A METHOD FOR TARGETING ACETYLCHOLINESTERASE MOLECULES TO THE NEUROMUSCULAR SYNAPSE

GOVERNMENT SUPPORT

Work described herein may have been supported in part by NIH Grant number ROl

AG05917 from the National Institute on Aging (NIA). The U.S. Government may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention is directed generally to the field of molecular genetics. More particularly, the present invention describes novel DNA constructs, novel chimeric fusion proteins encoded by the novel DNA constructs, and novel methods for treating animals including humans by administering the novel chimeric fusion proteins.

BACKGROUND OF THE INVENTION

Since the introduction of biological and chemical weapons during the First World War, intensive research and development by the super power nations has led to the creation of large stockpiles of chemical weapons and new technologies for the delivery of these weapons. These weapons of mass destruction are so named because of their ability to kill enormous numbers of people in a short period. Weapons of this nature are attractive to both developing countries and terrorist groups because they are easier and cheaper to acquire than nuclear weapons. Moreover, the technologies and synthetic methodologies for manufacturing some chemical weapons are openly available or easily accessible to the public. Terrorists may seek to obtain greater status or bargaining power against their more developed enemies by demonstrating that they have the technological capabilities required to develop, produce, and deliver chemical and biological warfare agents.

Although the use of biological and chemical weapons is banned by international treaty, these weapons are thought to be in the stockpiles of several extremist nations and terrorist organizations. The release of the nerve gas sarin in the Tokyo subway system in 1995, killing 12 and wounding over 1000, demonstrated the consequences of chemical warfare technology in the hands of terrorists. In the United States, the threat and fear of potential terrorist attacks using biological and chemical weapons has been particularly elevated since the attacks of September 11, 2001.

Nerve gases, such as sarin, soman, tabun, and VX, are classified chemically as organophosphate compounds. Organophosphates are characterized as stable, easily dispersed, and highly toxic, with toxicity taking effect rapidly both when absorbed through the skin and via respiration. The threat of organophosphate poisoning is not limited to exposure to nerve gases, as commercial pesticides such as malathion and parathion are also organophosphate compounds that are toxic upon exposure. In the United States, approximately 20,000 reported organophosphate exposures occur per year; however, it is estimated that only 1% of field worker illness from pesticide exposure is reported.

Internationally, organophosphate poisoning occurs in virtually every country in the world. The United Nations reports that over 30,000 organophosphate-related fatalities occur worldwide each year. Third world countries have less legislation regarding safe agricultural use of pesticides; therefore, a much higher incidence of poisoning exists among field workers and the public who buy produce from these fields. Organophosphate compounds exert their toxic, often fatal, effects by irreversibly inactivating acetylcholinerase (AChE), the enzyme that terminates neurotransmission at the neuromuscular synapse. The normal function of cholinergic synapses requires that the neurotransmitter acetylcholine (ACh) be hydrolyzed by AChE within several milliseconds to terminate neurotransmission. Organophosphate poisoning occurs when the inactivated AChE is unable to break down ACh, leading to ACh

accumulation throughout the autonomic nervous system, the somatic nervous system, and the brain, resulting in overstimulation of the acetylcholine receptors. Decreasing muscle strength leading to paralysis occurs when motor plates remain depolarized by persisting levels of

acetylcholine.

In general, there are two classes of acetylcholine receptors, muscarinic and nicotinic acetylcholine receptors. Muscarinic receptors are located on the heart, eyes, glands, GI tract, and respiratory system. The brain and spinal cord both contain muscarinic and nicotinic receptors. Prolongation of neurotransmission results in extensive damage to the target cells and, frequently, death of the organism. The inhibition of AChE by nerve gases generally results in death by asphyxiation within a few minutes, as control is lost over respiratory muscles.

The forms of AChE responsible for terminating neurotransmission consist of two types of subunits, catalytic subunits responsible for hydrolyzing the acetylcholine and non- catalytic subunits responsible for localizing the enzyme to the correct location on the cell surface, the cholinergic synapse (reviewed in e.g. Massoulie et ai, Progress in Neurobiology 41 :31-91 (1993); Rotundo, J. Neurocytology, 2003). AChE exists in different oligomeric forms, as illustrated in Figure 1. The major secretable forms of the enzyme found in most tissues consist of monomers, dimers, or tetramers of a common catalytic subunit, referred to as the globular forms, or the asymmetric or collagen-tailed forms found predominantly in skeletal muscle in higher vertebrates that consist of one to three tetramers covalently linked to a triple helical collagen-like tail (CoIQ). The catalytic subunits that make up the various forms of AChE are enzymatically identical in the sense that they all have the same catalytic site and differ only in their carboxy-terminal 30-40 amino acids that determine which type of post-translational modifications related to localization occur.

The collagenic tail is responsible for attaching the catalytic subunits to the extracellular matrix located between the nerve terminal and the muscle fiber membrane (Figure 2). It is expressed only in vertebrates and has been cloned from several species, including Torpedo (Krejci et al., J. Neurosci. 19:10672-10679 (1991)), rat (Krejci et al., The Journal of Bio. Chem. 272(36):22840-22847 (1997)), and human (Ohno et al, Proc. Natl. Acad. ScL USA 95: 9654-9659 (1998); Donger et al., Am. J. Hum. Genet. 63:967-975 (1998)). There is extensive amino acid sequence identity between species, especially in regions critical for self-assembly and association with other proteins. The CoIQ has been divided into several functional domains (Krejci et al., 1991), some of which have now been characterized. Near the amino terminus is a region called the proline-rich domain, or PRAD, that is responsible for attachment of the tetramers (Bon et ah, The Journal of Bio. Chem. 272(5):3016-3021 (1997); Bon and Massoulie, The Journal of Bio. Chem. 272(5):3007-3015 (1997); Simon et al, EMBO J. 6:1865-1873 (1998)). Co-expression of the CoIQ subunit alone with "T" type catalytic subunits (AChEx), is sufficient to generate all the oligomeric forms of AChE found in nerves and muscle, indicating that no additional proteins are necessary for assembly of the collagen-tailed forms (Krejci et al., 1991; Legay et al., Journal ofNeurochem. 60:337-345 (1993); Ohno et al, 1998).

