A New Approach To Treatment of LSD’s; Messenger RNA (mRNA) Therapy

Messenger RNA (mRNA) is a group of molecules that convey genetic information from DNA in the nucleus into the cytosol of the cell. There, this information is used by ribosomes to make protein. So mRNA’s that make specific proteins can be constructed and inserted into a cell, which has the machinery to make the specific protein. This forms the basis of mRNA therapy.

Not surprisingly, the concept is not new, but early attempts were unsuccessful for several reasons. These are now gradually being overcome, and mRNA therapy has become one of the most exciting new therapeutic developments seen for many years, with a wide range of applications. For example, it can be used to make vaccines far more quickly and effectively. The possibility of anti-cancer vaccines is also being explored. 

Importantly, mRNA therapy provides solutions to some of the drawbacks of gene therapy.

Here is a useful review of mRNA therapy.

Next-Generation Therapeutics mRNA as a Novel Therapeutic Option for Single-Gene Disorders

Monogenic disorders, such as LSD’s, are prime candidates for mRNA therapy. There has been very encouraging progress in this area. Earlier this year, researchers from Translate Bio, Shire Pharmaceuticals and Biomere successfully treated a mouse model of Fabry disease using mRNA therapy, using the liver as the source of enzyme. Here is their paper

Improved Efficacy in a Fabry Disease Model Using a Systemic mRNA Liver Depot System as compared to ERT

It is probably only a matter of time before mRNA therapy is used to treat more LSD’s. For the time being, however, it is unclear whether it can be sued to treat LSD’s that affect the CNS. We shall have to wait and see.


Cervical Spinal Cord Compression in MPS IVA; Clinical Signs In The Absence of MRI Changes.

Cervical spine instability is a well-known complication of mucopolysaccharidosis IVA (Morquio A disease). If left untreated it nearly always results in spinal cord compression  and progressive neurological deterioration. Timely surgery can be not only life-saving but may prevent  irreversible damage to the cervical cord.

MRI scan of the cervical spine can detect early cord compression, allowing surgeons to intervene early. It has become an essential part of monitoring MPS IVA.

In a recent paper, Broomfield and colleagues at the Royal Manchester Children’s Hospital have described their experience of cervical spine surgery in MPS IVA over an eighteen-year period. Their findings of course confirm previously reported experience as above. However, they also include six patients who developed clinical signs of cord compression in the absence of any MRI changes. This is an important observation and a timely message. It underlines the importance of careful clinical examination, as well as the danger of over-reliance on MRI scans.

Here is the full reference with the link to the paper (pdf kindly supplied by Dr Broomfield).

Outcomes from 18 years of cervical spine surgery in MPS IVA; a single centre’s experience. Broomfield, A., Zuberi, K., Mercer, J. et al. Childs Nerv Syst (2018) 34: 1705. 

MPS II Update

There have been a number of interesting publications over the last year on MPS II.

Whiteman and Kimura (Shire Inc.) have written a comprehensive overview of the development of idursulfase therapy. Here is the full article

Development of idursulfase therapy for MPS II 2017

Muenzer et al reported the results of follow up of patients receiving ERT. Data from the Hunter Outcome Survey (HOS) was used for this purpose. A total of 639 patients (excluding females, those patients who had received HSCT, and patients enrolled in the phase 1/2 [TKT018] or phase 2/3 [TKT024] clinical trials) who had been followed up for at least 6 months on ERT were reported. Continuing improvements were observed in various visceral parameters. However, results of cognitive function were not reported. The authors acknowledge some shortcomings of this study. For example, collection of urine for GAG analysis is more difficult in patients with severe neurological involvement.

Here is the full paper

Clinical outcomes in idursulfase-treated patients with MPS II. 3- year data from Hunter Outcome Survey (HOS)

Burton et al report survival in MPS II patients. Again, their data is drawn from HOS. Two key findings are

  1. A 54% increase in survival in treated vs untreated patients
  2. A five fold higher risk of death in patients with cognitive impairment.

