Abstract Committee Review

I suggest rewording of 'ethically concerning, and poorly predictive of human"

concerning might be inappropriate here and I would replace by challenging.

I am unsure on what basis the author claim that mice "are poorly predictive of human". I think this is more wishful thinking than factual. I would remove this part of the sentence.

Thank you for the feedback. I have made changes to the abstract to clarify the points highlighted. Please see attached and below for the details:

• I suggest rewording of 'ethically concerning, and poorly predictive of human" concerning might be inappropriate here and I would replace by challenging.
• I am unsure on what basis the author claim that mice "are poorly predictive of human". I think this is more wishful thinking than factual. I would remove this part of the sentence.

Before: Current regulatory and product-development workflows still rely heavily on animal-based assays, which are slow, ethically concerning, and poorly predictive of human neuromuscular physiology.

Now: Current regulatory and product-development workflows still rely heavily on animal-based assays, which are slow, ethically challenging and do not fully recapitulate the human NMJ response to BoNT because of differences in receptor sequences, ganglioside expression, and neuronal uptake kinetics. BoNT/B, for example, is relatively less effective in humans than in mice due to an amino-acid change in synaptotagmin II that reduces high-affinity binding of BoNT/B (and some related toxins).

As requested, I replaced concerning by challenging and replaced poorly predictive to clarify the pharmacological differences between the mouse and human response to different subtypes of BoNT. Here are the references for the mentioned differences.

1. Sabine Pellett ¹, William H Tepp ¹, Eric A Johnson ^1,* Botulinum Neurotoxins A, B, C, E, and F preferentially enter cultured human motor neurons compared to other cultured human neuronal populations. FEBS Lett. 2019 Jul 4;593(18):2675–2685. doi: 10.1002/1873-3468.13508
2. Frank Neuschäfer-Rube ¹, Andrea Pathe-Neuschäfer-Rube ¹, Gerhard P Püschel ¹ Discrimination of the Activity of Low-Affinity Wild-Type and High-Affinity Mutant Recombinant BoNT/B by a SIMA Cell-Based Reporter Release Assay. Toxins (Basel) 2022 Jan 17;14(1):65. doi: 10.3390/toxins14010065.
3. Peng, L. et al. Botulinum neurotoxin D-C uses synaptotagmin I and II as receptors, and human synaptotagmin II is not an effective receptor for type B, D-C and G toxins. J. Cell Sci. 125, 3233–3242 (2012).

1. Strotmeier, J., Willjes, G., Binz, T. & Rummel, A. Human synaptotagmin-II is not a high affinity receptor for botulinum neurotoxin B and G: increased therapeutic dosage and immunogenicity. FEBS Lett. 586, 310–313 (2012).
2. Coffield, J. A. et al. In vitro characterization of botulinum toxin types A, C and D action on human tissues: Combined electrophysiologic, pharmacologic and molecular biologic approaches. J. Pharmacol. Exp. Ther. 280, 1489–1498 (1997).

Thank you very much for organizing this event and valuable comments.

Margaret