WO2014197837A1 - Materials and methods for producing a biological toxin - Google Patents

Abstract

The current invention provides a method of producing a heterologous biological toxin or subunit thereof in a bacterium, such as a non-pathogenic bacterium. The current invention also provides a method of producing botulinum neurotoxin in a bacterium other thanClostridium spp. The current invention further provides a method of producing botulinum neurotoxin inLactococcus lactis. Furthermore, the current invention provides a hybrid toxin comprising a first subunit of a first biological toxin and a carrier which may be a second subunit from a second biological toxin. Additionally, the current invention provides a method of treating a disease in a subject by administering a pharmaceutically effective amount of a hybrid toxin to the subject, wherein the hybrid toxin is specifically designed for the treatment of the disease in the subject.

Description

DESCRIPTION

MATERIALS AND METHODS FOR PRODUCING A BIOLOGICAL TOXIN

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. Provisional Application Serial No. 61/831,920, filed June 6, 2013, which is hereby incorporated by reference herein in its entirety, including any figures, tables, nucleic acid sequences, amino acid sequences, or drawings.

BACKGROUND OF THE INVENTION

Biological toxins require strict safeguards against exposure through inhalation, absorption through skin or mucous membranes, ingestion, or percutaneous injury because these toxins are extremely hazardous, even in minute quantities. However, appropriate use of biological toxins provides medical treatment options for various disorders.

For example, over the past three decades, there has been a continuing fascination with botulinum neurotoxin (BoNT) and the opportunities for use of this potentially deadly neurotoxin as a treatment in certain diseases. BoNT interferes with the release of acetylcholine from presynaptic nerve endings and prevents muscle contractions and/or glandular secretion. Since BoNT is used primarily as a focal injectable treatment, resulting in fewer side effects than systemically administered medications, it has evolved to become the treatment of choice for management of many forms of dystonia, limb spasticity, cosmetic glabellar lines, hyperhidrosis, and sialorrhea [1]. The use of OnabotulinumtoxinA (Botox Cosmetic) to address migraines was approved by the FDA in 2010 [2], and its use to treat bladder hyperactivity was approved in 2011 [3-5]. BoNT also has a broad spectrum of off-label indications, including focal hand dystonia, lower limb spasticity [6,7], management of chronic anal fissures [4], and tension headaches [8]. The commercially available neurotoxins include three main brands of BoNT/A: OnabotulinumtoxinA (BOTOX®), AbobotulinumtoxinA (DYSPORT®), IncobotulinumtoxinA (XEOMIN®), and also a brand of BoNT type B, RimabotulinumtoxinB (MYOBLOC® or NEUROBLOC®) [9]. Each formulation has specific clinical recommendations which have been formulated based on the available science, and on differing biochemical profiles.

An issue with production of BoNT has been the use the pathogenic Clostridium spp. to manufacture the drug. The process is costly, labor intensive, requires special equipment, and is extremely hazardous, particularly in view of the fact that disinfection includes procedures to destroy the Clostridium spores. While production of other biological toxins may present similar problems with pathogenicity and there are always safety concerns in manufacturing biological toxins, working with spores is particularly problematic. Therefore, a safer method of manufacturing biological toxins in non- pathogenic bacteria is desirable.

BRIEF SUMMARY OF THE INVENTION

The current invention provides a method of producing a biological toxin in nonpathogenic bacteria. The current invention also provides a method of producing botulinum neurotoxin (BoNT) in a bacterium other than Clostridium spp. A further aspect of the invention provides a method of producing a biological toxin in a lactic acid bacterium (LAB), such as Lactococcus lactis.

The current invention also provides a method of producing hybrid toxins (also referred to herein as chimeric toxins or fusion proteins), wherein the hybrid toxins comprise multiple subunits from different biological toxins. An example of a hybrid toxin of the current invention comprises a first subunit from a first biological toxin and a carrier, such as a second subunit from a second biological toxin.

Furthermore, another aspect of the invention provides a method of treating a disorder in a subject in need thereof by administering an effective amount of a biological toxin to the subject. Another aspect of the invention provides a method of treating a disease in a subject by administering an effective amount of a hybrid toxin comprising multiple subunits to the subject, wherein the multiple subunits of the hybrid toxin are selected to treat the disease in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Cloning of light chain (Lc) and (He) of BoNT in the expression vectors for expression of BoNT in Lactococcus lactis. Figure 2. Schematic representation of a hybrid toxin comprising a catalytic Lc (payload) subunit and a targeting He (carrier).

DETAILED DESCRIPTION OF THE INVENTION The correct processing of biological toxins by the toxin producing cell is believed to be critical for obtaining biologically active toxin. For example, stringent need for correct processing of the holotoxin, operated by specific proteases, led experts to believe that functional botulinum neurotoxin (BoNT) can only be produced in Clostridium botulinum or the closely related Clostridium argentinense. The current invention provides a method of producing biological toxins, for example, BoNT, in a nonpathogenic host, thereby greatly improving the safety associated with manufacturing hazardous toxins using pathogenic bacteria and dramatically decreasing the costs of both equipment and personnel. The current invention also provides a method of producing BoNT in a bacterium other than Clostridium spp.

One aspect of the current invention is a method of producing a biological toxin, or a subunit thereof, using a bacterium other than Clostridium botulinum. The method of the current invention comprises:

a) introducing in to the bacterium an expressible nucleic acid encoding the biological toxin or subunit; and

b) culturing the bacterium containing the expressible nucleic acid encoding the biological toxin or subunit under the conditions that allow expression of the biological toxin.

Optionally, the method further comprises c) purifying the biological toxin or subunit from the culture of the bacterium.

Preferably, a non-pathogenic bacterium is used; however, pathogenic bacterium may be desired in some circumstances.

As used herein, the term "biological toxin" refers to a poisonous biomolecule, for example a protein, which is produced by living cells or organisms and is capable of causing a disorder when introduced into the body of an animal, for example, a human. The source for the biological toxin can be any organism, for example, plants, animals, bacteria, fungi, protists, lower eukaryotes, and higher eukaryotes. In some embodiments, the biological toxin is a "wild-type" toxin, such as botulinum toxin, or other toxin produced by a bacterium other than Clostridium botulinum. {e.g., a non-pathogenic bacterium such as a lactic acid bacteria or a Bacillus), wherein the toxin is heterologous to the non-Clostridrium bacterium. In some embodiments, the biological toxin is a hybrid toxin, representing a genetic fusion of one or more biological toxins or one or more subunits thereof (a payload) and one or more carriers. The hybrid toxins may be produced using Clostridium spp. (e.g., Clostridium botulinum) or a bacterium other than Clostridium botulinum (e.g., a non-pathogenic bacterium such as a lactic acid bacteria or a Bacillus). In some embodiments, the biological toxin comprises one or more serotypes of botulinum toxin A-G.

As used herein, the term "carrier" refers to a molecule that targets a "payload" molecule such as a biological toxin, to a target cell or cell type. The carrier may be homologous (natural) to the payload or heterologous to the payload. Typically, the target cell is not a natural target cell. In some embodiments, the carrier is a subunit of a second biological toxin. In some embodiments, the first subunit of the first biological toxin is the active subunit of the first biological toxin and the carrier is a targeting subunit of a second biological toxin. In some embodiments, the first subunit of the first biological toxin is the active subunit of botulinum neurotoxin and the carrier is selected from among a targeting subunit of Tetanus toxin, adenovirus knob, reovirus protein sigma 1, Shiga-like toxin binding moiety, or rabies attachment protein.

Through genetic fusion, one or more carriers and one or more payloads can be arranged at any location along the length of the chimeric construct and oriented with respect to one another. For example, the fusion protein of carrier and payload can comprise the carrier on the N-terminus and the payload on the C-terminus of the fusion protein (carrier-payload), or the payload on the N-terminus and the carrier on the C- terminus of the fusion protein (payload-carrier). The arrangement of the domains (payload and carrier) can be controlled by their arrangement in the encoding nucleic acid molecules.

Optionally, a cleavable linker can be inserted between the payload and the carrier. An example of a cleavable linker is the target polypeptide of the BoNT light chain itself [13]. A cleavable linker can be cleaved under the conditions for which it is designed and/or utilized. For example, a cleavable linker can be cleaved by an enzyme endogenous to the subject or by an exogenous enzyme that is administered to the subject before, during, or after administration of the toxin.

In another embodiment, a linker that is resistant to cleavage can be genetically inserted between the payload and the carrier. For example, a cleavage resistant SNAP25- BoNT light chain cleavable linker or a mutant SNAP25 which is active yet resistant to cleavage may be utilized, as identified in [14]. As used herein, term "heterologous" in connection with a biological toxin or subunit thereof means the toxin or subunit is a type not normally produced by that bacteria. In the context of a carrier, a carrier is heterologous to its payload if the carrier and payload do not naturally occur within the same molecule.

As used herein, the term "hybrid toxin" refers to a toxin comprising multiple (two or more) subunits, wherein the subunits originate in different biological toxins and are not naturally present together in a biological toxin. For example, in some embodiments, a hybrid toxin of the current invention comprises the active subunit of BoNT and targeting subunit of Tetanus toxin.

As used herein, the term "non-pathogenic bacterium" means a bacterium that is generally not capable of causing a disease in human or non-human animal. It should be understood, however, that a non-pathogenic bacterium of the invention can become an opportunistic pathogen under some circumstances and occasionally cause a disease, for example, in an immuno-compromised animal.

The bacterium may be cultured in a culture vessel (dish, plate, bottle, or other suitable substrate) of any scale (large or small), as an open or closed culture. In some embodiments, the bacterium is cultured in a bioreactor, such as a batch, fed-batch, or continuous bioreactor.

The biological toxin produced by the method of the current invention can comprise one or more subunits. An example of the biological toxin that can be produced by the method of the current invention is botulinum neurotoxin.

Methods of purifying a polypeptide expressed in bacteria are well known to a person of ordinary skill in the art and these methods are within the purview of the current invention.

Many biological toxins follow a multi-subunit configuration, for example, the AB configuration, where A subunit is the catalytic subunit exerting the cellular damage, while B subunit targets the holotoxin to specific sites in host tissues. B subunit can also act as a chaperone in some cases [10, 11]. Such biological toxins are called binary toxins. Examples of such binary biological toxins are BoNT, Anthrax toxin, Tetanus toxin, ricin, Shiga-like toxin, Shiga toxin, etc. Additional binary toxins are well known to a person of ordinary skill in the art and are within the purview of the current invention. Typically, the biological activity of the A subunit is 5-6 logs lower when it acts alone compared to when it acts in concert with the targeting subunit. For example, BoNT serotypes A-G follow this design [12]. BoNT A-G are produced as 150KDa inactive holopeptides that undergo enzymatic cleavage thereby removing a short peptide, and thus releasing an active toxin composed of a 50KDa catalytic light chain (Lc) and a lOOKDa heavy chain targeting moiety (He). Lc and He remain in contact through a disulfide bridge.

