1. Field of the Invention [0001] The present invention is directed to the fields of molecular biology, neurobiology, pheromone reception, cellular activation, signal transduction, G-protein coupled receptors and animal behavior. In particular, the invention relates to a novel olfactory receptor- ligand pair and methods of their use. The invention also relates to methods of screening for modulators of the receptor-ligand pair and uses of modulators so identified. The invention also relates to receptors that function in the same manner as the novel receptor-ligand pair identified herein and nucleotides encoding such receptors.

2. Description of Related Art [0002] The olfactory system provides sensory information about the chemical composition of the external world. In mammals, olfactory chemoreception initiates at the level of olfactory sensory neurons that are located in the main olfactory epithelium (MOE) and the epithelium of the vomeronasal organ (VNO). The MOE typically and primarily mediates the detection of volatile odorants. The VNO typically mediates the detection of nonvolatile odorants, such as pheromones, though both systems are generally known as subsystems of the olfactory system as a whole. In most mammals, intraspecies communication relies heavily on the vomeronasal system, and on its ability to detect pheromones (Halpern, 1987). These are chemical signals that provide information about gender, dominance, and reproductive status between individuals of the same species (Sorensen, 1996). Pheromones elicit in the recipients innate and stereotyped reproductive and social behaviors along with profound neuroendocrine and physiological changes (Jemiolo et al. 1989).

[0003] In mammals, the VNO resides in a blind-ended pouch within the septum of the nose. Axonal projections from the VNO converge to form the vomeronasal nerve and reach

target cells within the accessory olfactory bulb. The VNO is connected to and transmits information to specialized centers of the limbic system, including the vomeronasal amygdala, the bed nucleus of the stria terminalis, and specific nuclei of the ventromedial hypothalamus involved in reproduction and aggression (Halpern, 1987). Pheromones activate the VNO which results in behavioral and endocrine responses that do not involve higher cognitive centers of the brain (Halpern, 1987). Specific pheromones and odorants have been shown to generate action potentials and to induce calcium entry in vomeronasal sensory neurons (VSNs) (Leinders-Zufall et al., 2000; Sambrook et al., 2001). These activated cells begin the intercellular signalling that typically results in the modulation of organism behavior.

[0004] VSNs express members of either of two large unrelated families of G protein- coupled seven transmembrane receptors, the Vlr (Dulac and Axel, 1995) and V2r superfamilies (Ryba and Tirindelli, 1997; Matsunami and Buck, 1997; Herrada and Dulac, 1997). These are the only candidate pheromone receptors known in mammals. In the mouse, individual VSNs express a single member of the Vlr gene repertoire, which is constituted of about 150 members (Rodriguez et al., 2002). Expression of Vlr receptors is crucial to the normal development of the vomeronasal sensory system: VSNs from genetically engineered mice which activate a given Vlr locus but which are prevented to express the corresponding Vlr receptor, project aberrantly to the brain; in addition, these neurons apparently die within a few weeks (Rodriguez et al., 1999; Belluscio et al., 1999). Mice lacking an entire Vlr gene cluster comprising 16 Vlr genes are impaired in their ability to respond adequately to pheromonal cues, both in terms of behavior and electrical activity (Del Punta et al., 2002).

[0005] Because few mammalian pheromones have been identified at the molecular level, ligand-receptor interactions are difficult to define (Sorensen, 1996). Indeed, although the vomeronasal receptor genes encode putative pheromone receptors, there has been as yet no direct evidence that any of these molecules is a receptor for a pheromone. It has also not been known if each VNO receptor recognizes a distinct ligand or if several receptors recognize the same ligand. Additionally it has not been known whether specific G proteins are involved in signal transduction of specific pheromonal stimuli via specific G-protein coupled receptors. Despite this lack of knowledge, these molecules have served as useful molecular markers whose expression tends to correlate with that of the two families of vomeronasal receptor genes.

[0006] There is therefore a long felt and significant need for the direct demonstration that an identified pheromone activates a VSN through a receptor-dependent process and the identification of a receptor-agonist or receptor-ligand pair in the vomeronasal system. There is a

further need for the methods and compositions necessary to exploit the identification of such a pair in developing compounds with which to modulate vomeronasal responses.

SUMMARY OF THE INVENTION [0007] The inventors are the first to have identified a receptor-ligand pair within the vomeronasal system. The inventors have therefore disclosed the identification of the agonist 2- heptanone for the mouse Vlrb2 vomeronasal receptor as the first highly specific receptor- pheromone pair. Therefore, in one embodiment, the invention provides a composition of matter comprising a receptor-ligand complex of vomeronasal receptor Vlrb2 and the ligand 2- heptanone. A"complex"a may include an ionic or other non-covalent inteaction between the receptor and the ligand. "Complex"may also refer to a ligand that is covalently attached to the receptor. In one embodiment, the vomeronasal receptor is purified. In another embodiment, the receptor is embedded in a membrane. In yet a further embodiment, the vomeronasal receptor is immobilized on a solid substrate.

[0008] In another embodiment, the vomeronasal receptor of the receptor-ligand complex is expressed in a cell that normally does not express the receptor. In a further aspect, the expression of the receptor is such that a higher level of expression is achieved in comparison to physiological levels of expression. The cells that express the receptor may be any cell of the olfactory system, and in particular embodiments, the cells are vomeronasal sensory cells or odorant sensory cells, or even in cells in which endogenous expression of Vlrb2 does not occur.

Alternatively, the cells may endogenously express Vlrb2, but within the context of the invention, the level of Vlrb2 expression obtained by the methods and compositions of the invention is higher than endogenous expression of Vlrb2.

[0009] In one embodiment, such overexpression of Vlrb2 is achieved by expression of exogenous polynucleotides encoding all or part of Vlrb2 as provided in SEQ ID NO: 2. In another embodiment, overexpression is achieved through particular recombinant polynucleotide constructs containing polynucleotides encoding all or part of Vlrb2. In a further embodiment, the recombinant vomeronasal receptor is expressed in a cell in which the endogenous expression of the receptor is inhibited or eliminated. The cells expressing Vlrb2 may occur in vitro or in vivo. If in vivo, the cells may occur within paricular tissues or organs. In particular embodiments, the cells occur within a olfactory neuro-epithelium.

[0010] Accordingly, this invention provides a method for modulating the maternal behavior of a subject comprising obtaining the overexpression or exogenous expression of Vlrb2 in the sensory neurons of the subject and administering effective amounts of a 2-heptanone-

containing composition. Similarly, the invention provides a method for modulating the social behavior of a subject comprising obtaining the overexpression or exogenous expression of Vlrb2 in the sensory neurons of the subject and administering effective amounts of a 2-heptanone- containing composition. Similar methods emcompassed by the invention include methods for modulating the reproductive functions, fertility, or behavior of a subject. The inventors specifically contemplate the modulation of any behavior in a subject in which it is known that activation of vomeronasal, odorant, or other cells results in a modulation of that behavior.

[0011] In further embodiment, the invention provides a method for screening for additional Vlr agonists, antagonists, or ligands and modulators of vomeronasally mediated responses. By way of non-limiting example, such methods would include a method of identifying a modulator of a vomeronasal receptor mediated response, which comprises contacting a composition comprising the Vlrb2: 2-heptanone receptor-ligand complex with a candidate modulator and determining the effect of the modulator on the vomeronasal receptor mediated response.

[00121 In yet another embodiment, the invention provides for methods for identifying a novel receptor for 2-heptanone comprising obtaining a nucleic acid encoding a candidate receptor; expressing the candidate receptor in a cell, contacting the receptor with 2-heptanone and determining if the cell is activated thereby. In particular embodiments, the endogenous expression of the candidate receptor or of the Vlrb2 receptor is inhibited or eliminated.

Inhibition or elimination of endogenous expression of either the candidate receptor or of the Vlrb2 receptor may be achieved by techniques well known to those of skill in the art (see, e. g.

Rodriguez et al. 1999). In additional aspects, the methods for identifying a novel receptor or for identifying novel ligands or modulators utilize the Vlrb2: 2-heptanone receptor ligand pair as a positive control for activation by a known ligand.

[0013] In further aspects, the methods of the invention further comprise the step of identifying a receptor or candidate receptor expressing cell through expression of an exogenous nucleotide sequence encoding a marker. In particular embodiments, the marker is a protein. In still further particular embodiments, the marker protein is green fluorescent protein (GFP). In particular embodiments, the expression of the marker protein is in lieu of expression of the endogenous candidate receptor or Vlrb2.

[0014] In still further aspects the invention is contemplated to include receptors identified by the methods of the invention. In particular aspects, the receptors so identified are characterized as encoded by polynucleotide sequences hybridizing at low stringency conditions with a polynucleotide fragment corresponding to the nucleotide sequence of SEQ ID NO: 1; and

polynucleotide sequences which, but for the degeneracy of the genetic code, would hybridize to such polynucleotide sequences or those of SEQ ID NO: 1.

[0015] Following long-standing patent law, the words"a"and"an,"when used in conjunction with the word"comprising"in the claims or specification, denotes one or more.

BRIEF DESCRIPTION OF THE DRAWINGS [0016] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0001] FIG. 1A. Vlrb2-expressing VSNs respond to 2-heptanone in dissociated VSNs from the VG line (scale bar: 15 pm).

[0018] FIG. 1B. Dissociated VSNs from the VG line (same frame as in FIG. 1A) expressing GFP (brightly fluoresent cells in middle of figure) allowed Vlrb2-expressing neurons to be identified (scale bar: 15 llm).

