EP1216300A1 - Procedes et compositions relatifs aux sous-unites beta1a de canaux sodiques - Google Patents

Procedes et compositions relatifs aux sous-unites beta1a de canaux sodiques

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Publication number
EP1216300A1
EP1216300A1 EP00967234A EP00967234A EP1216300A1 EP 1216300 A1 EP1216300 A1 EP 1216300A1 EP 00967234 A EP00967234 A EP 00967234A EP 00967234 A EP00967234 A EP 00967234A EP 1216300 A1 EP1216300 A1 EP 1216300A1
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EP
European Patent Office
Prior art keywords
βla
sodium channel
subunit
cell
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00967234A
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German (de)
English (en)
Inventor
Lori L. Isom
Kristin Kazen-Gillespie
Kathryn E. Rogers
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Janssen Pharmaceuticals Inc
University of Michigan
Original Assignee
Ortho McNeil Pharmaceutical Inc
University of Michigan
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Application filed by Ortho McNeil Pharmaceutical Inc, University of Michigan filed Critical Ortho McNeil Pharmaceutical Inc
Publication of EP1216300A1 publication Critical patent/EP1216300A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates generally to sodium channel proteins and more particularly to the ⁇ subunits of voltage gated sodium channel proteins, to DNA sequences encoding these subunits, to the polypeptide products of recombinant expression of these DNA sequences, to peptides whose sequences are based on amino acid sequences deduced from these DNA sequences, and to procedures relating to the development of drugs that influence function of such proteins.
  • Ion channels from mammalian systems are the subject of intensive scientific investigation because of the importance and variety of their biochemical functions.
  • Ion channels are now understood to be polypeptide or protein structures with tertiary- quaternary structures forming interior pores embedded in plasma cell membranes, that control the flow of ionic cmrents.
  • Current work shows there are ion channel families comprised of voltage-gated sodium, potassium, and calcium channels, as well as the ligand-gated acetylcholine receptors, glycine receptors, and gamma ammobutyric acid receptors.
  • Voltage-gated sodium channels have been the subject of numerous studies and much is known about these channels and their component parts. These transmembrane proteins are responsible for the early sodium permeability increases that underlie initial depolarization of the action potential in many excitable cells such as muscle, nerve, and cardiac cells. More specifically, sodium channels are composed of a central pore-forming ⁇ subunit (260 kDa) and two auxiliary subunits, ⁇ l (36 kDa) and ⁇ 2 (33kDa), which do not form the pore yet play critical roles in channel modulation and expression. The ⁇ l subunit is of particular interest because a mutation in the ⁇ l gene (Scnlb) has been implicated to play a role in fibrillar seizures and generalized epilepsy, GEFS+
  • ⁇ l subunits can be classified as members of the V-set of the Ig- superfamily which includes many cell adhesion molecules, ⁇ 1 and type IIA ⁇ subunit co-expression has been well-characterized in Xenopus oocytes and in mammalian cells.
  • CHL Chinese hamster lung
  • the present invention describes a novel splice variant of the sodium channel ⁇ 1 subunit and various methods and compositions for exploiting this finding.
  • the present invention contemplates an isolated nucleic acid comprising a region, or the complement thereof, encoding a sodium channel ⁇ l A subunit or an allelic variant or mutant thereof.
  • the sodium channel ⁇ l A subunit coding region encodes a primate sodium channel ⁇ lA subunit.
  • the sodium channel ⁇ l A is a splice variant of sodium channel ⁇ l subunit. More particularly, the splice variant results from an intron retention of the sodium channel ⁇ l subunit encoding nucleic acid.
  • the nucleic acid has a sequence of SEQ ID NO:l (GenBankTM AF182949).
  • the nucleic acid is selected from the group consisting of genomic DNA, complementary DNA and RNA.
  • the nucleic acid is a complementary DNA and further comprises a promoter operably linked to said region, or the complement thereof, encoding said sodium channel ⁇ lA subunit.
  • the nucleic acid may comprise a polyadenylation signal operably linked to said region encoding said sodium channel ⁇ l A subunit encoding region.
  • the nucleic acid may also comprise an origin of replication.
  • the nucleic acid may be a viral vector selected from the group consisting of retrovirus, adenovirus, he ⁇ esvirus, vaccinia virus and adeno-associated virus. More specifically, the nucleic acid is packaged into a viral particle or the nucleic acid may be packaged into a liposome or a dendrimer formulation.
  • the present invention contemplates an isolated oligonucleotide of between about 10 and about 50 consecutive bases of a nucleic acid, or complementary thereto, encoding a sodium channel ⁇ lA subunit.
  • the oligonucleotide may be 17, 18, 19, 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,
  • these consecutive bases may be derived from the coding region of SEQ ID NO: 1.
  • a sodium channel ⁇ lA subunit-encoding nucleic acid operably linked to a first promoter.
  • preferred promoters may be selected from the group consisting of
  • the expression construct may be a lentiviral, adenoviral, adeno-associated viral, vaccinia viral, he ⁇ es viral or retroviral expression construct. More particularly, the ⁇ l A encoding nucleic acid sequence is as set forth in SEQ ID NO: 1. In other embodiments, the ⁇ lA encoding expression construct encodes a protein of SEQ ID NO:2.
  • the sodium channel ⁇ lA subunit has the amino acid sequence as set forth in SEQ ID NO:2.
  • the peptide is conjugated to a carrier molecule.
  • the carrier molecule may be any molecule commonly used to generate antibodies.
  • the carrier molecule may be KLH and BSA.
  • the sodium channel ⁇ l A subunit has the amino acid sequence as set forth in SEQ ID NO:2.
  • the antibody provides a monoclonal antibody that binds immunologically to a sodium channel ⁇ l A subunit.
  • the antibody does not bind immunologically to other sodium channel subunit polypeptides.
  • the antibody further comprises a detectable label.
  • the label may be selected from the group consisting of a fluorescent label, a chemiluminescent label, a radiolabel and an enzyme.
  • a hybridoma cell that produces a monoclonal antibody that binds immunologically to a sodium channel ⁇ lA subunit. In prefened embodiments, the antibody does not bind immunologically to other sodium channel subunit polypeptides.
  • the present invention provides a polyclonal antisera, antibodies of which bind immunologically to a sodium channel ⁇ l A subunit.
  • the antisera may be generated from any animal normally used by those of skill in the art to produce antisera.
  • the antisera is derived from an animal selected from the group consisting human, mouse, horse, dog, goat, rabbit, rat, and sheep.
  • Additional embodiments of the present invention provide a method of screening for a modulator of sodium channel activity comprising providing a cell co- expressing a sodium channel ⁇ lA subunit polypeptide with a sodium channel ⁇ subunit; contacting said cell with a candidate modulator substance; and determining the effect of said candidate substance on the sodium channel function in said cell.
  • the determining comprises comparing the sodium cunent density of the cell in the presence of said candidate substance with sodium current density of said cell in the absence of said candidate substance wherein an alteration of said density is indicative of said candidate substance being a modulator. More particular embodiments describe the candidate substance as being selected from a small molecule library. In preferred embodiments, the candidate substance alters the expression of said sodium ⁇ l A subunit. The method is such that the cell may be contacted in vitro or in vivo.
  • a modulator of sodium channel activity identified by a method comprising providing a cell co-expressing a sodium channel ⁇ lA subunit polypeptide with a sodium channel ⁇ subunit; contacting said cell with a candidate modulator substance; and determining the effect of said candidate substance on the sodium channel function in said cell.
  • the modulator is an inhibitor of sodium channel ⁇ lA subunit activity.
  • the modulator is an activator of sodium channel ⁇ lA subunit activity.
  • the modulator is an inhibitor of sodium channel activity.
  • the candidate substance modulates the expression of sodium channel ⁇ 1 A subunit.
  • the modulator is an activator of sodium channel activity.
  • the modulator may be a naturally occurring modulator of sodium channel ⁇ lA activity or is a modulator synthesized from rational drug design.
  • Other aspects of the present invention contemplate a method for transforming a cell comprising contacting the cell with a nucleic acid expression construct (i) encoding a sodium channel ⁇ 1 A subunit and (ii) a promoter active in said cell, wherein said promoter is operably linked to the region encoding said sodium channel ⁇ lA subunit, under conditions permitting the uptake of said expression construct by said cell.
  • the cell is a brain cell, lung cell, muscle cell, adrenal cell, fibroblast cell, or a cardiac cell.
  • the expression construct is encapsulated in a liposome or a dendrimer.
  • the expression construct is a viral vector selected from the group consisting of retrovirus, adenovirus, adeno- associated virus, vaccinia virus and he ⁇ esvirus.
  • the nucleic acid is encapsulated in a viral particle.
  • a method for decreasing the number of fibrillar seizures in an individual comprising administering to said individual a modulator of a sodium channel ⁇ lA subunit in an amount effective to change the sodium channel activity in said individual.
  • the modulator is identified according to a method comprising providing a cell expressing a sodium channel ⁇ 1 A subunit polypeptide; contacting said cell with a candidate substance; and determining the effect of said candidate substance on the activity of said sodium channel ⁇ lA subunit.
  • the modulator is an anti-epileptic agent.
  • the modulator is an inhibitor of sodium channel ⁇ lA subunit.
  • the modulator affect the level of expression of the sodium channel ⁇ lA subunit.
  • the modulator decreases the expression of sodium channel ⁇ lA subunit.
  • a method for treating fibrillar seizures in a subject comprising altering the activity or level of sodium channel ⁇ lA subunits of a cell in said subject.
  • the altering comprises contacting said cell with a sodium channel ⁇ lA subunit under conditions permitting the uptake of said sodium channel ⁇ 1 A subunit by said cell.
  • the contacting comprises contacting said cell with a nucleic acid expression construct (i) encoding a sodium channel ⁇ lA subunit and (ii) a promoter active in said cell, wherein said promoter is operably linked to the region encoding said sodium channel ⁇ l A subunit, under conditions permitting the uptake of said expression construct by said cell.
  • the subject is a human.
  • Another embodiment contemplates a method for decreasing neuropathic pain in an individual comprising administering to said individual a modulator of a sodium channel ⁇ lA subunit in an amount effective to change the sodium channel activity in said individual.
  • the modulator decreases the expression of sodium channel ⁇ l A subunit of the cells of said individual.
  • a method for treating neuropathic pain in a subject comprising altering the activity or level of sodium channel ⁇ lA subunits of a cell in said subject.
  • sodium channel ⁇ l A is involved in certain cardiac functions relating to the sodium channel.
  • neuropathic pain also could be employed to ameliorate disorders in cardiac function, e.g., cardiac arrhythmia and the like.
  • altering the activity or level of sodium channel ⁇ lA subunits of a cell in a subject would be useful in treating cardiac anhythmia.
  • FIG. 1A-FIG. ID Sequence analysis of ⁇ lA
  • FIG. 1 A Deduced amino acid sequence of ⁇ 1 A. Solid box: signal peptide that becomes cleaved in the mature protein. Arrow: site of alternate splice resulting in retention of intron 3. Asterisks: N- linked glycosylation sites. Dotted box: transmembrane segment predicted from Kyte- Dolittle hydropathy profile. Solid underline: amino acid sequence used to synthesize ⁇ lA-MAP.
  • FIG. IB Sequence analysis of ⁇ lA - Comparison of ⁇ lA and ⁇ l amino acid sequences. Upper sequence: ⁇ lA.
  • FIG. IC Sequence analysis of ⁇ lA - Putative membrane topologies of ⁇ lA and ⁇ l .
  • ⁇ lA is predicted to encode a type I membrane protein with similar topolgy to ⁇ l .
  • the disulfide-linked immunoglobulin fold (from the indicated carboxy-terminal disulfide bond to the amino terminus; traced by " ⁇ ") is common to both ⁇ l and ⁇ lA.
  • ⁇ lA contains a novel 66-amino acid juxtamembrane region, 19-amino acid transmembrane segment, and 39-amino acid intracellular domain.
  • FIG. ID Sequence analysis of ⁇ l A - Sequence homology to known proteins. Results of BLAST-P search of the Swissprot database using the novel, carboxy-terminal domain of ⁇ 1 A beginning at residue 130 as the query sequence.
  • FIG. 2A-FIG. 2D Effects of ⁇ lA on the functional properties of whole cell sodium currents.
  • FIG. 2A Voltage-dependent sodium currents recorded in a SNallA cell (top traces) and a SNallA ⁇ lA cell (bottom traces). Cunents were elicited by depolarizations to -40, -30, -20, -10, 0 and +10 mV, from a prepulse potential of -100 mV.
  • FIG. 2B Mean activation (filled symbols) and inactivation
  • n 15; SNaIIA ⁇ lA-7, -8.1 ⁇ 2.13, -7.9 ⁇ 0.48, 6; SNaIIA ⁇ lA-8, -11.6 ⁇ 1.21, - 7.4 ⁇ 0.31, 11; SNaIIA ⁇ lA-16, -17.0 ⁇ 1.41, -7.18 ⁇ 0.39, 11; inactivation: SNallA,
  • FIG. 2C Inactivation time constants ( ⁇ jna.tivation) determined from fits of current decay for SNallA (O), SNaIIA ⁇ lA-7 (D), SNaIIA ⁇ lA-8 ( ⁇ ) and SNallA ⁇ l A-l 6 (V), plotted as a function of test potential.
