[BobbyB]

ALS TDF Results Well Received at the Yokohama ALS/MND Conference

John McCarty, PhD
12/19/2006

The Motor Neurone Disease Association’s 17th Annual International Symposium on ALS/MND was held this year in Yokohama, Japan and is the major conference in the world on topics of research, treatment and care in ALS. As usual, ALS TDF sent a team of researchers to attend the lectures and to meet with other scientists and clinicians (our collective impressions will be posted as a report in the next weeks). This year, however, two of our researchers – Sean Scott and Jerry De Zutter – also made significant presentations as part of session 7A: Interpretation of Data from SOD1 Models.

This session centered on the critical question of how to optimally use the ALS mouse – based on the SOD1 G93A mutation. Other talks included a discussion on information potentially gained by studying the different mouse background strains and the results from the ENMC (European Neuromuscular Centre) workshop that set guidelines for using the ALS mouse as preclinical studies to support clinical trials in humans.

The information presented by Sean Scott, VP of Drug Development at ALS TDF, had the potential to be controversial. Data generated at TDF in the last 4 years working with the ALS mouse has established that a number of preclinical studies with some high profile drug candidates could not be repeated. Statistical analysis at TDF, using our rich database of ALS mouse data, demonstrated that unless the studies were tightly controlled, noise in the experimental system would swamp most signals and could be interpreted as a positive result.

The conclusions reached by the TDF research team are currently under review for publication as a paper that should serve as a benchmark for ALS researchers. Furthermore, the conclusions were also incorporated at the ENMC Workshop (held earlier in 2006); their report was the subject of the last talk of the 7A session from Dr. Ludolph of the University of Ulm in Germany. Overall, the conclusions that the ALS mouse model can be informative but that studies need to be much more rigorously performed to be used as preclinical support for drug trials were well accepted and applauded by the audience of ALS researchers.

Jerry De Zutter’s talk was also met with interest and enthusiasm as he reported on TDF’s preliminary studies designed to increase information flow from ALS mouse studies. Typical mouse studies rely on large scale observations such as weight, functional muscle use and survival to gauge efficacy and – due to system noise – require fairly large numbers of mice for an interpretable signal. A major initiative at TDF has been to identify molecular-based markers to report disease progression. Such markers would reflect better the events occurring in cells as the disease takes hold – thus potentially yielding substantially more information and hopefully with more streamlined studies.

To date, Dr. De Zutter, Director of Gene Expression Core, working with his colleagues have identified a panel of genes that are significantly altered in response to the disease in the mouse. TDF will continue to identify more such genes and to establish their use in monitoring drug effects in the ALS mouse as TDF continues the critical mission of evaluating drugs to support ALS clinical trials.

The titles and abstracts of the presentations from Sean and Jerry can be found below as well as in the following link:

http://www.mndassociation.org/resear...ract_book.html

C38 DESIGN, POWER AND INTERPRETATION OF STUDIES IN THE STANDARD MURINE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
SCOTT S, KELLY N, KRANZ J, LINCECUM J, RAMASUBBU J, VIEIRA F, HEYWOOD J
ALS Therapy Development Foundation, Cambridge,MA, USA

Identification of SOD1 as the mutated protein in a significant subset of familial amyotrophic lateral sclerosis (FALS) cases has led to the generation of transgenic rodent models of autosomal dominant SOD1 FALS. Mice carrying 23 copies of the human SOD1G93A SOD1G93A transgene are considered the standard model for FALS and ALS therapeutic studies. To date, there have been at least 50 publications describing therapeutic agents that extend the lifespan of this mouse. However, no therapeutic agent has shown corresponding, substantial, clinical efficacy. We used computer modelling and statistical analysis of 5429 SOD1G93A mice from our efficacy studies to identify several critical confounding biological variables that must be appreciated and should be controlled for when designing and interpreting efficacy studies. Having identified these biological variables we subsequently instituted parameters for optimal study design in the SOD1G93A mouse model. We repeated several of the major animal studies (minocycline, creatine, ritonavir, celecoxib, sodium phenyl butyrate, ceftriaxone, WHI-P131, thalidomide, and riluzole) using an optimal study design and found no survival benefit in the SOD1G93A mouse for any compounds administered by their previously reported routes and doses. The presence of these uncontrolled confounding variables in the screening system, and the failure of these drugs to demonstrate efficacy in adequately designed and powered repeat studies, leads us to conclude that the majority of published effects are most likely measurements of noise in the distribution of survival means as opposed to actual drug effect.

C39 AN ARRAY-BASED APPROACH TO IDENTIFY BIOMARKERS FOR ALS
DE ZUTTER J, LINCECUM J, VIEIRA F, WANG M, SCOTT S
ALS Therapy Development Foundation, Cambridge, MA, USA

Background: The SOD1 G93A transgenic mouse model is the standard tool for the investigation of new therapeutic candidates for ALS. These transgenic mice are asymptomatic at 60 days of age, and display progressively worsening symptoms from approximately 90 days until death, which occurs on average at 136 +/-10 days. Our studies suggest that a minimum of 24 animals are required per group because of variability in the survival of the animal. As such, it would be highly desirable to have a surrogate endpoint that could facilitate drug screening using fewer animals per group.

Objective: The primary goal of this study is to establish a panel of biomarkers for disease progression in the SOD1 G93A mouse. In achieving this goal we will further our understanding of the molecular mechanisms in ALS and also generate a diagnostic tool for evaluating therapeutic candidates. In addition, if a disease progression-modifying compound also induces altered expression levels of a particular gene, then a target validation approach will be initiated to determine if that gene and/or gene product is a
therapeutic target.

Method: We analyzed spinal cord tissue from SOD1 G93A mice at various time points during the mouse lifespan. We used a real time-quantitative PCR based, targeted array approach to systematically examine expression levels of functionally categorized genes. More focused RT-qPCR assays were performed to confirm expression changes observed by the array approach.

Results: Over the time course examined, we detected increasing RNA levels of: the glial marker Gfap (4.1-fold), proteasome subunits Psmb8 and Psmb9 (5.8- and 3.8- fold, respectively), apoptosis associated cyclinD1 (3.7- fold), and inflammatory mediators Tnf alpha (9.5-fold), Trem2 (11.6-fold) and Tyrobp (12.6-fold), among others. Decreasing RNA levels were measured for, among others: the neuronal markers NSE/Eno2 and NF-M/Nef3 (2.0- and 3.1-fold, respectively), the molecular motor subunits Kif3 alpha and Dnchc1 (1.7-fold and 2.3-fold, respectively). The genes, whose RNA reproducibly changed over the SOD1 G93A mouse lifespan, were categorized as progressively changing (over the course of disease) or late stage (changed at or near end stage of disease). To assemble the biomarker panel we included or excluded genes based on their temporal expression. To follow up results that suggest a gene’s product is a target for therapeutic intervention, immunoblotting, ELISA and in situ hybridization experiments were performed to examine protein levels of genes of interest.

Conclusion: The end product of this effort is a panel of genes which may be used as a diagnostic tool to monitor disease progression in the SOD1 G93A mouse and to examine the effects of therapeutic candidates on that progression. By-products of this work are potential targets for therapeutic intervention in ALS.