[0019] FIG. 1C. Ca++ imaging on Vlrb2-expressing VSNs loaded with Fura-2AM (10 uM) (AF/F). 2-heptanone (10-8M) induced an intracellular Ca++ increase (representative trace, 12 neurons tested, all responded). The other bath-applied pheromones and odorant mixes (mix A: cineole, myrtenal, eugenol; mix B: citral, anisol, fenchone; 2,5-dimethylpyrazine (dmp), a-farnesene (a-F), P-famesene (P-F), pentyl acetate (pa), all at 10-8M) did not evoke any response (n=12).

[0020] FIG. 1D. Structural analogues of 2-heptanone, 2-heptanol and 4-heptanone, did not elicit responses (representative trace, 12 neurons tested).

[0021] FIG. 2A. 2-heptanone currents in Vlrb2-expressing VSNs. Representative whole-cell patch clamp recording (10 neurons tested, holding potential:-40 mV) of 1 s pulses of mix A, 2-heptanone, dmp, a-F and ß-F, all at 10-8M. Only 2-heptanone induced an inward current in Vlrb2-expressing neurons.

[0022] FIG. 2B. Concentration-response of 2-heptanone. Current responses were obtained at-40 mV in the presence of 1-s pulse of increasing concentrations of 2-heptanone (10- 13 to 10-6 M) on whole-cell patch from Vlrb2-expressing VSNs. Currents were normalized with the maximal current at saturating 2-heptanone concentration (10-6 M) before averaging (n=5 for each concentration), I max (10-6 M 2-heptanone) = 130 + 36 pA. The continuous line is the best fit of the Hill equation (I/Imax = c/(c+Klx2)) to the data: Kl/2 = 1.4 + 0.2 x 10''° M 2-heptanone.

[0023] FIG. 3A. Vlrb2 expression is required for response to 2-heptanone in neuronal cells from the DV mutant mouse line (scale bar: 15 pm).

[0024] FIG. 3B. Visualization of neurons of FIG. 3A expressing the vlrb2 locus and GFP but not the Vlrb2 receptor (i. e. cells from the DV mutant mouse line) (scale bar: 15 Zm).

[0025] FIG. 3C. Ca++ increases (AF/F) were not evoked by bath applied 2- heptanone (10-8M). Cell viability was assessed by KCl (50 mM) (representative trace, 6 neurons tested).

[0026] FIG. 3D. Representative whole-cell patch clamp recording (4 neurons tested, holding potential:-40 mV), showing that 2-heptanone induced-currents were not observed in GFP-expressing VSNs from the DV line. A KCl pulse (50 mM, 1 s) evoked an inward current of 160 pA in the same neuron.

DETAILED DESCRIPTION OF THE INVENTION 1. The Vomeronasal System [0027] The VNO mediates mainly the detection of nonvolatile odorants, such as pheromones. These are chemical signals that provide information about gender, dominance, and reproductive status between individuals of the same species (Sorensen, 1996). Pheromones elicit in the recipients innate and stereotyped reproductive and social behaviors along with profound neuroendocrine and physiological changes.

[0028] In mammals, the VNO resides in a blind-ended pouch within the septum of the nose. Axonal projections from the VNO converge to form the vomeronasal nerve and reach target cells within the accessory olfactory bulb. The VNO is exclusively connected to specialized centers of the limbic system, including the vomeronasal amygdala, the bed nucleus of the stria terminais, and specific nuclei of the ventromedial hypothalamus involved in reproduction and aggression (Halpern, 1987). Pheromones activate the VNO which results in behavioral and endocrine responses that do not involve higher cognitive centers of the brain (Halpern, 1987).

[0029] However, it is inappropriate to consider the VNO as the exclusive site of pheromone detection (Johnston, 1998), because some mammals such as the rabbit (Hudson and Distel, 1986) and the pig (Dorries et al., 1997) are able to detect pheromones via the main

olfactory system. Furthermore, fish lack a VNO but express V2R homologs within their olfactory epithelium (Naito et al., 1998; Cao et al., 1998).

[0030] Recent studies show evidence supportive of a functional vomeronasal system in humans. Nevertheless, some studies suggest that adult human VNO may display species- specific, gender-dimorphic and stereospecific responses to vomeropherin ligands (Monti-Bloch et al., 1994). However, the existence of pheromones and a functional vomeronasal system in humans remains controversial (Preti and Wysocki, 1999). Despite an undisputed presence of VNO-like structure during early human embryogenesis, it regresses after birth to become vestigial in adults (Humphrey, 1940; Stensaas et al., 1991). The existence of a known receptor- ligand pair, i. e. the Vlrb2 : 2-heptanone pair provided by the present invention enables the further analysis and modulation of the vomeronasal system in most vertebrates, and especially in primates, at least via ex vivo technologies as described below.

2. Receptors, Activation, and Signalling [0031] The functional and anatomical dichotomy between the main and vomeronasal (or accessory) olfactory systems above reflected in the proteins that are the putative receptors for their respective sensory stimuli. In the main olfactory system, odorant receptor genes encode seven transmembrane proteins (sometimes known as 7TM proteins, or G-protein coupled receptors, or GPCRs) and are members of a multigene family that may comprise as many as 1000 genes in rat and mouse (Buck and Axel, 1991).

[0032] In the VNO, two families of genes encoding seven-transmembrane proteins have been proposed to encode pheromone receptors. The first family of vomeronasal receptor genes consists of at least 150 genes that are expressed selectively in vomeronasal sensory neurons of the apical zone of the epithelium of the VNO (Rodriguez et al. 2002). The second family of putative vomeronasal receptor genes comprises 30-140 genes that are expressed in vomeronasal sensory neurons of the basal zone (Ryba and Tirindelli, 1997; Matsunami and Buck, 1997; Herrada et al., 1997). There are no conserved motifs between the two families of vomeronasal receptors, and vomeronasal receptors have no sequence homology with odorant receptors (Rodriguez et al. 2002).

[0033] An individual VNS typically expresses one or only a few members of the two large superfamilies of putative receptor genes. (Dulac and Axel, 1995; Rodriguez et al. 1999).

Neurons in the apical and basal zones express G protein subunits (respectively, G-alpha-i2 and G-alpha-o) and project their axons to distinct regions in the mouse accessory olfactory bulb (Berghard and Buck, 1996). The existence of segregated fibers and corresponding G proteins

suggests that distinct pheromone signals are likely to elicit electrical stimulation of restricted populations of VNO sensory neurons in order to generate distinct behavioral responses (Herrada et al., 1997).

[0034] The mouse vomeronasal receptor Vlrb2 is encoded by the sequence of SEQ ID NO: 1, the amino acid translation of which is provided as SEQ ID NO: 2 (Rodriguez et al.

1999). Vlrb2 has also been previously known to those of skill in the art as Vr ; 2 or Vlr5 (Rodriguez et al. 1999). The tissue and cellular specificity of Vlrb2 expression has been thoroughly documented (Rodriguez et al. 1999). Interestingly, the expression of Vlrb2 is monoallelic. That is, only one of the pair of alleles that exist within Vlrb2 expressing cells is actually expressed. Biallelic expression is nonexistent or extremely rare for Vlrb2 (Rodriguez et al., 1999). Whatever the mechanism for this monoallelic expression, the net result is expression of a single receptor type in the expressing cell. This specificity of expression is in addition to the specificity of receptor responses to the binding of particular ligands, agonists, or antagonists.

[0035] Despite the great diversity in ligands and putative vomeronasal receptors, this fundamental scheme of events forms the basis for the multiplicity of different cell signaling phenomena that ultimately results in the modulation of various physiological, endocrine, and behavioral responses. Binding of a ligand, such as 2-heptanone to a protein receptor such as Vlrb2 to form a receptor-ligand complex on the surface of a neuronal cell typically initiates a chain of events that results in the depolarization of the neuronal cell membrane. This phenomenon is well known to those of skill in the art and its biochemical basis is also well understood. For example, in U. S. Patent 5,668, 006, G-protein coupled receptors are reported to control many physiological functions, such as mediating transmembrane signaling from external stimuli (vision, taste and smell), endocrine function (pituitary and adrenal), exocrine function (pancreas), heart rate, lipolysis, and carbohydrate metabolism. The molecular cloning of a number of such receptors have revealed many structural and genetic similarities, permitting classification of the G protein coupled receptor superfamily into five distinct groups.

[0036] U. S. Patent 5,691, 188 describes how upon binding to the receptor, the receptor-ligand complex presumably undergoes a conformation change leading to activation of the G protein. G proteins are described as being comprised of three subunits: a guanylnucleotide binding alpha subunit; a beta subunit; and a gamma subunit. G proteins cycle between two forms, depending on whether GDP or GTP is bound to the alpha subunit thereto. When GDP is bound, the G protein exists as an heterotrimer, the G-alpha-beta-gamma complex. When GTP is bound, the a subunit dissociates, leaving a G-beta-gamma complex. Importantly, when a G-alpha-beta-gamma complex operatively associates with an activated G protein coupled

receptor in a cell membrane, the rate of exchange of GTP for bound GDP is increased and, hence, the rate of dissociation of the bound G-alpha subunit from the G-beta-gamma complex r increases. The free G-alpha-subunit and G-beta-gamma complex are capable of transmitting a signal to downstream elements of a variety of signal transduction pathways resulting in the activation of the cell.