An aspect of the current invention also provides a method of producing separate subunits of a biological toxin using two or more bacteria, wherein the biological toxin comprises multiple subunits. In another embodiment, the current invention provides a method of producing a biological toxin comprising a first subunit and a carrier, such as a second subunit. The method of producing a biological toxin comprising a first subunit and a carrier according to the current invention, comprises:

a) introducing into a first bacterium an expressible nucleic acid encoding the first subunit;

b) introducing into a second bacterium an expressible nucleic acid encoding the carrier {e.g., second subunit); and

c) culturing the first and the second bacteria containing the expressible nucleic acid encoding the first subunit and the carrier under conditions that allow expression of the first subunit and the carrier.

Optionally, the method further comprises: d) purifying the first subunit and the carrier from the culture of the first bacterium and the second bacterium. Optionally, the method further comprises: e) assembling the first subunit and the carrier to produce the biological toxin.

The first bacterium and the second bacterium can be cultured in the same culture vessel {i.e. co-cultured). Alternately, the first and the second bacteria can be cultured in separate culture vessels. For example, the first non-pathogenic bacterium may be cultured in a first bioreactor and the second bacterium may be cultured in a second bioreactor.

In an embodiment of the invention, the first bacterium and the second bacterium belong to the same species and strain. For example, the bacterium used to manufacture a multisubunit biological toxin is Lactococcus lactis.

Alternately, the first bacterium and the second bacterium can belong to different species and strains. When the first bacterium and the second bacterium belong to different species and strain, in one embodiment, the first bacterium belongs to the genus Lactococcus and the second bacterium belongs to the genus Bacillus. In an embodiment of the invention, the first bacterium is Lactococcus lactis and the second bacterium is Bacillus subtilis.

Table 1 lists examples of toxins and subunits that can be utilized in producing biological toxins, compositions, microorganisms, and methods of the invention, and corresponding references and GenBank accession numbers, including sequences, are incorporated herein by reference in their entirety.

Table 1. Exemplified Toxins and Subunits

GenBank Protein

Toxin name Reference

Accession Number

Botulinum Thompson, D.E. et al., "The complete amino neurotoxin type A acid sequence of the Clostridium botulinum holotoxin from P10845.4 type A neurotoxin, deduced by nucleotide

Clostridium sequence analysis of the encoding gene," Eur. J. botulinum Biochem., 189 (1), 73-81 (1990).

Botulinum

Seong, H.Y. et al., "Effects of minor arginyl neurotoxin type A

and isoleucyl tRNA on the expression of light chain from AA021363

botulinum toxin light chain in Escherichia coli," Clostridium

Unpublished.

botulinum

Heavy chain of

Botulinum

neurotoxin Type A

Botulinum Lacy, D.B., et al., "Crystal structure of

comprises amino

neurotoxin type A botulinum neurotoxin type A and implications acids 448-1295 of

heavy chain from for toxicity," Nature Structural Biology, 5, 898 the holotoxin. The

Clostridium - 902 (1998).

heavy chain

botulinum

comprises a

translocation

domain and a

subunit from phage-borne stx genes and their flanking

The amino acid sequences of the toxins and subunits exemplified in Table 1 are provided below. MPFWKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPE EGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTS IVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFG HEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHEL IHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQEN EFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFS VDK LKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGF NLRNTNLAANFNGQNTEI NMNFTKLK FTGLFEFYKLLCVRGIITSKTKSLDKG YNKALNDLCIKV NWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQ YYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGK YELDKYTMFHYLRAQE FEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVK VNKATEAAMFLGWVEQLVY DFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIA IPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDL IRKKMKE ALENQ AE ATKAIIN YQ YNQ YTEEEK NINFNIDDL S SKLNE SINKAMIN INKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLK DKV NTLSTDIPFQLSKYVDNQRLLSTFTEYIK IINTSILNLRYESNHLIDLSRYAS KINIGSKVNFDPIDK QIQLFNLESSKIEVILK AIVYNSMYENFSTSFWIRIPKYFN SISL NEYTIINCME NSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYI NRWIFVTITNNRL NSKIYINGRLIDQKPISNLGNIHAS NIMFKLDGCRDTHRYI WIKYFNLFDKELNEKEIKDL YDNQ SN S GILKDF WGD YLQ YDKP Y YMLNL YDPN KYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIV RNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKND QGITNKCKMNLQD NGNDIGFIGFHQF NI AKLVASN WYNRQIERS SRTLGC S W EFIPVDDGWGERPL (Botulinum neurotoxin type A holotoxin from Clostridium botulinum; Accession no. P10845.4; SEQ ID NO: l)

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQVKAFKIHNKIWVIPERDTFTNPEE GDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSI VRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFG HEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHEL IHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQEN EFRLYYYNKFKDIASTLNKASIVGTTASLQYMK VFKEKYLLSEDTSGKFSVDKL KFDKLYKMKTEIYTEDDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGF NLRNTNLAANFNGQNTEINNMNFTKLK FTGLFEFYKLLCVRGIITSKTKSLDEG YNK (Botulinum neurotoxin type A light chain from Clostridium botulinum; Accession no. AA021363; SEQ ID NO:2)

MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKP EDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMI INGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNEN ETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPAL ILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPS TDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYS IDVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEE GFNISDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCKSVKAPGICIDVDN EDLFFIADKNSFSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISKIELPSENTES LTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTSSFDDALLFSN KVYSFFSMDYIKTANKVVEAGLFAGWVKQIVNDFVIEANKSNTMDKIADISLIVP YIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLIPVVGAFLLESYIDNK KIIK TIDNALTKR EKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEII KYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLA VEKLLDFDNTLKK LLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDLSIYTND TILIEMFNKYNSEIL NIILNLRYKD NLIDLSGYGAKVEVYDGVELNDK QFKLT SSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYK DGIQNYIHNEYTIINCMKNN SGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTIT NL NAKIYI NGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYK IQSYSEYLKDFWGNPLMYNKEYYMFNAGNK S YIKLKKDSPVGEILTRSKYNQN SKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYF KKEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFY ESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE (Botulinum neurotoxin type B holotoxin from Clostridium botulinum; Accession no. AAA23211; SEQ ID NO:3)

MPVTINNFNYNDPID NNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKP

EDFNKSSGIFNRDVCEYYDPDYLNTNDKK IFLQTMIKLFNRIKSKPLGEKLLEMI

INGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNEN ETIDIGIQNHFASREGFGGIMQMKFCPEYVSVF NVQENKGASIFNRRGYFSDPAL ILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPS TDKSIYDKVLQNFRGIVDRLNKVLVCISDPSININIYK KFKDKYKFVEDSEGKYSI DVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIK LLDNEIYTIEEG FNISDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCKSVKAPGICIDVDN

(Botulinum neurotoxin type B light chain from Clostridium botulinum; Accession no. AAB22656; SEQ ID NO:4)

MNIKKEFIKVISMSCLVTAITLSGPVFIPLVQGAGGHGDVGMHVKEKEK KDENK

PvKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKA IGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQ SSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIK ASDSDGQDL LFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNY MDKFNEQEWLSLEELKDQRMLARYEKWEKIKQHYQHWSDSLSEEGRGLLKKL QIPIEPK DDIIHSLSQEEKELLKRIQIDSSDFLSTEEKEFLKKLQIDIRDSLSEEEKEL LNRIQVDSSNPLSEKEKEFLKKLKLDIQPYDINQRLQDTGGLIDSPSINLDVRKQY KRDIQNIDALLHQSIGSTLYNKIYLYENMNI NLTATLGADLVDSTDNTKINRGIF NEFK NFKYSISSNYMIVDINERPALDNERLKWRIQLSPDTRAGYLENGKLILQR IGLEIKDVQIIKQSEKEYIRIDAKVVPKSKIDTKIQEAQLNINQEWNKALGLPKYTK LITFNVHNRYASNIVESAYLILNEWKNNIQSDLIKKVTNYLVDGNGRFVFTDITLP NIAEQYTHQDEIYEQVHSKGLYVPESRSILLHGPSKGVELRNDSEGFIHEFGHAVD DYAGYLLDK QSDLVTNSKKFIDIFKEEGSNLTSYGRTNEAEFFAEAFRLMHSTD HAERLKVQK APKTFQFINDQIKFIINS (Lethal Factor of Anthrax toxin from Bacillus anthracis; Accession no. P15917; SEQ ID NO:5)

MTRNKFIPNKF SII SF S VLLF AIS S S Q AIE VN AMNEH YTESDIKRNHKTEKNKTEKE KFKDSI NLVKTEFTNETLDKIQQTQDLLKKIPKDVLEIYSELGGEIYFTDIDLVEH KELQDLSEEEK SMNSRGEKVPFASRFVFEKKRETPKLIINIKDYAINSEQSKEVY YEIGKGISLDIISKDKSLDPEFLNLIKSLSDDSDSSDLLFSQKFKEKLELNNKSIDINF IKENLTEFQHAFSLAFSYYFAPDHRTVLELYAPDMFEYMNKLEKGGFEKISESLK KEGVEKDRIDVLKGEKALKASGLVPEHADAFKKIARELNTYILFRPVNKLATNLI KSGVATKGLNEHGKSSDWGPVAGYIPFDQDLSKKHGQQLAVEKGNLENK SITE HEGEIGKIPLKLDHLRIEELKENGIILKGKKEIDNGK YYLLESNNQVYEFRISDEN NEVQYKTKEGKITVLGEKFNWRNIEVMAK VEGVLKPLTADYDLFALAPSLTEI K QIPTKRMDKVVNTPNSLEKQKGVTNLLIKYGIERKPDSTKGTLSNWQKQMLD RLNEAVKYTGYTGGDVVNHGTEQDNEEFPEKDNEIFIINPEGEFILTK WEMTGR FIEK ITGKDYLYYFNRSYNKIAPGNKAYIEWTDPITKAKINTIPTSAEFIK LSSIR RSSNVGVYKDSGDKDEFAK ESVKKIAGYLSDYYNSANHIFSQEKKRKISIFRGIQ AYNEIENVLKSKQIAPEYK YFQYLKERITNQVQLLLTHQKSNIEFKLLYKQLNFT ENETDNFEVFQKIIDEK (Edema Factor of Anthrax toxin from Bacillus anthracis; Accession no. AAA79215; SEQ ID NO:6)

MKKRKVLIPLMALSTILVSSTGNLEVIQAEVKQENRLLNESESSSQGLLGYYFSDL NFQAPMVVTSSTTGDLSIPSSELENIPSENQYFQSAIWSGFIKVKKSDEYTFATSAD NHVTMWVDDQEVINKASNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTD SQNKKEVISSDNLQLPELKQKSSNSRKKRSTSAGPTVPDRDNDGIPDSLEVEGYT VDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVS PEARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVH GNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTAR LNANIRYVNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKN LAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVR VDTGSNWSEVLPQIQETTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEA LKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIK QLAELNATNIYTVLDKIK LNAKMNILIRDKRFHYDR NIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKI LSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFK YNDKLPLYISNPNY KVNVYAVTKENTIINPSENGDTSTNGIKKILIFSK GYEIG (Protective Antigen of Anthrax toxin from Bacillus anthracis; Accession no. AAF86457; SEQ ID NO:7)