  • FIG. 2D Mean V I 2 values for activation (filled symbols) and inactivation (open symbols) for cell lines
  • FIG. 3A and FIG. 3B Effect of ⁇ l A on the level of expression of functional sodium channels.
  • FIG. 3 A Current densities for SNallA and
  • FIG. 3B Amplitude- frequency histogram for SNallA (black bars) and SNallA ⁇ lA (white bars; data for all three SNallA ⁇ lA cell lines were combined). Currents were evoked by depolarization to +10 mV. The bars indicate the number of cells with peak cunents that fell within different amplitude ranges.
  • the present invention describes the isolation, molecular cloning and functional characterization of a new protein involved in voltage-gated sodium channel formation.
  • the data presented herein show that ⁇ l A is a splice variant of the ⁇ l gene.
  • ⁇ lA contains an identical amino terminal and extracellular Ig-fold region as the ⁇ l protein; however, this region is followed by a significantly different extracellular juxtamembrane domain, predicted transmembrane region, and predicted intracellular
  • the inventors show that the ⁇ lA mRNA is expressed early in embryonic brain development and then disappears after birth.
  • Western blot analysis of membrane preparations using an antibody to a unique, extracellular region of ⁇ l A not found in ⁇ l showed that ⁇ lA protein is expressed in adult rat heart, skeletal muscle, and adrenal gland but was not detected in adult brain or spinal cord.
  • ⁇ lA functions to increase channel expression at the plasma membrane when coexpressed with ⁇ llA subunits in CHL fibroblasts.
  • DRG dorsal root ganglion
  • ⁇ lA functions to increase channel expression at the plasma membrane when coexpressed with ⁇ llA subunits in CHL fibroblasts.
  • mean steady state inactivation curves for ⁇ lA-expressing cell lines were shifted to more positive potentials than the mean inactivation curves for cells expressing ⁇ alone.
  • Previous studies showed that coexpression of ⁇ and ⁇ 1 subunits in CHL cells shifted the voltage-dependence of inactivation to more negative potentials compared to ⁇ alone (Isom et al, 1995b).
  • the novel, carboxy-terminal domains of ⁇ l A likely is important for electrophysiological function. It has been shown previously that the extracellular domain of ⁇ 1 is essential for expression and function of the ⁇ l complex in Xenopus oocytes (Chen and Cannon, 1995; McCormick et al, 1998). The inventors suggest that the extracellular domain of ⁇ 1 is essential for expression and function of the ⁇ l complex in Xenopus oocytes (Chen and Cannon, 1995; McCormick et al, 1998). The inventors suggest that the extracellular
  • Ig fold common to ⁇ l and ⁇ lA, is essential for the observed increases in channel expression levels.
  • this report introduces a novel splice variant of ⁇ l, ⁇ lA, and adds to the understanding of ⁇ l structure/function relationships in terms of channel expression/stabilization and electrophysiology.
  • the present invention exploits these findings to provide a variety of novel methods and compositions related to voltage-gated sodium channels.
  • the novel protein and DNA sequences described herein and variants thereof may be used in the production of antibodies, expression vectors, in methods for producing recombinant proteins, methods of producing recombinant cells, screening assays and identification of modulators of sodium channels using such assays.
  • the sodium channel plays a central role in a number of biologically significant areas.
  • the present inventors contemplate that the sodium channel is involved in neuropathic pain and as such modulators of the sodium channel ⁇ l A subunit may be identified that modulate neuropathic pain.
  • these channels are important in certain aspects of cardiac function, and the present invention may be employed in various ways of altering, adjusting or otherwise treating disorders stemming form changes in cardiac function.
  • the sodium ⁇ l A subunit may be involved in altering the amount, degree, or severity of fibrillar seizures in an individual.
  • the present invention describes methods and compositions that may be used in modifying, altering or otherwise managing these disorders as well as providing details of the use of the sodium channel ⁇ lA subunit related compositions in various research settings.
  • the localized cell surface density and the functional properties of sodium channels are a key determinant of the threshold for action potential generation and the frequency of firing of neurons.
  • Myelination of both central and peripheral axons permits rapid saltatory conduction of action potentials through a cooperation between the ax on itself and specialized glial cells which envelope the axon with multiple insulating layers. These cells are the ohgodendrocytes in the central nervous system (CNS), and Schwann cells in the peripheral nervous system (PNS).
  • Interruptions in the myelin sheath known as nodes of Ranvier, contain locally high concentrations of voltage-gated sodium channels, the membrane proteins which are responsible for initiation and propagation of the action potential.
  • Brain and muscle sodium channel ⁇ subunit isoforms interact with multiple members of the syntrophin family, cytoplasmic peripheral membrane proteins which contain a PDZ domain (Gee et al, 1998).
  • Cell adhesion molecules present in the node of Ranvier such as neuro fascin and NrCAM, have also been proposed to participate in linking axonal proteins with glial processes (Davis et al, 1996).
  • sodium channel ⁇ subunit isoforms are differentially localized in neurons.
  • ⁇ lLA is located axonally and ⁇ l is located somatodendritically in hippocampal slices (Westenbroek et al, 1989).
  • information regulating sodium channel localization may be encoded in the amino acid sequence of various subunit isoforms.
  • Sodium channels isolated from brain are heterotrimeric structures composed of a central, pore-containing oc-subunit and two auxiliary subunits, ⁇ l and ⁇ 2, which do not form the pore but play critical roles in channel gating, voltage-dependence of activation and inactivation, expression levels, and localization.
  • At least 4 genes encoding sodium channel ⁇ subunits are expressed in the CNS: ⁇ l (Noda et al, 1986a; Noda et al, 1986b), ⁇ ll/IIA (Auld et al, 1988; Noda et al, 1986a; Noda et al, 1986b), ⁇ lll (Kayano et al.
  • RNA encoding ⁇ subunits is sufficient to direct the synthesis of functional sodium channels inXenopus oocytes (Goldin et al, 1986; Noda et al, 1986b), but co- injection of low molecular weight brain mRNA from brain or skeletal muscle is required for rapid inactivation (Auld et al, 1988; Joho et al, 1990, Krafte et al, 1988; Ukomadu et al, 1992; Zhou et al, 1991). These results suggested a possible role for the low molecular weight ⁇ subunits in sodium channel function.
  • Glial-derived extracellular matrix molecules such as tenascin-C (TN-C) and tenascin-R (TN-R), play important roles in cell interactions in the developing nervous system, such as neuronal migration, neuritogenesis, and neuronal regeneration (Bartsch, 1996; Chiquet-Ehrismann et al, 1994; Erickson, 1993; Schachner et al, 1994).
  • TN-R is expressed predominantly by ohgodendrocytes during the onset and early phases of myelin formation, and remains expressed by some ohgodendrocytes in the adult (Bartsch et al, 1993; Fuss et al, 1991 ; Fuss et al, 1993; Pesheva et al, 1989; Wintergerst et al, 1993) as well as some neurons and interneurons in the spinal cord, retina, cerebellum, and hippocampus (Bartsch et al, 1993, Fuss et al, 1993; Wintergerst et al, 1993).
  • TN-R co-localizes with other glial-derived molecules, such as myelin-associated glycoprotein (MAG) and a phosphacan-related molecule, at high density in myelinated CNS nerves (Xiao et al, 1997).
  • MAG myelin-associated glycoprotein
  • TN-R is a multi-functional molecule that promotes neurite outgrowth when presented as a uniform substrate, inhibits growth cone advance when offered as a sha ⁇ substrate boundary, and induces axonal defasciculation in vitro, resulting from interaction with one of its neuronal receptors, F3/contactin (Lochter and Schachner, 1993; Lochter et al, 1994; Pesheva et al, 1993, Taylor et al, 1993).
  • Cell adhesion molecules of the Ig superfamily interact homophilically and heterophilically to transduce signals between adjacent cells or adjacent axons where they participate in axonal fasciculation.
  • Cell adhesion molecules of the LI family have been studied extensively. For example, transfection of neuroglian, a member of the LI family, into Drosophila S2 cells results in homophilic binding and cellular aggregation (Hortsch et al, 1998; Malhotra et al, 1998). It appears that the ⁇ subunits behave as classical CAMs in addition to playing roles as channel modulators, ⁇ subunits participate in modulation of the voltage-dependence of channel activation and inactivation, channel gating mode, as well as channel expression levels at the plasma membrane.
  • the ⁇ subunits are members of the Ig superfamily and play roles in cellular adhesion and repulsion.
  • sodium channel ⁇ subunits are multifunctional proteins with possible roles independent of the ion channel complex.
  • the inventors propose that the sodium channel ⁇ subunits function as true CAMs, acting as bridges between the extra- and intracellular neuronal environments.
  • the present invention provides detailed characterization, cloning and sequencing of an important and novel sodium channel ⁇ subunit.
  • a splice variant of the sodium channel ⁇ 1 subunit that results from an intron retention in the ⁇ 1 subunit encoding gene.
  • This splice variant is refened to herein as ⁇ l A.
  • Co-expression of ⁇ lA with an ⁇ -subunit results in an increase in sodium current density compared to cells expressing ⁇ alone.
  • This increase in cunent density reflected two effects of ⁇ lA: 1) an increase in the proportion of cells expressing detectable sodium cunents, and 2) an increase in the level of functional sodium channels in expressing cells.
  • the present invention also relates to fragments of the polypeptide that may or may not retain the ⁇ -subunit regulatory (or other) activity of ⁇ l A. Fragments, including the N-terminus of the molecule may be generated by genetic engineering of translation stop sites within the coding region (discussed below). Alternatively, treatment of the ⁇ lA molecule with proteolytic enzymes, known as proteases, can produce a variety of N-terminal, C-terminal and internal fragments. Examples of fragments may include contiguous residues of the ⁇ lA sequence given in FIG.
  • IB of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 100, 200, or more amino acids in length.
  • fragments may be purified according to known methods, such as precipitation (e.g., ammonium sulfate), HPLC, ion exchange chromatography, affinity chromatography (including immunoaffinity chromatography) or various size separations (sedimentation, gel electrophoresis, gel filtration).
  • ⁇ lA encodes a 272 amino acid polypeptide.
  • the predicted molecular weight of this molecule is 50kDa, with a resulting pi of 6.42.
  • this molecule may be used as a standard in assays where molecule weight and pi are being examined.
  • ⁇ 1 A a splice variant of ⁇ 1 , is the result of the retention of intron 3 containing an in frame stop codon. This alternate splicing event produces a novel carboxy- terminus that includes an extracellular region, a transmembrane segment, and a short intracellular domain.
  • the retained intron contains an alternate, in- frame termination codon as well as a polyadenylation signal.
  • Waxman and coworkers have previously reported that intron 5 of ⁇ 1 can be retained, creating a novel isoform which is expressed in rat brain, optic nerve, sciatic nerve, and skeletal muscle.
  • This isoform contains a 86 nucleotide insert encoded by intron 5 in the 3' untranslated region (Oh and Waxman, 1994; Dib-Hajj and Waxman, 1995). While the intron retention reported previously does not alter the ⁇ 1 coding sequence, the present data describe a very significant coding sequence change resulting in a novel carboxy terminus.
  • Sodium channel ⁇ subunits are members of the immunoglobulin (Ig) superfamily and contain extracellular cell adhesion molecule (CAM) domains. Because CAMs interact with extracellular protein ligands as well as with other Ig proteins, Ig proteins, Ig proteins, and others
  • the ⁇ subunits likely act in a similar fashion.
  • the inventors suggest that the transmembrane and/or the intracellular domains of the sodium channel ⁇ subunits are responsible for transducing a signal from the extracellular environment to cytoskeletal or signaling molecules inside the cell.
  • Tenascin-R (TN-R) an extracellular matrix protein that is secreted by ohgodendrocytes during formation of CNS nodes of
  • Ranvier is a functional modulator of sodium channel ⁇ and ⁇ subunits of TN-R binding and can be used to study the effects of varying the subunit sequence.
  • GPI glycophosphatidyinositol
  • the Ig loop region of the ⁇ lA and the other ⁇ subunits is responsible for mediating homophilic binding and the intracellular COOH-terminal regions of these molecules are then responsible for transducing a signal resulting in ankyrin recruitment to the plasma membrane.
  • a truncated ⁇ subunit lacking the COOH- terminal intracellular domain is predicted to display homophilic binding but be incapable of ankyrin recruitment.
  • mutants to test this Drosophila S2 cells.
  • recombinant fragments of TN-R can be used to inhibit homophilic binding between ⁇ subunits expressed in S2 cells. This will allow determination of whether these two binding events are mediated by the same or different extracellular amino acid motifs in the ⁇ subunits.
  • Some CAMs belonging to the Ig superfamily interact heterophilically with other CAMs to transduce signals across the plasma membrane to the cytoskeleton and other signaling molecules. This interaction can be intercellular or trans, such that different CAMs on adjacent cells interact, or it can be intracellular or cis, such that adjacent molecules on the same plasma membrane interact to produce a signal.
  • Neurofascin, ankyrin G , and sodium channels have been shown to be colocalized at CNS nodes of Ranvier. Thus, neurofascin and ⁇ subunits may interact via cis heterophilic binding.
  • Drosophila S2 cell model system it will be possible to test for aggregation and ankyrin recruitment by mixing ⁇ 1 -, ⁇ 2-, and/or neuro fascin- transfected cells, after one of the cell lines has been labeled with Dil.
  • Dil By performing coimmunoprecipitations on cells that are cotransfected with a ⁇ subunit plus neurofascin it will be possible to test for cis interactions.
