This Weekend, November 7th - the CINRG Fall Meeting

Just a quick note - this weekend the Cooperative International Neuromuscular Research Group based out of Children's National Medical Center - and the clinical arm of the Research Center for Genetic Medicine - is having their third annual meeting here in DC.

Coordinators, docs, and physical therapists from all over the world will be here.

I am excited because some of my favorite people will be here, Carrie Miceli and Stan Nelson from UCLA, many of the Foundation to Eradicate Duchenne group like Joel Wood, and my dear friends, Tina and Dan Carson. Annie Kennedy from MDA will be here as well. Katie Bushby and Nick Catlin will be here from the UK and foundation leaders, Debra Miller and Pat Furlong from cureDuchenne and Parent Project plus George Vella from Charley's fund will join us. Of course Bob Leshner and Avital Cnaan from CNMC and Lakshmi from India. Although it is a CINRG meeting, Eric Hoffman, as the head of GenMed, of which CINRG is the clinical branch, will be here to lend his support.

The agenda includes the Natural History study, strength testing measures, cardiac drug trial plans, duchenne mutation data comparisons, non-hormonal steroid drug development, various aspects of exon-skipping, and updates from our friends 'across the pond'.

There is a treatment goal in sight and as it unfolds, I am more and more impressed by the duchenne community coming together from around the world, making it happen with compassion and good-will.

Enough said.

Morpholinos 101: An Introduction

Dear Friends,

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.

Jon, thank you for this wonderful insight into morpholinos and duchenne dystrophy. After reading this post, I think we should name you Morpholino Man!
____________________________________________________________________

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.

Men over 40 with duchenne

Now you can join the likes of Mark Twain and say: "The report of my death is an exaggeration." There are over 400 men with duchenne muscular dystrophy over 40. Who are you?

What do you have to say for yourselves? Where are you 'oh miracles of the living'?

I'm thinking about writing a piece on you, your lives and philosophy but first I have to find you. To that end please take the survey below - send this link to your friends, your friend's friends, and anyone who might be able to answer the call!

If you know of someone who doesn't have a computer and would like to participate, please feel free to send me their contact information.

Click Here to take survey

A Poem for Uncle Petey

Below is a poem that Petey wrote about his uncle, who died Saturday after battling ALS. Even though there are allusions to family connections - karate kid, movies, Stephen Segal - it's easy to follow. One more note - Uncle Petey was the president of the regional CWA for twenty years or so - hence the 'union' references.

Uncle Petey

I remember that grin
Even when the disease loomed
I knew how it must have grown heavy
But his strength was there
I could see it

I regret not seeing enough of a union man
That a President recognized
And a family grew with
Who dressed up like Dumbledor
And quoted Steven Segal
That danced at a daughters wedding
And honored his beliefs

He is a passed titan
Who is still alive through warm hearts that mourn
That celebrate a man still immortal
Telling stories to us in our minds campfire

Petey was the true American dream man
Hit by something deadly
But never stopped swinging

I'm not a religious man
But I know he would say a prayer now
He would be strong with his big heart
And he still is as we know

So Uncle Petey, I know you're listening
And your still grinning in your Dumbledor hat
Saying "Sweep the leg Johnny"
And watching all the best action movies
Starting a union in the afterlife

No strangers in Bangalore: Muscular Dystrophy Conference

Deb Robbins, Parent Project Australia, reports on the first Asian PP Conference in Bangalore, India. I love her down-to-earth, funny, and wise-beyond way of looking at things.

Over 500 delegates attended the First Asian DMD Conference organized by United Parent Projects Muscular Dystrophy, Health Care Global Foundation and NIMHANS at St John’s Auditorium in Bangalore. Parents also came from Iran, Israel, Sri Lanka, Italy, The Netherlands and France. Stakeholders around the world were able to view the lectures via live streaming organized by Duchenne Parent Project Italy. International spectators were able to send chat questions to be answered by the panels along with those from the conference floor.

This was no local seminar but a national conference boasting an international program. Prof Jean-Claude Kaplan, Dr Annemieke Aartsma, Dr Madhuri Hegde, Debby Schrans for Dr Hendriksen, Dr Raghavendra Rao, Dr Lakshmi Raman and Dr Herve Laouenan explained their research. Dr Rao’s reasons for the suspected value of yoga for Duchenne were particularly innovative. Clinicians included Dr Nathalie Goemans from Belgium, Dr Jes Rahbek from Denmark, Dr John Bach, Dr Karim Wahbi and Prof Douglas Biggar. Though only one therapist presented, 160 practitioners attended Helen Posselt’s workshop.

Everywhere was evidence of dedication –the geneticists in Dr Lakshmi’s lab giving up their leisure time for family fun days and disseminating information to doctors surgeries on weekends. And no problem was any trouble for Dr Rao, the local organizer, to solve.

Particularly inspiring were the calls to rally together to abate the progression of Duchenne for one and for all, by Dr Ajaikumar (HCG), Dr Elizabeth Vroom (Conference Convenor & UPPMD President) and Hafiz Issadeen from Sri Lanka. All presenters urged parents everywhere to be proactive in both care and advocacy. With better care and promising research around the corner, it is more important than ever to plan for longer and more comfortable lives for our children than we could have imagined 15 years ago. The Parent Project Muscular Dystrophy group of India was inaugurated at the end of the two day conference and these core parent advocates networked with presenters and UPPMD stakeholders on the last evening.
In Bangalore as at home, I enjoyed swapping practical care tips and the trials and blessings of life with DMD, with other parents. More than one parent asked me to read specific medical information about their child confusing me for one of the doctors. They were desperate for more information though we’d just sat through hours of presentations. Yet the children and youths running, waddling or wheeling around the auditorium were as serene and ‘wise-eyed’ as Australian, European and American boys confirming my own theories about just who is saving who?