MPITI NFRYSDPV NDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPE DFNPPSSLIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIIN AIPYLGNSYSLLDKFDTNSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGPVLNK EVRGIVLRVDNKNYFPCRDGFGSIMQMAFCPEYVPTFDNVIENITSLTIGKSKYFQ DPALLLMHELIHVLHGLYGMQVSSHEIIPSKQEIYMQHTYPISAEELFTFGGQDAN LISIDIK DLYEKTLNDYKAIANKLSQVTSCNDPNIDIDSYKQIYQQKYQFDKDSN GQYIVNEDKFQILYNSIMYGFTEIELGKKFNIKTRLS YFSMNHDPVKIPNLLDDTIY NDTEGFNIESKDLKSEYKGQNMRVNTNAFRNVDGSGLVSKLIGLCKKIIPPTNIRE NLYNRTASLTDLGGELCIKIK EDLTFIAEK SFSEEPFQDEIVSYNTKNKPLNFNY SLDKIIVDYNLQSKITLPNDRTTPVTKGIPYAPEYKSNAASTIEIHNIDDNTIYQYLY AQKSPTTLQRITMTNSVDDALINSTKIYSYFPSVISKVNQGAQGILFLQWVRDIIDD FTNESSQKTTIDKISDVSTIVPYIGPALNIVKQGYEGNFIGALETTGVVLLLEYIPEIT LPVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYKLVKAKWLGTVNTQFQKR SYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQIADEINNLK KLEEKANKAMINI NIFMRESSRSFLVNQMINEAK QLLEFDTQSK ILMQYIKANSKFIG ITELKKLESKINKVFSTPIPFSYSK LDCWVDNEEDIDVILK STILNLDINNDIISDI SGFNSSVITYPDAQLVPGINGKAIHLV NESSEVIVHKAMDIEYNDMF NFTVSF WLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKG NLIWTLKDSAG EVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIR ED NITLKLDRC NNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNP LRYDTEYYLIPVASSSKDVQLK ITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFII KRYTP NEIDSFVKSGDFIKLYVSY NNEHIVGYPKDGNAF NLDRILRVGYNAP GIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIA SNWYFNHLKDKILGCDWYFVPTDEGWTND (Tetanus toxin from Clostridium tetani; Accession no. P04958; SEQ ID NO:8)

MKPGGNTIVIWMYAVATWLCFGSTSGWSFTLEDNNIFPKQYPIINFTTAGATVQS YTNFIRAVRGRLTTG AD VRHEIP VLPNRVGLPINQRFIL VEL SNH AEL S VTL ALD V TNAYVVGYRAGNSAYFFHPDNQEDAEAITHLFTDVQNRYTFAFGGNYDRLEQL AGNLRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFIICIQMISEAARFQYIEGE MRTRIRYNRRSAPDPSVITLENSWGRLSTAIQESNQGAFASPIQLQRRNGSKFSVY DVSILIPIIALMVYRCAPPPSSQFSLLIRPVVPNFNADVCMDPEPIVRIVGRNGLCV DVRDGRFHNGNAIQLWPCKSNTDANQLWTLKRDNTIRSNGKCLTTYGYSPGVY VMIYDCNTAATDATRWQIWDNGTIINPRSSLVLAATSGNSGTTLTVQTNIYAVSQ GWLPTNNTQPFVTTIVGLYGLCLQANSGQVWIEDCSSEKAEQQWALYADGSIRP QQNRDNCLTSDSNIRETVVKILSCGPASSGQRWMFKNDGTILNLYSGLVLDVRAS DPSLKQIILYPLHGDPNQIWLPLF (Ricin toxin from Ricinus communis (castor bean); Accession no. P02879; SEQ ID NO:9)

MKIIIFRVLTFFFVIFSVNVVAKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT SLLMIDSGTGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYR FADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS GTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLT LNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASD EFPSMCPADGRVRGITHNKILWDSSTLGAILMRRTISS (Shiga toxin A subunit from Shigella dysenteriae; Accession no. CAC05622; SEQ ID NO: 10) MKKTLLIAASLSFFSASALATPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRW NLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR (Shiga toxin B subunit from Shigella dysenteriae; Accession no. CAC05623; SEQ ID NO: l 1)

MKCILFKWVLCLLLGFSSVSYSREFMIDFSTQQSYVSSLNSIRTEISTPLEHISQGTT SVSVINHTPPGSYFAVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNTATNTFYRF SDFTHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLVSSYLALMEFSG NTMTRDASRAVLRFVTVTAEALRFRQIQGEFRQALSETAPVYTMTPEEVDLTLN WGRISNVLPEFRGEGGVRVGRISFNNISAILGTVAVILNCHHQGARSVRAVNEEIQ PECQITGDRPVIRINNTLWESNTAAAFLNRRAHSLNTSGE

(Shiga-like toxin type II - A subunit from Escherichia coli; Accession no. AAA16360; SEQ ID NO: 12)

MK MFMAVLFALVSVNAMAADCAKGKIEFSKYNEDDTFTVKVDGKEYWTSRW NLQPLLQSAQLTGMTVTIKSSTCESGSGFAEVQFNND

(Shiga-like toxin type II -B subunit from Escherichia coli; Accession No. CAA85781; SEQ ID NO: 13)

In some embodiments, the toxin or toxin subunit includes the initiation methionine, whereas in other embodiments, it does not. For example, the proteins shown above include the initiation methionine, but the invention nevertheless includes those proteins that lack the initiation methionine. This can also be true for the other proteins of different types, different subtypes, and even the different species described elsewhere in the specification.

In some embodiments the toxin or toxin subunit comprises or consists of one or more toxins or toxin subunits listed in Table 1. In some embodiments the toxin or toxin subunit comprises or consists of one or more polypeptides selected from among SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l l, SEQ ID NO: 12, or SEQ ID NO: 13.

Functional variants and functional fragments of biological toxins or toxin subunits, and nucleic acids encoding them, may be utilized in the various aspects of the invention. Functional variants of toxins and toxin subunits retain the same activity exhibited in vitro and/or in vivo by the native (non- variant) toxin or subunit (as a toxin or toxin constituent), though the activity may differ in extent or degree. Functional fragments of toxins or toxin subunits retain the same activity exhibited in vitro and/or in vivo by the full-length toxin or subunit (as a toxin or toxin constituent), though the activity may differ in extent or degree. Functional variants may include one or more sequence alterations anywhere in the polypeptide, e.g., one or both ends and/or in the middle portion. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional variant, resulting in a silent alteration. In some embodiments, conservative substitutions for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs (see Table 2). Conservative substitutions also include substitutions by amino acids having chemically modified side chains that do not eliminate the biological function (as a toxin or toxin constituent) of the resulting variant. It should be understood that although functional fragments and variants retain the activity exhibited by the full-length toxin or subunit or the non-variant toxin or subunit, respectively, truncation or alteration can confer new activities and/or properties and such functional fragments and variants are within the perview of the invention.

Table 2.

Class of Amino Acid Examples of Amino Acids

Nonpolar Ala, Val, Leu, He, Pro, Met, Phe, Trp

Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin

Acidic Asp, Glu

Basic Lys, Arg, His

Thus, the invention includes the use of nucleic acids that encode functional variants and functional fragments of specific biological toxins or toxin subunits. A nucleic acid or fragment thereof has substantial identity with another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide bases. A polypeptide or fragment thereof has substantial identity with another if, when optimally aligned, there is an amino acid sequence identity of at least about 60%>, 61%>, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acids.

In some embodiments, the functional variant or functional fragment is a functional variant or functional fragment of a toxin or toxin subunit listed in Table 1. In some embodiments, the functional variant or functional fragment is a functional variant or functional fragment of a polypeptide selected from among SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l l, SEQ ID NO: 12, or SEQ ID NO: 13.

The invention includes the use of variant polypeptides comprising amino acid sequences exhibiting between at least (or at least about) 20.00%> to 99.99%> (inclusive) identity to the full length, native, or naturally occurring polypeptide. The aforementioned range of percent identity is to be taken as including, and providing written description and support for, any fractional percentage, in intervals of 0.01%, between 20.00%) and, up to, including 99.99%>. These percentages are purely statistical and differences between two polypeptide sequences can be distributed randomly and over the entire sequence length. Thus, variant polypeptides can have 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with a referenced amino acid sequence or with functional fragments thereof. In one embodiment, a variant polypeptide exhibits at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the reference polypeptide. The percent identity can be calculated with reference to the full-length polypeptide or the length of the fragment of a particular SEQ ID NO: that is identified.

As indicated above, the invention includes the use of polynucleotides and polypeptides having substantial identity or substantial similarity (substantial homology) to toxin and toxin subunit sequences disclosed herein. Sequence identity means the degree of sequence relatedness between two polypeptides or two polynucleotides sequences as determined by the identity of the match between two strings of such sequences. Identity can be readily calculated. While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Methods commonly employed to determine identity between two sequences include, but are not limited to those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the two sequences tested. Such methods are codified in computer programs. Examples of computer program methods to determine identity between two sequences include, but are not limited to, GCG (Genetics Computer Group, Madison Wis.) program package (Devereux, J., et ah, Nucleic Acids Research, 1984, 12(1)387), BLASTP, BLASTN, and FASTA. The well-known Smith Waterman algorithm may also be used to determine identity.

Alternatively, substantial homology or similarity exists when a nucleic acid or fragment thereof will hybridize to another nucleic acid (or a complementary strand thereof) under selective hybridization conditions, to a strand, or to its complement. Selectivity of hybridization exists when hybridization which is substantially more selective than total lack of specificity occurs. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions will generally include temperatures in excess of 30 degrees C, typically in excess of 37 degrees C, and preferably in excess of 45 degrees C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is more important than the measure of any single parameter. Thus, as herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. Such hybridization techniques are well known to those of skill in the art. Stringent hybridization conditions are as defined above or, alternatively, conditions under overnight incubation at 42 degrees C. in a solution comprising: 50%> formamide, 5 X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5 X Denhardt's solution, 10%> dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 X SSC at about 65 degrees C.

Methods of purifying a polypeptide expressed in bacteria are well known to a person of ordinary skill in the art and these methods are within the purview of the current invention for purification of the biological toxin or one or more subunits thereof (e.g., extraction, precipitation and differential solubilization, ultracentrifugation, chromatography (size exclusion, charge, hydrophobicity, ion exchange, affinity (e.g., metal binding, immunoaffinity, purification of tagged protein), high performance liquid chromatography). Purification of the biological toxin or subunit(s) may also include one or more concentration steps (e.g., lyophylization, ultrafiltration, denaturing-condition electrophoresis, non-denaturing-condition electrophoresis). Similarly, the methods of assembling different subunits of a protein comprising multiple subunits are also well known to a person of ordinary skill in the art and are within the purview of this invention.

