# US 9,518,263 B2 · Signal activated RNA interference

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- Number: US 9,518,263 B2
- Document ID: US9518263B2
- Status: Granted
- Kind: B2
- Application number: 14/691,256
- Filed: 2015-04-20
- Published: 2016-02-18
- Granted: 2016-12-13
- Earliest claimed: 2007-12-10
- Assignees: California Institute of Technology
- Applicants: California Institute of Technology
- Inventors: Si-ping Han; Robert D. Barish; William A. Goddard, III
- Web page: https://patentgrep.com/patent/US9518263B2

## CPC

- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C07H 21/02: with ribosyl as saccharide radical
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 2310/11: Antisense
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/141: MicroRNAs, miRNAs
- C12N 2310/3517: Marker; Tag
- C12N 2310/3519: Fusion with another nucleic acid
- C12N 2310/53: partially self-complementary or closed
- C12N 2320/10: in screening processes
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)

## Abstract

The invention provides compositions and methods for signal activated RNA interference (saRNAi), preferably in vivo. The invention provides polynucleotides that switches between an inactive form and an active form upon covalent or non-covalent interaction with one or more specific chemical signals, such as disease-specific mRNA, miRNA, or other cellular RNA products with sequences that characterize diseased states of the cell. The interaction between the subject polynucleotides and the signals is preferably mediated by hybridization, which exposes, facilitates the formation, and/or allows the formation of a substrate that can be processed by proteins of the RNAi pathway (such as Dicer). The input and output of multiple different polynucleotides of the invention can form an in vivo signaling network. In addition, the multiple input signals can be integrated to modulate the activity of the subject polynucleotides.

## Claims

### Claim 1

A signal-activatable polynucleotide construct for enzyme-assisted molecular delivery, the construct comprising:
a targeting domain and
a sensor domain covalently bound to the targeting domain, the sensor domain comprising:
a signal detection region complementary to at least one signal polynucleotide,
a first chimeric sequence comprising at least one RNA region,
a second chimeric sequence comprising a DNA region complementary to an RNA region of the at least one RNA regions of the first chimeric sequence, and
a masking segment complementary to the first chimeric sequence;
wherein the sensor domain is configured such that:
in absence of the signal polynucleotide,
the masking segment hybridizes with the first chimeric sequence,
the DNA region of the second chimeric sequence is in a configuration not processable by RNase H, and
the signal detection region is presented for binding to the signal polynucleotide; and
in presence of the signal polynucleotide,
the signal detecting region hybridizes with the signal polynucleotide,
the masking segment is displaced from the first chimeric sequence, and
the DNA region of the second chimeric sequence hybridizes with the complementary RNA region of the at least RNA region of the first chimeric sequence to provide an RNase H binding site presented for binding to RNase H, and
the sensor domain is configured to allow release of the targeting domain from the sensor domain upon cleavage of the RNase H binding site by the RNase H.

### Claim 2 (depends on 1)

The signal activatable construct of claim 1, wherein the second chimeric sequence and the targeting domain are located at opposite sides of the first chimeric sequence.

### Claim 3 (depends on 1)

The signal activatable construct of claim 1, wherein the inactive conformation the DNA region of the second chimeric sequence hybridizes with an RNA-DNA region of the second chimeric sequence.

### Claim 4 (depends on 1)

The signal activatable construct of claim 1, wherein the complementary binding of the at least one signal polynucleotide to the signal detection region results in a duplex polynucleotide having approximately 20 consecutive base pairs.

### Claim 5 (depends on 1)

The signal-activated polynucleotide construct of claim 1, wherein the construct comprises one or more 2′-o-methyl modifications that inhibit PKR and/or TLR activation.

### Claim 6 (depends on 1)

The signal-activatable polynucleotide construct of claim 1, wherein the signal activatable construct is in a configuration such that at 37° C. in physiological conditions, a form of the signal activatable construct in absence of the signal polynucleotide is at least about 5 kcal/mole, lower in free energy than a form of said signal activatable construct in presence of the signal polynucleotide.

### Claim 7 (depends on 1)

The signal-activatable polynucleotide construct of claim 1, comprising two or more single-stranded polynucleotides.

### Claim 8 (depends on 7)

The signal-activatable polynucleotide construct of claim 7, wherein nucleotides with chemical modifications, if any, are largely present on one or more said single-stranded polynucleotides.

### Claim 9 (depends on 7)

The signal-activatable polynucleotide construct of claim 7, wherein the signal-activatable polynucleotide is not activated in the presence of the signal polynucleotide when one or more said single-stranded polynucleotides are removed from the construct.

### Claim 10 (depends on 7)

The signal-activatable polynucleotide construct of claim 7, wherein two or more of said single-stranded polynucleotides are covalently linked by a linker moiety (such as PEG) or a bond other than canonical polynucleotide linkage.

### Claim 11 (depends on 1)

The signal-activatable polynucleotide construct of claim 1, configured to have two or more signal polynucleotides bind simultaneously or sequentially to different portions of said signal detection region.

### Claim 12 (depends on 1)

The signal-activatable polynucleotide construct of claim 1, wherein the targeting domain comprises a guide sequence and a sense sequence forming a duplex region of no more than 19 base pairs in length.

### Claim 13 (depends on 12)

The signal-activatable polynucleotide construct of claim 12, wherein the duplex region comprises one or more chemical modifications that confer resistance to nuclease degradation.

### Claim 14 (depends on 12)

The signal-activatable polynucleotide construct of claim 12, wherein in the absence of the signal polynucleotide, the guide sequence and the sense sequence form a double-stranded region comprising one or more chemical modifications that inhibits Dicer cleavage and/or productive RISC incorporation.

### Claim 15 (depends on 12)

The signal-activatable polynucleotide construct of claim 12, wherein the guide sequence is substantially complementary to a transcript of a target gene.

### Claim 16 (depends on 1)

The signal-activatable polynucleotide construct of claim 1, wherein the targeting domain is configured to interfere with a target intracellular process of the cells through RNAi in presence of the signal polynucleotide.

### Claim 17 (depends on 1)

The signal-activatable polynucleotide construct of claim 1, wherein the targeting domain comprises siRNA, microRNA, and/or additional duplex structure is suitable to be used in connection with RNA interfering.

### Claim 18 (depends on 1)

The signal-activatable polynucleotide construct of claim 1, wherein in the absence of the signal polynucleotide, generation of the siRNA or miRNA and/or productive incorporation of said siRNA or miRNA into RISC is inhibited by at least about 2-fold, 5-fold, 10-fold, or 20-fold or more.

### Claim 19 (depends on 1)

A method to inhibit expression of a target gene, comprising providing an effective amount of the signal-activated polynucleotide construct of claim 1 and the said signal polynucleotide.

### Claim 20 (depends on 19)

The method of claim 19, wherein said target gene is within a cell.

### Claim 21 (depends on 20)

The method of claim 20, wherein the cell is contacted with the signal-activated polynucleotide construct in vitro.

### Claim 22 (depends on 20)

The method of claim 20, wherein the cell is that of a human, a non-human primate, a non-primate mammal, a rodent, a livestock animal, a bird, an insect, a fly, a worm, or a plant.

### Claim 23 (depends on 1)

A pharmaceutical composition comprising an effective amount of one or more of the signal-activated polynucleotide construct of claim 1 and a pharmaceutically acceptable amount of excipients, carriers, or diluents.

### Claim 24 (depends on 1)

A vector encoding the signal-activated polynucleotide construct of claim 1.

### Claim 25 (depends on 1)

A cell comprising the signal-activated polynucleotide construct of claim 1.