US20130323838A1

US20130323838A1 - TRIGGERED RNAi

Info

Publication number: US20130323838A1
Application number: US13/896,235
Authority: US
Inventors: Niles A. Pierce; Peng Yin
Original assignee: California Institute of Technology CalTech
Current assignee: California Institute of Technology CalTech
Priority date: 2008-02-27
Filing date: 2013-05-16
Publication date: 2013-12-05
Also published as: US8497364B2; US20090247615A1

Abstract

The present application relates to methods and compositions for triggering RNAi. Triggered RNAi is highly versatile because the silencing targets are independent of the detection targets. In some embodiments, methods of silencing or modulating the expression of a target gene are provided. The methods generally comprise providing an initiator to a cell comprising a detection target and a silencing target gene, wherein the detection target is different from the silencing target gene, wherein binding of the detection target to the initiator initiates formation of an inactivator double-stranded RNA (inactivator dsRNA). The inactivator dsRNA can silence or modulate the expression of the silencing target gene.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser. No. 12/395,489, filed Feb. 27, 2009, which claims priority to U.S. Provisional Application No. 61/067,249, filed Feb. 27, 2008, each of which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention
The present application relates generally to nucleic acid systems for triggering RNAi for silencing of one or more silencing targets in the presence of a detection target.
2. Description of the Related Art
RNA interference (RNAi) is a conserved biological response to double-stranded RNA (dsRNA) that results in sequence-specific silencing of target gene expression (Fire et al. 1998 Nature 391: 806-811). In recent years, RNAi has been adopted as an important experimental tool to study and manipulate gene expression and has generated great interest as a potential therapeutic approach (Dillon et al. 2005 Annual Review of Physiology 67: 147-173, incorporated herein by reference in its entirety).
Until now, in the practice of using RNAi for gene silencing, an RNA duplex is introduced into the cell cytoplasm such that one of the strands in the duplex matches the sequence of the target mRNA to be silenced. In the traditional RNAi pathway, an “inactivator dsRNA” is introduced into the cell's cytoplasm and is processed by the Dicer enzyme, producing small interfering RNAs (siRNAs). (See, Tijsterman and Plasterk 2004 Cell 117(1): 1-3, incorporated herein by reference in its entirety). The siRNAs are then processed by the RNA-induced silencing complex (RISC complex), which unwinds the siRNA and retains one strand as a targeting co-factor. The RISC-siRNA complex recognizes and induces the degradation of the target mRNA, which is complementary to the siRNA strand retained by the RISC complex. The cleaved mRNA is then further degraded by natural mechanisms. The traditional RNAi mechanism implements the logical operation: if gene q is detected, silence gene q. Thus, using the standard RNAi technique, the detection and silencing steps are restricted to operating on the same gene.

SUMMARY OF THE INVENTION

In some embodiments, methods of forming an inactivator double-stranded RNA (dsRNA) are provided. The methods generally comprise contacting a sample comprising a detection target with an initiator comprising a detection target binding region and an initiator region, wherein the detection target binding region can interact with the detection target, and wherein the detection target can be different from the silencing target gene; contacting the sample with a first substrate monomer and a second substrate monomer; wherein the first substrate monomer comprises a second substrate monomer complement region and the second substrate monomer comprises a first substrate monomer complement region, wherein the second substrate monomer complement region can be complementary to the first substrate monomer complement region; wherein the first substrate monomer comprises a silencing target complement region and an initiator complement region that can be complementary to the initiator region of the initiator, wherein upon binding of the detection target binding region to the detection target, the initiator region of the initiator can be made available to bind to the initiator complement region of the first substrate monomer; and wherein the second substrate monomer comprises a silencing target region that can be complementary to the silencing target complement region of the first substrate monomer, wherein following binding of the initiator complement region of the first monomer to the initiator region of the initiator, the silencing target complement region of the first substrate monomer hybridizes to the silencing target region of the second substrate monomer to form an inactivator dsRNA. In some embodiments, the inactivator dsRNA can be formed by hybridization of the silencing target complement region to the silencing target region. In some embodiments the sample further comprises a silencing target gene. In some embodiments, the inactivator dsRNA can be used to silence or modulate expression of a target gene. In some embodiments, the inactivator dsRNA can be processed to silence the silencing target gene. In some embodiments, the initiator comprises a nucleic acid hairpin monomer. In some embodiments, the first substrate monomer comprises a nucleic acid hairpin monomer that comprises a first hairpin loop region. In some embodiments, the first hairpin loop region comprises the second substrate monomer complement region. In some embodiments, the second substrate monomer comprises a nucleic acid hairpin monomer that comprises a second hairpin loop region. In some embodiments, the second hairpin loop region comprises the first substrate monomer complement region. In some embodiment the method further comprises contacting the sample with at least one accessory molecule comprising a DNA sequence that binds to a region flanking the initiator binding site of the detection target. In some embodiments, the inactivator dsRNA can be processed by Dicer. In some embodiments, the inactivator dsRNA can be processed to produce a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNA comprises a 27-base pair RNA duplex substrate. In some embodiments, the inactivator dsRNA can be an siRNA. In some embodiments, the inactivator dsRNA can be a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNA can activate protein kinase R (PKR). In some embodiments, the detection target can be a nucleic acid. In some embodiments, the silencing target gene can be an mRNA comprising a sequence different from the sequence of the detection target. In some embodiments, the detection target can be associated with a disease or disorder. In some embodiments, the detection target can be an mRNA associated with a cancer. In some embodiments, the detection target can be a viral nucleic acid. In some embodiments, the detection target can be an mRNA molecule associated with a disease or disorder. In some embodiments, the silencing target gene can be an mRNA. In some embodiments, the mRNA can be fluorescent protein RNA. In some embodiments, the mRNA can be a housekeeping gene mRNA. In some embodiments, the mRNA can be a protein kinase R (PKR) mRNA. In some embodiments, the initiator comprises a recognition molecule such that upon binding of the recognition molecule to the detection target, a portion of the initiator can be made available to bind to the initiator complement region of the first substrate monomer. In some embodiments, the recognition molecule can be an aptamer. In some embodiments, the detection target can be a nucleic acid. In some embodiments, the detection target can be selected from the group consisting of polypeptides, carbohydrates, lipids and small molecules. In some embodiments, the first substrate monomer comprises a sticky end region. In some embodiments, the methods can comprise additional monomers and/or complexes.
In other embodiments, methods of forming an inactivator dsRNA are provided that generally comprise contacting a sample comprising a detection target with an initiator comprising a detection target binding region and an initiator region, wherein the detection target binding region can interact with the detection target, and wherein the detection target can be different from the silencing target gene; and contacting the sample with a first substrate monomer and a second substrate monomer. In some embodiments, the first substrate monomer comprises a second substrate monomer complement region and the second substrate monomer comprises a first substrate monomer complement region, wherein the second substrate monomer complement region can be complementary to the first substrate monomer complement region; the first substrate monomer comprises a silencing target complement region and an initiator complement region that can be complementary to the initiator region of the initiator, wherein following binding of the detection target binding region to the detection target, the initiator region of the initiator can be made available to bind to the initiator complement region of the first substrate monomer; and the second substrate monomer comprises a silencing target region that can be complementary to the silencing target complement region of the first substrate monomer, wherein following binding of the initiator complement region of the first monomer to the initiator region of the initiator, the silencing target complement region of the first substrate monomer hybridizes to the silencing target region of the second substrate monomer to form an inactivator dsRNA. In some embodiments, the inactivator dsRNA can be formed by hybridization of the silencing target complement region to the silencing target region. In some embodiments the sample further comprises a silencing target gene. In some embodiments, the inactivator dsRNA can be used to silence or modulate the expression of the target gene. In some embodiments, the inactivator dsRNA can be processed to silence the silencing target gene, for example by Dicer. In some embodiments, the initiator comprises a nucleic acid hairpin monomer. In some embodiments, the first substrate monomer comprises a nucleic acid hairpin monomer that comprises a first hairpin loop region. In some embodiments, the first hairpin loop region comprises the second substrate monomer complement region. In some embodiments, the second substrate monomer comprises a nucleic acid hairpin monomer that comprises a second hairpin loop region. In some embodiments, the second hairpin loop region comprises the first substrate monomer complement region. In some embodiment the method further comprises contacting the sample with at least one accessory molecule comprising a DNA sequence that binds to a region flanking the initiator binding site of the detection target. In some embodiments, the inactivator dsRNA can be processed by Dicer. In some embodiments, the inactivator dsRNA can be processed to produce a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNA comprises a 27-base pair RNA duplex substrate. In some embodiments, the inactivator dsRNA can be an siRNA. In some embodiments, the inactivator dsRNA can be a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNA can activate protein kinase R (PKR). In some embodiments, the detection target can be a nucleic acid. In some embodiments, the silencing target gene can be an mRNA comprising a sequence different from the sequence of the detection target. In some embodiments, the detection target can be associated with a disease or disorder. In some embodiments, the detection target can be an mRNA associated with a cancer. In some embodiments, the detection target can be a viral nucleic acid. In some embodiments, the detection target can be an mRNA molecule associated with a disease or disorder. In some embodiments, the silencing target gene can be an mRNA. In some embodiments, the mRNA can be fluorescent protein RNA. In some embodiments, the mRNA can be a housekeeping gene mRNA. In some embodiments, the mRNA can be a protein kinase R (PKR) mRNA. In some embodiments, the initiator comprises a recognition molecule such that upon binding of the recognition molecule to the detection target, a portion of the initiator can be made available to bind to the initiator complement region of the first substrate monomer. In some embodiments, the recognition molecule can be an aptamer. In some embodiments, the detection target can be a nucleic acid. In some embodiments, the detection target can be selected from the group consisting of polypeptides, carbohydrates, lipids and small molecules. In some embodiments, the first substrate monomer comprises a sticky end region. In some embodiments, the methods can comprise additional monomers and/or complexes.
In other embodiments, methods of forming an inactivator dsRNA are provided that generally comprise contacting a sample comprising a detection target with an initiator comprising a detection target binding region and an initiator region, wherein the detection target binding region can interact with the detection target, and wherein the detection target can be different from the silencing target gene; contacting the sample with a first substrate complex and a second substrate complex: wherein the first substrate complex comprises a second substrate complex complement region and the second substrate complex comprises a first substrate monomer complement region, wherein the second substrate complex complement region can be complementary to the first substrate complex complement region; wherein the first substrate complex comprises a silencing target region and an initiator complement region that can be complementary to the initiator region of the initiator, wherein upon binding of the detection target binding region of the initiator to the detection target, the initiator region of the initiator can be made available to bind to the initiator complement region of the first substrate complex; and wherein the second substrate complex comprises a silencing target complement region, wherein following binding of the initiator complement region to the initiator region, the silencing target region of the first substrate complex can be made available to bind to the silencing target complement region of the second substrate complex to form an inactivator dsRNA. In some embodiments, the inactivator dsRNA can be formed by hybridization of the silencing target complement region to the silencing target region. In some embodiments the sample further comprises a silencing target gene. In some embodiments, the inactivator dsRNA can be used to silence or modulate the expression of a target gene. In some embodiments, the inactivator dsRNA can be processed to silence the silencing target gene. In some embodiments, the first substrate complex comprises a nucleic acid duplex. In some embodiments, the second substrate complex comprises a nucleic acid duplex. In some embodiments, the first substrate complex comprises a region complementary to a region of the second substrate complex. In some embodiments, the first substrate complex comprises two monomers. In some embodiments, the first substrate complex comprises a first bulge loop region. In some embodiments, the second substrate complex comprises two monomers. In some embodiments, the second substrate complex comprises a second bulge loop region, wherein a portion of the first bulge loop region can be complementary to the second bulge loop region. In some embodiments, the initiator comprises a nucleic acid hairpin monomer. In some embodiments, the inactivator dsRNA can be processed by Dicer. In some embodiments, the inactivator dsRNA can be processed to produce a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNA comprises a 27-base pair RNA duplex substrate. In some embodiments, wherein the inactivator dsRNA can be an siRNA. In some embodiments, the inactivator dsRNA can be a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNA activates protein kinase R (PKR). In some embodiments, the methods can comprise additional monomers and/or complexes. In some embodiments, the silencing target gene can be an mRNA. In some embodiments, the mRNA can be fluorescent protein RNA.
In other embodiments, methods of forming an inactivator dsRNA are provided that generally comprise contacting a sample comprising a detection target with an initiator comprising a detection target binding region and an initiator region, wherein the detection target binding region can interact with the detection target, and wherein the detection target can be different from the silencing target gene; contacting the sample with a first substrate complex and a second substrate complex. In some embodiments, the first substrate complex comprises a second substrate complex complement region and the second substrate complex comprises a first substrate monomer complement region, wherein the second substrate complex complement region can be complementary to the first substrate complex complement region; the first substrate complex comprises a silencing target region and an initiator complement region that can be complementary to the initiator region of the initiator, wherein upon binding of the detection target binding region of the initiator to the detection target, the initiator region of the initiator can be made available to bind to the initiator complement region of the first substrate complex; and the second substrate complex comprises a silencing target complement region, wherein following binding of the initiator complement region to the initiator region, the silencing target region of the first substrate complex can be made available to bind to the silencing target complement region of the second substrate complex to form an inactivator dsRNA. In some embodiments, the inactivator dsRNA can be formed by hybridization of the silencing target complement region to the silencing target region. In some embodiments the sample further comprises a silencing target gene. In some embodiments, the inactivator dsRNA can be used to silence or modulate the expression of a target gene. In some embodiments, the inactivator dsRNA can be processed to silence the silencing target gene. In some embodiments, the first substrate complex comprises a nucleic acid duplex. In some embodiments, the second substrate complex comprises a nucleic acid duplex. In some embodiments, the first substrate complex comprises two monomers. In some embodiments, the first substrate complex comprises a first bulge loop region. In some embodiments, the second substrate complex comprises two monomers. In some embodiments, the second substrate complex comprises a second bulge loop region, wherein a portion of the first bulge loop region can be complementary to the second bulge loop region. In some embodiments, the initiator comprises a nucleic acid hairpin monomer. In some embodiments, the inactivator dsRNA can be processed by Dicer. In some embodiments, the inactivator dsRNA can be processed to produce a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNA comprises a 27-base pair RNA duplex substrate. In some embodiments, wherein the inactivator dsRNA can be an siRNA. In some embodiments, the inactivator dsRNA can be a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNA activates protein kinase R (PKR). In some embodiments, the methods can comprise additional monomers and/or complexes. In some embodiments, the silencing target gene can be an mRNA. In some embodiments, the mRNA can be fluorescent protein RNA.
In other embodiments, methods of forming an inactivator dsRNA are provided that comprise contacting a sample comprising a detection target gene with an initiator comprising a detection target binding region and an initiator region, wherein the detection target binding region interacts with the detection target, and wherein the detection target can be different from the silencing target gene; contacting the sample with a first substrate complex and a second substrate complex: wherein the first substrate complex comprises a silencing target complement region and an initiator complement region that can be complementary to the initiator region of the initiator, wherein upon binding of the detection target binding region of the initiator to the detection target, the initiator region of the initiator binds to the initiator complement region of the first substrate complex; wherein the second substrate complex comprises a silencing target region, wherein following binding of the initiator complement region to the initiator region, the silencing target region of the second substrate complex binds to the silencing target complement region of the first substrate complex to form an inactivator dsRNA. In some embodiments, the inactivator dsRNA can be formed by hybridization of the silencing target complement region to the silencing target region. In some embodiments the sample further comprises a silencing target gene. In some embodiments, the inactivator dsRNA can be used to silence or modulate the expression of a target gene. In some embodiments, the inactivator dsRNA can be processed to silence the silencing target gene. In some embodiments, the first substrate complex comprises a nucleic acid duplex. In some embodiments, the first substrate complex comprises two monomers. In some embodiments, the second substrate complex comprises a nucleic acid duplex. In some embodiments, the second substrate complex comprises two monomers. In some embodiments, the initiator comprises a nucleic acid hairpin monomer. In some embodiments, the inactivator dsRNA can be processed by Dicer. In some embodiments, the inactivator dsRNA can be processed to produce a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNA comprises a 27-base pair RNA duplex substrate. In some embodiments, the inactivator dsRNA can be an siRNA. In some embodiments, the inactivator dsRNA can be a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNA can activate protein kinase R (PKR). In some embodiments, the methods can comprise additional monomers and/or complexes. In some embodiments, the silencing target gene can be an mRNA. In some embodiments, the mRNA can be fluorescent protein RNA.
In other embodiments, methods of forming an inactivator dsRNA are provided that generally comprise contacting a sample comprising a detection target gene with an initiator comprising a detection target binding region and an initiator region, wherein the detection target binding region interacts with the detection target, and wherein the detection target can be different from the silencing target gene; and contacting the sample with a first substrate complex and a second substrate complex. In some embodiments, the first substrate complex comprises a silencing target complement region and an initiator complement region that can be complementary to the initiator region of the initiator, wherein upon binding of the detection target binding region of the initiator to the detection target, the initiator region of the initiator binds to the initiator complement region of the first substrate complex; and the second substrate complex comprises a silencing target region, wherein following binding of the initiator complement region to the initiator region, the silencing target region of the second substrate complex binds to the silencing target complement region of the first substrate complex to form an inactivator dsRNA. In some embodiments, the inactivator dsRNA can be formed by hybridization of the silencing target complement region to the silencing target region. In some embodiments the sample further comprises a silencing target gene. In some embodiments, the inactivator dsRNA can be used to silence or modulate the expression of a target gene. In some embodiments, the inactivator dsRNA can be processed to silence the silencing target gene. In some embodiments, the first substrate complex comprises a nucleic acid duplex. In some embodiments, the first substrate complex comprises two monomers. In some embodiments, the second substrate complex comprises a nucleic acid duplex. In some embodiments, the second substrate complex comprises two monomers. In some embodiments, the initiator comprises a nucleic acid hairpin monomer. In some embodiments, the inactivator dsRNA can be processed by Dicer. In some embodiments, the inactivator dsRNA can be processed to produce a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNA comprises a 27-base pair RNA duplex substrate. In some embodiments, the inactivator dsRNA can be an siRNA. In some embodiments, the inactivator dsRNA can be a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNA can activate protein kinase R (PKR). In some embodiments, the methods can comprise additional monomers and/or complexes. In some embodiments, the silencing target gene can be an mRNA. In some embodiments, the mRNA can be fluorescent protein RNA.
In other embodiments, methods of forming an inactivator dsRNA are provided that generally comprise contacting a sample comprising a detection target with an initiator comprising a detection target binding region, an initiator region, and a silencing target complement region wherein the detection target binding region can interact with the detection target, and wherein the detection target can be different from the silencing target gene; and contacting the sample with a substrate monomer, wherein the substrate monomer comprises an initiator complement region that can be complementary to the initiator region of the RNA hairpin initiator monomer and a silencing target region that can be complementary to the silencing target complement region to form an inactivator dsRNA. In some embodiments, the inactivator dsRNA can be formed by hybridization of the silencing target complement region to the silencing target region. In some embodiments, the hybridization of the silencing target complement region to the silencing target region occurs subsequent to interaction of the detection target to the detection target binding region In some embodiments the sample further comprises a silencing target gene. In some embodiments, the inactivator dsRNA can be used to silence or modulate the expression of a target gene. In some embodiments, the inactivator dsRNA can be processed to silence the silencing target gene. In some embodiments, the initiator and substrate monomer co-exist stably in the absence of a detection target. In some embodiments, the initiator comprises a nucleic acid hairpin monomer. In some embodiments, the substrate monomer comprises a nucleic acid hairpin monomer. In some embodiments, the inactivator dsRNA can be processed by Dicer. In some embodiments, the inactivator dsRNA can be processed to produce a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNA comprises a 27-base pair RNA duplex substrate. In some embodiments, the inactivator dsRNA can be an siRNA. In some embodiments, the inactivator dsRNA can be a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNA can activate protein kinase R (PKR). In some embodiments, the methods can comprise additional monomers and/or complexes. In some embodiments, the silencing target gene can be an mRNA. In some embodiments, the mRNA can be fluorescent protein RNA.
In the embodiments described herein, the methods can further comprise contacting the sample with at least one accessory molecule comprising a DNA sequence that binds to a region flanking the initiator binding site of the detection target. In any of the embodiments described herein, the detection target can be a nucleic acid. In any of the embodiments described herein, the silencing target gene can be an mRNA comprising a sequence different from the sequence of the detection target. In some of the embodiments described herein, the detection target can be associated with a disease or disorder. For example, the detection target can be an mRNA associated with a cancer. In other embodiments the detection target can be a viral nucleic acid. The detection target can also be an mRNA molecule associated with a disease or disorder. In any of the embodiments described herein, the silencing target gene can be an mRNA. The mRNA can be, for example, a protein kinase R (PKR) mRNA. The mRNA can be, for example, fluorescent protein mRNA. The mRNA can also be a housekeeping gene mRNA. In any of the embodiments described herein, the initiator can comprise a recognition molecule such that upon binding of the recognition molecule to the detection target, a portion of the initiator can be made available to bind to the initiator complement region of the first substrate monomer. The recognition molecule can be, for example, an aptamer. In any of the embodiments described herein, the detection target can be a nucleic acid. In any of the embodiments described herein, the detection target can be selected from the group consisting of polypeptides, carbohydrates, lipids and small molecules. In any of the embodiments described herein, a monomer or complex can comprise a sticky end region. In some embodiments, the methods can comprise additional monomers and/or complexes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts a summary of logical operation of the triggered RNAi pathway. Each letter (i.e., p and q) represents a region of nucleic acids. Letters marked with an asterisk (*) are complementary to the corresponding unmarked letter.

