May 2016 Update


A 501(c)3 Non-Profit Biomedical Research Company
3050A Berkmar Drive
Charlottesville, VA 22901

James P. Bennett, Jr. M.D. Ph.D.
President and Chief Scientific Officer
434-242-7275 (cell)

Update on our Research

May, 2016


Next-generation RNA sequencing shows that ALS spinal cord has disordered inflammatory signaling

In collaboration with National Disease Resource Interchange (NDRI), a non-profit distributor of disease tissue specimens, and Dr. David Brohawn, a recent Ph.D. graduate in Human Genetics and student of Dr. Bennett, we have established that spinal cords from individuals who died from amyotrophic lateral sclerosis (ALS) show abnormal gene expression profiles that indicated disordered inflammation signaling. We used a relatively new technology called “RNA sequencing” (RNA-seq) that provided levels of all the 20,000+ genes’ RNA’s from each of 7 ALS and 8 CTL, age-matched spinal cord samples.

Doing this research required a substantial investment in bioinformatics approaches. We elected to use several algorithms to identify what are known as “differentially expressed genes”, or DEG’s, that are genes significantly changed in ALS compared to CTL specimens. We used three different algorithms (none is ideal) and determined a consensus set of DEG’s. Determining which cellular processes are associated with these DEG’s revealed that abnormal inflammation signaling stood out.

We then used a completely different network approach to analyze the same primary sequencing data, known as “weighted gene coexpression network analysis” (WGCNA) to identify which genes were significantly associated with ALS. By asking which cell processes were represented by these genes, we came to the same answer- inflammation.

In the brain and spinal cord, inflammation is mediated by molecules secreted by astrocytes and microglia, two types of glial cells that make up the majority of cells in the nervous system. Astrocytes can be “activated” to secrete these inflammatory molecules, and microglia arise from peripheral cells called macrophages that cross into the nervous system and take up residence. Microglia can also be activated by infection, trauma, stroke and other stressors to secrete inflammatory chemicals. It turns out that inflammation markers are present very early in degenerative brain diseases, and there is an emerging consensus that abnormal inflammation signaling may be a genesis event in neurodegeneration.

One of the major inflammatory proteins is known as “tumor necrosis factor”, or TNF. Our programs identified TNF as an upstream mediator of abnormal inflammation signaling. TNF secretion can be reduced by curcumin, a natural product and component of turmeric. TNF secretion can also be reduced by bupropion, a chemical/drug used for decades to treat depression. It is possible to measure TNF in blood serum, as well as parts of TNF receptors.

Thus, these results are highly translational, meaning that drugs can be given to humans to see if TNF and/or TNF receptor levels in serum or spinal fluid can be reduced.  There are other “biomarkers” of inflammation that can also be assayed, resulting in a comprehensive approach that can be applied to therapy development.

Please see the attached preliminary manuscript “RNAseq Analyses Identify Tumor Necrosis Factor-mediated Inflammation as a Major Abnormality in ALS Spinal Cord” for any details:

Microneurotrophins and ALS Therapy

ALS is a cruel disease that kills innocent people rapidly and has no effective treatment to slow its course. Working with another non-profit, ALS Worldwide, and collaborators at other institutions, we have pursued development of microneurotrophins (MNT’s) to treat ALS. MNT’s are small molecule derivatives of the naturally occurring brain steroid dehydroepiadrostenedione (DHEA), that do not possess any of DHEA’s interactions with sex steroid receptors but do interact potently with neurotrophin protein receptors. Neurotrophins (NT’s) are large protein molecules that help neurons develop and maintain resilience to insults.

Thus, MNT’s are small molecule NT agonists and represent a major advance in therapeutics. MNT’s provide many beneficial effects on neurons; their “pleitropic” nature makes them attractive as therapeutics.

Unfortunately we do not have any predictive animal or cell models for ALS. Our collaborators and we have devised novel ways to estimate therapeutic activity of MNT’s. By examining multiple MNT’s in several of these novel assays, we have selected one lead compound to develop further and take before the FDA. We are finalizing all the details and hope to start human studies in late 2016.

