# US 11,299,749 B2 · Cell-specific expression of modRNA

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- Number: US 11,299,749 B2
- Document ID: US11299749B2
- Status: Granted
- Kind: B2
- Application number: 16/354,814
- Filed: 2019-03-15
- Published: 2019-07-04
- Granted: 2022-04-12
- Earliest claimed: 2016-09-16
- Assignees: Icahn School of Medicine at Mount Sinai
- Applicants: Icahn School of Medicine At Mount Sinai
- Inventors: Lior Zangi; Ajit Magadum
- Web page: https://patentgrep.com/patent/US11299749B2

## CPC

- C12N 15/85: for animal cells
- A61K 31/7084: Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- C12N 5/0657: {Cardiomyocytes; Heart cells}
- C12N 5/0676: {Pancreatic cells}
- 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)}
- C12N 15/67: General methods for enhancing the expression
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Disclosed is an expression regulatory system for cell-specific transcription (expression) of a protein of interest, for example a cell cycle inducer that reactivates proliferation in adult or neonatal cardiomyocytes or insulin-producing beta cells. The expression regulatory system comprises a first nucleic acid that encodes a microRNA recognition element that specifically binds a target cell miR, and a translation suppressor protein; and a second nucleic acid that comprises a suppressor protein interaction motif that binds the translation suppressor protein, and a gene that encodes a protein of interest. When a cell of interest is co-transfected with the first and second nucleic acids of the system, the protein of interest expressed in a cell-specific fashion.

## Claims

### Claim 1

An expression regulatory system for cell-specific expression of a gene of interest (GOI) comprising:
a first nucleic acid that encodes a microRNA (miR) recognition element near its 3′UTR that specifically binds a target cell-specific miR, and a translation suppressor protein; and
a second nucleic acid that comprises a suppressor protein interaction motif that binds the translation suppressor protein, and a gene that encodes a protein of interest, wherein the protein of interest is a cell cycle inducer protein; wherein
(a) said first nucleic acid comprises the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4,
(b) said second nucleic acid comprises the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8,
or both (a) and (b).

### Claim 2 (depends on 1)

The system of claim 1, wherein said first and second nucleic acids are modified RNA (modRNA).

### Claim 3 (depends on 1)

The system of claim 1, wherein the target cell is a cardiomyocyte and the target cell miR is selected from the group consisting of miR1, miR208 and miR1 plus miR208.

### Claim 4 (depends on 1)

The system of claim 1, wherein the translation suppressor protein is L7Ae and the suppressor protein interaction motif is a k-motif.

### Claim 5 (depends on 1)

The system of claim 1, wherein the gene further encodes a reporter protein or selection marker.

### Claim 6 (depends on 5)

The system of claim 5, wherein the reporter protein or selection marker is a fluorescent protein or antibiotic resistance marker.

### Claim 7 (depends on 5)

The system of claim 5, wherein the reporter protein or selection marker is selected from the group consisting of green fluorescence protein (GFP), nuclear GFP (nGFP), inactive human CD25, (ihCD25) and inactive mouse CD25 (imCD25).

### Claim 8 (depends on 1)

The system of claim 1, wherein the cell cycle inducer protein is selected from the group consisting of Lin28 and Pkm2.

### Claim 9 (depends on 1)

The system of claim 1, wherein said first nucleic acid comprises the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

### Claim 10 (depends on 1)

The system of claim 1, wherein said second nucleic acid comprises the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

### Claim 11 (depends on 1)

A composition comprising first and second nucleic acids of claim 1.

### Claim 12 (depends on 11)

The composition of claim 11, wherein said nucleic acids are obtained by in vitro transcription.

### Claim 13 (depends on 11)

The composition of claim 11, wherein a first vector comprises the first nucleic acid and a second vector comprises the second nucleic acid.

### Claim 14 (depends on 1)

A vector comprising first and second nucleic acids of claim 1.

### Claim 15

A method for expressing a protein in a cell of interest, the method comprising contacting said cell with a modRNA encoding an anti-miR specific for a miR target of said cell of interest, wherein said protein of interest is a cell cycle inducer protein, said cell of interest is a cardiomyocyte (CM), and said modRNA encoding an anti-miR specific for a cardiomyocyte miR target comprises the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

### Claim 16

A method for inducing/reactivating proliferation of neonatal or adult cardiomyocytes in vitro or in vivo following myocardial infarction (MI), the method comprising contacting a portion of said cardiomyocytes with a first modRNA that encodes a recognition element for a cardiomyocyte-specific miR and a translation suppressor protein and a second modRNA that encodes a k-motif to bind the translation suppressor protein and a cell cycle inducer gene, wherein said miR is miR1, miR208, or a combination of miR1 and miR208.

### Claim 17 (depends on 16)

The method of claim 16, wherein the cell cycle inducer gene is selected from the group consisting of Lin28 and Pkm2 and Cyclin D2.

### Claim 18 (depends on 16)

The method of claim 16, wherein the inducing/reactivating proliferation comprises inducing/reactivating proliferation of adult cardiomyocytes in vivo following myocardial infarction (MI).