[0037] In the case of neuronal cells responding to the binding of ligands to their receptors, the transmision of the signal to downstream elements typically may result in the depolarization of the neuronal membrane (defined as a change in the cell membrane potential to a more positive charge) and may produce an action potential (a rapid, transient depolarization of the cell membrane) that transmits the signal along the cell membrane and eventually may result in the inter-cellular transmision of the signal via synapses. For endogenous, or exogenous Vlr receptors expressed within the VNO or its biological equivalent, activation through the binding of a ligand and receptor to form a ligand-receptor complex thus may typically lead to the modulation of neurological, endocrine, and behavioral responses (among others) through the neuronal connections typical of the VNO or its equivalent structures.

3. Physiological, Endocrine, and Behavioral Responses [0038] Vomeronasal mediated information is not directly transmitted to the higher cortical areas typically thought to be involved in odor perception and discrimination. Rather, vomeronasal neurons are linked to the amygdala and hypothalamus, which control hormone levels, emotions, basis drives and instinctive behaviors such as sexual arousal and agression (Halpem, 1987). Modulation of the VNO in male rodents alters a variety of endocrine-mediated responses to female pheromones including androgen surges, vocalization, territorial marking, and inter-male aggression. Modulation of the VNO in female rodents delays or prevents activation of reproduction, and may abolish the effects of over-crowding on sexual maturation, and reduce maternal responses to intruders (Wysocki et al., 1991). Thus, modulation of the vomeronasal signaling system results in the modulation of innate behavioral or physiological responses (Halpern, 1987) [0039] Therefore, activation or modulation of the activation of VNS through the binding of a ligand with its receptor in a cell so situated as to initiate or modulate intercellular signalling of the vomeronasal system directly results in the modulation of a number of responses known to be controled by input from the vomeronasal organ. Likewise, compounds that modulate the activation of cells expressing vomeronasal receptors through the binding of ligands

are expected to modulate the resultant physiological, endocrine, and behavioral responses known to be controled by the vomeronasal system.

4. Ligands and Modulators [0040] A ligand is a naturally occuring or synthetic compound that binds to a protein receptor. A modulator is defined as any substance that alters function or response. Typically, a modulator is a naturally occuring or synthetic compound that acts to alter a typical response observed upon binding of a ligand with its receptor to form a receptor-ligand complex. A modulator, as well as a ligand (of course) may act as an agonist. An agonist is a molecule or substance that can activate a receptor protein or enzyme. A modulator may act as an antagonist.

An antagonist is a molecule that binds to or otherwise interacts with a receptor to inhibit the activation of the receptor mediated signalling pathway or cell of which the receptor is a part.

[0041] Naturally, and as well known in the art, modulators in the context of the present invention may act directly upon the receptor-ligand complex, the components or enzymes of the G-protein coupled signalling mechanism, the cellular response, intercellular signaling, or other involved component of the vomeronasal system activated by the receptor- ligand complex.

5. Methods of Screening [0042] The present invention further comprises methods for identifying modulators of the responses generated by the Vlrb2: 2-heptanone receptor-ligand pair. These assays may comprise random screening of large libraries of candidate substances. Alternatively, the assays may be used to focus on particular classes of compounds selected with an eye towards structural attributes that are believed to make them more likely to modulate the vomeronasal receptor mediated response.

[0043] To identify a modulator, one generally will determine in the presence and absence of the candidate substance one or more characteristics of the response such as the presence, magnitude, frequency, strength, persistance, or other desirable characteristic of a vomeronasal receptor mediated response. For example, a method generally comprises: (a) providing a candidate modulator; (b) admixing the candidate modulator with an isolated composition or cell, or a suitable experimental animal;

(c) measuring one or more characteristics of the response expected in the composition, cell or animal in step (b); and (d) comparing the characteristic measured in step (c) with the characteristic of the compound, cell or animal in the absence of said candidate modulator, wherein a difference between the measured characteristics indicates that said candidate modulator is, indeed, a modulator of the response.

The methods may be conducted in cell free systems, in isolated cells, cultured cells, cell or tissue sections, including sections of whole organs or section of whole body parts or whole organisms, or in living organisms including transgenic animals.

[0044] It will, of course, be understood that all the screening methods of the present invention are useful in themselves notwithstanding the fact that effective candidates may not be found. The invention provides methods for screening for such candidates, not solely methods of finding them.

[0045] As used herein the term"candidate substance"refers to any molecule that may potentially inhibit or enhance vomeronasal receptor mediated activity. The candidate substance may be a protein or fragment thereof, a small molecule, or even a nucleic acid molecule. It may prove to be the case that the most useful pharmacological compounds will be compounds that are structurally related or unrelated to the identified receptor-ligand pair, i. e. the Vlrb2 : 2-heptanone pair. Using lead compounds to help develop improved compounds is know as"rational drug design"and includes not only comparisons with know inhibitors and activators, but predictions relating to the structure of target molecules.

[0046] The goal of rational drug design is to produce structural analogs of biologically active compounds, polypeptides or target compounds. 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. In one approach, one would generate a three-dimensional structure for a target molecule, or a fragment thereof. This could be accomplished by x-ray crystallography, computer modeling or by a combination of both approaches.

[0047] It also is possible to use antibodies to ascertain the structure of a target compound activator or inhibitor. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a

mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically-or biologically-produced peptides. Selected peptides would then serve as the pharmacore. Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.

[0048] On the other hand, one may simply acquire, from various commercial sources, small molecule libraries that are believed to meet the basic criteria for useful drugs in the identification of useful compounds. Screening of such libraries, including combinatorially generated libraries (e. g., peptide libraries), is a rapid and efficient way to screen large number of related (and unrelated) compounds for activity. Combinatorial approaches also lend themselves to rapid evolution of potential drugs by the creation of second, third and fourth generation compounds modeled upon active, but otherwise undesirable compounds.

[0049] Candidate compounds may include fragments or parts of naturally-occurring compounds, or may be found as active combinations of known compounds, which are otherwise inactive. 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 pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. Thus, it is understood that the candidate substance identified by the present invention may be peptide, polypeptide, polynucleotide, small molecule inhibitors or any other compounds that may be designed through rational drug design starting from known inhibitors or stimulators.

[0050] Other suitable modulators include antisense molecules, ribozymes, and antibodies (including single chain antibodies), each of which would be specific for the target molecule. For example, an antisense molecule that bound to a translational or transcriptional start site, or splice junctions, would be ideal candidate inhibitors. This and other techniques of gene suppression are well known in the art. Inhibition or regulation of gene expression may also be achieved by methods such as RNA interference (RNAi). A review of this technique is found in the published U. S. Patent Application 2002008635 Al by Tuschl, et al. (July 4,2002), which describes the use of interfering RNA as a means to modulate or control expression of a target gene or genes.

[0051] In addition to the modulating compounds initially identified, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the structure of the modulators. Such compounds, which may include

peptidomimetics of peptide modulators, may be used in the same manner as the initial modulators. An inhibitor according to the present invention may be one which exerts its inhibitory or activating effect upstream, downstream or directly on the receptor-ligand complex.

6. In vitro Assays [0052] A quick, inexpensive and easy assay to run is an in vitro assay. Such assays generally use isolated molecules, can be run quickly and in large numbers, thereby increasing the amount of information obtainable in a short period of time. A variety of vessels may be used to run the assays, including test tubes, plates, dishes and other surfaces such as dipsticks, beads, silicon wafers and even chips.

[0053] One example of a cell free assay is a binding assay. While not directly addressing function, the ability of a modulator to bind to a target molecule in a specific fashion is strong evidence of a related biological effect. For example, binding of a molecule to a target may, in and of itself, be inhibitory, due to steric, allosteric or charge-charge interactions. The target may be either free in solution, fixed to a support, expressed in or on the surface of a cell.

Either the target or the compound may be labeled, thereby permitting determination of binding.

Usually, the target will be the labeled species, decreasing the chance that the labeling will interfere with or enhance binding. Competitive binding formats can be performed in which one of the agents is labeled, and one may measure the amount of free label versus bound label to determine the effect on binding.

[0054] A 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. Bound polypeptide is detected by various methods.

[0055] Additionally, functional G protein coupled receptors may be immobilized through covalent bonding of C-or N-reactive groups attached to the chosen solid substrate.

Alternatively, functional G-protein coupled receptors may be immobilized by biotinylation of the extracellular domain of the receptor followed by attachment to a solid substrate, such as a chip or sensor chip, via a mixed self-assembled monolayer of biotinylated thiols and an excess of gama- hydroxy-undecanethiol to which streptavidn is bound (Bieri et al. 1999). This method results in a stable, active G-protein coupled receptor layer whose activity upon binding of the appropriate ligand may be directly monitored.

7. In cyto Assays [0056] The present invention also contemplates the screening of compounds for their ability to modulate vomeronasal receptor mediated responses in cells. Various cell lines or primary cell cultures can be utilized for such screening assays, including cells specifically engineered for this purpose. Rodriguez et al. 1999 and in WO 01/25431 describe gene targeting technology for introduction of mutations or"knock-out"mutations into putative receptor genes.

For neuronal cells, the methods disclosed therein provide for the effective identification and isolation (if desired) of individual neurons that would normally express the knocked-out allele.

[0057] Depending on the assay, culture may be required. The cell is examined using any of a number of different physiologic assays. Alternatively, molecular analysis may be performed, for example, looking at protein expression, mRNA expression (including differential display of whole cell or polyA RNA) and others.

