# US 11,110,125 B2 · Coordinating gene expression using RNA destabilizing elements

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- Number: US 11,110,125 B2
- Document ID: US11110125B2
- Status: Granted
- Kind: B2
- Application number: 16/272,679
- Filed: 2019-02-11
- Published: 2019-08-15
- Granted: 2021-09-07
- Earliest claimed: 2018-02-13
- Assignees: Chimera Bioengineering, Inc.
- Applicants: Chimera Bioengineering, Inc.
- Inventors: Benjamin Wang; Gusti Zeiner; Krista McNally
- Web page: https://patentgrep.com/patent/US11110125B2

## CPC

- C12N 15/85: for animal cells
- A61K 38/208: {IL-12}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/36: Immune checkpoint inhibitors
- A61K 40/4202: {Receptors, cell surface antigens or cell surface determinants}
- A61K 40/4211: {CD19 or B4}
- A61P 35/00: Antineoplastic agents
- C07K 14/195: from bacteria
- C07K 14/52: Cytokines; Lymphokines; Interferons
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/82: Translation products from oncogenes
- C07K 16/18: against material from animals or humans
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2818: {against CD28 or CD152}
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- 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 15/62: DNA sequences coding for fusion proteins
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2830/002: inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
- 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
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Control Devices are disclosed including RNA destabilizing elements (RDE), and RNA control devices, combined with transgenes, including Chimeric Antigen Receptors (CARs) in eukaryotic cells. RDEs can be combined with RNA control devices to make RDEs that include ligand mediated control. These smart RDEs and other RDEs can be used to optimize expression of transgenes, e.g., CARs, in the eukaryotic cells so that, for example, effector function is optimized. CARs and transgene payloads can also be engineered into eukaryotic cells so that the transgene payload is expressed and delivered at desired times from the eukaryotic cell.

## Claims

### Claim 1

A method of expressing a plurality of transgenes, comprising the steps of: obtaining a primary T-cell comprising a receptor, a first heterologous nucleic acid comprising a polynucleotide encoding a first transgene, and a polynucleotide encoding a first RNA destabilizing element (RDE), wherein the first RDE is an AU rich element, a second heterologous nucleic acid comprising a polynucleotide encoding a second transgene, and a polynucleotide encoding a second RDE, wherein the second RDE is an AU rich element, wherein the first heterologous nucleic acid is transcribed to make a first transcript encoding the first transgene operably linked to the first RDE, wherein a glycolytic enzyme with RDE binding activity-binds to the first RDE and regulates expression of the first transgene, and the second heterologous nucleic acid is transcribed to make a second transcript encoding the second transgene operably linked to the second RDE, wherein the glycolytic enzyme with RDE binding activity-binds to the second RDE and regulates expression of the second transgene;
and binding the receptor on the primary T-cell to a ligand wherein binding of the ligand by the receptor activates the primary T-cell and the primary T-cell increases glycolytic activity whereby the glycolytic enzyme with RDE binding activity catalyzes a step of glycolysis, wherein activation of glycolysis in the primary T-cell reduces the amount of the glycolytic enzyme with RDE binding activity available to bind the first RDE and reduces the amount of the glycolytic enzyme with RDE binding activity available to bind the second RDE which produces increased expression of the first and second transgenes.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first transgene encodes a cytokine, a FasL, an antibody, a growth factor, a chemokine, an enzyme that cleaves a polypeptide or a polysaccharide, a granzyme, a perforin, a reporter, or a checkpoint inhibitor.

### Claim 3 (depends on 2)

The method of claim 2, wherein the second transgene encodes a cytokine, a FasL, an antibody, a growth factor, a chemokine, an enzyme that cleaves a polypeptide or a polysaccharide, a granzyme, a perforin, a reporter, or a checkpoint inhibitor.

### Claim 4 (depends on 1)

The method of claim 1, wherein a polypeptide encoded by the first transgene is secreted from the primary T-cell.

### Claim 5 (depends on 4)

The method of claim 4, wherein the secreted polypeptide acts on a target cell.

### Claim 6 (depends on 4)

The method of claim 4, wherein a polypeptide encoded by the second transgene is secreted from the primary T-cell.

### Claim 7 (depends on 6)

The method of claim 6, wherein the secreted polypeptide acts on a target cell.

### Claim 8 (depends on 6)

The method of claim 6, wherein the target cell is a cancer cell or a bacterium.

### Claim 9 (depends on 8)

The method of claim 8, further comprising the step of killing the target cell.

### Claim 10 (depends on 1)

The method of claim 1, wherein the first RDE is an AU 2 or an AU 101, and the second RDE is an AU 5 or an AU 21.

### Claim 11 (depends on 1)

The method of claim 1, wherein the first RDE is an AU 2 or an AU 101, and the second RDE is an AU 19 or an AU 22.

### Claim 12 (depends on 1)

The method of claim 1, wherein the first RDE is an AU 2 or an AU 101, and the second RDE is an AU 3, an AU 20, an AU 10, an AU 7, or an AU 23.

### Claim 13 (depends on 1)

The method of claim 1, wherein the receptor is a chimeric antigen receptor or a T-cell receptor.