Here is the full paper

Survival in idursulfase treated and untreated patients with mucopolysaccharidosis type II data from the Hunter Outcome Survey (HOS)

These findings are not surprising, but they emphasise the need for early diagnosis of, and more effective treatments for, the brain involvement in MPS II.

Escolar et al reported a scoring system for early diagnosis of CNS disease. They found that seven early clinical markers and a severity score index of CNS involvement can be used for initial screening of children who might benefit from CNS-directed therapies. This paper has not received the attention that it should have; the severity score described should be more widely used.

Here is the full paper

Early clinical markers of central nervous system involvement in mucopolysaccharidosis type II

As far as treatment of brain involvement in MPS is concerned, Scarpa et al have written this timely review of recent developments in the field.

Here is their full paper

Treatment of brain disease in the mucopolysaccharidoses 2017


MPS II: Analysis of the mouse brain throws new light on some aspects of neurological involvement.

Mucopolysaccharidosis type II is often associated with challenging, disruptive behaviour and varying rates of cognitive decline. However, the underlying mechanisms are only poorly understood.

Now, several teams from Padova, Italy, have published findings that shed new light on this area. They performed an analysis of the MPS II mouse brain, using RNA sequencing (RNA-Seq). A detailed description is beyond the scope of this post, but basically RNA-Seq is an example of next generation sequencing that is widely used to study gene expression by transcriptome profiling. The transcriptome is the complete set of transcripts in a cell, and their quantity, for a specific developmental stage or physiological condition. Understanding the transcriptome is essential for interpreting the functional elements of the genome and revealing the molecular constituents of cells and tissues, and also for understanding development and disease.

The researchers investigated genes known to be involved in many aspects of neurological function. They found that there was dysregulation of many of these genes, for example calcium homeostasis, axon guidance, mitochondrial function, oxidative stress and circadian rhythm.

Several of these, for example mitochondrial function and oxidative stress, are involved in different stages of the Parkinson Disease pathway. A particularly important finding was the upregulation of alpha-synuclein, the accumulation of which is responsible for synucleopathies such as Parkinson’s disease (PD). For many years, Gaucher disease was the only lysosomal storage disorder (LSD) thought to be associated with PD. However, in recent years such as association has been described in other LSD’s such as Niemann-Pick disease type I and sphingomyelinase deficiency. Accumulation of alpha-synuclein has been demonstrated in the cortex of patients with MPS III. While PD has not yet been described in MPS II patients, it seems that it is only a matter of time before such an association is reported.

Alpha-synuclein also contributes to the development of Alzheimer’s disease. Its accumulation may therefore be of relevance to the cognitive decline that is seen in many MPS II patients.

It is quite possible that patients with MPS II will be found to have similar patterns of gene dysregulation to those seen in the mouse model. Were this to be found to be the case, it may well have implications for counselling and management.

However, a note of caution is required. The MPS II mouse has a single mutation;  whereas human MPS II is caused by many mutations in the IDS gene. So even assuming that similar gene dysregulation to that seen in the mouse will be seen in human MPS II, we dont know how this will be linked to the individual mutations seen in patients. For example, will the gene controlling circadian rhythm be affected more severely in patients with certain MPS II mutations than others?

We also need to bear in mind that, while some aspects of human behaviour may be controlled by the genome, others may well be related to as yet unidentified epigenetic factors. Epigenetics is the term given to the modification of gene expression rather than the genetic code itself. The human epigenome has not yet been fully characterised; work on this is under way.

It may therefore be some time before we have answers to these important questions. Nevertheless, these are important findings that are likely to greatly improve our understanding of the complex neurological and neuropsychiatric features seen in MPS II.

Sangamo Receives Fast Track Designation From The FDA For In Vivo Genome Editing Product Candidates For The Treatment Of MPS I And MPS II

Sangamo Therapeutics announced on July 13th that it had received fast-track approval from the FDA for its genome editing program for MPS I and MPS II.

