Department of Health Funded Project: Progress Report, August 2005

Department of Health Funded Project:
“Restoring Dystrophin Expression in Duchenne Muscular Dystrophy: A UK Consortium for Preclinical Optimisation and a Phase I/II Clinical Trial Using Antisense Oligonucleotides”

Progress Report 3: August 2005

A joint meeting with members of the Scientific Advisory Board and the MDEX Consortium was held on the 25th May 2005.  Each of the lead investigators presented an update of progress within their research group together with milestones for the next quarter.  These were reviewed and discussed by members of the Scientific Advisory Board who were happy with the progress made to date.



You can download this report as a Word document: DoH progress report 08/05 (86 kb) [doc]

Contents:


Department of Health report:

The second quarterly progress report has been submitted to the Department of Health and below is a summary for this quarter together with objectives for the next reporting period.

1. Chemistry of the molecular patches:

The aim of researchers working in the chemistry section is to optimise the formulation of the molecular patches for maximum effect. The Dickson/Graham laboratory is developing a series of different patches and has been studying exons 43, 44, 45, 46, 51 and 53. They are varying the genetic composition of the patches which will then be analysed to see which formulation is most effective. Small fragments of genetic material have been generated and sent to a commercial company for analysis. Results are expected within the next quarter.

Molecular patches made by our Dutch collaborators and Dr Steve Wilton in Australia have been obtained. Testing and comparison of these with patches produced by the UK MDEX Consortium is underway in both the Muntoni/Morgan and Dickson/Graham laboratories. Systems used to validate the efficiency and efficacy of the molecular patches is being optimised.

In order to test a variety of different patches, muscle cells from patients having the appropriate genetic errors need to be cultured and stored. This work is ongoing in the Muntoni/Morgan labs and will provide a stock of cultures for future testing of the patches.

The Wells laboratory has performed a literature survey of the different chemistries of molecular patches and their current application in any medical condition. This information includes the chemistries used, the efficiency of the molecular patch and whether they had been used in any clinical trials. The reason for this study was to ensure that the chemistry of the patch used in the clinical trial will give the most optimal result. Research is this area is continually advancing and during this year of developing the chemistry, the Consortium aims to develop a formulation that will give the highest levels of skipping and be safe for use in humans.

A major consideration when designing molecular patches is to prevent their breakdown. Based on new results from key players in this field the MDEX Consortium, together with the Scientific Board has decided that the backbone of the patch will be a morpholino and not 2’-O-methyl as initially suggested. This means that our chemistry will differ slightly from that of the Dutch but importantly will allow us to compare the efficiencies of two different chemistries in a clinical trial. This quarter the Consortium will check whether patches that work well with a 2’-O-methyl backbone are equally or more effective when combined with a morpholino backbone. Some of the main reasons for deciding on this backbone are that morpholinos have already been used in clinical trials (unrelated to muscular dystrophy) and have a high safety profile. It also means that we will not have to use a carrier to get them into muscle cells and they are cheaper to manufacture.

The Wood laboratory has begun work on developing improved methods for targeting the delivery of molecular patches to muscle and increasing their stability. A couple of interesting compounds have been identified and will be chemically joined to the genetic structure of the patch and then tested for efficacy.

Although this group will begin developing methods for systemic delivery, more research in this area will be needed and the three charities have identified this as a priority area for obtaining additional funding and support.

2. Toxicology work:

In preparation for a clinical trial the molecular patch to be used needs to pass through a series of stringent safety tests. Initial toxicology experiments are being conducted in the Wells laboratory to determine the dose and volume of molecular patches that will eventually be used in the clinical trials. These studies together with more detailed ones, done by specialist laboratories, are required by the regulatory authorities to assess the safety of the molecular patches before they are used in the clinical trial.
In preparation for these studies, the Wells group is compiling a document for the Consortium outlining the existing toxicological information we have and the additional information and testing that will be required to take molecular patches forward to clinical trial.

As part of this work the Wells laboratory is developing a spliceOmouse which will be used to identify where the molecular patches go after they have been injected and whether dystrophin protein is produced. The genetic sequences which will be used to generate the spliceOmouse are currently being evaluated in muscle cell cultures.

3. Preparatory Clinical Studies: (Hammersmith and Newcastle teams)

In order to determine the best muscle to be injected in the clinical trials it is important to obtain information that will inform the clinician of the appearance of muscle in different age groups of Duchenne boys. The Muntoni team has now fine-tuned muscle magnetic resonance imaging (MRI) as a way to study these small muscles and the protocol has been shared with the Newcastle group.

