Anti-Sense - Recently Approved Gene Therapy
Using an FDA-approved method, the goal of this research is to reduce polyglucosan bodies in patients with APBD in order to safely slow down the progression of the disease. Polyglucosans are an abnormal form of glycogen that cannot be broken down and used for fuel in the body. Instead they build up to form clumps that can damage cells, especially nerve cells.
This project should result in decreased levels of polyglucosans, representing a new treatment approach for APBD and a possibly cure.
Two major avenues are being explored:
1. Antisense oligonucleotides (ASO).
ASOs are small sequences of DNA that block disease processes by interfering with the production of a particular protein that is their target. In APBD, ASOs would be injected directly into the central nervous system where they can knock down their specific target, glycogen synthase (an enzyme involved in converting glucose to glycogen).
The ASO sequences discovered by ISIS Pharmaceuticals will be injected into APBD mouse models generated by Dr. Akman and Lafora Disease mouse models generated by Dr. Minassian. The goal is to use ASOs in periodic injections for APBD patients to reduce the build up of polyglucosan bodies by glycogen synthase and thus slow down the disease.
2. Triple-helix-forming oligos (TFO).
These are very stable molecules that bind specifically to the DNA sequence they target. TFOs will be used in the same way as ASOs but will have highly specific targeted effects, giving them tremendous potential as gene therapy.
TFOs have already been tested and shown to be able to pass into the nuclei of nerve cells by Dr. Kakhlon. He is now working in collaboration with the Israeli company GeneArrest to use them in periodic injections for APBD patients to reduce polyglucosan bodies by targeting the enzyme glycogen synthase and thus slowing down the disease. APBDRF is also funding the testing of TFOs on glycogen branching enzyme (GBE) in collaboration with Dr. Tropak.
Two research centers and two Pharmaceutical companies are workingon the Antisence project. Hadassah University Hospital and Columbia University.
May 2013 Interview with Dr. Or Kahlon
Is there a potential for human trial 2014?
At the moment, in vivo screening of antisense oligos (ASO) against PTG injected intracerebroventricularly (ICV, i.e., directly to the brain) into the hippocampus, cortex and cerebellum of mice has been completed. Some ASO were indeed shown to reduce PTG mRNA in these brain regions. Nevertheless, Isis Pharmaceuticals is still trying to improve the knockdown by increasing the dosage. Therefore, we expect that in about a month, these ASOs will be ready for ICV injection to APBD mice. At least two injections, 3 months apart, are planned and then the effects on different APBD biomarkers in the mice will be tested. This should be completed within a year and if the results are satisfactory then yes, human trials should commence before the end of 2014. These human trials will be based on PTG knockdown. Isis Pharmaceuticals has also recently initiated experiments for knocking down Glycogen Synthase 1 (GYS1, the glycogen synthase variant expressed in brain, muscle and heart), which is expected to be more effective than PTG knockdown. Therefore, human trials involving GYS1 ASO are expected to start approximately a year after the the PTG ASO-based trials.
How would an APBD Registry help facilitate the beginning of the Trial?
Once the specific ASO is ready for use, it will be a great medical advantage if it could be tested on a variety of APBD patients with different underlying genetics (homozygous or heterozygous for Y329S, or with a different GBE mutation) and with variable natural histories of the disease. Such a variation can serve to test the efficacy of any drug which will be proven more potent if it can correct disease symptoms from a variety of patients.
Why has ISIS Pharmaceuticals decided to also do in ASO on glycogen synthase GS? Isn't there a concern about reducing glycogen to too low a level?
Following a discussion held at Isis Pharmaceuticals headquarters in Carlsbad, CA between Dr. Minassian, myself and Isis Pharmaceuticals representatives it was decided that to initiate experiments aimed at GYS1 knockdown. GYS1 knockdown, or even knockout, is well tolerated in both mice and humans, especially if hypoglycemia is kept in check. Anyway hypoglycemia should only be expected if the liver form of GYS (GYS2) is targeted and not GYS1. Patients completely lacking GYS have no symptoms except for child cardyomyopathy. Humans exhibiting 50% GYS activity (which is the level expected from an ASO-mediated GS knockdown) are completely healthy. We presume that as long as they are not starved, and are exposed to a variety of foods, humans and animals can tolerate GYS deficiency Therefore, we now think that we can target GYS1 directly instead of targeting other proteins, such as PTG, which are only ancillary proteins to GYS activity. Isis has only recently embarked on a GYS1 knockdown project and, as for PTG, they are doing this in two stages - first in vitroin mouse hepatocytes and only then in vivo as described above. Therefore we expect the potentially more effective GYS1 ASO to be ready a year and a half from now at the earliest (once it has gone through all the stages described above for the PTG ASO).
Isis has also decided to study glycogenin, what is the significance of that?
Glycogenin is a protein which serves as a primer for the initiation of glycogen biosynthesis. It could therefore also be beneficial to knock down glycogenin and thus inhibit the initiation of glycogen which is bound to take on the form of polyglucosan in tissues from APBD patients where GBE is deficient. Therefore, reducing glycogenin is also expected to reducepolyglucosan levels and thus glycogenin is a valid target for knock down.
What will it take for ISIS Pharmaceuticals to be convinced to do a human trial on APBD?
I think that once we have convincing results from mice, Isis Pharmaceutical will be convinced to move on to the next step which is clinical trials in patients. All this will of course will be done in accordance with the FDA guidelines and regula6tions.
What other antisense-based technologies are being developed for curing APBD?
In collaboration with the Israeli biotechnology company GeneArrest, we have initiated a project in which we will capitalize on Triple Helix Forming Oligos (TFOs) designed to target GYS1. In contrast to ASOs, TFOs are nucleic-amino acid polymers which target the DNA and not the mRNA and are considered much more stable and specific than ASOs. We are now only at the beginning of this project. We have shown that the TFOs permeate the cell membrane and can accumulate in the nuclei of mouse brain cells. With funding from the APBDRF we plan to move to the next stage which includes designing TFO specifically targeting GYS1 and demonstrating GYS1 knockdown at the levels of expression and activity. As with the ASOs, we will then move on to test the effects of the GYS1 TFO on APBD mouse models.
What your donation will fund:
Recent donations have used to purchase glycogensynthase TFO as discussed.
The Coddon Family Foundation-$10,000
French MDA Grant- 100,000 euro
Prusiner-Abramsky award for $50,000.
Anonymous Israeli donor $20,000
Anonymous US donor $10,000
We would like to thank Isis Pharmaceuticals and GeneArrest for funding much of the ongoing research not included in our estimates.