Developing and preserving APBD mouse models are critical in discovering the genetic basis for preventing and curing APBD.
6) Human Trials for APBD Read More
Dr. Rafael Schiffmann’s human trial uses an oil called triheptanoin or C7 to supply energy to brain cells instead of glycogen, which cannot be broken down for energy in patients with APBD.
7) Breadth of all work APBDRF is supporting Read More
Creating a Patient Registry:
A patient registry for APBD is now open. Dr. Edwin Kolodny speaks about the importance of creating patient registries here. Join the APBD patient registry.
1) Dr. Akman Studies Glycogen Branching Enzyme Mutations in the Jewish-American Population:
Dr. Akman and Dr. DiMauro have submitted a proposal to determine the frequency of glycogen branching enzyme (GBE) mutations in the Jewish-American population. Their proposal has two specific aims.
Aim 1: To determine how often the typical Y329S genetic mutation occurs in samples provided by the main Jewish DNA repository in New York (DOR Yeshorim) using methods of analyzing DNA sequences. To date 700 DNA samples have been received.
Aim 2: To find a second GBE1 mutation in patients with clinically typical APBD but only one proven Y329S mutation instead of the usual two, using whole genome sequencing (Columbia University Genome Center New York, NY). See Manifesting Heterozygote phenomenon **explanation below.
2) Dr. Minassian completes the first whole genome sequencing of an APBD patient and will start sequencing a second very shortly, with the aim of identifying the second missing mutation (January 2013):
3) Dr. Kakhlon finds area as a marker of genetic variability on the 2nd allele – continues his work on the Manifesting Heterozygote phenomenon ** with a 20K grant from anonymous donor and support from the APBDRF (February 2013)
**Manifesting Heterozygote Phenomenon
APBD is most commonly caused by mutations in the GBE1 gene. This leads to a deficiency of glycogen branching enzyme (GBE) which plays a part in storing glucose as glycogen – a polymer of glucose. The disease is inherited by an autosomal recessive pattern, meaning that both copies of the brancher enzyme gene in each cell have an error. Usually APBD patients inherit one mutated copy from each parent.
The mutations in both copies of the brancher enzyme gene can be identified in most APBD patients. However, using standard methods of gene sequencing, in about 40% of patients only one mutation can be identified, even though these patients have the same degree of GBE deficiency and the same disease severity as people with mutations in both copies of the gene.
It is thought that the other copy of the brancher enzyme gene is also abnormal even though the abnormality cannot be detected using the common methods of gene sequencing. This mysterious abnormality of the brancher enzyme gene could be due to the complete absence of the gene, a mutation in part of the area that regulates the synthesis of the gene, or more likely a mutation in the part of the gene responsible for building the messenger RNA – a nucleic acid that serves as a template for making the brancher enzyme protein.
Dr. Kakhlon has found an area of genetic variability on this 2nd copy of the gene. With a grant from an anonymous donor, he continues his work on the manifesting heterozygote phenomenon.
4) Dr. Minassian begins work to validate genetic variability (March 2013).
5) Dr. Natovicz publishes surprising APBD carrier gene frequency 1:34.5
In August 2012, Dr. Marvin Natowicz published provocative results of an unexpectedly high frequency of APBD gene mutation in people of Ashkenazi Jewish background. His research found the mutation has a carrier frequency of 1 in 34.5 Ashkenazi Jews. Before this, APBD was considered a rare condition, with only 50 affected individuals described in the medical literature. There are now about 100 known cases. If Dr. Natovicz‘s results are validated, little known APBD would have a similar carrier frequency to Tay-Sachs disease.
High Throughput Screening (HTS) of small molecules
Finding a small molecule medication that safely slows down the enzyme glycogen synthase involved in converting glucose to glycogen will provide a treatment for Adult Polyglucosan Disease. Furthermore, by slowing further accumulation it is hoped that the body will then be able to clear polyglucosans that have already built up. Thus, stopping the disease would not only be a treatment, but could ultimately be a cure.
