Compositions and methods for the Delivery of Biologically Active RNAs

Abstract

The present invention provides novel compounds, compositions, and methods for the delivery of biologically active RNA molecules to cells. Specifically, the invention provides novel nucleic acid molecules, polypeptides, and RNA-protein complexes useful for the delivery of biologically active RNAs to cells and polynucleotides encoding the same. The invention also provides vectors for expressing said polynucleotides. In addition, the invention provides cells and compositions comprising the novel compounds and vectors, which can be used as transfection reagents. The invention further provides methods for producing said compounds, vectors, cells, and compositions. Additionally, vectors and methods for delivering biologically active RNA molecules to cells and/or tissues are provided. The novel compounds, vectors, cells, and compositions are useful, for example, in delivering biologically active RNA molecules to cells to modulate target gene expression in the diagnosis, prevention, amelioration, and/or treatment of diseases, disorders, or conditions in a subject or organism.

1 - 36 . (canceled) 
     
     
         37 . A method for secreting a biologically active RNA from a cell, comprising:
 administering to a cell an expression vector comprising
 a first polynucleotide that encodes a biologically active RNA sequence and a recognition RNA sequence, and 
 a second polynucleotide that encodes a polypeptide comprising an RNA binding domain that binds the recognition RNA sequence and a non-classical secretory domain sequence, 
   expressing the first and second polynucleotides,   secreting the biologically active RNA sequence through a non-classical secretory pathway.   
     
     
         38 . The method of  claim 37 , wherein the cell is a bacterial cell, a protozoan cell, an insect cell, a yeast cell, a plant cell, a fungal cell or a mammalian cell. 
     
     
         39 . The method of  claim 37 , wherein biologically active RNA sequence is selected from the group consisting of an aptamer, a ribozyme, an antisense nucleic acid, a short interfering RNA (siRNA), a double stranded RNA (dsRNA), a short hairpin RNA (shRNA), a micro-RNA (miRNA), and a transcript encoding one or more biologically active peptides. 
     
     
         40 . The method of  claim 37 , wherein the non-classical secretory domain sequence nucleotide is selected from the group consisting of Galectin-1, Galectin-3, Interleukin 1α (IL-1α), Interleukin 1β (IL-1β), Hydrophilic Acylated Surface Protein B (HASPB), High Mobility Group Box Protein 1 (HMGB1), Fibroblast Growth Factor 1 (FGF-1), Fibroblast Growth Factor 2 (FGF-2), Interleukin 2 (IL-2), Secretory Transglutaminase, Annexin-1, Thioredoxin, Rhodanese, and Plasminogen Activator. 
     
     
         41 . The method of  claim 37 , wherein the polypeptide encoded by the second polynucleotide further comprises a cell penetrating peptide. 
     
     
         42 . The method of  claim 41 , wherein the cell penetrating peptide sequence is selected from the group consisting of penetratin, transportin, model amphiphatic peptide (MAP), trans-activator of transcription (TAT), homeodomain of antennepedia (AntP), feline herpes virus (FHV) coat protein, and transportin 10 (TP10). 
     
     
         43 . A method for modulating a signaling pathway or the expression of a target gene of a target cell comprising:
 administering to a host cell a vector comprising
 a first polynucleotide that encodes a biologically active RNA sequence and a recognition RNA sequence, and 
 a second polynucleotide that encodes a polypeptide an RNA binding domain that binds the recognition RNA sequence, a non-classical secretory domain sequence, and a transport peptide sequence that facilitates delivery of the biologically active RNA sequence to neighboring cells and tissues, 
   expressing the vector in the host cell,   secreting the biologically active RNA from the host cell,   delivering the biologically active RNA sequence to a neighboring cell, wherein the biologically active RNA modulates a signaling pathway or the expression of a target gene of the target cell.   
     
     
         44 . The method of  claim 43 , wherein the transport peptide sequence is a cell penetrating peptide. 
     
     
         45 . The method of  claim 44 , wherein the cell penetrating peptide sequence is selected from the group consisting of penetratin, transportin, model amphiphatic peptide (MAP), trans-activator of transcription (TAT), homeodomain of antennepedia (AntP), feline herpes virus (FHV) coat protein, and transportin 10 (TP10). 
     
     
         46 . The method of  claim 43  wherein the host cell and target cell are in a subject having, suspected of having, or at risk of developing arthritis, macular degeneration, muscular dystrophy, Huntington's disease, coronary heart disease, or a cancerous disease or condition. 
     
     
         47 . The method of  claim 43 , wherein biologically active RNA sequence is selected from the group consisting of an aptamer, a ribozyme, an antisense nucleic acid, a short interfering RNA (siRNA), a double stranded RNA (dsRNA), a short hairpin RNA (shRNA), a micro-RNA (miRNA), and a transcript encoding one or more biologically active peptides. 
     
