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Fc gamma riia-specific nucleic acid interference

Fc gamma riia-specific nucleic acid interference description/claims

This application claims the benefit of the filing date of U.S. Provisional Application No. 60/519,899, filed Nov. 14, 2003, entitled “Inhibition of phagocytosis with RNAi specific for cell surface receptors”, and of U.S. Provisional Application No. 60/564,459, filed Apr. 22, 2004, entitled “Short interfacing (si) RNA sequence(s) specific for the SH2 tyrosine kinase, Syk, inhibits phagocytosis by human macrophages”. The teachings of both of the above-mentioned applications are incorporated by reference herein in their entirety.

Immunoglobulins are typically composed of two fundamental domains, the constant domain (Fc) and the variable domain. While the variable domain interacts with target antigens, the constant domain mediates a variety of biological events by interacting with other proteins of the host organism. Receptors for the Fc portion of IgG, Fcγ receptors, play an essential role in the protection of the organism against foreign antigens by removing antigen-antibody complexes from the circulation. Receptors are present on monocytes, macrophages, neutrophils, natural killer (NK) cells, platelets, and T and B lymphocytes, and they participate in diverse functions such as phagocytosis of immune complexes, NK cell ADCC, platelet activation, and modulation of antibody production by B cells.

Fcγ receptors also play a role in a number of diseases characterized by a hyperactive immune system or other undesirable immunological activity. Fcγ receptors participate in a number of autoimmune and inflammatory diseases. As an example, Fcγ receptors are implicated in immune thrombocytopenia. The pathogenic mechanism of immune thrombocytopenia involves antibody-mediated destruction of platelets in the reticuloendothelial system through Fcγ receptors (FcγRs) expressed on tissue macrophages, particularly in the spleen and liver. FcγRs signal via immunoreceptor tyrosine-based activation motifs (ITAMs) that are located either in the cytosolic domains of the receptors themselves (FcγRIIA), or within associated γ (FcγRI and FcγRIIIA) or ξ (FcγRIIIA) subunits. Following clustering of the FcγRs and their associated γ subunits by bound IgG ligands, tyrosine residues within the ITAMs become phosphorylated. The tyrosine-phosphorylated residues of the ITAMs serve as high affinity binding sites for Syk, a tyrosine kinase that contains tandem SH2 domains, which propagates intracellular signaling processes. In humans, FcγRIIA and FcγRIII are the primary activating receptors.

The FcγRIIB receptor has an inhibitory role. FcγRIIB recruits the SHIP kinase and abrogates signaling triggered by activating Fcγ receptors. The overall cellular response depends in part on the ratio of signaling mediated by inhibiting and activating receptors.

An object of the present disclosure is to provide nucleic acid agents that inhibit FcγRIIA expression and to provide methods of using such agents for therapeutic purposes.

The disclosure provides, in part, RNAi constructs that target FcγRIIA and decrease FcγRIIA expression. Such constructs may be used in essentially any method where it is desirable to decrease the level of FcγRIIA protein. In particular, the disclosed nucleic acids will be useful in treating various disorders related to immune system function, such as immune thrombocytopenia, heparin-induced thrombocytopenia and asthma.

