# US 12,215,323 B2 · Exogenous control of mammalian gene expression through aptamer-mediated modulation of polyadenylation

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- Number: US 12,215,323 B2
- Document ID: US12215323B2
- Status: Granted
- Kind: B2
- Application number: 17/144,456
- Filed: 2021-01-08
- Published: 2021-10-07
- Granted: 2025-02-04
- Earliest claimed: 2015-11-12
- Assignees: Baylor College of Medicine
- Applicants: Baylor College of Medicine
- Inventors: Laising Yen; Liming Luo; Pei-Wen Chao
- Web page: https://patentgrep.com/patent/US12215323B2

## CPC

- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 15/115: Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers}
- C12N 15/67: General methods for enhancing the expression
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)

## Abstract

Embodiments of the disclosure concern the use of expression constructs in which at least one polyA signal is embedded upstream of an expressible transcript, such as within a 5′ UTR for the transcript, for example. In certain embodiments, the polyA signal is comprised within a ligand-binding aptamer, and the binding of the ligand to the aptamer, or lack thereof, dictates the outcome for the expressible transcript. In specific embodiments, absence of the ligand causes the expressed transcript having a polyA in its 5′ UTR to be expressed but then degraded, whereas presence of the ligand causes inhibition of degradation upon expression of the expressible transcript. More than one ligand-binding aptamer may be present on the same expression construct.

## Claims

### Claim 1

A system for modulating gene expression, comprising a polyA aptamer polynucleotide that comprises in a 5′ to 3′ direction:
a) at least one ligand-binding aptamer comprising at least one polyA cleavage signal therein; and
b) an expressible polynucleotide that is a reporter gene,
wherein:
(i) the at least one ligand-binding aptamer resides within the 5′ untranslated region (UTR) of the expressible polynucleotide;
(ii) the polyA aptamer polynucleotide comprises at least one U/UG rich region, at least one G rich region, or both of at least one U/UG rich region and at least one G rich region; and
(iii) in a 5′ to 3′ direction, the at least one poly A cleavage signal resides upstream of the at least one U/UG rich region and/or the at least one G rich region.

### Claim 2 (depends on 1)

The system of claim 1, wherein the system comprises a polynucleotide that expresses a ligand that binds the at least one ligand-binding aptamer.

### Claim 3 (depends on 2)

The system of claim 2, wherein the polynucleotide that expresses the ligand is the same polyA aptamer polynucleotide that comprises the aptamer and expressible polynucleotide.

### Claim 4 (depends on 2)

The system of claim 2, wherein the polynucleotide that expresses the ligand is a different polynucleotide than the polyA aptamer polynucleotide that comprises the aptamer and expressible polynucleotide.

### Claim 5 (depends on 2)

The system of claim 2, wherein the polyA aptamer polynucleotide comprises two, three, or more polyA signals in the 5′ UTR of the expressible polynucleotide.

### Claim 6 (depends on 1)

The system of claim 1, wherein the ligand-binding aptamer comprises one, two, three, or more U/UG rich regions.

### Claim 7 (depends on 1)

The system of claim 1, wherein in a 5′ to 3′ direction of the polyA aptamer polynucleotide the ligand-binding aptamer resides upstream of one, two, or more U/UG rich regions.

### Claim 8 (depends on 2)

The system of claim 2, wherein in a 5′ to 3′ direction of the polyA aptamer polynucleotide the ligand-binding aptamer resides upstream of one, two, or more G rich regions.

### Claim 9 (depends on 1)

The system of claim 1, wherein in a 5′ to 3′ direction of the polyA aptamer polynucleotide the aptamer comprises two polyA signals and two U/UG rich regions.

### Claim 10 (depends on 1)

The system of claim 1, wherein the polyA aptamer polynucleotide comprises 2, 3, 4, 5, or more aptamers.

### Claim 11 (depends on 10)

The system of claim 10, wherein a first aptamer and a second aptamer are in the same orientation in a 5′ to 3′ linear direction.

### Claim 12 (depends on 10)

The system of claim 10, wherein a first aptamer and a second aptamer are in a different orientation in a 5′ to 3′ linear direction.

### Claim 13 (depends on 10)

The system of claim 10, wherein the polyA aptamer polynucleotide comprises one G-rich region.

