Research on Therapies

Therapies for GNE Myopathy: Current Status and Future Prospects

A number of approaches for development of a cure for GNE myopathy are currently available. These therapeutic possibilities are being developed for a large number of different diseases, including genetic disorders. None of these have reached a stage where they are ready to be used by patients. Initial studies do indicate that all therapeutic approaches appear to be safe and non toxic, suggesting that these therapeutic platforms can be developed for treatment of different diseases. Sialic acid supplementation as a cure for GNE myopathy is in extended phase two trial and is likely to be the fastest to reach the market. The National Institutes of Health in Bethesda, Maryland, U.S.A. has just started recruiting patients for phase one/phase two trial of Dex-M74, a ManNac substrate.

1. Gene Therapy

HIBM or GNE myopathy is caused by a defective GNE gene that has reduced activity, as a result of which the level of sialic acid, an essential sugar, is low in cells. For unknown reasons physiological effect is more pronounced in muscle cells leading to progressive muscle degeneration. Mutations in the GNE gene are responsible for this defect. Gene therapy approaches offer a possibility of delivering a corrected copy of the gene in muscle cells so that active GNE protein is made and sialic acid levels are restored. Gene delivery can be done in many ways. However, the first two approaches are more common.

  • Viruses such as adeno associated virus (AAV), lentivirus, etc. are being considered as vehicles for carrying the healthy DNA to the muscle cells to correct the affected gene.
  • Use of lipids and other synthetic molecules in which the DNA can be encapsulated.
  • Naked DNA can also be delivered, but may be amenable to degradation.
  • Messenger RNA (mRNA) therapy which uses mRNA of the gene in place of DNA along with non viral delivery system

A brief description of these approaches is given below. So far FDA has not approved any gene therapy product for patient treatment. Most of the trials with genetic diseases are either phase I or phase I/II combined trials. Many of these have been completed, but progression to phase III trials does not seem to have happened. From the available information only one potential gene therapy treatment seems to have reached phase III. This is for “Leber's congenital amaurosis”, a rare eye disease. A gene therapy trial utilizing AAV vector with a functional GNE gene may start soon. We hope that safety and efficacy of some of the vectors (AAV, non viral systems) are proven favourably and preclinical data are generated so that gene therapy trial could be tried for GNE patients.

Viruses as vector for gene therapy

AAV is turning out to be a front runner because of multiple reasons, such as its ability to enter into, and function in non dividing cells. It shows low immune response, low integration rates and is generally non pathogenic. On the other hand lentiviruses do lead to multiple integrations in host DNA that can cause some problems. Size of the DNA that can be packaged into the virus (to make virus particles in the test tube in a way that they can enter cells just like normal viruses, but will not cause disease) is also a limiting factor; for example, AAVs can package only 4-5 kb DNA. That is why it is very difficult to pack the whole gene of DMD in one virus as the gene is very large. Alternate methods are being developed for packaging fragments and assembling inside cells (please see for details Genomic version of GNE gene is about 62 kb and mRNA (the part that has information about making proteins) is about 5.1 kb. Open reading frame of GNE (absolutely minimal part of the gene which with some help can make GNE protein in cells) is 2.2 kb, which is a size suitable for packaging in AAV vector. Thus packing should not a problem in the case of GNE. Data is required to check packing efficiency of GNE in different versions of AAV vectors.
One of the major issues in using viral vector is the need for giving multiple injections, which could trigger potentially undesirable immune response against the viral vector. Vectors are being designed where immune response is negligible so that the gene could be delivered a number of times. AAV has been modified in a recent Phase I trial for muscular dystrophy to further reduce its immune response and enhance ability to make functional protein in muscle cells (AAV vector 2.5). This vector was well tolerated and could deliver functional proteins (Bowles et al. Molecular Therapy, Vol 20, page 443, 2012). The trial was conducted by injecting into muscles of one side of the body and using the other side as control. AAV2.5 vector appears to be much more advanced than the patent filed by Israel GNE group.

Use of lipids

Cationic lipids are thought to be good carriers as they protect the DNA during delivery and are efficiently taken up by cells. However, to be most effective in gene therapy the DNA needs to reach the nucleus of the cell, which is the inner compartment. While viral vectors deliver the DNA to the nucleus, lipids deliver it to the outer compartment- the cytoplasm. Lipids are also thought to be more toxic. Lipid carrier was used in a study with a single GNE myopathy patient (Nemunaitis et al. Human Gene Therapy vol 22, page 1331, 2011). One intra-muscular and one intra-venous injection was given using “lipoplex”, a lipid mixture. Gene delivery did take place, but it is not clear as to how many cells received the gene. Better cationic lipids have now been developed which could be more effective. Unfortunately, no animal data seems to be available to indicate how efficiently the vector is taken up by different cells. Most of the information is available as conference posters where no details are given.