There is a strong body of evidence that the collagen-tailed form of AChE is the predominant form of the enzyme at the neuromuscular synapse (reviewed in Massoulie et al, 1993; Taylor, The Journal of Bio. Chem. 266:4025-4028 (1991); Rotundo and Fambrough, Function and Molecular Structure of Acetylcholinesterase in Myology (A.G. Engel and C. Franzini-Armstrong, eds., McGraw-Hill, 1994; Kimbell et al, J. Biol. Chem. 279:10997- 11005 (2004)) (Figure 3). The CoIQ AChE molecule is highly concentrated at sites of nerve- muscle contact (Hall, J. Neurobiol. 4:343-361 (1973)) where it is attached to the synaptic basal lamina (Lwebuga-Mukasa et al., Biochemistry 15: 1425-1434 (1976); McMahan et al.,

Nature 271:172-174 (1978)) via strong, possibly covalent, interactions (Rossi and Rotundo, J. Biol Chem. 268:19152-19159 (1993)). In mice whose CoIQ gene has been deleted by homologous recombination, there is no AChE at the neuromuscular junction (Feng et al., 1999). Thus CoIQ is clearly a targeting subunit responsible for localization and attachment of AChE to the neuromuscular synapse.

Organophosphate compounds inactivate AChE by phosphorylating the active site serine hydroxyl group on the enzyme, leading to the loss of ability to hydrolyze the acetylcholine substrate. Current strategies for treatment of individuals exposed to organophosphate compounds include reactivation of inactivated AChE using oxime reactivators, prophylactic administration of muscarinic antagonists such as atropine, and placement of the victim on a ventilator if necessary. Oxime reactivators such as 2-pyridine aldoxime methiodide (2-PAM) restore the function of inactivated AChE by displacing the covalently bound organophosphate molecule from the inactivated enzyme. However, poisoning by some nerve agents, such as soman, is complicated by the inhibited enzyme going through an "aging" process (Strayer reaction) that renders it incapable of being reactivated by any oxime. Atropine binds to muscarinic acetylcholine receptors to protect against excess acetylcholine-mediated neurotransmission resulting from AChE inhibition. However, atrophine treatment has no direct effects on the inactivated AChE, the nerve gas, or on nicotinic acetylcholine receptors. In cases of severe nerve gas poisoning, large doses of atrophine need to be taken until the level of functional AChE is restored. Moreover, in spite of ongoing developments in these types of treatments, the fatality rate could remain as high as 35% with large-scale exposure during a military conflict.

Another strategy employed to reduce the deadly effects of nerve gas exposure is to pretreat individuals at risk of exposure to organophosphate compounds with active site antagonists such as pyridostygmine bromide (PB). However, this strategy has its own

harmful drawbacks. PB is a carbamate compound that is thought to protect AChE by reversibly binding to ("carbamylating") it, so that the nerve agent cannot bind to it. It may also assist in protection against nerve agent by "desensitizing" ACh receptors. However, PB treatment may lead to bromide intoxication from prolonged consumption of excessive doses of bromide, causing protean symptoms, particularly psychiatric, cognitive, neurological, and dermatologic (and some believe this may be the cause of the "Gulf War Syndrome").

The most current research efforts to reduce the effects of exposure to nerve agents that inhibit AChE focus on the development of scavenging enzymes that stoichiometrically inactivate the nerve agent, or catalytic scavenging enzymes capable of hydrolyzing nerve agents in situ, in both cases reducing the effectiveness of the nerve agent. To this end, various forms of recombinant AChE, butyrylcholinesterase, paraoxonase, and other enzymes have been developed and studied for their effectiveness (Broomfield et al., 1991; Allon et al., 1998; Billeck et al., 1999; Broomfield et al., 1999). However, at best these molecules would be administered systemically and would inactivate unreacted organophosphates but leave untouched the inactivated AChE molecules. Thus there still remains little that can be done for victims that have been exposed to high levels of organophosphates.

Conventional methods of treatment for victims of nerve agent or pesticide poisoning are thus limited to controlling the damage caused by nerve agent exposure and/or limited in effectiveness only with certain organophosphate compounds. Moreover, the costs associated with such treatments are not limited to the financial costs required to deliver massive doses of the drugs in cases of severe poisoning but also include the costs to individuals suffering from deleterious side effects resulting from treatment.

There is a need for a method of treating organophosphate poisoning that directly treats the inactivated AChE molecules. There is also a need for a more cost effective and

efficacious treatment for nerve agent exposure. The invention is directed to these and other important ends.

SUMMARY OF THE INVENTION

The present invention provides novel DNA constructs and recombinant proteins encoded by the DNA constructs that will be useful for the treatment of individuals exposed to organophosphate compounds, such as nerve gases and pesticides. The present invention also provides methods of treating organophosphate poisoning in which the inactivated acetylcholinesterase molecules at the neuromuscular synapse are directly replaced with a functional catalytically active form of acetylcholinesterase. In preferred embodiments, the functional form of acetylcholinesterase comprises the native acetylcholinesterase catalytic subunit fused to a segment of collagen-like tail targeted to the neuromuscular synapse.

In one embodiment of the invention, the method of treating an animal exposed to organophosphates comprises replacing inactivated acetylcholinerasterase with a functional catalytically active form of acetylcholinesterase.

In another embodiment, a method of treating organophosphate poisoning is provided wherein inactivated acetylcholinerasterase is replaced with a functional catalytically active form of acetylcholinesterase.

In another embodiment, the method of treating an animal exposed to organophosphates comprises administering to the animal a therapeutically effective amount of a functional catalytically active form of acetylcholinesterase targeted to the neuromuscular synapse.

In another embodiment, the method of treating an animal exposed to organophosphates comprises administering to the animal a therapeutically effective amount

of recombinant chimeric acetylcholinesterase comprising a segment of collagen-like tail targeted to the neuromuscular synapse.

In another embodiment, the method of treating an animal exposed to organophosphates comprises administering to the animal a therapeutically effective amount of recombinant chimeric acetylcholinesterase comprising a catalytic acetylcholinesterase subunit fused to a segment of collagen-like tail targeted to the neuromuscular synapse.

In another embodiment, a method of replacing inactivated cholinesterase in an animal in need thereof is provided wherein a functional catalytically active form of acetylcholinesterase is administered to the animal.

In another embodiment, a method of replacing inactivated cholinesterase in an animal in need thereof is provided wherein a therapeutically effective amount of recombinant chimeric acetylcholinerase comprising a segment of collagen-like tail targeted to the neuromuscular synapse is administered to the animal.

