Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing
Reexamination Certificate
1999-12-21
2001-09-11
McGarry, Sean (Department: 1636)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
C424S093200, C424S093400, C424S184100, C435S252100, C435S252300, C514S002600, C514S04400A
Reexamination Certificate
active
06287556
ABSTRACT:
INTRODUCTION
1. Field of the Invention
The field of this invention is microbial-based intracellular delivery of agents to eukaryotic cells.
2. Background
The efficient delivery of macromolecules to the cytosol of mammalian cells is of fundamental importance in such processes as the generation of transfected phenotypes and the study of protein function and localization. Furthermore, delivery of macromolecules to the cytosol is also important for the induction of cell-mediated immunity and is a significant challenge facing the rational design of vaccines to intracellular pathogens. Numerous methodologies currently exist for delivering macromolecules to mammalian cells. These include but are not limited to: mechanical techniques such as electroporation (1) and microinjection (2); fuision methodologies such as fusion with vesicles and liposomes (2); chemical treatments employing the use of ATP or EDTA (3) or the external addition of molecules mixed with pore-forming toxins such as &agr;-toxin of
Staphylococcus aureus
(4). Many of these methods have a disadvantage in that the molecule to be delivered may require laborious purification (i.e. protein) or the delivery method is limited to use in vitro. In order to overcome these obstacles, investigators have sought to use biological vectors that can enter tissues, cells and specific cellular compartments for the delivery of macromolecules. These vectors are often derived from either retroviruses or bacteria that have evolved to invade and replicate in specific hosts, organs, or cell types. While retroviral vectors have been used for the delivery and subsequent expression of DNA in host cells (5-7), bacterial vectors have been exploited primarily for the delivery of antigenic proteins and more recently adapted for the delivery of DNA to mammalian cells (8-12).
Relevant Literature
Lee et al. (1997) U.S. Pat. No. 5,643,599 and Lee et al. (1996)
J. Biol. Chem
. 271, 7249-7252 describe hemolysin loaded liposomes for intracellular delivery of macromolecules. Dietrich, G., et al. (1998)
Nature Biotech
. 16, 138-139 and Ikonomidis, G., et al. (1997)
Vaccine
15, 433-440 describe the use of
Listeria monocytogenes
as a macrophage delivery vehicle. Sizemore, D. R., Branstrom, A. A., & Sadoff, J. C. (1995)
Science
270, 299-302 and Courvalin, P., et al. (1995)
C. R. Acad. Sci. III
318, 1207-1212 describe the use of attenuated Shigella and invasive strains of
Shigella flexneri
and
E. coli
, respectively, as a DNA delivery vehicle. Hess, J., et al. (1998)
Proc. Nati. Acad. Sci. U.S.A
. 95, 5299-5304; and Darji, A., et al. (1995)
J. Biotechnol
. 43, 205-212 describe the expression of listeriolysin in several heterologous systems: an invasive
E. coli, Mycobacterium bovis
and
Listeria innocua
, respectively. Moriishi et al. (1996)
FEMS Immunol. Med. Microbiol
. 16, 213-222, 217 describe the transformation of an
E. coli
with a plasmid encoding listeriolysin. Sanderson, S., Campbell, D. J., & Shastri, N. (1995)
J. Exp. Med
. 182, 1751-1757, describe the cloning of a
Listeria monocytogenes
genomic library in
E coli
. Higgins and Portnoy (1998)
Nature Biotech
. 16, 181-185 review bacterial delivery of DNA.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to intracellular delivering of agents to eukaryotic cells. The compositions include microbial delivery vehicles such as nonvirulent bacteria comprising a first gene encoding a nonsecreted foreign cytolysin operably linked to a heterologous promoter and a second gene encoding a different foreign agent. In particular embodiments, the bacteria may be variously invasive to the target cell, autolysing within target cell endosomes and preferably, a laboratory strain of
E. coli
. The cytolysin may lack a functional signal sequence, and is preferably a listeriolysin. The foreign agent may be a nucleic acid or protein, and is frequently bioactive in and therapeutic to the target eukaryote. In addition, the invention provides eukaryotic cells comprising the subject nonvirulent bacteria and nonhuman eukaryotic host organisms comprising such cells. The invention also provides methods for introducing foreign agents into eukaryotic cells comprising the step of contacting the cell in vivo or in vitro with the subject bacteria under conditions whereby the agent enters the cell. In particular embodiments, the bacterium is endocytosed into a vacuole of the cell, undergoes lysis and the cytolysin mediates transfer of the agent from the vacuole to the cytosol of the cell.
REFERENCES:
patent: 4888170 (1989-12-01), Curtiss, III
patent: 5294441 (1994-03-01), Curtiss, III
patent: 5387744 (1995-02-01), Curtis, III et al.
patent: 5424065 (1995-06-01), Curtis, III et al.
patent: 5824538 (1998-10-01), Branstrom et al.
patent: 5855879 (1999-01-01), Curtis, III
patent: 5855880 (1999-01-01), Curtiss, III et al.
patent: 5877159 (1999-03-01), Powell et al.
Branch, “A good antisense molecule is hard to find”, TIBS 23, pabes 45-50, Feb. 1998.*
Anderson, “Human Gene Therapy” Nature, vol. 392, pp. 25-30, Feb. 1998.*
Darji et al., Cell 91:765-775 (1997).
Higgins et al., Molecular Microbiology 31(6):1631-1641 (1999).
Higgins et al., Nature Biotechnology 16(2):138-139 (1998).
Ikonomidis et al., Journal of Experimental Medicine 180(6):2209-2218 (1994).
Dietrich G. et al. “Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated Listeria monocytogenes” Nature Biotechnology vol. 16, Jan. 1998, pp. 181-185.
Higgins Darren E.
Portnoy Daniel A.
Lacourciere Karen A
McGarry Sean
Osman Richard Aron
The Regents of the University of California
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