Optionally, the biological toxins of the invention will be labeled, so as to be directly detectable, or will be used in conjunction with secondary labeled immunoreagents which will specifically bind the toxin and/or target of the toxin. Labeled toxins may be detected and tracked in vitro or in vivo. In some embodiments, the label will have a light detectable characteristic. Potential labels include fluorophores, such as fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin and allophycocyanin. Other labels of interest may include dyes, enzymes, chemiluminescers, particles, radioisotopes, or other directly or indirectly detectable agent. The label may be attached to a toxin subunit, or non-toxin subunit, or other constituent of the biological toxin.

In an embodiment of the invention, assembling the first subunit and the carrier to produce the hybrid toxin is performed in a container by providing appropriate conditions to facilitate assembly of the biological toxin. The appropriate conditions maintained for assembling the first subunit and the carrier comprise appropriate temperature, pH, salt concentration, redox environment, and, optionally, protein folding agent. Chemical manipulations to assemble different protein subunits via a plethora of heterobifunctional crosslinking agents are well-known to a person of ordinary skill in the art and such manipulations are within the purview of this invention. For example, commercially available kits can be used to assemble different protein subunits into hybrid toxins.

An ideal preparation of a biological toxin or subunit thereof should be stable, should have low immunogenicity, should have a specific function in determined cells, should have adequate potency with controllable effects, should have long-lasting benefits, and should exert minimal or no side effects. For example, in case of botulinum toxin, the ability to design biological toxin recombinant mixtures may also enhance heavy chain (He) terminal binding to the presynaptic protein array, aiding in the control of specific vesicular and trans-membrane "SNAP (Soluble NSF Attachment Protein) Receptor" (SNARE proteins). The clinical uses for biological toxins, for example, BoNT, are still limited; however, clinical interest in the potential role of the biological toxins, for example, neurotoxins, in multiple disorders is increasing. For example, BoNT is likely to be useful for treatment of disorders beyond movement disorders, pain, and other current indications. The understanding of the pathophysiologic mechanisms by which biological toxins, for example, neurotoxins, affect specific target cells, for example, neuronal cells, and the differential effects seen with different serotypes, will aid in tailoring diagnoses and treatments individualized for a patient. To this effect, the current invention provides biological toxins and/or hybrid toxins for such individualized diagnosis and treatment.

The current invention also provides a method of producing a hybrid toxin using a bacterium. The hybrid toxin of the current invention can comprise of a first subunit of a first biological toxin and a carrier, such as a second subunit of a second biological toxin. According to the current invention, the method of producing the hybrid toxin comprises: a) introducing in to the bacterium an expressible nucleic acid encoding the first subunit of the first biological toxin and the carrier (for example, a second subunit of a second biological toxin); and

b) culturing the bacterium under conditions that allow the expression of the first subunit of the first biological toxin and the carrier. Optionally, the method further comprises c) purifying first subunit of the first biological toxin and the carrier, and, optionally d) assembling first subunit of the first biological toxin and the carrier into the hybrid toxin.

In an embodiment, the hybrid toxin produced by the method of the current invention comprises the first subunit of the first biological toxin which is the active subunit of the first biological toxin and the carrier which is a targeting subunit of a second biological toxin. In another embodiment, the hybrid toxin produced by the method of the current invention comprises the active subunit of botulinum neurotoxin and the targeting subunit of Tetanus toxin. In an even further embodiment, the hybrid toxin produced by the method of the current invention comprises the active subunit of Tetanus toxin and the targeting subunit of botulinum toxin. In an embodiment of the invention, expressing the first subunit and the carrier in a bacterium produces the hybrid toxin in the bacterium without the need of assembling the two or more components (first subunit and carrier). In another embodiment, the first subunit of the first biological toxin and the carrier are purified from the culture of the bacterium and assembled into the hybrid toxin.

Furthermore, the current invention provides a method of producing a hybrid toxin, wherein the hybrid toxin comprises of a first subunit of a first biological toxin and a carrier, such as a second subunit of a second biological toxin, the method comprising: a) introducing into a first bacterium an expressible nucleic acid encoding the first subunit of the first biological toxin;

b) introducing into a second non-pathogenic bacterium an expressible nucleic acid encoding the carrier; and

c) culturing the first and the second bacteria in one or more culture vessels under conditions that allow for the expression of the first subunit of the first biological toxin and the carrier.

Optionally, the method further comprises d) purifying the first subunit of the first biological toxin and the carrier from the one or more bioreactors; and e) assembling the first subunit of the first biological toxin and the carrier to produce the hybrid toxin.

In an embodiment of the invention, the first bacterium and the second bacterium can be cultured in the same culture vessel, i.e. co-cultured. Alternately, the first and the second bacteria can be cultured in separate culture vessels, for example, the first bacterium is cultured in a first bioreactor and the second bacterium is cultured in a second bioreactor.

In another embodiment of the invention, the first bacterium and the second bacterium belong to the same species and strain. In an embodiment of the invention, the bacterium used to produce a multi-subunit biological toxin is Lactococcus lactis.

Alternately, the first bacterium and the second bacterium can belong to different species and strain. When the first bacterium and the second bacterium belong to different species and strain, the first bacterium can belong to the genus Lactococcus and the second bacterium can belong to the genus Bacillus. In an embodiment of the invention, the first bacterium is Lactococcus lactis and the second bacterium is Bacillus subtilis.

In an embodiment of the invention, assembling the first subunit and the carrier to produce the hybrid toxin is performed in a container by providing appropriate conditions to facilitate assembly of the hybrid toxin. The appropriate conditions maintained for assembling the first subunit and the carrier comprise appropriate temperature, pH, salt concentration, redox environment, and, optionally, protein folding agent. Chemical manipulations to assemble different protein subunits via a plethora of heterobifunctional crosslinking agents are well-known to a person of ordinary skill in the art and such manipulations are within the purview of this invention. For example, ad hoc kits that are commercially available from different sources can be used to assemble different protein subunits into hybrid toxins.

The current invention also provides a hybrid toxin comprising a first subunit of a first biological toxin and a carrier, such as a second subunit of a second biological toxin. In an embodiment, the hybrid toxin of the current invention comprises the active subunit of the first biological toxin and a carrier that is the targeting subunit of a second biological toxin. In another embodiment, the hybrid toxin comprises the active subunit of botulinum neurotoxin and the targeting subunit of Tetanus toxin. In a further embodiment, the hybrid toxin comprises the active subunit of Tetanus toxin and the targeting subunit of botulinum toxin.

The current invention provides specifically tailored toxins for individualized treatment. The novel approach to provided biological toxins as provided by the current invention further expands the applications of biological toxins by providing a quick and relatively safe way to "swap" the properties of different components of biological toxins, as well as to introduce novel ligand moieties that will expand and refine targeting options and ultimately expand the many potential medical applications.

Other aspects of the invention provide hybrid proteins and compositions containing one or more of the hybrid proteins. In these aspects of the invention, a hybrid protein comprises a heterologous sequence fused to the naturally occurring protein. The additional amino acid domain or domains may be located upstream (N-terminal) or downstream (C-terminal) from the sequence of the natural protein. The heterologous sequence may further provide increased stability, targeting or bioavailability of the hybrid protein; or facilitate purification or production of compositions comprising the hybrid proteins. Non-limiting examples of additional amino acid sequences useful for purification or stabilization of the hybrid proteins of the current invention include a maltose binding protein (MBP) sequence, glutathione-S-transferase (GST) sequence, a His tag sequence, a multimerization domain, or the constant region of an immunoglobulin (Ig) molecule.

In certain aspects of the invention, various subdomains of different bacterial toxins are operably linked to a promoter. The term "operably linked" indicates that the polypeptide and additional amino acid domain are associated through peptide linkage, either directly or indirectly, e.g., via spacer residues. In this manner, the hybrid toxins of the current invention can be produced recombinantly, e.g., by direct expression in a host cell. Also, if needed, the additional amino acid sequence included in the hybrid proteins may be eliminated, either at the end of the production/purification process or in vivo, e.g., by means of an appropriate endo-/exopeptidase. For example, a spacer sequence included in the hybrid toxins may comprise a recognition site for an endopeptidase (such as a caspase) that can be used to separate by enzymatic cleavage the desired polypeptide from the additional amino acid domain, either in vivo or in vitro.

In a particular embodiment, the additional amino acid residues in the hybrid toxins comprise the constant region of an immunoglobulin, such as the Fc portion of a human immunoglobulin. The sequence of the Fc portion may be derived, for instance, from an IgG, preferably from a human IgG. The Ig sequence may also be modified to reduce effector function or to increase the stability of a resulting dimer. The amino acid sequence derived from the constant region of an immunoglobulin may be linked to the C- terminus or to the N-terminus of the naturally occurring full-length protein or subunit.

In a further embodiment, the additional amino acid residues in the hybrid toxins comprise a multimerization domain, allowing complexes to be formed between two or more hybrid toxins of this invention, or between one or more hybrid toxins of this invention and a distinct protein. An example of such multimerization domain includes a leucine zipper. The multimerization domain may be linked to the C-terminus or to the N- terminus of the naturally occurring protein, or both.

An aspect of the invention also provides a polynucleotide that encodes a hybrid toxin of the current invention. The hybrid polynucleotide may include various modifications relative to naturally occurring nucleic acids. The modifications that can be made to naturally occurring nucleic acids include alteration in the naturally occurring nucleotide sequence (e.g. substitutions, insertions, deletions, or a combination thereof of one or more nucleotides), fusion with a heterologous nucleotide sequence, attachment of labels, chemical modification of the nucleotides, incorporation of nucleotide analogues, or a combination of two or more of the foregoing modifications.

Further aspects of the invention provide compositions comprising a hybrid toxin of the current invention and one or more pharmaceutically acceptable constituents. The pharmaceutically acceptable constituents include, but are not limited to, excipients, carriers, diluents, fillers, permeation enhancers, solubilizers, chelators, and adjuvants. In certain embodiments, the pharmaceutically acceptable carrier is a non-naturally occurring compound. Examples of pharmaceutically acceptable carriers are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention. In another embodiment, the pharmaceutical composition of the current invention is sterile.