8. In vivo Assays l0058] In vivo assays involve the use of various animal models, including transgenic animals that have been engineered to have specific defects, or carry markers that can be used to measure the ability of a candidate substance to reach and effect different cells within the organism. Due to their size, ease of handling, and information on their physiology and genetic make-up, mice are a preferred embodiment, especially for transgenics in the context of the present invention. However, other animals are suitable as well, especially vertebrates, and including rats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, and any other member of the Rodentia, as well as fish, ferrets, cats, dogs, sheep, goats, pigs, cows, horses, and monkeys.

Assays for modulators and receptors may be conducted using an animal model derived from any of these species.

[0059] In such assays, one or more candidate substances are administered to an animal, and the ability of the candidate substance (s) to alter one or more characteristics, as compared to a similar animal not treated with the candidate substance (s), identifies a modulator.

The characteristics may be any of those discussed above with regard to the receptor-ligand complex, cell, physiology and behavior, immune response, etc. Determining the effectiveness of a compound in vivo may involve a variety of different criteria. Also, measuring toxicity and dose response can be performed in animals in a more meaningful fashion than in in vitro or in cyto assays. Similarly, expression of candidate receptors may be accomplished in an animal model derived from the above listed species.

9. Pharmaceutical Compositions [0060] Pharmaceutical compositions of the present invention comprise an effective amount of one or more modulators of Vlrb2 : 2-heptanone complex activity or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases"pharmaceutical or pharmacologically acceptable"refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one modulator or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e. g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0061] As used herein, "pharmaceutically acceptable carrier"includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e. g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.

Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0062] The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient (s) in a composition and appropriate dose (s) for the individual subject.

[0063] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0. 1% of an active compound. In other embodiments, the active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5

microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 56 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non- limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

[0064] In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e. g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0065] In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e. g., glycerol, propylene glycol, liquid polyethylene glycol, etc), lipids (e. g., triglycerides, vegetable oils, liposomes) and combinations thereof. 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 by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

[0066] In other embodiments, one may use eye drops, nasal solutions or sprays, aerosols or inhalants in the present invention. Such compositions are generally designed to be compatible with the target tissue type. In a non-limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in preferred embodiments the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5. 5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug

stabilizers, if required, may be included in the formulation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.

[0067] In certain embodiments the modulator is prepared for administration by such routes as oral ingestion. In these embodiments, the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e. g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be incorporated directly with the food of the diet. Preferred carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof. In other aspects of the invention, the oral composition may be prepared as a syrup or elixir. A syrup or elixir, may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.

[0068] In certain preferred embodiments an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof. In certain embodiments, a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof ; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof ; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof ; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. ; or combinations thereof. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.

10. Polynucleotides and Nucleic Acids [0069] Certain embodiments of the present invention concern a nucleic acid. In certain aspects, a nucleic acid comprises a wild-type or a mutant vlrb2 nucleic acid. In particular aspects, the nucleic acid encodes for or comprises a transcribed nucleic acid. In other aspects, the nucleic acid comprises a nucleic acid segment of SEQ ID NO : 1, or a biologically functional equivalent thereof. In particular aspects, the nucleic acid encodes a protein, polypeptide, peptide.

[0070] The term"nucleic acid"is well known in the art. A"nucleic acid"as used herein will generally refer to a molecule (i. e. , a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. A nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e. g., an adenine"A, "a guanine"G,"a thymine"T"or a cytosine"C") or RNA (e. g., an A, a G, an uracil"U"or a C). The term"nucleic acid" encompass the terms"oligonucleotide"and"polynucleotide, "each as a subgenus of the term "nucleic acid. "The term"oligonucleotide"refers to a molecule of between about 3 and about 100 nucleobases in length. The term"polynucleotide"refers to at least one molecule of greater than about 100 nucleobases in length.

[0071] These definitions generally refer to a single-stranded molecule, but in specific embodiments will also encompass an additional strand that is partially, substantially or fully complementary to the single-stranded molecule. Thus, a nucleic acid may encompass a double- stranded molecule or a triple-stranded molecule that comprises one or more complementary strand (s) or"complement (s)" of a particular sequence comprising a molecule. As used herein, a single stranded nucleic acid may be denoted by the prefix"ss, "a double stranded nucleic acid by the prefix"ds, "and a triple stranded nucleic acid by the prefix"ts." [0072] A nucleic acid may be made by any technique known to one of ordinary skill in the art, such as for example, chemical synthesis, enzymatic production or biological production. Non-limiting examples of a synthetic nucleic acid (e. g., a synthetic oligonucleotide), include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques such as described in EP 266,032, or via deoxynucleoside H-phosphonate intermediates as described by Froehler et al., 1986 and U. S.

Patent 5,705, 629, each incorporated herein by reference. In the methods of the present invention, one or more oligonucleotide may be used. Various different mechanisms of oligonucleotide synthesis have been disclosed in, for example, U. S. Patents 4,659, 774, 4,816, 571,5, 141,813, 5,264, 566,4, 959,463, 5,428, 148,5, 554,744, 5,574, 146,5, 602,244, each of which is incorporated herein by reference.

[0073] A non-limiting example of an enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCRTM (see for example, U. S.

Patents 4,683, 202 and 4,682, 195, each incorporated herein by reference), or the synthesis of an oligonucleotide described in U. S. Patent 5,645, 897. A non-limiting example of a biologically produced nucleic acid includes a recombinant nucleic acid produced (i. e. , replicated) in a living cell, such as a recombinant DNA vector replicated in bacteria.

[0074] As used herein, the term"complementary"or"complement (s)" also refers to a nucleic acid comprising a sequence of consecutive nucleobases or semiconsecutive nucleobases (e. g., one or more nucleobase moieties are not present in the molecule) capable of hybridizing to another nucleic acid strand or duplex even if less than all the nucleobases do not base pair with a counterpart nucleobase. In certain embodiments, a"complementary"nucleic acid comprises a sequence in which about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, to about 100%, and any range derivable therein, of the nucleobase sequence is capable of base-pairing with a single or double stranded nucleic acid molecule during hybridization. In certain embodiments, the term"complementary"refers to a nucleic acid that may hybridize to another nucleic acid strand or duplex in stringent conditions, as would be understood by one of ordinary skill in the art.

[0075] In certain embodiments, a"partly complementary"nucleic acid comprises a sequence that may hybridize in low stringency conditions to a single or double stranded nucleic acid, or contains a sequence in which less than about 70% of the nucleobase sequence is capable of base-pairing with a single or double stranded nucleic acid molecule during hybridization.

[0076] As used herein, "hybridization","hybridizes"or"capable of hybridizing"is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature. The term"anneal"as used herein is synonymous with "hybridize. "The term"hybridization,""hybridize (s)" or"capable of hybridizing"encompasses the terms"stringent condition (s)" or"high stringency"and the terms"low stringency"or"low stringency condition (s)." [00771 As used herein"stringent condition (s)" or"high stringency"are those conditions that allow hybridization between or within one or more nucleic acid strand (s) containing complementary sequence (s), but precludes hybridization of random sequences.

Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand.

Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like.

[0078] Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about

70°C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid (s), the length and nucleobase content of the target sequence (s), the charge composition of the nucleic acid (s), and to the presence or concentration of formamide, tetramethylammonium chloride or other solvent (s) in a hybridization mixture.

[0079] It is also understood that these ranges, compositions and conditions for hybridization are mentioned by way of non-limiting examples only, and that the desired stringency for a particular hybridization reaction is often determined empirically by comparison to one or more positive or negative controls. Depending on the application envisioned it is preferred to employ varying conditions of hybridization to achieve varying degrees of selectivity of a nucleic acid towards a target sequence. In a non-limiting example, identification or isolation of a related target nucleic acid that does not hybridize to a nucleic acid under stringent conditions may be achieved by hybridization at low temperature and/or high ionic strength. Such conditions are termed"low stringency"or"low stringency conditions", and non-limiting examples of low stringency include hybridization performed at about 0.15 M to about 0.9 M NaCI at a temperature range of about 20°C to about 50°C. Of course, it is within the skill of one in the art to further modify the low or high stringency conditions to suite a particular application.

[0080] As used herein"wild-type"refers to the naturally occurring sequence of a nucleic acid at a genetic locus in the genome of an organism, or a sequence transcribed or translated from such a nucleic acid. Thus, the term"wild-type"also may refer to an amino acid sequence encoded by a nucleic acid. As a genetic locus may have more than one sequence or alleles in a population of individuals, the term"wild-type"encompasses all such naturally occurring allele (s). As used herein the term"polymorphic"means that variation exists (i. e., two or more alleles exist) at a genetic locus in the individuals of a population. As used herein "mutant"refers to a change in the sequence of a nucleic acid or its encoded protein, polypeptide or peptide that is the result of the hand of man.

[0081] The present invention also concerns the isolation or creation of a recombinant construct, a recombinant protein or polypeptide, or a recombinant host cell through the application of recombinant nucleic acid technology known to those of skill in the art or as described herein. A recombinant construct or host cell may comprise a nucleic acid encoding all or part of a VRI receptor, and may express a VR1 protein, peptide or polypeptide, or at least one biologically functional equivalent thereof.