     
         48 . The method of  claim 43 , wherein the non-classical secretory domain sequence nucleotide is selected from the group consisting of Galectin-1, Galectin-3, Interleukin 1α (IL-1α), Interleukin 1β (IL-1β), Hydrophilic Acylated Surface Protein B (HASPB), High Mobility Group Box Protein 1 (HMGB1), Fibroblast Growth Factor 1 (FGF-1), Fibroblast Growth Factor 2 (FGF-2), Interleukin 2 (IL-2), Secretory Transglutaminase, Annexin-1, Thioredoxin, Rhodanese, and Plasminogen Activator. 
     
     
         49 . The method of  claim 37 , wherein the host cell and target cell are in a subject having, suspected of having, or at risk of developing arthritis and the biologically active RNA sequence is an Oncostatin M (OSM) inhibitory aptamer. 
     
     
         50 . The method of  claim 37 , wherein the host cell and target cell are in a subject having, suspected of having, or at risk of developing macular degeneration and the biologically active RNA sequence is a Vascular Endothelial Growth Factor (VEGF) inhibitory aptamer. 
     
     
         51 . The method of  claim 37 , wherein the host cell and target cell are in a subject having, suspected of having, or at risk of developing muscular dystrophy and the biologically active RNA sequence is a Myostatin inhibitory aptamer. 
     
     
         52 . The method of  claim 37 , wherein the host cell and target cell are in a subject having, suspected of having, or at risk of developing coronary heart disease and the biologically active RNA sequence is a von Willebrand factor (vWF) inhibitory aptamer. 
     
     
         53 . The method of  claim 37 , wherein the host cell and target cell are in a subject having, suspected of having, or at risk of developing a cancerous disease or condition and the biologically active RNA sequence is selected from the group consisting of a Human Epidermal Growth Factor Receptor 3 (HER3) inhibitory aptamer and a β-catenin inhibitory aptamer. 
     
     
         54 . The method of  claim 37 , wherein the host cell and target cell are in a subject having, suspected of having, or at risk of developing arthritis and the biologically active RNA sequence is an shRNA targeting the Matrix Metalloproteinase-2 (Mmp2) transcript. 
     
     
         55 . The method of  claim 37 , wherein the host cell and target cell are in a subject having, suspected of having, or at risk of developing macular degeneration and the biologically active RNA sequence is an shRNA targeting the Vascular Endothelial Growth Factor Receptor (VEGFR) transcript. 
     
     
         56 . The method of  claim 37 , wherein the host cell and target cell are in a subject having, suspected of having, or at risk of developing muscular dystrophy and the biologically active RNA sequence is an shRNA targeting the myostatin transcript. 
     
     
         57 . The method of  claim 37 , wherein the host cell and target cell are in a subject having, suspected of having, or at risk of developing Huntington's disease and the biologically active RNA sequence is an shRNA targeting the huntingtin (HTT) transcript. 
     
     
         58 . The method of  claim 37 , wherein the host cell and target cell are in a subject having, suspected of having, or at risk of developing coronary heart disease and the biologically active RNA sequence is an shRNA targeting the apolipoprotein B (ApoB) transcript. 
     
     
         59 . The method of  claim 37 , wherein the host cell and target cell are in a subject having, suspected of having, or at risk of developing a cancerous disease or condition and the biologically active RNA sequence is selected from the group consisting of an shRNA targeting the Epidermal Growth Factor Receptor (EGFR) transcript, an shRNA targeting the Harvey Rat Sarcoma viral oncogene homolog (H-Ras) transcript, an shRNA targeting the B-cell lymphoma-2 (Bcl-2) transcript, an shRNA targeting the surviving transcript, an shRNA targeting the Focal Adhesion Kinase (FAK) transcript, an shRNA targeting the Signal Transducer and Activator of Transcription-3 (STAT-3) transcript, an shRNA targeting the Src transcript, an shRNA targeting the RET receptor tyrosine kinase transcript, an shRNA targeting the Kinesin Spindle Protein (KSP) transcript, and an shRNA targeting the Multiple Drug Resistance (MDR) transcript. 
     
     
         60 . The method of  claim 37 , wherein the vector further comprises a first promoter sequence and a termination sequence, and a second promoter sequence and a polyA addition sequence, wherein the first polynucleotide is operably linked to the first promoter sequence and the termination sequence and wherein the second polynucleotide is operably linked to the second promoter sequence and the polyA addition sequence 
     
     
         61 . The method of  claim 43 , wherein the vector further comprises a first promoter sequence and a termination sequence, and a second promoter sequence and a polyA addition sequence, wherein the first polynucleotide is operably linked to the first promoter sequence and the termination sequence and wherein the second polynucleotide is operably linked to the second promoter sequence and the polyA addition sequence.