This invention relates to novel recombinant clostridial neurotoxins exhibiting increased duration of effect and to methods for the manufacture of such recombinant clostridial neurotoxins. These novel recombinant clostridial neurotoxins comprise a random coil domain, and the methods comprise the steps of inserting a nucleic acid sequence coding for a random coil domain into a nucleic acid sequence coding for a parental clostridial neurotoxin and expression of the recombinant nucleic acid sequence comprising the random coil domain-coding sequence in a host cell. The invention further relates to novel recombinant single-chain precursor clostridial neurotoxins used in such methods, nucleic acid sequences encoding such recombinant single-chain precursor clostridial neurotoxins, and pharmaceutical compositions comprising the recombinant clostridial neurotoxin with increased duration of effect.

Provided is a preparation method for a modified toxin polypeptide. The preparation method comprises: step 1): expressing a modified toxin polypeptide precursor; step 2): enriching the toxin polypeptide precursor; and step 3): activating the toxin polypeptide precursor to obtain a modified toxin polypeptide.

A single chain, polypeptide fusion protein, comprising: a non-cytotoxic protease, which protease is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent (eg clostridial neurotoxin L-chain or IgA protease); a galanin Targeting Moiety that is capable of binding to a Binding Site on the nociceptive sensory afferent, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the nociceptive sensory afferent (eg GALR1, GALR2, or GALR3 receptor); a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the non-cytotoxic protease and the galanin Targeting Moiety; a translocation domain that is capable of translocating the protease from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent (eg HN domain of clostridial neurotoxin); a first spacer located between the non-cytotoxic protease and the protease cleavage site, wherein said first spacer comprises an amino acid sequence of from 4 to 25 amino acid residues; and a second spacer located between the galanin Targeting Moiety and the translocation domain, wherein said second spacer comprises an amino acid sequence of from 4 to 35 amino acid residues. Nucleic acid sequences encoding the polypeptide fusion proteins, methods of preparing same and uses thereof are also described (eg of treating, preventing or ameliorating pain).

Compositions and methods of modulating cellular function and treatment of disease in mammals comprising locally administering a regulated SNARE inhibitor and a translocating agent to the mammal. Regulated SNARE inhibitors include clostridial neurotoxins, tetanus neurotoxin and their free light chain portions and IgA protease. Translocating agents include acids, encapsulating vectors, and transduction domains.

A method for purifying a clostridial neurotoxin comprising contacting a cation exchange resin with a composition comprising a clostridial neurotoxin, wherein the contacting step is performed at at least pH 7.3, wherein the step of contacting a cation exchange resin with a composition comprising said clostridial neurotoxin occurs prior to conversion of the clostridial neurotoxin from a single chain form into a dichain form. Also provided are uses of a buffer having a pH value that is ?1 pH unit or higher than the calculated pi of a clostridial neurotoxin, purification intermediates and clostridial neurotoxins obtainable by the invention, wherein the clostridial neurotoxin is in a single chain form.

Provided is a preparation method for a modified toxin polypeptide. The preparation method comprises: step 1): expressing a modified toxin polypeptide precursor; step 2): enriching the toxin polypeptide precursor; and step 3): activating the toxin polypeptide precursor to obtain a modified toxin polypeptide.

The present invention provides an engineered clostridial toxin comprising at least one amino acid modification, wherein said at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least 0.2 pI units higher than the pI of an otherwise identical clostridial toxin lacking said at least one amino acid modification. Also provided are corresponding uses of the engineered clostridial toxin in therapy.

This invention relates to novel recombinant clostridial neurotoxins exhibiting increased duration of effect and to methods for the manufacture of such recombinant clostridial neurotoxins. These novel recombinant clostridial neurotoxins comprise a random coil domain, and the methods comprise the steps of inserting a nucleic acid sequence coding for a random coil domain into a nucleic acid sequence coding for a parental clostridial neurotoxin and expression of the recombinant nucleic acid sequence comprising the random coil domain-coding sequence in a host cell. The invention further relates to novel recombinant single-chain precursor clostridial neurotoxins used in such methods, nucleic acid sequences encoding such recombinant single-chain precursor clostridial neurotoxins, and pharmaceutical compositions comprising the recombinant clostridial neurotoxin with increased duration of effect.

The present invention provides an engineered clostridial toxin comprising at least one amino acid modification, wherein said at least one amino acid modification increases the isoelectric point (pI) of the engineered clostridial toxin to a value that is at least 0.2 pI units higher than the pI of an otherwise identical clostridial toxin lacking said at least one amino acid modification. Also provided are corresponding uses of the engineered clostridial toxin in therapy.

The present disclosure relates to the use of the non-toxic proteolytic C fragment of tetanus toxin and plasmids encoding such protein fragment to increase muscle mass and/or muscle strength in a subject in need thereof. As such, methods of ameliorating the severity of a pathological condition characterized, at least in part, by a decreased amount, development, or metabolic activity of muscle are provided. The disclosed compositions and method are also useful for the treatment of condition in which increase in muscle mass and muscle strength are desirable, including cosmetic uses.