FIGS. 2A-2I schematically illustrate one embodiment of a triggered RNAi system. There are three synthetic nucleic acid hairpin strands, A, B, and C shown. Hairpins A, B and C of FIGS. 2A-2I co-exist metastably and do not induce RNAi action on their own. The mRNA “detection target” is D (comprising sequence regions b*-x*-a*). When D is present in the system, it activates A, which in turn catalyzes B and C to form the RNA duplex B.C.

FIG. 3 schematically illustrates one embodiment of a triggered RNAi system. As illustrated in FIG. 3A, Duplex B.C from FIG. 2I contains a duplex substrate that can be processed by Dicer. As illustrated in FIG. 3B, the duplex substrate portion of B.C is processed by Dicer to induce RNAi action. As illustrated in FIG. 3C, the RNAi action silences the mRNA “silencing target” S (comprising sequence regions c-y-z). Note that the mRNA detection target D and the mRNA silencing target S have completely independent sequences.

FIG. 4 schematically illustrates another embodiment of a triggered RNAi system using the three synthetic nucleic acid hairpin strands, A, B, and C shown. In the absence of the mRNA “detection target” T, hairpins A, B and C of FIG. 4 co-exist metastably and do not induce RNAi action on their own. The hairpin loops on B and C possess regions that are complementary to one another that allow the formation of B.C-1. T comprises sequence regions u*-v*-y*-s*. When T is present in the system, it activates A, and the complex T.A is formed. T.A, in turn, reacts with B.C-1 to form T.A.B.C. This leads to the formation of the RNA duplex B.C-2. Duplex B.C-2 is recognized and processed by Dicer to silence an mRNA “silencing target” comprising sequence regions (z)*-(x)*.

FIG. 5 schematically illustrates another embodiment of a triggered RNAi system using the three synthetic nucleic acid hairpin strands, A, B, and C, shown. In the absence of the mRNA “detection target” T, hairpins A, B and C of FIG. 5 co-exist metastably and do not induce RNAi action on their own. The hairpin loops on B and C possess regions that are complementary to one another that allow the formation of B.C-1. T comprises sequence regions u*-v*-y*-s*. When T is present in the system, it activates A, and the complex T.A is formed. T.A, in turn, interacts with B.C-1 to form T.A.B.C. The presence of a toehold region, r, on B further facilitates the interaction between T.A and B.C-1 to form T.A.B.C. This leads to the formation of the RNA duplex B.C-2. Duplex B.C-2 is recognized and processed by Dicer to silence an mRNA “silencing target” comprising sequence regions (z)*-(x)*.

FIG. 6 schematically illustrates another embodiment of a triggered RNAi system using the five synthetic nucleic acid strands, A, B, C, D, and E, shown. FIG. 6 illustrates a detection target T that interacts with and opens an initiator monomer (initiator) A. In the absence of the mRNA “detection target” T, hairpin A, complex C.E, and complex B.D co-exist metastably and do not induce RNAi action on their own. The bulge loops on C.E and B.D possess regions (y and y*, respectively) that are complementary to one another. T comprises sequence regions (a)-(w)-(b)-(s). When T is present in the system, it activates A, and the complex T.A is formed. T.A, in turn, reacts with C.E and B.D to form T.A.B.C.D.E. This leads to the formation of the RNA duplexes B.C and D.E. Duplex D.E is recognized and processed by Dicer to silence an mRNA “silencing target” comprising sequence regions (x)-(z).

FIG. 7 schematically illustrates another embodiment of a triggered RNAi system. There are five synthetic nucleic acid strands, A, B, C, D, and E, shown. In the absence of the mRNA “detection target” T, hairpin A, complex B.C, and complex D.E co-exist metastably and do not induce RNAi action on their own. T comprises sequence regions (a*)-(x*)-(b*). When T is present in the system, it interacts with A to form the complex T.A. T.A, in turn, reacts with B.C to form T.A.B and C. C is now available to react with D.E to form E and the duplex C.D. Duplex C.D is recognized and processed by Dicer to silence an mRNA “silencing target” comprising sequence regions (y)-(c).

FIG. 8 schematically illustrates another embodiment of a triggered RNAi system. There are 2 synthetic nucleic acid hairpin strands, A and B, shown. In the absence of the mRNA “detection target” T, hairpin A, and hairpin B co-exist stably and do not induce RNAi action on their own. T comprises sequence regions (x)-(a). When T is present in the system, it interacts with A to form the complex T.A. T.A, in turn, reacts with B to form T.A.B. T.A.B is recognized and processed by Dicer to silence an mRNA “silencing target” comprising sequence regions (b)*-(y)*-(c)*.

FIG. 9 schematically illustrates another embodiment of a triggered RNAi system. There are five synthetic nucleic acid strands, A, B, C, D, and E, shown. In the absence of the mRNA “detection target” T, hairpin A, complex C.E and complex B.D co-exist metastably and do not induce RNAi action on their own. The bulge loops on C.E and B.D possess regions (y and y*, respectively) that are complementary to one another. T comprises sequence regions (a)-(w)-(b)-(s). When T is present in the system, it interacts with A to form the complex T.A. T.A, in turn, reacts with C.E and B.D to form T.A.B.C.D.E. This leads to the formation of the RNA duplexes B.C and D.E. Duplex D.E serves as siRNA which results in the silencing of an mRNA “silencing target” comprising sequence regions (x)-(z).