In another embodiment, a method of replacing inactivated cholinesterase in an animal in need thereof is provided wherein a therapeutically effective amount of recombinant chimeric acetylcholinerase comprising a catalytic acetylcholinesterase subunit fused to a segment of collagen-like tail targeted to the neuromuscular synapse is administered to the animal.

In some forms of the invention, the segment of collagen-like tail comprises heparin binding domains and the C-terminal domain of wild type CoIQ.

The invention also provides for recombinant acetylcholinesterase polypeptides comprising a first polypeptide comprising the heparin binding domains and a second polypeptide comprising the C-terminal domain of CoIQ.

In yet another embodiment, the recombinant chimeric acetylcholinesterase polypeptides are those encoded by the nucleotide sequences comprising SEQ ID NOs: 11-20.

In another embodiment, the recombinant chimeric acetylcholinesterase polypeptides have amino acid sequences described by SEQ ID NOs: 1-10.

In accordance with the invention, the functional catalytically active acetylcholinesterase is administered by any suitable route of administration, including parenteral, oral, and intravenous routes of administration.

Therapeutically effective amounts of acetylcholinesterase may be administered in any

suitable dose range, including dose ranges from about 1 μg/kg to about 10 mg/kg of body weight per day or about 0.01 mg/kg to 1 mg/kg of body weight per day.

It is a further object of the invention to provide a method of reversing motor and plate depolarization due to persisting levels of acetylcholine at the neuromuscular synapse by introducing into the synapse a therapeutically effective amount of a functional catalytically active form of acetylcholinesterase.

Another embodiment of the invention provides a method of reversing motor and plate depolarization due to persisting levels of acetylcholine at the neuromuscular synapse, comprising introducing into the synapse a therapeutically effective amount of recombinant chimeric acetylcholinerase comprising a segment of collagen-like tail targeted to the neuromuscular synapse.

Still another embodiment of the invention provides a method of reversing motor and plate depolarization due to persisting levels of acetylcholine at the neuromuscular synapse, comprising introducing into the synapse a therapeutically effective amount of recombinant chimeric acetylcholinerase comprising a catalytic acetylcholinesterase subunit fused to a segment of collagen-like tail targeted to the neuromuscular synapse.

It is yet another object of the invention to provide a kit for treating organophosphate poisoning, comprising a therapeutically effective amount of a functional catalytically active form of acetylcholinesterase targeted to the neuromuscular synapse.

In one embodiment, a kit for treating organophosphate poisoning is provided, comprising a therapeutically effective amount of recombinant chimeric acetylcholinerase comprising a segment of collagen-like tail targeted to the neuromuscular synapse. In another embodiment, the recombinant chimeric acetylcholinerase further comprises a catalytic acetylcholinesterase subunit fused to the segment of collagen-like tail targeted to the neuromuscular synapse.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the oligomeric forms of AChE in animals. The major secretable forms of AChE consist of monomers (Gi), dimers (G ₂ ), and tetramers (G ₄ ) of the AChE catalytic subunit. The collagen-tailed (CoIQ) synaptic form of AChE (Aj ₂ ) attaches to the extracellular matrix (ECM) at the synapse.

FIG. 2 illustrates the localization of AChE (small circles) at sites of nerve-muscle contact in animals.

FIG. 3 illustrates the structure of the functional AChE form present at the neuromuscular junction and the organization of a collagenic tail (CoIQ) polypeptide chain. Following the leader sequence (LS), the N-terminal domain (NTD) of the CoIQ chain contains a proline- rich attachment domain (PRAD) responsible for attachment of the tetrameric forms of theAChE catalytic subunit. The collagenic domain contains two heparin sulfate binding

domains (HSBD 1 and HSBD 2). The C-terminal domain (CTD) of the CoIQ chain contains a trimerization domain and a cysteine rich domain.

FIG. 4 illustrates DNA construct embodiments according to the invention which may be used to create various mouse chimeric proteins for targeting in intact animals. The numbers refer to the nucleotides in the coding sequence.

FIG. 5 illustrates DNA construct embodiments according to the invention which may be used to create various human chimeric proteins for targeting in intact animals. The numbers refer to the amino acids in the mature protein.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to specific embodiment and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alteration and further modifications of the invention, and such further applications of the principles of the invention as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the invention relates.

All terms as used herein are defined according to the ordinary meanings they have acquired in the art. Such definitions can be found in any technical dictionary or reference known to the skilled artisan, such as the McGraw-Hill Dictionary of Scientific and Technical Terms (McGraw-Hill, Inc.), Molecular Cloning: A Laboratory Manual (Cold Springs Harbor, New York), and Remington 's Pharmaceutical Sciences (Mack Publishing, PA). These references, along with those references and patents cited herein are hereby incorporated by reference in their entirety.

The present invention is directed to a novel method of treating organophosphate poisoning that involves replacing inactivated synaptic acetylcholinesterase molecules with functional catalytically active acetylcholinesterase (AChE). "Organophosphates" refer to compounds capable of inactivating AChE by phosphorylating the serine hydroxyl group located on the active site of AChE. Phosphorylation occurs to inactivate the enzyme AChE when a covalent bond forms between the organophosphate molecule and the AChE molecule. Exemplary organophosphates include insecticides such as malathion, parathion, diazinon, fenthion, dichlorvos, and chlorpyrifos and nerve gases such as soman, sarin, tabun, and VX.

In accordance with the invention, the inactivated AChE is replaced with a catalytically active form of AChE that localizes to the neuromuscular junction. As used herein, "neuromuscular junction" refers to a neuromuscular synapse, or a site of nerve-muscle contact. A "cholinergic synapse" refers to a neuromuscular synapse wherein neurotransmission is mediated by acetylcholine. "Neurotransmission" refers to the transmission of nerve impulses, or traveling nerve signals that lead to localized muscle cell contraction, across the synapse. "Catalytically active AChE" refers to a form of acetylcholinesterase capable of hydrolyzing acetylcholine.

In accordance with the invention, the catalytically active AChE is a collagen-tailed form containing specific sites of attachment for localization of the replacement enzymes to the neuromuscular synapse. In a specific aspect of the invention, the replacement AChE molecules are novel recombinant human AChE chimeras consisting of the catalytic AChE subunit fused to various length segments of the collagenic tail subunit (Figure 4). In a further aspect of the invention, the collagenic tail segment of the recombinant AChE chimeras comprises the heparin-binding domains and the C-terminal domain (CTD) of the wild-type CoIQ subunit.

