Ipsen Biopharm Limited

Harvard College

The Broad Institute, Inc.

Ipsen Bioinnovation Limited

Matthew Beard

David R Liu

Philip Marks

Michael S Packer

Aimee Cossins

Bryony Gray

Verity Cadd

Travis R Blum

The disclosure provides amino acid sequence variants of Botulinum neurotoxin (BoNT) proteases that cleave (VAMP1, VAMP2, VAMP7, VAMP8, SNAP25, SNAP23, PTEN, etc.) and methods of evolving the same. In some embodiments, proteases described by the disclosure are useful for cleaving proteins found in a cell, that is in an intracellular environment. In some embodiments, proteases described by the disclosure are useful for treating diseases associated with increased or aberrant VAMP7, VAMP8, SNAP23 or PTEN expression or activity, for example, cancer and neurological disorders. Some aspects of this disclosure provide methods for generating BoNT protease variants by continuous directed evolution.

Evolution of BoNT peptidases

A VAMP epitope suitable for generating an antibody against a VAMP C-terminal neurotoxin cleavage product. Method of using such an epitope to generate an antibody against cleaved VAMP. Method of using such an antibody to assay for cleavage of a VAMP by clostridial neurotoxin.

Cellular vamp cleavage assay

The invention provides a nucleic acid sequence comprising a sequence of contiguous nucleotides, wherein said sequence of contiguous nucleotides has at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 1, and wherein said sequence of contiguous nucleotides encodes a single-chain BoNT/E1 protein. The present invention also provides methods for producing soluble single-chain BoNT/E1 protein in anhost cell, together with methods for producing soluble di-chain BoNT/E1 protein.

Recombinant clostridium botulinum neurotoxins

Growing community of inventors

Abingdon, United Kingdom

Matthew Beard