FIG. 10 schematically illustrates another embodiment of a triggered RNAi system. There are five synthetic nucleic acid strands, A, B, C, D, and E, shown. In the absence of the mRNA “detection target” T, hairpin A, complex B.C, and complex D.E co-exist metastably and do not induce RNAi action on their own. T comprises sequence regions (a*)-(x*)-(b*). When T is present in the system, it interacts with A to form the complex T.A. T.A, in turn, reacts with B.C to form T.A.B and C. C is now available to react with D.E to form E and the duplex C.D. Duplex C.D serves as siRNA which results in the silencing of an mRNA “silencing target” comprising sequence regions (y)-(c).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Various embodiments disclosed herein are generally directed towards compositions and methods for triggered RNAi.
At present, there are a limited number of techniques that exist for RNAi. Various RNAi techniques have been restricted in utility because it has not been possible to use RNAi to silence targets in response to the presence of other, perhaps unrelated molecules. While up to 100-fold enhancements in silencing potency have been achieved by using longer RNA duplexes that are processed into siRNAs by Dicer prior to incorporation into RISC (Kim et al., 2005, Nat. Biotechnol. 23(2): 222-226), the ability for catalytic amplification of RNAi has been limited. Some of the embodiments described herein overcome these and other limitations.
A new approach to sequence-specific silencing of a silencing target, triggered RNAi, has been developed based on the triggered hybridization of nucleic acid molecules, typically starting from monomer hairpins (U.S. Patent Publication No. 2008/0214488 and PCT Publication No. WO 2008/106658, which are hereby incorporated by reference in their entireties). In the triggered RNAi process, a detection event is transduced into the formation of a distinct inactivator dsRNA. Monomers can be used that generate inactivator dsRNAs in the presence of a detection target. The inactivator dsRNAs can be used for gene silencing. For example, in some embodiments, the inactivator dsRNAs can be processed by Dicer into siRNAs. Once formed, the siRNAs can lead to silencing of one or more silencing targets. As a result, triggered RNAi is much more versatile than traditional RNAi, because the sequence of the silencing target is not limited to the sequence of the detection target. In some embodiments, detection targets other than nucleic acid molecules can trigger RNAi. In some embodiments, the triggered RNAi system can be catalytic, and the detection binding event can be amplified to silence one or more silencing targets.
Triggered RNAi can be used for, for example, the specific detection of targets (“detection targets”) associated with a disease or disorder and the specific silencing of independent targets (“silencing targets”) to treat the disease or disorder. The detection targets, which can be found, for example, exclusively in diseased cells or to a greater extent in diseased cells than in healthy cells, act as a trigger for RNAi silencing of silencing targets. Silencing targets may be, for example, genes involved in the disease or disorder that may be unrelated to the detection target, although in some embodiments it may be related. In this way, a disease or disorder can be treated and/or prevented by targeting diseased cells and silencing selected genes. The triggered RNAi methods described herein are not limited treating diseases or disorders. The methods described herein can be used in any context where it is desirable to silence a gene or mRNA when activation of a molecule is detected. In other embodiments, silencing targets may be, for example, mRNA encoding a reporter molecule, such as, for example, GFP. The mRNA can be, for example, endogenous or exogenous mRNA.
Methods and compositions for triggered RNAi are provided. A schematic depiction of the logical operation of one embodiment of a triggered RNAi process is shown in FIG. 1. In FIG. 1, an mRNA detection target p is present in a sample or cell. Detection of the detection target p by the triggered RNAi pathway results in silencing of mRNA silencing target q. In various embodiments, methods of silencing a target gene are provided. In some embodiments, the methods comprise providing an initiator, a first monomer, and a second monomer to a sample comprising a detection target p and a silencing target gene q. Preferably, the detection target p is different from the silencing target gene q. The initiator can bind to a portion of the detection target p. In preferred embodiments, binding of the detection target p can induce a conformational change in the initiator and expose a region of the initiator that can recognize and bind the first monomer. In some embodiments, binding of the initiator to the first monomer can induce a conformational change in the first monomer and expose a region of the first monomer that can recognize and bind the second monomer. In some embodiments, binding of the first monomer to the second monomer can produce an inactivator dsRNA which can silence the silencing target gene q. Preferably, the sequence of the mRNA silencing target q is independent of the detection target p.
One embodiment of a method for triggered RNAi is generally outlined in FIGS. 2 and 3. In FIGS. 2A-2C, a detection target (denoted “D”) has a region a* which pairs with region a of hairpin A and induces a strand displacement interaction resulting in the formation of complex D.A. The complex D.A. has a newly exposed single-stranded tail that contains the sequence y*-c*-b*, hereinafter referred to as the initiator region “I”. As shown in FIGS. 2D-F, the initiator region I of the D.A complex nucleates at the sticky end b of hairpin B, and induces a strand displacement interaction to open B and expose the single-stranded tail z*-y*-c* (for visual simplicity, only the I portion of the complex D.A is depicted). As shown in FIG. 2H, the I.B complex nucleates at the sticky end c of hairpin C (FIG. 2G) and opens C. As shown in FIG. 2I, hairpin C subsequently displaces I to form complex B.C. As shown in FIG. 3A, complex B.C contains a duplex substrate that can be processed by Dicer. The duplex substrate portion of B.C is recognized by Dicer and processed to induce RNAi action (FIGS. 3A-B). This results in the silencing of a silencing target gene (denoted “S”) comprising the sequence c-y-z (FIG. 3C). The silencing target gene S is independent of the mRNA detection target D. I is available to start another cycle amplifying reaction. Other embodiments of methods for triggered RNAi are outlined in FIGS. 4-10. In some embodiments, non-hairpin monomers can be used (see, e.g., FIG. 7). In other embodiments, nucleic acid complexes can be used. In some embodiments, dicer substrates are formed. In other embodiments, RISC substrates (e.g., siRNA) are formed. Some embodiments describe catalytic methods. One example of a catalysis is when multiple dicer substrates can be created with one target (see, e.g., FIG. 4).
In some embodiments, methods are provided for triggered RNAi silencing of multiple genes. Such triggered RNAi methods would implement the logical operation: if detection target p is detected, silence genes q₁, q₂, q₃. . . . For example, in some embodiment, an inactivator dsRNA can be processed to silence multiple gene targets. These methods can be used, for example, to create silencing networks in which multiple genes are being detected, each leading to the silencing of some number of other genes. One or more of the mRNA detection targets and mRNA silencing targets could coincide.
In other embodiments, methods are provided for triggered RNAi activation of PKR. Protein kinases and other cell-signaling messengers are activated during an immune response. In vitro studies show that PKR is activated when two PKR molecules dimerize and phosphorylate each other in complex with RNA duplexes longer than approximately 30 bp, and that the strength of the activation increases with duplex length up to ˜85 bp (Manche, L. et al. Molecular and Cellular Biology 12:5238-5248 (1992); Wu, S., and R. J. Kaufman. The Journal of Biological Chemistry 272:1291-1296 (1997); which are incorporated herein by reference in their entirety). Activation of PKR can lead to selective cell death, which is a strategy envisioned for the development of therapies for cancer (Shir, A. and A. Levitski. Nature Biotechnology 20:895-900 (2002); Friedrich, I. et al. Seminars in Cancer Biology 14:223-230 (2004); Friedrich, I. et al. Molecular Therapy 12:969-975 (2005); which are incorporated herein by reference in their entirety). Exploiting these activation properties, it was demonstrated that binding of anti-sense RNA to a region that spans the splice point of an oncogenic mRNA fusion entity resulted in creation of a 39-base pair duplex within cancer cells, selectively killing them (Shir, A., and A. Levitzski. 2002). The 39-bp duplex formed by the oncogenic fusion entity and the anti-sense RNA partially activated the protein kinase PKR, which is involved in inhibition of protein synthesis and cell death.
The composition of the triggered RNAi monomers can be designed such that none of the monomers alone contain a sufficiently long dsRNA component to activate PKR, but the inactivator dsRNA formed by the triggered RNAi process contains a component that can activate PKR. dsRNAs for activation of PKR are described in, for example, U.S. patent application Ser. No. 11/544,306, filed Oct. 6, 2006, published as U.S. Publication No. 20070087334, which is herein incorporated by reference in its entirety.
In other embodiments, methods are provided for triggered RNAi to avoid activation of the protein kinase PKR. As part of an antiviral immune response in many mammalian cells, PKR is activated by long double-stranded RNA, leading to the inhibition of protein synthesis and to cell death (Jagus et al. 1999; Williams 1999). Triggered RNAi can be used to avoid activating this pathway. For example, hairpins for triggered RNAi silencing of PKR can be designed to silence PKR mRNA upon detection of a selected detection target.
In other embodiments, methods are provided for triggered RNAi involving detection of a non-mRNA target. For example, triggered RNAi could be activated by using a conformation-switching aptamer that exposes an initiator region upon binding to a target protein or small molecule. The initiator region would then catalyze the formation of the dsRNA substrates as with the triggered RNAi mechanism shown in FIGS. 2 and 3. The experimental selection of conformation switching aptamers has been previously demonstrated (See, for example, Jhaveri et al., 2000, Nat. Biotechnol. 18: 1293-1297.
In some embodiments, triggered RNAi can be combined with an nucleic acid exponential amplifier scheme, such as the scheme disclosed in U.S. Publication No. 2005-0260635, filed Mar. 22, 2005, to achieve even higher amplification.
In some embodiments, methods are provided for triggered RNAi silencing of exogenous mRNA. In some embodiments, methods are provided for triggered RNAi silencing of an mRNA encoding a reporter molecule, such as, for example, green fluorescence protein (GFP) and other imaging or diagnostic markers. The detection of a detection target of interest can thus result in a change of the reporter signal via triggered RNAi.
As will be appreciated by one of skill in the art, triggered RNAi can have great benefit, including diagnostic and therapeutic uses. The introduction of triggered mechanical transduction between the diagnosis and treatment modalities of a single therapeutic is conceptually tantalizing because it allows independent optimization of specificity and potency, providing unprecedented flexibility in diagnosing and treating disease one cell at a time. The use of distinct diagnosis and treatment domains allows independent optimization of specificity and potency; the introduction of triggered mechanical transduction between these domains provides active suppression of the drug's activity until a positive diagnosis is achieved at the molecular level. These dynamic drugs would provide unprecedented flexibility in diagnosing and treating disease one cell at a time.
The above and additional embodiments are discussed in more detail below, after a brief discussion of some of the terms used in the specification

Definitions

The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control. It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, etc discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.
Unless otherwise defined, scientific and technical terms used in connection with the invention described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used, for example, for nucleic acid purification and preparation, chemical analysis, recombinant nucleic acid, and oligonucleotide synthesis. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the instant specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of described herein are those well known and commonly used in the art.
As utilized in accordance with the embodiments provided herein, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
The term “nucleic acid” refers to natural nucleic acids, artificial nucleic acids, analogs thereof, or combinations thereof. Nucleic acids may also include analogs of DNA or RNA having modifications to either the bases or the backbone. For example, nucleic acid, as used herein, includes the use of peptide nucleic acids (PNA). The term “nucleic acids” also includes chimeric molecules.
As used herein, the terms “polynucleotide,” “oligonucleotide,” and “nucleic acid oligomers” are used interchangeably and mean single-stranded and double-stranded polymers of nucleic acids, including, but not limited to, 2′-deoxyribonucleotides (nucleic acid) and ribonucleotides (RNA) linked by internucleotide phosphodiester bond linkages, e.g. 3′-5′ and 2′-5′, inverted linkages, e.g. 3′-3′ and 5′-5′, branched structures, or analog nucleic acids. Polynucleotides have associated counter ions, such as H⁺, NH₄ ⁺, trialkylammonium, Mg²⁺, Na⁺and the like. A polynucleotide can be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof. Polynucleotides can be comprised of nucleobase and sugar analogs. Polynucleotides typically range in size from a few monomeric units, e.g. 5-40 when they are more commonly frequently referred to in the art as oligonucleotides, to several thousands of monomeric nucleotide units. Unless denoted otherwise, whenever a polynucleotide sequence is represented, it will be understood that the nucleotides are in 5′ to 3′ order from left to right and that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine.
As used herein “double stranded RNA,” and “dsRNA” are used interchangeably and refer to a polynucleotide having two complementary strands of RNA. An “inactivator dsRNA” is a dsRNA that can silence a silencing target. In some embodiments, an inactivator dsRNA can be processed by Dicer to form siRNAs. In some embodiments, an inactivator dsRNA can comprise an siRNA. In some embodiments, an inactivator dsRNA can silence a silencing target by activating PKR.
An “RNA duplex substrate” is an inactivator dsRNA that can processed by Dicer and/or RISC.
The term “hairpin” and refers to a structured formed by intramolecular base pairing in a single-stranded polynucleotide ending in an unpaired loop. A “hairpin loop” refers to a single stranded region that loops back on itself and is closed by a single base pair.
“Interior loop,” “internal loop,” and “bulge loop” are used interchangeably and refer to a loop in which the single-stranded portion of a loop is bordered by two base pairs, one on each side.
The term “sticky end” refers to a nucleic acid sequence that is available to hybridize with a complementary nucleic acid sequence. A “sticky end” is located at an end of a double-stranded nucleic acid. The secondary structure of the “sticky end” is preferably such that the sticky end is available to hybridize with a complementary nucleic acid under the appropriate reaction conditions without undergoing a conformational change. In some embodiments the sticky end is preferably a single stranded nucleic acid.
“Monomers” as used herein refers to individual nucleic acid oligomers. Typically, each monomer comprises at least one region that is complementary to at least one portion of another monomer being used for the triggered RNAi process. The makeup of the monomers for some embodiments is described in more detail below. Two or more monomers can associate (e.g. by hybridization) to form a complex. For example, a complex can comprise two nucleic acid monomers that share a region of complementarity. In embodiments described herein using one or more monomers, complexes can be substituted for monomers such that the embodiments can use monomers or complexes or combinations of monomers and complexes. Complexes can comprise two or more nucleic acid strands. Complexes can comprise two complementary or substantially complementary strands. Complexes can comprise various secondary structures. For example, complexes can comprise a bulge loop structure that is closed by two nucleic acid base pairs.
“Triggered RNAi monomers” are monomers that are able to assemble to form an inactivator dsRNA upon exposure to an activated initiator.
“Substrate monomers” are monomers that can form a duplex comprising an inactivator dsRNA. Typically two substrate monomers form an RNA duplex having a dsRNA region which can be used by RNAi machinery to silence a silencing target.
An “initiator region” is a region on a molecule that is able to initiate the formation of RNA duplexes from monomers. Preferred initiator regions comprise a sequence that is complementary to the initiator complement region of a substrate monomer.
An “initiator” is a molecule that is able to interact with a detection target and subsequently expose an initiator region. Preferred initiators comprise a detection target binding region that is complementary to or otherwise recognize a portion of a detection target, and an initiator region. Before the initiator interacts with a detection target, the initiator region is not exposed and therefore not available to hybridize to an initiator complement region. In some embodiments the detection target binding region can comprise a sticky end. Other embodiments of the initiator comprise a recognition molecule that binds or interacts with a detection target. In some embodiments the initiator can comprise an aptamer that recognizes a specific molecule, and the aptamer can comprise the detection target binding region.
Interaction of the detection target to the detection target binding region or to the recognition molecule of the initiator begins the triggered RNA process by exposing the initiator region. An “activated initiator” is an initiator that is bound to a detection target. In some embodiments, the initiator can comprise a monomer. In some embodiments, the initiator can be a monomer linked to a recognition molecule.
The initiator region of the initiator is preferably only available to hybridize with the initiator complement region of a substrate monomer when an initiator has been activated by an interaction between the initiator and the detection target. In some embodiments, the initiator hybridizes with the initiator complement region of a second monomer which in turn interacts with a substrate monomer. The substrate monomer preferably comprises a silencing target complement region that is able to hybridize to a silencing target region of another substrate monomer. Preferably, the silencing target complement region of a substrate monomer is different from the detection target binding region of an initiator. In some embodiments, the silencing target complement region of a substrate monomer can be the same as or overlap with the initiator complement region.
In addition, the silencing target complement region of a first substrate monomer is preferably only available to hybridize with the silencing target region of another substrate monomer when the first substrate monomer has already hybridized to an initiator, or an intervening monomer that is made available by the initiator, as discussed in more detail below.
A “detection target” is a molecule of interest, or a combination of molecules of interest, whose presence can activate an initiator that can initiate RNAi. Preferred detection targets can be associated with a disease or disorder. In some embodiments, the detection target can be a molecule, or combination of molecules, that is recognized by the initiator, such that the initiator region of the initiator is made available to interact with another monomer. The detection target can be a nucleic acid or any other type of molecule.
An “initiator binding site” is a site on a detection target that can bind to an initiator. Preferably, the initiator binding site is complementary to the detection target complement site of an initiator.
A “silencing target” is a gene of interest, or a combination of genes of interest, that can be silenced by triggered RNAi. Silencing refers to the modulation of the expression of a nucleic acid (e.g., a gene or mRNA). Silencing can refer, for example, to a decreased expression, a reduction of expression, a prevention of expression, or an inhibition of expression of a nucleic acid (e.g., an mRNA silencing target). Preferably, silencing targets can be mRNA. The mRNA can be endogenous or exogenous mRNA.
“Metastable monomers” refer to monomers that, in the absence of a detection target, are kinetically disfavored from associating with other monomers comprising complement regions. In the absence of a detection target, stable monomers are at equilibrium and do not with other monomers comprising complement regions.
As used herein, “polymerization” refers to the association of two or more monomers to form a polymer. The “polymer” may comprise covalent bonds, non-covalent bonds or both. For example, in some embodiments two species of monomers are able to hybridize to form a polymer comprising an RNA duplex substrate.
A “target cell” is a cell that contains or may contain a detection target for triggered RNAi. Examples of target cells include, for example without limitation, cells that contain a nucleic acid signature for a disease, such as, for example, mutant mRNA or fusion mRNA entities. Other examples include, but are not limited to, cells that contain higher-than-background levels of mRNA, peptides, polypeptides, antibodies or fragments thereof, signal cascade molecules, viral particles, bacteria and parasitic organisms.