The existence of specific binding sites for the precise localization of AChE molecules to the neuromuscular synapse, suggested by the highly organized pattern of AChE molecules attached to the basal lamina of the neuromuscular junction, was shown with a molecular transplantation assay in which the CoIQ AChE isolated from one species was "transplanted" to the neuromuscular junction of another (Rotundo et al., 1997). Quail wild-type globular and collagen-tailed AChE forms were immunoaffinity purified and tested for their ability to attach to frog neuromuscular junctions pretreated with high-salt detergent buffers. The neuromuscular junctions were visualized by labeling acetylcholine receptors with a fluorescent probe specific for the skeletal muscle nicotinic acetylcholine receptor. Binding of exogenous quail AChE was determined using a species-specific monoclonal antibody. When frog neuromuscular synapses were incubated with globular quail AChE forms, there was no detectable binding above background levels. On the other hand, incubation of the frog muscle sections with the asymmetric CoIQ AChE form led to immunolabeling of the attached quail AChE in over 80% of the frog synaptic sites.

The molecules on the cell surface that serve as acceptors for AChE during the process of synaptic localization were identified as perlecan, an abundant heparin-sulfate proteoglycan (Peng et al., 1999). Wild-type CoIQ AChE is known to bind to heparin via its collagen-like tail and this may be part of the mechanism for sequestering it on the muscle cell surface or at the neuromuscular junction (Inestrosa and Perelman, TIPS 10:325-329 (1989); Rossi and Rotundo, /. Biol. Chem. 271(4): 1979-1987 (1996); Rotundo et al., 1997). Pretreatment of Xenopus muscle cells with heparin abolished the binding of exogenous quail CoIQ AChE to the cell surface in a molecular transplantation assay. Transplanted AChE colocalized with perlecan on the cell surface in both clustered and diffuse states of AChE distribution, as determined by AChE treated muscle cells double-labeled with anti-avian AChE monoclonal antibody and a polyclonal anti-perlecan antibody. Furthermore, purified CoIQ AChE and not

purified globular AChE bound to isolated perlecan-antibody Sepharose beads in vitro, and in a Biacore instrument, indicating that AChE binds to perlecan via its collagen-like tail.

Perlecan in turn binds to dystroglycan, an important transmembrane protein linking the extracellular matrix to the myofibrilar apparatus through dystrophin or utrophin, both highly concentrated at the neuromuscular junction. Mice lacking perlecan and dystroglycan in their muscle were found not to have AChE at their neuromuscular junctions (Jacobson et al., J. Cell. Biol. 152:435-450 (2001); Arikawa-Hirasawa et al., Nat. Neurosci. 5:119-123 (2002)). Thus, the CoIQ subunit is absolutely necessary to mediate attachment of the AChE catalytic subunits to muscle cell surface perlecan, which is absolutely required for the localization of AChE to the neuromuscular junction.

The heparin binding sites in the collagenic tail are necessary, but not sufficient for AChE attachment to the synapse. The cysteine-rich C-terminal globular domain of the collagenic tail is also required for binding of AChE to the neuromuscular junction (Figure 3). Using mutant human CoIQ subunits isolated from patients with congenital myasthenia (also known as endplate AChE deficiency), the molecular transplantation technique was used to identify regions of CoIQ critical for binding to the synapse (Kimbell et al., 2004). Wild-type and mutated recombinant human CoIQ AChE expressed in COS cells and partially purified on heparin columns were tested for insertion competence into heterologous frog neuromuscular junctions.

The C-terminal domain CoIQ mutants R315X, Q317X, D342E, R410P, and R410Q were found to compromise anchoring of the asymmetric AChE to the synaptic basal lamina. Insertion incompetence of two truncation mutations, R315X and Q371X, suggests that essential residues for anchoring of CoIQ are located downstream of codon 371, but this does not exclude importance of residues upstream of codon 371, as indicated by the insertion incompetence of D342E. As aspartate and glutamate are similar residues, that D342 but not

E342 CoIQ inserts into the synaptic basal lamina indicates that D342 is likely an evolutionally conserved key residue that is essential for anchoring CoIQ. Insertion incompetence of R410P and R410Q also indicates that R410 plays an essential role in harboring CoIQ.

With the major requirements for the attachment of AChE to the neuromuscular synapse defined, novel chimeric AChE molecules comprising the catalytic AChE subunit fused to various length segments of the CoIQ subunit will be constructed from cloned and expressed AChE catalytic and CoIQ subunits. hi accordance with the invention, chimeric AChE proteins used for targeting to the neuromuscular synapse include proteins comprising various regions of the collagenic tail, such as proteins with amino acid sequences shown by SEQ ID NOs: 1-10 (see Table 1). In one embodiment, the nucleotide sequences used to create DNA constructs encoding the mouse chimeric proteins include those shown in Figure 4, having the nucleic acid sequences shown by SEQ ID NOs: 11-15 (Table 1). In another embodiment, the nucleotide sequences used to create DNA constructs encoding the human chimeric proteins include those shown in Figure 5, having the nucleic acid sequences shown by SEQ ID NOs: 16-20 (Table 1). In a further embodiment of the invention, the nucleotide sequences used to create the human chimeric proteins include those comprising the human AChE (Prody et al, 1987; Soreq et al, 1990) and human CoIQ sequences (Ohno et al, 1998; Donger et al, 1998). The terms "encoding" and "coding" refer to the process by which a nucleotide sequence, through the mechanisms of transcription and translation, provides the information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce a polypeptide. The nucleotide sequences of the invention may be either synthesized in vitro or isolated from a biological source. Such methods of synthesis or isolation are well known to the skilled artisan.

