Intracellular delivery of small molecules, proteins, and nucleic acids

Abstract

An amino acid sequence Arg-Lys-Met-Leu-Lys-Ser-Thr-Arg-Arg-Gln-Arg-Arg (SEQ ID NO:1) functions as a protein transduction domain (PTD) and is capable of delivering small molecules, proteins, and nucleic acids to an intracellular compartment of a cell. An amino terminal lysine linker improves the efficiency of the PTD. A nuclear localization signal can be used to target the PTD to a cell's nucleus. The PTD can be used in PTD-cargo moiety complexes that can reversibly immortalize cells and increase cell viability in culture.

1 - 3 . (canceled) 
     
     
         4 . An isolated and purified polynucleotide encoding Arg-Lys-Met-Leu-Lys-Ser-Thr-Arg-Arg-Gln-Arg-Arg (SEQ ID NO:1). 
     
     
         5 . A vector comprising the polynucleotide of  claim 4 . 
     
     
         6 . A host cell comprising the vector of  claim 5 . 
     
     
         7 - 13 . (canceled) 
     
     
         14 . A polynucleotide encoding a fusion protein comprising Arg-Lys-Met-Leu-Lys-Ser-Thr-Arg-Arg-Gln-Arg-Arg (SEQ ID NO:1) linked to a polypeptide cargo moiety. 
     
     
         15 . A vector comprising the polynucleotide of  claim 14 . 
     
     
         16 . A host cell comprising the vector of  claim 15 . 
     
     
         17 . A method of delivering a cargo moiety to an intracellular compartment of a cultured cell comprising the step of:
 contacting a cell in vitro with a complex comprising a polypeptide with a PTD linked to a cargo moiety, wherein the PTD comprises Arg-Lys-Met-Leu-Lys-Ser-Thr-Arg-Arg-Gln-Arg-Arg (SEQ ID NO:1), whereby the cargo moiety is delivered to an intracellular compartment of the cell.   
     
     
         18 . A method of reversibly immortalizing a cell in culture comprising the step of:
 contacting a cell in vitro with a complex comprising a polypeptide with a PTD linked to a cargo moiety, wherein the PTD comprises Arg-Lys-Met-Leu-Lys-Ser-Thr-Arg-Arg-Gln-Arg-Arg (SEQ ID NO:1) and wherein the cargo moiety is an immortalization protein, whereby the cell is reversibly immortalized.   
     
     
         19 . The method of  claim 18  further comprising the step of removing the complex from the cell culture medium to reverse immortalization. 
     
     
         20 . The method of  claim 18  wherein the cell is a primary cell. 
     
     
         21 . The method of  claim 20  wherein the primary cell is selected from the group consisting of adipocytes, astrocytes, cardiac muscle cells, chondrocytes, endothelial cells, epithelial cells, fibroblasts, gangliocytes, glandular cells, glial cells, hematopoietic cells, hepatocytes, keratinocytes, myoblasts, neural cells, osteoblasts, ovary cells, pancreatic beta cells, renal cells, smooth muscle cells, and striated muscle cells. 
     
     
         22 . The method of  claim 18  wherein the immortalization protein is selected from the group consisting of SV40 large T antigen and telomerase. 
     
     
         23 . A cell reversibly immortalized by the method of  claim 18 . 
     
     
         24 . A method of increasing viability of a cell in culture, comprising the step of:
 contacting a cell in vitro with a complex comprising a polypeptide with a PTD linked to a cargo moiety, wherein the cargo moiety is an anti-apoptotic protein.   
     
     
         25 . The method of  claim 24  wherein the anti-apoptotic protein is selected from the group consisting of mutant p53 and Bcl X L. 
     
     
         26 . The method of  claim 24  wherein the cell is a primary cell. 
     
     
         27 . The method of  claim 24  wherein the PTD comprises Arg-Lys-Met-Leu-Lys-Ser-Thr-Arg-Arg-Gln-Arg-Arg (SEQ ID NO:1). 
     
     
         28 . The method of  claim 26  wherein the primary cell is selected from the group consisting of adipocytes, astrocytes, cardiac muscle cells, chondrocytes, endothelial cells, epithelial cells, fibroblasts, gangliocytes, glandular cells, glial cells, hematopoietic cells, hepatocytes, keratinocytes, myoblasts, neural cells, osteoblasts, ovary cells, pancreatic beta cells, renal cells, smooth muscle cells, and striated muscle cells. 
     
     
         29 - 30 . (canceled)