The modular expression of biological toxins in bacteria paves the way to a wide variety of applications. For example, the modular expression of the current invention can be used to deliver active peptides or drugs into specific target cells or tissues, including but not limited to, nervous system, brain, heart, muscle, bone, liver, kidney, pancreas, stomach, intestines, skin, lungs, thymus, adrenal gland, spleen, prostate, testis, ovaries, pituitary gland, thyroid gland, parathyroid gland, pineal gland, other endocrine glands, other exocrine glands, blood, lymphatic tissue, and eyes. An aspect of the current invention provides a method of targeting a hybrid toxin to a target cell by producing a composition comprising an active subunit (payload) and a targeting subunit (carrier) in a non-pathogenic bacterial cell. For example, the method of producing the hybrid toxins of the current invention can be modified to express different combinations of catalytic light chains and targeting heavy chains of binary biological toxins and multisubunit biological toxins. For example, a catalytic Lc (payload) of a first biological toxin can be directed to a target tissue using a targeting He (carrier) from a second biological toxin thereby targeting the Lc of the first biological toxin to a target cell which is not a natural target cell for the first biological toxin. For example, botulinum Lc can be targeted by its own carrier, i.e., botulinum He. Alternately, Tetanus Lc can be targeted to a specific target tissue by botulinum He. This aspect of the invention can also be manipulated to target therapeutic moieties other than biological toxins, for example, nerve growth factors, whose therapeutic applications are limited by the lack of sites- specific delivery system. An example of this aspect of the invention is described in Fig. 2.

In an embodiment of the invention the payload/carrier combination is designed to target the payload to a target cell which is not a natural target cell for the payload by combining the payload with the carrier that can deliver the payload to the target cell. For example, Tetanus Lc can be combined with botulinum He to manufacture a combination useful for delivering Tetanus Lc to a target tissue specific for botulinum He.

The carriers used according to current invention include, but are not limited to, BoNT, tetanus toxin binding domains, Adenovirus knob protein from different variants, protein sigma 1 attachment protein from reovirus type 1 , shiga-like toxin binding moiety, and rabies attachment protein.

The current invention further provides a method of delivering a biological toxin to a subject, comprising administering to the subject a toxin of the invention {e.g., a hybrid toxin) or a toxin produced according to a method of the invention. In some embodiments, the biological toxin is administered for treatment of a disorder. Thus, another aspect of the invention is a method of treating a disorder in a subject. Treatment of a disorder is intended broadly to include treatment for cosmetic purposes {e.g., as a temporary remedy for wrinkles). In some embodiments, the method of treating a subject according to the invention comprises administering to the subject a pharmaceutically effective amount of a biological toxin, such as botulinum toxin, wherein the toxin is produced using a bacterium other than Clostridium botulinum. In another aspect, the method of treating a subject according to current invention comprises administering to the subject a pharmaceutically effective amount of the hybrid toxin of the current invention. In an embodiment, the method of treating a disorder in a subject comprises administering a pharmaceutically effective amount of a hybrid toxin comprising a first subunit of a first biological toxin and a carrier, such as a second subunit of a second biological toxin, wherein the first subunit of the first biological toxin and the carrier are specifically selected to produce the hybrid toxin which treats the disease in the subject.

Examples of disorders that may be treated with an appropriate biological toxin, such as botulinum toxin, include, but are not limited to, movement disorders {e.g., Parkinson's disease, spasms, and distonias, such as idiopathic focal dystonias, tardive dystonia, and hemifacial spasm/post-facial nerve palsy synkinesis), upper motor neuron syndrome, pain, fibromyalgia, ophthalmological disorders (such as concomitant and nonconcomitant misalignment), involuntary eyelid twitching, involuntary muscle contractions, buttock deformity, anal fissure, hyperhidrosis (excessive sweating), cervical dystonia, migraine and other headache disorders, and achalasia (see, for example, [15] - [20], which are incorporated herein by reference in their entirety).

Biological toxins may be administered to a human or non-human animal subject by any route suitable to effect the intended treatment (for example, applied topically, systemically {e.g., intravenously) or by direct injection into or on a target anatomical site). For example, the biological toxin may be administered intramuscularly, transcutaneously or subcutaneously, intradermally, transconjunctivally, or directly into glands. Delayed or controlled release delivery systems may be used (see, for example [21], which is incorporated herein by reference in its entirety).

Compositions containing the biological toxins of the invention, optionally with one or more diluents and/or other active or non-active constituents, may be formulated for their intended route of administration. Preferably, compositions are suitable for topical application to the skin or mucosa, or for injection. In some embodiments, the composition is a liquid. In some embodiments, the composition is a pharmaceutically acceptable cream, salve, ointment, or gel. Compositions of the invention can be hydrophilic or hydrophobic. The composition can be an aqueous composition, although other suitable solvents, such as alcohols or other organic solvents, may be used. A combination of solvents may be used. Topically administered compositions may also comprise a matrix in which a biological toxin of the invention is dispersed so that the toxin is held in contact with the skin in order to administer the toxin transdermally.

The current invention further provides a bacterium containing an expressible genetic material encoding a heterologous biological toxin (heterologous to the bacterium). In an embodiment of the invention, the bacterium is a non-pathogenic bacterium. In an embodiment of the invention, the bacterium is a bacterium other than Clostridium spp. In another embodiment of the invention, the bacterium contains an expressible nucleic acid encoding botulinum neurotoxin. The bacterium can be a gram-positive bacterium or a gram-negative bacterium. The bacterium can be a lactic acid bacterium, for example, lactic acid bacterium belonging to a genus selected from the group consisting of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus (e.g., S. lutetiensis), Carnobacterium, Enterococcus, Oenococcus, Tetragenococcus, Vagococcus, and Weisella. If the lactic acid bacterium belongs to the genus Lactococcus, it can be selected from the group consisting of L. lactis, L. lactis subsp. lactis, L. lactis subsp. cremoris, L. lactis subsp. hordniae, L. lactis subsp. tructae, L. garvieae, L. plantarum, L. raffinolactis, L. piscium, L. chungangensis, and L. fujiensis. In an embodiment, the lactic acid bacterium of the current invention is Lactococcus lactis subs, lactis IL1403.

The non-pathogenic bacterium of the current invention can belong to the genus

Bacillus, for example, non-pathogenic bacterium selected from the group consisting of B. alcalophilus, B. alvei, B. aminovorans, B. amyloliquefaciens, B. aneurinolyticus, B. aquaemaris, B. atrophaeus, B. boroniphilus, B. brevis, B. caldolyticus, B. centrosporus, B. circulans, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, B. larvae, B. laterosporus, B. lentus, B. licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B. pantothenticus, B. polymyxa, B. pseudoanthracis, B. pumilus, B. schlegelii, B. sphaericus, B. sporothermodurans, B. subtilis, B. thermoglucosidasius, B. vulgatis, and B. weihenstephanensis. In an embodiment of the current invention, B. subtilis is used for the production of the biological toxin according to the method of the current invention.

In a further aspect of the invention, the bacterium used in the method overproduces one or more stabilizing agents. The stabilizing agents that can be used for that purpose include, but are not limited to, trehalose. In an embodiment, the bacterium used in the method of the current invention is genetically engineered to overproduce stabilizing agents. For example, trehalose biosynthetic operon from Escherichia coli can be cloned and introduced into a bacterium which improves the bacterium's tolerance to desiccation. Such bacterium having improved tolerance to desiccation can be used in the method of the current invention.

In a further aspect of the invention, the sequence of the nucleic acid encoding the biological toxin or subunit is optimized for expression in a bacterium of interest. In an embodiment, the sequence of the nucleic acid encoding the biological toxin or subunit is optimized for expression in a gram-negative bacterium or a gram-positive bacterium. Methods of optimizing the sequence of nucleic acid for expression in a gram-positive or a gram-negative bacterium are well known to a person of ordinary skill in the art of molecular biology and are within the purview of this invention. Examples of tools and methods for codon optimization that may be utilized include, but are not limited to, Fulgsang A., "Codon Optimizer: a freeware tool for codon optimization," Protein Expression and Purification, 2003, 21 :247-249; Puigbo P. et ah, "OPTIMIZER: a web server for optimizing the codon usage of DNA sequences," Nucleic Acids Research, Web Server Issue, 2007, 35:W126-131; Burgess-Brown N.A. et ah, "Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study," Protein Expression and Purification, 2008, 59:94-102; and U.S. Patent No. 5,082,767 (Hatfield G.W. et al., "Codon Pair Utilization"), which are each incorporated herein by reference in their entirety.

In a further aspect of the invention, the expression of the biological toxin or subunit in a bacterium is under the control of a promoter. The promoter can be a constitutive promoter or an inducible promoter. In an embodiment of the invention, the promoter is constitutively active in a gram-positive or a gram-negative bacterium. In another embodiment of the invention, the promoter is constitutively active in a gram- positive bacterium. In a further embodiment of the invention, the constitutive promoter is a composite sequence derived from Gram-positive promoters, for example, P24 coupled to pi 70, the latter of which becomes activated under acidic conditions typically at the end of the growth period for LAB, thus extending the time for protein production. In an embodiment of the invention, the inducible promoter used to control nucleic acid expression (polypeptide production) is a nisin promoter.

In a further aspect of the invention, the nucleic acid encoding the biological toxin or subunit is designed to contain an mRNA stabilization sequence. The mRNA stabilization sequence can be present as an untranslated mRNA sequence. The untranslated mRNA stabilization sequence can be present at the 5 ' or the 3 ' region of the mRNA. Specific sequences and locations of the sequences that stabilize mRNA are well known to a person of ordinary skill in the art and are within the purview of the current invention.

The current invention also provides a method of treating a disorder in a subject, wherein the method comprises administering to the subject a pharmaceutically effective amount of the hybrid toxin of the current invention. An aspect of the current invention provides a method of treating a disorder in a subject, the method comprising administering to the subject a pharmaceutically effective amount of the hybrid toxin which is specifically designed to treat the disorder in the subject.

Exemplified Embodiments

Embodiment 1. A method of producing a biological toxin, or a subunit thereof, using a bacterium other than Clostridium botulinum, the method comprising: a) introducing into the bacterium an expressible nucleic acid encoding the biological toxin or subunit; and

b) culturing the bacterium containing the introduced nucleic acid under conditions that allow expression of the nucleic acid and production of the biological toxin or subunit.

Embodiment 2. The method of embodiment 1, further comprising: c) purifying the biological toxin or subunit from the culture of the bacterium.

Embodiment 3. The method of embodiment 1, wherein the biological toxin comprises multiple subunits.

Embodiment 4. The method of embodiment 1, wherein the biological toxin is botulinum neurotoxin.

Embodiment 5. The method of embodiment 1, wherein the bacterium is a non-pathogenic bacterium. Embodiment 6. The method of embodiment 1, wherein the bacterium is a gram-positive bacterium or a gram-negative bacterium.

Embodiment 7. The method of embodiment 1, wherein the bacterium is a lactic acid bacterium.

Embodiment 8. The method of embodiment 7, wherein the lactic acid bacterium belongs to the genus selected from the group consisting of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Carnobacterium, Enterococcus, Oenococcus, Tetragenococcus, Vagococcus, and Weisella.

Embodiment 9. The method of embodiment 7, wherein the lactic acid bacterium is a member of the genus Lactococcus.

