Introduction: Neurogenic and idiopathic detrusor overactivity (DO) is currently treated with onabotulinumtoxin A (onaBoNT/A) as third-line therapy. However, BoNT/A requires repeated injections into the bladder and is known to have diminished treatment effect after 5 years. Novel, chimeric toxins may provide an alternative to BoNT/A in the setting of treatment failure. Chimeric BoNT/A-B is a construct that consists of the light chain (catalytic domain) of BoNT/A, paired with a receptor binding domain of BoNT/B. This receptor binding domain binds to synaptotagmin-1, recently shown to be the dominant BoNT receptor in the bladder. Here, we aim to determine the effect of a chimeric BoNT/A-B construct in mouse bladder tissues. Methods: Gene fragments encoding the light chain and translocation domain of BoNT/A, as well as the receptor binding domain for BoNT/B, were produced via PCR and cloned into a vector to produce chimeric BoNT/A-B. To assess ex vivo smooth muscle response, wild type (WT) mouse bladders were harvested and placed in Kreb's buffer. Each bladder was cut into three longitudinal strips and mounted in tissue chambers at 37 C with continuous bubbled mixture of carbogen. Bladder strips were attached to a force transducer (Fig. A) and underwent nerve-evoked contraction studies via electrical field stimulation (EFS; 1-64Hz, 30V 0.5 ms pulse width, 10 sec duration), with the addition of 0.3 or 1 nM BoNT/A-B, to generate a frequency response curve. Results: Ex vivo contraction studies in the presence of 1 nM BoNT/A-B showed a reduction in 80% of nerve-evoked contractions by 100 minutes; BoNT/A had produced the same degree of reduction at only 300 minutes, reflecting the rapid onset of BoNT/A-B. At a lower dose of 0.3 nM, BoNT/A-B reduced nerve-evoked contractions by 40% at 100 minutes; BoNT/A had minimal effect (Fig. B). All bladder weights were similar, and data were normalized to the cross-sectional area of the bladder strip. Carbachol dose-response curves served as controls to ensure bladder strips were viable and contractile pre- and post-exposure. Conclusions: We demonstrate chimeric BoNT/A-B has a faster onset and great potency at lower doses in smooth muscle ex vivo. This novel toxin may provide improved neuromuscular paralysis smooth muscle disorders, such as overactive and neurogenic bladder. SOURCE OF Funding: H.T. is supported by the NIH T32 Grant, AUA Urology Care Foundation Research Scholar Award, the SUFU Chemodenervation Grant, and the Office of Faculty Development at Harvard Medical School. M.D. is supported by grants from the NIH and holds the Investigator in the Pathogenesis of Infectious Disease award from the Burroughs Wellcome Fund. M.P.S. received support from the U.S. Department of Veterans Affairs.