[0082] Herein certain embodiments, a"gene"refers to a nucleic acid that is transcribed. In certain aspects, the gene includes regulatory sequences involved in transcription,

or message production or composition. In particular embodiments, the gene comprises transcribed sequences that encode for a protein, polypeptide or peptide. As will be understood by those in the art, this functional term"gene"includes both genomic sequences, RNA or cDNA sequences or smaller engineered nucleic acid segments, including nucleic acid segments of a non-transcribed part of a gene, including but not limited to the non-transcribed promoter or enhancer regions of a gene. Smaller engineered gene nucleic acid segments may express, or may be adapted to express using nucleic acid manipulation technology, proteins, polypeptides, domains, peptides, fusion proteins, mutants and/or such like.

[0083]"Isolated substantially away from other coding sequences"means that the gene of interest forms the significant part of the coding region of the nucleic acid, or that the nucleic acid does not contain large portions of naturally-occurring coding nucleic acids, such as large chromosomal fragments, other functional genes, RNA or cDNA coding regions. Of course, this refers to the nucleic acid as originally isolated, and does not exclude genes or coding regions later added to the nucleic acid by the hand of man.

[0084] The nucleic acid (s) of the present invention, regardless of the length of the sequence itself, may be combined with other nucleic acid sequences, including but not limited to, promoters, enhancers, polyadenylation signals, restriction enzyme sites, multiple cloning sites, coding segments, and the like, to create one or more nucleic acid construct (s). As used herein, a "nucleic acid construct"is a nucleic acid engeneered or altered by the hand of man, and generally comprises one or more nucleic acid sequences organized by the hand of man.

[0085] In a non-limiting example, one or more nucleic acid constructs may be prepared that include a contiguous stretch of nucleotides identical to or complementary to SEQ ID NO : 1. A nucleic acid construct may be about 3, about 5, about 8, about 10 to about 14, or about 15, about 20, about 30, about 40, about 50, about 100, about 200, about 500, about 1,000, about 2,000, about 3,000, about 5,000, about 10,000, about 15,000, about 20,000, about 30,000, about 50,000, about 100,000, about 250,000, about 500,000, about 750,000, to about 1,000, 000 nucleotides in length, as well as constructs of greater size, up to and including chromosomal sizes (including all intermediate lengths and intermediate ranges), given the advent of nucleic acid constructs such as, a yeast artificial chromosome are known to those of ordinary skill in the art. It will be readily understood that"intermediate lengths"and"intermediate ranges, "as used herein, means any length or range including or between the quoted values (i. e. , all integers including and between such values). Non-limiting examples of intermediate lengths include about 11, about 12, about 13, about 16, about 17, about 18, about 19, etc. ; about 21, about 22, about 23, etc. ; about 31, about 32, etc. ; about 51, about 52, about 53, etc. ; about

101, about 102, about 103, etc. ; about 151, about 152, about 153, etc. ; about 1,001, about 1002, etc,; about 50,001, about 50,002, etc; about 750,001, about 750,002, etc. ; about 1,000, 001, about 1,000, 002, etc. Non-limiting examples of intermediate ranges include about 3 to about 32, about 150 to about 500,001, about 3,032 to about 7,145, about 5,000 to about 15,000, about 20,007 to about 1,000, 003, etc.

[0086] In particular embodiments, the invention concerns one or more recombinant vector (s) comprising nucleic acid sequences that encode a Vlrb2 protein, polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO : 2. In other embodiments, the invention concerns recombinant vector (s) comprising nucleic acid sequences that encode a Vlrb2 protein, polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in SEQ ID NO : 2. In particular aspects, the recombinant vectors are DNA vectors.

[0087] The term"a sequence essentially as set forth in SEQ ID NO : 1" or"a sequence essentially as set forth in SEQ ID NO : 2" means that the sequence substantially corresponds to a portion of SEQ ID NO : 1 or SEQ ID NO : 2, respectively, and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO : 2.

Thus, "a sequence essentially as set forth in SEQ ID NO : 1" encompasses nucleic acids, nucleic acid segments, and genes that comprise part or all of the nucleic acid sequences as set forth in SEQ ID NO : 1.

[0088] The term"biologically functional equivalent"is well understood in the art and is further defined in detail herein. Accordingly, a sequence that has between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID N0 : 2 will be a sequence that is"essentially as set forth in SEQ ID N0 : 2," provided the biological activity of the protein, polypeptide or peptide is maintained.

[0089] Biological function equivalents may also describe particular cells, tissues, or organs wherein at least one biological function is shared among the equivalents. Thus, cells, tissues, or organs that respond, transduce, or otherwise process or transmit signals as the VNO does would be biologically functional equivalents for those functions.

(0090] In certain other embodiments, the invention concerns at least one recombinant vector that includes within its sequence a nucleic acid sequence essentially as set forth in SEQ ID NO: 1. In particular embodiments, the recombinant vector comprises DNA sequences

that encode protein (s), polypeptide (s) or peptide (s) exhibiting Vlrb2 activity when part of a receptor-ligand complex.

[0091] 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 and serine, and also refers to codons that encode biologically equivalent amino acids. Codon usage for various organisms and organelles can be found in the literature of the art and are well known, allowing one of skill in the art to optimize codon usage for expression in various organisms using the disclosures herein.

Thus, it is contemplated that codon usage may be optimized for other animals, as well as other organisms such as a prokaryote (e. g., an eubacteria, an archaea), an eukaryote (e. g., a protist, a plant, a fungi, an animal), a virus and the like, as well as organelles that contain nucleic acids, such as mitochondria, chloroplasts and the like, based on the preferred codon usage as would be known to those of ordinary skill in the art.

[0092] It will also be understood that amino acid sequences or nucleic acid sequences may include additional residues, such as additional N-or C-terminal amino acids or 5' or 3'sequences, or various combinations thereof, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein, polypeptide or peptide activity where expression of a proteinaceous composition is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5'and/or 3'portions of the coding region or may include various internal sequences, i. e. , introns, which are known to occur within genes.

[00931 Excepting intronic and flanking regions, and allowing for the degeneracy of the genetic code, nucleic acid sequences that have between about 70% and about 79%; or more preferably, between about 80% and about 89%; or even more particularly, between about 90% and about 99%; of nucleotides that are identical to the nucleotides of SEQ ID NO: 1 will be nucleic acid sequences that are"essentially as set forth in SEQ ID NO: 1." l0094l It will also be understood that this invention is not limited to the particular nucleic acid or amino acid sequences of SEQ ID NO : 1 or SEQ ID NO : 2. Recombinant vectors and isolated nucleic acid segments may therefore variously include these coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, and they may encode larger polypeptides or peptides that nevertheless include such coding regions or may encode biologically functional equivalent proteins, polypeptide or peptides that have variant amino acids sequences.

[00951 The nucleic acids of the present invention encompass biologically functional equivalent Vlrb2 proteins, polypeptides, or peptides. Such sequences may arise as a consequence of codon redundancy or functional equivalency that are known to occur naturally within nucleic acid sequences or the proteins, polypeptides or peptides thus encoded.

Alternatively, functionally equivalent proteins, polypeptides or peptides may be created via the application of recombinant DNA technology, in which changes in the protein, polypeptide or peptide structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced, for example, through the application of site-directed mutagenesis techniques as discussed herein below, e. g., to introduce improvements or alterations to the antigenicity of the protein, polypeptide or peptide, or to test mutants in order to examine Vlrb2 protein, polypeptide or peptide activity at the molecular level.

[0096] Fusion or chimeric proteins, polypeptides or peptides may be prepared, e. g., where the vlrb2 coding regions are aligned within the same expression unit with other proteins, polypeptides or peptides having desired functions. Non-limiting examples of such desired functions of expression sequences include purification or immunodetection purposes for the added expression sequences, e. g., proteinaceous compositions that may be purified by affinity chromatography or the enzyme labeling of coding regions, respectively.

[0097] As used herein an"organism"may be a prokaryote, eukaryote, virus and the like. As used herein the term"sequence"encompasses both the terms"nucleic acid"and "proteincecous"or"proteinaceous composition. "As used herein, the term"proteinaceous composition"encompasses the terms"protein","polypeptide"and"peptide."As used herein "artificial sequence"refers to a sequence of a nucleic acid not derived from sequence naturally occurring at a genetic locus, as well as the sequence of any proteins, polypeptides or peptides encoded by such a nucleic acid. A"synthetic sequence", refers to a nucleic acid or proteinaceous composition produced by chemical synthesis in vitro, rather than enzymatic production in vitro (i. e. , an"enzymatically produced"sequence) or biological production in vivo (i. e., a"biologically produced"sequence).

11. Vectors [0098] The term"vector"is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be"exogenous, "which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence

in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e. g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Maniatis et al., 1990 and Ausubel et al., 1996, both incorporated herein by reference).

[0099] The term"expression vector"refers to any type of genetic construct comprising a nucleic acid coding for a RNA capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes.

Expression vectors can contain a variety of"control sequences, "which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. a. Promoters and Enhancers [00100] A"promoter"is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate the specific transcription a nucleic acid sequence. The phrases "operatively positioned,""operatively linked, ""under control, "and"under transcriptional control"mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.

