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  Compostions and methods for enhancing delivery of nucleic acids into cells and for modifying expression of target genes in cellsUSPTO Application #: 20060040882Title: Compostions and methods for enhancing delivery of nucleic acids into cells and for modifying expression of target genes in cells Abstract: Polynucleotide delivery-enhancing polypeptides are admixed or complexed with, or conjugated to, nucleic acids for enhancing delivery the nucleic acids into cells. The transported nucleic acids are active in target cells as small inhibitory nucleic acids (siNAs) that modulate expression of target genes, mediated at least in part by RNA interference (RNAi). The siNA/polypeptide compositions and methods of the invention provide effective tools to modulate gene expression and alter phenotype in mammalian cells, including by altering phenotype in a manner that eliminates disease symptoms or alters disease potential in targeted cells or subject individuals to which the siNA/polypeptide compositions are administered. (end of abstract) Agent: Peter J. Knudsen, Esq. Patent Counsel - Bothell, WA, US Inventors: Lishan Chen, Kunyuan Cui, Yuching Chen, Sasha J. Mayer USPTO Applicaton #: 20060040882 - Class: 514044000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), O-glycoside, , Nitrogen Containing Hetero Ring, Polynucleotide (e.g., Rna, Dna, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20060040882. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCES TO RELATED APPLICATIONS [0001] This application claims the priority benefit of U.S. Provisional Patent Application No. 60/568,027, filed May 4, 2004, U.S. Provisional Patent Application No. 60/570,512, filed May 12, 2004, U.S. Provisional Patent Application No. 60/570,513, filed May 12, 2004, U.S. Provisional Patent Application No. 60/613,416, filed Sep. 27, 2004, U.S. Provisional Patent Application No. 60/656,572 filed Feb. 25, 2005, and U.S. Provisional Patent Application No. 60/667,833, filed Apr. 1, 2005, the disclosures of which are incorporated herein by reference in their entirety. TECHNICAL FIELD [0002] The invention relates to methods and compositions for delivering nucleic acids into cells. More specifically, the invention relates to procedures and preparations for delivering double-stranded polynucleotides into cells to modify expression of target genes to alter a phenotype, such as a disease state or potential, of the cells. BACKGROUND OF THE INVENTION [0003] Delivering nucleic acids into animal and plant cells has long been an important object of molecular biology research and development. Recent developments in the areas of gene therapy, antisense therapy and RNA interference (RNAi) therapy have created a need to develop more efficient means for introducing nucleic acids into cells. [0004] A diverse array of plasmids and other nucleic acid "vectors" have been developed for delivering large polynucleotide molecules into cells. Typically these vectors incorporate large DNA molecules comprising intact genes for the purpose of transforming target cells to express a gene of scientific or therapeutic interest. [0005] The process by which exogenous nucleic acids are delivered artificially into cells is generally referred to as transfection. Cells can be transfected to uptake a functional nucleic acid from an exogenous source using a variety of techniques and materials. The most commonly used transfection methods are calcium phosphate transfection, and electroporation. A variety of other methods for tranducing cells to deliver exogenous DNA or RNA molecules have been developed, including viral-mediated transduction, cationic lipid or liposomal delivery, and numerous methods that target mechanical or biochemical membrane disruption/penetration (e.g., using detergents, microinjection, or particle guns). [0006] RNA interference is a process of sequence-specific post transcriptional gene silencing in cells initiated by a double-stranded (ds) polynucleotide, usually a dsRNA, that is homologous in sequence to a portion of a targeted messenger RNA (mRNA). Introduction of a suitable dsRNA into cells leads to destruction of endogenous, cognate mRNAs (i.e., mRNAs that share substantial sequence identity with the introduced dsRNA). The dsRNA molecules are cleaved by an RNase III family nuclease called dicer into short-interfering RNAs (siRNAs), which are 19-23 nucleotides (nt) in length. The siRNAs are then incorporated into a multicomponent nuclease complex known as the RNA-induced silencing complex or "RISC". The RISC identifies mRNA substrates through their homology to the siRNA, and effectuates silencing of gene expression by binding to and destroying the targeted mRNA. [0007] RNA interference is emerging a promising technology for modifying expression of specific genes in plant and animal cells, and is therefore expected to provide useful tools to treat a wide range of diseases and disorders amenable to treatment by modification of endogenous gene expression. [0008] There remains a long-standing need in the art for better tools and methods to deliver siRNAs and other small inhibitory nucleic acids (siNAs) into cells, particularly in view of the fact that existing techniques for delivering nucleic acids to cells are limited by poor efficiency and/or high toxicity of the delivery reagents. Related needs exist for improved methods and formulations to deliver siNAs in an effective amount, in an active and enduring state, and using non-toxic delivery vehicles, to selected cells, tissues, or compartments to mediate regulation of gene expression in a manner that will alter a phenotype or disease state of the targeted cells. BRIEF DESCRIPTION OF THE DRAWINGS [0009] FIG. 1 illustrates peptide-mediated uptake of siNAs complexed or conjugated to a polynucleotide delivery-enhancing polypeptide of the invention. [0010] FIG. 2 further illustrates peptide-mediated uptake of siNAs complexed or conjugated to a polynucleotide delivery-enhancing polypeptide of the invention. [0011] FIG. 3 shows