1) Lower Polyglucosan Bodies by lowering Glycogen Synthase: Read More
A) Dr. Akman finds 15 drugs that reduce polyglucosan accumulation (February 2013): Read More
Validation in progress by Dr. Akman (APBD mouse embryonic fibroblasts) and Dr. Kakhlon (patient-derived skin fibroblasts and APBD mouse brain cultures)
Previous research on Lafora Disease showed that decreasing the production of glycogen stopped the formation of polyglucosan, nerve cell death and seizures that characterize that disease. After two years the mice are still completely healthy, confirming that removing polyglucosans and polyglucosan bodies cures the disease in mice.
In this non-targeted approach used by both Dr. Akman and Dr. Kakhlon, tens of thousands of small molecules designed by computational chemists to be “druggable” (i.e., stable, predicted to dock to protein surfaces, soluble etc.), some of which are FDA approved drugs are being screened for their ability to reduce polyglucosan levels. The small molecules are screened in three models: mouse embryonic fibroblasts, brain cultures from APBD mouse models generated by Dr. Akman and skin cells from APBD patients. Using more than one model allows positive hits to be confirmed.
Dr. Akman has already found positive hits. These are being analyzed by Dr. Dima Kozakov, a chemist from Boston University, in order to find the group of atoms in the drug molecule that are responsible for the biological and pharmacological interactions and to try to improve them by modifying their chemistry.
B) Dr. Kakhlon begins High Content/Throughput Screening of HCS/ HTS 20k drugs. Funded by the French Muscular Dystrophy Association (AFM) and APBDRF $26K (March 2013): Read More
Cell-based High content screening (HCS) for discovery of new drugs for treatment/relief of APBD is a newer method of investigating cell processes and their chemical or genetic changes using intact live cells. Unlike biochemical screening, they detect responses in the structure and function of networks of normal and diseased cells. Cell-based high-content screening of small-molecule libraries is used to identify new therapeutic compounds.
The Tel Aviv University’s Cell Screening Facility for Personalized Medicine (Prof. Weil’s laboratory for Neurodegenerative Diseases and Personalized Medicine) will perform the following projects:
1) Improve a cell-based test for high content analysis (HCA) using connective tissue cells (fibroblasts) from an APBD human source (patient).
2) Perform high throughput screening (HTS) of a 20,000 small-molecules library using an automated liquid handling system (Freedom Evo Tecan).
3) Identify compound hits by selecting those compounds tested in item #1 above that are able to significantly reduce polyglucosan bodies.
4) Select molecules able to increase PTG a protein the helps activate glycogen synthase levels as potential diabetes therapy candidates.
5) Perform a separate second HTS and hit identification similar to the first screening procedure using positive hits from the same compound library but a different cell-based HCA test. This activity will depend on the development of the cell-based HCA test using mouse nerve cell cultures isolated from APBD mice.
C) Dr. Minassian also begins High Throughput Screening using a biochemical assay. All hits in the three groups will be shared and cross-checked, and then tried in mouse models to determine whether they will cure the disease in the mouse: Read More
Previous research on Lafora Disease (another polyglucosan body disease due to a mutation in a different gene unrelated to glycogen synthesis) showed that decreasing the production of glycogen stopped the formation of polyglucosan, nerve cell death and seizures that characterize that disease. After two years the Lafora mice are still completely healthy, confirming that removing polyglucosan and polyglucosan bodies cure the disease.
2) Lower Polyglucosan Bodies by increasing Glycogen Branching Enzyme
A) Dr. Tropak has tested 1200 drugs (February 2013): Read More
Dr. Tropak is testing drugs to develop a cure for APBD by enhancing glycogen branching enzyme (GBE) activity. In his work he will identify small molecules that bind to and stabilize the mutated GBE in active form of the enzyme in order to correct the deficiency that results in APBD. A “library” of chemical compounds that are already being used to treat other disorders will be screened to find those that stabilize GBE. This should result in decreased levels of polyglucosan, representing a new treatment approach for APBD.
