Peptide-mediated gene transfer

Abstract

A methodology that allows for highly efficient transfer and stable integration of DNA into both established eukaryotic cell lines and primary cells, including non-dividing cells such as human peripheral blood monocytes and macrophages, entails the use of a synthetic polypeptide comprised of a peptide domain which corresponds to a nuclear localization signal sequence and a DNA binding domain which is rich in basic amino acids, separated by a hinge region of neutral acid which prevents stearic interference between the two domains.A synthetic polypeptide that allows for highly efficient transfer of DNA into eukaryotic cells, including, for example, non-dividing cells such as human peripheral blood monocytes and macrophages.<?insert-end id="INS-S-00001" ?>

Description

RELATED APPLICATION[0001]This application was filed as a reissue of Ser. No. 08 / 240,514, filed May 14, 1994, now U.S. Pat. No. 5,670,347.<?insert-end id="INS-S-00002" ?>BACKGROUND OF THE INVENTION[0002]The present invention is directed to a methodology for highly efficient, stable integration of DNA into a eukaryotic genome. More specifically, the present invention is directed to the use of a synthetic polypeptide, containing a nuclear localization signal to complex with a DNA molecule and to facilitate its transportation and integration into the nuclear genome of a mammalian or other eukaryotic cell, for example, in the context of producing cell lines with an extended life.[0003]DNA-CaPO4 co-precipitation was the first method developed to introduce genes into mammalian cells. (“Gene” in this regard denotes a structural DNA segment, i.e., a DNA that codes for a polypeptide, and comprehends oncogenes as well as DNAs coding for a known expression product.) The co-precipitation m...

AI Technical Summary

Benefits of technology

"The present invention provides a method for efficient gene transfer in a wide range of cell types, including primary cells from various mammalian species. The invention also provides cell lines that have extended life in culture. Additionally, the invention provides a screening system for identifying sequences that influence the expression of cloned genes in various primary cell types from different species. The invention involves a transfection vector comprising a synthetic polypeptide linked electrostatically to a DNA structural sequence, which is designed to efficiently transfer genes into cells. The vector contains a polymeric chain of basic amino acid residues, an NLS peptide, and a hinge region of neutral amino acids that connects the polymeric chain and the NLS peptide. The NLS peptide is located at the amino terminus of the polypeptide and the polymeric basic amino acid chain at the carboxyl terminus. The invention also provides an extended life cell line that is the product of transfecting a mammalian cell with a vector described above. The mammalian cell can be selected from a variety of cell types, such as human and rabbit cells."

Problems solved by technology

The co-precipitation method was applicable only to certain cell types, however, and could not be used to introduce genes into a wide variety of cell lines, especially those of hematopoietic origin.

In addition to giving poor results with respect to stable gene expression, transfer methods based on such DNA-complexing agents often were effective only with established cell lines, and did not work very well with primary cells isolated from various mammalian species.

Even though these vectors were effective with respect to various mammalian cells, there were many restrictions on a wider application of the retroviral gene-transfer technique.

All these involve very time-consuming experimental manipulations.

Additional safety features have been introduced into some of the newer versions of retroviral vectors, yet are not available for all types of viral vectors.