  • a mutation in the Ig loop region of ⁇ l has recently been implicated in frbrillar seizures and generalized epilepsy (Wallace et al, 1998).
  • Mutations in LI -CAM are linked to hydrocephalus, motor neuron defects, agenesis of the co ⁇ us callosum, and the cerebrospinal tract.
  • Mutations in myelin P o a CAM with homology to ⁇ 1 , may be involved in Charcot- Marie-Tooth disease. Alterations in sodium channel clustering and localization are inherent in demyelinating disease such as multiple sclerosis. Given the present disclosure of a novel ⁇ subunit of the sodium channel containing a COOH terminus that is different from those described for ⁇ 1 and ⁇ 2, there will be an increase the basic understanding of the molecular and cellular biology of sodium channel ⁇ subunits, especially their roles as CAMs, and lead to the future development of therapeutic agents for these and related diseases.
  • ⁇ lA-expressing cell lines also exhibited subtle functional differences compared to the parent SNallA cell line. For example, inactivation curves in SNaHA ⁇ 1 A cell lines were shifted to slightly more positive potentials than inactivation curves for SNallA cells. This finding differs from previous results showing that coexpression of ⁇ 1 and ⁇ llA in CHL cells shifts inactivation to potentials approximately 10 mV more negative than for cells expressing ⁇ alone. However, there is a significant difference in the ⁇ l and ⁇ lA proteins in that the latter contains a novel 55 residue long juxtamembrane portion which may explain this difference in steady state inactivation.
  • TTX-sensitive sodium channel ⁇ subunits expressed in brain (type I Scala, Smith and Goldin, 1998; type II/Scn2a, Isom et al, 1992; Isom et al, 1995; type III/Scn3a, Patton et al, 1994; type VI/PN4/Scn8a, Smith et al, 1998) and skeletal muscle (Skm 1/Scn4a Wallner et al, 1993) have been shown to be modulated by co- expression of ⁇ l subunits in heterologous systems.
  • TTX-resistant sodium channel ⁇ subunits expressed in cardiac myocytes (Skm 2/Hl/Scn5a; Qu et al, 1995) and peripheral nerve (PN3/SNS/Scnl0a; Sangemeswaran et al, 1996; Klugbauer et al, 1995; Akopian et al, 1996) are much less sensitive or insensitive to modulation by ⁇ 1 when co-expressed either in Xenopus oocytes or mammalian cells. It is proposed that tissues in which TTX-resistant channels are expressed may also express novel auxiliary subunits, possibly ⁇ lA.
  • TTX-resistant channels are modulated by ⁇ lA.
  • ⁇ lA When the present application refers to the function of ⁇ 1 A or "wild-type" activity, it is meant that the molecule in question has the ability to modulate the sodium density of a cell co-expressing the sodium ⁇ -subunit.
  • the cellular signaling mediated by these pores is influenced by the ⁇ l A.
  • Other phenotypes that may be considered to be regulated by the normal ⁇ l A gene product include peripheral nerve sodium channels (i.e., SCN10A, SCN11A); cardiac sodium channels (SCN5A);
  • neuropathic pain and cardiac arrythmias such as Long QT may be involved
  • cell adhesion/repulsion thus including axonal fasciculation defasciculation, growth cone guidance, and synaptic remodeling following epileptic seizures. Determination of which molecules possess this activity may be achieved using assays familiar to those of skill in the art. For example, transfer of genes encoding ⁇ l A, or variants thereof, into cells that do not have a functional sodium channel, and hence exhibit impaired anion transport, will identify, by virtue of increasing the sodium cunent density in these cells, those molecules having ⁇ lA function.
  • the assays using TN-R, ankyrin and/or neurofascin binding discussed herein also may be employed to show ⁇ 1 A function seeing as these molecules are known to interact with ⁇ subunits. Additional studies may be performed comparing such assays from cells co-expressing ⁇ lA subunits and an ⁇ -subunit with cells co-expressing other ⁇ subunits (e.g. , ⁇ 1 , ⁇ 2 and the like) with an ⁇ -subunit thereby giving a quantitative and qualitative difference in the function/activity of a ⁇ l A as compared to e.g., a ⁇ l or ⁇ 2 subunit.
  • ⁇ lA is belongs to the Ig superfamily of CAMs.
  • ⁇ lA is predicted to encode a type I membrane protein with similar topolgy to ⁇ l (FIG. IC).
  • ⁇ lA contains a novel 66-amino acid juxtamembrane region, 19-amino acid transmembrane segment, and 39-amino acid intracellular domain.
  • the disulfide- linked immunoglobulin fold is common to both ⁇ l and ⁇ lA.
  • the ⁇ subunits are known to regulate the function of the pore-forming ⁇ -subunit of the sodium channel. Given that there is a substantial difference in the COOH terminus of the ⁇ lA as compared to e.g.
  • ⁇ l it will be desirable to determine the effect of this novel COOH terminus on the function in the sodium channel-regulating role of ⁇ lA. This also may be a fruitful approach to developing screening assays for the absence of ⁇ lA function or in the development of therapies, for example, in targeting the sodium current density and other functions of ⁇ l A, targeting the substrate upon which ⁇ l A acts (e.g., a subunit, ankyrin, TN-R), and the like.
  • Variants of ⁇ lA Amino acid sequence variants of the polypeptide can be substitution, insertion or deletion variants.
  • Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity, and are exemplified by the variants lacking a transmembrane, COOH-terminus, the Ig fold or other region described above.
  • Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell.
  • Insertion mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an immunoreactive epitope or simply a single residue. Terminal additions, called fusion proteins, are discussed below.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • amino acids of a protein may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below. Table 1 shows the codons that encode particular amino acids.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte &
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte & Doohttle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (- 0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those that are within ⁇ 1 are particularly prefened, and those within ⁇ 0.5 are even more particularly prefened.
  • Patent 4,554,101 the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine *-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Another embodiment for the preparation of polypeptides according to the invention is the use of peptide mimetics.
  • Mimetics are peptide-containing molecules that mimic elements of protein secondary structure.
  • the present inventors have identified that the ⁇ lA is a splice variant of ⁇ l subunit of voltage-gated sodium channels. This provides a starting point for further mutational analysis of the molecule.
  • domain switching involves the generation of chimeric molecules using different but, in many cases, related polypeptides.
  • the inventors have predicted the functionally significant regions of these molecules. It is possible, then, to switch related domains of these molecules in an effort to determine the criticality of these regions to the ⁇ subunit activity and function.
  • These molecules may have additional value in that these "chimeras" can be distinguished from natural molecules, while possibly providing the same function.
  • Ig fold domain Another structural aspect of ⁇ lA that provides fertile ground for domain switching experiments is the Ig fold domain. This domain may be substituted for Ig folds from other members of the Ig superfamily domains in order to alter the specificity of this function. A further investigation of the homology between ⁇ lA and other members of the Ig superfamily is warranted by this observation.
  • a specialized kind of insertional variant is the fusion protein.
  • This molecule generally has all or a substantial portion of the native molecule, linked at the N- or C- terminus, to all or a portion of a second polypeptide.
  • fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.
  • Another useful fusion includes the addition of a immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification.
  • fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions.
  • One particular fusion of interest would include a deletion construct lacking the carboxy terminal site of ⁇ l A but containing other regions that could bind the substrate molecule. Indeed any of the distinct regions of the ⁇ lA protein can be removed or replaced in order to determine their function in relation to the whole protein. Fusion to a polypeptide that can be used for purification of the substrate- ⁇ l A complex would serve to isolate the substrate for identification and analysis.
  • Protein Purification It will be desirable to purify ⁇ lA or variants thereof. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non- polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
  • purified protein or peptide is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state.
  • a purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity.
  • substantially purified is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
  • Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
  • a prefened method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number.”
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • High Performance Liquid Chromatography is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
  • Gel chromatography is a special type of partition chromatography that is based on molecular size.
  • the theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size.
  • the sole factor determining rate of flow is the size.
  • Gel chromatography is unsu ⁇ assed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adso ⁇ tion, less zone spreading and the elution volume is related in a simple matter to molecular weight.
  • Affinity chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind. This is a receptor-ligand type interaction.
  • the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
  • a particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography.
  • Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins.
  • Lectins are usually coupled to agarose by cyanogen bromide.
  • Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins.
  • Other lectins that have been include lentil lectin, wheat germ agglutinin, which has been useful in the purification of N-acetyl glucosaminyl residues, and Helix pomatia lectin.
  • Lectins themselves are purified using affinity chromatography with carbohydrate ligands.
  • Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and
  • L-fucose will bind to lectins from lotus.
  • the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
  • affinity chromatography One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.
  • the present invention also describes smaller ⁇ lA-related peptides for use in various embodiments of the present invention.
  • the peptides of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques.
  • Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, (1984); Tarn et al, (1983); Merrifield, (1986); and Barany and Merrifield (1979).
  • Short peptide sequences, or libraries of overlapping peptides usually from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides.
  • recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • the present invention also provides for the use of ⁇ 1 A proteins or peptides as antigens for the immunization of animals relating to the production of antibodies. It is envisioned that either ⁇ l A, or portions thereof, will be coupled, bonded, bound, conjugated or chemically-linked to one or more agents via linkers, polylinkers or derivatized amino acids. This may be performed such that a bispecific or multivalent composition or vaccine is produced. It is further envisioned that the methods used in the preparation of these compositions will be familiar to those of skill in the art and should be suitable for administration to animals, i.e., pharmaceutically acceptable. Preferred agents are the carriers are keyhole limpet hemocyannin (KLH) or bovine serum albumin (BSA).
  • KLH keyhole limpet hemocyannin
  • BSA bovine serum albumin
  • the present invention also provides, in another embodiment, nucleic acid sequences encoding ⁇ lA.
  • the present invention is not limited in scope to these genes, however, as one of ordinary skill in the art could, using these nucleic acids, readily identify related homologs in various other species (e.g., rat, dog, rabbit, monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species).
  • the present invention is not limited to the specific nucleic acids disclosed herein.
  • a " ⁇ lA encoding nucleic acid” may contain a variety of different bases and yet still produce a conesponding polypeptide that is functionally indistinguishable, and in some cases structurally, from the polypeptide disclosed herein as SEQ ED NO:2.
  • any reference to a nucleic acid should be read as encompassing a host cell containing that nucleic acid and, in some cases, capable of expressing the product of that nucleic acid.
  • cells expressing nucleic acids of the present invention may prove useful in the context of screening for agents that induce, repress, inhibit, augment, interfere with, block, abrogate, stimulate or enhance the function of ⁇ l A or voltage gated sodium channel activity in general.
  • nucleic Acids Encoding ⁇ lA The nucleic acid given in FIG. IC and disclosed in SEQ ID NO:l represent the ⁇ l A encoding nucleic acids of the present invention. Nucleic acids according to the present invention may encode an entire ⁇ lA protein as shown in SEQ ID NO:2, a domain of ⁇ l A that expresses a particular function attributable to ⁇ l A, or any other fragment of the ⁇ l A sequences set forth herein.
  • the nucleic acid may be derived from genomic DNA, /. e. , cloned directly from the genome of a particular organism. In prefened embodiments, however, the nucleic acid would comprise complementary DNA (cDNA).
  • a cDNA plus a natural intron or an intron derived from another gene such engineered molecules are sometime refened to as "mini-genes.”
  • mini-genes This is particularly of note seeing as the ⁇ 1 A protein results from a splice variant of ⁇ 1 gene.
  • the ⁇ lA protein results from a retention of intron 3 of the ⁇ l gene.
  • these and other nucleic acids of the present invention may be used as molecular weight standards in, for example, gel electrophoresis.
  • cDNA is intended to refer to DNA prepared using messenger RNA (mRNA) as template.
  • mRNA messenger RNA
  • a nucleic acid encoding a ⁇ l A refers to a nucleic acid molecule that has been isolated free of total cellular nucleic acid.
  • the invention concerns a nucleic acid sequence essentially as set forth in SEQ ID NO: 1.
  • the term “as set forth in SEQ ID NO: 1” means that the nucleic acid sequence substantially conesponds to a portion of SEQ ID NO: 1.
  • the term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine (Table 1 , below), and also refers to codons that encode biologically equivalent amino acids, as discussed in the following pages.
  • sequences that have at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotides of SEQ ID NO:l will be sequences that are "as set forth in SEQ ID NO: 1." Sequences that are essentially the same as those set forth in SEQ ID NO:l may also be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:l under standard conditions.
  • the DNA segments of the present invention include those encoding biologically functional equivalent ⁇ lA proteins and peptides, as described above. Such sequences may arise as a consequence of codon redundancy and amino acid functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
  • functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques or may be introduced randomly and screened later for the desired function, as described below.
  • nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementary rules.
  • complementary sequences means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:l under relatively stringent conditions such as those described herein.
  • Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single-stranded molecules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands.
  • a partially complementary sequence is one that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid; it is refened to using the functional term "substantially homologous.”
  • the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization and the like) under conditions of low stringency.
  • a substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence or probe to the target sequence under conditions of low stringency.
  • low stringency conditions are such that nonspecific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding, the probe will not hybridize to the second non- complementary target sequence.
  • sequences of the present invention may encode the entire ⁇ lA protein or functional or non-functional fragments thereof.