In certain aspects, the disclosure provides RNAi constructs for inhibiting the expression of FcγRIIA. Such nucleic acids may comprise (a) an antisense polynucleotide strand that hybridizes to at least a portion of a FcγRIIA transcript and inhibits FcγRIIA expression; and (b) a sense polynucleotide that hybridizes to said antisense polynucleotide. The antisense and sense strands may be two separate nucleic acid strands, or the strands may be joined by a linker or by a stretch of further nucleic acid. For example, the RNAi construct may be a single stranded nucleic acid containing two regions that are complementary, thereby forming a hairpin nucleic acid with a stretch of double-stranded helix. Such hairpin nucleic acids are often processed into an siRNA inside a cell. Optionally, the portion of the nucleic acid that forms a double helix is about 19 to about 23 base pairs in length. The antisense polynucleotide strand may be complementary to a sequence of the human FcγRIIA mRNA of SEQ ID NO: 1, or that of another animal of interest. The RNAi construct may be designed so as to have little or no effect on FcγRIIB expression. For example, the antisense strand may be designed to have no more than 15 consecutive nucleotides that are complementary to an FcγRIIB mRNA sequence, and preferably the antisense strand has no more than 10, no more than 5, or no more than 3 consecutive nucleotides that are complementary to an FcγRIIB mRNA sequence. The antisense polynucleotide strand may be complementary to at least 5, 6, 7, 8, 10, 15, 20 or more nucleotides of a sequence selected from the group consisting of: SEQ ID Nos. 4 through 13. The antisense polynucleotide may consist of a sequence that is complementary to a sequence selected from the group consisting of: SEQ ID Nos. 4-13. The sense and antisense strands may be essentially any suitable nucleic acid, including RNA, DNA or other nucleic acid species that are not readily categorized as DNA or RNA. The sense and antisense strands need not be formed of the same nucleic acids. Either or both strands may include one or more modifications to the typical nucleic acid structure. For example, the antisense strand may comprise one or more of the following modifications: (a) a modification to the sugar-phosphate backbone; (b) a modification to a base portion of a nucleotide; and (c) a conjugated hydrophobic moiety. The sense strand may be similarly modified.

RNAi constructs may be formulated for administration to an organism. A pharmaceutical preparation for delivery of an RNAi construct to an organism may comprise a pharmaceutically acceptable carrier and an RNAi construct that inhibits expression of FcγRIIA. The preparation may be suitable for any desirable mode of administration. In certain instances, such as for the treatment of asthma or other disorders of the airway system, the preparation may be designed for administration by inhalation (e.g., aerosolized or intranasal).

In certain aspects, the disclosure provides methods for decreasing expression of FcγRIIA in a cell. Such methods may comprise contacting the cell with a composition comprising an RNAi construct that inhibits FcγRIIA expression.

In certain aspects, the disclosure provides methods for decreasing expression of FcγRIIA in one or more cells of an individual. Such methods may comprise administering to the individual a composition comprising a double-stranded nucleic acid that inhibits FcγRIIA expression. The individual may be diagnosed with a condition associated with excess Fc receptor activity or with any of a variety of disorders related to the immune system, such as asthma, immune thrombocytopenia or an autoimmune disease.

In an additional aspect, the disclosure provides improved assays for measuring phagocytosis of target material in phagocytic cells. The methods employ phagocytic cells from a cell line, particularly a cell of a hematopoietic lineage, such a monoblastic cell line or other immune cell line. Desirable cells will generally express one or more Fcγ receptors that mediate phagocytosis of opsonized material, such as FcγRIIA. A method may comprise (a) exposing target material to antibody to generate prepared target material; (b) exposing the phagocytic cells to the prepared target material; and (c) selectively detecting the prepared target material that is internalized by the phagocytic cells. The target material will often be cells, particularly platelets. However, phagocytic cells are generally take up materials non-specifically, and therefore other materials, such as microbeads, may be used as a target material. Target material may be labeled in a variety of ways to facilitate detection, and target material may be pre-labeled or labeled upon exposure to antibody. The label may, for example, be a fluorescent label. In a preferred embodiment, the label is a fluorescent label, and selectively detecting the prepared target material comprises selectively quenching any extracellular label. Thus, detection of the fluorescent signal necessarily detects signal from unquenched fluorophore within the phagocytic cells.

FIG. 1: A diagram comparing the sequences of human FcγRIIA and FcγRIIB, showing the location of probes for Northern blots that distinguish between the two transcripts.

FIG. 2: Using probes designed to hybridize with FcγRIIA only, or designed to hybridize with both FcγRIIA and FcγRIIB, siRNA transfection of THP-1 cells was shown to allow the selective knockdown of FcγRIIA.

FIG. 3: FcγRIIA-specific siRNA inhibited antibody mediated platelet phagocytosis.

• Provisional Patent
• Utility Patent

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