Progress Update: 3rd Gen Oligonucleotide Targeting DUX4 in Mouse Model

by Yi-Wen Chen
See grant: Investigation of a third generation oligonucleotide targeting DUX4 using a new mouse model of FSHD
Updated 14-Feb-2018 (added more comprehensive information)

This report summarizes our research activities in the first six months supported by the Friends of FSH Research. Based on the data collected from our in vitro studies, we selected three of the 3GAs prescreened by Idera Pharmaceutical for additional in vivo characterizations. The selected 3GAs most efficiently suppressed DUX4 expression in immortalized human FSHD myoblasts and corrected the myogenic defects in FSHD myoblasts. The in vivo efficacy of the 3GAs was evaluated using the FLExDUX4 mouse model created by collaborator, Dr. Peter Jones (Jones & Jones, 2018). The first study was to show that the 3GAs could enter muscles after subcutaneous injections (s.c.). Fluorescein tagged 3GA (3GA-F) was manufactured by Idera Pharmaceutical and shipped to us. The FLExDUX4 mice received one s.c. injection of 3GA-F. Twenty-four hours after the injection, we collected and snap-froze muscles (tibialis anterior, gastrocnemius, soleus, quadriceps, bicep, triceps, deltoid, masseter, and diaphragm muscles) and other organs (livers and kidneys). The tibialis anterior (TA) muscles, livers and kidneys were cryosectioned (5μm) to determine the localization of the 3GA-F. The muscle sections were also immunofluorescently stained with anti-dystrophin antibodies to visualize the sarcolemmal membrane. Our results showed that the 3GA-F was found in all 3 organs. In the TA muscles, the 3GA-F was found to be localized inside myofibers and in the extracellular matrix. With one injection, we did not see suppressive effects on DUX4 mRNA within 24 hours as expected.

To increase the amount of 3GAs in the muscles, we conducted a study with six s.c. injections (one injection every other day) of one of the 3GAs (3GA-2) which performed the best in our preliminary studies. Three weeks old male FLExDUX4 mice (n=4) received s.c. injections every other day for six injections. Control FLExDUX4 mice (n=4) received s.c. injections of phosphate-buffered saline (PBS). We also collected samples from four wildtype littermates to show baseline expression levels of DUX4 (no expression) and downstream genes. Muscles were collected 24 hours after the last injection. Our data showed significant knockdown of DUX4 in the triceps examined (42% reduction).

Two challenges we have encountered during this period of study. The first is the very low expression of DUX4 in the FLExDUX4 mice. Both Dr. Jones’ and our groups observed that the well-known DUX4 downstream genes, such as mouse Zcan4c and Trim36, were not up-regulated by this level of DUX4 in the model, therefore we have to rely on the expression of DUX4 solely for efficacy evaluation. To allow easier assessments, we are considering use FLExDUX4/ACTA1-Cre mice for the next experiments. Without transgene induction, the FLExD/ACTA1-Cre mice leak DUX4 at a slightly higher level, which induces the downstream genes. The reduction of the downstream genes can be used to confirm the DUX4 knockdown. The second challenge is that the founder of the Idera Pharmaceutical recently retired and the reorganization of the company delayed the production of 3GAs. The problem is recently resolved and the company is currently synthesizing large quantity of 3GAs for our long term trial. We will confirm the effect of DUX4 suppression and evaluate functional improvement in the FLExDUX4 mice. We have observed both pathological changes in the muscles and muscle functional deficits in the FLExDUX4 mice. We anticipate that the treatments will reverse these pathological effects of DUX4.