This is excellent news. If the trials are successful, patients will have a permanent, stable production of circulating enzyme via the liver. It may even be possible to do away with intravenous infusions of ERT altogether. I had briefly covered the technology involved in an earlier post.

However, a few caveats are worth noting.

The current trials are aimed at patients with the attenuated forms of the disease, that is to say, patients who do not have CNS involvement. Since conventional ERT does not cross the blood-brain barrier, it is unclear whether enzyme produced by gene editing will fare any better.

In MPS II, the deficient enzyme, iduronate sulfatase, requires activation by FGE (formylglycine generating enzyme). This is controlled by the gene SUMF1. Since this gene functions normally in MPS II, FGE is already available in the cells. So one must assume that no further supplies of FGE will be required to activate the iduronate sulfatase that is produced by gene editing.

Ultimately, this approach should be seen primarily as a means of permanently replacing conventional ERT. Treatment of the neurological forms will almost certainly require other approaches. Many of these, such as fusion proteins and gene therapy, have shown early promise, but more work needs to be done.




Cognitive endpoints for therapy development for neuronopathic mucopolysaccharidoses: Results of a consensus procedure

The results of a consensus meeting of 15 international experts on cognitive endpoints in MPS has just been published. A modified Delphi technique was used. Briefly this is a systematic forecasting method that involves structured interaction among a group of experts on a subject. It typically includes at least two rounds of experts answering questions and giving justification for their answers, providing the opportunity between rounds for changes and revisions. The multiple rounds, which are stopped after a pre-defined criterion is reached, enable the group of experts to arrive at a consensus forecast on the subject being discussed.

There was a very high level of consensus. Of the 12 statements considered, there was 93% consensus on 11 and 100% consensus on the other 11.

This a very timely paper in view of the increasing number of clinical trials, both in progress and being planned, for the neuronopathic forms of MPS disorders, and the plethora of assessment tools currently available.

The statements are given below (copied directly from the paper). For those who are interested in further details, here is the link to the full paper. 

For trials evaluating the effect of treatment in children with MPS I, II or III aged up to 3years (age equivalent), the recommended instrument to measure cognitive outcomes is the Bayley-III.

For trials evaluating the effect of treatment in children with MPS I, II or III of all ages, the recommended instrument to measure adaptive behavior is the Vineland, using the extended interview format.

For (multinational) trials evaluating the effect of treatment in children with MPS I aged 3years and over (age equivalent), the recommended instruments to measure cognitive outcome are the Wechsler tests. We recognize the utility of the Kaufman Assessment Battery for Children, Second Edition (KABC-II) and the Differential Ability Scales, Second Edition (DAS-II) in particular populations because of their reduced fine motor demand and less emphasis on speed of performance compared with the Wechsler tests.

For (multinational) trials evaluating the effect of treatment in children with MPS II or III aged 3–18years (age equivalent), the recommended instruments to measure cognitive outcome are either the DAS-II or the KABC-II. The use of only the non-verbal domain/index may be appropriate if this is necessary to ensure consistent application between trial sites across multiple countries. Other factors to consider in the selection of the measure and on whether to use the entire test or only the non-verbal domain are: the need for verbal interaction; the time required to administer the test; fine motor requirements; availability of normative data; availability of translations.

In a set of trials within the same program, we recommend using the same test protocol for all trial sites worldwide; including, if possible, the same test editions or, if not, the most recent.

We acknowledge the usefulness and value of historical data that elucidate the natural history of MPS I, II and III, including standardized cognitive and developmental outcome measures other than those recommended in these consensus statements.

We strongly recommend building and sustaining an infrastructure to share natural history data.

We acknowledge that in multinational trials it may be necessary and appropriate to use one set of psychometrically sound normative data; however, this is only recommended for a non-verbal outcome measure. If a specified tool has not been validated in a country, we recommend the parallel use of a country-specific instrument to establish concurrent validity.

We recommend the use of a standard written translation of the measurement instrument, including the administration instructions, produced by a professional translator with experience with standardized tests. Such a professional translation should always be accompanied by a back-translation. We also recommend cross-cultural adaptation. Lastly, we recommend that a local psychologist/psychometrician should review the fidelity of the translation and of the cross-cultural adaptation.

