# US 11,999,964 B2 · Synthetic mammalian signaling circuits for robust cell population control

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,999,964 B2
- Document ID: US11999964B2
- Status: Granted
- Kind: B2
- Application number: 17/460,120
- Filed: 2021-08-27
- Published: 2022-03-03
- Granted: 2024-06-04
- Earliest claimed: 2020-08-28
- Assignees: California Institute of Technology
- Applicants: California Institute of Technology
- Inventors: Yitong Ma; Mark W. Budde; Michaelle N. Mayalu; Michael B. Elowitz
- Web page: https://patentgrep.com/patent/US11999964B2

## CPC

- C12N 9/80: acting on amide bonds in linear amides {(3.5.1)}
- C12N 15/85: for animal cells
- C07K 14/415: from plants
- C12N 5/10: Cells modified by introduction of foreign genetic material
- C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)}
- C12N 9/86: acting on amide bonds in cyclic amides, e.g. penicillinase {(3.5.2)}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12Y 113/12003: Tryptophan 2-monooxygenase (1.13.12.3)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/95: containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2501/30: Hormones
- C12N 2501/48: Regulators of apoptosis
- C12N 2800/107: for mammalian
- C12N 2810/10: Vectors comprising a non-peptidic targeting moiety
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- C12Y: ENZYMES

## Abstract

Disclosed herein include circuits, compositions, nucleic acids, populations, systems, and methods enabling cells to sense, control, and/or respond to their own population size. In some embodiments, an orthogonal communication channel allows specific communication between engineered cells. Also described herein, in some embodiments, is an evolutionarily robust ‘paradoxical’ regulatory circuit architecture in which orthogonal signals both stimulate and inhibit net cell growth at different signal concentrations. In some embodiments, engineered cells autonomously reach designed densities and/or activate therapeutic or safety programs at specific density thresholds. Methods of treatment are also provided in some embodiments.

## Claims

### Claim 1

A nucleic acid composition, comprising:
a first polynucleotide encoding a first synthase, wherein the first synthase comprises an indole-3 acetic acid hydrolase capable of catalyzing the synthesis of an orthogonal signal from a first precursor molecule;
a second polynucleotide encoding a second synthase, wherein the second synthase is capable of catalyzing the synthesis of the first precursor molecule from a second precursor molecule; and
a third polynucleotide encoding a transporter capable of transporting the orthogonal signal across a cell membrane;
wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide are operably linked to a ubiquitous mammalian promoter.

### Claim 2 (depends on 1)

The nucleic acid composition of claim 1, comprising: a fourth polynucleotide encoding a signal-binding protein comprising the F-box transport inhibitor response 1 (TIR1) wherein the TIR1 protein is capable of binding the orthogonal signal; a fifth polynucleotide encoding a first fusion protein comprising a signal-responsive domain and a pro-growth protein; and optionally a sixth polynucleotide encoding a second fusion protein comprising a signal-responsive domain and a pro-death protein, wherein the signal-responsive domain comprises an auxin inducible degron (AID); and wherein the signal-binding protein bound to the orthogonal signal is capable of reducing the stability of the first and/or second fusion protein comprising a signal-responsive domain; wherein the fourth polynucleotide, the fifth polynucleotide, and the sixth polynucleotide are operably linked to a ubiquitous mammalian promoter.

### Claim 3 (depends on 2)

The nucleic acid composition of claim 2, wherein the auxin inducible degron (AID) comprises a minimal auxin inducible degron (mAID).

### Claim 4 (depends on 2)

The nucleic acid composition of claim 2, wherein the pro-growth protein is essential for survival and/or cell cycle progression, optionally a cell-cycle regulator, and/or wherein the pro-growth protein is essential for survival and/or cell cycle progression in the presence of an exogenous agent, optionally the exogenous agent is an antibiotic, further optionally the pro-growth protein provides antibiotic resistance.

### Claim 5 (depends on 2)

The nucleic acid composition of claim 2, wherein the pro-death protein is capable of halting cell growth and/or inducing cell death, and/or wherein the pro-death protein is capable of halting cell growth and/or inducing cell death in the presence of a pro-death agent.

### Claim 6 (depends on 2)

The nucleic acid composition of claim 2, wherein the signal-binding protein bound to the orthogonal signal is capable of triggering the ubiquitylation and proteasomal degradation of the protein comprising a signal-responsive domain.

### Claim 7 (depends on 1)

The nucleic acid composition of claim 1, wherein the indole-3 acetic acid hydrolase comprises iaaH, aux2 and/or AMI1.

### Claim 8 (depends on 1)

The nucleic acid composition of claim 1, wherein the transporter is auxin exporter PIN2, optionally Arabidopsis thaliana PIN2.

### Claim 9 (depends on 1)

The nucleic acid composition of claim 1, wherein the second precursor molecule is an endogenous molecule of a cell, optionally L-tryptophan.

### Claim 10 (depends on 1)

The nucleic acid composition of claim 1, wherein the orthogonal signal is an auxin.

### Claim 11 (depends on 1)

The nucleic acid composition of claim 1, wherein the nucleic acid composition is comprised within one or more vectors; optionally at least one of the one or more vectors is a viral vector, a plasmid, a naked DNA vector, a lipid nanoparticle, or any combination thereof; and further optionally the viral vector is an AAV vector, a lentivirus vector, a retrovirus vector, an integration-deficient lentivirus (IDLV) vector.