Novel Therapies' Funding Summary

Fractional diffusion imaging as a biomarker for pathology in Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy is a genetic muscle wasting disease which affects approximately 1 in 3600 boys in the UK. The disease is caused by a mis-expressed protein which prevents efficient muscle contraction precluding the body from building and repairing muscles. This results in progressively weakened muscles and greatly reduced life expectancy. One challenge in developing effective therapies for DMD is the need to spatially map the changes to the tissue caused by the pathology. DMD is known to affect the size of muscle fibres, how they are arranged, and (importantly) their permeability. Measuring each of these is challenging, and usually required invasively taking a tissue sample which can be stained an imaged under a microscope. This technique is powerful, but its invasiveness means that it cannot be performed too often.

We are developing new types of MRI which can detect the microscopic changes caused by DMD without having to take a tissue sample from the patient. Current imaging approaches provide measurements of the amount of fat present in muscle tissue. This is highly effective in later stages of the disease when muscle tissue is being replaced by fat but is less effective at earlier stages, where the muscle tissue is changing but has not yet been replaced with fat. Our new approach measures changes in the microscopic structure of muscle tissue and as such is sensitive to changes during the earlier stage of the disease.

This means that the progression of the disease can be measured more frequently and the impact of therapies monitored more closely. The new approaches we have developed work well in laboratory tests, but need refinement before they can be applied in clinical settings. In particular, we are developing sequences which acquire data quickly and efficiently whilst also maximising the sensitivity of the scan to disease progression and investigating how well these new images reveal changes in tissue.

Developing microRNAs as biomarkers and therapeutic targets in children with spinal muscular atrophy

Molecules such as microRNAs in circulating body fluids have been used as informative non-invasive biomarkers to indicate disease severity and predict response to therapy. Spinal muscular atrophy (SMA) is a devastating neuromuscular disease affecting motor neurons in the spinal cord. Infants with the most common type, type 1 SMA, have aggressive disease progression and can barely live longer than 2 years. Encouragingly, Spinraza TM (also known as Nusinersen), a new genetic therapy has shown very promising efficacy in clinical trials and has recently been approved by the regulators FDA in the US and EMA in Europe. Great Ormond Street hospital has the largest UK paediatric neuromuscular centre. To date, there are more than 20 patients are receiving Spinraza treatment here. While Spinraza is effective in slowing down disease progression, it is not a complete cure. Some affected children respond well, while in others the response is limited. This indicates the need of informative biomarkers to predict drug response for this condition.

Our team has previously identified a particular molecular signature in patients’ blood and SMA mice. In particular, we found that miRNA is dysregulated in SMA. In this study, we will measure the change of miRNAs in cerebrospinal fluid and blood samples collected during the treatment in patients before and after Spinraza administration. We will use the most state-of-the-art techniques, such as next generation sequencing, for the discovery. This is a unique opportunity as spinal fluid from SMA patients has never been studied before. We will correlate the laboratory data with clinical information on patients’ motor function improvement. While these studies may provide further insight on the response to therapy in patients receiving Spinraza, they also pave the way for the assessment of the efficacy of any new therapies in the future and may lead to the development of new treatment for this condition.

Novel genetic therapies for motile cilia disease

Primary ciliary dyskinesia (PCD) is a rare, inherited lung disease caused by abnormal cilia, the hair-like extensions of specialized cells that beat to create movement of fluids in the body. Chronic and progressive lung disease confers significant morbidity to affected children who can suffer with other symptoms such as cardiac disease. PCD affects an estimated 1 per 10,000 individuals and our team at the UCL Great Ormond Street Institute of Child Health has long-term involvement with the well characterised UK PCD cohort, supported by a specialised NHS service since 2006. Our genetic screening of >500 patients shows that most of PCD arises from severe ‘knock-out’ gene mutations that stop production of the gene-encoded proteins. 45% of patients carry so-called ‘splice site’ and ‘premature termination’ mutations leading to loss of cilia proteins. These mutations are amenable to rescue using a promising new RNA-based technology, antisense oligonucleotide (AON) therapy.

