FORCE platform overcomes barriers of oligonucleotide delivery to muscle and corrects myotonic dystrophy features in preclinical models

Abstract

Background: We developed the FORCE^TM platform to overcome limitations of oligonucleotide delivery to muscle and enable their applicability to neuromuscular disorders. The platform consists of an antigen-binding fragment, highly specific for the human transferrin receptor 1 (TfR1), conjugated to an oligonucleotide via a cleavable valine-citrulline linker. Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by expanded CUG triplets in the DMPK RNA, which sequester splicing proteins in the nucleus, lead to spliceopathy, and drive disease progression.

Methods: Multiple surrogate conjugates were generated to characterize the FORCE platform. DYNE-101 is the conjugate designed to target DMPK and correct spliceopathy for the treatment of DM1. HSA^LR and TfR1^hu/mu;DMSXL^Tg/Tg mice were used as models of myotonic dystrophy, the latter expresses human TfR1 and a human DMPK RNA with >1,000 CUG repeats. Cynomolgus monkeys were used to determine translatability of DYNE-101 pharmacology to higher species.

Results: In HSA^LR mice, a surrogate FORCE conjugate achieves durable correction of spliceopathy and improves myotonia to a greater extent than unconjugated ASO. In patient-derived myoblasts, DYNE-101 reduces DMPK RNA and nuclear foci, consequently improving spliceopathy. In TfR1^hu/mu;DMSXL^Tg/Tg mice, DYNE-101 reduces mutant DMPK RNA in muscle, thereby correcting splicing. Reduction of DMPK foci in cardiomyocyte nuclei accompanies these effects. Low monthly dosing of DYNE-101 in TfR1^hu/mu;DMSXL^WT/Tg mice or cynomolgus monkeys leads to a profound reduction of DMPK expression in muscle.

Conclusions: These data validate FORCE as a drug delivery platform and support the notion that DM1 may be treatable with low and infrequent dosing of DYNE-101.

Fig. 1. FDC reduces WT Dmpk expression in mouse skeletal muscle to a greater extent than ADC and enhances ASO delivery via a TfR1-mediated mechanism.

WT male mice were administered by tail IV injection on day 0 with 10 mg/kg ASO-equivalents of a FDC4 (blue bars), ADC1 (green bars) or vehicle (gray bars), or b ASO1 (red bars), FDC5 (teal bars), and FDC4 (blue bars), and sacrificed on day 14 for analysis of gene expression. Total RNA extracted from the indicated muscles was reverse-transcribed and Dmpk RNA expression was measured by qPCR using Ppib as the reference gene. Expression in each muscle from mice injected with FDC4 or ADC1 was normalized to Dmpk expression in the corresponding muscle of mice administered with vehicle. Data are means + SD; data were analyzed by one-way ANOVA followed by uncorrected Fisher’s LSD test, statistically significant differences from vehicle are indicated with exact P values. Each circle represents an individual mouse.

Fig. 2. FAB02 maintains monomeric structure under thermal and chemical stress conditions.

Representative size exclusion-ultra performance liquid chromatography traces from 10 µL injections of 1.0 mg/ml FAB02 native or previously subjected to stressed conditions. Thermal stress was evaluated by freeze/thaw cycles (−80 °C to room temperature (RT)) (a) and to high temperature (50 °C) (b). Chemical stress consisted of forced oxidation with 0.5% hydrogen peroxide (c) and by exposure to 1% ammonium bicarbonate (d) at RT at the indicated time points.

Fig. 3. Internalization of FAB02 is mediated by TfR1.

Representative images of WT and TfR1^−/− HeLa cells (a) treated for 4 h with FAB02 labeled with AlexaFluor 647 (pseudocolored to green). Cells were stained with wheat germ agglutinin labeled with AlexaFlour 488 (membrane, pseudocolored to red,) and DAPI (nuclei). White bars within images indicate size. b WT (blue bars) and TfR1^−/− (black bars) HeLa cells were treated for 24 h with FAB02 labeled with a pH-sensitive dye (CypHer5E). Cells were harvested and analyzed by flow cytometry for median fluorescent intensity normalized to the degree of labeling (Dye/Ab). Data are medians + SD. Each circle represents a technical replicate from two independent experiments. c Representative images of human cardiomyocytes treated for 30 min with FAB02 labeled with AlexaFluor 647 (pseudocolored to green), fixed, stained with DAPI (nuclei) and RAB5 (30 min sample, red). The image with merged AlexaFluor 647, RAB5, DAPI signals is an enlargement of the white boxed area. White bars within images indicate size.

Fig. 4. FDC1 suppresses Dmpk mRNA in skeletal and cardiac muscle of TfR1^hu/mu mice to a greater extent than ASO1.

TfR1^hu/mu mice were injected via tail vein on days 0 and 7 with vehicle (gray bars), 10 mg/kg ASO1 (red bars), or FDC1 delivering 10 mg/kg ASO-equivalent of ASO1 (blue bars) and sacrificed 14 days later for analysis of gene expression. Total RNA extracted from the indicated muscles was reverse-transcribed and Dmpk RNA expression was measured by qPCR using Ppib as the reference gene. Expression in each muscle from mice treated with FDC1 or ASO1 was normalized to Dmpk expression in the corresponding muscle of mice treated with vehicle. Data are means + SD; data were analyzed by one-way ANOVA followed by uncorrected Fisher’s LSD test, statistically significant differences from vehicle or between ASO1 and FDC1 are indicated with exact P values. Each circle represents an individual mouse.