Exemplary Embodiments

As noted above, RNAi has been adopted as an important tool to study and manipulate gene expression. However, until now, silencing of genes has been limited by the sequence of the inactivator dsRNA. For example, silencing targets are limited to the sequences present in the inactivator dsRNA introduced to the cell or sample.
In some embodiments, methods are provided for triggered RNAi silencing of one or more genes based on the detection of at least one detection target. Thus, in some embodiments, the triggered RNAi approach implements the logical operation: if detection target p is detected, silence gene q. In other embodiments, methods are provided for triggered RNAi silencing of multiple genes. Such triggered RNAi processes would implement the logical operation: if detection target p is detected, silence genes q₁, q₂, q₃. . . .
In some embodiments of triggered RNAi, three or more metastable monomer hairpins are used. The hairpins may comprise loops that are protected by long stems. Hairpin monomers can comprise hairpin loops that are closed by a single base pair. In some embodiments, the loops can be resistant to invasion by complementary single-stranded nucleic acids. This stability allows for the storage of potential energy in the loops. Potential energy is released when a triggered conformational change allows the single-stranded bases in the loops to hybridize with a complementary strand, preferably in a second hairpin monomer. Each monomer is caught in a kinetic trap, preventing the system from rapidly equilibrating. See, for example, Dirks, R. and N. Pierce, Proc. Natl. Acad. Sci. USA 101(43): 15275-15278 (2004), and U.S. patent application Ser. No. 11/087,937, filed Mar. 22, 2005 (published as U.S. Publication No. 2005-0260635 on Nov. 24, 2005), each of which is incorporated herein by reference in its entirety. Thus, pairs of substrate monomers are unable to form an inactivator dsRNA in the absence of a detection target. In some embodiments, the inactivator dsRNA is a Dicer substrate. In other embodiments, the inactivator dsRNA is a RISC substrate (e.g., siRNA itself).
In some embodiments, introduction of one or more detection targets causes the monomers to undergo a series of hybridization events to form an RNA duplex substrate that can be processed by Dicer and subsequently lead to silencing of one or more silencing targets. In other embodiments, a RISC substrate (e.g., siRNA itself) is formed. In some embodiments, triggered RNA monomers can polymerize in the presence of detection targets such as cancer related mRNA sequences or viral nucleic acids and form an RNA duplex substrate. Triggered RNAi leads to gene silencing of one or more silencing targets in cells comprising the detection target, for example diseased cells comprising a disease-associated detection target.
Some embodiments involve methods of treating a patient suffering from a disease or disorder such as, for example, a cancer, autoimmune diseases (e.g., type 1 diabetes and multiple sclerosis), or a viral infection.
For example, in some embodiments, the death of cancer cells containing oncogenic mRNA mutations is triggered, leaving healthy cells untouched. Genetic fusions are common somatic oncogenic mutations and can provide suitable targets for therapy (e.g., triggered RNA therapy) because the mutant oncogenic sequence is up to 50% orthogonal to the wild type sequence. For example, the Philadelphia chromosome, arising from a BCR-ABL fusion, has been established as the unique cause of chronic myeloid leukemia (CML). Other conserved oncogenic fusions are found, for example, in EGFR and in EWS. Single point mutations that lead to activation of an oncogene and single point mutations that inactivate a tumor suppressor are examples of additional targets. One example of an oncogenic single point mutation is the BRAF^T1799Amutation (V600E), found in approximately 6.4% of all human tumors. In some embodiments, the detection target binding region is substantially complementary to a detection target region of a nucleic acid or other molecule that is associated with the disease.
Embodiments disclosed herein can also be used to treat autoimmune diseases (e.g., type 1 diabetes and multiple sclerosis). For example, the destruction of insulin-producing cells in diabetes and the destruction of myelin sheaths in multiple sclerosis is believed to be mediated by autoreactive T-cells that target a specific autoantigen associated with the disease. These diseases can be treated according to embodiments described herein by triggering the death of autoreactive T-cells that bind a specific autoantigen by detecting the mRNA sequences coding for the complementarity determining region 3 (CDR3) of the T-cell receptors specific to that autoantigen. For multiple sclerosis, this can be achieved by triggering apoptosis within autoreactive T-cells that attack the myelin sheath, known to be a patient-specific, oligoclonal population.
Embodiments disclosed herein can also be used to treat autoimmune diseases (e.g., dengue fever). One disease control strategy for this type of mosquito-borne viral infection is to drive genes conferring disease refractoriness into native mosquito populations. To achieve higher than Mendelian replacement frequencies, a maternal-effect selfish genetic element was recently engineered that achieves rapid population replacement in Drosophila. As an alternative (or enhancement) to conferring disease refractoriness, the selfish gene could be linked to a transgene that expresses a triggered RNAi therapeutic. Upon detection of the dengue mRNA, the mechanical therapeutic would trigger the RNAi-mediated silencing of an endogenous housekeeping gene to kill the infected mosquito cell. Triggering cell death rather than attempting to knock down the viral mRNA directly can increase the difficulty of evolving away from the therapy by ensuring that cells containing even a small fraction of the targeted viral mRNA are still marked for death.
In some embodiments the methods comprise administering to target cells in the patient, such as tumor cells, an effective amount of triggered RNAi monomers, one of the monomers being an initiator having a detection target binding region. For example, the detection target binding region of an initiator may be at least 90% complementary to a detection target region of a nucleic acid that is associated with the disease, such as an mRNA associated with the cancer or a viral-associated nucleic acid. In some embodiments, the detection target binding region can be completely complementary to a detection target region of a nucleic acid that is associated with the disease. In some embodiments, the initiator is an aptamer that recognizes a disease-associated molecule. In some embodiments an aptamer is identified that is able to specifically bind a detection target. The detection target is not limited to a nucleic acid but may be, for example, a polypeptide or small molecule. In some embodiments the aptamer is linked to an initiator region in such a way that the initiator region is unavailable to stimulate formation of inactivator dsRNAs in the absence of detection target binding to the aptamer. Preferably, conformation changes in the aptamer secondary structure expose the initiator region. In some embodiments, such an aptamer initiator is a hairpin nucleic acid that comprises an initiator region that is complementary to the sticky end of a substrate monomer and a region of the substrate monomer adjacent to the sticky end, a loop region and an aptamer sequence. The hairpin aptamer initiator may also comprise a region that enhances the stability of the hairpin in the absence of aptamer binding to the detection target.
Detection targets can be any molecule of interest to be used in conjunction with triggered RNAi. In some embodiment a detection target can be an oncogenic mRNA such as an mRNA associated with, for example, a glioma or Ewing's sarcoma. Such mRNAs are disclosed in, for example, Shir, A., and A. Levitzski, Nature Biotechnology 20:895-900 (2002); Dohjima, T. et al., Molecular Therapy 7: 811-816 (2003), each of which is herein incorporated by reference in its entirety. In other embodiments, the detection target can be a fusion sequence associated with tumors. Examples of fusion sequences are provided in, for example, Kim et al., Nucleic Acids Research 34: D22-D24 (2006), which is herein incorporated by reference in its entirety. In some embodiments, the detection target can be endogenous mRNA or naturally occurring mRNA. In other embodiments, the detection target can be exogenous mRNA or mRNA provided to the cells.
In some embodiments, triggered RNAi monomers may be designed to form an inactivator dsRNA in the presence of, for example, a viral gene transcript. In some embodiments, a patient to be treated can be infected with, for example, the human immunodeficiency virus. A target cell is removed from a patient. In a preferred embodiment, the target cell is a CD34-positive hematopoietic stem cell. Such stem cells can be purified by one of skill in the art. Methods for such purification are known and taught for example in U.S. Pat. Nos. 4,965,204; 4,714,680; 5,061,620; 5,643,741; 5,677,136; 5,716,827; 5,750,397 and 5,759,793. Triggered RNAi monomers that are able to form an inactivator dsRNA which can silence one or more silencing targets in the presence of a target in the viral genome are transfected into the isolated CD34-positive stem cells. The treated stem cells are then reintroduced into the patient.