  • the hybridizing segments may be shorter oligonucleotides. Sequences of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length.
  • exemplary oligonucleotides of 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be used, although others are contemplated. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or 3431 bases and longer are contemplated as well. Such oligonucleotides will find use, for example, as probes in Southern and Northern blots and as primers in amplification reactions.
  • hybridization refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
  • a hybridization complex may be formed in solution or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which cells have been fixed for in situ hybridization).
  • hybridization solution may be varied to generate conditions of either low or high stringency different from, but equivalent to, the above listed conditions.
  • substitution of amino acids by site-directed mutagenesis it is appreciated that lower stringency conditions are required. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature.
  • a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C.
  • hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
  • hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 10 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C.
  • Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 ⁇ M MgCl , at temperatures ranging from approximately 40°C to about 72°C.
  • Formamide and SDS also may be used to alter the hybridization conditions.
  • One method of using probes and primers of the present invention is in the search for genes related to ⁇ lA or, more particularly, homologs of ⁇ lA from other species. The existence of a rat homolog strongly suggests that other homologs of the human ⁇ l A will be discovered in species more closely related, and perhaps more remote, than mouse.
  • the target DNA will be a genomic or cDNA library, although screening may involve analysis of RNA molecules. By varying the stringency of hybridization, and the region of the probe, different degrees of homology may be discovered.
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.
  • the technique further provides a ready ability to prepare and test sequence variants, inco ⁇ orating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is prefened, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
  • site-directed mutagenesis is performed by first obtaining a single- stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein.
  • An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared.
  • This primer is then annealed with the single-stranded DNA preparation, taking into account the degree of mismatch when selecting hybridization conditions, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • E. coli polymerase I Klenow fragment DNA polymerizing enzymes
  • a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation.
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence anange
  • sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained.
  • recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • mutant ⁇ l A proteins may not be non-functional. Rather, they may have aberrant functions that cannot be overcome by replacement gene therapy, even where the "wild-type” molecule is expressed in amounts in excess of the mutant polypeptide.
  • Antisense treatments are one way of addressing this situation.
  • Antisense technology also may be used to "knock-out" function of ⁇ 1 A in the development of cell lines or transgenic mice for research, diagnostic and screening pu ⁇ oses.
  • Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences. By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules.
  • the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA.
  • G:C cytosine
  • A:T thymine
  • A:U uracil
  • Inclusion of less common bases such as inosine, 5-mefhylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and or stability.
  • Antisense RNA constructs, or DNA encoding such antisense RNA's may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
  • Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used.
  • complementary or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated.
  • an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
  • a non-homologous region e.g., ribozyme; see below
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
  • a genomic clone will need to be used.
  • the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
  • Ribozymes Another approach for addressing the "dominant negative" mutant protein is through the use of ribozymes. Although proteins traditionally have been used for catalysis of nucleic acids, another class of macromolecules has emerged as useful in this endeavor. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cook, 1987; Gerlach et al, 1987; Forster and
  • Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et al, 1981).
  • U.S. Patent No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991 ; Sarver et al, 1990).
  • ribozymes elicited genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme.
  • expression vectors are employed to express the ⁇ lA polypeptide product, which can then be purified and, for example, be used to vaccinate animals to generate antisera or monoclonal antibodies with which further studies may be conducted.
  • the expression vectors are used in gene therapy. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
  • expression construct is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • the transcript may be translated into a protein, but it need not be.
  • expression includes both transcription of a gene and translation of mRNA into a gene product.
  • expression only includes transcription of the nucleic acid encoding a gene of interest.
  • the nucleic acid encoding a gene product is under transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • under transcriptional control means that the promoter is in the conect location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
  • Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. At least one module in each promoter functions to position the start site for
  • RNA synthesis The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of direction the expression of the nucleic acid in the targeted cell.
  • such a promoter might include either a human or viral promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of the coding sequence of interest.
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given pu ⁇ ose.
  • a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.
  • Promoters that may be useful include insulin, elastin, amylase, pdr-1, pdx-1 and glucokinase, MMTV, MT-1, ecdysone and RuBisco, c-fos, TNF-alpha, C-reactive protein (Arcone et al, 1988), haptoglobin (Oliviero et al, 1987), serum amyloid A2,
  • IL-8 alpha- 1 acid glycoprotein (Prowse and Baumann, 1988), alpha- 1 antitypsin, lipoprotein lipase (Zechner et al, 1988), angiotensinogen (Ron et al, 1991), fibrinogen, c-jun (inducible by phorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide), collagenase (induced by phorbol esters and retinoic acid), metallothionein (heavy metal and glucocorticoid inducible), Stromelysin (inducible by phorbol ester, interleukin-1 and EGF), alpha-2 macroglobulin and alpha- 1 antichymo trypsin.
  • promoters that could be used according to the present invention include Lac-regulatable, chemotherapy inducible (e.g. MDR), and heat (hyperthermia) inducible promoters, Radiation-inducible (e.g., EGR (Joki et al, 1995)), Alpha-inhibin, RNA pol III tRNA met and other amino acid promoters, U 1 snRNA (Bartlett et al. , 1996), MC- 1 , PGK, -actin and alpha-globin.
  • MDR chemotherapy inducible
  • heat hyperthermia inducible promoters
  • Radiation-inducible e.g., EGR (Joki et al, 1995)
  • Alpha-inhibin e.g., RNA pol III tRNA met and other amino acid promoters
  • U 1 snRNA Bartlett et al. , 1996)
  • MC- 1 , PGK -actin and al
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.
  • enhancers The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. The list of viral promoters, cellular promoters/enhancers and inducible promoters/enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct is extensive and well known to those in the art.
  • Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.
  • a terminator are also serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • the cells contain nucleic acid constructs of the present invention, a cell may be identified in vitro or in vivo by including a marker in the expression construct. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • enzymes such as he ⁇ es simplex vims thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed.
  • Immunologic markers also can be employed. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.
  • IRES internal ribosome binding sites
  • IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
  • IRES elements from two members of the picanovirus family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well as an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames.
  • each open reading frame can be transcribed together, each separated by an IRES, creating polycistronic messages.
  • IRES element By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
  • Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker.
  • the expression construct In order to effect expression of sense or antisense nucleic acid constructs, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states.
  • One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).
  • the first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986).
  • papovaviruses simian virus 40, bovine papilloma virus, and polyoma
  • adenoviruses Rosgeway, 1988; Baichwal and Sugden, 1986.
  • the viral vectors used herein may be adenoviral, such as described in U.S. Patent No. 5,824,544; U.S. Patent No. 5,707,618; U.S. Patent No. 5,693,509; U.S. Patent No. 5,670,488; U.S. Patent No. 5,585,362 retroviral, such as described inU.S. Patent No. 5,888,502; U.S. Patent No. 5,830,725; U.S. Patent No. 5,770,414; U.S. Patent No. 5,686,278; U.S. Patent No. 4,861,719; an adeno-associated viral, such as described inU.S. Patent No. 5,474,935; U.S. Patent No. 5,139,941 ; U.S. Patent No. 5,622,856; U.S. Patent No. 5,658,776; U.S. Patent No. 5,773,289; U.S. Patent No.
  • Non-viral vectors Delivery of the expression constructs through non-viral vectors also is contemplated. Such delivery may employ mi croinj ection (U.S. Patent No. 5,612,205), electroporation (U.S. Patent No. 5,507,724; U.S. Patent No. 5,869,326; U.S. Patent No. 5,824,547; U.S. Patent No. 5,789,213; U.S. Patent No. 5,749,847; U.S. Patent No.
  • nucleic acid encoding the gene of interest may be positioned and expressed at different sites.
  • the nucleic acid encoding the gene may be stably integrated into the genome of the cell.
  • nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA.
  • nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • Cell Culture and Propagation Primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression construct, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well documented and are disclosed herein by reference (Freshner, 1992).
  • One embodiment of the foregoing involves the use of gene transfer to immortalized cells for the production of proteins.
  • the gene for the protein of interest may be transfened as described above into appropriate host cells followed by culture of cells under the appropriate conditions.
  • the gene for virtually any polypeptide may be employed in this manner.
  • the generation of recombinant expression vectors, and the elements included therein, are discussed above.
  • the protein to be produced may be an endogenous protein normally synthesized by the cell in question.
  • Examples of useful mammalian host cell lines are Vero and HeLa cells and cell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, NIH3T3, RIN and MDCK cells.
  • a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and process the gene product in the manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to insure the conect modification and processing of the foreign protein expressed.
  • a number of selection systems may be used including, but not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively.
  • anti- metabolite resistance can be used as the basis of selection for dhfr, that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside G418; and hygro, that confers resistance to hygromycin.
  • Animal cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i.e., a monolayer type of cell growth).
  • Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products.
  • suspension cultured cells have limitations, such as tumorigenic potential and lower protein production than adherent cells.
  • the culture is usually mixed with one or more agitators, based on bladed disks or marine propeller patterns. Agitator systems offering less shear forces than blades have been described. Agitation may be driven either directly or indirectly by magnetically coupled drives. Indirect drives reduce the risk of microbial contamination through seals on stiner shafts.
  • the airlift reactor also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture.
  • the gas stream enters a riser section of the reactor and drives circulation. Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor.
  • the main advantage of this design is the simplicity and lack of need for mechanical mixing. Typically, the height-to-diameter ratio is 10:1.
  • the airlift reactor scales up relatively easily, has good mass transfer of gases and generates relatively low shear forces.
  • the antibodies of the present invention are particularly useful for the isolation of antigens by immunoprecipitation.
  • Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein.
  • For the isolation of membrane proteins cells must be solubilized into detergent micelles.
  • Nonionic salts are prefened, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.
  • Antibodies and their uses are discussed further, below.
  • the present invention contemplates an antibody that is immunoreactive with a ⁇ l A molecule of the present invention, or any portion thereof.
  • An antibody can be a polyclonal or a monoclonal antibody.
  • an antibody is a monoclonal antibody.
  • Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988).
  • a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal.
  • an immunogen comprising a polypeptide of the present invention
  • a wide range of animal species can be used for the production of antisera.
  • an animal used for production of antisera is a non- human animal including rabbits, mice, rats, hamsters, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a prefened choice for production of polyclonal antibodies.
  • Antibodies both polyclonal and monoclonal, specific for isoforms of antigen may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art.
  • a composition containing antigenic epitopes of the compounds of the present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds of the present invention.
  • Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
  • the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods and in immunohistochemical procedures such as tissue staining, as well as in other procedures which may utilize antibodies specific to ⁇ lA- related antigen epitopes. Additionally, it is proposed that monoclonal antibodies specific to the particular ⁇ l A of different species may be utilized in other useful applications In general, both polyclonal and monoclonal antibodies against ⁇ l A may be used in a variety of embodiments.
  • Anti- ⁇ lA antibodies will also be useful in immunolocahzation studies to analyze the distribution of ⁇ lA during various cellular events, for example, to determine the cellular or tissue-specific distribution of ⁇ lA polypeptides under different points in the cell cycle.
  • a particularly useful application of such antibodies is in purifying native or recombinant ⁇ 1 A, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.
  • a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
  • exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • Means for conjugating a polypeptide to a carrier protein include glutaraldehyde, -maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal).
  • polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
  • MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified ⁇ lA protein, polypeptide or peptide or cell expressing high levels of ⁇ lA
  • a selected immunogen composition e.g., a purified or partially purified ⁇ lA protein, polypeptide or peptide or cell expressing high levels of ⁇ lA
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep, or frog cells is also possible.
  • the use of rats may provide certain advantages (Goding, 1986), but mice are prefened, with the
  • BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies are selected for use in the mAb generating protocol.
  • B-cells B-lymphocytes
  • These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are prefened, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
  • a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately 5 x 10 7 to 2 x 10 8 lymphocytes.
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
  • any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984).
  • the immunized animal is a mouse
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 ratio, though the ratio may vary from about 20: 1 to about 1 : 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v)
  • Fusion procedures usually produce viable hybrids at low frequencies, around 1 x 10 "6 to 1 x 10 "8 . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium.
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • Exemplary and prefened agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimi dines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • the prefened selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
  • HPRT hypoxanthine phosphoribosyl transferase
  • This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs.
  • the cell lines may be exploited for mAb production in two basic ways.
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration.
  • the individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
  • mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
  • the present invention also contemplates the use of ⁇ lA and active fragments, and nucleic acids coding thereof, in the screening of compounds for sodium channel modulator activity.
  • activity may be a stimulatory activity in which the activity of the sodium channel is increased or an inhibitory activity in which the activity of the sodium channel is decreased.
  • the modulator may be active in stimulating ⁇ lA activity, overcoming the lack of ⁇ l A or blocking the effect of a mutant ⁇ lA molecule.
  • assays may make use of a variety of different formats and may depend on the kind of "activity" for which the screen is being conducted.
  • Contemplated functional "read-outs” include binding to a compound, inhibition of binding to a substrate, ligand, receptor or other binding partner by a compound, or a functional readout such as monitoring sodium current density.
  • agents known to alter the activity of sodium channels include but are not limited to blockers and activators of sodium channels.
  • the present section is directed to identifying additional modulators of sodium channel function using the findings of the present invention.
  • the active compounds may include fragments or parts of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive. However, prior to testing of such compounds in humans or animal models, it may be necessary to test a variety of candidates to determine which have potential. Accordingly, in screening assays to identify useful agents which modulate sodium channel function, it is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds.