Assessors must be qualified in administering neurodevelopmental measurement instruments and have experience in their use, preferably with the disease being evaluated. Assessors need to be trained in person to perform the specific measurements in the protocol and should be subject to periodic quality control and auditing of scoring.

We recommend analyzing and reporting age-equivalent scores in all trials, and standard scores where possible.

When transitioning from one test to another because of developmental or chronological age, we recommend administering the two tests concurrently at least once during the same visit (on separate days) to compare the test results.

As far as I am aware, this is the first time a consensus paper on this important subject has been written. The authors make the point that new assessment tools are likely to be published in the future, and stress the importance of reviewing and, if necessary, revising these recommendations every 3 years.

A potentially new therapeutic target in Pompe disease

Pompe disease affects cardiac and skeletal muscle. Accumulation of glycogen, build up of autophagic material, and muscle atrophy are all well-described features. Enzyme replacement therapy has dramatically improved the outlook. However, it is not perfect.

Muscle atrophy can occur in the absence of glycogen build up and defective autophagy. The cause is not clear; the precise cell-signalling mechanisms involved have yet to be elucidated.

Interest has focused recently on mTOR (mechanistic target of rapamycin). This is a serine-threonine kinase that is a regulator of cell growth, including muscle mass. mTOR exists as two complexes, mTORC1 and mTORC2. Of these, mTORC1 is closely associated with the lysosome, where it is activated. Upon activation, it suppresses autophagy and facilitates cell growth and protein synthesis. A detailed discussion is beyond the scope of this post but please see this paper.

mTOR and lysosomal regulation

The close association of mTORC1 with the lysosome has led to speculation that it might be a useful therapeutic target in LSD’s. Now, teams from the National Institutes of Health and Boston have published the results of their systematic studies of mTOR activity in Pompe muscle cells. They have shown that mTOR activity is reduced, and that this is at least in part due to the action of tuberous sclerosis complex 2 (TSC2) (please see this link for a detailed discussion of TSC2). The group have further gone on to show that the effect of TSC2 on mTOR activity can be reversed by arginine. This effect was seen in vitro and, critically, in vivo; oral administration of arginine to Pompe mice resulted in clearing of autophagy and increased muscle mass. The full paper can be found here:-

Modulation of mTOR signaling as a strategy for the treatment of Pompe disease

Clearly there is a long way to go. The muscle atrophy seen in patients can be very severe, and there may well be a point beyond which nothing can reverse it. It is also unclear how effective such an intervention would be on its own in clinical practice.

However, at the very least, these are very interesting findings, in that they suggest that there may well be alternative approaches to the treatment of this devastating condition. The authors have made a strong case for clinical translation. They also very reasonably point out that, given the central regulatory role of mTOR in lysosomal function, other LSD’s may well stand to benefit from such approaches.


Early diagnosis of cardiac involvement in MPS

Cardiomyopathy is seen in all forms of MPS. Valvular and myocardial disease are both seen. Enzyme replacement therapy appears to have little, if any, effect on the valve disease. However, the myocardium does seem to respond, though not completely.

Echocardiography has been the standard method of evaluating cardiac involvement for many years. More recently, MRI has been shown to be more sensitive. However, there are practical limitations to MRI. In young children general anaesthesia may be required. At the very least, this is inconvenient, and it carries increased risk in MPS patients.

A relatively new tool is speckle tracking echocardiography (STE). It provides information which is not available with any of the currently used echocardiographic parameters. For a detailed discussion of STE please see this article.

Speckle-tracking echocardiography

Researchers in Naples have studied myocardial function in a small group of children with MPS using STE. Abnormalities were detected in various aspects of ventricular function. Importantly, some of these were seen even in the absence of any abnormalities in the standard echocardiogram.

Here is the link to their paper.

This suggests that STE may detect myocardial dysfunction at an early stage, even before it is seen on standard echocardiography.