Fig. 5. FDC2 displays superior efficacy compared to unconjugated ASO2 in HSA^LR mice.

HSA^LR mice were injected IV with vehicle (black bars/circles), 10 or 20 mg/kg unconjugated ASO2 (red bars/circles), or 10 or 20 mg/kg ASO-equivalent doses of FDC2 (blue bars/circles) and sacrificed 28 days later. a ACTA1 RNA expression measured by RT-qPCR of total RNA from quadriceps (top) or gastrocnemius (bottom) using Gtf2b as the reference gene. Expression in each muscle was normalized to ACTA1 expression in the corresponding muscle of mice injected with vehicle. Data are means + SD; data were analyzed by one-way ANOVA followed by uncorrected Fisher’s LSD test, statistically significant differences from vehicle are indicated with exact P values. Each circle represents an individual mouse. b Overall splicing derangement calculated using murine DM splicing index, horizontal lines are means ± SD, data were analyzed by one-way ANOVA followed by uncorrected Fisher’s LSD test, statistically significant differences from vehicle are indicated with exact P values. Each circle represents an individual mouse. WT splicing data (green line) previously published. c Grading of EMG myotonic discharges. Data are means + SEM; data were analyzed by Kruskal–Wallis test with Dunn’s multiple comparisons test, statistically significant differences from vehicle are indicated with exact P values. Each circle represents an individual mouse.

Fig. 6. DYNE-101 reduces mutant human DMPK RNA expression in the nucleus and corrects splicing in muscle of a DM1 mouse model.

a TfR1;DMSXL mice enable assessment of DYNE-101 ability to target mutant human DMPK RNA via TfR1-mediated uptake. b DMPK, Malat1, Birc5, and Gapdh expression measured by RT-qPCR in total RNA from nuclear (N) and cytoplasmic (C) fractions from gastrocnemius of TfR1^hu/mu;DMSXL^WT/Tg mice. Data are means +  SD. Each circle represents independent measurements except for analysis of Birc5 and GAPDH expression, where n = 1. c DMPK expression in fractions from gastrocnemius of TfR1^hu/mu;DMSXL^WT/Tg mice administered with 10 mg/kg ASO-equivalent of DYNE-101 (blue bars) relative to mice injected with vehicle (black bars). Data are means + SD. Each circle represents independent measurements. d DMPK expression measured by RT-qPCR in total RNA from muscles of TfR1^hu/mu;DMSXL^Tg/Tg female mice administered weekly with 10 mg/kg ASO-equivalent of DYNE-101 (blue bars), 28 days after initial injection, relative to expression in corresponding muscles of mice injected with vehicle (black bars). Data are means + SD; data were analyzed by unpaired t test with Welch’s correction, statistically significant differences from vehicle are indicated with exact P values. Each circle represents an individual mouse. e Representative images of nuclei (blue) and DMPK foci (red) in cardiomyocytes used to calculate foci area. Insets display myofibrils (gray) in greyscale. Total DMPK foci area in heart of TfR1^hu/mu;DMSXL^Tg/Tg mice 28 days after initial injection. Data are means + SD, analyzed by unpaired t test: *P < 0.05. Each circle represents an individual mouse. f Composite splicing index in muscle of TfR1^hu/mu mice injected with vehicle (gray circle) or TfR1^hu/mu;DMSXL^Tg/Tg mice injected with vehicle (black circle) or DYNE-101 (blue circle), 28 days after initial injection. Horizontal lines are means ± SD; data were analyzed by one-way ANOVA followed by uncorrected Fisher’s LSD test, statistically significant differences from TfR1^hu/mu mice injected vehicle or between TfR1^hu/mu;DMSXL^Tg/Tg mice treated with vehicle and DYNE-101 are indicated with exact P values. Each circle represents an individual mouse.

Fig. 7. DYNE−101 low repeat monthly dosing reduces DMPK expression in muscle of TfR1^hu/mu;DMSXL^WT/Tg mice.

Mutant human DMPK expression measured by RT-qPCR in muscle of TfR1^hu/mu;DMSXL^WT/Tg male and female mice a 1 month after a single IV injection with DYNE−101 (blue bars) or b 1 month following the last of 4 monthly doses at the indicated ASO-equivalent doses (blue bars), relative to expression in the same muscles of mice injected with vehicle (black bars). Data are means + SD; data were analyzed by one-way ANOVA followed by uncorrected Fisher’s LSD test, statistically significant differences from vehicle or between DYNE-101 dose levels are indicated with exact P values. Each circle represents an individual mouse.

Fig. 8. DYNE−101 low repeat monthly dosing reduces WT DMPK expression in the muscle of cynomolgus monkeys.

WT DMPK expression in muscle of male cynomolgus monkeys a 4 weeks after a single IV infusion with DYNE−101 (blue bars) or b or 4 weeks following the last of 2 monthly infusions (blue bars), relative to expression in the same muscle of monkeys infused with vehicle (gray bars), shown for each ASO-equivalent dose. Data are means + SD; data were analyzed by one-way ANOVA followed by uncorrected Fisher’s LSD test, statistically significant differences from vehicle are indicated with exact P values. Each circle represents an individual monkey.