Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-07-28
2003-05-06
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S091100, C435S091310, C435S440000, C435S252100, C536S023100, C536S024500
Reexamination Certificate
active
06558954
ABSTRACT:
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, “Genetics”, 2nd ed. (Holt Rinehart Winston, New York, 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 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 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 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 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 (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 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 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.
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 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 drug-sensitive 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 drug-sensitive 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 drug-resistant 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 drug-sensitive 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.
As demonstrated by the examples, in a preferred embodiment the nucleic acid is an oligonucleotide which binds to the porphyrin in a stoichiometric ratio, and greatly enhances uptake by cells. The examples demonstrate delivery to a variety of bacteria. The disclosed method and compositions have utility in delivery of nucleic acids to bacterial cells, both in culture and in natural environments such as in plants and animals (including humans). Delivery of nucleic acids to bacteria has many known utilities, including genetic manipulation of bacteria and for diagnostic purposes, and the disclosed method can be used to effect any of these known purposes. Delivery of nucleic acids to bacteria infecting animals and humans can be used for therapeutic effect, for example, by altering, weakening, or killing the bacteria to which the nucleic acid is delivered.
Ribozymes can be used to cleave genetic elements which are required for cell viability or which confer drug resistance on eukaryotic pathogens. In a preferred embodiment, the ribozyme is RNAase P, which is directed to cleave the genetic element by external guide sequences (“EGS”) targeted to the element. EGSs (Forster and Altman,
Science
249:783-786 (1990); Yuan et al.,
Proc. Natl. Acad. Sci. USA
89:8006-8010 (1992)) directing RNAase P to cleave are oligonucleotides that, in combination with an RNA molecule to be cleaved, form a structure recognized by RNAse P as a substrate. Preferred prokaryotic EGSs have nucleotides complement
George Shaji T.
Goldberg Allan R.
Takle Garry B.
Holland & Knight LLP
McGarry Sean
Yale University
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