to probe gene function in development.
The function of a Morpholino depends on where on an RNA it is targeted. Morpholinos can block translation, modify splicing or block micro-RNA maturation and function. More exotic applications, like blocking ribozyme activity or triggering translational frameshifts, have also been explored. A challenge for any antisense oligo is getting the oligo into the cells where it can function.
Developmental biologists microinject Morpholinos right into the cytosol of a cell, into the egg or early zygote. This won't work for medical applications, such as treatment of a DMD patient, because practially you can't microinject into every cell where the oligo effect is needed. In most organisms, injecting Morpholino oligos into the bloodstream isn't useful because the oligos don't cross the cell membranes. They do, however, filter through the kidney and so you end up with expensive urine -- but no useful biological effects. DMD may be a special case, as dystrophic muscle is leakier than healthy muscle. Morpholinos have been shown in dogs to enter leaky muscle cells from the blood, but the doses required for good activity were pretty large.
Companies working with Morpholinos have been trying to develop modifications of the oligos (broadly called "delivery moieties") to help them enter cells from the bloodstream. Likely the two solutions that have been developed enter cells as they endocytose (form little sacks from their outer membrane and bring these sacks, with their contents, into the inside of the cell) and then the delivery moieties help the oligos escape from the endosomes (the little sacks). AVI BioPharma Inc. has developed what they call a PPMO (peptide-conjugated phosphorodiamidate Morpholino oligo) which does enter cells from the blood -- my wife, Dr. Hong Moulton, is the inventor of the PPMO (proud husband!). Gene Tools developed a system that has some structural similarities to the PPMO (Dr. Yongfu Li of Gene Tools invented the Vivo-Morpholino); both the PPMO and our Vivo-Morpholinos have guanidinium groups in their delivery moieties, and it is these groups that are thought to interact with the membrane of an endosome and make it leaky. Because the dose required of a PPMO or a Vivo-Morpholino is much lower than the dose required of an unmodified Morpholino to reach the same level of cellular activity, I expect that the most effective treatments for human genetic diseases will be developed using these or similar delivery-enabled oligos.
Here is a recent review that I wrote with Dr. Shan Jiang of Gene Tools describing the PPMO and Vivo-Morpholino. The references in this paper provide a good path into the recent DMD literature using Morpholinos. This is open-access, you don't have to pay to view the paper. Just follow this link and then click on the PDF link on that page.
PPMO and Vivo-Morpholino Review
Moulton JD, Jiang S. Gene Knockdowns in Adult Animals: PPMOs and Vivo-Morpholinos. Molecules. 2009 Mar 25;14(3):1304-23.
Here's the Gene Tools website:
Gene Tools
This site is loaded with information about using Morpholinos for research.
If you'd rather hear about Morpholinos than read about them, here are some mp3 files discussing Morpholinos:
Morpholino discussions
Here's the searchable online Morpholino publication database I maintain at Gene Tools:
Morpholino publication database
Here's AVI BioPharma:
Here is a link to the clinical trial page for the Morpholino trial underway in England:
Francesco Muntoni's UK Morpholino Clinical Trial link
Here is the Wikipedia page for Morpholino oligos:
Morpholino Wikipedia page
Here is my blog, where I post notes about new Morpholino publications and whatever else I want (yes, it really is on MySpace):
Jon Moulton's blog
I tweet new Morpholino citations as I find them in the scientific literature:
Morpholino Tweet
If anyone reading this is filled with the desire to start doing Morpholino experiments, you might start here:
Build-your-own Morpholino Experiments
Thanks for coming along for this tour of Morpholino technology. Morpholinos are doing many useful things for biological research -- their application to DMD starts Morpholinos down the path to realizing their potential as therapeutics for genetic and viral diseases. The best outcome will be if the oligos help some boys and their families who deserve real hope and improved health.
