PHENOTYPIC CONVERSION OF CELLS
MEDIATED BY EXTERNAL GUIDE
CROSS-REFERENCE TO RELATED 5
This application is a continuation-in-part of International Application PCT/US97/04000, filed Mar. 14, 1997. This application claims benefit of U.S. Provisional Application No. 60/023,675, filed Aug. 16, 1996. 10
BACKGROUND OF THE INVENTION
The present invention is generally in the fields of delivery of nucleic acids to bacteria and of using external guide sequences to convert the phenotype of pathogens.
Many techniques have been developed for getting nucleic acids into bacterial cells. Early techniques made use of naturally occurring systems of genetic transfer, including bacteriophage and bacterial conjugation (Goodenough, 20 "Genetics", 2nd ed. (Holt Rinehart Winston, N.Y., 1978), pages 397-436). Bacteriophage can transfer not only viral nucleic acid, but occasionally also transfer bacterial nucleic acid accidently encapsidated in the phage (a process referred to as transduction). In bacterial conjugation, Hfr (high 25 frequency of recombination) cells contained genes that mediated an orderly, linear transfer of a bacterial chromosome or other genetic element from one bacterial cell to another. Both systems were generally limited to the transfer of nucleic acid already present in a bacterial cell. With the 30 advent of recombinant DNA technology, both the need and means for getting nucleic acids into bacterial cells increased. In vitro manipulation and adaptation of natural episomal plasmids and bacteriophage allowed the formation of novel nucleic acids and the cloning of genes. However, effective 35 use of recombinant technology was limited to those cells for which methods of introduction of nucleic acids had been developed.
Although some cells will spontaneously take up some nucleic acids, efficient uptake required the development of 40 methods to increase the permeability and/or for physical manipulation of cells. Examples of these techniques include transformation (via increased permeability), transfection (transformation of viral vectors), electroporation (electrically stimulated uptake), and microinjection 45 (physical injection of nucleic acids). For example, efficient uptake of nucleic acids by the popular laboratory bacterium Escherichia coli requires salt treatments to increase the permeability of the cell wall and membrane followed by a heat shock to cause the cell to actually internalize adhered 50 nucleic acid. Electroporation is commonly used for eukaryotic cells and uses an electric shock to cause internalization of nucleic acids. For cells with a cell wall (such as yeast, most plant cells, and many bacteria), uptake of nucleic acids generally requires that the cell wall be enzymatically 55 digested. Nearly all methods of getting nucleic acids into bacterial cells requires treatment or manipulation of the cells. Such manipulations are difficult or impossible to use on bacterial cells in their natural environment, especially for those bacteria colonizing or infected plants and animals. go
Drug resistance in pathogens are a problem of clinical importance. The use, and misuse, of antibiotics and other drugs meant to control the growth of pathogens has led to an increasing number of organisms having resistance to the effects of the drugs. The standard approach to this problem 65 has consisted of attempts to discover new drugs to which the organisms are sensitive, an expensive and time-consuming
process. To further complicate matters, organisms continue to mutate to acquire resistance to newly developed drugs.
It is therefore an object of the present invention to provide a method and compositions for delivering nucleic acids to bacterial cells.
It is also an object of the present invention to provide a method for converting drug-resistant bacterial cells to drugsensitive bacterial cells.
It is also an object of the present invention to provide a means for killing or reducing the viability of eukaryotic pathogens.
It is also an object of the present invention to provide a means for converting drug-resistant eukaryotic cells to drugsensitive eukaryotic cells.
SUMMARY OF THE INVENTION
Disclosed are a method and compositions for delivering nucleic acids to bacterial cells. The method does not require manipulation of the bacteria and is therefore particularly suited to delivery of nucleic acids to bacteria in natural environments, including to bacteria inside animals bodies. The method generally involves conjugating the nucleic acid to be delivered with a cationic porphyrin and bringing the conjugate and the target bacterial cells into contact. Both the porphyrin and conjugated nucleic acid are taken up by the bacterial cells and the nucleic acid can then have a biological effect on the cells. Preferred nucleic acids for delivery using the disclosed method include external guide sequences, ribozymes, plasmids and other vectors, and antisense nucleic acids.
Specifically disclosed is a method for converting drugresistant bacterial cells to drug-sensitive cells by delivery of external guide sequences to the cells which then promote cleavage of RNA molecules involved in conferring the drug-resistant phenotype on the cells. The drug-resistant phenotype of the cells is thus converted to a drug-sensitive phenotype. The drug-sensitive cells are then susceptible to drug therapy. Also disclosed is a method and compositions for killing or reducing the viability of eukaryotic pathogens, or converting drug-resistant eukaryotic cells to drugsensitive cells. The method involves the delivery of external guide sequences, ribozymes, or vectors encoding external guide sequences or ribozymes, to the eukaryotic cells. Preferred target eukaryotic cells for the disclosed method include algae, protozoa, fungi, slime mold, and cells of helminths.
The use, and misuse, of antibiotics and other drugs meant to control the growth of pathogens has led to an increasing number of organisms having resistance to the effects of the drugs. Such resistance often results from the acquisition of a gene or genes conferring resistance on the organism, or an increase in the prevalence of certain alleles, which confer resistance on the organism, through selective pressure. The disclosed method and compositions can be used to attack such genetically based drug resistance.
Porphyrins or phthalocyanins (referred to jointly herein as "porphyrins" unless otherwise stated) or other macrocyclic compounds are useful in the disclosed method and compositions. The system is extremely simple, since the two principle components are a porphyrin having a net overall positive charge, as defined in more detail below, and the nucleic acid to be delivered, which has a net overall negative charge. The porphyrin binds the compound to be delivered and enhances uptake of the nucleic acid. The porphyrin also enhances the stability of the complexed nucleic acid against nuclease disgestion.