  • the present invention is directed to a method for determining the ability of a candidate substance to modulate the activity of the sodium channel, the method including generally the steps of
  • a candidate substance as being capable of modulating sodium channel function, one would measure or determine the sodium cunent density of the cell that co-expresses a ⁇ lA channel subunit with a sodium channel ⁇ -subunit in the absence of the added candidate substance. One would then add the candidate substance to the cell and re-determine the sodium current density in the presence of the candidate substance. A candidate substance which produces an alteration in the sodium cunent density relative to the density in its absence is indicative of a candidate substance with modulatory capability.
  • the candidate screening assay is quite simple to set up and perform, and is related in many ways to the assay discussed above for determining sodium current density.
  • Effective amounts are those amounts effective at reproducibly altering sodium cunent density in comparison to the normal levels in the absence of the candidate substance. Compounds that achieve significant appropriate changes in activity will be used. If desired, a battery of compounds may be screened in vitro to identify other agents for use in the present invention.
  • a significant alteration in the sodium channel function e.g., as measured using sodium cunent density, are represented by an increase/decrease in sodium current density levels of at least about 30%-40%, and most preferably, by changes of at least about 50%, with higher values of course being possible.
  • the agents will often be selected on the basis of their known properties or by structural and/or functional comparison to those agents already demonstrated to be effective. Therefore, the effective amounts will often be those amounts proposed to be safe for administration to animals in another context, for example, as disclosed herein.
  • the agents identified may be those which bind to the ⁇ l A molecule or fragment thereof.
  • the polypeptide or fragment may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the polypeptide or the compound may be labeled, thereby permitting determining of binding.
  • the assay may measure the inhibition of binding of ⁇ l A to a natural or artificial substrate or binding partner.
  • Competitive binding assays can be performed in which one of the agents ( ⁇ lA, binding partner or compound) is labeled.
  • the polypeptide will be the labeled species.
  • One may measure the amount of free label versus bound label to determine binding or inhibition of binding.
  • Another technique for high throughput screening of compounds is described in WO 84/03564. Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with ⁇ lA and washed. Bound polypeptide is detected by various methods.
  • Purified ⁇ l A can be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies to the polypeptide can be used to immobilize the polypeptide to a solid phase.
  • fusion proteins containing a reactive region may be used to link the ⁇ l A active region to a solid phase.
  • ⁇ 1 A Various cell lines containing wild-type or natural or engineered mutations in ⁇ 1 A can be used to study various functional attributes of ⁇ 1 A and how a candidate compound affects these attributes. Methods for engineering mutations are described elsewhere in this document.
  • the compound would be formulated appropriately, given its biochemical nature, and contacted with a target cell. Depending on the assay, culture may be required. The cell may then be examined by virtue of a number of different physiologic assays. Alternatively, molecular analysis may be performed in which the function of ⁇ 1 A, or related pathways, may be explored. This may involve assays such as those for protein expression, enzyme function, substrate utilization, mRNA expression (including differential display of whole cell or polyA RNA) and others.
  • the present invention also encompasses the use of various animal models.
  • identity seen between human and rat ⁇ lA provides an excellent opportunity to examine the function of ⁇ l A in a whole animal system where it is normally expressed.
  • Treatment of animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal.
  • Administration will be by any route the could be utilized for clinical or non-clinical pu ⁇ oses, including but not limited to oral, nasal, buccal, rectal, vaginal or topical.
  • administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
  • systemic intravenous injection regional administration via blood or lymph supply and intratumoral injection.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides or compounds with which they interact (agonists, antagonists, inhibitors, binding partners, etc.). By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules.
  • drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules.
  • ⁇ l A or a fragment thereof This could be accomplished by x-ray crystallography, computer modeling or by a combination of both approaches.
  • An alternative approach, "alanine scan” involves the random replacement of residues throughout molecule with alanine, and the resulting affect on function determined.
  • Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.
  • drugs which have improved ⁇ lA activity or which act as stimulators, inhibitors, agonists, antagonists or ⁇ lA or molecules affected by ⁇ l A function.
  • sufficient amounts of ⁇ lA can be produced to perform crystallographic studies.
  • knowledge of the polypeptide sequences permits computer employed predictions of structure- function relationships.
  • Sodium channel blockers are a well characterized group of pharmaceutical agents especially in the control of anhythmia.
  • These drugs include but are not limited to quinidine, phenytoin, mexiletine, tocainide, procainamide, disopyramide, moricizine, propafenone, flecainide, and the like.
  • quinidine phenytoin
  • mexiletine mexiletine
  • tocainide procainamide
  • disopyramide moricizine
  • propafenone flecainide
  • Sodium channel blockers are a well characterized group of pharmaceutical agents especially in the control of anhythmia.
  • these drugs include but are not limited to quinidine, phenytoin, mexiletine, tocainide, procainamide, disopyramide, moricizine, propafenone, flecainide, and the like.
  • those of skill in the art are refened to Goodman and Gilman's "Pharmaceutical
  • anesthetic agents that may be used to block these channels include but are not limited to lidocaine, benzocaine, bupivacaine, cocaine, etidocaine, mepivacaine, promoxine, prilocaine, procaine, proparacaine, ropivacaine and tetracaine.
  • lidocaine benzocaine
  • bupivacaine cocaine, etidocaine, mepivacaine, promoxine, prilocaine, procaine, proparacaine, ropivacaine and tetracaine.
  • sodium channel blockade also useful in sodium channel blockade are the toxins based on tetrodotoxin and saxitoxin, two of the most potent poisons known. These agents may be used in rational drug design to produce a therapeutic that is less toxic but still an effective modulator of the sodium channel.
  • certain sodium channel blockers also are known to be effective in anti- convulsant therapy. These agents include but are not limited to phenytoin, carbamazepine, valproate, lamotrigine and topiramate.
  • assays may measure various properties such as repulsion form TNR, cell adhesion assays, immunocytochemical assays and the like.
  • TNR repulsion form
  • cell adhesion assays cell adhesion assays
  • immunocytochemical assays immunocytochemical assays
  • Tissue culture 4-well or 24-well dishes are coated with methanol-solubilized nitrocellulose according to Lagenaur and Lemmon (1987).
  • 2.5 ⁇ l aliquots of TN-R (15 nM) and TN-C (15 nM) or GST-fusion domains of TN-R (25 nM) are applied to the nitrocellulose/poly-DL-ornithine (PO)-coated surfaces of the dishes and incubated for 2 h at 37 °C in a humidified atmosphere as described previously (Xiao et al, 1996).
  • the dishes are then washed three times with Ca 2+ - and Mg 2+ -free Hank's balanced salt solution (CMF-HBSS).
  • CMF-HBSS Hank's balanced salt solution
  • the coating efficiency is determined as described previously (Xiao et al, 1996). Substrate boundaries are marked in ink. The source of nitrocellulose (Schleicher and Schuell, catalog #401188, BA85, 0.45 ⁇ m) is important to obtain consistent results in this assay. Parental 1610 or transfected cells are plated at a density of 10 5 cells/ml. After 20 h, the cells are fixed with 2.5% glutaraldehyde and stained with Coomassie blue (Sigma Chemical Co., St. Louis,
  • Sodium channels appear to recognize substrates such as TN-R by an initial adhesion.
  • An exemplary adhesion assay that may be employed is described as follows. Tissue culture 4-well or 24-well dishes are coated with methanol-solubilized nitrocellulose according to Lagenaur and Lemmon (1987) and air-dried under a sterile hood. For adhesion assays, 2.5 ⁇ l spots of different TN-R fragments or GST (each at, a concentration of 25 ⁇ M) are applied to the nitrocelluose-coated surfaces of the dishes and incubated for 2 h at 37 °C in a humidified atmosphere.
  • the dried spots are washed with PBS and then flooded with CMF-HBSS containing 2% heat-inactivated fatty acid free BSA (Sigma) and incubated 2 h to block residual non-specific protein binding sites.
  • the dishes are then washed with PBS and cells from the various cell lines are plated at a density of 10 5 cells/ml in 0.5 ml of growth medium containing 10% BSA.
  • CMF-HBSS containing 2.5% glutaraldehyde.
  • a mixture of EGF-L, EGF-S, and FN6-8 can be added to the culture medium. After fixation, cultures are stained with 0.5% toluidine blue in 2.5% sodium carbonate. Cells adhering to the various spots of TN-R fragments can then be photographed and counted.
  • Stable clones of transfected S2 cells are induced overnight in the presence of 0.7 mM CuSO4 followed by aggregation for 4 h at room temperature on a rotary shaker.
  • Cell aggregates containing at least 10 cells are then analyzed by phase contrast microscopy for homophilic aggregation or by a combination of phase contrast and fluorescent microscopy for heterophilic aggregation.
  • Immunocytochemistry of Drosophila ankyrin distribution in S2 cells is performed following aggregation experiments.
  • Cells are fixed with 2% paraformaldehyde, and permeabilized with 0.5% Triton X-100.
  • Mouse anti- Drosophila ankyrin is used as the primary antibody followed by incubation with fluorescein isothiocyanate-conjugated secondary antibody. Slides are then viewed with a Bio-Rad MRC 600 confocal scanning laser microscope.
  • Immunoprecipitations can be performed from S2 cells following protein synthesis induction but without inducing cell aggregation (cells not shaken). Cells are then pelleted and solubilized in 1.25% Triton X-100 and the soluble fraction is incubated overnight with the appropriate antibody. Protein- A-sepharose is then added and the incubation continued for 2 h. Immunoprecipitates are eluted from the Protein- A-sepharose with SDS-PAGE sample buffer and separated on 10 % SDS-PAGE gels. Proteins are then transferred to nitrocellulose and probed with the appropriate second antibody.
  • ⁇ lA and the conesponding gene may be employed as a diagnostic or prognostic indicator of such seizures in infants. More specifically, point mutations, deletions, insertions or regulatory perturbations relating to ⁇ lA may be the cause of these seizures. If it can be predicted that an individual is predisposed to such seizures, then a prophylactic course of treatments can be designed.
  • One embodiment of the instant invention comprises a method for detecting variation in the expression of ⁇ 1 A. This may comprise determining that level of ⁇ 1 A or determining specific alterations in the expressed product. Obviously, this sort of assay has importance in the diagnosis of related disease.
  • the biological sample can be any tissue or fluid. Various embodiments include cells of the skin, muscle, fascia, brain, prostate, breast, endometrium, lung, head & neck, pancreas, small intestine, blood cells, liver, testes, ovaries, colon, skin, stomach, esophagus, spleen, lymph node, bone marrow or kidney.
  • fluid samples such as peripheral blood, lymph fluid, ascites, serous fluid, pleural effusion, sputum, cerebrospinal fluid, lacrimal fluid, stool or urine.
  • Nucleic acid to be used in such an analysis is isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al, 1989).
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA.
  • RNA is used, it may be desired to convert the RNA to a complementary DNA.
  • the RNA is whole cell RNA; in another, it is poly-A RNA.
  • the nucleic acid is amplified.
  • the specific nucleic acid of interest is identified in the sample directly using amplification or with a second, known nucleic acid following amplification.
  • the identified product is detected.
  • the detection may be performed by visual means (e.g., ethidium bromide staining of a gel).
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994).
  • alteratrons should be read as including deletions, insertions, point mutations and duplications. Point mutations result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those occurring in non-germ line tissues. Germ line tissue mutations can occur in any tissue and are inherited. Mutations in and outside the coding region also may affect the amount of ⁇ lA produced, both by altering the transcription of the gene or in destabilizing or otherwise altering the processing of either the transcript (mRNA) or protein, or altering the ratio of expression of one subunit in comparison to another. It is contemplated that mutations in the ⁇ l A gene may be identified in accordance with the present invention.
  • FISH fluorescent in situ hybridization
  • PFGE direct DNA sequencing
  • SSCA single- stranded conformation analysis
  • ASO allele-specific oligonucleotide
  • dot blot analysis denaturing gradient gel electrophoresis, RFLP and PCRTM-SSCP.
  • primer as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template- dependent process. Typically, primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is prefened. Probes are defined differently, although they may act as primers. Probes, while perhaps capable of priming, are designed to binding to the target DNA or RNA and need not be used in an amplification process.
  • the probes or primers are labeled with radioactive species ( 32 P, 14 C, 35 S, 3 H, or other label), with a fluorophore (rhodamine, fluorescem) or a chemillumiscent (luciferase).
  • radioactive species 32 P, 14 C, 35 S, 3 H, or other label
  • fluorophore rhodamine, fluorescem
  • chemillumiscent luciferase
  • PCRTM polymerase chain reaction
  • PCRTM two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence.
  • An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides.
  • a reverse transcriptase PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing
  • RNA into cDNA are well known and described in Sambrook et al, 1989.
  • Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641 filed December 21, 1990. Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • Q ⁇ Replicase described in PCT Application No. PCT/US87/00880, may also be used as still another amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence that can then be detected.
  • restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha-thio]-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention, Walker et al, (1992).
  • Strand Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
  • a similar method called Repair Chain Reaction (RCR)
  • RCR Repair Chain Reaction
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • SDA Strand Displacement Amplification
  • CPR cyclic probe reaction
  • a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample.
  • the reaction Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated. Still another amplification methods described in GB Application No. 2 202
  • modified primers are used in a PCRTM-like, template- and enzyme-dependent synthesis.