This approach has been successful for other diseases in blocking the mutation and restoring normal protein function to patient’s cells. Antisense drugs are suitable for targeted rescue of both these classes of mutation and we aim to evaluate their therapeutic effects in PCD gene-mutated cells by: (1) developing a cell model of PCD mutations (2) optimising the effects of custom antisense therapy for mutational rescue (3) testing successful antisense therapy in relevant patient cells. This proof-of-principle study uses well established, previously trialled, therapeutic technology of minimal immunogenicity that does not involve genome integration or alteration associated with ethical concerns and oncogenic risk. AON therapy has significant potential for investigation as a novel treatment of lung disease with promise of rapid translation to the clinic and trials in PCD patients. Our aim to alleviate this chronic debilitating disease fits with NIHR GOSH Biomedical Research Centre remit to treat diseases of child health.

Genetic therapy for congenital melanocytic naevi

Congenital melanocytic naevus (CMN) syndrome is the association of very extensive darkly pigmented moles in the skin birth, which can cover 80% of the skin surface, with problems in the brain and a predisposition to the skin cancer melanoma. It is currently entirely untreatable by any method, and when melanoma arises in these children it is always fatal, usually within 6 months. Great Ormond Street Hospital has the largest cohort of patients with this disease in the world.

In 2013, our team established that the commonest cause CMN syndrome is a genetic mutation (mistake) which occurs randomly in the womb. The gene is called NRAS, and it is an important gene in all body cells, controlling cell growth. Mutations in NRAS are known to be able to cause melanoma in the general population. Since 2015 we have been developing and optimising a way of targeting the mutation in the skin so that it prevents it being active. We have confirmed that this works in cells in the laboratory, and now we are proposing to test the potential treatment more thoroughly, looking for side effects and making the best delivery system for the treatment into the skin. In parallel our international collaborator would test them in two animal models.

Repurposing Drug-X as a broad spectrum antibiotic targeting gram positive infection

The fight against antibiotic resistance is a global priority. We have made a major discovery that an existing drug (Drug-X) used to treat a non-infectious condition also has strong antibiotic effects on a wide range of bacteria that cause life threatening infections including mycobacterial (TB-like bacteria) and Gram-positive pathogens (such as MRSA, Listeria and C.difficile).

This project will move forward the repurposing of Drug-X as an antibiotic. To progress repurposing rapidly and to address a current major clinical need we will focus on developing Drug-X for treatment of the rapid growing Mycobaterium abscessus complex, which has emerged as a major pathogen with devastating consequences in infected cystic fibrosis children. Response of M.abscessus complex bacteria to current prolonged antibiotic therapy is poor with progressive loss of lung function and increased mortality, so a new antibiotic is desperately needed. Encouragingly we have shown Drug-X disrupts the extremely tenacious M.abscessus complex biofilm in addition to killing bacteria. It is essential to treat M.abcessus with more than one antibiotic to prevent it becoming resistant. This study will use the state of the art ‘hollow fibre’ system that will allow us to determine the best antibiotic/s to combine with Drug-X, and the optimum dose of Drug-X in a subsequent clinical trial.

Drug-X has a side effect in that it lowers blood pressure in some patients that may preclude its first line use for infections other than M.abcessus. This project will allow us to make analogues of Drug-X that retain their antibiotic properties but lack the part of the drug molecule that we have identified causes lowering of blood pressure. If successful this will allow us to develop new molecule that will be a completely new antibiotic that could treat a wide range of lethal gram positive bacterial infections.

Developing a hepatocyte-specific antisense oligonucleotide therapy for hereditary tyrosinemia type 1

Tyrosinemia type 1 (HT1) is caused by genetic changes in the fumarylacetoacetate hydrolase (FAH) gene, which blocks the breakdown of the amino-acid Tyrosine. This leads to the accumulation of toxic molecules, resulting in lethal liver failure and kidney dysfunction. Children with HT1 can be treated with nitisinone (NTBC), a drug inhibiting an enzyme called HPD, which prevents the accumulation of toxic molecules. However, this treatment requires lifelong daily intake of NTBC in order to maintain a therapeutic effect.

