Genetic Therapy Testing

DUX4-DBD Suppression in HEK293T Cells

We tested multiple versions of our genetic therapy in both C2C12 and HEK293T cells. Our tests involved co-transfecting cells with a reporter plasmid, DUX4-fl plasmid, and DUX4-DBD plasmid for competitive inhibition of DUX4-fl. The reporter produced fluorescent proteins at a rate correlated with DUX4-fl binding. As DUX4-DBD concentration increased, DUX4-fl binding and reporter activation decreased across trials.

Figure 1: The mScarlet fluorescence from the DUX4 reporter decreases with increased ratios of DUX4-DBD to DUX4-fl when co-transfected, showing suppressed DUX4-fl binding to the DUX4 reporter’s binding sites.

Figure 2: RFP decreases with decreased activation of the reporter by DUX4-fl while GFP increases with the amount of DUX4-DBD transfected.

Flow Cytometry Data

We performed flow cytometry on HEK293T cells to quantify single-cell fluorescent protein expression. As DUX4-DBD concentrations rose, mScarlet reporter output decreased while GFP expression increased.

Figure 3: B8-.5xfl C1-.5xfl, .5xDBD C6-.5xfl, 5x DBD, C12, .5xfl, 12.5 DBD Green name: 25x DBD:FL ratio Orange name: 10x DBD:FL ratio Red name: 1x DBD:FL ratio Blue name: 0x DBD:FL ratio

C2C12 Cell Line Analysis

A similar trend was observed in C2C12 cells, although transfection efficiency was lower. Fluorescence assays confirmed DUX4-DBD-mediated suppression in these cells, aligning with results from HEK293T assays.

Figure 4: The mScarlet fluorescence decreases with decreased activation of the reporter by DUX4-fl.

Figure 5: RFP decreases with decreased activation of the reporter by DUX4-fl while GFP increases with the amount of DUX4-DBD transfected.

Introduction of KRAB to DUX4-DBD

We modified our DUX4-DBD plasmid by adding the KRAB repressor domain, which recruits chromatin-modifying proteins. In HEK293T cells, the DBD-KRAB construct further suppressed reporter activation compared to DUX4-DBD alone.

Figure 6: The mScarlet fluorescence decreases with decreased activation of the reporter by DUX4-fl. The DUX4-DBD + Krab construct represses reporter activation via DUX4-fl at a higher rate than the DUX4-DBD construct.

Future Plans

Post Wiki freeze, we aim to validate DUX4-DBD+KRAB in C2C12 cells with additional technical replicates. We also plan to test the construct in FSHD patient-derived cell lines and in vivo models using AAV gene therapy.