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Delivery of functional protein sequences by translocating polypeptides

Inactive Publication Date: 2005-09-22
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013] In still another embodiment, the present invention provides method(s) for modulating expression of a target gene product in a cell in culture that is transfected with the target gene under control of one or more regulatory elements by contacting the cell under suitable conditions with one or more regulatory agents attached to a translocating polypeptide, whereby the one or more regulatory agents are translocated into the cell and interact therein with the one or more regulatory elements, thereby modulating expression of the target gene product by the cell.

Problems solved by technology

A variety of transfection methods (e.g. lipids, calcium phosphate) exist in the marketplace; however, these methods rarely result in more than 50% of cells expressing a gene carried on a plasmid with which the cells are transfected.
Since most cells do take up exogenous DNA, inefficient transfections do not appear to be due to inability of the DNA complex to enter the cell.
Indeed, observations of directly injected lipid-DNA complexes suggest that movement from the endosomes to the cytoplasm and nucleus is the most important limitation to successful transfections (J. H. Richardson et al., Proc. Natl. Acad. Sci.

Method used

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  • Delivery of functional protein sequences by translocating polypeptides
  • Delivery of functional protein sequences by translocating polypeptides
  • Delivery of functional protein sequences by translocating polypeptides

Examples

Experimental program
Comparison scheme
Effect test

example 1

Introduction of VP22 Fusion Protein into Cells in Culture by Transfection

[0085] The complete open reading frame (ORF) encoding the VP22 protein was cloned into the eukaryotic expression vector pcDNA3.1 / myc-His (Invitrogen, San Diego, Calif.), to create the vector pVP22 / Myc-His FIG. 5; SEQ ID NO:1), in which the ORF of the fusion partner can be inserted into a multiple cloning site located between the VP22 ORF and sequences encoding the C-terminal Anti-myc epitope and a poly His tag. The anti-myc epitope provides for easy detection of recombinant protein with Anti-myc antibody, and the poly His tag is useful for purification. Alternatively, the vector used was modified by covalent coupling of the Vaccinia Virus Topoisomerase I protein to linearized vector DNA (e.g., pVP22 TOPO® TA Cloning® Kit (Invitrogen)). In this type of vector, the ORF of a gene product of interest (i.e., a “fusion partner”) was cloned as a PCR product into the vector. An example of such a Topoisomerase-adapted...

example 2

Transfection of Cells with a Gene Fusion Followed by Mixing with Untransfected Cells

[0089] To demonstrate how VP22 may be used to modulate expression of a functional gene product, a system for delivery of the site specific DNA recombinase Flp, was developed. COS cells expressing a VP22-Flp recombinase fusion protein were prepared as described above and mixed with CHO cells that had been transfected with a reporter plasmid pFIN4 / lacZ (FIG. 1). In the reporter plasmid, a segment of DNA that includes a transcriptional terminator, the Bovine Growth Hormone polyadenylation signal (Goodwin and Rottman, J. Biol. Chem. 267:16330-4, 1992), is flanked by frt sites (recombination sites recognized by the recombinase Flp) to separate the CMV promoter and an otherwise operatively associated reporter gene encoding β-galactosidase as reporter. Cells transfected with pFIN4 / lacZ did not express β-galactosidase due to the presence of the transcriptional terminator placed between the frt sites.

[0090...

example 3

Transfection of Cells with a Gene Fusion Followed by Preparation of a Cell Free Lysate from the Transfected Cells

[0093] In these studies, a cell free lysate was prepared from cells transfected with pVP22 / myc-His as follows: COS cells were grown to 50% confluence in a 100 mm dish (approximately 106 cells). Cells were transfected with 20 μg of pVP22 / myc-His DNA using Pfx-6. Forty hours post-transfection, the cell monolayer was washed twice with PBS and then collected by scraping into 10 ml PBS. Cells were centrifuged at 500 g for 5 min and the PBS was aspirated from the cell pellet, which was then frozen on dry ice. Frozen cell pellets were stored at −80° C. prior to preparation of lysates. The cell pellet was thawed on ice following addition of 0.5 ml ice cold lysis buffer (10 mM HEPES, pH 7.9, 400 mM NaCl, 0.1 mM ethylene diaminetetraacetic acid (EDTA), 0.5 mM dithiothreitol (DTT), 5% glycerol). The lysate was then vortexed briefly and centrifuged at 10000×g for 5 min at 4° C.

[00...

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Abstract

The invention provides methods for modulating a cellular process by contacting a cell in culture with a cell process-modifying molecule attached to a translocating polypeptide. For example, in one embodiment, a cell in culture is transfected with a target gene by contacting the cell in culture with a polynucleotide (that contains the target gene) attached to a translocating polypeptide. In another embodiment, expression of a target gene product in a cell in culture that contains a target gene under control of one or more regulatory elements is modulated by contacting the cell in culture with one or more regulatory agents attached to a translocating polypeptide. The one or more regulatory agents are translocated into the cell in culture and interact therein with the one or more regulatory elements to modulate expression of the target gene product by the cell.

Description

FIELD OF INVENTION [0001] The present invention relates to methods for translocating polynucleotides and polypeptides between cells. More particularly, the present invention relates to use of translocating proteins to deliver a cell process-modifying molecule into the cell where the cell process-modifying molecule interacts specifically with a responsive target site. BACKGROUND OF THE INVENTION [0002] Translocating proteins are defined by their ability to cross biological membranes, such as cell membranes. A number of translocating proteins, have been described, including VP22 from Herpes Simplex Virus type 1 (G. Elliot and P. O'Hare, Cell 88, 223-233 (1997)), a fragment of the Antennapedia protein from Drosophila (Antp) (D. Derossi et al., Journal of Biological Chemistry 269, 10444-10450 (1994)), and Protein H from Streptococcus pyogenes (Axcrona et al., Manuscript in preparation (1999)). [0003] Antennapedia is a homeoprotein with a DNA binding domain composed of three alpha helice...

Claims

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Application Information

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IPC IPC(8): A61K48/00C07K14/035C12N15/85C12N15/87
CPCA61K48/0025C07K14/005C07K2319/00C12N15/85C12N2840/20C12N2710/16622C12N2800/108C12N2800/30C12N15/87
Inventor DALBY, BRIANBENNETT, ROBERT
Owner LIFE TECH CORP
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