Recombinant expression vectors may be constructed by incorporating the above- recited nucleotide sequences within a vector according to methods well known to the skilled artisan and as described, for example, in Sambrook et al, Molecular Cloning: A Laboratory

Manual, Cold Springs Harbor Laboratory, 2 ^nd ed., Cold Springs Harbor, New York (1989). Other references describing molecular biology and recombinant DNA techniques include, for example, DNA Cloning 1: Core Techniques, (D. N. Glover, et al., eds., IRL Press, 1995); DNA Cloning 2: Expression Systems, (B. D. Hames, et al., eds., IRL Press, 1995); DNA Cloning 3: A Practical Approach, (D. N. Glover, et al., eds., IRL Press, 1995); DNA Cloning 4: Mammalian Systems, (D. N. Glover, et al., eds., IRL Press, 1995); Oligonucleotide Synthesis (M. J. Gait, ed., IRL Press, 1992); Nucleic Acid Hybridization: A Practical Approach, (S. J. Higgins and B. D. Hames, eds., IRL Press, 1991); Transcription and Translation: A Practical Approach, (S. J. Higgins & B. D. Hames, eds., IRL Press, 1996); R.

I. Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4 ^tn Edition (Wiley-Liss,

1986); and B. Perbal, A Practical Guide To Molecular Cloning, 2 ^nd Edition, (John Wiley & Sons, 1988); and Current Protocols in Molecular Biology (Ausubel et al, eds., John Wiley & Sons), which is regularly and periodically updated.

Suitable expression vectors according to the invention are, for example, bacterial or yeast plasmids, wide host range plasmids and vectors derived from combinations of plasmid and phage or virus DNA. Vectors derived from chromosomal DNA are also included. Furthermore, an origin of replication and/or a dominant selection marker can be present in the vector according to the invention. The vectors according to the invention are suitable for transforming, transfecting, or infecting a host cell. Exemplary plasmid vectors for expression include pTargeT (Invitrogen) and pcDNA3.1 (Invitrogen) for expression in mammalian cells and pPICZα (Invitrogen) for expression in yeast.

The recombinant DNA constructs encoding the AChE chimeras may be expressed in any cells suitable for use as host cells for recombinant DNA expression, including bacterial host cells, yeast and other fungi, plant or animal hosts such as Chinese Hamster Ovary cells or monkey cells. Thus, a host cell which comprises the DNA or expression vector according to the invention is also within the scope of the invention. Suitable host cells transformed with the DNA constructs can be fermented and subjected to conditions which facilitate the expression of the heterologous DNA, leading to the formation of large quantities of the desired protein. Non-limiting examples of preferred host cells suitable for protein expression in accordance with the invention include yeast (Pichia pastoήs) and simian COS cells.

The desired chimeric AChE proteins may be purified prior to administration to an animal. The optional purification procedure for the chimeric proteins present in the host cell extract or culture medium may be based on the properties of the biomolecules, such a size, charge and function. Methods of purification include centrifugation, electrophoresis, chromatography, dialysis or a combination thereof. As known in the art, electrophoresis may be utilized to separate the proteins in the sample based on size and charge. Electrophoretic procedures are well known to the skilled artisan, and include isoelectric focusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), agarose gel electrophoresis, and other known methods of electrophoresis.

The purification step may be accomplished by a chromatographic fractionation technique, including size fractionation, fractionation by charge and fractionation by other properties of the biomolecules being separated. As known in the art, chromatographic systems include a stationary phase and a mobile phase, and the separation is based upon the interaction of the biomolecules to be separated with the different phases. In some forms of the invention, column chromatographic procedures may be utilized. Such procedures include partition chromatography, adsorption chromatography, size-exclusion chromatography, ion-

exchange chromatography and affinity chromatography. Such methods are well known to the skilled artisan. For example, if the desired protein has a known binding affinity domain, such as the heparin binding domains of the chimeric proteins of the present invention, the proteins may be purified by affinity chromatography using heparin agarose columns. An affinity tag may also be engineered into the desired protein for purification purposes. For example, the DNA constructs of the invention may encode for 6-Histidine tags (His tags) attached to the CoIQ AChE fusion proteins to facilitate protein purification on nickel affinity columns.

In one embodiment of the invention, the purified chimeric proteins are tested for binding to the neuromuscular junction with the in vitro transplantation assay described in Rotundo et al., 1997 and Kimbell et al., 2004. A microscope slide to which thin sections of muscle tissue are attached is enclosed in a small chamber. Test solutions containing different forms of the enzyme are added to the slide. The neuromuscular junctions are visualized by labeling acetylcholine receptors with a probe specific for the synapse, such as the fluorescent snake toxin alpha-bungarotoxin. Binding of the exogenous chimeric AChE can be determined by any methods known in the art. For example, a species-specific antibody that recognizes the exogenous protein can be added to the slide, followed by the addition of a secondary antibody conjugated to a molecule that enables visualization of the antibody. In one embodiment of the invention, the secondary antibody is conjugated to a fluorescent molecule, such as fluorescein, enabling the localization of bound AChE by immunofluorescence. In another, the AChE molecules are localized using a fluorescently- tagged snake venom molecule, fasciculin 2, developed by Rotundo and colleagues (Peng et al., 1999), that binds specifically to the AChE catalytic subunit in a 1:1 ratio. The extent of binding of the chimeric protein to the neuromuscular junction can be determined in terms of binding constants and total numbers of AChE molecules bound per neuromuscular junction.

The antibodies to be used in the binding assays described herein may be polyclonal antibodies and may be obtained by procedures which are well known to the skilled artisan, including injecting purified wild-type AChE into various animals and isolating the antibodies produced in the blood serum. The antibodies may be monoclonal antibodies whose method of production is well known to the art, including, for example, injecting purified wild-type AChE into a mouse, isolating the spleen cells producing the anti-serum, fusing the cells with tumor cells to form hybridomas and screening the hybridomas. Once the antibody is provided, a bound chimeric AChE can be detected and/or quantitated by the immunofluorescence assays previously described herein.

Chimeric AChE forms that bind to the neuromuscular junction in the in vitro assays will then be used for in vivo testing by injection into CoIQ null mice that are devoid of AChE at their neuromuscular junctions. CoIQ deficient mice can be developed by methods known to the skilled artisan. In one embodiment, the CoIQ null mice to be used are described in Feng et al., J. Cell Biol. 144:1349-1360 (1999). Since there is no endogenous AChE at the neuromuscular synapses of these mice, successful insertion of chimeric AChE in vivo can be detected using the same methods used in the in vitro studies, i.e. antibody detection and the fluorescent snake toxin fasciculin 2.