[00101] A promoter generally comprises a sequence that 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, for example, 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. Additional 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 been shown to contain functional elements downstream of the start site as well. To bring a coding sequence"under the control of'a promoter, one positions the 5'end of the transcription initiation site of the transcriptional reading frame"downstream"of (i. e. , 3'of) the chosen promoter. The"upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

[00102] 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 cooperatively or independently to activate transcription. A promoter may or may not be used in conjunction with an"enhancer, "which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[00103] A promoter may be one naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5'non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as"endogenous. "Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not "naturally occurring,"i. e. , containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. For example, promoters that are most commonly used in recombinant DNA construction include the p-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein (see U. S. Patents 4,683, 202 and 5,928, 906, each incorporated herein by reference). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[00104] Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, (see, for example Sambrook et al. 2001). The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the

introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

[00105] Additionally any promoter/enhancer combination (as per, for example, the Eukaryotic Promoter Data Base EPDB, www. epd. isb-sib. ch/) could also be used to drive expression. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. 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.

[00106] Table 1 lists non-limiting examples of elements/promoters that may be employed, in the context of the present invention, to regulate the expression of a RNA. Table 2 provides non-limiting examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus.

Table 1 Promoter and/or Enhancer Promoter/Enhancer References Immunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983; Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al., 1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al. ; 1990 Immunoglobulin Light Chain Queen et al., 1983; Picard et al., 1984 T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al. ; 1990 HLA DQ a and/or DQ P Sullivan et al., 1987 ß-Interferon Goodbourn et al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2 Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin et al., 1990 MHC Class II 5 Koch et al., 1989 MHC Class 11 HLA-Dra Sherman et al., 1989 ß-Actin Kawamoto et al., 1988; Ng et al. ; 1989 Muscle Creatine Kinase (MCK) Jaynes et al., 1988; Horlick et al., 1989; Johnson et al., 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase I Ornitz et al., 1987 Table 1 Promoter and/or Enhancer Promoter/Enhancer References Metallothionein (MTII) Karin et al., 1987; Culotta et al., 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987 Albumin Pinkert et al., 1987; Tronche et al., 1989,1990 a-Fetoprotein Godbout et aL, 1988; Carnpere et al., 1989 y-Globin Bodine et al., 1987; Perez-Stable et al., 1990 ß-Globin Trudel et al., 1987 c-fos Cohen et al., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlund et al., 1985 Neural Cell Adhesion Molecule Hirsch et al., 1990 (NCAM) a-Antitrypsin Latimer et al., 1990 H2B (TH2B) Histone Hwang et al., 1990 Mouse and/or Type I Collagen Ripe et al., 1989 Glucose-Regulated Proteins Chang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsen et al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 Troponin I (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et al., 1989 (PDGF) Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerji et al., 1981 ; Moreau et al., 1981 ; Sleigh et al., 1985; Firak et al., 1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al., 1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinka et al., 1980,1981 ; Tyndell et al., 1981; Dandolo et al., 1983; de Villiers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbell and/or Villarreal, 1988 Table 1 Promoter and/or Enhancer Promoter/Enhancer References Retroviruses Kriegler et al., 1982,1983 ; Levinson et al., 1982; Kriegler et al., 1983,1984a, b, 1988; Bosze et al., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al., 1988; Celander et al., 1988; Choi et al., 1988; Reisman et al., 1989 Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos and/or Wilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987 Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et al., 1987; Spandau et al., 1988; Vannice et al., 1988 Human Immunodeficiency Virus Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al., 1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddock et al., 1989 Cytomegalovirus (CMV) Weber et al., 1984; Boshart et al., 1985; Foecking et al., 1986 Gibbon Ape Leukemia Virus Holbrook et al., 1987; Quinn et al., 1989 TABLE 2 Inducible Elements Element Inducer References MT II Phorbol Ester (TFA) Palmiter et al., 1982; Heavy metals Haslinger et al., 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouse mammary Glucocorticoids Huang et al., 1981; Lee et tumor virus) al., 1981; Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta et al., 1985; Sakai et al., 1988 P-Interferon Poly (rI) x Tavernier et al., 1983 TABLE 2 Inducible Elements Element Inducer References Poly (rc) Adenovirus 5 E2 EIA Imperiale et al., 1984 Collagenase Phorbol Ester (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b Murine MX Gene Interferon, Newcastle Hug et al., 1988 Disease Virus GRP78 Gene A23187 Resendez et al., 1988 a-2-Macroglobulin IL-6 Kunz et al., 1989 Vimentin Serum Rittling et al., 1989 MHC Class I Gene H-2xb Interferon Blanar et al., 1989 HSP70 EIA, SV40 Large T Taylor et al., 1989, Antigen 1990a, 1990b Proliferin Phorbol Ester-TPA Mordacq et al., 1989 Tumor Necrosis Factor a PMA Hensel et al., 1989 Thyroid Stimulating Thyroid Hormone Chatterjee et al., 1989 Hormone a Gene

[00107] The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, is well known to those of skill in the art. Nonlimiting examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murine epididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997), and human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996). b. Initiation Signals and Internal Ribosome Binding Sites [00108] A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences.

Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be"in-frame"

with the reading frame of the desired coding sequence to ensure translation of the entire insert.

The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

[00109] In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. 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 picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. 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 (see U. S. Patents 5,925, 565 and 5,935, 819, each herein incorporated by reference). c. Multiple Cloning Sites [00110] Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector (see, for example, Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997. )"Restriction enzyme digestion"refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. "Ligation"refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology. d. Splicing Sites [00111] Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression (see, for example, Chandler et al., 1997, herein incorporated by reference.)

e. Termination Signals [00112] The vectors or constructs of the present invention will generally comprise at least one termination signal. A"termination signal"or"terminator"is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.

[00113] In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3'end of the transcript. RNA molecules modified with this polyA tail appear to be more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

[00114] Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation. f. Polyadenylation Signals [00115] In expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the 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. Preferred embodiments include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, convenient and known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport. g. Origins of Replication [00116] In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed"ori"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

h. Selectable and Screenable Markers [00117] In certain embodiments of the invention, cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

[00118] Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used 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 and screenable markers are well known to one of skill in the art. i. Plasmid Vectors [00119] In certain embodiments, a plasmid vector is contemplated for use to transform a host cell. In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. In a non-limiting example, E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, for example, promoters which can be used by the microbial organism for expression of its own proteins.

[00120] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with

these hosts. For example, the phage lambda GEM-11 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as, for example, E. coli LE392.

[00121] Further useful plasmid vectors include pIN vectors (Inouye et al., 1985); and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage. Other suitable fusion proteins are those with ß-galactosidase, ubiquitin, and the like.

[00122] Bacterial host cells, for example, E. coli, comprising the expression vector, are grown in any of a number of suitable media, for example, LB. The expression of the recombinant protein in certain vectors may be induced, as would be understood by those of skill in the art, by contacting a host cell with an agent specific for certain promoters, e. g., by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, generally of between 2 and 24 h, the cells are collected by centrifugation and washed to remove residual media. j. Viral Vectors [00123] The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells (e. g., mammalian cells). Non-limiting examples of virus vectors that may be used to deliver a nucleic acid of the present invention are described below.

1. Adenoviral Vectors [00124] A particular method for delivery of the nucleic acid involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors. "Adenovirus expression vector"is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell-specific construct that has been cloned therein.

Knowledge of the genetic organization of adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992). The use of Adenoviral vectors in the provision of nucleotide sequences encoding odorant receptors is examplified by the dislcosure of Firestein et al. (U. S.

Patent 6,218, 358).

2. AAV Vectors [00125] The nucleic acid may be introduced into the cell using adenovirus assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994).

Adeno-associated virus (AAV) is an attractive vector system for use in present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broad host range for infectivity (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details concerning the generation and use of rAAV vectors are described in U. S. Patents 5,139, 941 and 4,797, 368.

3. Retroviral Vectors [00126] Retroviruses are useful because of their ability to integrate nucleotide sequences into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell-lines (Miller, 1992).

[00127] In order to construct a receptor vector, a nucleic acid (e. g., one encoding a receptor of interest) is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into a special cell line (e. g., by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).

[00128] Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function.

Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U. S. Patents 6,013, 516 and 5,994, 136). Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by multiply attenuating

the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.

[00129] Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences.

For example, recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U. S. Patent 5,994, 136. One may target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell-type. By inserting a sequence (including a regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific.

4. Other Viral Vectors [00130] Other viral vectors may be employed as vaccine constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), sindbis virus, cytomegalovirus and herpes simplex virus may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Couparet al., 1988; Horwich et al., 1990).

5. Delivery Using Modified Viruses [00131] A nucleic acid to be delivered may be housed within an infective virus that has been engineered to express a specific binding ligand. The virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell. A novel approach designed to allow specific targeting of retrovirus vectors was developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.

[00132] Another approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al., 1989).