Please see attached paper about MNT testing. Note that it also takes advantage of our use of neural stem cells, discussed in a subsequent section.

rhTFAM and Mitochondrial Therapy of Neurodegenerative Diseases

Mitochondrial transcription factor A (TFAM) is a naturally occurring, critical protein necessary for replication of mitochondrial DNA (mtDNA, hundreds-thousands of copies in each cell) and synthesis of mitochondrial RNA’s (mtRNA’s) from mtDNA’s. Recombinant human TFAM (rhTFAM) is natural TFAM protein engineered to pass quickly through cell membranes and rapidly localize to the inside of mitochondria (the “mitochondrial matrix”) where it is changed to natural TFAM. rhTFAM stimulates mitochondrial energy production and cellular synthesis of mitochondrial components.

All major adult degenerative brain diseases show some level of mitochondrial energy deficiency, and rhTFAM has great potential as a modified natural product to restore energy production in the brains and spinal cords of persons afflicted with these diseases. Developing rhTFAM for these purposes requires that special synthesis procedures be followed (“good manufacturing practice”, GMP) and the the resulting very pure rhTFAM be tested for toxicity in animals before being given to humans, using rigorous testing procedures (“good laboratory practice, GLP).  Both GMP and GLP are very expensive, approaching 1 million dollars for each.

rhTFAM is a “shovel-ready” therapeutic, needing only funding for the GMP and GLP parts before being given to humans. It has great potential for restoring mitochondrial function in brains, retinas and muscles of persons with degenerative diseases.

Please see attached papers about rhTFAM that have been done by our group. (First Paper, Second paper)

Adult Stem Cells for Therapy Development

Adult stem cells, in contrast to embryonic stem cells, are made from adult tissues. Initially skin cells were used, but many investigators, including us, use peripheral blood mononuclear cells (PBMC’s) that are transformed to a primitive embryonic state by a process known as genetic reprogramming. Some use viruses to do this reprogramming, we use small electric currents to create microscopic holes in cell membranes so that a small piece of circular DNA, called a plasmid, can enter the cells and begin the reprogramming.

After reprogramming and time in cell culture, the resulting cells are known as “induced pluripotential stem cells”, or iPSC’s. We take the iPSC’s and convert them to neural stem cells (NSC’s), which are like the primitive cells before your brain began to differentiate into nerve cells or glia.

NSC’s are not tumor cells but do reproduce (replicate) in cell culture. By adding special proteins to the culture media, NSC’s can be turned into astrocytes or neurons.

We use NSC’s as a platform for drug development. By introducing plasmids into NSC’s and expressing a DEG (see above) we can determine if the DEG has adverse effects on the NSC. We then can see if a drug ameliorates these adverse DEG effects. If we examine disease-specific DEG’s in this manner, we can test new drugs for their ability to help cells resist adverse events of DEG expression.

If we make the NSC’s from a series of individuals, we can examine the ability of drugs to help (or not) those individuals. Our ultimate goal is to use NSC’s made from individuals to help predict which drugs might be helpful (or not) for that particular person. By this means we can “personalize” medicine recommendations.

This approach is very similar conceptually to identifying drugs that could work against a particular tumor taken from a person.

Please see attached papers about our use of NSC and motor neurons made from NSC’s. (First Paper, Second Paper)

Do iPSC’s Grow Better in a 3-D Matrix?

Traditional cell culture uses 2-dimensional plastic surfaces, whereas the natural environment where our neurons and glia live is 3-dimensional.  Many have wondered whether a more natural state will be created by growing iPSC’s or their derivatives in a 3-D scaffold, to try and approximate the more natural growing conditions.

To that end we are conducting experiments with 2-D compared to 3-D growth conditions. We will use RNA-seq to compare 2-D to 3-D to control brain tissues to attempt to answer this question.

Pending the outcome, we may switch to using 3-D scaffolds for NSC-related drug development and testing.

Attached Papers: