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Title:  Sodium channel in dorsal root ganglia
United States Patent: 
7,067,629
Issued: 
June 27, 2006

Inventors:
 Dib-Hajj; Sulayman (East Lyme, CT); Waxman; Stephen G. (New Haven, CT)
Assignee: 
Yale University (New Haven, CT)
Appl. No.: 
388470
Filed: 
March 17, 2003


 

Woodbury College's Master of Science in Law


Abstract

A novel tetrodotoxin resistant sodium channel is described, along with isolated nucleotides that encode this receptor. Methods for identifying agents that modulate the Na.sup.+ current through the receptor are provided, as well as related therapeutic and diagnostic methods.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel gene that we have discovered, called NaN. NaN encodes a previously unidentified protein, referred to herein as NaN, that belongs to the .alpha.-subunit voltage-gated sodium channel protein family and that produces a TTX-R sodium current. Such channels underlie the generation and propagation of impulses in excitable cells like neurons and muscle fibers. NaN is a novel sodium channel, with a sequence distinct from other, previously identified, channels. The preferential expression of NaN on sensory, but not other neurons, makes it a very useful target for diagnostic and/or therapeutic uses in relation to acute and/or chronic pain pathologies.

Characterization of the NaN Sodium Channel:

The present invention relates to a previously unidentified, voltage-gated sodium channel a-subunit (NaN), predicted to he TTX-R, voltage-gated, and preferentially expressed in sensory neurons innervating the body (dorsal root ganglia or DRG) and the face (trigeminal ganglia). The predicted open reading frame (ORF), the part of the sequence coding for the NaN protein molecule, has been determined with the putative amino acid sequence from different species (rat, mouse, human) presented in FIGS. 2, 7B, 8B or 11B (SEQ ID NO: 3, 5, 8 or 42) (see Original Patent).

All of the relevant landmark sequences of voltage-gated sodium channels are present in NaN at the predicted positions, indicating that NaN belongs to the sodium channel family. But NaN is distinct from all other previously identified Na channels, sharing a sequence identity of less than 53% with each one of them. NaN is distinct from SNS, the only other TTX-R Na.sup.+ channel subunit that has been identified, until our discovery, in PNS. We have identified and cloned NaN without using any primers or probes that are based upon or specific to SNS. Moreover, NaN and SNS share only 47% similarity of their predicted open reading frame (ORF), comparable to the limited similarity of NaN to all subfamily 1 members.

The low sequence similarity to existing .alpha.-subunits clearly identifies NaN as a novel gene, not simply a variant of an existing channel. Sequence variations compared to the other voltage-gated channels indicate that NaN may be the prototype of a novel and previously unidentified, third class of TTX-R channels that may possess distinct properties compared to SNS. NaN and SNS, which are present in nociceptive DRG and trigeminal neurons, may respond to pharmacological interventions in different ways. The preferential expression of NaN in sensory DRG and trigeminal neurons provides a target for selectively modifying the behavior of these nerve cells while not affecting other nerve cells in the brain and spinal cord. A further elucidation of the properties of NaN channels will be important to understand more fully the effects of drugs designed to modulate the function of the "TTX-R" currents which are characteristic of DRG nociceptive neurons and which contribute to the transmission of pain messages, and to abnormal firing patterns after nerve injury and in other painful conditions.

NaN Nucleic Acids:

Nucleic acid molecules of the invention include the nucleotide sequences set forth in FIG. 1, FIG. 7A, FIG. 8A, FIG. 11A (see Original Patent)as well as nucleotide sequences that encode the amino acid sequences of FIG. 2, FIG. 7B, FIG. 8B and 11B (see Original Patent). Nucleic acids of the claimed invention also include nucleic acids which specifically hybridize to nucleic acids comprising the nucleotide sequences set forth in FIG. 1, FIG. 7A, 8A or FIG. 11A, or nucleotide sequences which encode the amino acid sequences of FIG. 2, FIG. 7B, FIG. 8B or FIG. 11B. A nucleic acid which specifically hybridizes to a nucleic acid comprising that sequence remains stably bound to said nucleic acid under highly stringent or moderately stringent conditions. Stringent and moderately stringent conditions are those commonly defined and available, such as those defined by Sambrook et al. [59] or Ausubel et al. [60]. The precise level of stringency is not important, rather, conditions should be selected that provide a clear, detectable signal when specific hybridization has occurred.

Hybridization is a function of sequence identity (homology), G+C content of the sequence, buffer salt content, sequence length and duplex melt temperature (T[m]) among other variables. See, Maniatis et al.[62]. With similar sequence lengths, the buffer salt concentration and temperature provide useful variables for assessing sequence identity (homology) by hybridization techniques. For example, where there is at least 90 percent homology, hybridization is commonly carried out at 68.degree. C. in a buffer salt such as 6.times.SCC diluted from 20.times.SSC. See Sambrook et al. [59]. The buffer salt utilized for final Southern blot washes can be used at a low concentration, e.g., 0.1.times.SSC and at a relatively high temperature, e.g., 68.degree. C., and two sequences will form a hybrid duplex (hybridize). Use of the above hybridization and washing conditions together are defined as conditions of high stringency or highly stringent conditions. Moderately stringent conditions can be utilized for hybridization where two sequences share at least about 80 percent homology. Here, hybridization is carried out using 6.times.SSC at a temperature of about 50 55.degree. C. A final wash salt concentration of about 1 3.times.SSC and at a temperature of about 60 68.degree. C. are used. These hybridization and washing conditions define moderately stringent conditions.

In particular, specific hybridization occurs under conditions in which a high degree of complementarity exists between a nucleic acid comprising the sequence of an isolated sequence and another nucleic acid. With specific hybridization, complementarity will generally be at least about 70%, 75%, 80%, 85%, preferably about 90 100%, or most preferably about 95 100%.

As used herein, homology or identity is determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al. Proc. Natl. Acad. Sci. USA 87: 2264 2268 (1990) and Altschul, S. F. J. Mol. Evol. 36: 290 300(1993), both of which are herein incorporated by reference) which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al. (Nature Genetics 6: 119 129 (1994)) which is herein incorporated by reference. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al. Proc. Natl. Acad. Sci. USA 89: 10915 10919 (1992), herein incorporated by reference). For blastn, the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively.

The nucleic acids of the present invention can be used in a variety of ways in accordance with the present invention. For example, they can be used as nucleic acid probes to screen other cDNA and genomic DNA libraries so as to select by hybridization other DNA sequences that encode homologous NaN sequences. Contemplated nucleic acid probes could be RNA or DNA labeled with radioactive nucleotides or by non-radioactive methods (for example, biotin). Screening may be done at various stringencies (through manipulation of the hybridization Tm, usually using a combination of ionic strength, temperature and/or presence of formamide) to isolate close or distantly related homologs. The nucleic acids may also be used to generate primers to amplify cDNA or genomic DNA using polymerase chain reaction (PCR) techniques. The nucleic acid sequences of the present invention can also be used to identify adjacent sequences in the genome, for example, flanking sequences and regulatory elements of NaN. The nucleic acids may also be used to generate antisense primers or constructs that could be used to modulate the level of gene expression of NaN. The amino acid sequence may be used to design and produce antibodies specific to NaN that could be used to localize NaN to specific cells and to modulate the function of NaN channels expressed on the surface of cells.

Vectors and Transformed Host Cells:

The present invention also comprises recombinant vectors containing and capable of replicating and directing the expression of nucleic acids encoding a NaN sodium channel in a compatible host cell. For example, the insertion of a DNA in accordance with the present invention into a vector using enzymes such as T4 DNA ligase, may be performed by any conventional means. Such an insertion is easily accomplished when both the DNA and the desired vector have been cut with the same restriction enzyme or enzymes, since complementary DNA termini are thereby produced. If this cannot be accomplished, it may be necessary to modify the cut ends that are produced by digesting back single-stranded DNA to produce blunt ends, or by achieving the same result by filling in the single-stranded termini with an appropriate DNA polymerase. In this way, blunt-end ligation may be carried out. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini. Such linkers may comprise specific oligonucleotide sequences that encode restriction site recognition sequences.

Any available vectors and the appropriate compatible host cells may be used [59, 60]. Commercially available vectors, for instance, those available from New England Biolabs Inc., Promega Corp., Stratagene Inc. or other commercial sources are included.

The transformation of appropriate cell hosts with an rDNA (recombinant DNA) molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. Frog oocytes can be injected with RNA and will express channels, but in general, expression in a mammalian cell line (such as HEK293 or CHO cells) is preferred. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed, see, for example, Cohen et al. [61]; and [62]. With regard to transformation of vertebrate cells with vectors containing rDNAs, electroporation, cationic lipid or salt treatment methods are typically employed [63, 64].

Successfully transformed cells, i.e., cells that contain an rDNA molecule of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using conventional methods [65, 66] or the proteins produced from the cell assayed via an immunological method. If tags such as green fluorescent protein are employed in the construction of the recombinant DNA, the transfected cells may also be detected in vivo by the fluorescence of such molecules by cell sorting.

For transient expression of recombinant channels, transformed host cells for the measurement of Na.sup.+ current or intracellular Na.sup.+ levels are typically prepared by co-transfecting constructs into cells such as HEK293 cells with a fluorescent reporter plasmid (such as pGreen Lantern-1, Life Technologies, Inc.) using the calcium-phosphate precipitation technique [27]. HEK293 cells are typically grown in high glucose DMEM (Life Technologies, Inc) supplemented with 10% fetal calf serum (Life Technologies, Inc). After 48 hrs, cells with green fluorescence are selected for recording [28].

For preparation of cell lines continuously expressing recombinant channels, the NaN construct is cloned into other vectors that cany a selectable marker in mammalian cells. Transfections are carried out using the calcium phosphate precipitation technique [27]. Human embryonic kidney (HEK-293), chinese hamster ovary (CHO) cells, derivatives of either or other suitable cell lines are grown under standard tissue culture conditions in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The calcium phosphate-DNA mixture is added to the cell culture medium and left for 15 20 hr, after which time the cells are washed with fresh medium. After 48 hrs, antibiotic (G418, Geneticin, Life Technologies) is added to select for cells which have acquired neomycin resistance. After 2 3 weeks in G418, 10 20 isolated cell colonies are harvested using sterile 10 ml pipette tips. Colonies are grown for another 4 7 days, split and subsequently tested for channel expression using whole-cell patch-clamp recording techniques and RT-PCR.

Method of Measuring Na.sup.+ Current Flow:

Na.sup.+ currents are measured using patch clamp methods [29], as described by Rizzo et al. [30] and Dib-Hajj et al. [28]. For these recordings data are acquired on a Macintosh Quadra 950 or similar computer, using a program such as Pulse (v 7.52, HEKA, German). Fire polished electrodes typically (0.8 1.5 MW) are fabricated from capillary glass using a Sutter P-87 puller or a similar instrument. In the most rigorous analyses, cells are usually only considered for analysis if initial seal resistance is <5 Gohm, they have high leakage currents (holding current <0.1 nA at -80 mV), membrane blebs, and an access resistance <5 Mohm. Access resistance is usually monitored throughout the experiment and data are not used if resistance changes occur. Voltage errors are minimized using series resistance compensation and the capacitance artifact is canceled using computer controlled amplifier circuitry or other similar methods. For comparisons of the voltage dependence of activation and inactivation, cells with a maximum voltage error of .+-.10 mV after compensation are used. Linear leak subtraction is usually used for voltage clamp recordings. Membrane currents are typically filtered at 5 KHz and sampled at 20 KHz. The pipette solution contains a standard solution such as: 140 mM CsF, 2 mM MgCl.sub.2, 1 mM EGTA, and 10 mM Na-HEPES (pH 7.3). The standard bathing solution is usually 140 nM NaCl, 3 mM KCl, 2 mM MgCl.sub.2, 1 mM CaCl.sub.2, 10 mM HEPES, and 10 mM glucose (pH 7.3).

Voltage clamp studies on transformed cells or DRG neurons, using methods such as intracellular patch-clamp recordings, can provide a quantitative measure of the sodium current density (and thus the number of sodium channels in a cell), and channel physiological properties. These techniques, which measure the currents that flow through ion channels such as sodium channels, are described in Rizzo et al. [21]. Alternatively, the blockage or enhancement of sodium channel function can be measured using optical imaging with sodium-sensitive dyes or with isotopically labeled Na. These methods which are described in Rose, et al., (J. Neurophysiology, 1997 in press) [67] and by Kimelberg and Walz [31], measure the increase in intracellular concentration of sodium ions that occurs when sodium channels are open.

Measurement of Intracellular Sodium ([Na.sup.+].sub.i)

The effects of various agents on cells that express Na.sup.+ can be determined using ratiometric imaging of [Na.sup.+].sub.i using SBFI or other similar ion-sensitive dyes. In this method, as described by Sontheimer et al. [32], cytosolic-free Na.sup.+ is measured using an indicator for Na.sup.+, such as SBFI (sodium-binding benzofuran isophthalate; [33]) or a similar dye. Cells are first loaded with the membrane-permeable acetoxymethyl ester form of the dye (which is dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM). Recordings are obtained on the stage of a microscope using a ratiometric imaging setup (e.g., from Georgia Instruments). Excitation light is provided at appropriate wavelengths (e.g., 340:385 nm). Excitation light is passed to the cells through a dichroic reflector (400 nm) and emitted light above 450 nm is collected. Fluorescence signals are amplified, e.g., by an image intensifier (GenIISyS) and collected with a CCD camera, or similar device, interfaced to a frame grabber. To account for fluorescence rundown, the fluorescence ratio 340:385 is used to assay cytosolic-free Na.sup.+.

For calibration of SBFI's fluorescence, cells are perfused with calibration solutions containing known Na.sup.+ concentrations (typically 0 and 30 mM, or 0, 30, and 50 mM [Na.sup.+]), and with ionophones such as gramicidin and monensin (see above) after each experiment. As reported by Rose and Ransom [34], the 345/390 nm fluorescence ratio of intracellular SBFI changes monotonically with changes in [Na.sup.+].sub.i. Experiments are typically repeated on multiple (typically at least 4) different coverslips, providing statistically significant measurements of intracellular sodium in control cells, and in cells exposed to various concentrations of agents that may block, inhibit or enhance Na.sup.+.

Method to Measure Na.sup.+ Influx via Measuring .sup.22Na or .sup.86Rb.

.sup.22Na is a gamma emitter and can be used to measure Na.sup.+ flux [31], and .sup.86Rb.sup.+ can be used to measure Na.sup.+/K.sup.+-ATPase activity [32]. .sup.86Rb.sup.+ ions are taken up by the Na.sup.+/K.sup.+-ATPase-like K.sup.+ ions, but have the advantage of a much longer half-life than .sup.42K.sup.+[35]. Thus, measurement of the unidirectional ouabain-sensitive .sup.86Rb.sup.+ uptake provides a quantitative method for assaying Na.sup.+/K.sup.+-ATPase activity which provides another indicator of the electrical firing of nerve cells. Following incubation of cells expressing NaN with the isotope .sup.22Na.sup.+, the cellular content of the isotope is measured by liquid scintillation counting or a similar method, and cell protein is determined using a method such as the bicinchoninic acid protein assay [36] following the modifications described by Goldschmidt and Kimelberg [37] for cultured cells. .sup.22Na and .sup.86Rb.sup.+ fluxes are determined in the presence and absence of agents that may block, inhibit, or enhance NaN. This permits determination of the actions of these agents on NaN.

Method to Identify Agents that Modulate NaN-Mediated Current:

Several approaches can be used to identify agents that are able to modulate (i.e., block or augment) the Na.sup.+ current through the NaN sodium channel. In general, to identify such agents, a model cultured cell line that expresses the NaN sodium channel is utilized, and one or more conventional assays are used to measure Na.sup.+ current. Such conventional assays include, for example, patch clamp methods, the ratiometric imaging of [Na.sup.+].sub.i, and the use of .sup.22Na and .sup.86Rb as described above.

In one embodiment of the present invention, to evaluate the activity of a candidate compound to modulate Na.sup.+ current, an agent is brought into contact with a suitable transformed host cell that expresses NaN. After mixing or appropriate incubation time, the Na.sup.+ current is measured to determine if the agent inhibited or enhanced Na.sup.+ current flow.

Agents that inhibit or enhance Na.sup.+ current are thereby identified. A skilled artisan can readily employ a variety of art-recognized techniques for determining whether a particular agent modulates the Na.sup.+ current flow.

Because Na.sup.+ is preferentially expressed in pain-signaling cells, one can also design agents that block, inhibit, or enhance Na.sup.+ channel function by measuring the response of laboratory animals, treated with these agents, to acute or chronic pain. In one embodiment of this aspect of the invention, laboratory animals such as rats are treated with an agent for instance, an agent that blocks or inhibits (or is thought to block or inhibit) NaN. The response to various painful stimuli are then measured using tests such as the tail-flick test and limb withdrawal reflex, and are compared to untreated controls. These methods are described in Chapter 15 of Reference [38]. In another embodiment of this aspect of the invention, laboratory animals such as rats are subjected to localized injection of pain-producing inflammatory agents such as formalin [39], Freunds adjuvant [40] or carageenan, or are subjected to nerve constriction [41,42] or nerve transection [43] which produce persistent pain. The response to various normal and painful stimuli are then measured, for example, by measuring the latency to withdrawal from a warm or hot stimulus [38] so as to compare control animals and animals treated with agents that are thought to modify NaN.