Thus, chimeric proteins of the invention will be useful for administration to humans suffering from organophosphate poisoning characterized by inactivated acetylcholinesterase, as described above. The fusion of the C-terminal domain of CoIQ with the catalytic subunit of AChE will produce a chimeric molecule with the abilities to both hydrolyze the acetylcholine and bind to the neuromuscular synapse in vivo. When administered to a human, these novel chimeras targeted to the neuromuscular synapse will effectively neutralize organophosphate poisoning by replacing inactivated AChE at the synapse. The proteins provided herein can be formulated into pharmaceutical compositions by

admixture with pharmaceutically acceptable nontoxic excipients and carriers. The formulations of the invention are useful for parenteral administration, for example, intravenous, subcutaneous, intramuscular, intraventricular, intracranial, intracapsular, intraspinal, intraci sternal, or intraperitoneal administration. In accordance with the invention, the preferred routes of administration are intravenous, intramuscular, and subcutaneous. The compositions can be formulated for parenteral administration to humans or other animals in therapeutically effective amounts (e.g., amounts which eliminate or reduce the patient's pathological condition) to provide therapy for the organophosphate poisoning described above. Exemplary formulations include aqueous solutions or lyophilized powders for dilution.

The compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington's Pharmaceutical Sciences. Formulations for parenteral administration may contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, and the like. In particular, biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of the proteins. Other potentially useful parenteral delivery systems for the chimeric proteins include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of the compounds described herein in a therapeutic composition will vary depending upon a number of factors, including the dosage of the drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration. In general terms, the compounds of this invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v

compound for parenteral administration. Typical dose ranges would be from about 1 μg/kg

to about 10 mg/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 1 mg/kg of body weight per day.

Reference will now be made to specific examples illustrating the constructs and methods above. It is to be understood that the examples are provided to illustrate preferred embodiments and that no limitation to the scope of the invention is intended thereby.

Examples

Example 1: Mutation analysis of CoIQ

Novel DNA constructs encoding single amino acid mutations in the C-terminal domain (CTD) of human CoIQ were cloned along with constructs encoding the wild-type catalytic AChEr subunit. Wild-type or mutant CoIQ and the wild-type AChE catalytic subunit were then coexpressed in COS cells to determine the segments of CoIQ necessary for binding to the neuromuscular synapse.

(A) Construction of expression vectors

Human AChE ₇ and COLQ cDNAs were cloned and introduced into a CMV-based mammalian expression vector pTargeT (Invitrogen), as described in Ohno et al., 1998. Each CTD mutation, as well as S312G/R410Q double mutations, was introduced into COLQ cDNA using the QuikChange site-directed mutagenesis kit (Stratagene). Artificial HSBD mutants were constructed similarly. pTargeT-ColQ-HSBD-del carries the deletion of RKG at codons 130-132 in the first HSBD and deletion of KRG at codons 235-237 in the second HSBD. pTargeT-ColQ-HSBD-NQGG harbors 8 missense codons: NQGG substitution for RKGR at codons 130-133 in the first HSBD and NQGG substitution for KRGK at codons 235-238 in the second HSBD. For each construct, presence of the desired mutation and absence of unwanted artifacts were confirmed by sequencing the entire insert.

(B) Transfection and AChE extraction

Plasmids encoding the wild-type or mutant form of human CoIQ were cotransfected into COS cells with a plasmid encoding the wild-type human AChEj catalytic subunit using the DEAE dextran method as described in Duval et al, EMBO J. 11:3255-3261 (1992). Five μg of each plasmid DNA were added to COS cells in 100 mm culture dishes in 3 ml

Dulbecco's Modified Eagle Medium (DMEM) (Gibco) containing 10% fetal bovine serum (FBS) and chloroquine and incubated for 3 hours at 37°C. Cells were then washed three times with PBS, 6 ml of fresh DMEM +10% FCS was added, and the dishes were incubated at 37°C for 72 hours. To extract the expressed enzyme, cells were scraped from dishes in 1 ml of 50 mM Tris-HCl buffer, pH 7.4 (from now on "Tris-HCl buffer" refers to 50 mM Tris pH 7.4) containing 0.5% TX-100, 1.0 mM EDTA, 1 M NaCl, leupeptin (1 μg/ml) and pepstatin (2 μg/ml). The crude extract was vortexed in 1.5 ml microcentrifuge tubes, centrifuged at 10,000 x g for 30 min and the supernatant was stored in 50% glycerol at -2O ⁰ C until use. Aliquots (200 μl) of the AChE-containing supernatant were centrifuged on 5-20% sucrose gradients in Tris-HCl buffer containing 1 M NaCl, 0.5% TX-100, and 1 mM EDTA and the fractions collected were assayed for AChE activity using the Ellman method to confirm that the collagen-tailed AChE form is expressed. Bacterial β-galactosidase (-16. IS) was included in the gradients as a marker for the CoIQ-AChE form.

(C) Isolation of CoIQ-AChE on heparin agarose columns

Extracts from COS cells expressing wild-type or mutated collagen-tailed AChE were vortexed and centrifuged at 10,000 x g for 30 min, the supernatant was diluted to 0.2 M NaCl with Tris-HCl buffer containing 0.5% TX-100, 1.0 mM EDTA, leupeptin (1 μg/ml) and pepstatin (2 μg/ml), and loaded onto columns containing 250 μl heparin agarose (Sigma) that had been pre-equilibrated with 10 volumes of Tris-HCl buffer containing 0.2 M NaCl. The columns were washed with 5 volumes Tris-HCl buffer containing 0.2 M NaCl and the

asymmetric forms eluted with 1 ml of Tris-HCl buffer containing 1 M NaCl. The eluate containing the asymmetric AChE forms was concentrated with YM-30 Centricon concentrators (Amersham Biosciences) and diluted in Tris-HCl buffer containing 0.2 M NaCl. To verify that indeed only the collagen-tailed AChE forms were being retained on the column and eluted, the eluates were analyzed by velocity sedimentation. β-Galactosidase (-16. IS) was included in the gradients as a marker for the CoIQ-AChE form. This procedure showed that only the collagen-tailed AChE forms are retained on the heparin column and specifically eluted with 1 M NaCl.