Ethical approval to perform such studies has to be obtained and an application was made in the middle of April by the Hammersmith and Newcastle teams. Ethical approval was finally granted at the beginning of August and the first patient has already been recruited into the study. Boys with DMD will be recruited at the time of loss of ambulation or before they undergo scoliosis surgery to have leg muscle MRI’s. This will help to study the preservation of their individual foot muscles. During surgery a muscle biopsy from a foot muscle will also be obtained for analysis.

Another ethics application is being prepared for studying the effect of molecular patches in cultured muscle cells. Skin cells will be obtained from boys with Duchenne and then converted into muscle cells in the laboratory. The reason for using skin cells is that it is a far less invasive technique and will generate a larger collection of cultures that can be used for testing. Skin cells will be obtained from children undergoing diagnosis and also from non-ambulant DMD boys undergoing rehabilitation or scoliosis surgery.

A comprehensive clinical, pathological and genetic database is being generated to contain specific genetic information on individual DMD boys and will indicate whether muscle biopsies and/or skin cultures are available. This comprehensive list will help to identify cell cultures of DMD boys with mutations that can be used to evaluate how efficient a designed molecular patch is in skipping the mutation. Data of 277 DMD and BMD patients have so far been entered into the database.

The initial document of frequently asked questions and answers has been updated to contain a section with practical information for families who are willing to participate in the study. This document will be distributed in both the Hammersmith and Newcastle neuromuscular clinics and be posted onto the websites of the Muscular Dystrophy Campaign, Parent Project UK, Duchenne Family Support Group and the European Neuromuscular Centre website. The aim is to increase awareness of the study and facilitate the recruitment of patients.

A letter to all consultant paediatric neurologists and paediatricians with an interest in neuromuscular disorders in the UK and Scotland has been prepared and distributed. The aim is to identify how many patients could potentially benefit from the exon skipping approach in UK and could theoretically be recruited, if carrying the relevant mutations, into the study. A proforma has been prepared to collect anonymous information on DMD boys with skippable mutations of exons 51 or 53 with only their date of birth as additional information. The deadline for collection of this information is the end of July 2005. This information will be forwarded and collected by the London group.

The objectives for the next reporting period are:

1. Chemistry of the molecular patches:

The objectives of the Dickson/Graham laboratory for the next quarter are:

  • Continued work on optimising the formulation and conditions for getting the molecular patches into human muscle cells.
  • The manufacture and testing of newly developed molecular patches which have undergone initial screening and show promise.
  • Testing molecular patches for exon 51 with the new morpholino backbone both in cultured muscle cells and animal models.

The objectives of the Imperial College laboratories are:

  • To optimise getting the molecular patches into cultured human muscle cells and test the efficiency of these in terms of how much dystrophin protein is formed. Results are expected from a comparative study using the Dutch molecular patch for exon 51 and the one produced by the Dickson/Graham laboratory.
  • To test a variety of different molecular patches in cells cultures from Duchenne boys.
  • To optimise systems to quantify the amount the amount of dystrophin produced.

The objectives of the Wood laboratory for the next quarter are:

  • To improve and optimise methods for getting the molecular patches into muscle cells.
  • To work towards the identification of additional chemistries to improve the stability of molecular patches and allow for systemic delivery.
  • To develop and fine tune tests to analyse the different chemistries that will be produced.
  • Promising chemistries will be tested in animal models using local injection into muscle.

2. Assessing toxicology, delivery and longevity of AO in mouse models:

The objectives of the Wells laboratory for the next quarter are:

  • To further progress work on the formation of the SpliceOmouse.
  • To complete dose and volume experiments to determine the quantity of molecular patch to be administered in the clinical trial.
  • To complete the document outlining the existing toxicological information and the proposed additional testing required to take molecular patch therapy forward to the clinical trial.

3. Preparatory clinical studies:

The objectives of the Hammersmith and Newcastle teams are:

  • To finish the MRI optimisation work, so that patient muscle can be studied using MRI.
  • Once approval for the muscle biopsy and MRI studies are obtained, work will begin to obtain consent from patients to take muscle biopsies at the time of a surgical procedure.
  • To submit an ethics application to take skin biopsies from patients coming to clinic. The skin cells will then be converted to muscle cells and stored for future testing of molecular patches.

The objectives for the first year of the project are well on target.

MDEX Consortium
August 2005

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