B) Dr. Tropak to test Dr. Kakhlon’s computational peptides
The computational chemist Amit Michaeli from Hebrew University is designing peptides (short amino acid sequences) that can either 1) bind to the mutated Glycogen Branching Enzyme (GBE) and stabilize it, or 2) destabilize the enzyme Glycogen Synthase (GS). Dr. Tropak plans to test the effects of GBE stabilizing peptides on GBE activities. He also plans to test whether peptides predicted to stabilize GBE enable it to resist change in its chemical or physical structure at relatively high temperatures. This would suggest the ability to stabilize GBE and thus counter the effect of the Y329S gene mutation in APBD.
The Antisense Project:
Aims to reduce polygclucosan bodies in patients with APBD in order to slow down the disease progression. Two major avenues are being explored:
1) Antisense oligonucleotides (ASO).
ASOs are small sequences of single-stranded DNA that block disease processes by changing the production of a particular protein. ASOs would be injected directly into the central nervous system where they can specifically knock down their target, glycogen synthase. ISIS Pharmaceuticals has already screened, scaled up and tested the safety and side effects of ASOs against protein targeting glycogen (PTG) in normal mice. PTG is a scaffold protein assisting glycogen synthase (GS) to carry out its function. ISIS Pharmaceuticals is now carrying out the same procedure for glycogen synthase and glycogenin. The ASO sequences discovered by ISIS Pharmaceuticals will be injected to 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 polyglucosan bodies buildup by glycogen synthase and thus slow down the disease.
2) Triple-helix-forming oligos (TFO).
These are very stable compounds that bind specifically to the DNA sequence they target. TFOs will be used in the same way as ASOs. The only difference is that they are more stable and specific than ASOs. TFOs have already been tested for their ability to pass into the nuclei of nerve cells, which has recently been demonstrated by Dr. Kakhlon. Dr. Kakhlon is working in collaboration with the Israeli company GeneArrest. Their goal is to use TFOs in periodic injections for APBD patients to reduce polyglucosan bodies by inhibiting GS and to slow down the disease. APBDRF is also funding the testing of TFOs on glycogen branching enzyme (GBE) in collaboration with Dr. Tropak.
Developing and Preserving Mouse Models:
1) Dr. Akman’s Mouse Model Chart
We are grateful to Dr. Akman for sharing the chart of the five mouse models he has worked on for many years
Phenotypes of mouse Gbe1 genetic variants
Ranked based on the severity of disease
How suitable model is it for APBD research?
|Gbe1 Exon7 knockout(Ready to be submitted for sperm preservation)
|Not suitable for APBD Research.Represents Andersen disease.
|Y329 knock in with PGK-Neo Cassette‡.(will be ready to be submitted for sperm preservation in 2 months)
6 to 7 months
In all tissues
|Very suitable for APBD research both for the Y329S mutation analysis and severe GBE deficiency.Disease starts as early as 4 months of age.This genotype is good for both histological and behavioral evaluation.
|Wild Type with PGK-Neo Cassette(Available in Houston only from Dr. W. Craigen)
||8 to 9 months
||PresentIn all tissues
||Very suitable for APBD with severe GBE deficiency.Disease starts as early as 4 months of age.This genotype is good for both histological and behavioral evaluation.
|Y329 knock in only(Ready to be submitted for sperm preservation)
||More than 1 year*
||PresentIn all tissuesExcept brain accumulation starts late yet it is more severe at 4 months of age
||Suitable for APBD due to GBE deficiency.Best model for APBD possibly late onset of the disease however Mice are ONLY for histological evaluation.Behavioral analysis requires aging mice beyond 1 year which can be costly.
|Wild Type without PGK-Neo Cassette(Available in Houston only from Dr. W. Craigen)
||2 years or more
||Not Suitable for APBD research there is no GBE deficiency.Animals can be used as a control to Y329S knock in mice.