Embodiment 10. The method of embodiment 8, wherein the lactic acid bacterium is selected from the group consisting of L. lactis, L. lactis subsp. lactis, L. lactis subsp. cremoris, L. lactis subsp. hordniae, L. lactis subsp. tructae, L. garvieae, L. plantarum, L. raffinolactis, L. piscium, L. chungangensis, and L. fujiensis .

Embodiment 11. The method of embodiment 10, wherein the lactic acid bacterium is Lactococcus lactis.

Embodiment 12. The method of embodiment 1, wherein the bacterium belongs to the genus Bacillus.

Embodiment 13. The method of embodiment 12, wherein the bacterium belongs to a species selected from the group consisting of B. alcalophilus, B. alvei, B. aminovorans, B. amyloliquefaciens, B. aneurinolyticus, B. aquaemaris, B. atrophaeus, B. boroniphilus, B. brevis, B. caldolyticus, B. centrosporus, B. circulans, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, B. larvae, B. laterosporus, B. lentus, B. licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B. pantothenticus, B. polymyxa, B. pseudoanthracis, B. pumilus, B. schlegelii, B. sphaericus, B. sporothermodurans, B. subtilis, B. thermoglucosidasius, B. vulgatis, and B. weihenstephanensis.

Embodiment 14. The method of embodiment 13, wherein the bacterium is Bacillus subtilis.

Embodiment 15. A method of producing a biological toxin comprising a first subunit and a second subunit, the method comprising: a) introducing into a first bacterium an expressible nucleic acid encoding the first subunit;

b) introducing into a second bacterium an expressible nucleic acid encoding the second subunit; and

c) culturing the first and the second bacteria containing the expressible nucleic acids under conditions that allow expression of the first subunit and the second subunit.

Embodiment 16. The method of embodiment 15, further comprising: d) purifying the first subunit and the second subunit from the culture of the first bacterium and the second bacterium.

Embodiment 17. The method of embodiment 16, further comprising: e) assembling the first subunit and the second subunit to produce the biological toxin.

Embodiment 18. The method of embodiment 15, wherein the first bacterium and/or the second bacterium are non-pathogenic bacterium.

Embodiment 19. The method of embodiment 15, wherein both the first bacterium and the second bacterium are cultured in the same culture vessel.

Embodiment 20. The method of embodiment 15, wherein the first bacterium and the second bacterium are cultured in separate culture vessels.

Embodiment 21. The method of embodiment 15, wherein the first bacterium and the second bacterium belong to the same species and strain.

Embodiment 22. The method of embodiment 15, wherein both the first bacterium and the second bacterium are lactic acid bacteria.

Embodiment 23. The method of embodiment 15, wherein both the first bacterium and the second bacterium are Lactococcus lactis.

Embodiment 24. The method of embodiment 15, wherein the first bacterium and the second bacterium belong to different species and strain.

Embodiment 25. The method of embodiment 24, wherein the first bacterium belong to the genus Lactococcus and the second bacterium belong to the genus Bacillus.

Embodiment 26. The method of embodiment 17, wherein assembling the first subunit of the first biological toxin and the second subunit of the second biological toxin to produce the biological toxin is performed in a container by providing appropriate conditions. Embodiment 27. The method of embodiment 26, wherein the appropriate conditions comprise appropriate temperature, pH, salt concentration, redox environment, and protein folding agent.

Embodiment 28. A bacterium containing an expressible nucleic acid encoding a heterologous biological toxin or subunit thereof.

Embodiment 29. The bacterium of embodiment 28, wherein the bacterium is non-pathogenic.

Embodiment 30. The bacterium of embodiment 28, wherein the biological toxin is botulinum neurotoxin.

Embodiment 31. The bacterium of embodiment 28, wherein the bacterium is other than Clostridium spp.

Embodiment 32. The bacterium of embodiment 28, wherein the bacterium is a gram-positive bacterium or a gram-negative bacterium.

Embodiment 33. The bacterium of embodiment 28, wherein the bacterium is a lactic acid bacterium.

Embodiment 34. The bacterium of embodiment 33, wherein the lactic acid bacterium belongs to a genus selected from the group consisting of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Carnobacterium, Enterococcus, Oenococcus, Tetragenococcus, Vagococcus, and Weisella.

Embodiment 35. The bacterium of embodiment 34, wherein the lactic acid bacterium belongs to the genus Lactococcus.

Embodiment 36. The bacterium of embodiment 35, wherein the lactic acid bacterium is selected from the group consisting of L. lactis, L. lactis subsp. lactis, L. lactis subsp. cremoris, L. lactis subsp. hordniae, L. lactis subsp. tructae, L. garvieae, L. plantarum, L. raffinolactis, L. piscium, L. chungangensis, and L. fujiensis .

Embodiment 37. The bacterium of embodiment 36, wherein the lactic acid bacterium is Lactococcus lactis.

Embodiment 38. The bacterium of embodiment 36, wherein the lactic acid bacterium is Lactococcus lactis IL1403.

Embodiment 39. The bacterium of embodiment 28, wherein the bacterium belongs to the genus Bacillus. Embodiment 40. The bacterium of embodiment 39, wherein the bacterium is selected from the group consisting of B. alcalophilus, B. alvei, B. aminovorans, B. amyloliquefaciens, B. aneurinolyticus, B. aquaemaris, B. atrophaeus, B. boroniphilus, B. brevis, B. caldolyticus, B. centrosporus, B. circulans, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, B. larvae, B. laterosporus, B. lentus, B. licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B. pantothenticus, B. polymyxa, B. pseudoanthracis, B. pumilus, B. schlegelii, B. sphaericus, B. sporothermodurans, B. subtilis, B. thermoglucosidasius, B. vulgatis, and B. weihenstephanensis.

Embodiment 41. The bacterium of embodiment 40, wherein the bacterium is

Bacillus subtilis.

Embodiment 42. A method of producing a hybrid toxin using a bacterium, wherein the hybrid toxin comprises of a first subunit of a first biological toxin and a carrier, the method comprising:

a) introducing into the bacterium a expressible nucleic acid encoding the first subunit of the first biological toxin and the carrier; and

b) culturing the bacterium in conditions that allow for the expression of the first subunit of the first biological toxin and the carrier.

Embodiment 43. The method of embodiment 42, further comprising c) purifying the hybrid toxin.

Embodiment 44. The method of embodiment 42, wherein the bacterium is non-pathogenic.

Embodiment 45. The method of embodiment 42, wherein the bacterium belongs to the genus Lactococcus.

Embodiment 46. The method of embodiment 42, wherein the bacterium is

Lactococcus lactis.

Embodiment 47. The method of embodiment 42, wherein the carrier is a subunit of a second biological toxin.

Embodiment 48. The method of embodiment 42, wherein the first subunit of the first biological toxin is the active subunit of the first biological toxin and the carrier is a targeting subunit of a second biological toxin. Embodiment 49. The method of embodiment 42, wherein the first subunit of the first biological toxin is the active subunit of botulinum neurotoxin and the carrier is selected from among a targeting subunit of Tetanus toxin, adenovirus knob, reovirus protein sigma 1 , Shiga-like toxin binding moiety, or rabies attachment protein.

Embodiment 50. The method of embodiment 42, wherein the first subunit of the first biological toxin is the active subunit of Tetanus toxin and the carrier is the targeting subunit of botulinum toxin.

Embodiment 51. A method of producing a hybrid toxin, wherein the hybrid toxin comprises of a first subunit of a first biological toxin and a carrier, the method comprising:

a) introducing into a first bacterium an expressible nucleic acid encoding the first subunit of the first biological toxin;

b) introducing into a second bacterium an expressible nucleic acid encoding the carrier; and

c) culturing the first and the second bacteria in one or more bioreactors under conditions that allow for the expression of the first subunit of the first biological toxin and the carrier.

Embodiment 52. The method of embodiment 51 , further comprising d) purifying the first subunit of the first biological toxin and the carrier from the one or more bioreactors.

Embodiment 53. The method of embodiment 52, further comprising e) assembling the first subunit of the first biological toxin and the carrier to manufacture the hybrid toxin.

Embodiment 54. The method of embodiment 51, wherein the first bacterium and/or the second bacterium is non-pathogenic.

Embodiment 55. The method of embodiment 51, wherein the first bacterium and the second bacterium are cultured in the same culture vessel.

Embodiment 56. The method of embodiment 51, wherein the first bacterium and the second bacterium are cultured in different bioreactors.

Embodiment 57. The method of embodiment 51, wherein the first and the second bacteria belong to the same species and strain. Embodiment 58. The method of embodiment 51, wherein both the first bacterium and the second bacterium are lactic acid bacteria.

Embodiment 59. The method of embodiment 51, wherein both the first bacterium and the second bacterium are Lactococcus lactis.

Embodiment 60. The method of embodiment 51 , wherein the first bacterium and the second bacterium belong to different species or strain.

Embodiment 61. The method of embodiment 51, wherein the first bacterium belongs to the genus Lactococcus and the second bacterium belongs to the genus Bacillus.

Embodiment 62. The method of embodiment 52, wherein assembling the first subunit of the first biological toxin and the carrier to manufacture the hybrid toxin is performed in a container by providing appropriate conditions.

Embodiment 63. The method of embodiment 62, wherein the appropriate conditions comprise appropriate temperature, pH, salt concentration, redox environment, and, optionally, protein folding agent.

Embodiment 64. The method of embodiment 51, wherein the carrier is a subunit of a second biological toxin.

Embodiment 65. The method of embodiment 51, wherein the carrier is selected from among a targeting subunit of Tetanus toxin, adenovirus knob, reovirus protein sigma 1 , Shiga-like toxin binding moiety, or rabies attachment protein.

Embodiment 66. A hybrid toxin comprising a first subunit of a first biological toxin and a carrier.

Embodiment 67. The hybrid toxin of embodiment 66, wherein the carrier is a subunit of a second biological toxin.

Embodiment 68. The hybrid toxin of embodiment 66, wherein the first subunit of the first biological toxin is the active subunit of the first biological toxin and the carrier is the targeting subunit of the second biological toxin.

Embodiment 69. The hybrid toxin of embodiment 66, wherein the first subunit of the first biological toxin is the active subunit of botulinum toxin and the carrier is selected from among a targeting subunit of the Tetanus toxin, adenovirus knob, reovirus protein sigma 1 , Shiga-like toxin binding moiety, or rabies attachment protein. Embodiment 70. The hybrid toxin of embodiment 66, wherein the first subunit of the first biological toxin is the active subunit of Tetanus toxin and the carrier is the targeting subunit of botulinum toxin

Embodiment 71. A method of delivering a toxin, toxin subunit, or hybrid toxin to a subject, comprising administering to the subject the toxin, toxin subunit, or hybrid toxin of any preceding embodiment.

Embodiment 72. A method of treating a disorder in a subject, the method comprising administering to the subject a pharmaceutically effective amount of the toxin, subunit or hybrid toxin of any preceding embodiment.