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labeled probes are added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989; Gingeras et al, PCT Application WO 88/10315).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR 3SR
  • the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • amplification techniques involve annealing a primer which has target specific sequences.
  • DNA RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
  • the double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6.
  • an RNA polymerase such as T7 or SP6.
  • the RNA's are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6.
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isofhermally without addition of enzymes at each cycle.
  • the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Miller et al, PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "RACE” and "one-sided PCRTM" (Frohman, M.A., In: PCRTM PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Academic Press, N.Y., 1990; Ohara et al, 1989.
  • Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide may also be used in the amplification step of the present invention (Wu et al, 1989).
  • Southern/Northern Blotting Blotting techniques are well known to those of skill in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provide different types of information, although cDNA blotting is analogous, in many aspects, to blotting of RNA species.
  • a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a nitrocellulose filter.
  • a suitable matrix often a nitrocellulose filter.
  • the different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter.
  • the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Because the probe is designed to base pair with the target, the probe will bind a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above.
  • a probe usually labeled
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al. (1989).
  • chromatographic techniques may be employed to effect separation. There are many kinds of chromatography which may be used in the present invention: adso ⁇ tion, partition, ion-exchange and molecular sieve, and many specialized techniques for using them, including column, paper, thin-layer and gas chromatography.
  • Products may be visualized in order to confirm amplification of the marker sequences.
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
  • visualization is achieved indirectly.
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.
  • detection is by a labeled probe.
  • the techniques involved are well known to those of skill in the art and can be found in many standard books on molecular protocols. See Sambrook et al. (1989).
  • chromophore or radiolabel probes or primers identify the target during or following amplification.
  • U.S. Patent No. 5,279,721 discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids.
  • the apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
  • the amplification products described above may be subjected to sequence analysis to identify specific kinds of variations using standard sequence analysis techniques.
  • oligonucleotide primers may be designed to permit the amplification of sequences throughout the ⁇ lA encoding nucleic acid that may then be analyzed by direct sequencing.
  • kits All the essential materials and reagents required for detecting and sequencing ⁇ 1 A and variants thereof may be assembled together in a kit. This generally will comprise preselected primers and probes. Also included may be enzymes suitable for amplifying nucleic acids, including various polymerases (RT, Taq, SequenaseTM etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe.
  • RT Reverse transcription
  • RT-PCRTM relative quantitative PCRTM
  • PCRTM the number of molecules of the amplified target DNA increase by a factor approaching two with every cycle of the reaction until some reagent becomes limiting. Thereafter, the rate of amplification becomes increasingly diminished until there is no increase in the amplified target between cycles.
  • a graph is plotted in which the cycle number is on the X axis and the log of the concentration of the amplified target DNA is on the Y axis, a curved line of characteristic shape is formed by connecting the plotted points. Beginning with the first cycle, the slope of the line is positive and constant. This is said to be the linear portion of the curve. After a reagent becomes limiting, the slope of the line begins to decrease and eventually becomes zero. At this point the concentration of the amplified target DNA becomes asymptotic to some fixed value. This is said to be the plateau portion of the curve.
  • the concentration of the target DNA in the linear portion of the PCRTM amplification is directly proportional to the starting concentration of the target before the reaction began.
  • concentration of the amplified products of the target DNA in PCRTM reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundances of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCRTM products and the relative mRNA abundances is only true in the linear range of the PCRTM reaction.
  • the final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. Therefore, the first condition that must be met before the relative abundances of a mRNA species can be determined by RT- PCRTM for a collection of RNA populations is that the concentrations of the amplified PCRTM products must be sampled when the PCRTM reactions are in the linear portion of their curves. The second condition that must be met for an RT-PCRTM experiment to successfully determine the relative abundances of a particular mRNA species is that relative concentrations of the amplifiable cDNAs must be normalized to some independent standard.
  • RT-PCRTM The goal of an RT-PCRTM experiment is to determine the abundance of a particular mRNA species relative to the average abundance of all mRNA species in the sample.
  • mRNAs for ⁇ - actin, asparagine synthetase and lipocortin II were used as external and internal standards to which the relative abundance of other mRNAs are compared.
  • RT-PCRTM is performed as a relative quantitative RT-PCRTM with an internal standard in which the internal standard is an amplifiable cDNA fragment that is larger than the target cDNA fragment and in which the abundance of the mRNA encoding the internal standard is roughly 5-100 fold higher than the mRNA encoding the target.
  • This assay measures relative abundance, not absolute abundance of the respective mRNA species.
  • Absolute mRNA abundance can be used as a measure of differential gene expression only in normalized samples. While empirical determination of the linear range of the amplification curve and normalization of cDNA preparations are tedious and time consuming processes, the resulting RT-PCRTM assays can be superior to those derived from the relative quantitative RT-PCRTM assay with an internal standard.
  • chip-based DNA technologies such as those described by Hacia et al. ( 1996) and Shoemaker et al.
  • Antibodies of the present invention can be used in characterizing the ⁇ lA content of healthy and diseased tissues, through techniques such as ELISAs and Western blotting. This may provide a screen for the presence or absence of a disorder or as a predictor of future dysfunction.
  • the use of antibodies of the present invention, in an ELISA assay is contemplated.
  • anti- ⁇ lA antibodies are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate.
  • the immobilizing surface is contacted with the sample to be tested in a manner conducive to immune complex (antigen/antibody) formation.
  • the occunence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for ⁇ l A that differs the first antibody.
  • Appropriate conditions preferably include diluting the sample with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween ® . These added agents also tend to assist in the reduction of nonspecific background.
  • BSA bovine gamma globulin
  • PBS phosphate buffered saline
  • the layered antisera is then allowed to incubate for from about 2 to about 4 hr, at temperatures preferably on the order of about 25° to about 27°C. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material.
  • a prefened washing procedure includes washing with a solution such as PBS/Tween ® , or borate buffer.
  • the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
  • an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
  • one will desire to contact and incubate the second antibody-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hr at room temperature in a PBS-containing solution such as PBS/Tween*).
  • the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol pu ⁇ le or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
  • a chromogenic substrate such as urea and bromocresol pu ⁇ le or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label.
  • Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
  • the preceding format may be altered by first binding the sample to the assay plate. Then, primary antibody is incubated with the assay plate, followed by detecting of bound primary antibody using a labeled second antibody with specificity for the primary antibody.
  • the antibody compositions of the present invention will find great use in immunoblot or Western blot analysis.
  • the antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof. In conjunction with immunoprecipitation, followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background.
  • Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard. 11. Methods for Treating ⁇ lA Related Disorders
  • the present invention also involves, in another embodiment, the treatment of disorders such as epilepsy and fibrillar seizures.
  • disorders such as epilepsy and fibrillar seizures.
  • the types of disorders that may be treated, according to the present invention are limited only by the involvement of ⁇ l A. By involvement, it is not even a requirement that ⁇ l A be mutated or abnormal - the overexpression of this protein may actually overcome dysfunction of sodium channel function within the cell.
  • ⁇ l A based therapy including therapies with the modulators identified in the section above, immunotherapeutics, protein therapy, gene-based therapies and combination therapies.
  • Immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy aberrant cells.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells.
  • Immunotherapy could be used as part of a combined therapy, in conjunction with ⁇ l A-targeted gene therapy.
  • the general approach for combined therapy is discussed below.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • Another therapy approach is the provision, to a subject, of ⁇ l A polypeptide, active fragments, synthetic peptides, mimetics or other analogs thereof.
  • the protein may be produced by recombinant expression means or, if small enough, generated by an automated peptide synthesizer.
  • Formulations would be selected based on the route of administration and pu ⁇ ose including, but not limited to, liposomal formulations and classic pharmaceutical preparations.
  • One of the therapeutic embodiments contemplated by the present inventors is the intervention, at the molecular level, in the events involved in the malfunction of voltage gated sodium channels.
  • the present inventors intend to provide, to a cell having an ⁇ subunit forming the sodium channel pore, an expression construct capable of providing ⁇ l A to that cell.
  • the lengthy discussion above of expression vectors and the genetic elements are also useful in this embodiment.
  • Particularly prefened expression vectors are viral vectors such as adenovirus, adeno-associated virus, he ⁇ esvirus, vaccinia virus and retrovirus. Also prefened is liposomally- encapsulated expression vector.
  • the present invention identifies a sodium channel ⁇ subunit that can be used to modulate the activity of the sodium channel, it is contemplated that protein and gene-based therapies centered on this discovery may be combined with other known modulators of sodium channel function to achieve a desirable change in sodium channel function in these settings.
  • compositions of the present invention To achieve such useful modulation of the sodium channel activity using the methods and compositions of the present invention, one would generally contact a "target" cell with a ⁇ lA expression construct, protein, or other ⁇ lA based therapeutic identified herein and at least one other agent. These compositions would be provided in a combined amount effective to alter the voltage gated sodium channel function of the cell. This process may involve contacting the cells with the ⁇ 1 A based therapeutic and the agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the ⁇ lA based therapeutic and the other includes the agent.
  • the ⁇ lA based treatment may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the other agent and ⁇ l A based composition are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and ⁇ lA based composition would still be able to exert an advantageously combined effect on the cell.
  • A/B/A B/A/B B/B/A A/AB B/AA A/B/B B/B/A B/B/A/B A/A/B/B A/B/A B A B/B/A B/B/A/A B/A/B/A B/A A B B/B/B/A
  • both agents are delivered to a cell in a combined amount effective to alter the sodium cunent density in said cell.
  • compositions - expression vectors, virus stocks, proteins, antibodies and drugs - in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • One will generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells. Buffers also will be employed when recombinant cells are introduced into a patient.
  • Aqueous compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are refened to as inocula.
  • “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well know in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be inco ⁇ orated into the compositions.
  • the active compositions of the present invention may include classic pharmaceutical preparations.
  • compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra.
  • the active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium stearate, and gelatin.
  • Sterile injectable solutions are prepared by inco ⁇ orating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • pharmaceutically acceptable earner includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be inco ⁇ orated into the compositions.
  • the polypeptides of the present invention may be inco ⁇ orated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared inco ⁇ orating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be inco ⁇ orated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
  • the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
  • the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • the solution For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic
  • transgenic animals are produced which contain a functional transgene encoding a functional ⁇ lA polypeptide or variants thereof.
  • Transgenic animals expressing ⁇ lA transgenes, recombinant cell lines derived from such animals and transgenic embryos may be useful in methods for screening for and identifying agents that induce or repress function of ⁇ l A.
  • Transgenic animals of the present invention also can be used as models for studying malfunctions of voltage gated sodium channels.
  • a ⁇ l A transgene is introduced into a non- human host to produce a transgenic animal expressing a human or murine ⁇ l A gene.
  • the transgenic animal is produced by the integration of the transgene into the genome in a manner that permits the expression of the transgene.
  • Methods for producing transgenic animals are generally described by Wagner and Hoppe (U.S. Patent No. 4,873,191) and Brinster et al. 1985) and in "Manipulating the Mouse Embryo; A
  • ⁇ 1 A ⁇ 1 A gene flanked by genomic sequences is transfened by microinj ection into a fertilized egg. The microinjected eggs are implanted into a host female, and the progeny are screened for the expression of the transgene.
  • Transgenic animals may be produced from the fertilized eggs from a number of animals including, but not limited to reptiles, amphibians, birds, mammals, and fish.
  • transgenic mice are generated which overexpress ⁇ 1 A or express a mutant form of the polypeptide.
  • the absence of a ⁇ l A in "knock-out" mice permits the study of the effects that loss of ⁇ l A protein has on a cell in vivo.
  • Knock-out mice also provide a model for the development of ⁇ l A-related disorders.
  • transgenic animals and cell lines derived from such animals may find use in certain testing experiments.
  • transgenic animals and cell lines capable of expressing wild-type or mutant ⁇ 1 A may be exposed to test substances. These test substances can be screened for the ability to enhance wild-type ⁇ lA expression and or function or impair the expression or function of mutant ⁇ lA.
  • ⁇ l A transcript identified by library screening was expressed by rat adrenal gland
  • a region of ⁇ l A from the amino terminus was amplified past the region in which the amino acid sequence changed from identity to non-identity to ⁇ l, or the putative splice site, by reverse-transcriptase polymerase chain reaction (RT-PCR) using adult rat adrenal gland total RNA as template and ⁇ lA3 (5'-GAAGATGAGCGCTTTGAGG-3'(SEQ ID NO:3), primer sequence common to ⁇ l and ⁇ lA) and ⁇ lA5 (5'-GAGAGACACAGCAAGC (SEQ ID NO:4), primer sequence unique to ⁇ lA) as oligonucleotide forward and reverse primers, respectively.
  • RT-PCR reverse-transcriptase polymerase chain reaction
  • Rat adrenal gland cDNA was synthesized from total RNA using Superscript II (Gibco/BRL, Gaithersburg, MD) according to the manufacturer's instructions in a total volume of 20 ⁇ l. 2.3 ⁇ g of total rat adrenal RNA (purified using Trizol reagent, Gibco/BRL) was used in the reaction.
  • the PCR conditions were as follows: 1 ⁇ l of cDNA, 0.5 ⁇ M of each primer, 200 ⁇ M of each dNTP (Boehringer Mannheim), 5 ⁇ l of Mg 2+ -free 10X PCR buffer (Perkin Elmer-Roche Molecular Systems, Branchburg, NJ), and 1.5 mM MgCl 2 were mixed in a total volume of 50 ⁇ l.