(D) Transplantation of AChE to frog neuromuscular junctions

The method of Rotundo et ai, 1997, was used with minor modifications. Frog (Rana pipiens) gastrocnemius muscles were excised, snap frozen by immersion of the muscle in 2- methylbutane cooled with liquid nitrogen, and stored frozen at -80 ⁰ C. Ten μm cross sections from the belly of the muscle were cut in a Leica CM 1900 cryostat at -20 ⁰ C, adhered to Gold Seal microscope slides, and stored at -8O ⁰ C until used. For analysis, the slides were brought to room temperature, excess moisture removed, and plastic wells for transplantation and immunological staining affixed to the slides using rubber cement. Stock asymmetric AChE solutions used for transplantation were eluted from heparin columns with Tris-HCl buffer containing 1 M NaCl and concentrated. For addition to sections, the stock solutions were diluted in Tris-HCl buffer containing IM NaCl and 5 mg/ml each of bovine serum albumin, chicken ovalbumin and gelatin (all from Sigma Chem. Co., St. Louis, MO) to give 1-2 ng AChE /well final, and the NaCl concentration adjusted to 0.5M. The ionic strength of the enzyme solution (100 μl/well) was adjusted stepwise over a period of 3 hrs to 0.3M to prevent aggregation of collagen tailed AChE forms. After addition of the enzyme preparation, the wells were sealed with coverslips, placed in a humidified chamber and incubated overnight at room temperature. The sections were then washed for two hours with

Tris-HCl buffer containing 0.2 M NaCl and the protein cocktail described above prior to processing for immunofluorescence. The specific binding is not disrupted by high salt concentrations (1 M NaCl) or heparin (500 μg/ml).

(E) Immunofluorescence

Colocalization of AChE with AChR was visualized with a Leica DMRA fluorescence

microscope. AChRs were labeled with rhodamine labeled α-bungarotoxin (1 μg/ml) and

human AChE was detected with a primary mouse anti-human AChE monoclonal AE3 that does not recognize frog AChE (as described in Fambrough et al., P roc. Natl. Acad. ScL USA 79:1078-1082 (1982)) (10 μg/ml) followed by a FITC-conjugated rabbit anti-mouse second antibody (10 μg/ml). Muscle sections were fixed in 4% paraformaldehyde in PBS, mounted in glycerol containing p-phenylendiamine (1 mg/ml), 0.02 M sodium bicarbonate, pH 9.5 and 0.06 x PBS, and sealed with clear nail polish.

(F) In-vitro perlecan binding assay

Plasmids encoding the above-described HSBD mutants of human CoIQ cDNAs were cotransfected into COS cells with the plasmid encoding the wild-type human AChEj catalytic subunit and the CoIQ-AChE was isolated and tested for its ability to bind immobilized perlecan. The microtiter plate perlecan binding assay developed by Vigny et al., J. Biol. Chem. 258: 8794-8798 (1983), was used with 10 ng perlecan, the heparan sulfate proteoglycan isolated from the mouse EHS sarcoma cells (Sigma, St. Louis, MO), per well. CoIQ-AChE harboring mutations HSBD-del and HSBD-NQGG as well as wild-type CoIQ and G1/G2 globular forms of AChE were added to the polystyrene microtitre plates in a total volume of 200 μl. Plates were incubated overnight at 4°C, washed with Tris-HCl buffer containing 0.2 M or 1 M NaCl, and the bound AChE activity was determined by the Ellman

method. In contrast to wild-type collagen-tailed AChE, the globular G]/G ₂ and G ₄ AChE forms exhibited little or no binding to immobilized perlecan in vitro.

Example 2: Catalytically active CoIQ AChE chimeras

Novel DNA constructs comprising the catalytic AChE _T subunit fused to various length subunits of the C-terminal domain of CoIQ are constructed from cloned and expressed mouse (Figure 4) or human (Figure 5) AChE catalytic and CoIQ subunits. SEQ ED NOs: 11- 15 correspond to the nucleotide sequences of DNA constructs comprising cloned and expressed mouse AChE catalytic and CoIQ subunits; SEQ DD NO.: 16-20 correspond to the nucleotide sequences of DNA constructs comprising cloned and expressed human AChE catalytic and CoIQ subunits. SEQ ID NO: 11 corresponds to a mouse, and SEQ ID NO: 16 corresponds to a human AChE construct encoding a full-length trimeric CoIQ tail, comprising the collagenic region ("COL," containing the heparin binding domains), the trimerization domain ("TD"), and the C-terminal domain ("CTD"). SEQ ID NO: 12 corresponds to a mouse, and SEQ ID NO: 17 corresponds to a human AChE construct encoding a single-stranded CoIQ tail, by deletion of the trimerization domain from the full- length CoIQ coding region. SEQ ID NO: 13 corresponds to a mouse, and SEQ ID NO: 18 corresponds to a human AChE construct encoding a truncated trimeric CoIQ tail, comprising the trimerization domain and the C-terminal domain. SEQ ID NO: 14 corresponds to a mouse, and SEQ ID NO: 19 corresponds to a human AChE construct encoding a truncated trimeric CoIQ tail, comprising the trimerization domain and the C-terminal domain with two C-terminal cysteines (Cys 293 and Cys 295 in the mouse and Cys 291 and Cys 293 in the human) excluded. SEQ ID NO: 15 corresponds to a mouse, and SEQ ID NO: 20 corresponds to a human AChE construct encoding a truncated trimeric CoIQ tail comprising the C- terminal domain only. SEQ ID NOs: 1-5 correspond to the amino acid sequences of the resulting fusion proteins obtained by expression of the mouse DNA constructs in appropriate

host cells; SEQ ID NOs: 6-10 correspond to the amino acid sequences of the resulting fusion proteins obtained by expression of the human DNA constructs in appropriate host cells.

(A) Construction of expression vectors

Mouse or human AChEr and COLQ cDNAs are cloned and introduced into a CMV- based mammalian expression vector pTargeT (Invitrogen), as described in Ohno et ah, 1998. CTD mutations are introduced into COLQ cDNA using the QuikChange site-directed mutagenesis kit (Stratagene). Deletion of the trimerization domain only or of the trimerization domain and the collagenic region from the CoIQ tail is accomplished by amplifying the desired sequence by the polymerase chain reaction using the full length CoIQ as a template and sequence specific primers designed to introduce unique restrictions enzyme sites at the ends of the desired sequence for inserting into the constructs containing the AChE catalytic subunit at the 5' end (see Figure 4). For each construct, presence of the desired mutation and absence of unwanted artifacts is confirmed by sequencing the entire insert.