Monomers

Two or more distinct species of nucleic acid monomers can be utilized in a triggered RNAi process. In some embodiments, preferably three or more distinct species of nucleic acid monomers are utilized in a triggered RNAi process. In the methods described herein, the monomers can be, for example, RNA, DNA or RNA-DNA hybrid monomers. Each monomer species typically comprises at least one region that is complementary to a portion of another monomer species. However, the monomers are designed such that they are kinetically trapped and the system is preferably unable to equilibrate in the absence of an activated initiator that can disrupt the secondary structure of one of the monomers. Thus, the monomers are preferably unable to form an inactivator dsRNA in the absence of an activated initiator. Hybridization of an initiator region of an activated initiator to a substrate monomer, or to an intervening monomer that in turn reacts with a substrate monomer, initiates a reaction of kinetic escapes by the monomer species resulting in formation of an inactivator dsRNA. In the examples below, substrate monomers and/or complexes interact in the presence of an activated initiator to form an inactivator dsRNA.
In some embodiments, the initiator can be a monomer. An initiator is preferably unable to trigger formation of an inactivator dsRNA from the other monomers in the absence of a detection target. In some embodiments, the initiator can comprise a hairpin monomer. In some embodiments, the initiator can comprise an aptamer. In some embodiment, the aptamer can comprise the detection target binding region. In some embodiments, the initiator can comprise a recognition molecule. In some embodiments, the initiator can be a conformation-switching aptamer that exposes an initiator region upon binding to a target protein or small molecule.
In some embodiments, the initiator can comprise a substrate monomer.
In some embodiments, one or more monomer species are employed that have a hairpin structure. In some embodiments, three monomer species are employed that have a hairpin structure. In other embodiments, one monomer species is employed that has a hairpin structure.
In some embodiments, the hairpin monomers can comprise loops protected by long stems. In other embodiments, monomers with a different secondary structure are provided. However, in some embodiments, the secondary structure is preferably such that the initiator monomers are metastable or stable in the absence of a detection target and the substrate monomers do not react under the reaction conditions in the absence of an activated initiator that is able to initiate formation of inactivator dsRNA. In some embodiments, at least three monomer species or complexes or combinations thereof including an initiator, a first substrate monomer and a second substrate monomer are used. In some embodiments, in the presence of a detection target, the secondary structure of an initiator can change such that it is able to hybridize to a first substrate monomer species. This in turn preferably leads to a change in the secondary structure of the first substrate monomer, which is then able to hybridize to a second substrate monomer species and form an RNA duplex comprising an inactivator dsRNA region. In some embodiments, the inactivator dsRNA can be an RNA duplex substrate for RNAi, such as a substrate for Dicer. In other embodiments, inactivator dsRNA is siRNA (RISC substrate). In some embodiments, the second substrate monomer is able to displace the first substrate monomer from the initiator once the first substrate monomer has hybridized to the second substrate monomer. In this way, the activated initiator can be recycled and used to initiate formation of more RNA duplex substrates for Dicer. Thus, in the presence of one or more detection targets, multiple RNA duplex substrates can be produced.
A number of criteria can be used to design the monomers to achieve the desired properties. These include, for example and without limitation, sequence symmetry minimization, the probability of adopting the detection target secondary structure at equilibrium, the average number of incorrect nucleotides at equilibrium relative to the target structure, hybridization kinetics, and the silencing target sequence. The composition of the monomers is not limited to any particular sequences or number of bases, and is designed based on the particular detection and silencing targets of the particular triggered RNAi reaction.
Monomers can be synthesized using standard methods, including commercially available nucleic acid synthesizers or obtained from commercial sources such as Integrated DNA Technologies (Coralville, Iowa).
In some embodiments, the monomers are RNA monomers. In some embodiments, the monomers can be RNA-DNA hybrids. In some embodiments, the monomers comprise a protein-binding region, or other recognition molecule. In some embodiments, the monomers can contain a fluorophore, luminescent molecule, colorimetric compound or other component that allows the resulting polymers to be visualized. In embodiments using one or more monomers, complexes can be substituted for monomers such that the embodiments can use monomers or complexes or combinations of monomers and complexes.
In some embodiments, at least three RNA hairpin monomers are utilized as illustrated in FIGS. 2 and 3. In the depicted embodiment, the monomers are denoted “A” (FIG. 2B), “B” (FIG. 2E) and “C” (FIG. 2G). The monomers each preferably comprise a sticky end, a hairpin loop region at the opposite end of the sticky end, and two “stems regions,” a first stem region and a second stem region, that together can form a duplex region. The small letters represent sequence segments. Letters marked with an asterisk (*) are complementary to the corresponding unmarked letter.
In preferred embodiments, the first stem region of a monomer can hybridize to the second stem region of the monomer to form the hairpin structure. For example, as shown in FIG. 2B, the monomer (A) comprises a first stem region comprising a region (x-b) that is able to hybridize to the second stem region (b*-x*). In some embodiments, in the absence of a detection target, the first and second stem regions of each monomer are generally hybridized to form a duplex region of the monomer.
In the depicted embodiment, a monomer (A) (hereinafter referred to as the “initiator”) comprises a “detection target binding region” (comprising the sequence a-x-b) and an “initiator region” comprising the sequence y*-c*-b*. In the depicted embodiment, a first substrate monomer (B) comprises an “initiator complement region” b-c-y and a “silencing target complement region” (comprising the sequence z*-y*-c*). In the depicted embodiment, a second substrate monomer C comprises a “silencing target region” (comprising the sequence c-y-z) and a “recycling region” having the same sequence as the initiator region of the initiator (comprising the sequence y*-c*-b*).
The detection stage of some embodiments of triggered RNAi is depicted in FIGS. 2A-2C. A region (b*-x*-a*) of the detection target (D) and the detection target binding region (a-x-b) of the initiator (A) are typically substantially complementary. That is, the region (b*-x*-a*) of the detection target (D) is able to hybridize to the detection target binding region (a-x-b) of the initiator (A).
The initiator (A) preferably comprises a sticky end a, which is a portion of the detection target binding region (a-x-b). Sticky end a of the initiator is complementary to a sequence segment a* of a detection target (D; FIG. 2A). In some embodiments, the detector complement region of an initiator can comprise a sticky end and a portion of the first stem region of the initiator. For example, in the depicted figure, the initiator (A) has a detection target binding region a-x-b, where a is a sticky end, and x-b is portion of the first stem region of the initiator.
Preferably, upon hybridization of the detection target to the sticky end of the detector complement region of the initiator, one arm of the hairpin structure is displaced. This opens the hairpin. In the depicted embodiment, the detection target (D) nucleates at the sticky end a of the initiator (A) by pairing segment a* with a. This induces a strand displacement interaction resulting in the hybridization of the detection target (D) at a region b*-x*-a* to the detection target binding region a-x-b of the initiator (A) to form the first complex (D.A) (FIG. 2C). The detection target may be, for example, any molecule in whose presence suppression of a target gene is desired, such as a cellular component (such as, for example, a nucleic acid sequence) that is found only in target diseased cells or to a lesser extent in healthy cells, as discussed in more detail below. Detection targets include, without limitation, polypeptides, such as antibodies and antibody fragments, nucleic acids, aptamers, and small molecules. The use of an initiator bound to a recognition molecule is described in more detail below.
In the depicted embodiment, the first complex (D.A) has a newly exposed single-stranded tail that comprises the initiator region (I) having sequence y*-c*-b* of the initiator (A) (FIG. 2C).
In some embodiments, the initiator region of an initiator can comprise a portion of the loop region and a portion of the second stem region of the initiator. For example, in the depicted embodiment, the initiator region (I) of the initiator (A) has a sequence y*-c*-b*, where y*-c* is a portion of the loop region and b* is a portion of the second stem region of the initiator. In the absence of a detection target, the first and second stem regions of the initiator are generally hybridized to form a duplex region of the initiator, and the initiator region of the initiator is generally not available for hybridization to another monomer.
The inactivator dsRNA formation stage of some embodiments of triggered RNAi is depicted in FIGS. 2D-2I. In the depicted embodiment, the first substrate monomer (B) (FIG. 2E) preferably comprises a sticky end b that is complementary to a sequence segment b* of the initiator region (I) of the first complex (D.A) that becomes accessible upon detection target binding to the initiator and opening of the hairpin (FIG. 2D). (For visual simplicity, only the I portion of complex D.A is depicted in FIGS. 2D, 2F, 2H and 21. However, I remains a portion of complex D.A.) The first substrate monomer (B) has an initiator complement region which is substantially complementary to the initiator region of the initiator. In some embodiments, the initiator complement region of the first substrate monomer comprises a sticky end and a portion of the first stem region of the first monomer. For example, in the depicted embodiment, the first substrate monomer (B) has an initiator complement region b-c-y, where b is a sticky end and c-y is a portion of the first stem region.
Preferably, upon hybridization of the exposed initiator region of the first complex to the sticky end of the initiator complement region of the first substrate monomer, one arm of the hairpin structure is displaced. This opens the hairpin. In the depicted embodiment, the initiator region (I) of the first complex (D.A) nucleates at the sticky end b of the first substrate monomer (B) and induces a strand displacement interaction resulting in the hybridization of the initiator region I (y*-c*-b*) of the first complex (D.A) to the initiator complement region (b-c-y) of the first substrate monomer (B) and formation of a second complex (I.B) (FIG. 2F).
In the depicted embodiment, the second complex (I.B) has a newly exposed single-stranded tail that comprises a silencing target complement region (z*-y*-c*) (FIG. 2F).
In some embodiments, the silencing target complement region preferably can comprise a portion of the loop region and a portion of the second stem region of the first substrate monomer. For example, in the depicted embodiment, the silencing target complement region of the first substrate monomer (B) has a sequence z*-y*-c*, where z* is a portion of the loop region and y*-c* is a portion of the second stem region of the first substrate monomer.
In the absence of an activated initiator, the first and second stem regions of the first substrate monomer are generally hybridized to form a duplex region of the first substrate monomer, and the silencing target complement region is generally not available for hybridization to another monomer. The silencing target complement region of the first substrate monomer is exposed by the opening of the hairpin through binding of the initiator I.
The second substrate monomer (C) (FIG. 2G) preferably comprises a sticky end c that is complementary to a sequence segment c* of the silencing target complement region (z*-y*-c*) of the first substrate monomer (B) that becomes accessible upon the binding of the initiator region (I) of the first complex (D.A) to the first substrate monomer (B) (FIG. 2F). The second substrate monomer has a silencing target region which is substantially complementary to the silencing target complement region of the first substrate monomer. In some embodiments, the silencing target region of a second substrate monomer comprises a sticky end and a portion of the first stem region of the second substrate monomer. For example, in the depicted embodiment, the second monomer (C) has a silencing target region c-y-z, where c is a sticky end and y-z is a portion of the first stem region.
Preferably, upon hybridization of the silencing target complement region of the first substrate monomer to the sticky end of the silencing target region of the second substrate monomer, one arm of the hairpin structure is displaced. This opens the hairpin. In the depicted embodiment, the silencing target complement region of the second complex (I.B) nucleates at the sticky end c of the second substrate monomer (C) and induces a strand displacement interaction resulting in the hybridization of the silencing target complement region (z*-y*-c*) of the second complex (I.B) to the silencing target region (c-y-z) of the second substrate monomer (C) and opening the second substrate monomer (C) (FIG. 2H). In the depicted embodiment, the second substrate monomer (C) has a newly exposed single-stranded tail that comprises the recycling region (y*-c*-b*).
In some embodiments, the second substrate monomer can comprise a recycling region having the same sequence as the initiator region of the initiator. In some embodiments, the recycling region can comprise a portion of the loop region and a portion of the second stem region of the second substrate monomer. For example, in the depicted embodiment, the second substrate monomer has a recycling region y*-c*-b*, where y*-c* is a portion of the loop region and b* is a portion of the second stem region of the second substrate monomer. In the absence of a second complex comprising the initiator and the first substrate monomer, the first and second stem regions of the second substrate monomer are generally hybridized to form a duplex region of the second substrate monomer, and the recycling region is generally not available for hybridization to another monomer. The recycling region of the second substrate monomer is exposed by the opening of the hairpin. The exposed recycling region can bind the initiator complement region of the first substrate monomer, thereby displacing the first complex comprising the initiator such that the initiator can be recycled to react with another first substrate monomer.
In the depicted embodiment, after the hairpin is opened to expose the recycling region of the second substrate monomer, the second substrate monomer (C) subsequently displaces the initiator region (I) of the second complex (I.B) to form a third complex (B.C) (FIG. 2I). The third complex (B.C) comprises an inactivator dsRNA. In the depicted embodiment, the inactivator dsRNA is an RNA duplex substrate that can be processed by the RNAi mechanism in the cell. For example, the inactivator dsRNA can be of a size that can be processed by Dicer. In the depicted embodiment, the RNA duplex substrate is a 27-bp RNA duplex substrate comprising the sequence z*-y*-c*. However, the length of the RNA duplex substrate can vary, and will depend on the design of the substrate monomers used to form the duplex substrate. The composition of the inactivator RNAs is discussed in more detail below.
The sequence of the inactivator dsRNA will depend on the sequence of the portion of the triggered RNA monomers used to form the RNA duplex substrate. The sequence of the triggered RNA monomers can be designed to provide an inactivator dsRNA having a desired sequence. Preferably, the sequence of the inactivator dsRNA comprises at least a portion of the sequence of the silencing target. In some embodiments the entire sequence of the inactivator dsRNA corresponds to a portion of the silencing target sequence.
In some embodiments, after displacement by the recycling region of the second substrate monomer, the displaced initiator region (I) can be used in further inactivator dsRNA formation reactions.
The silencing stage of some embodiments of triggered RNAi is depicted in FIGS. 3A-3C. In the depicted embodiment, the RNA duplex substrate (27-bp RNA duplex substrate) of the third complex (B.C) is recognized by Dicer and processed to induce RNAi action (FIGS. 3A-3B). This results in the suppression of the mRNA silencing comprising the target sequence (c-y-z) (FIG. 3C).
FIGS. 4 and 5 illustrate embodiments in which a detection target T interacts with and opens an initiator monomer (initiator) A, that in turn reacts with a first substrate monomer B of complex B.C-1 to open up B, that in turn reacts with a second substrate monomer C of complex B.C-1 to form an inactivator dsRNA duplex B.C-2.
In some embodiments, at least three nucleic acid (e.g., RNA or DNA) hairpin monomers are utilized as illustrated in FIG. 4. In the depicted embodiment, the monomers are denoted “A”, “B”, and “C”. In the absence of the detection target, monomers A, B, C, and the silencing target co-exist metastably. Monomers A (initiator) and B (first substrate monomer) preferably each comprise a sticky end, a hairpin loop region at the opposite end of the sticky end, and two “stems regions,” a first stem region and a second stem region, that together can form a duplex region. Monomer C (second substrate monomer) preferably comprises a hairpin loop region and two “stems regions,” a first stem region and a second stem region, that together can form a duplex region. The small letters represent sequence segments. Letters marked with an asterisk (*) are complementary to the corresponding unmarked letter.
In some embodiments, the first stem region of a monomer can hybridize to the second stem region of the monomer to form the hairpin structure. For example, as shown in FIG. 4, the monomer (A) comprises a first stem region comprising a region (v-y) that is able to hybridize to the second stem region (v*-y*). In some embodiments, in the absence of a detection target, the first and second stem regions of each monomer are hybridized to form a duplex region of the monomer.
In FIG. 4, a monomer (A) (hereinafter referred to as the “initiator”) comprises a “detection target binding region” (comprising the sequence u-v-y-s) and an “initiator region” comprising the sequence x*-y*. In the depicted embodiment, a first substrate monomer (B) comprises an “initiator complement region” x-y and a “silencing target complement region” (comprising the sequence (x*)-(z*)). In the depicted embodiment, a second substrate monomer C comprises a “silencing target region” (comprising the sequence (x)-(z)) and a “recycling region” having the same sequence as the initiator region of the initiator (comprising the sequence x*-y*).
In the depicted embodiment, monomer B comprises a region (y-(z*)) complementary to a region of monomer C (y*-(z)). As illustrated in FIG. 4, the hairpin loop region of B comprises y-(z*) and the hairpin loop region of C comprises y*-(z). This results in the formation of complex B.C-1.
The detection stage of some embodiments of triggered RNAi is depicted in FIG. 4. A region (u*-v*-y*-s*) of the detection target (T) and the detection target binding region (u-v-y-s) of the initiator (A) are typically substantially complementary. For example, the region (u*-v*-y*-s*) of the detection target (T) is able to hybridize to the detection target binding region (u-v-y-s) of the initiator (A).
The initiator (A) preferably comprises a sticky end u, which is a portion of the detection target binding region (u-v-y-s). Sticky end u of the initiator is complementary to a sequence segment u* of a detection target (T; FIG. 4). In some embodiments, the detector complement region of an initiator can comprise a sticky end and a portion of the first stem region of the initiator. For example, in the depicted figure, the initiator (A) has a detection target binding region u-v-y-s, where u is a sticky end, and v-y is portion of the first stem region of the initiator. In other embodiments, the detector complement region of an initiator can comprise a sticky end, a portion of the first stem region of the initiator, and a portion of the hairpin loop region (for example, region s in FIG. 4). Preferably, upon hybridization of the detection target to the sticky end of the detector complement region of the initiator, one arm of the hairpin structure is displaced. This opens the hairpin. In the depicted embodiment, the detection target (T) nucleates at the sticky end u of the initiator (A) by pairing segment u* with u. This induces a strand displacement interaction resulting in the hybridization of the detection target (T) at a region u*-v*-y*-s* to the detection target binding region u-v-y-s of the initiator (A) to resulting in the formation of complex T.A (step (1) in FIG. 4). In the depicted embodiment, T.A has a newly exposed single-stranded tail that contains the sequence v*-y*-x*-t.
The inactivator dsRNA formation stage of some embodiments of triggered RNAi is depicted in FIG. 4. In the depicted embodiment, the single-stranded tail of the T.A complex nucleates at the segment y in the hairpin loop region of monomer B, and induces a strand displacement interaction to open B (step (2)). The exposed region of B nucleates at the hairpin loop of C, thereby opening C. This subsequently results in the formation of duplex B.C-2 and the T.A complex is displaced from monomer B (step (3)). Complex B.C-2 comprises an inactivator dsRNA. For example, the inactivator dsRNA can be of a size that can be processed by Dicer. In the depicted embodiment, complex B.C-2 contains a 25-bp RNA duplex substrate that can be processed by dicer (Integrated DNA Technologies, 2007) in the silencing stage. However, the length of the RNA duplex substrate can vary, and will depend on the design of the substrate monomers used to form the duplex substrate. The composition of the inactivator RNAs is discussed in more detail below.
As illustrated in FIG. 5, in some embodiments, monomer B comprises an external toehold region, r, at the 5′ end of monomer B which can further facilitate the reaction between complex T.A and complex B.C-1 to open B. As illustrated in FIG. 5, the single-stranded tail of the T.A complex nucleates at the segment r in the sticky end of monomer B, and induces a strand displacement interaction to open B (step (2)).
The silencing stage of some embodiments of triggered RNAi is depicted in FIG. 4. In the depicted embodiment, the RNA duplex substrate (25-bp RNA duplex substrate) of complex B.C-2 is recognized by Dicer and processed to induce RNAi action. This results in the suppression of the mRNA target sequence x-z (which is independent of the mRNA detection target u*-v*-y*-s*).
In some embodiments, one nucleic acid (e.g., RNA or DNA) hairpin monomer (A) and two nucleic acid complexes (B.D and C.E) are utilized as illustrated in FIG. 6. FIG. 6 is illustrative of embodiments in which a detection target T interacts with and opens an initiator monomer (initiator) A, that in turn reacts with a monomer (or strand) D of a first substrate complex B.D. to open up D, that in turn reacts with a monomer (or strand) E of a second substrate complex C.E to form an inactivator dsRNA duplex D.E.
Monomer A (initiator) preferably comprise a sticky end, a hairpin loop region at the opposite end of the sticky end, and two “stems regions,” a first stem region and a second stem region, that together can form a duplex region. Complexes B.D and C.E each preferably comprise a bulge loop region, a first stem region, and a second stem region, that together can form a duplex. The stem regions are substantially complementary.
In FIG. 6, a monomer (A) (hereinafter referred to as the “initiator”) comprises a “detection target binding region” (comprising the sequence a*-w*-b*-s*) and an “initiator region” comprising the sequence b-x. In the depicted embodiment, a first substrate complex (B.D) comprises an “initiator complement region” b*-x* and a “silencing target region” (comprising the sequence (x)-(z)). In the depicted embodiment, a second substrate complex C.E comprises a “silencing target complement region” (comprising the sequence (x*)-(z*)). Complex B.D preferably comprises nucleic acid monomers B and D. In the depicted embodiment, regions x* and z* of monomer B are complementary to regions (x) and (z) of monomer D. The monomers are associated into a complex in the absence of a target. Complex C.E preferably comprises nucleic acid monomers C and E. In the depicted embodiment, regions x and z of monomer C are complementary to regions (x*) and (z*) of monomer E. The monomers are associated into a complex in the absence of a target.
In the depicted embodiment, complex B.D comprises a bulge loop region (y*)) complementary to a bulge loop region of complex C.E (y). This results in the formation of complex D.B.C.E. In the absence of the detection target, monomer A, complex B.D, complex C.E, and the silencing target co-exist metastably.
The detection stage of some embodiments of triggered RNAi is depicted in FIG. 6. A region ((a)-(w)-(b)-(s)) of the detection target (T) and the detection target binding region (a*-w*-b*-s*) of the initiator (A) are typically substantially complementary. For example, the region ((a)-(w)-(b)-(s)) of the detection target (T) is able to hybridize to the detection target binding region (a*-w*-b*-s*) of the initiator (A).
The initiator (A) preferably comprises a sticky end a*, which is a portion of the detection target binding region (a*-w*-b*-s*). Sticky end a* of the initiator is complementary to a sequence segment a of a detection target (T; FIG. 6). In some embodiments, the detector complement region of an initiator can comprise a sticky end and a portion of the first stem region of the initiator. For example, in the depicted figure, the initiator (A) has a detection target binding region (a*-w*-b*-s*), where a* is a sticky end, and w*-b* is portion of the first stem region of the initiator. In other embodiments, the detector complement region of an initiator can comprise a sticky end, a portion of the first stem region of the initiator, and a portion of the hairpin loop region (for example, region s* in FIG. 6). Preferably, upon hybridization of the detection target to the sticky end of the detector complement region of the initiator, one arm of the hairpin structure is displaced. This opens the hairpin. In the depicted embodiment, the detection target (T) nucleates at the sticky end a* of the initiator (A) by pairing segment a with a*. This induces a strand displacement interaction resulting in the hybridization of the detection target (T) at a region ((a)-(w)-(b)-(s)) to the detection target binding region a*-w*-b*-s* of the initiator (A) to resulting in the formation of complex T.A (step (1) in FIG. 6). In the depicted embodiment, T.A has a newly exposed single-stranded tail that contains the sequence w-b-x-t.
The inactivator dsRNA formation stage of some embodiments of triggered RNAi is depicted in FIG. 6. In the depicted embodiment, the single-stranded tail of the T.A complex nucleates at the segment b* on monomer B and partially displaces monomer D from monomer B (step (2)). This subsequently results in the formation of duplex B.C and duplex D.E, and the T.A complex is displaced from monomer B (step (3)). Complex D.E comprises an inactivator dsRNA. For example, the inactivator dsRNA can be of a size that can be processed by Dicer. In the depicted embodiment, complex D.E contains a 27-bp RNA duplex substrate that can be processed by dicer (Integrated DNA Technologies, 2007) in the silencing stage. However, the length of the RNA duplex substrate can vary, and will depend on the design of the substrate monomers used to form the duplex substrate. The composition of the inactivator RNAs is discussed in more detail below.
The silencing stage of some embodiments of triggered RNAi is depicted in FIG. 6. In the depicted embodiment, the RNA duplex substrate (27-bp RNA duplex substrate) of complex D.E is recognized by Dicer and processed to induce RNAi action. This results in the suppression of the mRNA target sequence x-z (which is independent of the mRNA detection target a-w-b-s). In some embodiments, different dimensions of the segments can be utilized (FIG. 9). The different dimensions of the segment results in the catalytic formation of a 19 bp RNA duplex that serves as siRNA at the end of step (3), which results in the silencing of the silencing target in step (4) (FIG. 9).
In some embodiments, one nucleic acid (e.g., RNA or DNA) hairpin monomer (A) and two nucleic acid complexes (B.C and D.E) are utilized as illustrated in FIG. 7. FIG. 7 is illustrative of embodiments in which a detection target T interacts with and opens an initiator monomer (initiator) A, that in turn reacts with a monomer (or strand) B of a first substrate complex B.C to displace C, that in turn reacts with a monomer (or strand) D of a second substrate complex D.E to form an inactivator dsRNA duplex C.D.
Monomer A (initiator) preferably comprise a sticky end, a hairpin loop region at the opposite end of the sticky end, and two “stems regions,” a first stem region and a second stem region, that together can form a duplex region. Complex B.C preferably comprises a first stem region and a second stem region that together can form a duplex. Complex D.E preferably comprises a first stem region that can form a duplex.
In FIG. 7, a monomer (A) (hereinafter referred to as the “initiator”) comprises a “detection target binding region” (comprising the sequence a-x-b) and an “initiator region” comprising the sequence (b)-y. In the depicted embodiment, a first substrate complex (B.C) comprises an “initiator complement region” b*-x* and a “silencing target complement region” (comprising the sequence (y)*-(c)*). In the depicted embodiment, a second substrate complex D.E comprises a “silencing target region” (comprising the sequence (y)-(c)). Complex B.C preferably comprises nucleic acid monomers B and C. In the depicted embodiment, regions y and c of monomer B are complementary to regions (y)* and (c)* of monomer C. Complex D.E preferably comprises nucleic acid monomers D and E. In the depicted embodiment, region (y) of monomer D is complementary to regions y* of monomer E.
In the absence of the detection target, monomer A, complex B.C, complex D.E, and the silencing target co-exist metastably.
The detection stage of some embodiments of triggered RNAi is depicted in FIG. 7. A region ((a*)-(x*)-(b*)) of the detection target (T) and the detection target binding region (a-x-b) of the initiator (A) are typically substantially complementary. For example, the region ((a*)-(x*)-(b*)) of the detection target (T) is able to hybridize to the detection target binding region (a-x-b) of the initiator (A).
The initiator (A) preferably comprises a sticky end a, which is a portion of the detection target binding region (a-x-b). Sticky end a of the initiator is complementary to a sequence segment a of a detection target (T; FIG. 7). In some embodiments, the detector complement region of an initiator can comprise a sticky end and a portion of the first stem region of the initiator. For example, in the depicted figure, the initiator (A) has a detection target binding region (a-x-b), where a is a sticky end, and x-b is portion of the first stem region of the initiator. Preferably, upon hybridization of the detection target to the sticky end of the detector complement region of the initiator, one arm of the hairpin structure is displaced. This opens the hairpin. In the depicted embodiment, the detection target (T) nucleates at the sticky end a of the initiator (A) by pairing segment a* with a. This induces a strand displacement interaction resulting in the hybridization of the detection target (T) at a region ((a*)-(x*)-(b*)) to the detection target binding region (a-x-b) of the initiator (A) to resulting in the formation of complex T.A (step (1) in FIG. 7). In the depicted embodiment, T.A has a newly exposed single-stranded tail that contains the sequence s-y*-b*-x *.
The inactivator dsRNA formation stage of some embodiments of triggered RNAi is depicted in FIG. 7. In the depicted embodiment, the single-stranded tail of the T.A complex nucleates at the segment b on monomer B and displaces monomer C from monomer B (step (2)). The displaced monomer C further nucleates at the segment c of the D.E complex and displaces monomer E from monomer D, resulting in the duplex C.D (step (3)). Complex C.D comprises an inactivator dsRNA. For example, the inactivator dsRNA can be of a size that can be processed by Dicer. In the depicted embodiment, complex C.D contains a 27-bp RNA duplex substrate that can be processed by dicer (Integrated DNA Technologies, 2007) in the silencing stage. However, the length of the RNA duplex substrate can vary, and will depend on the design of the substrate monomers used to form the duplex substrate. The composition of the inactivator RNAs is discussed in more detail below.
The silencing stage of some embodiments of triggered RNAi is depicted in FIG. 7. In the depicted embodiment, the RNA duplex substrate (27-bp RNA duplex substrate) of complex C.D is recognized by Dicer and processed to induce RNAi action. This results in the suppression of the mRNA target sequence y-c (which is independent of the mRNA detection target b*-x*-a*). In some embodiments, different dimensions of the segments can be utilized (FIG. 10). the different dimensions of the segment results in the catalytic formation of a 19 bp RNA duplex that serves as siRNA at the end of step (3), which results in the silencing of the silencing target in step (4) (FIG. 10).
In some embodiments, at least two nucleic acid (e.g., RNA or DNA) hairpin monomers are utilized as illustrated in FIG. 8. In the depicted embodiment, the monomers are denoted “A” and “B”. In the absence of the detection target, monomer A, monomer B, and the silencing target co-exist stably. FIG. 8 is illustrative of embodiments in which a detection target T interacts with and opens an initiator monomer (initiator) A, that in turn reacts with a first substrate monomer B to form an inactivator dsRNA duplex T.A.B.
Monomers A (initiator) preferably comprises a sticky end, a hairpin loop region at the opposite end of the sticky end, and two “stems regions,” a first stem region and a second stem region, that together can form a duplex region. Monomer B (substrate monomer) preferably comprises a sticky end, a hairpin loop region at the opposite end of the sticky end, and one “stems region” that can form a duplex region.
In FIG. 8, a monomer (A) (hereinafter referred to as the “initiator”) comprises a “detection target binding region” (comprising the sequence (a)*-(x)*), an “initiator region” (comprising the sequence (b)-(y)-(c)), and a “silencing target complement region” (comprising the sequence (b)-(y)-(c)). In the depicted embodiment, the initiator region and the silencing target complement region comprise identical sequences. In some embodiments, the initiator region and the silencing target complement region comprise substantially identical sequences. In the depicted embodiment, a substrate monomer B comprises an “initiator complement region” (comprising the sequence (b)*-(y)*-(c)*) and a “silencing target region” (comprising the sequence (b)*-(y)*-(c)*). In the depicted embodiment, the initiator complement region and the silencing target region comprise identical sequences. In some embodiments, the initiator complement region and the silencing target region comprise substantially identical sequences.
The detection stage of some embodiments of triggered RNAi is depicted in FIG. 8. A region ((x)-(a)) of the detection target (T) and the detection target binding region ((a)*-(x)*) of the initiator (A) are typically substantially complementary. For example, the region ((x)-(a)) of the detection target (T) is able to hybridize to the detection target binding region ((a)*-(x)*) of the initiator (A).
The initiator (A) preferably comprises a sticky end (a)*, which is a portion of the detection target binding region ((a)*-(x)*). Sticky end (a)* of the initiator is complementary to a sequence segment (a) of a detection target (T; FIG. 8). In some embodiments, the detector complement region of an initiator can comprise a sticky end and a portion of the first stem region of the initiator. For example, in the depicted figure, the initiator (A) has a detection target binding region (a)*-(x)*, where (a)* is a sticky end, and (x)* is portion of the first stem region of the initiator Preferably, upon hybridization of the detection target to the sticky end of the detector complement region of the initiator, one arm of the hairpin structure is displaced. This opens the hairpin. In the depicted embodiment, the detection target (T) nucleates at the sticky end (a)* of the initiator (A) by pairing segment (a) with (a)*. This induces a strand displacement interaction resulting in the hybridization of the detection target (T) at a region (x)-(a) to the detection target binding region (a)*-(x)* of the initiator (A) to resulting in the formation of complex T.A (step (1) in FIG. 8). In the depicted embodiment, T.A has a newly exposed single-stranded tail that contains the sequence b*-s-c-y-b-x.
The inactivator dsRNA formation stage of some embodiments of triggered RNAi is depicted in FIG. 8. In the depicted embodiment, the single-stranded tail of the T.A complex nucleates at the segment b* on monomer B and opens monomer B (step (2)). This subsequently results in the formation of complex T.A.B (step (2)). Complex T.A.B comprises an inactivator dsRNA. For example, the inactivator dsRNA can be of a size that can be processed by Dicer. In the depicted embodiment, complex T.A.B contains a 27-bp RNA duplex substrate that can be processed by dicer (Integrated DNA Technologies, 2007) in the silencing stage. However, the length of the RNA duplex substrate can vary, and will depend on the design of the substrate monomers used to form the duplex substrate. The composition of the inactivator RNAs is discussed in more detail below.
The silencing stage of some embodiments of triggered RNAi is depicted in FIG. 8. In the depicted embodiment, the RNA duplex substrate (27-bp RNA duplex substrate) of complex T.A.B is recognized by Dicer and processed to induce RNAi action. This results in the suppression of the mRNA target sequence (b)*-(y)*-(c)* (which is independent of the mRNA detection target (x)-(a)).
The mRNA silencing target sequence can be a portion of the sequence of any gene for which silencing is desirable in the presence of the detection target. The silencing target can be a sequence which may or may not be associated with a disease or disorder. Silencing targets are discussed in detail below.
In some embodiments, the result is an RNA duplex comprising an inactivator dsRNA. In some embodiments, the inactivator dsRNA can comprise an RNA duplex substrate that can be processed by Dicer. Dicer processing of the RNA duplex substrate produces small interfering RNAs (siRNAs). The siRNAs can then be processed by the RISC complex, which unwinds the siRNA and retains one strand as a targeting co-factor. The RISC-siRNA complex recognizes and induces the degradation of the silencing target mRNA, which is complementary to the siRNA strand retained by the RISC complex.
The length of the hairpin loop, stem regions and sticky ends of the monomers can be adjusted, for example to ensure kinetic stability in particular reaction conditions and to adjust the rate of polymerization in the presence of initiator. In some embodiments the length of the sticky ends is the same as the length of the hairpin loops. In other embodiments the sticky ends are longer or shorter than the hairpin loops. In some embodiments, if the hairpin loops are longer than the sticky ends, the hairpin loops can comprise a region that is complementary to the sticky end of a monomer.
In some embodiments the length of the hairpin loops is short relative to the stems. For example, the stems may be two or three times as long as the hairpin loops.
The loop regions are preferably between about 1 and about 100 nucleotides, and more preferably between about 3 and about 30 nucleotides. In one embodiment the loops and sticky ends of a pair of hairpin monomers are about 6 nucleotides in length and the stems are about 18 nucleotides long.
The regions (e.g., a, b, c, x, y and z) are not limited to any particular sequences or number of bases, and are designed based on the particular detection and silencing targets of the triggered RNAi reaction. For example, in FIGS. 2 and 3, sample segment lengths expressed as numbers of bases can be, for example without limitation, a=7, b=7, c=7, x=8, y=8, and z=12. Sample compositions (RNA or DNA) for segments of the first, second and third monomers (A, B and C) can be, for example without limitation: A: a-x-b-y*-c*-b*-x*; B: b-c-y-b*-(z*-y*-c*); C: (c-y-z)-b-y*-c*-b*-z*-y*, where RNA segments are contained inside parentheses and DNA segments are not contained inside parenthesis. For example, in FIG. 4, sample segment lengths expressed as numbers of bases can be, for example without limitation, u=10, v=10, y=10, s=2, t=2, and p=2, x-z=13-12 or 15-10 or 16-9. For example, in FIG. 5, sample segment lengths expressed as numbers of bases can be, for example without limitation, r=6, and p=2, x-y-z=10-15-7 or 14-11-3 or 16-9-1. For example, in FIG. 6, sample segment lengths expressed as numbers of bases can be, for example without limitation, a=10, w=13, y=10, s=2, t=2, b=6, x=13, z=14, and y=17. For example, in FIG. 7, sample segment lengths expressed as numbers of bases can be, for example without limitation, p=2, b=6, y+c=27, and c=3, 4, 5, or 6. For example, in FIG. 8, sample segment lengths expressed as numbers of bases can be, for example without limitation, a=9, b=5, c=8, x=15, y=14, and s=0, 1, 2, 3, or 4. The segment (s) is optional and can be designed to disrupt the interaction between (y) and (b*), which are not determined by target RNA sequences. For example, in FIG. 9, sample segment lengths expressed as numbers of bases can be, for example without limitation, a=10, w=13, s=2, t=2, b=6, x=10, z=9, y=10, 20, 30, or 40, and p=2. For example, in FIG. 9, sample segment lengths expressed as numbers of bases can be, for example without limitation, p=2, b=6, y+c=19, c=3, 4, 5, or 6.
Other refinements to the system stabilize the monomer hairpins to help prevent triggered RNAi in the absence of an activated initiator. This can be achieved, for example, via super-stable hairpin loop sequences (Nakano et al. Biochemistry 41:14281-14292 (2002), herein incorporated by reference in its entirety), with ostensible structural features that could further inhibit direct hybridization to the hairpin. In other embodiments hairpin loops are made to be self-complementary at their ends. This self-complementation “pinches” the hairpin loops, making them shorter. However, if the reactive sticky ends of each monomer are complementary to the loop regions on the opposite monomer, as described above, they will have a slight propensity to close up, thereby slowing down the reaction. This feature can be utilized if a slower reaction is desired. Completely self-complementary hairpins can also be used, for example if the monomer hairpins are forming dimers with interior loops that are more easily invaded than their hairpin counterparts.
Reaction conditions are preferably selected such that hybridization is able to occur, including between the detection target and the sticky end of an initiator, between the initiator region of an initiator and a sticky end of a first monomer, between the silencing target complement region of a first monomer and the sticky end of a second monomer, between the first and second stem regions of the monomers themselves, and between monomers that comprise a complex. At each step of monomer polymerization, energy is gained from the hybridization of the sticky end of the monomer. The reaction temperature does not need to be changed to facilitate the polymerization of triggered RNAi monomers. That is, the triggered RNAi monomer polymerization reactions are isothermic. They also do not require the presence of any enzymes. However, in some embodiments, Dicer is used to process the dsRNA substrate produced by polymerization of the monomers. In some embodiments, the RISC complex is used to unwind the siRNA and induce the degradation of the silencing target mRNA, which is complementary to the siRNA strand retained by the RISC complex.

