Functional expression of, and assay for, functional cellular...

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Reexamination Certificate

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C514S008100, C514S012200, C530S350000, C435S004000, C435S007100, C435S007200

Reexamination Certificate

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06218358

ABSTRACT:

BACKGROUND OF THE INVENTION
Olfactory transduction begins with the binding of an odorant ligand to a protein receptor on the olfactory neuron cell surface, thus initiating a cascade of enzymatic reactions that results in the production of a second messenger and the eventual depolarization of the cell membrane (
1
,
2
). This relatively straight-forward and common signalling motif is complicated by the fact that there are several thousand odorants, mostly low molecular weight organic molecules, and nearly one thousand different receptors (
3
,
4
). The receptors are members of the superfamily of membrane receptors characterized structurally by possessing seven transmembrane spanning helices, and functionally by being coupled to GTP-binding proteins. Other members of this superfamily recognize diverse ligands from peptides to biogenic amine neurotransmitters, hormones, drugs, and other organic compounds. The odorant receptor sub-family is the largest sub-family of G-protein coupled receptors (GPCRs) but remains in some ways the most enigmatic since no particular receptor has been definitively paired with any ligand. Strictly speaking vertebrate odorant receptors are classified as “orphan” receptors—receptors with no identified ligand (
5
,
6
).
This situation is especially problematic for understanding coding in the olfactory system and appreciating the nature of the neural image passed to the brain by the peripheral transducing cells (
7
). Current models of olfactory processing have been driven largely by genetic data based on the now well described patterns of receptor gene expression (
8
,
9
). Receptors can be grouped into sub-families based on sequence similarities, and subfamilies of receptors are known to be expressed within one of four restricted topographic zones in the nasal epithelium, although within these general zones expression patterns appear to be random (
10
,
11
). Further, all neurons expressing a particular receptor gene converge to a restricted target in the olfactory bulb (
12
,
13
). However, olfactory neurons typically generate physiological responses to multiple odorants (
14
,
15
) and if, as most evidence indicates, each cell expresses only one type of receptor (
11
,
16
), then the receptors must be able to bind a variety of molecules. Thus, while it may be attractive to hypothesize that the genetic categorization of receptor sequences reflects systematic differences in ligand specificities, and that genetic expression patterns underlie a spatial map for odorant sensitivity, experimental validation of these ideas requires knowing the correlation between receptor gene sequence and the encoded receptor protein's binding specificities, i.e. its receptive field.
Further progress in this area has been limited by the absence of a reliable and efficient system for expressing and assaying cloned odorant receptors. There appear to be two main obstacles to obtaining odorant receptor expression in a heterologous system. For one, expressed receptors must be properly targeted to, and inserted in, the plasma membrane, a process that may require specialized cellular machinery not available in heterologous cell culture expression systems. Secondly, even properly inserted receptors must couple to a second messenger system in order to produce a response that can be assayed (
17
). Olfactory specific isoforms of second messenger enzymes have been identified in olfactory neurons (
2
), raising the possibility that receptor-effector coupling may be highly specific, and that endogenous G-proteins in heterologous cell systems may be unable to produce a powerful enough response to be measured reliably.
In order to circumvent these two potential difficulties we have adopted an alternative strategy for odorant receptor expression. On the assumption that olfactory neurons themselves would be the most capable cells for expressing, targeting and coupling odorant receptors, we used the nasal epithelium as an expression system, driving expression of a particular receptor by including it in a recombinant adenovirus (Adv) and infecting rat nasal epithelia in vivo. Adenovirus vectors have been developed as a tool for efficient gene transfer in mammalian cells (
18
) and have shown promise in a variety of experimental and clinical applications (
19
-
23
). Here we show that this system effectively expresses a foreign odorant receptor gene that can be conveniently assayed for specific ligand activation by physiological methods. Additionally we have been able to identify a set of ligands that activate a particular receptor. This invention provides conclusive evidence that the putative odorant receptor genes cloned several years ago do indeed encode odorant receptors and, for the first time in a vertebrate, pairs a particular receptor of known amino acid sequence with a specific set of odorant ligands.
List of Definitions
The following definitions are provided for illustrative purposes only and are in no way to be construed as narrowing the scope of the instant invention in any way.
Depolarize—
a change in the cell membrane potential to a more positive voltage.
Action potential—
a rapid transient depolarization of the cell membrane lasting 1-5 milliseconds.
Agonist—
a molecule or substance that can activate a receptor protein or enzyme.
Antagonist—
a molecule that binds to or otherwise interacts with a receptor to inhibit the activation of that receptor or enzyme.
Ligand—
a naturally occurring or synthetic compound that binds to a protein receptor.
Odorant ligand—
a ligand compound that, upon biding to a receptor, leads to the perception of an odor including a synthetic compound and/or recombinantly produced compound including agonist and antagonist molecules.
Odorant receptor—
a receptor protein normally found on the surface of olfactory neurons which, when activated (normally by binding an odorant ligand) leads to the perception of an odor.
Olfactory receptor—
refers to the primary sensory neuron in the nasal epithelium which responds to odors or other ligands.
Receptor—
a membrane bound protein on the surface of cells that is capable of binding one or more ligands.
Functional interaction—
an interaction between a receptor and ligand that results in activation of cellular responses. These may include changes in membrane potential, secretion, action potential generation, activation of enzymatic pathways and long term structural changes in cellular architecture or function.
Differentiated—
refers to the final structural and functional attributes of a particular cell. The most mature state of a cell.
Cloned—
production or generation of a specific genetic sequence encoding a protein.
Recombinant—
a genetic construct in which a clone has been recombined in a novel genetic environment.
Recombinantly produced receptor—
a receptor protein produced by recombining its genetic sequence (clone) with other genes that induce the transcription and generation of the protein.
In vivo—
refers to preparations or methods performed in a living, intact animal or organism, whether conscious or not.
In situ—
refers to methods performed on tissue that remains in its normal place in the animal although the organism may be post mortem
In vitro—
refers to methods performed on tissues or cells that have been dissected free of an animal or are dissociated from the animal or organism. The most common example is cell culture.
SUMMARY OF THE INVENTION
The present invention relates to methods and materials for the expression and assaying of functional neuronal receptors in vivo. More particularly, the present invention relates to methods and materials for the expression and assaying of functional neuronal receptors, such as odorant receptors, in neuronal cells, such as olfactory cells, in animals, such as rats. More particularly still, the present invention relates to the use of recombinant viruses, such as recombinant adenioviruses, containing odorant receptor genes, to infect olfactory cells in an animal and express functional cloned odorant receptors which can then be assayed for specific interaction with

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