Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-04-14
2001-08-07
Yucel, Remy (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024100, C536S024300, C536S024500, C435S320100
Reexamination Certificate
active
06271359
ABSTRACT:
1. INTRODUCTION
The present invention relates to the discovery, identification and characterization of toxic agents which are lethal to pathogens and methods for targeting such toxic agents to a pathogen or pathogen infected cells in order to treat and/or eradicate the infection. In particular, the present invention relates to toxic agents which target bacteria at different stages of the bacterial life cycle, which are delivered alone or in combination to bacteria or bacteria-infected cells. The invention relates to toxic agents which are lethal to diseased cells and methods for targeting such toxic agents to a diseased cell in order to treat and/or eradicate the disease. The present invention relates to promoter elements which are pathogen-specific. The invention relates to promoter elements which are used to achieve pathogen-specific expression of the toxic agent(s) and/or ribozyme(s) of the present invention. The present invention relates to promoter elements which are tissue-specific. The invention relates to promoter elements which are used to achieve tissue-specific expression of the toxic agent(s) and/or ribozyme(s) of the present invention. Specifically, the invention relates to the delivery of one or more toxic gene products, antisense RNAs, or ribozymes, or combination thereof. The invention provides a novel system by which multiple pathogenic targets may be simultaneously targeted to cause the death of a pathogen, or cell infected with a pathogen. Further, the invention has important implications in the eradication of drug-resistant bacterium and bacterial pathogens The invention provides a novel system by which multiple targets may be simultaneously targeted to cause the death of a diseased cell. The invention also has important implications in the eradication of drug-resistant pathogens and drug-resistant diseased cells (such as cancer cells).
2. BACKGROUND
2.1. Antimicrobial Agents
Infectious diseases sicken or kill millions of people each year. Numerous antimicrobial therapies have been designed to target one or several infectious agents. These therapies show varying degrees of success in eradicating infection. However, the failure of many of these therapies to target specific infectious agents has lead to overuse or inappropriate use of the therapies, which in turn has lead to the development of drug resistant microbes. The development of drug resistance in many infectious agents has reduced the efficacy and increased the risk of using the traditional antimicrobial therapies.
Additionally, a majority of the art has focused on antibacterial agents which target proteins or molecules essential for viability of the bacterium. For example, many antibacterial agents act to disrupt the bacterial cell wall, or target an enzyme required in the cell wall synthesis pathway. However, there is need in the art for novel molecules and novel combinations of molecules that can act as lethal agents in bacteria and which may be delivered to a bacterial pathogen, without causing toxicity to the infected host. The present invention provides such novel products which may be used as toxic agents against pathogens such as bacteria.
2.2. Antisense
Antisense technology seeks to use RNA molecules which are complementary to (or antisense to) a cellular RNA, for the purpose of inhibiting a cellular RNA from being translated into the encoded protein. In this way, the expression of a specific protein is targeted for down regulation. However, a large number of difficulties exist in the art surrounding antisense technology. Commonly, delivery of an exogenous antisense molecule to the target cell is difficult or impossible to achieve. Further, antisense molecules do not consistently lead to a decrease in protein expression. For example, it has been shown that the expression of antisense RNA in transgenic mice did not invariably lead to a reduction in target RNA molecules, and when reduction in target RNA molecules did occur, it was not predictably paralleled by a reduction in protein. Even when protein levels were reduced sometimes no biological effect was detected (Whitton, J. Lindsay “Antisense Treatment of Viral Infection”
Adv. in Virus Res.
Vol. 44, 1994). Thus, there is a need in the art for a delivery system in which antisense molecules may be efficiently delivered to a target cell such as a bacterial pathogen.
2.3. Ribozymes
A ribozyme is a catalytic RNA molecule that cleaves RNA in a sequence specific manner. The use of ribozymes as potential gene regulators in mammalian cells and antiviral agents has been suggested, but are subject to serious questions regarding technical feasibility. For example, there are differences regarding how ribozymes can be introduced to target cells. In the case of eukaryotic cells, questions exist as to how ribozymes can be directed to the same subcellular compartments as their target RNAs. Other questions concern the effects of target RNA secondary structure on ribozyme activity. The art has not been successful in definitively answering these questions.
Furthermore, because ribozymes are a form of antisense technology, the problems encountered in applying antisense technology to disease treatment are also encountered in the use of ribozyme technology.
The experience in the art suggests that it is also not clear whether ribozymes work best when associated with only short non-specific flanking sequences, or when embedded in an unrelated larger RNA molecule (Whitton, 1994 supra). At present, sufficient data are not available, either in vitro or in cell culture to allow systematic comparison of the transactivities of free ribozymes with their embedded equivalents.
Another key technical concern in the use of ribozymes as antimicrobial agents is that the ribozyme must be introduced into and expressed by the targeted microbe so that the ribozyme(s) can cleave the targeted RNA(s) inside the microorganism. A second important concern is the tight coupling of transcription and translation in microorganisms which can prevent binding to and cleavage of the bacterial RNA targets. Additionally, bacterial RNAs often have a shorter half life than eukaryotic RNAs, thus lessening the time in which to target a bacterial RNA. The invention described herein addresses these concerns.
3. SUMMARY OF THE INVENTION
The present invention relates to toxic agents and methods for specifically targeting toxic agents to bacteria or bacteria-infected cells or other pathogens. Toxic agents of the present invention are directed to one or more targets and thus can be used alone or in combination to eradicate bacteria. Specifically, the invention relates to the delivery of one or more toxic proteins, antisense RNAs, multi-ribozymes, or combination thereof, to a cell, tissue, or subject containing an infectious bacteria or pathogen in order to eradicate such bacteria or pathogen.
In one embodiment, the invention relates to toxic agents which specifically target gene products essential for the survival or life cycle of a pathogen (such as replication, packaging etc). In one embodiment, the present invention relates to naturally occurring addiction system toxins which have been modified to be expressed in the absence of the addiction system antidote. In another embodiment, the present invention relates to naturally occurring addiction system toxins which have been modified to be expressed at higher levels than the addiction system antidote. In one example is an addiction system toxin (e.g., Doc) is used as a toxic agent and is uncoupled from its antidote. The invention also relates to antisense RNAs which target essential nucleotide sequences, such as DicF1 or a DicF1-like antisense molecule that specifically target a nucleotide sequence which encodes a protein essential for replication or survival. Further, the invention relates to modified antisense structures with increased stability to act as lethal agents when expressed in bacteria. The invention also relates to toxic sense molecules designed to target essential antisense molecules.
The present invention relates to promoter elements which
Clawson Gary
Dolan Joseph
Hoel Brian
Norris James
Pan Wei-Hua
MUSC Foundation for Research Development
Pennie & Edmonds LLP
Schmidt M
Yucel Remy
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