Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-07-27
2004-02-17
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S325000, C435S375000, C435S091100, C536S023100, C536S024500
Reexamination Certificate
active
06692910
ABSTRACT:
BACKGROUND OF THE INVENTION
Gene expression can be regulated at the post transcriptional level. For example, antisense RNA strategies have received great attention as a means for gene regulation. Antisense constructs, either in the form of oligonucleotides or as expressed antisense ribonucleic acids (RNAs), form colinear hybrids with target messenger ribonucleic acid (mRNA). In forming a double-stranded region on the mRNA, subsequent steps of protein synthesis may be interrupted by any of a variety of mechanisms. Interruption may occur by sterically blocking ribosome assembly or progression, sterically blocking intron/exon junctions and splice-sites needed for the processing of premature mRNA, or by invoking the cellular enzyme RNAse H that specifically cleaves mRNA in mRNA/DNA hybrids. However, the overall efficiency of antisense inhibition is quite variable and requires empirically testing a variety of constructs to obtain satisfactory results. Furthermore, it is often difficult to show an effect in stably transfected cells.
Ribozymes, RNA molecules that possess self-catalytic activity, have the theoretical advantage over antisense RNA strategies as a means for gene regulation because they are able to bind and cleave a target mRNA, dissociate, then find, bind and cleave another target mRNA. The specific target cleavage sites have a sequence requirement (Symons,
Ann. Rev. Biochem.,
61:641-671 (1992)). However, although ribozymes work well in vitro, their effectiveness in vivo has been limited.
Reduction of mRNA output from a gene has increasing importance in the development of strategies for somatic gene therapy as well as a molecular tool to study the function of a gene. Thus, there is a continued need to develop new and improved methods for gene regulation at the transcriptional level.
SUMMARY OF THE INVENTION
The present invention relates to the use of a mutant or modified U1 small nuclear ribonucleic acid (U1 snRNA) to regulate, reduce or inhibit gene expression in cells. Gene expression in a cell in accordance with the present invention is regulated, reduced or inhibited by introducing into the cell a mutant U1 snRNA which binds or hybridizes to a preselected site having a particular RNA sequence within a mRNA produced by a gene located in the cell. Upon binding to or hybridization with the preselected site, the mRNA becomes sequestered within the nucleus and as a result, the protein coded for by the mRNA is not produced.
Thus, in one embodiment, the invention is directed to a method of reducing expression of a gene in a cell comprising introducing into the cell a mutant U1 snRNA which binds to a preselected site in a mRNA produced by the gene. In a second embodiment, the invention is directed to a method of reducing protein production in a cell comprising introducing into the cell a mutant U1 snRNA which binds to a preselected site in a mRNA that codes for the protein. In a third embodiment, the invention is directed to a method of reducing output in a cell of a mRNA produced by a gene located in the cell comprising introducing into the cell a mutant U1 snRNA which binds to a preselected site in the mRNA.
The invention also relates to a method of delivery of a selected ribozyme to a target mRNA in a cell comprising introducing into the cell a ribozyme-U1 snRNA complex.
The invention further relates to a method of reducing expression of a gene in a human comprising (a) obtaining a biological sample containing cells from the human and maintaining the cells under conditions appropriate for cell viability; (b) introducing into the cells a mutant U1 snRNA which binds to a preselected site in a mRNA produced by the gene; and (c) returning the cells obtained in step (b) to the human.
The invention still further relates to methods for treating and/or prophylaxis of viral infections (e.g., human immunodeficiency virus (HIV) infection), dominant negative inherited diseases and disorders, and cancer due to overexpression of a cancer gene, in a human in need thereof. In one embodiment of this aspect of the invention, the method comprises (a) obtaining a biological sample containing cells from the human and maintaining the cells under conditions appropriate for cell viability; (b) introducing into the cells a mutant U1 snRNA which binds to a preselected site in a mRNA produced by a targeted gene; (c) maintaining the cells from step (b) under conditions appropriate for the mutant U1 snRNA to bind to the preselected site; and (d) returning the cells obtained in step (c) to the human. In a second embodiment of this aspect of the invention, the method comprises (a) obtaining a biological sample containing cells from the human and maintaining the cells under conditions appropriate for cell viability; (b) introducing into the cells a ribozyme-U1 snRNA complex comprising a selected ribozyme which is capable of cleaving a mRNA produced by a targeted gene and which is covalently linked to the 5′ end of a U1 snRNA; (c) maintaining the cells from step (b) under conditions appropriate for the selected ribozyme to cleave the mRNA; and (d) returning the cells obtained in step (c) to the human. In both embodiments of this aspect of the invention, the targeted gene is selected because its expression leads to undesirable and detrimental effects in the human (e.g., acquired immunodeficiency syndrome (AIDS), dominant negative inherited disease, cancer), thereby making it desirable to regulate, reduce or inhibit expression of the selected gene.
In a particular embodiment, a method for treating and/or prophylaxis of a viral infection, such as a HIV infection, in a human in need thereof comprises (a) obtaining a biological sample containing cells from the human and maintaining the cells under conditions appropriate for cell viability; (b) introducing into the cells a ribozyme-U1 snRNA complex comprising a selected ribozyme which is capable of cleaving a target viral mRNA and which is covalently linked to the 5′ end of a U1 snRNA; (c) maintaining the cells from step (b) under conditions appropriate for the selected ribozyme to cleave the target viral mRNA; and (d) returning the cells obtained in step (c) to the human. In a particular embodiment, the method for treating and/or prophylaxis of a HIV infection further comprises introducing into the cells a mutant U1 snRNA which binds to a preselected site in the 3′ long terminal repeat (LTR) proximal to the polyadenylation (PA) site (the 3′ terminal exon on the viral protein).
In a yet another embodiment of the invention, the methods of the present invention further comprise introducing into the cells a second or multiple mutant U1 snRNA, wherein each mutant U1 snRNA binds to a different preselected site in a mRNA produced by the same targeted gene.
The invention also includes compositions comprising a mutant U1 snRNA which binds to a preselected site in a mRNA produced by a gene in a cell and compositions comprising a ribozyme-U1 snRNA complex.
The invention further relates to methods of producing the compositions of the present invention. In two particular embodiments, the invention relates to methods of producing compositions comprising a mutant U1 snRNA which binds to a preselected site in a mRNA produced by a targeted gene in a cell and to methods of producing compositions comprising a ribozyme-U1 snRNA complex. The compositions of the invention can be prepared by selecting a non-consensus donor site in a mRNA produced by a target gene for binding the mutant U1 snRNA and then modifying the 5′-TACTTACCTG-3′ (SEQ ID NO: 1) sequence at the 5′ end of the U1 snRNA so that it is complementary to the non-consensus donor site selected. In the case of a ribozyme-U1 snRNA complex, the 5′-TACTTACCTG-3′ (SEQ ID NO: 1) sequence at the 5′ end of the U1 snRNA is modified so that it incorporates the ribozyme selected to be delivered to a mRNA produced by a target gene in a cell.
The invention also includes nucleic acid molecules which encode the mutant U1 snRNA of the invention. Also envisioned are host cell
Beckley Akin
Rowe David W.
Stover Mary Louise
Cummings & Lockwood
Schultz James Douglas
The University of Connecticut
Wang Andrew
LandOfFree
Inhibition of a target messenger RNA with a modified U1... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Inhibition of a target messenger RNA with a modified U1..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Inhibition of a target messenger RNA with a modified U1... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3331106