The preferred inhibitors and enhancers of NaN preferably will be selective for the NaN Na.sup.+ channel. They may be totally specific (like tetrodotoxin, TTX, which inhibits sodium channels but does not bind to or directly effect any other channels or receptors), or relatively specific (such as lidocaine which binds to and blocks several types of ion channels, but has a predilection for sodium channels). Total specificity is not required for an inhibitor or enhancer to be efficacious. The ratio of its effect on sodium channels vs. other channels and receptors, may often determine its effect and effects on several channels, in addition to the targeted one, may be efficacious [44].

It is contemplated that modulating agents of the present invention can be, as examples, peptides, small molecules, naturally occurring and other toxins and vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the modulating agents of the present invention. Screening of libraries of molecules may reveal agents that modulate NaN or current flow through it. Similarly, naturally occurring toxins (such as those produced by certain fish, amphibians and invertebrates) can be screened. Such agents can be routinely identified by exposing a transformed host cell or other cell which expresses a sodium channel to these agents and measuring any resultant changes in Na.sup.+ current

Recombinant Protein Expression, Synthesis and Purification:

Recombinant NaN proteins can be expressed, for example, in E. coli strains HB 101, DH5a or the protease deficient strain such as CAG-456 and purified by conventional techniques.

The peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art. In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.

Antibodies and Immunodetection:

Another class of agents of the present invention are antibodies immunoreactive with the Na.sup.+ channel. These antibodies may block, inhibit, or enhance the Na.sup.+ current flow through the channel. Antibodies can be obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of NaN, particularly (but not necessarily) those that are exposed extracellularly on the cell surface. Such immunological agents also can be used in competitive binding studies to identify second generation inhibitory agents. The antibodies may also be useful in imaging studies, once appropriately labeled by conventional techniques.

Production of Transgenic Animals:

Transgenic animals containing and mutant, knock-out or modified NaN genes are also included in the invention. Transgenic animals wherein both NaN and the SNS/PN3 gene are modified, disrupted or in some form modified are also included in the present invention. Transgenic animals are genetically modified animals into which recombinant, exogenous or cloned genetic material has been experimentally transferred. Such genetic material is often referred to as a "transgene". The nucleic acid sequence of the transgene, in this case a form of NaN, may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene. The transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.

The term "germ cell line transgenic animal" refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic animal to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic animals.

The alteration or genetic information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.

Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Pat. Nos. 4,736,866; 5,602,307; Mullins et al. (1993) Hypertension 22(4):630 633; Brenin et al. (1997) Surg. Oncol. 6(2)99 110; Tuan (ed.), Recombinant Gene Expression Protocols, Methods in Molecular Biology No. 62, Humana Press (1997)).

A number of recombinant or transgenic mice have been produced, including those which express an activated oncogene sequence (U.S. Pat. No. 4,736,866); express simian SV 40 T-antigen (U.S. Pat. No. 5,728,915); lack the expression of interferon regulatory factor 1 (IRF-1) (U.S. Pat. No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Pat. No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Pat. No. 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Pat. No. 5,720,936); have a reduced capacity to mediate cellular adhesion (U.S. Pat. No. 5,602,307); possess a bovine growth hormone gene (Clutter et al. (1996) Genetics 143(4):1753 1760); or, are capable of generating a fully human antibody response (McCarthy (1997) The Lancet 349(9049):405).

While mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species. Transgenic procedures have been successfully utilized in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al. (1997) Mol. Reprod. Dev. 46(4):515 526; Houdebine (1995) Reprod. Nutr. Dev. 35(6):609 617; Petters (1994) Reprod. Fertil. Dev. 6(5):643 645; Schnieke et al. (1997) Science 278(5346):2130 2133; and Amoah (1997) J. Animal Science 75(2):578 585).

The method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Pat. Nos. 5,489,743 and 5,602,307.
 


Claim 1 of 19 Claims

1. An isolated protein capable of producing a sodium current and encoded by a nucleic acid with at least 80% sequence identity to a nucleotide sequence comprising SEQ ID NO: 41.

____________________________________________
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