(B) Transfection and AChE extraction

Plasmids encoding the various length forms of mouse or human CoIQ are cotransfected into COS cells with a plasmid encoding the wild-type AChEj catalytic subunit using the DEAE dextran method previously described in Duval et al., 1992. Five μg of each plasmid DNA is added to COS cells in 100 mm culture dishes in 3 ml DMEM (Gibco) containing 10% FBS and chloroquine and incubated for 3 hours at 37°C. Cells are then washed three times with PBS, 6 ml of fresh DMEM +10% FCS is added, and the dishes are incubated at 37 ⁰ C for 72 hours. To extract the expressed enzyme, cells are scraped from dishes in 1 ml of 50 mM Tris-HCl buffer, pH 7.4 containing 0.5% TX-100, 1.0 mM EDTA, 1 M NaCl, leupeptin (1 μg/ml) and pepstatin (2 μg/ml). The crude extract is vortexed in 1.5 ml microcentrifuge tubes, centrifuged at 10,000 x g for 30 min and the supernatant is stored in

50% glycerol at -20 ⁰ C until use. Aliquots (200 μl) of the AChE-containing supernatant are chromatographed on the heparin columns and the high salt eluate centrifuged on 5-20% sucrose gradients in Tris-HCl buffer containing 1 M NaCl, 0.5% TX-100, and 1 mM EDTA and the fractions collected are assayed for AChE activity using the Ellman method. Bacterial β-galactosidase (-16. IS) is included in the gradients as a marker for the CoIQ-AChE form.

(C) Isolation of CoIQ-AChE on metal affinity resin columns

Extracts from COS cells expressing collagen-tailed AChE are vortexed and centrifuged at 10,000 x g for 30 min, the supernatant is diluted to 0.5 M NaCl with Tris-HCl buffer containing 0.5% TX-100, 1.0 mM EDTA, leupeptin (1 μg /ml) and pepstatin (2 μg/ml), and loaded onto columns containing 500 μl - 5 ml ProBond resin (Invitrogen), a nickel-containing resin that can bind 1-5 mg protein/ml that had been pre-equilibrated with 10 volumes of Native Binding Buffer (Invitrogen) containing 50 mM NaPO ₄ and 0.5 M NaCl, pH 8.0. The columns are washed with 10 volumes Native Wash Buffer (Invitrogen) containing 50 mM NaPO ₄ , 0.5 M NaCl and 20 mM imidazole and the asymmetric forms are eluted with 2 volumes of Native Elution Buffer (Invitrogen) containing 50 mM NaPO ₄ , 0.5 M NaCl and 20 mM imidazole. The eluate containing the asymmetric AChE forms is concentrated with YM-30 Centricon concentrators (Amersham Biosciences) and diluted in Tris-HCl buffer containing 0.2 M NaCl. To verify that indeed only the collagen-tailed AChE forms are being retained on the column and eluted, the eluates are analyzed by velocity sedimentation. β-Galactosidase (-16. IS) is included in the gradients as a marker for the CoIQ-AChE form. This procedure shows that only the collagen-tailed AChE forms are retained on the metal affinity column and specifically eluted.

(D) Transplantation of AChE to neuromuscular junctions of CoIO null mice

The method of Rotundo et al, 1997, is used with minor modifications. CoIQ null mice, described in Feng et al., 1999, are obtained from one of the original developers, Dr. Eric Krejci. CoIQ null mice muscles are excised, snap frozen by immersion of the muscle in 2-methylbutane cooled with liquid nitrogen, and stored frozen at -8O ⁰ C. Ten μm cross sections from the belly of the muscle are cut in a Leica CM 1900 cryostat at -20 ⁰ C, adhered to Gold Seal microscope slides, and stored at -80 ⁰ C until used. For analysis, the slides are brought to room temperature, excess moisture removed, and plastic wells for transplantation and immunological staining affixed to the slides using rubber cement. Stock asymmetric AChE solutions used for transplantation are eluted from nickel columns with Native Elution Buffer (Invitrogen) containing 50 mM NaPO ₄ , 0.5 M NaCl and 20 mM imidazole, and concentrated. For addition to sections, the stock solutions are diluted in Tris-HCl buffer containing IM NaCl and 5 mg/ml each of bovine serum albumin, chicken ovalbumin and gelatin (all from Sigma Chem. Co., St. Louis, MO) to give 1-2 ng AChE /well final, and the NaCl concentration is adjusted to 0.5M. The ionic strength of the enzyme solution (100 μl/well) is adjusted stepwise over a period of 3 hrs to 0.3M to prevent aggregation of collagen tailed AChE forms. After addition of the enzyme preparation, the wells are sealed with coverslips, placed in a humidified chamber and incubated overnight at room temperature. The sections are then washed for two hours with Tris-HCl buffer containing 0.2 M NaCl and the protein cocktail described above prior to processing for immunofluorescence or direct visualization. The specific binding is not disrupted by high salt concentrations (I M NaCl) or heparin (500 μg/ml).

(E) Immunofluorescence

Colocalization of AChE with AChR is visualized with a Leica DMRA fluorescence

microscope. AChRs are labeled with rhodamine labeled α-bungarotoxin (1 μg/ml) and

AChE is detected with a primary anti-AChE antibody, either anti-human AE3 (Fambrough et al., 1981) or anti-mouse Z3 (Brimijoin et al.) (10 μg/ml) followed by a FITC-conjugated rabbit anti-mouse second antibody (10 μg/ml). Muscle sections are fixed in 4% paraformaldehyde in PBS, mounted in glycerol containing p-phenylendiamine (1 mg/ml), 0.02 M sodium bicarbonate, pH 9.5 and 0.06 x PBS, and sealed with clear nail polish.

(F) Direct visualization of AChE at the neuromuscular synapse

Binding of exogenous mouse AChE to the neuromuscular synapse is visualized with a Leica DMRA fluorescence microscope. CoIQ null mice muscle sections are treated with Oregon Green-conjugated fasciculin 2, a fluorescent probe that binds irreversibly to AChE in vitro and in vivo, and Rhodamine-labeled α-bungarotoxin, both at 1 μg/ml. Muscle sections are fixed in 4% paraformaldehyde in PBS, mounted in glycerol containing p- phenylendiamine (1 mg/ml), 0.02 M sodium bicarbonate, pH 9.5 and 0.06 x PBS, and sealed with clear nail polish.

While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. In addition, all references cited herein are indicative of the level of skill in the art and are hereby incorporated by reference in their entirety.