Dr. Robert H. Brown, Jr. University of Massachusetts Medical
Recent work in the Brown Lab has focused on two aspects of HSAN1. One addresses therapy directly, the other indirectly through production of new mouse models. The first line of work, done by an outstanding graduate student, Huiya Yang, has been to suppress expression of one of the key mutant genes that causes HSAN1 – the long chain sub-unit of the serine palmitoyl transferase enzyme (SPTLC1). The underlying concept is that the mutant protein exerts a toxic effect on neurons by forming of a set of toxins, deoxysphingoid bases (DSBs). We believe that reducing the level of the mutant SPTLC1 gene will reduce levels of DSBs and slow or stop the neuropathy. We are exploring two approaches to gene silencing of DSBs. One project targets RNA using a class of compounds known as microRNA. This work has been co-mentored by an outstanding senior faculty member, Dr. Guangping Gao, who directs the Gene Therapy Center at UMass Medical School. Another targets RNA using anti-sense oligonucleotides (ASO); the ASO project has benefitted enormously from collaboration with Dr. Jonathan Watts, a faculty member in the RNA biology unit at UMass Medical. Dr. Yang has focused recently on the microRNA approach. For this, she has generated 24 different microRNAs that target different regions of RNA made from the SPTLC1 gene. She has screened these extensively in a cell line (HEK293T cells). We were pleased to find that many of the 24 candidate microRNAs suppress SPTLC1; four in particular are being advanced for further study. In the HEK293T cells, these four microRNAs suppress not only the SPTLC1 RNA but also the SPTLC1 protein.
One possible risk of this gene suppression therapy is that it may exceed too well. That is, if the chosen microRNA suppresses not only the diseased SPTLC1 gene but also the normal SPTLC1 gene there could in theory be adverse consequences from too little remaining SPTLC1 in cells. We have therefore taken an additional step, in which we silence the innate SPTLC1 gene but simultaneously replace it with an external version of the SPTLC1 gene that is modified so it cannot be attacked by the microRNA. This has required developing the modified SPTLC1 gene that resists the microRNAs. Huiya now has completed initial experiments modifying the SPTLC1 gene and created vectors that contain both the microRNA to turn off the innate SPTLC1 and also the external SPTLC1 gene that resists the microRNA. In pilot cell culture studies, this dual system appears to work well.
As an indirect approach for therapy development, we have undertaken experiments with our mouse core to generate new mouse models of HSAN1. In these new models, our goal is to put the HSAN1-causing mutations into the mouse genes. This differs from the previous models we have published in which we leave the mouse SPTLC1 genes intact but add external, so-called transgenes that have mutant SPTLC1. We are cautiously hopeful that we have at least one new line of mice with a knocked-in HSAN1 mutation. We will continue to work on a set of several mutations over the next months and look forward to a full report on these mice in the near term.
On behalf of all of us in the laboratory, I want to express once again our profound gratitude to the Deater Foundation for its ongoing support. We look forward to continued progress.