(*)In progress oldest animal is 1 year old Although animals have normal Gbe1 gene, amount of enzyme produced is severely reduced by PGK-Neo cassette. This mouse model is important because it emphasizes the importance of the amount of enzyme not only the point mutation. This model can be used in research that aims to up regulate the transcription of Gbe1.
(‡) PGK-Neo Cassette is a positive selection marker used to select mouse cells carrying specific (Y329S) mutation. it is meant to be removed after obtaining the mice with desired genotype. However, in our case it reduced the expression and silent the Gbe1 gene.
(**) In Dr. Minassian’s lab, several GBE deficient mice from the above table are being bred with mice deficient of PTG and glycogen synthase to determine whether reduction of glycogen synthase will cure the disease.
2) The Jackson Laboratory:
APBDRF is preserving the APBD mouse models for the future. The Jackson Laboratory has been contracted to perform sperm cryopreservation of two mouse lines, the #2 and #4 models from the above chart. The Jackson laboratory’s mission is to discover the genetic basis for preventing and curing human disease and to enable research and education in the global biomedical community. Founded in 1929, this nonprofit biomedical research institution has a tremendous wealth of information related to husbandry, genetics and biology associated with selecting and using laboratory mice in research to cure disease.
(7/22/13) Dr Akman has shipped the mice to Jackson lab. If they are successful our true Y329S and Y329S-PGK-Neo strains will be archived and protected against any accident or infection that can take place in animal rooms.
3) Dr. Minassian Mouse model projects: Read More
Both of the mouse model projects are in progress with initial results expected in 2013
Arresting APBD by downregulating Glycogen Synthase (GS)Arresting APBD by downregulating Glycogen Synthase (GS)
Previous research on Lafora Disease showed that decreasing the production of glycogen stopped the formation of polyglucosan, nerve cell death, and seizures that characterize the disease. After two years these mice are still completely healthy, confirming that preventing the formation of polyglucosans and polyglucosan bodies curing the disease in mice. Finding a small molecule medication that safely slows down the enzyme glycogen synthase involved in converting glucose to glycogen will provide a treatment for APBD. By stopping further accumulation it is hoped that the body will then be able to rid itself of polyglucosans that have already built up (shown in mouse model of APBD Akman et al., 2011). Thus, stopping the disease would not only be a treatment, but ultimately a cure.
1) Eliminating polyglucosans from the APBD mouse brain by activating neuronal amylase expression- The Vacuum Cleaner Mouse
Previous research conducted by Dr. Minassian generated mice that produce the enzyme amylase in nerve cells on demand after feeding them a molecule called doxycycline. Amylase is an enzyme that can digest polyglucosan bodies. It is produced by the pancreas and salivary glands. However amylase is not present in muscles and neuronal cells. This work was done through the APBDRF funding. Now that a second group of APBD mice are ready, Dr. Minassian will breed the amylase mice with the APBD mice and at six months of age activate amylase production to see if this clears polyglucosan bodies from the APBD mouse brain. If this is successful, it will not only be a treatment but a cure for the disease by reversing the lifelong accumulation of polyglucosan bodies. This proof of principle will open the door for a definitive treatment of APBD.
Dr. Minassian would then seek a way to get amylase into nerve cells in a single procedure. Since APBD is a disease of later adult years, if polyglucosan accumulations can be reversed at age 50, for example, it represents a cure for the duration of the patient’s life.
In collaboration with the laboratory of Dr. Melnyk in Toronto, Dr. Minassian has experiments underway to transport amylase into the brain using an inactivated bacterial toxin that crosses the blood brain barrier. In collaboration with Dr. Burghes and Dr. Kaspar at Ohio State University, Dr. Minassian is using viral vectors to transfer amylase into the brain. We hope that one of these approaches will work and will result in a cure for APBD.