Detection Targets

The detection target is preferably a nucleic acid or other molecule that is able to contact the initiator and trigger RNAi through the formation of an inactivator dsRNA. The detection target can comprise, but is not limited to, any of the following: a nucleic acid sequence, a peptide, a polypeptide, an antibody or fragment thereof, a signal cascade molecule, a lipid, a carbohydrate, a fused entity, a viral particle, a bacterium or a parasitic organism. In some embodiments, the detection target can be a portion of a nucleic acid associated with a disease or disorder.
In some embodiments the detection target is preferably a nucleic acid molecule. The nucleic acid detection target comprises a sequence that is complementary to a portion, such as, for example, a sticky end, of an initiator that is available for hybridization with the detection target while the initiator is in its kinetically stable state. The detection target also preferably comprises a sequence that is complementary to a portion of the initiator adjacent to the sticky end such that hybridization of the detection target to the sticky end causes a conformational change in the initiator and begins the triggered RNAi reaction. For example, the detection target may comprise a region that is complementary to the detection target binding region of the initiator, as described above and illustrated in FIG. 2A.
In some embodiments, the detection target binding region of the initiator is preferably at least 80%, more preferably at least 90%, 95% or higher, identical to at least a portion of detection target. In preferred embodiments, the detection target binding region is at least 2, 3, 4, 5, or 10 or more bases in length.
In various embodiments, the detection target can be, for example, a protein, a carbohydrate, a fatty acid, a hormone, or a polynucleotide. In some embodiments, the detection target can be a nucleic acid sequence. In some embodiments, the detection target can be at least a portion of the sequence of any gene for which silencing is desirable in the presence of the detection target. In some embodiments, the silencing target can be an mRNA. In other embodiments, the silencing target can be DNA. The silencing target can be a sequence which may or may not be associated with a disease or disorder.
In some embodiments, the detection target can be a nucleic acid, or a portion of a nucleic acid sequence that is necessary for the life cycle or replication of a virus, such as, for example, gene expression of the virus and the expression of a cellular receptor or co-receptor necessary for viral replication. In some particular embodiment of the invention, the virus is the human immunodeficiency virus (HIV). The detection target sequence may be, for example, selected from the group consisting of Rev, Gag, Pol, LTRs, TAR, RRE, att, pbs, ppt and other essential DNA and RNA cis-regulatory elements. In one embodiment of the invention, the detection target is an expressed region of the HIV viral genome, for example, a portion of the 9-kb transcript of the integrated HIV virus, or any of the variously spliced mRNA transcripts of HIV (Schwartz, S; Felber, B K; Benko, D M; Fenya, E M; Pavlakis, G N. Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1. J. Virol. 1990; 64(6): 2519-29). Target regions within the HIV transcripts can be chosen to correspond to any of the viral genes, including, for example, HIV-1 LTR, vif, nef, and rev.
In some embodiments, the detection target can be a sequence that is necessary for the life cycle or replication of a tumor cell. In other embodiments, the detection target can be a sequence that is indicative of a pre-cancerous state, such as, for example, an oncogene sequence. In some embodiments, the detection target can be a PKR sequence. In some embodiments, the detection target can be a marker such as a molecule associated with a specific cell type.

Silencing Targets

In preferred embodiments, the silencing target can be an mRNA. In various embodiments, the silencing target can be a nucleic acid sequence. In various embodiments, the silencing target can comprise at least a portion of the nucleic acid sequence of any gene for which silencing is desirable in the presence of the detection target. The silencing target can be a nucleic acid sequence which may or may not be associated with a disease or disorder.
In some embodiments, the silencing target can be a nucleic acid of at least a portion of a gene. In some embodiments, the gene can be a housekeeping gene. Housekeeping genes are known in the art, and a list of housekeeping genes is provided in, for example, Eisenberg et al., Trends in Genetics, 2003, 19(7): 362-365, which is hereby incorporated by reference in its entirety. In some embodiments, the gene can encode a structural protein such as, for example, actin or tubulin. In some embodiments, the silencing target sequence can be an mRNA encoding a motor protein, such as, for example, kinesin. In some embodiments, the gene can encode an enzyme involved in cell metabolism such as, for example, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, dehydrogenase ATP synthase, glycogen synthase kinase 3 alpha, and the like. In some embodiments, the gene can encode a ribosomal protein. In some embodiments, the gene can encode a cell cycle regulatory molecule such as, for example, p21, p19ARF, or cyclin. In some embodiments, the gene can encode an enzyme involved in protein degradation such as, for example, a ubiquitin activating enzyme or a ubiquitin-conjugating enzyme. In some embodiments, the gene can encode a kinase, such as, for example, protein kinase C. In some embodiments, the silencing target can be a cell death regulatory molecule such as, for example, apoptosis inhibitor 5 (API5) or a Bcl-2 family member. In some embodiments, the gene can encode a transcription factor. In some embodiments, the gene can encode a polymerase, such as RNA polymerase I. In some embodiments, the gene can encode an oncogene, such as c-myc, src or c-ras. In some embodiments, the gene can encode PKR. In some embodiments, the gene can encode an inhibitor of PKR.
In some embodiments, the silencing target can be a metabolic pathway gene.
In some embodiments, the silencing target can be a nucleic acid sequence that is necessary for the life cycle or replication of a virus, such as, for example, gene expression of the virus and the expression of a cellular receptor or co-receptor necessary for viral replication.
In some embodiments, the silencing target can be an mRNA encoding a reporter molecule, such as, for example, green fluorescence protein (GFP) and other imaging or diagnostic markers. The detection of a detection target of interest can thus result in a change of, for example, the fluorescence emission of GFP via triggered RNAi.