Plasmid DNA

Instead of viral vectors it is possible to use naked plasmid DNAs containing the desired gene under the control of suitable promoters which would allow the gene to be expressed into mRNA. These can be delivered to the tissues using specific approaches (such as US-1274 ), either naked or suitably encapsulated by lipids or other nanoparticle-based formulations. The advantage of this system is that it is easy to produce the DNA and scale up. However sometimes double stranded DNA does stimulate innate immune response.

mRNA therapy

Information in the gene is transcribed into mRNA which then makes the protein using cellular machinery. Therefore, instead of using DNA it is possible to correct the defect by making the corrected mRNA in the test tube and sending it into the cells. Since mRNA acts in the cytoplasm, and does not need to go to the nucleus, lipid carriers are better suited for mRNA delivery. This is a promising new approach and a lot of work is going on to optimize this approach; for example mRNA is being modified to increase its stability in the body. Trials are planned in the area of muscular dystrophy to repair damaged heart muscle, and in some vaccine trials to stimulate immunity ( Some examples, Kormann et al. Nature Biotechnology, vol 29, page 154, 2011: Wang et al. Molecular Therapy Vol 21, page 358, 2013; Zangi et al. Nature Biotechnology Vol 31, page 358, 2013). mRNAs have several advantages over plasmid DNA. They are relatively inert towards the immune system, and being short-lived are less likely to have adverse effects. However they will need to be administered to the patient on a continuous basis.
mRNAs are being tested as vaccine candidates for preventing/treating cancer and other diseases (, These approaches are at different phases of clinical trials. FDA approval may be granted soon for mRNA as vaccine/therapy for cancer (phase II trials are currently being done). There is a major attempt to use mRNA/short-RNA segments to treat DMD by preventing exon skipping ( Full length mRNA therapy for genetic disorders has not yet been attempted. However, developments in this technology and analysis of safety features will be useful for other applications, including treatment of GNE myopathy.
In addition, methods are being developed to correct gene mutations using oligonucleotides (short DNA chains). Most of the applications involve either correction of a mutation in a test tube, and then transferring the corrected cells, or exon skipping of a specific type of mutation. There is no literature to suggest that it may be applicable in GNE myopathy. However, some success stories are there. A chimeric RNA/DNA oligo was able to repair a genetic defect in a canine model of myotubular myopathy (Childers et al. Science Translation MedicineVol 6, Jan 22, 2014).

2. Stem cells

In an adult, most differentiated cells (such as muscle, brain, liver) do not multiply and their proliferating ability is restricted. However, these tissues do have a small number of dividing cells (stem cells) that have the ability to multiply and get differentiated into the respective tissue cells. These cells, though small in number, play a major role in tissue repair and regeneration. For therapeutic purposes one needs a larger number of stem cells. Fertilized embryos are a great source of stem cells (known as embryonic stem cells or ES cells), which under suitable conditions can convert into any cell type of the body. Since these can be cultivated and stored as ES cells, it is possible that these can be used for regeneration of any tissue. However, due to ethical reasons, and a ban on work with such cells, research has not progressed significantly. A new Nobel Prize-winning technology, known as induced pluripotent stem cells (iPS) allows generation of stem cells from cells of an adult individual, where ethical concerns are fewer. Unlike ES cells, these cells do not have the potential to differentiate into all cell types. Nevertheless, they do have the ability to get converted into some of the important cell types, such as muscle and neuronal cells. Some of the technical problems are to get sufficient number of iPS cells for use in therapy and to convert them into the desired cell type with high frequency. Both these problems are being addressed, and a recent report from Harvard University announced significant improvement in the efficiency of iPS cell formation ("", also Mali et al. Stem Cells Vol 26, page 1998, 2008). In this system skin cells from patients are taken and converted into iPS cells using a cocktail of molecules. iPS cells proliferate and can theoretically be kept in storage forever. Generating patient specific iPS cells eliminates the problem of rejection (like tissue grafting) inherent if cells are taken from unrelated individuals. However, there is still the problem of defective gene present in these cells since they came from the patient’s own tissue. In a recent breakthrough, Japanese scientists managed to correct the genetic defect of muscular dystrophy in iPS cells. The corrected cells were then converted into muscle cells that showed high level expression of the corrected gene ( In this regard the first clinical trial has started in Japan, where scientists are going to check efficacy and safety of human iPS cells.
Bone marrow is also a good source for stem cells, mostly known as mesenchymal stem cells (MSC). Adipose tissue, cord blood and tooth/gum can be other sources of MSCs. However, its ability to transform efficiently into non blood cells is not very clear. It is also believed that these cells may not survive for too long and whatever benefit one sees, may be short lived. Mostly autologous MSCs are being used and for therapy of genetic disorders there may be problem due to persistence of the genetic defect. Nevertheless, bone marrow cells are easier to handle and manipulate. Therefore, these are quite popular among stem cell biologists who are interested in developing therapies. Many clinical trials are starting to use these cells in DMD. In one of the studies from India human umbilical cord MSCs were administered to DMD patients and marginal improvement and stallings, of disease progression was observed ( It is also possible to use allogeneic MSCs (that is, from some other individual) for therapy. Though immune response and other complications can arise, it can avoid the problems associated with mutant GNE gene. No results from such trial has been published so far.
One of the major concerns about stem cells is that these can easily form cancer cells. Whether iPS cells or different versions of stem cells will show similar propensity for cancer, is not clear as yet. The studies thus far have not shown significant safety/toxicity problems.