  • the band was excised form the gel, subcloned into pCR2.1 (Invitrogen, Carlsbad, CA), and analyzed using ThermoSequenase (Amersham Pharmacia Biotech). The sequence obtained from this PCR clone was identical to that obtained from the original ⁇ lA clone plaque-purified from the adrenal cDNA library.
  • Intron 3 of the rat ⁇ l gene was amplified by PCR using rat genomic DNA as the template, oligonucleotides which encode ⁇ 1 coding sequence flanking intron 3, VVDK (SEQ ID NO:5) (5'- AGATCCACCTGGAGGTGGTGGACAAGG-3' (SEQ ID NO:6)) and ANRD (SEQ ID NO:6)
  • RNAse protection analysis 40 cycles of the following regimen were performed: 94°C for 10 sec, 58°C for 30 sec, 68°C for 4 min. plus 20 sec. added to each successive cycle. The samples were then held at 4°C until removal from the thermocycler (GeneAmp 2400, Perkin Elmer). The 5 kb PCR product was gel-purified and sequenced directly using oligonucleotide VVDK (SEQ ID NO: 5) as the sequencing primer. RNAse protection analysis:
  • a plasmid containing a RNAse protection probe template (pRPA-1) was constructed corresponding to nucleotides 364-533 in the ⁇ lA sequence. Briefly, a 169-nucleotide Alu I/Acc I fragment was excised from pBK. ⁇ l A and ligated into the Smal and Ace I sites of pBluescript (Stratagene). The resulting plasmid was then sequenced using ThermoSequenase (Amersham).
  • RNAse-free DNAse 2 units
  • RNAse-free water Free nucleotides were removed by ethanol precipitation with 0.5 M ammonium acetate and the final pellet was resuspended in 20 ⁇ l of RNAse-free water.
  • the probe (RPA-1) was quantitated by comparison of serial dilutions of the labeled probe with serial dilutions of control digoxigenin-labeled RNA supplied by Boehringer Mannheim following the manufacturer's instructions.
  • RNAse protection experiments were performed using the HybSpeed RPA kit from Ambion. Briefly, 20 ⁇ g of rat embryonic day 18 brain RNA were mixed with 1 ⁇ l of digoxigenin-labeled RPA-1 probe and 30 ⁇ g of yeast tRNA in 0.5 M ammonium acetate plus 2.5 volumes of ethanol. The reaction tubes were left at -20°C for 15 minutes and the RNA was precipitated by centrifugation in a microfuge at top speed. The RNA was resuspended in 10 ⁇ l of HybSpeed hybridization buffer that had been preheated to 95°C, vortexed vigorously, and the tubes were placed at 95°C for 3 minutes.
  • RNAse A and TI 10 U/ml of RNAse A and 400 U/ml RNAse TI
  • 150 ⁇ l of Hybspeed Inactivation/Precipitation mix were added to each reaction, and the RNA was precipitated and resuspended in 10 ⁇ l of Gel Loading Buffer 1.
  • the reactions were electrophoresed on a 1.5 mm thick 6% acrylamide TBE denaturing gel containing 7M urea in the Mini-Protean gel format (BioRad, Hercules,
  • Northern blot analysis of 20 ⁇ g of each sample was performed as previously described (Isom et al, 1995a) using a digoxigenin-labeled ⁇ lA antisense cRNA probe encoding nucleotides 428- 850 or a digoxigenin-labeled antisense cRNA probe specific to the 3' untranslated region of ⁇ l (nucleotides 1053-1508 of p ⁇ l .ClAa; Isom et al, 1992).
  • a plasmid containing ⁇ l cDNA including an in-frame amino terminal hemagglutinin (HA) epitope tag was obtained as a generous gift from the laboratory of R.A. Maue at Dartmouth University (Shah et al, 1996). This construct has been shown to express functional ⁇ l subunits in Xenpous oocytes.
  • the HA-tagged ⁇ l cDNA was recloned into the Eco RI and Not I sites of the mammalian expression vector pCIneo (Promega. Madison, WI) to create pCI. ⁇ l-HA.
  • pCI. ⁇ l-HA was subsequently digested with Ace I and Not I and agarose gel-purified to remove the 3' end of ⁇ l.
  • the Ace I restriction endonuclease site is common to ⁇ l and ⁇ lA.
  • pBK. ⁇ lA cDNA was digested with Ace I and Not I and gel-purified. The 3' end of ⁇ lA was then ligated into Ace 1/ Not I-digested pCI. ⁇ l-HA to create pCI. ⁇ lA-HA. The junctions were then sequenced to confirm that the segments of ⁇ l and ⁇ lA were successfully ligated in frame.
  • SNallA cells were transfected with pCI. ⁇ l A-HA using DOTAP as previously described (Isom et al, 1995b). Because SNallA cells are resistant to G418 as a result of the original transfection of the ⁇ llA subunit, pCI. ⁇ lA-HA was cotransfected with pSV2*Hyg to confer resistance to the antibiotic hygromycin. Drug selection with hygromycin (400 ⁇ g/ml) required approximately 1 week, Clonal cell lines were selected, analyzed by Northern blot, and expanded as previously described (Isom et al, 1995b).
  • MAP multiple antigenic peptide with amino acid sequence RWRDRWKEGDRLVSHRGQ (SEQ ID NO: 9), encoded by nucleotides 160 through
  • SuperSignal WestFemto chemiluminescent substrate solution (Pierce) was applied according to the manufacturer's instructions, the blot was placed between plastic sheet protectors, and exposed to Hyperfilm-ECL (Amersham) for the indicated times (typically 10 to 30 seconds) at room temperature.
  • Rat tissues were routinely fixed in 10% neutral buffered formalin, processed, embedded in paraffin blocks, sectioned onto slides.
  • the rabbit anti-rat ⁇ Al antibody was used at titer of 1 :900 for the human tissues and 1 :600 for the rat tissues. Briefly, slides were incubated (all incubations for 30 min.
  • Electrophysiological recordings on SNallA and SNallA ⁇ lA cells were performed by the patch clamp technique in the whole cell configuration (Hamill et al. , 1981), using an Axopatch 200B patch clamp amplifier and pCLAMP software (Axon Instruments). Data were filtered at 5 kH and digitally sampled at 50 kH. Series resistance was compensated 60-80%. Capacitive transients, elicited by voltage steps, were partially canceled using the internal clamp circuitry. Additional subtraction of transients and leak currents was obtained using the P/4 procedure (Armstrong and Bezanilla 1977). For whole cell recordings, recording pipettes were filled with 105 mM CsF, 10 mM CsCl, 10 mM NaCl, 10 mM EGTA, 10 mM HEPES, pH 7.4 with
  • the conductance values were normalized with respect to the maximal conductance, plotted as a function of V tes t, and fit with the Boltzman equation: l/(l+exp((V tes. - N ⁇ )lk), in which V/ 2 is the midpoint of the curve and k is a slope factor.
  • Steady state inactivation was examined by applying 100 ms long prepulses to potentials ranging from -100 to -10, in 5 mV steps, followed by a test pulse to 0 mV.
  • the peak amplitude of cunents evoked by the test pulses were normalized with respect to the largest currents, plotted as a function of prepulse potential and fit with the Boltzman equation.
  • Ipsilateral and contralateral DRG neurons were characterized as nociceptive if the diameter was ⁇ 25 ⁇ m; all others were designated as sensory (Waxman, et al., 1999). An average of 30-40 neurons were quantified for immunoreactivity and scored as follows: 0.0 for no staining, 1.0 for weak staining,
  • a rat adrenal gland cDNA library prepared in the ⁇ Express vector was screened with a digoxigenin-labeled cDNA probe encoding nucleotides 345-911 of p ⁇ l.ClAa (Isom et al, 1992).
  • a clone encoding a protein with a 5' region of identity to ⁇ l and a novel 3' region was identified by DNA sequencing. The identity of this clone was then confirmed independently by reverse transcriptase polymerase chain reaction (RT-PCR) from rat adrenal cDNA using the oligonuceotides ⁇ lA3 and ⁇ lA5 followed by DNA sequencing, as described in Experimental Procedures.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • ⁇ lA This clone, designated ⁇ lA, encoded a novel 253 amino acid protein of 29,055 daltons (predicted molecular mass of the mature protein with the signal sequence removed) which contains a predicted amino terminal region of identity to ⁇ l, residues met (-1) through lys (129), followed by a novel carboxy terminal region (FIG. 1 A and FIG. IB). Hydrophobicity analysis of the novel, carboxy-terminal region revealed an apparent
  • FIG. 1A and FIG. IC 66-amino acid extension of the extracellular region of ⁇ l followed by a 19-amino acid transmembrane domain and short, 39-amino acid intracellular carboxy-terminus.
  • the novel 3' region of ⁇ l A is structurally homologous to ⁇ l in that it predicts a transmembrane domain and short intracellular region, yet it contains little to no homology at the amino acid level (FIG. IB).
  • the amino terminal region common to ⁇ l and ⁇ lA contains the extracellular immunoglobulin fold.
  • ⁇ lA can thus be characterized as a cell adhesion molecule like ⁇ l and ⁇ 2.
  • LRP2 has been shown to be a cysteine-rich type I membrane protein that forms a multimeric complex with receptor-associated protein (RAP). LRP2 binds clusterin with high affinity and is localized to clathrin-coated pits, suggesting that it may be an endocytic receptor.
  • RAP receptor-associated protein
  • LRP2 interacts with extracellular matrix components, similar to sodium channel ⁇ l and ⁇ 2 subunits.
  • the BLAST-P search also revealed a 63-residue region of ⁇ 1 A with 26% identity to tensin, a protein that has been implicated as the anchor for actin filaments at focal adhesions and is thought to act as a link between the cytoskeleton and signal transduction proteins (Weigt et al, 1992).
  • the region of homology to ⁇ l A is located in the insertin domain of tensin. This domain has been shown to permit polymerization of actin filaments.
  • EXAMPLE 3 ⁇ l A is encoded by a retained intron in the ⁇ l gene
  • the genomic organization of the human sodium channel ⁇ l subunit gene has been reported previously (Makita et al, 1994).
  • the ⁇ l gene contains 5 introns and 6 exons.
  • the ⁇ lA cDNA is the result of retention of 13, creating a novel 3' end. It was determined that the site of divergence between the ⁇ l and ⁇ lA cDNAs was precisely located at the boundary between exon 3 andintron 3 of the ⁇ l gene. Furthermore, a consensus sequence for exon-intron boundaries in genomic DNA (Mount, 1982) was readily identified at this location.
  • EXAMPLE 4 ⁇ lA mRNA is expressed in embryonic brain and adult adrenal gland A comparison of the developmental time courses of ⁇ lA and ⁇ l mRNA expression in developing rat brain was determined using specific, non-cross- hybridizing antisense cRNA probes for ⁇ lA and ⁇ l, respectively.
  • Time-mated pregnant female Sprague-Dawley rats were anesthetized with 60 mg/kg Beuthanasia- D i.p., and the fetuses were surgically removed.
  • Embryonic day 9 fetuses were homogenized in their entirety in Trizol reagent to purify total RNA according to the manufacturer's instructions.
  • Northern blot analysis of 20 ⁇ g of each sample was performed as previously described (Isom et al, 1995a) using a digoxigenin-labeled ⁇ lA antisense RNA probe encoding nucleotides 428-850 or a digoxigenin-labeled antisense RNA probe specific to the 3' untranslated region of ⁇ l (nucleotides 1053-1508 of p ⁇ l.ClAa; Isom et al, 1992).
  • ⁇ lA is expressed early in development and disappears after birth.
  • ⁇ 1 expression is not detectable during embryonic brain development and becomes detectable as ⁇ lA mRNA expression is decreased.
  • a polyclonal antibody was generated against a MAP peptide containing the amino acid sequence RWRDRWKEGDRLVSHRGQ (SEQ ID NO:9) encoded by the retained portion of intron 3 found in the ⁇ l A cDNA clone.
  • Brain, spinal cord, heart, skeletal muscle, and adrenal gland membranes were prepared as described previously (Isom et al. 1995). Total protein in each membrane preparation was quantitated with the BCA Protein Assay Kit. 250 ⁇ g of each membrane preparation were separated by SDS-PAGE as previously described (Isom et al, 1995) and transferred to nitrocellulose.
  • Western blot analysis consisted of the following steps, all at room temperature: the blot was first washed for 10 minutes in
  • TBS-T (10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween-20) and then blocked for 1 hour in 5% non-fat dry milk in TBS-T at room temperature.
  • Primary anti- ⁇ l A antibody (1 :750 dilution) was applied in blocking solution for 30 minutes.
  • secondary antibody (horse radish peroxidase-conjugated goat anti-rabbit IgG, ICN) diluted to 1 : 100,000 was applied in blocking solution for 30 minutes.
  • the ⁇ l A protein was also localized to a small population of motor neurons, while ⁇ lA-expressing neurons were also observed in laminae II-V of the dorsal horn. All neuronal cell types of the dorsal root ganglia contained ⁇ l A, while fiber tracks and glial cells did not stain.
  • non-neuronal cells were also found to express ⁇ lA. These include endomysium membranes of individual muscle fibers in rat atria, other areas of the rat heart such as ventricles, lung alveoli and some bronchus columnar epithelial cells expressed ⁇ l A protein.
