US 12,385,911 B2 · Engineered red blood cell-based biosensors

Abstract

Disclosed are systems and methods for detecting extracellular ligands. The disclosed systems and method for detecting extracellular ligands typically comprise or utilize engineered red blood cells (eRBCs) that comprises modular extracellular sensors. The eRBCs may comprise: (i) a first exogenous extracellular sensor; the first extracellular sensor comprising: a) a ligand binding domain, b) a transmembrane domain, and c) a first fragment of a functional protein, and (ii) a second exogenous extracellular sensor; the second extracellular sensor comprising: a) a ligand binding domain, b) a transmembrane domain, and c) a second fragment of the functional protein. In the eRBCs, the ligand binding domain of the first exogenous sensor and the ligand binding domain of the second exogenous sensor bind to the same ligand to form a tertiary complex, and the first fragment of the functional protein and the second fragment of the functional protein interact in the tertiary complex to reconstitute functional activity of the functional protein. Suitable functional proteins for the disclosed eRBCs may include fluorescent proteins that emit fluorescence when the ligand binding domain of the first exogenous sensor and the ligand binding domain of the second exogenous sensor bind to the same ligand, and enzymatic proteins that exhibit enzymatic activity when ligand binding domain of the first exogenous sensor and the ligand binding domain of the second exogenous sensor bind to the same ligand.

Claims19

Dependent claims

A biosensor expressed in a blood cell, comprising:

(a) a first protein comprising:
(i) a first extracellular ligand binding domain,
(ii) a first transmembrane domain comprising a portion of an amino acid sequence from a red blood cell native membrane protein,
(iii) an intracellular first fragment of a functional protein, and
(iv) an amino acid linker connecting the first transmembrane domain and the intracellular first fragment of the functional protein;
and
(b) a second protein comprising:
(i) a second extracellular ligand binding domain,
(ii) a second transmembrane domain,
(iii) an intracellular second fragment of the functional protein, and
(iv) an amino acid linker connecting the second transmembrane domain and the intracellular second fragment of the functional protein;
wherein the first extracellular ligand binding domain and the second extracellular ligand binding domain bind to the same ligand, wherein the first fragment of the functional protein and the second fragment of the functional protein are capable of interacting to reconstitute activity of the functional protein, and wherein the amino acid linker of the first protein or the amino acid linker of the second protein comprises 7-25 amino acids.

9 more elements

A biosensor expressed in a blood cell, comprising:

(a) a first protein comprising:
(i) a first extracellular ligand binding domain,
(ii) a first transmembrane domain comprising a portion of an amino acid sequence from a red blood cell native membrane protein,
(iii) an extracellular linker comprising 7-25 amino acids and connecting the first extracellular ligand binding domain and the first transmembrane domain,
(iv) an intracellular first fragment of a functional protein, and
(v) an intracellular linker comprising 7-25 amino acids and connecting the first transmembrane domain and the intracellular first fragment of the functional protein;
and
(b) a second protein comprising:
(i) a second extracellular ligand binding domain,
(ii) a second transmembrane domain,
(iii) an extracellular linker comprising 7-25 amino acids and connecting the second extracellular ligand binding domain and the second transmembrane domain,
(iv) an intracellular second fragment of a functional protein, and
(v) an intracellular linker comprising 7-25 amino acids and connecting the second transmembrane domain and the intracellular second fragment of the functional protein;
wherein the first extracellular ligand binding domain and the second extracellular ligand binding domain bind to the same ligand, wherein the first fragment of the functional protein and the second fragment of the functional protein are capable of interacting to reconstitute activity of the functional protein, and wherein linkers comprise a sequence of amino acids selected from glycine, serine, and combinations thereof.

11 more elements

Dependencies are inferred from the claim text.

Engineered red blood cell-based biosensors

Life of this patent

Engineered red blood cell-based biosensors

Life of this patent

Abstract

Claims19