3. Sialic acid replacement

One of the features displayed by patients with GNE myopathy is reduced level of free and conjugated sialic acid in tissue and blood. In animal models it has been seen that supplementation with sialic acid or its precursor N-Acetyl mannosamine (ManNAc) restores sialic acid levels and suppresses disease phenotype. This has prompted development of therapeutics based on these molecules. The problem of sialic acid is that it gets cleared quickly from the system, that is, it is in circulation for a short time and only a small fraction is taken up by the cells due to its poor uptake. One of the solutions is to release it slowly in the intestine so that there is a sustained level of the molecule in circulation. The problem of uptake is still there even in slow release system. On the other hand, ManNAc is taken up by cells more readily, but has to be converted to sialic acid.
Another approach is to use modified sialic acid (such as an ester) which neutralizes the acidic charge of sialic acid. Because sialic acid has an acidic charge it has poor uptake, while the ester has a better absorptive capacity since the charge is neutralized. Once inside the cell the sialic acid ester is expected to be targeted by catalysts like esterases which break the ester bond and generate free sialic acid. This system is very well known and there are numerous examples of its use in cellular systems (for example, There is no reason why it should not work for GNE myopathy. Therefore, a slow release formulation of sialic acid ester could be a good treatment option. Ultragenyx has filed for a patent on modified sialic acid synthesis and its use in HIBM therapy (US patent application 20150038693). Analysis of the patent showed that there is lot of possibility of generating new molecules here. However, toxicology and safety studies need to be done as some of the esters are not naturally occurring molecules. There is a possibility that if esters are successful, the total dosage required may come down.

4. A way forward: Looking Into the Future For A Cure

We can think of a number of different strategies for developing a cure for GNE myopathy. We have to realise that some of the technology platforms discussed in this article (gene therapy and stem cells) may not be available in ready-to-use form very soon. We have to wait for phase two and/or phase three results of trials with these technologies in other diseases. Particularly, we have to see the safety and toxicity results. Safety and toxicity studies using normal animals are relatively easy, and to save costs these studies could be outsourced to an organization in Delhi or elsewhere in India. Obtaining animal model of GNE myopathy is an issue. However, there are many companies that make these models as per individual requirement, and given the funds, we could generate the animal models commercially. The problem is that we need to find and carefully select experienced people to carry out efficacy studies. For example, measurement of muscle strength in mice requires special expertise.
Ideally, gene-corrected autologous iPS cells (from respective patients) appear to be the best choice for long term cure. This technology has been developed in Kyoto and published in a paper last year. If Nishino’s group (who is a pioneer in GNE) can work with the Kyoto group for GNE myopathy we may be able to see some results sooner. HLA-matched heterologous (from different person) iPS cells also appear to be a possibility. The cost of treatment for heterologous system may be much lower. Since the cells can be prepared before, the amount of time for treatment may also be less. One issue is rejection of these cells, similar to what we see in organ grafts. In an ongoing trial in Europe heterologous stem cells seem to give long term benefit to heart patients (
Among all gene therapy methods, mRNA delivery appears to be the best choice at this point, since it is likely to have the least side effects. There is a company that can make this material on order. If we get access to animal models, and can generate funding, it could be possible to test this method. Finally, we can seriously think of modified forms of sialic acid in slow release formulation. India has well trained competent chemists, and its chemical industry has expertise to make any compound under GMP conditions. We can approach them for our specific requirement. These could then be tested for toxicity.

Sudha Bhattacharya
Alok Bhattacharya