Dr. Raphael Schiffmann is conducting a study in patients with APBD to test whether an insufficient supply of energy from glycogen to brain cells is a cause of APBD. While the role of glycogen is to supply energy, the abnormal glycogen that accumulates in patients with APBD cannot be broken down to supply this energy to cells.
The treatment being tested is an oil called triheptanoin or C7 which will be used instead of glycogen to supply energy to brain cells. In an initial study, five patients were stabilized and their ability to walk improved while taking C7. Based on these results a double-blind randomized controlled trial to compare C7 oil with regular vegetable oil will be done in at least 18 patients. This 3-year study funded by the Baylor Research Institute will be conducted in Dallas, Texas with sites in Israel and France.
Ultragenyx Pharmaceutical, Inc of Novato CA has purchased licensing rights for triheptanoin, the oil used in the study (A
Treatment Trial of Triheptanoin in Patients with Adult Polyglucosan Body Disease) being conducted here at Baylor Research Institute. As part of the agreement, the company has agreed to provide travel funds for study subjects (those currently enrolled and any future participants) as well as providing a more purified form of triheptanoin for the study. We are excited to have Ultragenyx support and would appreciate the APBD research foundation sharing this new information with the APBD community.
More human trials are coming up in the foreseeable future. Please read more about Human Trials
Breadth of all work APBDRF is supporting
Therapeutic approaches that the APBDRF is supporting to cure APBD can be divided into targeted (1) and non-targeted (2).
- Targeted agents can be subdivided into:
- Agents designed to lower GS levels and activity:i. Antisense Oligonucleotides (ASO): Isis Pharmaceuticals – Tamar R Grossmanii. Triple Helix Forming Oligonucleotides (TFO): Gene Arrest- Anwar Ryaiii. New compounds discovered by GS solvent mapping in Boston University by Dima Kozakoviv. Dr. Minassian’s collaborative work on amylase and using the CRISPR/Cas9 technology
- Agents designed to stabilize GBEi. Peptides predicted to fill the small cavity generated by the Y329S mutation: Amit Michaeli- Pepticom.ii. Small molecules screened by Differential Scanning Fluorimetry (DSF) for their capacity to increase the melting temperature of recombinant wild type GBE and GBE Y329S proteins. Both recombinant protein production and the DSF are done in the lab of Dr. Wyatt Yue at the University of Oxford who specializes in using in vitro and structural methods to evaluate small molecule binding to proteins and the specificity of this bindingiii. Lipid membranes. Their interaction with and effect on GBE and GBE Y329S (the mutation causing APBD) is investigated by Dr Pablo Escriba and his team at University of the Balearic Islands of Mallorca, Spain. Dr. Escriba’s research is aimed at the design and synthesis of new lipid molecules whose efficacy for the treatment of APBD in cell and/or animal models could be investigated.
- Non-targeted approach consists of our high-throughput screenings done at Prof. Miguel Weil’s Cell Screening Facility in Tel Aviv University managed by Dr. Leonardo Solmesky. In this facility, libraries of small molecules with diverse structures are screened for their capacity to lower polyglucosans regardless of the mechanism. In this non-targeted approach we are only interested in the end result – lowering of polyglucosan bodies. SEE VIDEO. Positive hits discovered by this high-throughput screenings will then be further studied in order to elucidate their mode of action. For example, in theory they could be GS inhibitors or GBE stabilizers, but might also be molecules which “open” polyglucosans, thus enabling their degradation by the enzyme which normally degrades glycogen (phosphorylase a). These positive hits will also be tested and validated in cell and animal models of APBD by Dr Berge Minassian (Sick Kids Hospital, University of Toronto) and Dr Orhan Akman (Columbia University).
The take home message is that no single approach is expected to provide a foolproof therapeutic on its own. We anticipate that a final therapeutic will consist of a combination of, for instance, a GBE stabilizer co-administered with GS-targeting ASO injection. This is why our effort is multifactorial.