Research at Day Lab

Cecil B. Day Laboratory for Neuromuscular Research

The Cecil B. Day Laboratory for Neuromuscular Research was founded in 1983 to investigate genetic defects that cause neuromuscular paralysis. The focus of the initial investigations was a rare form of muscular dystrophy known as Miyoshi myopathy. A second disease that has been extensively studied in the Day Lab is Lou Gehrig’s Disease, or amyotrophic lateral sclerosis (ALS). Other research topics include periodic paralysis, a sensory-motor neuropathy (HSN1) and a form of adrenoleukodystrophy (Lorenzo’s Oil Disease). In each disease category, the initial research goal has been to identify primary gene defects that can cause these disorders. In each case, investigators in the Day Laboratory have been fortunate to participate in the discovery of underlying gene defects. The laboratory is now generating animal and Petri dish models of each disease as a first step toward developing treatments.

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Development of RNAi Therapy for the treatment of amyotrophic lateral sclerosis (ALS)

Dr. Robert Brown, Professor and Chair, Department of Neurology is one of the world’s authorities on ALS. Dr. Brown’s laboratory identified one of the genes associated with ALS, cytosolic superoxide dismutase, or SOD1. Current views suggest that the mutation of SOD1 causes misfolding of the SOD1 protein and resulting toxicity to the neuron. 

Recent studies by Dr. Zuoshang Xu, Professor of Biochemistry and Molecular Pharmacology have demonstrated that RNAi molecules can silence mutant SOD1 in vivo and increase survival time in the mouse model of ALS. Drs. Brown and Xu have collaborated in the design of RNAi molecules for a trial in humans with mutant SOD1 associated ALS. First in human studies of RNAi in ALS are currently underway at UMass.

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Research update from Dr. Robert H. Brown, Jr. – July 2015

dr brown
Progress Report – ALS Investigations

U Mass Medical Center July 2015

The ALS laboratories at the U Mass Medical Center have made progress on several fronts, including therapy development, discovering some new ALS genes, and generating new animal models of ALS based on ALS genes.

I. Therapeutic Trials

A. Silencing Genes

1. SOD1. We have continued our studies to find ways to silence genes whose mutations cause ALS. Our most intensive effort has focused on silencing the SOD1 gene. Over the years we and others have documented that this gene can be effectively silenced in a Petri dish. Late last year, we published a report of studies from the laboratory of Zuoshang Xu here showing that this gene can be silenced in adult ALS mice, significantly prolonging survival. More recently, we have worked closely with Loreli Stoica in the laboratory of Miguel Sena-Esteves to document that by treating the ALS mice within a day of birth, one can delay the appearance of motor neuron disease for several months.

In parallel with these mouse studies, we have begun to test efficacy of the same intervention in large animals. With Florey Borel in the laboratory of Chris Mueller, we have been able to deliver the silencing reagents to the spinal cord and brains of monkeys. The delivery is achieved using a virus (adeno-associated virus or AAV) that has a remarkable capacity to permeate the central nervous system. We are fortunate in this regard to collaborate with the UMass Gene Therapy laboratory, headed by Dr. Guangping Gao, who is an expert in the biology of AAV. Using AAV as a delivery vehicle, we find that in some monkeys we achieve excellent permeation into the tissues of the brain and spinal cord and thereby also induce excellent silencing of the SOD1 gene.

These data have encouraged us to outline a pathway to a human trial. Toward that end, we have begun to work closely with colleagues (James Berry, Merit Cudkowicz) at the Massachusetts General Hospital to design the format for such a human trial. The transition from laboratory-based studies to a human trial is lengthy and costly, but the steps are clearly defined. We are extremely grateful to the Angel Fund for its commitment to this program; Angel Fund support has been absolutely pivotal in moving us toward this human trial of gene silencing. It should be noted that if these proof-of-concept studies are successful with the SOD1 gene, the path is paved for analogous trials with many other

ALS genes (and indeed for a variety of toxic mutant genes that cause other diseases).

2. Other approaches to gene silencing. Given the early promise of the SOD1 program, we are also embarking on two related programs. (1) We have now generated reagents that allow us to silence the most common ALS gene, known as C9orf72 or simply C9. Because the initial studies in cell cultures were promising, we have begun to use the AAV system to silence the C9 gene in mouse brain and spinal cord. This work is conducted by Gaby Toro with Chris Mueller. (2) We are continuing efforts to develop silencing reagents that do not require the use of a virus for delivery. These are built on small strands of DNA that target the genes we want to silence. These DNA-based molecules are chemically modified to enhance their stability and at the same time to facilitate their movement into the spinal cord. These studies, done by Havisha Karnam under the direction of Anastasia Khvorova, have produced a set of small DNA molecules that silence the target genes in cell culture and also show a capacity to permeate the nervous system. Testing underway now will ascertain

whether or not the target gene is silenced in the spinal cord.

B. Stem cell therapy trial

We are now into the first year of a two year trial of humN stem cells in ALS patients. This study, coming from the Israeli company Brainstorm Cell Therapeutics, uses bone marrow derived stem cells.

The patient’s bone marrow is harvested, the stem cells are prepared over one month in a cell culture laboratory, and the cells are then injected back into patients, via both the spinal fluid

and one arm muscle. This study is being conducted jointly with the Massachusetts General Hospital and the Mayo Clinic. The purpose of the study is to test safety of this approach. Of course, as in any safety study, we are hopeful that we WILL also see evidence of a clinical benefit. The full assessment, which will await follow-up of the last patients enrolled, will probably be available by mid-2016.