The strict need to take dozens of NTBC capsules every day for life, especially for those who began the treatment as new-borns and never experienced any symptoms, together with the extremely high cost of the drug, has resulted in increasing cases of patients not taking enough NTBC, and falling ill. Discontinuity of NTBC is associated with neurologic crises and liver cancer. Therefore, a new treatment that is more efficient, with the ability to block the HPD enzyme for longer, is needed. If developed, it would allow patients to take their treatment less frequently to monthly or even quarterly. We believe the lower frequency of treatment will avoid patients skipping their treatment and the occurrence of liver cancer and neurological crises, improve patient quality of life, and reduce the overall costs of managing HT1.

We here propose a 12-month study to develop an alternative therapy for HT1, using state-of-the-art antisense oligonucleotide (AON) technology. This new therapy is expected to have higher efficiency; better target the cells that most need the treatment -liver cells; and last longer, reducing the number and cost of the treatments needed. We expect the identification of the lead AON compound in this project will attract external funding to support subsequent studies to take this compound to the clinic.

Minimising visual system degeneration following hemidisconnection surgery in children – piloting an imaging informed intervention

Epilepsy is a medical condition in which the cells of the brain emit abnormal electrical activity causing seizures. Ongoing seizures damage the brain leading to decline of brain functions such as cognition. One treatment option is surgical removal of parts of the brain associated with the abnormal electrical activity. One such operation involves removal of one of the hemispheres of the brain, known as hemidisconnection. Although this operation may eliminate or reduce seizure activity it may result in damage to brain functions such as vision. This study consists of three parts: firstly, we will use imaging techniques to measure brain structure before and after surgery together with measures of visual function to determine short-term visual outcome and how this relates with brain structure. Secondly, we will recruit patients in whom hemidisconnection was performed more than 5 years ago to examine the long-term effects on visual function and brain structure, building on our pilot investigations. Thirdly we will work with visual rehabilitation training experts to set up a visual function intervention in a cohort of children undergoing hemidisconnection to establish feasibility and to lay the foundation for a trial aimed at achieving the best possible visual function shortly after surgery.

Antisense oligonucleotides for the treatment of ALDH7A1-deficiency

Humans rely on vitamin B6 for the proper functioning of their nervous, endocrine and immune systems. They are not able to make this micronutrient themselves and must therefore get it from their diet. There are many different dietary forms of vitamin B6 which our bodies convert to the active form of this vitamin. The active form is known as pyridoxal phosphate (PLP). PLP is essential for enzymes involved in metabolism of proteins, fats and carbohydrates to work properly.

Whilst a dietary deficiency of vitamin B6 is rare there are several genetic disorders which result in insufficient PLP within the cells of the body. PLP plays an important role in the brain therefore children with these disorders present with epilepsy.

The most common of these epilepsy disorders occurs when there are mutations in a gene that is responsible for making an enzyme called α-aminoadipic semialdehyde dehydrogenase. This enzyme is involved in the pathway which converts lysine, an amino acid which is present in the food we eat, into energy. Mutations in this gene result in an accumulation of metabolites which interact with PLP thereby reducing the amount of PLP available for the brain. The resulting epilepsy can be treated by giving supraphysiological doses of vitamin B6.

Unfortunately the compounds that accumulate, besides interacting with PLP, are also toxic to the brain. This toxicity results in intellectual disability and developmental problems. Treatment with B6 does not correct this. One way of preventing the accumulation of these compounds is to restrict the amount of lysine in the child’s diet. Children do not like this diet and the improvements seen have only been partial. Better treatments are therefore needed. We will investigate the use of antisense oligonucleotides as a way of preventing an accumulation of these toxic compounds in this disorder.

RNA interference of ENaC as a therapy for cystic fibrosis

Cystic fibrosis (CF) is an inherited disease caused by errors in the CF gene which normally makes a protein in cells lining the lung that allows salts to flow in and out. Cells lining the lung produce the mucus that entraps inhaled particles, such as bacteria, viruses and pollutants in the air, while the cilia, small, hair-like projections, wave backwards and forwards to sweep the trapped particles out of the lung. This helps to keep the lung clean and free of infections. In CF, the faulty ion channel means the thin layer of water in the lung is lost and the mucus becomes thicker and stickier and stops the cilia beating. This leads to bacterial infection and an immune response which can actually cause further damage to the lung. Our aim is to break this chain of events by preventing loss of the thin watery layer from the lung, allowing the cilia to sweep the lung clean and free of bacteria.