Diseases

Diseases contemplated for treatment in embodiments of the invention include any disease in which a detection target, such as a target associated with the disease is present in a cell and can initiate polymerization of triggered RNAi hairpin monomers, and wherein inhibition of translation of particular gene targets and/or cell death would be beneficial to a patient. The detection target can act on an initiator to trigger formation of RNA duplex substrates for RNAi machinery such as Dicer. Preferred embodiments include, but are not limited to, diseases in which the detection target is a nucleic acid molecule. In some embodiments, the nucleic acid molecule is an mRNA molecule associated with a disease or disorder, such as a mutant mRNA molecule. However, disease-associated RNAi detection targets can be, for example and without limitation, nucleic acid sequences, proteins, peptides, lipids, carbohydrates and small molecules.
In some embodiments, the disease to be treated is a type of cancer, such as, for example, leukemia, carcinoma, lymphoma, astrocytoma, sarcoma and particularly Ewing's sarcoma, glioma, retinoblastoma, melanoma, Wilm's tumor, bladder cancer, breast cancer, colon cancer, hepatocellular cancer, pancreatic cancer, prostate cancer, lung cancer, liver cancer, stomach cancer, cervical cancer, testicular cancer, renal cell cancer, and brain cancer.
In other embodiments, the disease to be treated is associated with infection by an intracellular parasite. For example, the intracellular parasite may be a virus such as, for example, an adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus, human herpes virus 6, varicella-zoster virus, hepatitis viruses, papilloma virus, parvovirus, polyomavirus, measles virus, rubella virus, human immunodeficiency virus (HIV), or human T cell leukemia virus. In other embodiments, the intracellular parasite may be a bacterium, protozoan, fungus, or a prion. More particularly, the intracellular parasite can be, for example, Chlamydia, Listeria, Salmonella, Legionella, Brucella, Coxiella, Rickettsia, Mycobacterium, Leishmania, Trypanasoma, Toxoplasma, and Plasmodium.
In other embodiments, silencing targets are not associated with a disease or disorder. For example, a silencing target may be used to inhibit or activate a reporter molecule, either in vitro or in vivo, in response to the presence of a detection target. Thus, methods disclosed herein can be used for detecting the presence of a detection target in a sample or cells.

Initiators

As discussed above, an initiator is a molecule that is able to interact with a detection target and subsequently expose an initiator region. The “activated initiator,” i.e., one in which the initiator region has been exposed, can initiate formation of inactivator dsRNAs by initiating polymerization of monomers. In some embodiments, an activated initiator can initiate formation of inactivator dsRNAs by initiating further hybridization of monomers. One exemplary embodiment of an activated initiator is shown in, for example, FIG. 2C. Other exemplary embodiments of activated initiators are shown for example, in FIGS. 3-10.
In various embodiments, the initiator comprises a detection target binding region such that an initiator region of the initiator is made available when a predetermined physical event occurs. In the preferred embodiments, that predetermined event can be the presence of a detection target such as one associated with a disease, disorder or any undesirable state. In each of these embodiments, the initiator preferably comprises a molecule that is responsive to the presence of the detection target. In preferred embodiments, the initiator region of the initiator is made available in the presence of the detection target. For example, the initiator may comprise a molecule that undergoes a conformational change in response to binding to the detection target. The conformational change can expose the initiator region of the initiator, which can subsequently stimulate formation of the dsRNA substrates.
As described above, in some embodiments, the detection target binding region of the initiator is preferably at least 80%, more preferably at least 90%, 95% or higher, complementary to at least a portion of a detection target. In preferred embodiments, the detection target binding region is at least 2, 3, 4, 5, or 10 or more bases in length. Furthermore, in some embodiments, the initiator region of the initiator is preferably at least 80%, more preferably at least 90%, 95% or higher, complementary to an initiator complement region of a substrate monomer. In preferred embodiments, the initiator region region is at least 2, 3, 4, 5, or 10 or more bases in length.
An aptamer is identified that is able to specifically bind a detection target molecule within a diseased cell. In some embodiments, the detection target binding region can be an aptamer.
The initiator preferably comprises a nucleic acid or other molecule that is able to contact a substrate monomer and trigger the formation of inactivator dsRNAs in the presence of a detection target. In some embodiments, the initiator comprises a sequence that is complementary to a portion, such as, for example without limitation, a sticky end, of a monomer, that is available for hybridization with the activated initiator while the monomer is in its kinetically stable state. In some embodiments, the initiator also preferably comprises a sequence that is complementary to a portion of the monomer adjacent to the sticky end such that hybridization of the monomer to the sticky end causes a conformational change in the monomer. For example, the initiator may comprise an initiator region that is complementary to the initiator complement region of a monomer, where the initiation region comprises a sticky end and a portion of a first stem region of the monomer adjacent to the sticky end, as described above and illustrated in FIG. 2B.
The structure of the initiator is preferably such that when the detection target is not present (or the other physical event has not occurred), the initiator region is not available to hybridize with an initiator complement region of a substrate monomer. The detection target frees the initiator region such that it can interact with an initiator complement region of a substrate monomer, triggering the formation of inactivator dsRNAs. In some embodiments, the detection target causes a conformational change in the initiator that allows the initiator to interact with the initiator complement region of a substrate monomer. Preferably, the initiator region is unavailable to hybridize to an initiator complement region unless the initiator is activated by binding to a detection target. In some embodiments, the initiator region of the initiator is substantially complementary to the initiator complement region of a substrate monomer. In some embodiments, the initiator complement region of a substrate monomer can comprise, for example, a sticky end and a portion of the first stem region of a substrate monomer.
In preferred embodiments, the initiator can comprise a detection target binding region that is complementary to an mRNA detection target sequence or a portion of an mRNA detection target sequence, for example, a portion of a mutant mRNA sequence that comprises a mutation associated with a disease or disorder. Embodiments also include specific combinations of mRNA sequences.
The initiator may comprise a nucleic acid initiator region that is linked to or associated with a recognition molecule, such as an aptamer, that is capable of interacting with a detection target. The initiator is designed such that the initiator region is unavailable to stimulate production of inactivator dsRNAs in the absence of the detection target. When the detection target interacts with the recognition molecule, the initiator region of the activated initiator is able to trigger RNAi. Preferably, the recognition molecule is one that is capable of binding the detection target.
Recognition molecules include, without limitation, polypeptides, antibodies and antibody fragments, nucleic acids, aptamers, and small molecules. The use of an initiator bound to an aptamer, where the aptamer serves as the detection target binding region.
Inactivator dsRNAs
In triggered RNAi, the silencing targets can be independent of the detection targets. Thus, triggered RNAi is considered an effective strategy for silencing genes in cells that contain a target molecule, where the target molecule may be unrelated to the gene that is silenced. Once an inactivator dsRNA forms within a target cell via the triggered RNAi mechanism, the inactivator dsRNA can lead to silencing of one or more desired silencing targets. In some embodiments, the inactivator dsRNA can be processed by Dicer, silencing of one or more desired silencing targets. Design of appropriate triggered RNAi hairpin monomers that do not themselves trigger RNAi in the absence of a detection target, but trigger RNAi upon binding a detection target, can be derived from detection target and silencing target sequences well known in the art and available from literature reviews and disclosed in, for example, various databases (e.g., NCBI).
The length and composition of the inactivator dsRNAs and RNA duplex substrates for Dicer can be controlled by the design of the substrate monomers used to form the inactivator dsRNAs. At least a portion of the inactivator dsRNA sequence is preferably homologous to at least a portion of the silencing target sequence. In some embodiments, the inactivator dsRNA formed is a RISC substrate that bypasses dicer action.
Design of RNA duplex substrates for Dicer is described in, for example, Amarzguioui et al., 2006, Nature Protocols 1, 508-517 and Vermeulen et al., 2005, RNA 11(5): 674-682. In some embodiments, the length of the RNA duplex substrate formed by substrate monomers is from about 12 to about 100 bp. In some embodiments, the length of the RNA duplex substrate formed by substrate monomers is from about 23 to about 30 bp. In some embodiments, the RNA duplex substrate can be about 25 to about 27 bp. In some embodiments, the RNA duplex substrate can be about 27 bp. In some embodiments, the RNA duplex substrate can be a blunt 27-mer duplex. In some embodiments, the RNA duplex substrate can comprise a single 2-base 3′-overhang. In some embodiments, the RNA duplex substrate can comprise a combination of a single 2-base 3′-overhang on one end and DNA bases on the opposing blunt end. In some embodiments, the RNA duplex substrate can comprise a single 1, 2, 3, 4 or 5-base 3′-overhang. RNA monomers may be designed to produce RNA duplex substrates, for example, by designing two complementary RNA strands where one strand has two additional non-complementary base pairs at an end.
In some embodiments, the RNA duplex substrate can comprise a 3′-overhang on the antisense. In some embodiments, the RNA duplex substrate can comprise a 3′-overhang resides on the sense (S) strand. In some embodiments, processing of the RNA duplex by Dicer can produce siRNAs comprising, for example, 21-nt passenger and guide strands that form a 19 bp duplex with 2-nt 3′ overhangs. See, e.g., Kim et al., 2007, Nature Reviews Genetics 8(3): 711-719. In some embodiments, processing of the RNA duplex by Dicer can produce siRNAs comprising, for example, 21-nt passenger and guide strands that form a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21-bp duplex with 0, 1, 2, 3, 4, 5-nt 3′ overhangs.
In some embodiments, the inactivator dsRNAs can be about 17 to about 27 bp long. In some embodiments, the inactivator dsRNAs can be about 25 to about 27 bp long. In some embodiments, the inactivator dsRNAs can comprise two 21-nt strands that form a 19 bp duplex with 2-nt 3′ overhangs. In some embodiments, the inactivator dsRNAs can comprise two 21-nt strands that form a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21-bp duplex with 0, 1, 2, 3, 4, 5-nt 3′ overhangs. In some embodiments, the inactivator can comprise an RNA duplex of about 10 to about 100 bases pairs with about 0 to about 100 nucleotides overhangs on any or all of the 3′ and 5′ ends of the duplex.

Gene Silencing Using Triggered RNAi

Because the silencing targets are independent of the detection targets, triggered RNAi is considered an effective strategy for silencing genes in cells containing a particular detection target by forming inactivator dsRNA which silences an independent gene. Once an inactivator dsRNA forms within a target cell, the inactivator dsRNA substrate leads to silencing of one or more silencing targets. In some embodiments, the inactivator dsRNA can be processed by Dicer, leading to subsequent silencing of desired silencing target(s). Design of appropriate triggered RNAi monomers that do not themselves trigger RNAi, but trigger RNAi upon binding a detection target, can be derived from detection target and silencing target sequences well known in the art and available from literature reviews and disclosed in, for example, various databases (e.g. NCBI).
Triggered RNAi can be performed, for example, using RNA hairpin monomers and/or complexed monomers (complexes) to detect a detection target, such as a target associated with a disease or a disorder and subsequently silence an independent silencing target. In some embodiments, the detection target can be associated with a cellular membrane. In other embodiments, the detection target can be a non-tethered entity. The detection target can be, for example, a nucleic acid associated with cancer, such as an mRNA associated with a cancer. In various embodiments, an RNA hairpin initiator with an initiator region hybridizes or binds the detection target. Binding of the detection target to the initiator causes a conformational change of the initiator and exposes the initiator region. Hybridization between the initiator complement region of a first substrate monomer and the exposed initiator region of the activated initiator causes a conformational change of the first substrate monomer to expose a sequence that is substantially complementary to the silencing target region of a second substrate monomer.
Hybridization between the exposed sequence of the first substrate monomer and the silencing target region of the second substrate monomer causes a conformational change of a second substrate monomer. This event forms an inactivator dsRNA. In some embodiments, the inactivator dsRNA can be a substrate for Dicer. Dicer can process the dsRNA substrate into siRNAs, which associate with the RISC complex and subsequently silence one or more silencing target. In other embodiments, the inactivator dsRNA is not processed by Dicer, but can still lead to silencing of one or more silencing targets. For example, the inactivator dsRNA can itself serve as a RISC substrate (e.g., as siRNA). Depending on the nature of the silencing target, the triggered RNAi action can lead to, for example, PKR activation, PKR inactivation, cell death, or extension of cell life.
In some embodiments, triggered RNAi can be used to treat a patient suffering from a disease or disorder. Because triggered RNAi can be specifically limited to those cells containing the detection target associated with the disease or disorder, the triggered RNAi monomers can be delivered to a multitude of cells, including healthy cells. Thus, general delivery of the monomers to a population of cells comprising cells containing a disease-associated detection target and wild-type cells is possible. Triggered RNAi takes place in diseased cells containing the detection target, leading to the silencing of one or more silencing targets in cells in therapeutic treatments. In a particular embodiment, triggered RNAi can occur in cells comprising an mRNA molecule associated with cancer, leading to silencing of housekeeping gene(s) and subsequently the death of cancerous cells.

Delivery of Triggered RNAi Monomers and Initiators to Target Cells

Initiators, triggered RNAi monomers, complexed monomers (complexes) and any accessory molecules, such as, for example, helper molecules, can be formulated with any of a variety of carriers well known in the art to facilitate introduction into a cell. Suitable carriers for delivery of nucleic acids to cells are well known in the art and include, for example, polymers, proteins, carbohydrates and lipids. For example, a cyclodextrin-containing polymer can be used for the delivery of the triggered RNAi monomers. Commercial transfection reagents known in the art, such as, for example, LNCaP (Altogen Biosystems) or lipofectamine RNAiMax (Invitrogen), can be used.
Delivery of nucleic acids can be accomplished, for example, as described by Heidel (Heidel, J. D. 2005. Targeted, systematic non-viral delivery of small interfering RNA in vivo. Doctoral thesis, California Institute of Technology. 128 p., herein incorporated by reference in its entirety). Also contemplated within the scope of the subject matter are gene delivery systems as described by Feigner et al. (Feigner et al. 1997. Hum Gene Ther 8:511-512, herein incorporated by reference in its entirety), including cationic lipid-based delivery systems (lipoplex), polycation-based delivery systems (polyplex) and a combination thereof (lipopolyplex). Cationic lipids are described, for example, in U.S. Pat. Nos. 4,897,355 and 5,459,127, each of the foregoing which is herein incorporated by reference in its entirety. Proteins can also be used for HCR delivery, such as synthetic neoglycoproteins (Ferkol et al. 1993. FASEB J 7:1081-1091; Perales et al. 1994. Proc Nat Acad Sci 91:4086-4090; each of which is incorporated herein by reference in its entirety), epidermal growth factor (EGF) (Myers, EPO 0273085, incorporated herein by reference in its entirety), and other ligands for receptor-mediated gene transfer (Wu and Wu. 1987. J Biol Chem 262(10):4429-4432; Wagner et al. 1990. Proc Natl Acad Sci USA 87(9):3410-3414; Ferkol et al. 1993. J. Clin Invest 92(5):2394-2300; Perales et al. 1994. Proc Natl Acad Sci USA 91(9):4086-4090; Myers, EPO 0273085; each of which is incorporated herein by reference in its entirety).
Viral and viral vector-like delivery systems generally known in the art, such as those described, for example, in U.S. Pat. No. 7,0333,834; U.S. Pat. No. 6,899,871; U.S. Pat. No. 6,555,367; U.S. Pat. No. 6,485,965; U.S. Pat. No. 5,928,913; U.S. patent application Ser. No. 10/801,648; U.S. patent application Ser. No. 10/319,074, and U.S. patent application Ser. No. 09/839,698, each of which is herein incorporated by reference, are also contemplated for use in the present subject matter. In addition, standard electroporation techniques can be readily adopted to deliver HCR monomers.
Delivery of triggered RNAi monomers can occur in vivo or ex vivo. In some embodiments, cells can be removed from a patient, transfected with the monomers and returned to the patient for therapeutic effects. In other embodiments, triggered RNAi monomers can be delivered to cells in vivo such as by, for example, injection of the monomers within a delivery vehicle into the bloodstream or by intramuscular, subcutaneous, or intraperitoneal means. An appropriate means of delivering triggered RNAi monomers to a desired population of cells can be identified by the skilled practitioner based on the particular circumstances without undue experimentation.

