Farnesyl diphosphate synthases have been shown to possess seven highly conserved regions (I-VII) in their amino acid sequences [Koyama et al. (1993) J. Biochem. (Tokyo) 113, 355-363]. Site-directed mutants of farnesyl diphosphate synthase from Bacillus stearothermophilus were made to evaluate the roles of the conserved aspartic acids in region VI and lysines in regions I, V, and VI. The aspartate at position 224 was changed to alanine or glutamate (mutants designated as D224A and D224E, respectively); aspartates at positions 225 and 228 were changed to isoleucine and alanine (D225I, D228A); lysine at position 238 was changed to either alanine or arginine (K238A, K238R). The lysines at positions 47 and 183 were changed to isoleucine and alanine (K471, K183A), respectively. Kinetic analyses of the wild-type and mutant enzymes indicated that the mutagenesis of Asp-224 and Asp-225 resulted in a decrease of Kcat values of approximately 10(4)- to 10(5)-fold compared to the wild type. On the other hand, D228A showed a Kcat value approximately one-tenth of that of the wild type, and the k(m) value for isopentenyl diphosphate increased approximately 10-fold. Both K471 and K183A showed k(m) values for isopentenyl diphosphate 20-fold larger and kcat values 70-fold smaller than the wild type. These results suggest that the two conserved lysines in regions I and V contribute to the binding of isopentenyl diphosphate and that the first and the second aspartates in region VI are involved in catalytic function. Aspartate-228 is also important for the binding of isopentenyl diphosphate rather than for catalytic reaction.

Objective The novel morpholino antisense oligonucleotide viltolarsen targets exon 53 of the dystrophin gene, and could be an effective treatment for patients with Duchenne muscular dystrophy (DMD). We investigated viltolarsen’s ability to induce dystrophin expression and examined its safety in DMD patients. Methods In this open‐label, multicenter, parallel‐group, phase 1/2, exploratory study, 16 ambulant and nonambulant males aged 5–12 years with DMD received viltolarsen 40 or 80 mg/kg/week via intravenous infusion for 24 weeks. Primary endpoints were dystrophin expression and exon 53 skipping levels. Results In western blot analysis, mean changes in dystrophin expression (% normal) from baseline to Weeks 12 and 24 were − 1.21 ( P = 0.5136) and 1.46 ( P = 0.1636), respectively, in the 40 mg/kg group, and 0.76 ( P = 0.2367) and 4.81 ( P = 0.0536), respectively, in the 80 mg/kg group. The increase in mean dystrophin level at Weeks 12 and 24 was significant in the 80 mg/kg group (2.78%; P = 0.0364). Patients receiving 80 mg/kg showed a higher mean exon 53 skipping level (42.4%) than those receiving 40 mg/kg (21.8%). All adverse events were judged to be mild or moderate in intensity and none led to study discontinuation. Interpretation Treatment with viltolarsen 40 or 80 mg/kg elicited an increasing trend in dystrophin expression and exon 53 skipping levels, and was safe and well tolerated. The decline in motor function appeared less marked in patients with higher dystrophin levels; this may warrant further investigation. This study supports the potential clinical benefit of viltolarsen.

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