We are very pleased to provide this update on progress toward devising methods to silence the SPTLC1 gene as a potential therapy for HSAN1. We have made progress in three areas. The key point is that we have now developed two types of reagents that can silence the SPTLC1 gene.
One type of reagent is composed of small strings of ~ 20 molecules of nucleic acids (antisense oligonucleotides or ASO’s) that have sequences complementary to specific sequences in the SPTLC1 gene. The concept is that these ASO’s bind RNA from the target gene and thereby activate enzymes that break-up the RNA, preventing it from making protein. As we have reported before, we have been fortunate to have Havisha Karnam as a graduate student working in conjunction with Anastasia Khvorova Ph.D. on this project. Dr. Khvorova is an internationally recognized expert in the chemistry of ASO’s. With Dr. Khvorova’s guidance, Havisha has used two types of chemistry (designated LNA gapmers and hsiRNA) to generate ASO’s that can silence SPTLC1. In particular, she has developed ASO’s that specifically target hamster SPTLC1 and not mouse, and reciprocally. She also now has ASO’s that target human SPTLC1. We need ASO’s that target hamster and not mouse because as you recall our mouse model of HSAN1 has the HSAN1 mutations in a hamster transgene added to each cell above and beyond the normal mouse SPTLC1 gene. In our last report, Havisha had made good progress in developing these reagents. However, over the last four months she has obtained optimized reagents and, most recently, has shown she can use them to shut off the hamster SPTLC1 gene (but as desired not the mouse gene) in neuronal cultures derived from the transgenic HSAN1 mice. As a positive control, Havisha has also shown that she can silence other genes in the spinal cords of normal mice (such as the normal huntingtin gene). We are very excited with this development because this is an important step toward testing anti-SPTLC1 ASO’S directly in the HSAN1 mice. These ASO’s are intended to be given into the spinal fluid. In patients, this would likely require multiple doses each year via LP.
The second type of reagent we have developed are microRNAs (miRs), the elements we described to you in our proposal last fall. These are very much like ASO’s except that these small strings of nucleic acids are made up of RNA (while the ASO’s above are made of hybrids of RNA and DNA, with chemical modifications). The miRs have been developed in conjunction with Dr. Chris Mueller, Dr. Li Yi and another graduate student, Gabrielle Toro. For this project, they have developed some new assays (using a technique called digital PCR) to be able to quantify with precision the levels individually of mouse, hamster and human SPTLC1 RNAs. In parallel they have also generated two miRs that target human SPTLC1 and one that targets hamster SPTLC1. They have also taken two of the reagents developed by Havisha (above) to target the hamster SPTLC1 and converted them from LNA gapmer and hsiRNA chemistries to make miRs. These new miRs against hamster and human have been devised in a form that let’s us put them into a virus commonly used by Chris Mueller, known as adeno-associated virus (AAV). (In fact, the head of the Gene Therapy Center at UMass patented scores of these AAVs). We and others have found that with two newer types of AAV (labeled AAV9 and AAVrh10) one can get excellent penetration of AAV into the spinal cord and brain, and thereby have the cargo carried by AAV released into the interior of brain and spinal.
Our goal in the next 2-3 months is to complete the process of packaging our new anti-SPTLC1 miRs
into the AAV and to use this system to treat our HSAN1 mice. In the long term, one hopes that this will be a useful clinical strategy in people. A particular advantage of AAV is that when it delivers new genes or miRs to the brain and spinal cord tissues, it permits extremely long-term (many years) expression of the cargo gene or miR. This means that, by contrast with the ASO therapy above, this AAV-mediated delivery of the anti-SPTLC1 miR could potentially result in years of treatment from a single injection.
Some describe this as “one and done”. A downside of this approach is that there is no way to retrieve the virus or turn off its cargo once it is delivered. These points not withstanding, our view is that this is a powerful therapeutic approach that warrants testing in any human disease in which a mutant gene is somehow adverse or toxic.
On a third front, a post-doc in my lab is also working on aspects of HSAN1 as one of two projects. Her goal is to get the assay for the deoxysphingoid bases (DSB) up and running here at UMass. We have been greatly assisted in this endeavor by Thorsten Hornemann who has provided protocols and details. Importantly, our plan is to test some samples that he also tests, so that we can be assured that we are measuring DSB levels identically to his lab. Hirosha is a careful scientist who is well on the way to getting this method running. This will help us enormously as we do more testing of our silencing therapies in the HSAN1 mice.
On a parallel note, I want to mention that we have been extremely fortunate to have a very capable
technician running our HSAN1 mouse colony. It is robust in numbers, suggesting that we should be in an excellent position to test the above reagents. I anticipate that the mouse-based testing will begin in the next 2-3 months.
On behalf of everyone here working on HSAN1, I want to offer my most sincere and heartfelt thanks for your support for our work. It has been quite consequential as we pursue the above two approaches to silencing the SPTLC1 gene. Also, we want to thank you for once again sponsoring the HSAN1 conference in Cambridge this spring. As before, it was scientifically illuminating and ultimately very motivating.
I look forward to remaining in close touch on all of these issues.
Dr. Robert H. Brown, MD, DPhil