Embodiment 73. A method of treating a disorder in a subject, the method comprising administering to the subject a pharmaceutically effective amount of the hybrid toxin of any preceding embodiment, wherein the first subunit of the first biological toxin and the carrier are specifically selected to treat the disorder in the subject.

Embodiment 74. A biological toxin or hybrid toxin produced by a method of any one of embodiments 1-27 or 42-65.

Embodiment 75. A composition comprising a biological toxin or hybrid toxin of any preceding embodiment.

Embodiment 76. The composition of embodiment 75, wherein the composition is a liquid, cream, salve, ointment, or gel.

Further Definitions

As used herein, the term "nucleic acid" refers to genetic material including, but not limited to, RNA, DNA, cDNA, etc. Further, expressible nucleic acid is the genetic material capable of being transcribed and translated into proteins. Examples of expressible nucleic acid include, but are not limited to, plasmids containing genes that are transcribed and translated into proteins by the host cell, genes that are incorporated in to the genome of a host cell that are transcribed and translated into proteins, RNA translated into proteins, DNA transcribed and translated into proteins in vitro, and RNA translated into proteins in vitro. Additional examples of expressible nucleic acid are well known to a person of ordinary skill in the art and are within the purview of the current invention. Expressible nucleic acid can encode one or more polypeptides. As used herein, the terms "oligopeptide", "polypeptide", "peptide" and "protein" are used interchangeably to refer to a series of residues of any length (two or more), typically L-amino acids, connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids. Linker elements can be joined to the polypeptides of the subject invention, for example, through peptide bonds or via chemical bonds (e.g., heterobifunctional chemical linker elements) as set forth below. Additionally, the terms "amino acid(s)" and "residue(s)" can be used interchangeably. Polypeptides according to the instant invention may also contain non-natural amino acids. In some embodiments, the polypeptides are not in their natural environment but may have been isolated or obtained by purification from natural sources or obtained from cultures or host cells prepared by genetic manipulation (e.g., the peptides, or fragments thereof, are recombinantly produced by host cells, or by chemical synthesis).

As used herein, the phrase "introducing a nucleic acid into a bacterium" comprises inserting the nucleic acid into one or more bacterium in such a way that the genetic material can be expressed into polypeptides. The genetic material introduced into the bacterium can exist independently of the bacterial genome, for example, a plasmid, or the genetic material introduced in to the bacterium can get incorporated into the bacterial genome.

For the purposes of this invention, the conditions that allow expression of expressible nucleic acids include, but are not limited to, providing an appropriate host for the expression, appropriate culture medium, temperature, pH, availability of inducers when inducible promoters are used, etc. Appropriate culture conditions that allow for the expression of nucleic acids are well known to a person of ordinary skill in the art and are within the purview of this invention.

As used herein, the term "bioreactor" refers to an apparatus for growing microorganisms, such as bacteria or yeast, under controlled conditions, and that are used in the production of a biological substance. Various types of bioreactors are well known to a person of ordinary skill in the art and are within the purview of the current invention. Bioreactors typically include a bioreactor vessel (a container that holds the media and cells) and ports for input and output of material. Bioreactors may also include an agitator, sparger, baffles, probes (temperature, dissolved oxygen, pH, pressure gauge), cooling jacket, condenser, back pressure valve, inlet filters, exhaust filters, valves, and a load cell. The culture media utilized can be defined or undefined as appropriate for the culture of the bacteria and production of the toxin. Examples of media include, but are not limited to, enriched media and selective media.

As used herein, the terms "treating," "treatment", and grammatical variations thereof refer to the cure of the disorder, mitigation of the disorder, alleviation or elimination one or more symptoms of the disorder, delay in the onset of the disorder, or prevention of the disorder or other undesirable condition.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting.

EXAMPLE 1 - PRODUCING BOTULINUM NEUROTOXIN IN LACTOCOCCUS LACTIS

The overall modular strategy used for DNA constructs is different from those reported in the literature for E. coli and the expression is optimized in Gram-positive bacteria and expressed in Lactococcus lactis as opposed to E. coli. Understanding that the Lc needs its own cognate heavy chain N-terminus (Hn) to execute effective delivery, other groups have fused Lc to Hn and have expressed heavy chain C-terminus (He; the binding moiety) as a separate protein. In contrast, according to an embodiment of the current invention, Lc is expressed as a separate protein and Hn is fused to He through a GGGCCC Apal site in-frame to provide for a Pro-Gly flexible linker.

The LAB expression was designed to operate using two different promoters allowing flexibility for expression of BoNT modules in two different embodiments. One embodiment uses the nisin promoter, which is inducible and leads to a more stable expression. The other promoter is unique and it is constitutive. This constitutive promoter is a composite sequence derived from a broad range of Gram-positive promoters, for example, P24 coupled to pi 70, which becomes activated under acidic conditions typically at the end of growth period for LAB. Each component of the promoter also contains untranslated sequences that stabilize the mR A.

The coding sequences for the genes are optimized for expression in Lactococcus lactis, which can also be modified for expression in any Lactococcus spp. or any related species. The starting material is pLactoBoNT/A-Lc, provided by Genescript in pUC57 and oriented 5 '-3 ' Hindlll-EcoRI. The synthetic DNA coding for catalytically inactive Lc contains a Notl-stuffer-Smal sequence downstream of the STOP codon to enable further cloning. Using the Xbal site in the vector polylinker sequence, the promoter-Lc cassette as a Bglll-Xbal fragment was cloned into the vector, pMSP3535H3. This cloning resulted in the removal of the resident nisin-inducible promoter and generated pBzMM200 (Fig. 1A). Insertion of Hn and He as separate genes downstream of Lc generated pBzMM201 (Fig. IB) which failed to produce any detectable He and was therefore discarded. As an alternative, Hn was provided with Notl and Apal ends, and He with Apal and Smal ends. Then, a tripartite ligation was set whereby pBzMM200, restricted with Notl and Smal was closed by bridging both Hn (Notl -Apal) and He (Apal- Smal). The results from this procedure are illustrated in the map for pGnMMl (Fig. 1C). This version of the modular Hn-Hc (heavy chain) was also expressed separately under the nisin-inducible promoter by adding Agel sites at both ends and cloning it into the pMSP3535H3 and cutting with Agel. This procedure generated pGnMM2 (Fig. ID). The same strategy was used for expressing He alone with pGnMM3 (Fig. IE) and, again, Lc together with Hn-Hc as inducible proteins with pGnMM5 (Figl . F).

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto. REFERENCES

I . Jankovic, J. Disease-oriented approach to botulinum toxin use. Toxicon 2009, 54, 614-623. 2. Olesen, J.; Tfelt-Hansen, P. Licence for Botox in so-called chronic migraine.

Lancet 2010, 376, 1825-1826; discussion 1826.

3. FDA approves Botox to treat specific form of urinary incontinence. Available online:

www.fda.gov/NewsEvents/Newsroorn/PressAnnouncements/ucm269509.htm (accessed on 24 October 2011).

4. Hasler, W.L. The expanding spectrum of clinical uses for botulinum toxin:

Healing of chronic anal fissures. Gastroenterology 1999, 116, 221-223.

5. Dykstra, D.D.; Sidi, A. A. Treatment of detrusor-sphincter dyssynergia with botulinum A toxin: A double-blind study. Arch. Phys. Med. Rehabil. 1990, 71, 24-26. 6. Snow, B.J.; Tsui, J.K.; Bhatt, M.H.; Varelas, M.; Hashimoto, S.A.; Calne, D.B.

Treatment of spasticity with botulinum toxin: a double-blind study. Ann. Neurol. 1990, 25, 512-515.

7. Das, T.K.; Park, D.M. Effect of treatment with botulinum toxin on spasticity.

Postgrad. Med. J. 1989, 65, 208-210.

8. Rollnik, J.D.; Tanneberger, O.; Schubert, M.; Schneider, U.; Dengler, R.

Treatment of tension-type headache with botulinum toxin type A: A double-blind, placebo-controlled study. Headache 2000, 40, 300-305.

9. Carruthers, A.; Carruthers, J. Botulinum toxin products overview. Skin Ther. Lett.

2008, 13, 1-4.

10. EXS. 2010;100:1-29. Toxins from bacteria. Henkel JS, Baldwin MR, Barbieri JT.

I I . Curr. Med Chem. 2008;15(5):459-69. Binary actin-ADP-ribosylating toxins and their use as molecular Trojan horses for drug delivery into eukaryotic cells. Barth H, Stiles BG. 12. Botulinum neurotoxin: a marvel of protein design. Montal M. Annu Rev Biochem.

2010; 79:591-617. doi: 10.1146/annurev.biochem.051908.125345. Review.

13. http://www.ncbi.nlm.nih.gov/pubmed/22141396, FEBS J. 2012; 279(3):515-23. doi: 10.1111/j.1742-4658.2011.08444.x. Stancombe PR, Masuyer G, Birch- Machin I, Beard M, Foster KA, Chaddock JA, Acharya KR.

14. Expression and purification of the light chain of botulinum neurotoxin A: a single mutation abolishes its cleavage of SNAP-25 and neurotoxicity after reconstitution with the heavy chain. http://www.ncbi.nlm.nih.gov/pubmed/7578132. 1995; 34(46): 15175-81. Zhou L, de Paiva A, Liu D, Aoki R, Dolly JO.

Uses of botulinum toxin injection in medicine today. BMJ. 2000, 320: 161-165. Munchau A, Bhatia KP.

Botulinum toxin: clinical use. Parkinsonism Relat. Disord. 2006; Sep. 12(6):331- 355. Epub 2006 Jul 25. Truong DD, Jost WH.

The use of botulinum toxin in physical medicine and rehabilitation. Bol. Asoc. Med. P R. 2006 Jan-Mar. 98(l):42-55. Cuevas-Tristan RL, Cruz-Jimenez M.

The current use of botulinum toxin. J. Clin. Neurosci. 2000; Sep. 7(5):389-394. Mahant N, Clouston PD, Lorentz IT.

Management of spasticity in adults: practical application of botulinum toxin. Eur. J. neural. 2006; Feb. 13 Suppl 1 :42-50. Pathak MS, Nguyen HT, Graham HK, Moore AP.

Botulinum toxin in movement disorders. Semin. Neurol. 2007; Apr. 72(2): 183- 194. Papapetropoulos S, Singer C.

Botulinum toxin implant. U.S. Patent Publication Number 20020028216. Published March 7, 2002. Donovan S.

Claims

CLAIMS We claim:

1. A method of producing a biological toxin, or a subunit thereof, using a bacterium other than Clostridium Botulinum, the method comprising:

a) introducing into the bacterium an expressible nucleic acid encoding the biological toxin or subunit; and

b) culturing the bacterium containing the introduced nucleic acid under conditions that allow expression of the nucleic acid and production of the biological toxin or subunit.

2. The method of claim 1, further comprising: c) purifying the biological toxin or subunit from the culture of the bacterium.