The only other LSD In which STE has been systematically studied is Fabry disease. This is the first such study to have been performed in MPS patients, and some limitations should be noted. Only a small number (15) were studied. There are limitations to STE at the moment and it has not yet been standardised, so that STE performed on different machines may yield different results. Finally, the clinical relevance of early myocardial involvement in MPS is unclear. For example, it does not in itself constitute an indication for ERT. In this respect it differs from Fabry disease. However, its sensitivity suggests that it may well detected responses to treatment earlier than standard echocardiography. Such studies, in large cohorts, need to be done.

So one needs to be cautious. Nevertheless, given its clear superiority to standard echocardiography, and the lack of requirement for a general anaesthetic, STE may well become a standard investigative tool in the not too distant future in MPS.

Genome editing for MPS I and MPS II using Zinc Finger Nucleases

Sangamo Therapeutics has announced plans to conduct clinical trials of genome editing using zinc finger nucleases (ZFN’s) for MPS I and MPS II in 2017. More details of the planned trials can be found on the clinicaltrials.gov website here.

Neither trial has started recruiting at the time of this post. Both trials will use the same technology (zinc finger nucleases, ZFN’s) to insert the transgenes for MPS I and MPS II respectively into the genome of hepatocytes (liver cells) using the albumin gene to control expression.


The liver is in many ways an ideal organ of choice for gene therapy, serving as a permanent “factory” for secreted proteins, including lysosomal enzymes, for delivery to other organs.

Two sets of agents have to be delivered to the body. The first is the ZFN’s to cut or cleave the DNA in the hepatocytes. The following video describes how they work in a way that is very easy to understand. It is about 12 minutes long and well worth watching. I should point out that there is one small mistake; please read Fok1 instead of Fokl (ie the number 1 not the letter l). However this really does not detract appreciably from the video.

Both strands of DNA are cleaved, so this break is called a double-stranded break. Once the DNA is cleaved, the required transgene (ie the genetic material that needs to be transferred) is inserted. So the second agent to be delivered is the transgene for MPS I or MPS II. In order to deliver the transgene to the liver, a vector is required. In the Sangamo trials, an adeno-associated virus (AAV) will be used. AAV’s have many properties that make them desirable for liver-directed gene therapy.

For a more detailed discussion of these topics please read these two articles.

Adeno-Associated Virus Gene Therapy for Liver Disease

The Liver as a Target Organ for Gene Therapy: State of the Art, Challenges, and Future Perspectives

Once the transgene is inserted, it is incorporated into the DNA by a process known as homologous recombination. 

Albumin is a protein that is produced in the liver and the albumin gene is very strongly expressed in hepatocytes. This “platform” forms the basis for the development of the methodology on which the trials are based, and which was developed by teams at the Childrens Hospital and Howard Hughes Institute in Philadelphia, and Sangamo Biosciences and published in this article.

In vivo genome editing of the albumin locus as a platform for protein replacement therapy.

The genetically engineered DNA will now start manufacturing the required protein, in this case the deficient enzyme. It is hoped that this will provide a permanent source of enzyme.

Both trials are restricted to adult patients with the attenuated forms of the disease. For the MPS I trial, this means patients with Hurler-Scheie, Scheie or Hurler post-HSCT. For the MPS II trial, it will be patients with the attenuated form of MPS II. This usually means patients who do not have central nervous system involvement (this is a personal observation).


Gene therapy for Pompe disease effective in mice

Single dose of gene therapy could augment or replace frequent enzyme infusions


January 26, 2017
Duke University Medical Center
After decades investigating a rare, life-threatening condition that cripples the muscles, researchers have developed a gene therapy they hope could enhance or even replace the only FDA-approved treatment currently available to patients. The therapy uses a modified virus to deliver a gene to the liver where it produces GAA, an enzyme missing in people with Pompe disease.
Here is the link to the published article
Low-dose liver targeted gene therapy for Pompe disease enhances therapeutic efficacy of ERT via immune tolerance induction