There is another protein that is important in CF lungs, called ENaC, that is more active than usual that increases the loss of water. Our goal is to switch off ENaC, and so reduce the water loss, making the mucus thinner and restoring the watery layer that bathes the cilia, allowing them to clean the lungs. This should prevent mucus blocking the air tubes in the lung, allowing patients to breathe more easily and preventing bacterial infections. We have made a drug called a short interfering RNA, or siRNA, and a nanoparticle to deliver it by inhalation, which we have shown in the lab switches off ENaC very efficiently. We now aim to show how this treatment works, alone and in combination with new CF drugs like Orkambi, so that it could benefit all CF patients.

Hereditary sensory and autonomic neuropathy type I (HSAN1, also known as HSN1) is a rare neurological condition affecting nerves lying outside the central nervous system (the brain and spinal cord). It leads to patients being unable to feel temperature, pressure and pain. Over time symptoms get worse often leading to painless injuries and mutilating skin ulcerations which can lead to amputation. This condition has been reported in residents of the United Kingdom, Europe, Australia, Canada and the United States. Currently, no effective treatment for HSAN1 is available.

The disorder is most commonly caused by genetic coding errors (genetic spelling mistake /mutations) in the SPTLC1 gene, which provides instructions for making one part of an enzyme involved in making certain fats which are important to keep nerves healthy. This genetic error leads to the production of a new substance, which is toxic to nerves, when the body breaks down some proteins.

Antisense oligonucleotides (ASOs) are short, synthetic, chemically-modified chains of nucleotides (the building blocks of RNA and DNA) that have the potential to target any gene of interest.

Dr. Zhou and her team are now developing a new therapy for HSAN1 by using ASOs to selectively reduce the production of the toxic substance. They are testing specifically-designed RNA compounds in cells grown from HSAN1 patients' skin. If successful, the lead compound(s) will move along the translational path from bench-to-bedside and eventually benefit the majority patients currently affected by mutations in the SPTLC1 gene.

Infants diagnosed with acute leukaemia have significantly inferior outcomes in comparison with older children diagnosed with this disease. Infant acute leukaemia is frequently caused by damage to DNA that results in the formation of abnormal cancer genes in developing blood cells. These cancer genes instruct the cells to make abnormal proteins that are necessary for the leukaemia cells to grow. If these abnormal proteins can be destroyed, the leukaemia cells will die and disease can be eliminated.

Despite remarkable improvements in the treatment of certain groups of childhood leukaemia over the past decades, there is still a desperate need for different therapies to treat these very young children. New therapies that destroy abnormal cancer proteins have shown great success in certain adult leukaemias and have been associated with favourable long-term outcomes.

In examining new approaches to destroy the abnormal cancer protein in infant acute leukaemia, our group discovered that the drug disulfiram (DSF) is able to cause efficient destruction of the target protein and thereby eliminate leukaemia cells. This exciting breakthrough could be a real game changer in the treatment of infant leukaemias. DSF has already passed all pharmacology and toxicology safety tests for use in human beings. Furthermore, all toxicities and side-effects are already known because the drug is already used in clinic for different indications. In this proposal we request the funds to validate the impact of this targeted treatment on cells derived from patient leukaemia samples. We predict that successful completion of this study could lead to clinical trials that will completely change the life of these sick and vulnerable babies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of Covid-19 disease and the ongoing global pandemic. The virus transmits through the respiratory route and enters respiratory cells by binding of a viral protein called Spike to the host cell protein Angiotensin-converting enzyme 2 (ACE2), while another host cell protein - Transmembrane Serine Protease 2 (TMPRSS2) – primes Spike to allow its interaction with ACE2. As such, Spike is the viral “key” to enter cells, while ACE2 and TMPRSS2 are the cellular “lock” and “gatekeeper” that allow SARS-CoV-2 entry.

In this project we are investigating a therapy to prevent the spread of SARS-CoV-2 by reducing the availability of these two cellular entry proteins and in turn blocking entry of the virus. Although vaccines have been successfully developed to prevent progression of severe Covid-19 disease, there are limitations to these vaccines blocking transmission of all the virus variants, and for use in individuals who have a weakened immune system. Our proposed gene therapy, targeting host proteins, would overcome and find a way round these limitations.