### Claim 14 (depends on 13)

The system of claim 13, wherein the G-rich region is in the 3′-most aptamer in a 5′ to 3′ direction of the polyA aptamer polynucleotide.

### Claim 15 (depends on 13)

The system of claim 13, wherein the G-rich region is in the second aptamer in a 5′ to 3′ direction of the polyA aptamer polynucleotide.

### Claim 16 (depends on 1)

The system of claim 1, wherein the at least one ligand-binding aptamer comprises two or more loops and wherein the number of nucleotides between two loops within the at least one ligand-binding aptamer is 10-25 nucleotides.

### Claim 17 (depends on 1)

The system of claim 1, wherein the ligand is a polypeptide, peptide, nucleic acid, small molecule, drug, metabolite, or a combination thereof.

### Claim 18 (depends on 1)

The system of claim 1, wherein the aptamer is between 14 and 250 nucleotides in length.

### Claim 19 (depends on 1)

The system of claim 1, wherein the polyA aptamer polynucleotide is at least part of a vector.

### Claim 20 (depends on 1)

The system of claim 1, wherein the expressible polynucleotide encodes the ligand.

### Claim 21 (depends on 1)

The system of claim 1, wherein expression of the expressible polynucleotide is regulated by a tissue-specific promoter.

### Claim 22

A polyA aptamer polynucleotide, wherein said polynucleotide comprises in a 5′ to 3′ direction:
a) at least one ligand-binding aptamer that comprises at least one polyA cleavage signal therein; and
b) an expressible polynucleotide that is a reporter gene,
wherein:
(i) the at least one ligand-binding aptamer resides within the 5′ untranslated region (UTR) of the expressible polynucleotide;
(ii) the polyA aptamer polynucleotide comprises at least one U/UG rich region, at least one G rich region, or both of at least one U/UG rich region and at least one G rich region; and
(iii) in a 5′ to 3′ direction, the at least one poly A cleavage signal resides upstream of the at least one U/UG rich region and/or the at least one G rich region.

### Claim 23 (depends on 22)

A cell comprising the polynucleotide of claim 22.

### Claim 24 (depends on 1)

A cell comprising the system of claim 1.

### Claim 25 (depends on 22)

A vector comprising the polynucleotide of claim 22.

### Claim 26 (depends on 1)

The system of claim 1, wherein the polyA aptamer polynucleotide comprises in a 5′ to 3′ direction:
a) three ligand-binding aptamers, wherein at least one of the ligand-binding aptamers is the at least one ligand-binding aptamer comprising at least one polyA cleavage signal therein;
b) at least one U/UG rich region and at least one G rich region; and
c) the expressible polynucleotide,
wherein the at least one of the ligand-binding aptamers comprising a polyA cleavage signal resides within the 5′ untranslated region (UTR) of the expressible polynucleotide.

### Claim 27 (depends on 26)

The system of claim 26, wherein the system comprises a polynucleotide that expresses a ligand that binds at least one of the ligand-binding aptamers.

### Claim 28 (depends on 26)

The system of claim 26, wherein the polyA aptamer polynucleotide comprises two, three, or more polyA cleavage signals in the 5′ UTR of the expressible polynucleotide.

### Claim 29 (depends on 26)

The system of claim 26, wherein the ligand-binding aptamer comprises two, three, or more U/UG rich regions.

### Claim 30 (depends on 26)

The system of claim 26, wherein in a 5′ to 3′ direction of the polyA aptamer polynucleotide:
at least one of the ligand-binding aptamers resides upstream of one, two, or more U/UG rich regions; or
at least one of the ligand-binding aptamers resides upstream of one, two, or more G rich regions.

### Claim 31 (depends on 26)

The system of claim 26, wherein the G-rich region is in the 3′-most ligand-binding aptamer in a 5′ to 3′ direction of the polyA aptamer polynucleotide.

### Claim 32 (depends on 26)

The system of claim 26, wherein the at least one of the three ligand-binding aptamers comprises two or more loops and wherein the number of nucleotides between two loops within said ligand-binding aptamer comprising two or more loops is 10-25 nucleotides.

### Claim 33 (depends on 26)

The system of claim 26, wherein a ligand-binding aptamer is between 14 and 250 nucleotides in length.