paw data for siRNA/peptide injected mice which demonstrate delayed RA progression in the treated mice comparable to that exhibited by Ramicade-treated subjects. [0012] FIG. 4 provides results of uptake efficacy and viability studies in mouse fibroblasts for PN73 rationally-designed derivative polynucleotide delivery-enhancing polypeptides of the invention. DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION [0013] The present invention satisfies these needs and fulfills additional objects and advantages by providing novel compositions and methods that employ a short interfering nucleic acid (siNA), or a precursor thereof, in combination with a polynucleotide delivery-enhancing polypeptide. The polynucleotide delivery-enhancing polypeptide is a natural or artificial polypeptide selected for its ability to enhance intracellular delivery or uptake of polynucleotides, including siNAs and their precursors. [0014] Within the novel compositions of the invention, the siNA may be admixed or complexed with, or conjugated to, the polynucleotide delivery-enhancing polypeptide to form a composition that enhances intracellular delivery of the siNA as compared to delivery resulting from contacting the target cells with a naked siNA (i.e., siNA without the delivery-enhancing polypeptide present). [0015] In certain embodiments of the invention, the polynucleotide delivery-enhancing polypeptide is a histone protein, or a polypeptide or peptide fragment, derivative, analog, or conjugate thereof. Within these embodiments, the siNA is admixed, complexed or conjugated with one or more full length histone proteins or polypeptides corresponding at least in part to a partial sequence of a histone protein, for example of one or more of the following histones: histone H1, histone H2A, histone H2B, histone H3 or histone H4, or one or more polypeptide fragments or derivatives thereof comprising at least a partial sequence of a histone protein, typically at least 5-10 or 10-20 contiguous residues of a native histone protein. In more detailed embodiments, the siRNA/histone mixture, complex or conjugate is substantially free of amphipathic compounds. In other detailed embodiments, the siNA that is admixed, complexed, or conjugated with the histone protein or polypeptide will comprise a double-stranded double-stranded RNA, for example a double-stranded RNA that has 30 or fewer nucleotides, and is a short interfering RNA (siRNA). In exemplary embodiments, the histone polynucleotide delivery-enhancing polypeptide comprises a fragment of histone H2B, as exemplified by the polynucleotide delivery-enhancing polypeptide designated PN73 described herein below. In yet additional detailed embodiments, the polynucleotide delivery-enhancing polypeptide may be pegylated to improve stability and/or efficacy, particularly in the context of in vivo administration. [0016] Within additional embodiments of the invention, the polynucleotide delivery-enhancing polypeptide is selected or rationally designed to comprise an amphipathic amino acid sequence. For example, useful polynucleotide delivery-enhancing polypeptides may be selected which comprise a plurality of non-polar or hydrophobic amino acid residues that form a hydrophobic sequence domain or motif, linked to a plurality of charged amino acid residues that form a charged sequence domain or motif, yielding an amphipathic peptide. [0017] In other embodiments, the polynucleotide delivery-enhancing polypeptide is selected to comprise a protein transduction domain or motif, and a fusogenic peptide domain or motif. A protein transduction domain is a peptide sequence that is able to insert into and preferably transit through the membrane of cells. A fusogenic peptide is a peptide that is able destabilize a lipid membrane, for example a plasma membrane or membrane surrounding an endosome, which may be enhanced at low pH. Exemplary fusogenic domains or motifs are found in a broad diversity of viral fusion proteins and in other proteins, for example fibroblast growth factor 4 (FGF4). [0018] To rationally design polynucleotide delivery-enhancing polypeptides of the invention, a protein transduction domain is employed as a motif that will facilitate entry of the nucleic acid into a cell through the plasma membrane. In certain embodiments, the transported nucleic acid will be encapsulated in an endosome. The interior of endosomes has a low pH resulting in the fusogenic peptide motif destabilizing the membrane of the endosome. The destabilization and breakdown of the endosome membrane allows for the release of the siNA into the cytoplasm where the siNA can associate with a RISC complex and be directed to its target mRNA. [0019] Examples of protein transduction domains for optional incorporation into polynucleotide delivery-enhancing polypeptides of the invention include: 1. TAT protein transduction domain (PTD) (SEQ ID NO: 1) KRRQRRR; 2. Penetratin PTD (SEQ ID NO: 2) RQIKIWFQNRRMKWKK; 3. VP22 PTD (SEQ ID NO: 3) DAATATRGRSAASRPTERPRAPARSASRPRRPVD; 4. Kaposi FGF signal sequences (SEQ ID NO: 4) AAVALLPAVLLALLAP, and SEQ ID NO: 5) AAVLLPVLLPVLLAAP; 5. Human .beta.3 integrin signal sequence (SEQ ID NO: 6) VTVLALGALAGVGVG; 6. gp41 fusion sequence (SEQ ID NO: 7) GALFLGWLGAAGSTMGA; 7. Caiman crocodylus Ig(v) light chain (SEQ ID NO: 8) MGLGLHLLVLAAALQGA; 8. hCT-derived peptide (SEQ ID NO: 9) LGTYTQDFNKFHTFPQTAIGVGAP; 9. Transportan (SEQ ID NO: 10) GWTLNSAGYLLKINLKALAALAKKIL; 10. Loligomer (SEQ ID NO: 11) TPPKKKRKVEDPKKKK; 11. Arginine peptide (SEQ ID NO: 12) RRRRRRR; and 12. Amphiphilic model peptide (SEQ ID NO: 13) KLALKLALKALKAALKLA. Continue reading... Full patent description for Compostions and methods for enhancing delivery of nucleic acids into cells and for modifying expression of target genes in cells Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Compostions and methods for enhancing delivery of nucleic acids into cells and for modifying expression of target genes in cells patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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