2. Vector Delivery and Cell Transformation [00133] Suitable methods for nucleic acid delivery for transformation of an organelle, a cell, a tissue or an organism for use with the current invention are believed to include virtually any method by which a nucleic acid (e. g. , DNA) can be introduced into an organelle, a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection (Wilson et al., 1989), by injection (U. S. Patents 5,994, 624,5, 981,274, 5,945, 100, 5,780, 448,5, 736,524, 5,702, 932,5, 656,610, 5,589, 466 and 5,580, 859), including microinjection (Harlan and Weintraub, 1985; U. S. Patent 5,789, 215); by electroporation (U. S. Patent 5,384, 253; Tur-Kaspa et al., 1986; Potter et al., 1984); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kanedaet al., 1989; Kato et al., 1991) and receptor- mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U. S. Patent Nos. 5,610, 042; 5,322, 783 5,563, 055,5, 550,318, 5,538, 877 and 5,538, 880); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U. S. Patent Nos. 5,302, 523 and 5,464, 765); by Agrobacterium-mediated transformation (U. S. Patent Nos. 5,591, 616 and 5,563, 055); by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U. S. Patent Nos. 4,684, 611 and 4,952, 500); by desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), and any combination of such methods. Through the application of techniques such as these, organelle (s), cell (s), tissue (s) or organism (s) may be stably or transiently transformed. a. Ex vivo Transformation [00134] Methods for tranfecting vascular cells and tissues removed from an organism in an ex vivo setting are known to those of skill in the art. For example, cannine endothelial cells have been genetically altered by retrovial gene tranfer in vitro and transplanted into a canine (Wilson et al., 1989). In another example, yucatan minipig endothelial cells were tranfected by retrovirus in vitro and transplated into an artery using a double-ballonw catheter (Nabel et al., 1989). Thus, it is contemplated that cells or tissues may be removed and tranfected ex vivo using the nucleic acids of the present invention. In particular aspects, the transplanted cells or tissues may be placed into an organism. In preferred facets, a nucleic acid is expressed in the transplated cells or tissues.

b. Injection [00135] In certain embodiments, a nucleic acid may be delivered to an organelle, a cell, a tissue or an organism via one or more injections (i. e. , a needle injection), such as, for example, subcutaneously, intradermally, intramuscularly, intervenously, intraperitoneally, etc.

Methods of injection of vaccines are well known to those of ordinary skill in the art (e. g., injection of a composition comprising a saline solution). Further embodiments of the present invention include the introduction of a nucleic acid by direct microinjection. Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985). c. Electroporation [00136] In certain embodiments of the present invention, a nucleic acid is introduced into an organelle, a cell, a tissue or an organism via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. In some variants of this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells (U. S. Patent 5,384, 253). Alternatively, recipient cells can be made more susceptible to transformation by mechanical wounding.

[00137] Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986) in this manner.

[00138] To effect transformation by electroporation in cells such as, for example, plant cells, one may employ either friable tissues, such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly.

In this technique, one would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounding in a controlled manner.

Examples of some species which have been transformed by electroporation of intact cells include maize (U. S. Patent 5,384, 253; Rhodes et al., 1995; D'Halluinet al., 1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soybean (Christou et al., 1987) and tobacco (Lee et al., 1989).

[00139] One also may employ protoplasts for electroporation transformation of plant cells (Bates, 1994; Lazzeri, 1995). For example, the generation of transgenic soybean plants by electroporation of cotyledon-derived protoplasts is described by Dhir and Widholm in International Patent Application WO 9217598. Other examples of species for which protoplast

transformation has been described include barley (Lazerri, 1995), sorghum (Battraw et al., 1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) and tomato (Tsukada, 1989). d. Calcium Phosphate [00140] In other embodiments of the present invention, a nucleic acid is introduced to the cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique. Also in this manner, mouse L (A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., 1990). e. DEAE-Dextran [00141] In another embodiment, a nucleic acid is delivered into a cell using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985). f. Sonication Loading [00142] Additional embodiments of the present invention include the introduction of a nucleic acid by direct sonic loading. LTK-fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et al., 1987). g. Liposome-Mediated Transfection [00143] In a further embodiment of the invention, a nucleic acid may be entrapped in a lipid complex such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is an nucleic acid complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen).

[00144] Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al., 1980).

[00145] In certain embodiments of the invention, a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane

and promote cell entry of liposome-encapsulated DNA (Kanedaet al., 1989). In other embodiments, a liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, a liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In other embodiments, a delivery vehicle may comprise a ligand and a liposome. h. Receptor Mediated Transfection [00146] Still further, a nucleic acid may be delivered to a target cell via receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in a target cell. In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present invention.

[00147] Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a nucleic acid-binding agent. Others comprise a cell receptor-specific ligand to which the nucleic acid to be delivered has been operatively attached.

Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990; Peraleset al., 1994; Myers, EPO 0273085), which establishes the operability of the technique. Specific delivery in the context of another mammalian cell type has been described (Wu and Wu, 1993). In certain aspects of the present invention, a ligand will be chosen to correspond to a receptor specifically expressed on the target cell population.

[00148] In other embodiments, a nucleic acid delivery vehicle component of a cell-specific nucleic acid targeting vehicle may comprise a specific binding ligand in combination with a liposome. The nucleic acid (s) to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the liposome membrane. The liposome will thus specifically bind to the receptor (s) of a target cell and deliver the contents to a cell. Such systems have been shown to be functional using systems in which, for example, epidermal growth factor (EGF) is used in the receptor-mediated delivery of a nucleic acid to cells that exhibit upregulation of the EGF receptor.

[00149] In still further embodiments, the nucleic acid delivery vehicle component of a targeted delivery vehicle may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding. For example, lactosyl-ceramide, a galactose-terminal asialganglioside, have been incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes (Nicolau et al., 1987). It is contemplated that the tissue-specific transforming constructs of the present invention can be specifically delivered into a target cell in a similar manner.

i. Microprojectile Bombardment [00150] Microprojectile bombardment techniques can be used to introduce a nucleic acid into at least one, organelle, cell, tissue or organism (U. S. Patents 5,550, 318; 5,538, 880; 5,610, 042; and PCT Application WO 94/09699). This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). There are a wide variety of microprojectile bombardment techniques known in the art, many of which are applicable to the invention.

[00151] Microprojectile bombardment may be used to transform various cell (s), tissue (s) or organism (s), such as for example any plant species. Examples of species which have been transformed by microprojectile bombardment include monocot species such as maize (PCT Application WO 95/06128), barley (Ritala et al., 1994; Hensgenset al., 1993), wheat (U. S.

Patent No. 5,563, 055), rice (Hensgens et al., 1993), oat (Torbet et al., 1995; Torbet et al., 1998), rye (Hensgens et al., 1993), sugarcane (Bower et al., 1992), and sorghum (Hagio et al., 1991); as well as a number of dicots including tobacco (Tomes et al., 1990; Buising and Benbow, 1994), soybean (U. S. Patent No. 5,322, 783), sunflower (Knittel et al. 1994), peanut (Singsit et al., 1997), cotton (McCabe and Martinell, 1993), tomato (VanEck et al. 1995), and legumes in general (U. S. Patent No. 5,563, 055).

[00152] In this microprojectile bombardment, one or more particles may be coated with at least one nucleic acid and delivered into cells by a propelling force. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold particles or beads. Exemplary particles include those comprised of tungsten, platinum, and preferably, gold. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA. DNA-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary.

[00153] For the bombardment, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.

[00154] An illustrative embodiment of a method for delivering DNA into a cell (e. g., a plant cell) by acceleration is the Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with cells, such as for example, a monocot plant cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectile aggregates and may contribute to a higher frequency of transformation by reducing the damage inflicted on the recipient cells by projectiles that are too large.

3. Host Cells [00155] As used herein, the terms"cell, ""cell line, "and"cell culture"may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence,"host cell"refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors. A host cell may be"transfected"or "transformed, "which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny. As used herein, the terms"engineered"and"recombinant"cells or host cells are intended to refer to a cell into which an exogenous nucleic acid sequence, such as, for example, a vector, has been introduced. Therefore, recombinant cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced nucleic acid.

[00156] In certain embodiments, it is contemplated that RNAs or proteinaceous sequences may be co-expressed with other selected RNAs or proteinaceous sequences in the same host cell. Co-expression may be achieved by co-transfecting the host cell with two or more distinct recombinant vectors. Alternatively, a single recombinant vector may be constructed to include multiple distinct coding regions for RNAs, which could then be expressed in host cells transfected with the single vector.

[00157] A tissue may comprise a host cell or cells to be transformed with a vector containing the receptor of interest. The tissue may be part or separated from an organism. In certain embodiments, a tissue may comprise, but is not limited to, adipocytes, alveolar, ameloblasts, axon, basal cells, blood (e. g., lymphocytes), blood vessel, bone, bone marrow,

brain, breast, cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial, epithelial, esophagus, facia, fibroblast, follicular, ganglion cells, glial cells, goblet cells, kidney, liver, lung, lymph node, muscle, neuron, ovaries, pancreas, peripheral blood, prostate, skin, skin, small intestine, spleen, stem cells, stomach, testes, anthers, ascite tissue, cobs, ears, flowers, husks, kernels, leaves, meristematic cells, pollen, root tips, roots, silk, stalks, and all cancers thereof.

[00158] In certain embodiments, the host cell or tissue may be comprised in at least one organism. In certain embodiments, the organism may be, but is not limited to, a prokayote (e. g., a eubacteria, an archaea) or an eukaryote, as would be understood by one of ordinary skill in the art. Preferred eukaryotes are vertebrates, particularly rodents and especially mice and rats and their relatives.

[00159] Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials. An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Cell types available for vector replication and/or expression include, but are not limited to, bacteria, such as E. coli (e. g., E. coli strain RR1, E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325), DH5a, JM109, and KC8, bacilli such as Bacillus subtilis ; and other enterobacteriaceae such as Salmonella typhimurium, Serratia marcescens, various Pseudomonas specie, as well as a number of commercially available bacterial hosts such as SURE Competent Cells and SOLOPACKT" Gold Cells (STRATAGENE, La Jolla). In certain embodiments, bacterial cells such as E. coli LE392 are particularly contemplated as host cells for phage viruses.