3. The method of claim 1, wherein the biological toxin comprises multiple subunits.

4. The method of claim 1 , wherein the biological toxin is botulinum neurotoxin.

5. The method of claim 1, wherein the bacterium is a non-pathogenic bacterium.

6. The method of claim 1, wherein the bacterium is a gram-positive bacterium or a gram-negative bacterium.

7. The method of claim 1 , wherein the bacterium is a lactic acid bacterium.

8. The method of claim 7, wherein the lactic acid bacterium belongs to the genus selected from the group consisting of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Carnobacterium, Enterococcus, Oenococcus, Tetragenococcus, Vagococcus, and Weisella.

9. The method of claim 7, wherein the lactic acid bacterium is a member of the genus Lactococcus.

10. The method of claim 8, wherein the lactic acid bacterium is selected from the group consisting of L. lactis, L. lactis subsp. lactis, L. lactis subsp. cremoris, L. lactis subsp. hordniae, L. lactis subsp. tructae, L. garvieae, L. plantarum, L. raffinolactis, L. piscium, L. chungangensis, and L.fujiensis.

11. The method of claim 10, wherein the lactic acid bacterium is Lactococcus lactis.

12. The method of claim 1, wherein the bacterium belongs to the genus Bacillus.

13. The method of claim 12, wherein the bacterium belongs to a species selected from the group consisting of B. alcalophilus, B. alvei, B. aminovorans, B. amyloliquefaciens, B. aneurinolyticus, B. aquaemaris, B. atrophaeus, B. boroniphilus, B. brevis, B. caldolyticus, B. centrosporus, B. circulans, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, B. larvae, B. laterosporus, B. lentus, B. licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B. pantothenticus, B. polymyxa, B. pseudoanthracis, B. pumilus, B. schlegelii, B. sphaericus, B. sporothermodurans, B. subtilis, B. thermoglucosidasius, B. vulgatis, and B. weihenstephanensis.

14. The method of claim 13, wherein the bacterium is Bacillus subtilis.

15. A method of producing a biological toxin comprising a first subunit and a second subunit, the method comprising:

a) introducing into a first bacterium an expressible nucleic acid encoding the first subunit;

b) introducing into a second bacterium an expressible nucleic acid encoding the second subunit; and

c) culturing the first and the second bacteria containing the expressible nucleic acids under conditions that allow expression of the first subunit and the second subunit.

16. The method of claim 15, further comprising: d) purifying the first subunit and the second subunit from the culture of the first bacterium and the second bacterium.

17. The method of claim 16, further comprising: e) assembling the first subunit and the second subunit to produce the biological toxin.

18. The method of claim 15, wherein the first bacterium and/or the second bacterium are non-pathogenic bacterium.

19. The method of claim 15, wherein both the first bacterium and the second bacterium are cultured in the same culture vessel.

20. The method of claim 15, wherein the first bacterium and the second bacterium are cultured in separate culture vessels.

21. The method of claim 15, wherein the first bacterium and the second bacterium belong to the same species and strain.

22. The method of claim 15, wherein both the first bacterium and the second bacterium are lactic acid bacteria.

23. The method of claim 15, wherein both the first bacterium and the second bacterium are Lactococcus lactis.

24. The method of claim 15, wherein the first bacterium and the second bacterium belong to different species and strain.

25. The method of claim 24, wherein the first bacterium belong to the genus Lactococcus and the second bacterium belong to the genus Bacillus.

26. The method of claim 17, wherein assembling the first subunit of the first biological toxin and the second subunit of the second biological toxin to produce the biological toxin is performed in a container by providing appropriate conditions.

27. The method of claim 26, wherein the appropriate conditions comprise appropriate temperature, pH, salt concentration, redox environment, and protein folding agent.

28. A bacterium containing an expressible nucleic acid encoding a heterologous biological toxin or subunit thereof.

29. The bacterium of claim 28, wherein the bacterium is non-pathogenic.

30. The bacterium of claim 28, wherein the biological toxin is botulinum neurotoxin.

31. The bacterium of claim 28, wherein the bacterium is other than Clostridium spp.

32. The bacterium of claim 28, wherein the bacterium is a gram-positive bacterium or a gram-negative bacterium.

33. The bacterium of claim 28, wherein the bacterium is a lactic acid bacterium.

34. The bacterium of claim 33, wherein the lactic acid bacterium belongs to a genus selected from the group consisting of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Carnobacterium, Enterococcus, Oenococcus, Tetragenococcus, Vagococcus, and Weisella.

35. The bacterium of claim 34, wherein the lactic acid bacterium belongs to the genus Lactococcus.

36. The bacterium of claim 35, wherein the lactic acid bacterium is selected from the group consisting of L. lactis, L. lactis subsp. lactis, L. lactis subsp. cremoris, L. lactis subsp. hordniae, L. lactis subsp. tructae, L. garvieae, L. plantarum, L. raffinolactis, L. piscium, L. chungangensis, and L.fujiensis.

37. The bacterium of claim 36, wherein the lactic acid bacterium is Lactococcus lactis.

38. The bacterium of claim 36, wherein the lactic acid bacterium is Lactococcus lactis IL1403.

39. The bacterium of claim 28, wherein the bacterium belongs to the genus Bacillus.

40. The bacterium of claim 39, wherein the bacterium is selected from the group consisting of B. alcalophilus, B. alvei, B. aminovorans, B. amyloliquefaciens, B. aneurinolyticus, B. aquaemaris, B. atrophaeus, B. boroniphilus, B. brevis, B. caldolyticus, B. centrosporus, B. circulans, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B. globigii, B. infernus, B. larvae, B. laterosporus, B. lentus, B. licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B. pantothenticus, B. polymyxa, B. pseudoanthracis, B. pumilus, B. schlegelii, B. sphaericus, B. sporothermodurans, B. subtilis, B. thermoglucosidasius, B. vulgatis, and B. weihenstephanensis.

41. The bacterium of claim 40, wherein the bacterium is Bacillus subtilis.

42. A method of producing a hybrid toxin using a bacterium, wherein the hybrid toxin comprises of a first subunit of a first biological toxin and a carrier, the method comprising:

a) introducing into the bacterium a expressible nucleic acid encoding the first subunit of the first biological toxin and the carrier; and

b) culturing the bacterium in conditions that allow for the expression of the first subunit of the first biological toxin and the carrier.

43. The method of claim 42, further comprising c) purifying the hybrid toxin.

44. The method of claim 42, wherein the bacterium is non-pathogenic.

45. The method of claim 42, wherein the bacterium belongs to the genus Lactococcus.

46. The method of claim 42, wherein the bacterium is Lactococcus lactis.

47. The method of claim 42, wherein the carrier is a subunit of a second biological toxin.

48. The method of claim 42, wherein the first subunit of the first biological toxin is the active subunit of the first biological toxin and the carrier is a targeting subunit of a second biological toxin.

49. The method of claim 42, wherein the first subunit of the first biological toxin is the active subunit of botulinum neurotoxin and the carrier is selected from among a targeting subunit of Tetanus toxin, adenovirus knob, reovirus protein sigma 1, Shiga-like toxin binding moiety, or rabies attachment protein.

50. The method of claim 42, wherein the first subunit of the first biological toxin is the active subunit of Tetanus toxin and the carrier is the targeting subunit of botulinum toxin.

51. A method of producing a hybrid toxin, wherein the hybrid toxin comprises of a first subunit of a first biological toxin and a carrier, the method comprising:

a) introducing into a first bacterium an expressible nucleic acid encoding the first subunit of the first biological toxin;

b) introducing into a second bacterium an expressible nucleic acid encoding the carrier; and

c) culturing the first and the second bacteria in one or more bioreactors under conditions that allow for the expression of the first subunit of the first biological toxin and the carrier.

52. The method of claim 51, further comprising d) purifying the first subunit of the first biological toxin and the carrier from the one or more bioreactors.

53. The method of claim 52, further comprising e) assembling the first subunit of the first biological toxin and the carrier to manufacture the hybrid toxin.

54. The method of claim 51, wherein the first bacterium and/or the second bacterium is non-pathogenic.

55. The method of claim 51, wherein the first bacterium and the second bacterium are cultured in the same culture vessel.

56. The method of claim 51 , wherein the first bacterium and the second bacterium are cultured in different bioreactors.

57. The method of claim 51, wherein the first and the second bacteria belong to the same species and strain.

58. The method of claim 51, wherein both the first bacterium and the second bacterium are lactic acid bacteria.

59. The method of claim 51, wherein both the first bacterium and the second bacterium are Lactococcus lactis.

60. The method of claim 51 , wherein the first bacterium and the second bacterium belong to different species or strain.

61. The method of claim 51, wherein the first bacterium belongs to the genus Lactococcus and the second bacterium belongs to the genus Bacillus.

62. The method of claim 52, wherein assembling the first subunit of the first biological toxin and the carrier to manufacture the hybrid toxin is performed in a container by providing appropriate conditions.

63. The method of claim 62, wherein the appropriate conditions comprise appropriate temperature, pH, salt concentration, redox environment, and, optionally, protein folding agent.

64. The method of claim 51 , wherein the carrier is a subunit of a second biological toxin.

65. The method of claim 51 , wherein the carrier is selected from among a targeting subunit of Tetanus toxin, adenovirus knob, reovirus protein sigma 1, Shiga-like toxin binding moiety, or rabies attachment protein.

66. A hybrid toxin comprising a first subunit of a first biological toxin and a carrier.

67. The hybrid toxin of claim 66, wherein the carrier is a subunit of a second biological toxin.

68. The hybrid toxin of claim 66, wherein the first subunit of the first biological toxin is the active subunit of the first biological toxin and the carrier is the targeting subunit of the second biological toxin.

69. The hybrid toxin of claim 66, wherein the first subunit of the first biological toxin is the active subunit of botulinum toxin and the carrier is selected from among a targeting subunit of the Tetanus toxin, adenovirus knob, reovirus protein sigma 1, Shiga-like toxin binding moiety, or rabies attachment protein.

70. The hybrid toxin of claim 66, wherein the first subunit of the first biological toxin is the active subunit of Tetanus toxin and the carrier is the targeting subunit of botulinum toxin.

71. A method of delivering a toxin, toxin subunit, or hybrid toxin to a subject, comprising administering to the subject the toxin, toxin subunit, or hybrid toxin of any preceding claim.

72. A method of treating a disorder in a subject, the method comprising administering to the subject a pharmaceutically effective amount of the toxin, subunit or hybrid toxin of any preceding claim.

73. A method of treating a disorder in a subject, the method comprising administering to the subject a pharmaceutically effective amount of the hybrid toxin of any preceding claim, wherein the first subunit of the first biological toxin and the carrier are specifically selected to treat the disorder in the subject.

74. A biological toxin or hybrid toxin produced by a method of any one of claims 1-27 or 42-65.

75. A composition comprising a biological toxin or hybrid toxin of any one of claims 1-27 or 42-70.

76. The composition of claim 75, wherein the composition is a liquid, cream, salve, ointment, or gel.