  • ⁇ l A a hemagglutinin (HA) epitope- tagged version of the ⁇ l A cDNA was constructed.
  • An epitope tag was included for potential use in the event that the polyclonal antibody production was unsuccessful.
  • the HA tag experiments was not necessary as this approach was successful in raising an anti- ⁇ l A antibody.
  • Stably transfected cells lines expressing ⁇ lA were created in the previously-characterized SNallA cell line. SNallA cells are a stable line expressing type IIA sodium channel ⁇ subunits in Chinese hamster lung (CHL) cells (Isom et al, 1995).
  • pcDNA3- ⁇ lA was co- transfected with pSV2*Hyg in a 10:1 ratio (pcDNA3- ⁇ lA: pSV2*Hyg) so that transfected clones could be selected with the antibiotic hygromycin.
  • a number of hygromycin-resistant colonies were analyzed by Northern blot for ⁇ 1 A mRNA expression. Positive clones were expanded and analyzed further by [ 3 H]-STX binding.
  • [ 3 H]-STX binding analysis revealed a significant increase in the expression levels of functional sodium channels at the plasma membrane of SNallA ⁇ l A- 16 cells as compared to the parent line, SNallA (Table 2).
  • Non-linear regression analysis of saturation binding showed a 4.4-fold increase in B max as compared to SNallA with no significant change in the K D (0.8 nM for SNallA vs. 0.9 nM for SNallA ⁇ l A- 16).
  • H-Saxitoxin binding analysis of ⁇ llA- and ⁇ HA/ ⁇ lA-expressing cell lines.
  • Whole cell saturation binding analysis of SNAIIA and SNallA- ⁇ lA cells was performed as previously described (Isom et al, 1995b) over a concentration range of 0.1 to 10 nM [ 3 H]STX with the addition of 10 ⁇ M unlabeled tetrodotoxin to assess non-specific binding.
  • H-Saxitoxin H-STX, 28 Ci/mmol
  • Binding data were normalized to protein concentration using the BCA Protein Assay kit. Saturation binding data were analyzed by non-linear regression using Prism to obtain K D and B max values.
  • FIG. 2 A shows currents, elicited by depolarizations to varying test potentials, recorded in a typical SNallA cell and a typical SNalLA ⁇ 1 A cell.
  • ⁇ 1 A did not dramatically alter the properties of voltage-activated sodium cunents. Nevertheless, cunents in ⁇ lA-expressing cell lines were subtly different from cunents in SNallA cells.
  • one effect of ⁇ 1 A association with ⁇ lLA may be a small positive shift in the voltage-dependence of steady state inactivation.
  • p 0.001
  • FIG. 3 A shows amplitudes of cunents evoked by depolarization to +10 mV in SNallA and SNallA ⁇ lA cell lines, converted to current densities to factor out any differences in cell surface area.
  • Cunent densities for SNallA cells were 6.9 pA/pF, whereas cunent densities were approximately 2.5 times greater for the three SNallA ⁇ l A cell lines (FIG. 3 A).
  • This difference reflected two distinct effects of ⁇ lA on sodium channel expression.
  • ⁇ lA greatly increased the proportion of cells with measurable whole cell sodium currents.
  • FIG. 3B plots the number of SNallA (black bars) or SNalLA ⁇ lA (white bars) cells with peak currents within different amplitude ranges.
  • this amplitude-frequency distribution was bimodal.
  • 40% of the cells (16 out of 40) cunents were indistinguishable from the small inward cunents recorded in untransfected CHL cells (i.e. ⁇ 100 pA).
  • ⁇ 100 pA In the remaining 60% of the cells, cunents, ranged from 500 pA to 5 nA, and thus were clearly due to expression of cloned type IIA channels.
  • all SNallA ⁇ l A cells expressed large sodium currents (FIG. 3A).
  • the frequency histogram for SNallA ⁇ lA followed a normal distribution with a modal current range of 2-3 nA.
  • EXAMPLE 8 ⁇ lA expression in neuropathic pain To better understand the relationship it may have in neuropathic pain, ⁇ lA subunit expression and localization was assessed in neural tissues after spinal nerve injury. In this example, spinal nerve ligation in rats was used to simulate injury.
  • Patterns of ⁇ l and ⁇ lA subunit expression levels differed from each other in the SNL-treated animals. Staining intensity by anti- ⁇ l A subunit antibodies in the DRG exhibited a post-surgical time-dependent increase. Labeling intensity in nociceptive neurons was slightly elevated at two days post-surgery, and labeling in both nociceptive and sensory populations was enhanced at two weeks. Importantly, and in parallel with the continued expression of allodynic behavior, ⁇ lA labeling remained elevated at eight weeks post-surgery. Labeling increases were more prominent in the L5 than in the L4 regions, and in the ipsilateral than the contralateral
  • EXAMPLE 9 Production of Mutants
  • the present Example briefly describes the generation of specific mutants contemplated by the present inventors. Such mutants will yield specific information about the function and properties of particular regions of the ⁇ lA protein.
  • the inventors propose that the transmembrane and/or COOH-terminal intracellular domains of the ⁇ subunits are responsible for transducing a signal from the extracellular site of TN-R interaction to cytoskeletal or signaling molecules inside the cell.
  • Truncation mutants that eliminate the intracellular COOH-terminal domains of ⁇ l and ⁇ 2 can thus be made and tested in the cell migration assays for adhesion and repulsion to determine the role of these domains in transducing signals from TN-R to the intracellular environment.
  • glycophosphatidyinositol (GPI) lipid anchors to the ⁇ 1 A or other ⁇ subunits will be fused with the extracellular domains of the ⁇ subunits.
  • the inventors also will construct expression vectors that contain point mutations of amino acids in ⁇ l A that are predicted to be located in the extracellular Ig fold. With these constructs it will be possible to determine whether the Ig fold contains the site for TN-R interaction. In addition these data will provide information as to which residues in the Ig fold are critical for ⁇ 1 A interaction. Conelating the amino acid sequences of ⁇ lA, ⁇ l and ⁇ 2 with the recently solved crystal structure of myelin P o (Shapiro et al, 1996) will allow rational choices regarding which mutations might interact with TN-R. These studies will involve the creation of stable cell lines. All cell lines, with the exception of those expressing point mutations in the extracellular regions, described below, will be cloned and characterized for mRNA and protein expression. Subunit truncation mutants
  • Truncation mutants of ⁇ lA may be constructed, sequenced, and transfected into Chinese hamster lung 1610 fibroblasts.
  • ⁇ lA cDNA is amplified by PCR.
  • the 3' reverse primer inco ⁇ orates a termination codon in place of a given residue such that the protein ends.
  • This mutant cDNA is then subcloned into an appropraite vector such as pCMVneo (Stratagene), pcDNA3.1Zeo (invitrogen,
  • transmembrane domains of ⁇ subunits can be determined by replacing them with GPI moieties.
  • a number of CAMs, including the TN-R receptor F3/contactin, have been shown to contain a GPI moiety which anchors the extracellular portion of the protein directly to the plasma membrane without a transmembrane domain.
  • Nascent proteins destined to be GPI-linked have been shown to contain both NH 2 - and COOH-terminal signal peptides (Udenfriend et al, 1995).
  • COOH-terminal signal peptide is cleaved and replaced with GPI by a putative transamidase enzyme in the endoplasmic reticulum, transamidase protein + GPI —> GPI-protein + COOH-terminal signal peptide.
  • the amino acid residue at which cleavage and GPI linkage occurs is termed the ⁇ site.
  • An extensive literature describes the hydrophobicity requirements for the COOH-terminal signal peptide, the length of the hydrophihc spacer region, as well as the identities of amino acid residues present at the ⁇ , ⁇ + 1, and ⁇ + 2 sites (reviewed by Udenfriend et al, 1995).
  • cDNAs can be constructed which contain a GPI linkage in place of their transmembrane domains by creating chimeras of the extracellular NH
  • Chimeras Chimeras can be generated by PCR using the following strategy: Briefly, the extracellular NH 2 -terminal regions of ⁇ 1 A or any other ⁇ subunit is amplified by PCR from extracellular epitope-tagged ⁇ lA, ⁇ l or ⁇ 2 plasmid templates. The PCR will inco ⁇ orate antisense oligonucleotide primers which code for IVSETVIP (SEQ ID NO: 10) for ⁇ l or PERDTVIP (SEQ ID NO: 11) for ⁇ 2 (a fusion protein between the 3' end of the ⁇ subunit extracellular region and the 5' end of the region of N-
  • IVSETVIP SEQ ID NO: 10
  • PERDTVIP SEQ ID NO: 11
  • the COOH-terminal region of N-CAM will also be amplified by PCR from chicken brain cDNA.
  • the PCRs inco ⁇ orate sense oligonucleotide primers that encode IVSETVIP (SEQ ID NO: 10) (for use with ⁇ l) or PERDTVIP (SEQ ID NO: 11) (for use with ⁇ 2), creating a fusion protein between the COOH-terminal residues of the extracellular region of ⁇ l, ⁇ 2 OR ⁇ lA and the 5' end of the COOH- terminal region of N-CAM).
  • the PCR products will be purified and mixed (extracellular ⁇ l + N-CAM in one reaction and extracellular ⁇ 2 + N-CAM in another reaction and extracellular ⁇ lA + N-CAM in another reaction) for use in a second round of PCR in which the components will anneal at the common IVSETVIP (SEQ ID NO:
  • the processed GPI-linked proteins expressed by transfected 1610 fibroblasts or other suitable cell line will each contain a COOH-terminal sequence that ends with TVIPA with an attached GPI lipid anchor.
  • GPI- ⁇ 2 are expressed at the plasma membrane as GPI-anchored proteins
  • transfected cells can be incubated with phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme which specifically cleaves the GPI moiety and releases the protein into the cell medium.
  • PI-PLC phosphatidylinositol-specific phospholipase C
  • the cells are then removed by centrifugation and ⁇ l, ⁇ lA or ⁇ 2 will be immunoprecipitated from the supernatant using antibodies specific to the extracellular epitope tags and analyzed by Western blot. If GPI-linked ⁇ l, ⁇ lA or ⁇ 2 is indeed expressed in the plasma membrane of transfected cells, then these lines can be tested in the cell migration assay.
  • mutants behave like cells transfected with full length ⁇ constructs, then it is concluded that the ⁇ subunit transmembrane domains are not necessary for transducing TN-R-mediated signaling events and that the ⁇ subunits behave similarly to F3/contactin. If these mutants are expressed but do not behave like wild type ⁇ subunits in the cell migration assay, then the conclusion is that the transmembrane domains are necessary for TN-R-mediated signal transduction.
  • ⁇ l, ⁇ lA and ⁇ 2 are structurally homologous to MP 0 and used the alignment of MP oand ⁇ l to make predictions about key residues in ⁇ l subunit interactions (Isom et al, 1995b; McCormick et al, 1998). Because ⁇ l, ⁇ lA, ⁇ 2 and MP oare structurally related CAMs, the alignment of their sequences can be used to make some predictions regarding possible interactions of the ⁇ subunits with TN-R.
  • Oligonucleotide-directed In Vitro Mutagenesis System Version 2 or Sculptor kits are used to mutate selected residues, as described previously (McCormick et al, 1998). Stable cells lines or transient transfections may be performed to generate cells to be tested for the mutant ⁇ subunits analysis.
  • the mutations may be tested for plasma membrane expression by transient transfection into 1610 fibroblasts as well as immunocytochemistiy using antibodies recently to ⁇ l, ⁇ 2, and ⁇ l A subunits. Mutants that express detectable protein at the plasma membrane can then be analyzed further in the cell migration assay. All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.
  • compositions and methods of this invention have been described in terms of prefened embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Makita et al Genomics 23, 628-634., 1994b. Makita et al, J Biol. Chem. 269, 7571-7578., 1994a.

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Abstract

La présente invention concerne une nouvelle sous-unité de canaux sodiques sensibles au voltage. Cette sous-unité est un variant d'épissure de la sous-unité β de canaux sodiques codant une nouvelle protéine. En outre, cette invention concerne des procédés et des compositions d'utilisation des acides nucléiques et protéines de cette sous-unité.
EP00967234A 1999-09-30 2000-09-29 Procedes et compositions relatifs aux sous-unites beta1a de canaux sodiques Withdrawn EP1216300A1 (fr)

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CA2411069A1 (fr) 2000-06-07 2001-12-13 Ortho-Mcneil Pharmaceutical, Inc. Sous-unites beta-1a humaines du canal sodium electriquement potentialise et procedes d'utilisation
SI1458386T1 (sl) 2001-12-27 2007-08-31 Ortho Mcneil Pharm Inc Aroil pirol heteroeril in metanoli, uporabni za zdravljenje motnje centralnega živčnega sistema
DE60224002T2 (de) * 2002-02-14 2008-11-06 Biofrontera Pharmaceuticals Ag Kathepsin Y Inhibitoren für die Entwicklung von Medikamenten zur Schmerzbehandlung

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EP1171589A1 (fr) * 1999-04-15 2002-01-16 Cambridge University Technical Services Limited Nouvelle famille de proteines sous-unite g(b) du canal sodique potentio-dependant, acides nucleiques codant pour elles et leur utilisation a des fins therapeutiques et diagnostiques
AU5010900A (en) * 1999-05-14 2000-12-05 Millennium Pharmaceuticals, Inc. Gene encoding a sodium channel beta-3 subunit protein

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