[00160] Examples of eukaryotic host cells for replication and/or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC 12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.

[00161] Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques

and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

4. Expression Systems [00162] Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote-and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.

[00163] The insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U. S. Patents 5,871, 986, 4,879, 236, and which can be bought, for example, under the name MAxBAcX 2.0 from INVITROGEN and BACPACKTM BACULOVTRUS EXPRESSION SYSTEM FROM CLONTECH@.

[00164] Other examples of expression systems include STRATAGENE's COMPLETE CONTROLTM Inducible Mammalian Expression System, which involves a synthetic ecdysone- inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INV1TROGEN), which carries the T- RExTM (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

[00165] It is contemplated that the proteins, polypeptides or peptides produced by the methods of the invention may be"overexpressed", i. e. , expressed in increased levels relative to its natural, endogenous, or physiological levels of expression in cells. Such overexpression may be assessed by a variety of methods, including radio-labeling and/or protein purification.

However, simple and direct methods are preferred, for example, detecting cell activation in response to an appropriate ligand such as 2-heptanone. Additional methods may be employed as known to those of skill in the art, including those involving SDS/PAGE and protein staining or western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein, polypeptide or peptide in comparison to the level in natural cells is indicative of overexpression, as is a relative abundance of the specific protein, polypeptides or peptides in relation to the other proteins produced by the host cell.

5. Proteins, Polypeptides, and Peptides [00166] The present invention also provides purified, and in preferred embodiments, substantially purified, proteins, polypeptides, or peptides. The term"purified proteins, polypeptides, or peptides"as used herein, is intended to refer to an proteinaceous composition, isolatable from mammalian cells or recombinant host cells, wherein the at least one protein, polypeptide, or peptide is purified to any degree relative to its naturally-obtainable state, i. e. , relative to its purity within a cellular extract. A purified protein, polypeptide, or peptide therefore also refers to a wild-type or mutant protein, polypeptide, or peptide free from the environment in which it naturally occurs.

[00167] The nucleotide and protein, polypeptide and peptide sequences for various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (http://www. ncbi. nlm. nih. gov/). The coding regions for these known genes may be amplified and/or expressed using the techniques disclosed herein or by any technique that would be know to those of ordinary skill in the art. Additionally, peptide sequences may be sythesized by methods known to those of ordinary skill in the art, such as peptide synthesis using automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, CA).

[00168] Generally, "purified"will refer to a specific protein, polypeptide, or peptide composition that has been subjected to fractionation to remove various other proteins, polypeptides, or peptides, and which composition substantially retains its activity, as may be assessed, for example, by the protein assays, as described herein below, or as would be known to one of ordinary skill in the art for the desired protein, polypeptide or peptide.

[00169] Where the term"substantially purified"is used, this will refer to a composition in which the specific protein, polypeptide, or peptide forms the major component of the composition, such as constituting about 50% of the proteins in the composition or more. In preferred embodiments, a substantially purified protein will constitute more than 60%, 70%, 80%, 90%, 95%, 99% or even more of the proteins in the composition.

[00170] A peptide, polypeptide or protein that is"purified to homogeneity, "as applied to the present invention, means that the peptide, polypeptide or protein has a level of purity where the peptide, polypeptide or protein is substantially free from other proteins and biological components. For example, a purified peptide, polypeptide or protein will often be sufficiently free of other protein components so that degradative sequencing may be performed successfully.

[00171] Various methods for quantifying the degree of purification of proteins, polypeptides, or peptides will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific protein activity of a fraction, or assessing the number of polypeptides within a fraction by gel electrophoresis.

[00172] To purify a desired protein, polypeptide, or peptide a natural or recombinant composition comprising at least some specific proteins, polypeptides, or peptides will be subjected to fractionation to remove various other components from the composition. In addition to those techniques described in detail herein below, various other techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity and other affinity chromatography steps; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques.

[00173] Another example is the purification of a specific fusion protein using a specific binding partner. Such purification methods are routine in the art. As the present invention provides DNA sequences for the specific proteins, any fusion protein purification method can now be practiced. This is exemplified by the generation of a specific protein-glutathione S-transferase fusion protein, expression in E. coli, and isolation to homogeneity using affinity chromatography on glutathione-agarose or the generation of a polyhistidine tag on the N-or C-terminus of the protein, and subsequent purification using Ni-affinity chromatography. However, given many DNA and proteins are known, or may be identified and amplified using the methods described herein, any purification method can now be employed.

[00174] Although preferred for use in certain embodiments, there is no general requirement that the protein, polypeptide, or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified protein, polypeptide or peptide, which are nonetheless enriched in the desired protein compositions, relative to the natural state, will have utility in certain embodiments.

[00175] 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. Inactive products also have utility in certain embodiments, such as, e. g., in determining antigenicity via antibody generation.

12. Pests [00176] Several pest species of vertebrates possess vomeronasal organs and thus respond to known pheromones and agonists of the vomeronasal system. As disclosed by Cook (publication WO 00/59545), disrupting the function of the vomeronasal organ in these species can lead to effective means of pest control. Such disruption may be achieved by the present invention through the methods providing for the identification of modulators of the VNS response mediated by the binding of 2-heptanone with the receptor Vlrb2. Identified modulators may thus be administered to members of the pest population by any suitable means known to the ordinary artisan (such as spraying or baits) to achieve the disruption of sexual physiology and behavior as disclosed in WO 00/59545.

EXAMPLES [00177] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1: Identification of Vlrb2-expressing neurons.

[00178] In order to isolate the Vlr properties which would be exclusively chemosensory and therefore to unequivocally link the detection of a known pheromone with the expression of a specific Vlr receptor, analyses of single vomeronasal sensory neurons (VSNs) was performed. Electrophysiological and calcium imaging techniques were applied to single VSNs expressing a known Vlr receptor (e. g. Vlrb2) at physiological levels.

[00179] Individual VSNs were identified in the mutant VG mouse line (Rodriguez et al., 1999) in which the translation of the endogenous Vlrb2 receptor, which is monoallelically transcribed, is coexpressed with GFP. VSNs expressing Vlrb2 are therefore readily identifiable, and repeated analysis of this defined population is possible (FIG. 1A, FIG. 1B). A similar strategy has been employed in the main olfactory system (Bozza et al., 2002).

[0001] To image the cellular activation of VSNs, freshly dissociated VSNs identified as described above were loaded with the Ca indicator Fura-2AM (10 uM) and imaged at

cellular resolution. VSNs were further selected for analysis of cellular activation according to the following criteria: 1) expression of GFP, 2) intact cell body and dendrite, and 3) response to 50 mM KC1.

EXAMPLE 2: All Vlrb2-expressing neurons specifically respond to 2-heptanone.

[00181] To test for cellular activation mediated through Vlr receptors, candidate ligands were applied to GFP-expressing VSNs isolated from male and female mice from the VG line. Candidate compounds were applied in mixes or individually, and intracellular calcium was monitored prior to, during, and after application of the candidates. In these cells, cellular activation is indicated by increase in intracellular calcium. Candidate ligands included known mouse pheromones and odorants: 2,5-dimethylpyrazine, a-farnesene, ß-farnesene, pentyl acetate, 2-heptanone, fenchone, anisol, citral, eugenol, cineole, myrtenal (each compound at 10- 8M) * [00182] All Vlrb2-expressing neurons tested (12 out of 12) responded to 2-heptanone (FIG. 1C). Responses to 2-heptanone were observed in only 0.8% of non-GFP-expressing neighboring VSNs, n=700. None of the 12 Vlrb2-expressing VSNs monitored responded to the other potential agonists. Other agonists did, however, induce intracellular calcium rises in neighboring non-GFP-expressingVSNs. Additionally, no Vlrb2-expressing VSN responded to analogues of 2-heptanone (2-heptanol and 4-heptanone) (FIG. 1D), indicating that the Vlrb2 mediated response is specific to 2-heptanone.

EXAMPLE 3: The Vlrb2 receptor mediates the cellular response to 2-heptanone.

[00183] VSNs from the Vlrb2-targeted mutant mouse line known as the delta-V, AV or DV line of Rodriguez et al. (1999) were used to confirm that the Vlrb2 receptor mediates the response to 2-heptanone. The DV line differs from the VG line by its lack of endogenous vlrb2 coding sequence. The coding sequence of vlrb2 is replaced by GFP. The mouse mutant line possesses, in part, a shorter lifespan resulting from this deletion. This mouse line nevertheless allows the identification of VSNs which would normally express the Vlrb2 receptor (FIG. 3A, FIG. 3B).

[00184] The same selection criteria as previously described were applied to select appropriate VSNs for analysis and testing. None of the tested VSNs from the DV line did respond with a Ca++ rise (n=6) (FIG. 3C) or inward current (n=4) to the potential agonists (mouse pheromones and odorants) nor to 2-heptanone (FIG. 3D). There is, therefore, a direct

association between the expression of Vlrb2 and the ability of a VSN to respond to the pheromone 2-heptanone. Interestingly, local field potentials induced by 2-heptanone are observed in whole VNO sensory epithelia from mice lacking one of the Vlr gene clusters, which comprises vlrb2 (Del Punta et al., 2002). Vlrb2 is therefore not the sole Vlr receptor able to recognize this ligand, but 2-heptanone is nevertheless the sole known ligand that activates responses through Vlrb2.

[00185] 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. While the compositions and methods of this invention have been described in terms of preferred 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.

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