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2020 BDSRA Australia Batten Disease Research Grant Recipients

Isogenic induced pluripotent stem cell (iPSC) models with CLN3 variants for high throughput drug screening

Cook Research Group (L to R): Sueanne Chear, Dr Sharn Perry and Prof. Anna King (absent: A/Prof. Anthony Cook)

Our goal is to identify new drugs to treat Batten disease caused by variants in the CLN3 gene. To do this, we have used advanced techniques in human stem cell biology and gene editing technology to develop a new human cell-based model of Batten Disease. Such models are needed to accelerate moving laboratory findings into effective and safe treatment options for people with this disease. Preliminary investigations with this new model indicate that when the stem cells are maturated in the laboratory into nerve cells, those cells with Batten disease CLN3 gene variants reveal changes in how these cells communicate with one another, as well as changes in structures within the cells involved in energy production or in degradation of proteins. These changes are consistent with what is known about Batten Disease from the study of human tissue sample and from animal models.

In this project, we will extend these preliminary findings to determine their robustness, and investigate further how it may be that CLN3 variants affect these features of nerve cells. Based on these findings, we plan to test over 350 drugs that are known to be activate in the brain for their ability to affect how cells with Batten Disease CLN3 variants function. This project will initiate a new program of research focused on determining the suitability of such drugs as new treatments for Batten disease.

Development and validation of a gene editing therapy for CLN2-related disease

Hewitt Research Group (L to R): Freya Patterson, Peter Tran and Prof. Alex Hewitt

Heralded as the most significant breakthrough in biology since PCR (a process developed in the early 1980’s that enables the amplification of DNA), the adaptation of the “CRISPR/Cas” system to mammalian cells is set to revolutionise treatments for inherited disease. The CRISPR/Cas system, used by bacteria to counter viral intrusion (somewhat akin to our adaptive immune system), can cut or edit DNA at specific sites. The clinical application of CRISPR/Cas technology opens the very real prospect of anticipatory cures to well-defined inherited diseases. We have engineered a novel enzyme, which essentially hides a gene editor inside the larger CRISPR/Cas enzyme. With this protein design we have found an improved on-target editing efficiency, and a reduced off-target footprint. As such, this protein appears more effective and safer. Additionally, it is small enough that it can be adequately packaged into an adeno-associated virus, thereby facilitating robust delivery to target cells. We now seek support to determine if this novel gene editing therapy can be directed to correct a disease-causing variant in a preclinical model of CLN2-related disease. This project opens the door to therapeutic gene editing and a definitive cure for Batten Disease.

Advancing gene therapy strategies and natural history studies in ovine CLN5 and CLN6 Batten disease

Mitchell Research Group (L to R): John Wynyard, Ashley Deane, Dr Nadia Mitchell and Dr Samantha Murray

Naturally occurring ovine (sheep) models of CLN5 and CLN6 Batten disease are well established at Lincoln University (NZ). We are developing gene therapies in these sheep models which are yielding encouraging results for translation to humans. Sheep have large human-like brains and disease progression in them closely follows that in affected children. We have shown that the single administration of CLN5 gene therapy to the fluid-filled spaces in the brain (the lateral ventricles) protects against stereotypical disease and brain atrophy. Treated sheep are still alive up to five years post-injection, three times longer than their natural life expectancy. We found that delivery of CLN5 gene therapy to the jelly-like fluid of the eye (the vitreous humour) also protected against visual loss.

This led us to test combined brain- and eye-directed gene therapy at different stages of CLN5 disease. Even when given at an advanced symptomatic stage, high doses of gene therapy halted disease progression, stopped brain atrophy and slowed visual loss. We are preparing these results for an Investigational New Drug application to the US Food and Drug Administration, with the aim to start the first CLN5 gene therapy clinical trial in humans next year. However, it will be important to test the equivalent doses and delivery routes in sheep in parallel with this clinical trial and to see if there are any long-term negative effects of the therapy. This project will undertake research to address these important questions. Additionally, we want to get a better understanding of the pathological changes in the brain, eye and spinal cord of affected sheep over the disease course, in order to provide robust, accurate data for translation to humans.

Membrane interaction of small molecules capable of improving Batten disease cell phenotypes

Associate Professor Ronald Clarke
Dr Alvaro Garcia

The CLN3 form of Batten disease is known to be due to a mutation in the CLN3 protein (also known as Battenin) which is located within an intracellular membrane. Recent research on mice which are deficient in the CLN3 protein has shown that the symptoms of their disease are alleviated by the small molecules carbenoloxolone, enoxolone, prednisolone and 7-ketocholesterol. Each of the molecules are steroid- or sterol-like, i.e., similar in structure and chemical behaviour to cholesterol. Although cholesterol is often associated with heart disease, it is a major constituent of animal cell membranes, where it plays an important beneficial role in optimizing the function of vital membrane proteins, including that of the sodium pump, whose activity controls cell volume. Considering the membrane location of CLN3 and the cholesterol-like structures of the molecules found to improve Batten disease symptoms, it appears highly likely that these molecules exert their effect through the membrane, either the membrane directly surrounding the CLN3 protein or a membrane located elsewhere in the cell. The aim of our proposed research is to explore this hypothesis and discover the role that membranes have in influencing the symptoms suffered by Batten’s disease sufferers.

Our research will use a combination of experimental and theoretical approaches to determine the effect of each molecule on electrical properties of membrane, membrane fluidity, the chemical forces within the membrane and both the location and orientation of each molecule within the membrane. By comparing the effect of each molecule on each of these properties with their known effects on Batten disease symptoms in mice, we will discover which membrane property needs to be modified in order to alleviate the symptoms of the disease. As well as investigating the lipid (or fat) component of the cell membranes, we will also study the effect that each molecule has on the model membrane protein, the sodium pump, which also plays a key role in the healthy function of nerve and muscle cells, i.e., cells which are particularly vulnerable in Batten disease. Our research thus represents a fundamental scientific study which will provide a rational foundation on which the design of improved drugs for the treatment of the CLN3 form of Batten disease can be based.