II. Gene Discovery in ALS

A. Profilin-1. As reported previously, Dr. John Landers (a former Angel Fund fellow) identified a new ALS gene called profilin-1. This was identified in a large family in Israel and France, studied

by Bob Brown along with clinicians from Israel. Dr. Landers and others have now documented that mutations in the profilin-1 gene can be detected in several families in the U.S. and Europe.

Several other laboratories have now robustly confirmed John’s observations.

B. Tubulin-4A. With Dr. Lander’s team, we have also identified another ALS gene, tubulin- 4A, which is mutated in a small subset of familial ALS cases. This is of interest because the gene, and the

protein it makes, are implicated in processes that control how well a nerve extends its axonal process to connect with other neurons or muscle. We are intrigued that these biological processes

implicated by the tubulin-4A finding are similar to those implicated by profilin-1.

C. TANK Binding Kinase-1 (TBK-1). Over the several years, Dr. Brown and Landers have been involved in several large consortia combining large sets of DNA from ALS cases, including non-familial (sporadic) ALS, to use high throughput DNA sequencing methods to search for new ALS genes. One

consortium, funded and coordinated Biogen, Inc., studied ~2,800 sporadic ALS cases and more than 6,000 controls, identifying a new ALS gene (TBK-1). The importance of this gene is still under

investigation, but early studies indicated that it interrelates with other ALS genes and thus begins to define another network of ALS genes.

D. ERB4. As part of a collaboration headed by colleagues in Japan, we have identified a small subset of ALS patients whose disease reflects mutations in a gene that makes a protein known as

receptor tyrosine kinase ERB4. The biological significance of this finding remains to be defined. However, it is already of interest that this gene is central importance in several fundamental

cellular processes that can involve nerve cells, including differentiation.

III. New ALS Models – in Animals and Stem Cells

A. C9orf72. Among the most useful tools in studies of any disease are cell and animal models of the disease. These permit one to test hypothesis about how the disease evolves and how it can be

treated. Several of us at UMMS have collaborated to generate a new mouse model of the C9orf72 gene defect that, as noted above, is the most commonly mutated ALS gene. Because these mice reproduce many of the molecular features of C9orf72-related ALS, we are hopeful that they will be valuable in the analysis of C9orf72. We already have begun preliminary treatment trials in mice using this model.

B. TDP43.  We have also been engaged in studying models of another ALS gene known as TDP43. Zuoshang Xu has generated a line of mice in which the activity of the TDP43 is fractionally

reduced. We were surprised to find that with no other molecular change these mice develop an adult-onset lethal paralysis, suggesting that one of the ways the TDP43 mutations cause ALS is by partially blunting the activity of this gene. In other studies, Jemeen Sreedharan has worked with Marc Freeman to develop a fruit-fly model of TDP43. They have then exploited the power of genetics in the fruit fly to discover new mutations that block this, thereby defining new pathways involved in TDP43 ALS. We are hopeful that knowledge of these pathways will provide new targets for therapy.

This is particularly important because the TDP43 protein has been implicated in many cases of ALS and a related disorder, frontotemporal dementia (FTD).

IV.   Related Studies of ALS Biology.

A. Axonal Transport in ALS. As described previously, we have continued to be interested in the possibility that mutant ALS genes may be toxic in part because they disturb the process of axonal transport in motor neurons. This is the mechanism whereby substances produced in the cell body of a motor neuron can be carried along the lengthy motor neuron process (the axon) to reach the distal terminal where the motor neuron contacts muscle and activates muscle contraction. In collaborations

with Drs. Scott Brady and Gerardo Morfini, Dr. Daryl Bosco and I have found that mutant SOD1 protein is toxic to the molecular apparatus that governs axonal transport. These studies were

performed at the Woods Hole Marine Biological Laboratories with Dr. Scott Brady, who has studied axonal transport in the squid giant axon for years. We are now attempting to find ways to relate

these studies in squid to measuring and modifying axonal transport in humans.

B. Stem Cell Biology and ALS. Several of us are now using stem cells to study the biology of different aspects of ALS. It is striking that one can now generate stem cells from skin cells of a human. This has allowed investigators like Dr. Fen-Biao Gao at UMMS and Dr. Kevin Eggan at Harvard,

to generate multiple lines of stem cells from a diversity of different ALS patients. With these, one can then generate motor neurons, meaning that one can now study in cell culture motor neurons

from living patients. Dr. Gao has done pivotal work using such stem cells to probe many aspects of ALS and fronto-temporal

dementia; he was the first person to make a tissue culture model of C9orf72 ALS-FTD. He has used these cultures to study many phenomena including toxic proteins made bythe C9orf72 gene and a type of regulatory RNA known as microRNA. Dr. Eggan has

used cells provided from the UMMS ALS clinic and elsewhere to develop stem cell models of ALS with which he has demonstrated a propensity toward enhanced electrical activity, consistent with the longstanding view that motor nerve hyperexcitability is one of the underpinnings of ALS. We have studied approaches to diminishing this using a drug known as mexilitine. Dr. Eggan and

collaborators have found that a different drug, retigabine, is also effective in suppressing motor neuron hyperexcitability. It is exciting that both mexilitine and retigabine are now being put into

trials in human ALS.

As the above documents, the ALS laboratories at U Mass Medical School are making significant progress in understanding ALS. While we are fortunate to receive funding from several sources, both federal and private, the funding provided by the Angel Fund is truly a cornerstone for our investigations; this is particularly true for the gene therapy and gene silencing program. All of

us in ALS research here are extremely grateful for the remarkably generous support provided by the

Angel Fund; we are optimistic that this will have a transformative impact as we all work toward finding a cure for this disease.

Respectfully submitted,

Robert H. Brown, Jr., D.Phil., M.D.

AF-report-12July2015

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