Initiation of Triggered RNAi

Activated initiators can serve as initiators of triggered RNAi. In embodiments based on detection of a disease-associated mRNA sequence, an initiator can be designed that only initiates formation of inactivator dsRNAs in the presence of the detection target mRNA sequence. If an mRNA detection target sequence is chosen that is only expressed in certain types of cells e.g. cells with a particular genetic mutation or nucleic acid signature, then triggered RNAi will only occur in the cells expressing the target sequence. Polymerization of triggered RNAi monomers will not occur in cells without the detection target mRNA sequence. Triggered RNAi creates inactivator dsRNAs, which leads to silencing of one or more silencing targets.
Detection targets contemplated for triggered RNAi include those associated with a disease or disorder, as well as other non-disease associated targets, such as a cell-type marker, as described herein and in the art. In some embodiments the detection target is a nucleic acid that is to be detected in a sample or a portion of a nucleic acid that is to be detected. In this case, the sequence of the detection target nucleic acid is taken into consideration in designing the triggered RNAi monomers and/or complexes. In some embodiments, the detection target can be a nucleic acid signature or specific mutation in a genetic sequence associated with a disease or disorder. Genetic mutations include, but are not limited to, point mutations, non-native genetic fusions, deletion of at least one base, insertion of at least one base, frame-shift mutations, and inversions. In other embodiments, the detection target is a combination of nucleic acid molecules associated with a disease or disorder.
Detection targets for triggered RNAi also include, but are not limited to, nucleic acid molecules, proteins, peptides, carbohydrates, lipids and small molecules. In preferred embodiments, the detection target binds a molecule, an initiator, which can initiate formation of inactivator dsRNAs. In some embodiments, the detection target can initiate formation of inactivator dsRNAs by, for example, binding directly to a substrate monomer.
Where the disease to be treated is a cancer, an mRNA detection target is typically one expressed in cancer cells and not in healthy cells or at least to a lesser extent in healthy cells. In some cases, a disease may be identified by the expression of several mRNA detection targets simultaneously. In this case, the triggered RNAi initiator sequence can be designed, for example, to initiate triggered RNAi only in the event that a specific combination of mRNAs is detected, for example, by detecting a portion of an mRNA fusion entity.
In some embodiments, triggered RNAi can be performed with RNA monomers instead of, or in conjunction with, DNA monomers. In some embodiments, triggered RNAi can be performed with RNA-DNA hybrid monomers. In some embodiments, RNAi can be performed with, for example, RNA hairpins with stems of approximate length 14 and loops of approximate length 4, which exhibit similar properties to DNA hairpins with stems of approximate length 18 and loops of approximate length 6. Stems and loops of other lengths are also possible.
The concentration of the monomers can be adjusted to ensure that a sufficient amount of inactivator dsRNA is produced to effectively silence one or more silencing targets. Because of the self-propagating nature of the reaction, each copy of the detection target can begin the formation of inactivator dsRNAs. Furthermore, recycling of an activated initiator molecule allows catalytic amplification of the initial detection target binding event. In a catalytic triggered RNAi scheme, the amount of inactivator dsRNA can be higher than the amount of detection target due to catalytic turnover of the activated initiator. Thus, the amount of detection target does not have to equal the amount of silencing target in order to achieve gene silencing, because each detection target can lead to silencing of multiple silencing targets. The embodiment depicted in FIGS. 2 and 3 is catalytic and hence amplifies the signal provided by the detection target to achieve sufficient amounts of inactivator dsRNA.
In some embodiments, triggered RNAi monomers can be used in conjunction with nucleic acid “helper” monomers to facilitate detection target recognition. In preferred embodiments, the nucleic acid “helper” monomers are DNA molecules. Triggered RNAi can be more difficult to initiate with a long mRNA or other nucleic acid detection target because secondary structure within the mRNA strand reduces accessibility of the target site, i.e., the portion of the detection target recognized by the detection target binding region, to the triggered RNAi monomers. The use of DNA “helper” monomers that bind to regions flanking the target site helps eliminate competing secondary structures that form between the target site and the flanking regions. RNAi is more effectively initiated as a result of the elimination of secondary structure formation within the detection target mRNA strand. Helper DNA strands can be from about 10 to about 100 bases in length. In some embodiments, the helper DNA strands are from about 10 to about 75 bases in length. In other embodiments, the helper DNA strands are from about 10 to about 50 bases in length. In other embodiments, the helper DNA strands are from about 10 to about 35 bases in length. In preferred embodiments, the helper DNA strands are from about 10 to about 25 bases in length.
In some embodiments, RNA monomers are used in conjunction with an initiator comprising a DNA probe molecule that contains an initiator region. The initiator region is exposed upon binding between the DNA probe molecule and an mRNA molecule detection target and subsequently initiates formation of inactivator dsRNA substrates. Single stranded regions on either side of the duplex region of the DNA probe compete with native base pairing within the detection target molecule to initiate the RNAi process.
The design of the triggered RNAi monomers can be adjusted such that they bind specifically to nucleic acid detection targets, mRNA or otherwise. In addition, the design of the substrate monomers can be adjusted such that they specifically silence one or more silencing targets. The design can be derived from sequences derived from literature reviews and disclosed in, for example, various databases (e.g. NCBI).

Compositions and Kits for Triggered RNAi and Therapeutic Benefit

Compositions and kits for triggered RNAi are contemplated for use within the scope of the subject matter. In some embodiments, the compositions comprise an initiator monomer (initiator), a first substrate monomer and a second substrate monomer. In some embodiments, the compositions comprise an initiator hairpin monomer, a first substrate monomer and a second substrate monomer. In some embodiments, the compositions comprise an initiator hairpin monomer, a first RNA hairpin substrate monomer and a second RNA hairpin substrate monomer. In some embodiments, the compositions comprise an initiator monomer, a first substrate complex and a second substrate complex. In some embodiments, the compositions comprise an initiator monomer and a first substrate monomer. Upon delivery to a target cell or sample and recognition of the detection target by the initiator, triggered RNAi is initiated causing the formation of inactivator dsRNAs and subsequent silencing of one or more silencing targets.
The compositions can also contain other components, such as, for example, accessory molecules that facilitate detection target recognition and aid the formation of initiator dsRNAs. Accessory molecules typically comprise nucleic acid molecules. In some embodiments, the accessory molecules are DNA helper strands that bind to regions flanking a detection target nucleic acid sequence. Preferably the accessory molecules are DNA helper strands that bind to regions flanking a the initiator binding site on a detection target.
Furthermore, the composition can comprise a carrier that facilitates the introduction of nucleic acids, such as, for example, RNA monomers and accessory nucleic acid molecules, into a cell, such as a cell containing a detection target associated with a disease or disorder. Carriers for delivery of nucleic acids into cells are well known in the art and examples are described above.
A kit for triggered RNAi typically comprises the compositions as described in detail above. In some embodiments the kits comprise an initiator monomer (initiator) and one or more monomers or complexes. In some embodiments, the kits comprise an initiator monomer and a first substrate monomer. In some embodiments, the kits comprise an initiator monomer, a first substrate monomer and a second substrate monomer. In some embodiments, the kits comprise an initiator monomer, a first substrate complex and a second substrate complex. In preferred embodiments, the kit is used to deliver triggered RNAi monomers and/or complexes to a population of cells comprising cells comprising a disease-associated detection target as well as healthy, wild-type cells. In some embodiments, the kit is used to deliver RNAi monomers to the tissues of a patient, wherein the tissues comprise cells comprising a detection target associated with a disease or disorder. In other embodiments, the kit is used to select for cells containing a detection target in vitro.
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

EXAMPLES

Example 1

Triggered RNAi for Inactivation of Target Gene in the Presence of a Detection Target Associated with a Disease

This example illustrates the use of RNAi to silence a target gene in the presence of a detection target.
Triggered RNAi can be used to silence a target gene by designing first, second and third monomers of a triggered RNA system to detect a detection target associated with a disease, or a cell type associated with a disease, and subsequently silence a target gene to treat the disease or disorder. Examples of triggered RNAi mechanisms are illustrated in FIGS. 2-10. For the basic triggered RNAi system illustrated in FIGS. 2 and 3, stock solutions can be prepared as follows: monomer 1 (hereinafter the “initiator”, depicted as “A” of FIG. 2A), monomer 2 (hereinafter the “first substrate monomer”, depicted as “B” of FIG. 2E), and monomer 3 (hereinafter the “second substrate monomer”, depicted as “C” of FIG. 2G), disease-associated detection target mRNA (either “detection target” comprising only a detection target sequence without flanking regions or “detection target fusion” comprising the detection target sequence with flanking regions), mRNA 1 (“healthy mRNA 1”), mRNA 2 (“healthy mRNA 2”), silencing target mRNA (“silencing target”) and helper DNA (“D1” and “D2” annealed together in one solution). Preferably, the silencing target can be labeled for ease of analysis. Reactions can be set up by combining the triggered RNAi monomers, helper DNA, and detection and silencing targets. The formation of inactivator dsRNAs and degradation of silencing target mRNA occurs in samples containing the disease-associated target mRNA, but not samples containing only healthy mRNAs. The samples can be analyzed by a variety of methods known in the art for the presence of the silencing target mRNA.
The initiator, first substrate monomer, and second substrate monomer are delivered to target cells using standard gene delivery methods. In the presence of the detection target, production of inactivator dsRNAs sufficient for silencing the silencing target is initiated. The inactivator dsRNAs silence the silencing target, thereby inactivating the silencing target gene.

Example 2

In Vivo Triggered RNAi Activation of Protein Kinase PKR

Triggered RNAi hairpins are delivered to target cells using standard gene delivery methods. In the presence of an intracellular mRNA detection target, production of inactivator dsRNAs sufficient for activating PKR is initiated. The inactivator dsRNAs activate PKR. Activated PKR slows the production of all the proteins within the cells and induces apoptosis. The triggering of this response slows and can reverse the growth of diseased or tumor cells.

Example 3

In Vivo Triggered RNAi Therapy of Diseases Caused by Fused Gene Mutations

Two or more genes are fused together to encode an oncogenic protein (Dohjima, T. et al. Molecular Therapy 7: 811-816 (2003), herein incorporated by reference in its entirety). Although the two or more genes are present in healthy cells, the overlap region between the genes is a distinct signature found only in tumor cells. Triggered RNAi hairpin monomers are designed to form inactivator dsRNAs in the presence of a detection target sequence included within the overlap region. The hairpin monomers include an initiator and substrate monomers. The hairpin monomers have stems ranging in length between 10 and 35 base pairs. The hairpin monomers are introduced in vivo into tumor cells by gene delivery methods known in the art. Recognition of the detection target by the initiator hairpin monomer activates the initiator, and the activated initiator initiates formation of inactivator dsRNAs. Inactivator dsRNAs are produced in the target cells, i.e., cells having the detection target. The inactivator dsRNAs are processed by Dicer to produces siRNAs. The siRNAs associate with RISC and subsequently inactivate one or more housekeeping genes, which slows the production of all the vital proteins within the cells and induces apoptosis. The triggering of this response slows and can reverse the growth of the tumor cells.

Example 4

Triggered RNAi-Aptamer Therapy of Disease

RNAi hairpin monomers are designed that link the aptamer to a hairpin monomer in such a way that in the absence of the detection target, the aptamer does not allow conformational change of the hairpin monomer and does not initiate formation of inactivator dsRNAs. The triggered RNAi hairpin monomers are introduced in vivo into cells by gene delivery methods known in the art. Recognition between the aptamer and the detection target allows conformational change of the hairpin monomer and exposes an initiator region. The initiator region can initiate production of an inactivator dsRNA in the target cells. The inactivator dsRNAs are processed by Dicer to produces siRNAs. The siRNAs associate with RISC and subsequently inactivates one or more housekeeping genes, which slows the production of all the vital proteins within the cells and induces apoptosis. The triggering of this response slows and can reverse the growth of the diseased cells or kills the diseased cells.

Example 5

Triggered RNAi Modulation of Green Fluorescent Protein GFP

This example illustrates the modulation of a reporter molecule signal in the presence of a detection target. Triggered RNAi monomers are added to samples. The samples can be in vitro samples or, triggered RNAi monomers can be delivered to target cells using standard gene delivery methods. In the presence of a detection target, production of inactivator dsRNAs is initiated. The inactivator dsRNAs modulate GFP mRNA levels. Thus, the presence of a detection target changes the fluorescence emission of the GFP.
While the present teachings have been described in terms of these exemplary embodiments, the skilled artisan will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the current teachings.
Although the disclosed teachings have been described with reference to various applications, methods, kits, and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings herein and the claimed invention below. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein.

Incorporation by Reference

All references cited herein, including patents, patent applications, papers, text books, and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

Equivalents

The foregoing description and Examples detail certain specific embodiments of the invention and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof.

Claims

What is claimed is:

1. A method of silencing a target gene, the method comprising:

contacting a sample comprising a detection target and a silencing target gene with an initiator comprising a detection target binding region and an initiator region, wherein the detection target binding region can interact with the detection target, and wherein the detection target is different from the silencing target gene;

contacting the sample with a first substrate complex and a second substrate complex:

wherein the first substrate complex comprises a second substrate complex complement region and the second substrate complex comprises a first substrate monomer complement region, wherein the second substrate complex complement region is complementary to the first substrate complex complement region;

wherein the first substrate complex comprises a silencing target region and an initiator complement region that is complementary to the initiator region of the initiator, wherein upon binding of the detection target binding region of the initiator to the detection target, the initiator region of the initiator is made available to bind to the initiator complement region of the first substrate complex; and

wherein the second substrate complex comprises a silencing target complement region, wherein upon binding of the initiator complement region to the initiator region, the silencing target region of the first substrate complex is made available to bind to the silencing target complement region of the second substrate complex;

wherein an inactivator double-stranded RNA (inactivator dsRNA) is formed by hybridization of the silencing target complement region to the silencing target region, and wherein the inactivator dsRNA is processed to silence the silencing target gene.

2. The method of claim 1, wherein the first substrate complex comprises a nucleic acid duplex.

3. The method of claim 1, wherein the second substrate complex comprises a nucleic acid duplex.

4. The method of claim 3 wherein the first substrate complex comprises a region complementary to a region of the second substrate complex.

5. The method of claim 1, wherein the first substrate complex comprises a first bulge loop region.

6. The method of claim 5, wherein the second substrate complex comprises a second bulge loop region, wherein a portion of the first bulge loop region is complementary to the second bulge loop region.

7. The method of claim 1, wherein the initiator comprises a nucleic acid hairpin monomer.

8. The method of claim 1, wherein the inactivator dsRNA is processed by Dicer.

9. The method of claim 8, wherein the inactivator dsRNA is processed to produce a 19 bp duplex with 2-nt 3′ overhangs.

10. The method of claim 8, wherein the inactivator dsRNA comprises a 27-base pair RNA duplex substrate.

11. The method of claim 1, wherein the inactivator dsRNA is an siRNA.

12. The method of claim 11, wherein the inactivator dsRNA is a 19 bp duplex with 2-nt 3′ overhangs.

13. The method of claim 1, wherein the inactivator dsRNA activates protein kinase R (PKR).