# US 12,180,485 B2 · Repressible control of gene expression

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- Number: US 12,180,485 B2
- Document ID: US12180485B2
- Status: Granted
- Kind: B2
- Application number: 18/066,482
- Filed: 2022-12-15
- Published: 2023-09-07
- Granted: 2024-12-31
- Earliest claimed: 2020-07-21
- Assignees: Trustees of Boston University
- Applicants: Trustees of Boston University
- Inventors: Wilson Wai Chun Wong; Yage Ding
- Web page: https://patentgrep.com/patent/US12180485B2

## CPC

- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/635: {Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline}
- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C07K 2319/00: Fusion polypeptide
- 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)

## Abstract

The technology described herein is directed to inducible and repressible polypeptides and polypeptide systems. In particular, described herein are split sequence-specific nucleases and split recombinases that are linked to drug-inducible or drug-repressible dimerization domains. In some embodiments, the polypeptides comprise sequestering domains and/or have their expression controlled by an inducible promoter. In multiple aspects described herein are polynucleotides, vectors, cells, and pharmaceutical compositions comprising said polypeptides or polypeptide systems. Also described herein are methods of using said polypeptide systems to modulate the expression of a target polypeptide or to treat a subject in need of a cell therapy.

## Claims

### Claim 1

A repressible split-nuclease polypeptide system comprising:
a) a first polypeptide comprising:
i) a first member of a repressible dimerization domain (RD1); and
ii) a first polypeptide fragment of a sequence-specific nuclease (N1); and
b) a second polypeptide comprising:
i) a second member of the repressible dimerization domain (RD2); and
ii) a second polypeptide fragment of the sequence-specific nuclease (N2); and
wherein the first and second members of the repressible dimerization domain come together in the absence of a repressor agent or repressor signal, resulting in protein complementation of the two nuclease polypeptide fragments to form the active nuclease protein in the absence of the repressor agent or repressor signal;
wherein the first and second members of the repressible dimerization domain do not come together in the presence of the repressor agent or repressor signal, resulting in no protein complementation of the two nuclease polypeptide fragments and no formation of the active nuclease protein in the presence of the repressor agent or repressor signal;
wherein the first (N1) and second (N2) nuclease polypeptide fragments are Cas13 endonuclease polypeptide fragments; and
wherein RD1 comprises a repressible protease and RD2 comprises a peptide domain, or RD2 comprises a repressible protease and RD1 comprises a peptide domain.

### Claim 2 (depends on 1)

The system of claim 1,
wherein the repressible protease specifically binds to the peptide domain in the absence of a specific protease inhibitor, wherein the repressible protease does not specifically bind to the peptide domain in the presence of a specific protease inhibitor.

### Claim 3 (depends on 1)

The system of claim 1, wherein the repressible protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3).

### Claim 4 (depends on 1)

The system of claim 1, wherein the peptide domain comprises ANR peptide (SEQ ID NO: 170).

### Claim 5 (depends on 1)

The system of claim 1, wherein the peptide domain comprises CP5-46-5D5E peptide (SEQ ID NO: 171).

### Claim 6 (depends on 2)

The system of claim 2, wherein the protease inhibitor is selected from grazoprevir, danoprevir, simeprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir.

### Claim 7 (depends on 1)

The system of claim 1, wherein the first (N1) and second (N2) nuclease polypeptide fragments are Cas13d endonuclease polypeptide fragments, wherein N1 and N2 are selected from the group consisting of:
a) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 1 with the C-terminus ending at amino acid 88 of SEQ ID NO: 1 or ending at amino acid 88 of a polypeptide that is at least 95% identical to SEQ ID NO: 1, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 1 with the N-terminus beginning at amino acid 89 of SEQ ID NO: 1 or beginning at amino acid 89 of a polypeptide that is at least 95% identical to SEQ ID NO: 1;
b) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 1 with the C-terminus ending at amino acid 263 of SEQ ID NO: 1 or ending at amino acid 263 of a polypeptide that is at least 95% identical to SEQ ID NO: 1, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 1 with the N-terminus beginning at amino acid 264 of SEQ ID NO: 1 or beginning at amino acid 264 of a polypeptide that is at least 95% identical to SEQ ID NO: 1;
c) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 1 with the C-terminus ending at amino acid 384 of SEQ ID NO: 1 or ending at amino acid 384 of a polypeptide that is at least 95% identical to SEQ ID NO: 1, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 1 with the N-terminus beginning at amino acid 385 of SEQ ID NO: 1 or beginning at amino acid 385 of a polypeptide that is at least 95% identical to SEQ ID NO: 1;
d) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 1 with the C-terminus ending at amino acid 404 of SEQ ID NO: 1 or ending at amino acid 404 of a polypeptide that is at least 95% identical to SEQ ID NO: 1, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 1 with the N-terminus beginning at amino acid 405 of SEQ ID NO: 1 or beginning at amino acid 405 of a polypeptide that is at least 95% identical to SEQ ID NO: 1;
e) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 1 with the C-terminus ending at amino acid 507 of SEQ ID NO: 1 or ending at amino acid 507 of a polypeptide that is at least 95% identical to SEQ ID NO: 1, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 1 with the N-terminus beginning at amino acid 508 of SEQ ID NO: 1 or beginning at amino acid 508 of a polypeptide that is at least 95% identical to SEQ ID NO: 1;
f) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 1 with the C-terminus ending at amino acid 559 of SEQ ID NO: 1 or ending at amino acid 559 of a polypeptide that is at least 95% identical to SEQ ID NO: 1, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 1 with the N-terminus beginning at amino acid 560 of SEQ ID NO: 1 or beginning at amino acid 560 of a polypeptide that is at least 95% identical to SEQ ID NO: 1;
g) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 1 with the C-terminus ending at amino acid 565 of SEQ ID NO: 1 or ending at amino acid 565 of a polypeptide that is at least 95% identical to SEQ ID NO: 1, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 1 with the N-terminus beginning at amino acid 566 of SEQ ID NO: 1 or beginning at amino acid 566 of a polypeptide that is at least 95% identical to SEQ ID NO: 1;
h) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 1 with the C-terminus ending at amino acid 576 of SEQ ID NO: 1 or ending at amino acid 576 of a polypeptide that is at least 95% identical to SEQ ID NO: 1, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 1 with the N-terminus beginning at amino acid 577 of SEQ ID NO: 1 or beginning at amino acid 577 of a polypeptide that is at least 95% identical to SEQ ID NO: 1;
i) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 1 with the C-terminus ending at amino acid 655 of SEQ ID NO: 1 or ending at amino acid 655 of a polypeptide that is at least 95% identical to SEQ ID NO: 1, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 1 with the N-terminus beginning at amino acid 656 of SEQ ID NO: 1 or beginning at amino acid 656 of a polypeptide that is at least 95% identical to SEQ ID NO: 1; and
j) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 1 with the C-terminus ending at amino acid 903 of SEQ ID NO: 1 or ending at amino acid 903 of a polypeptide that is at least 95% identical to SEQ ID NO: 1, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 1 with the N-terminus beginning at amino acid 904 of SEQ ID NO: 1 or beginning at amino acid 904 of a polypeptide that is at least 95% identical to SEQ ID NO: 1.

### Claim 8 (depends on 1)

The system of claim 1, wherein the first (N1) and second (N2) nuclease polypeptide fragments are Cas13a endonuclease polypeptide fragments, wherein N1 and N2 are selected from the group consisting of:
a) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 2 with the C-terminus ending at amino acid 416 of SEQ ID NO: 2 or ending at amino acid 416 of a polypeptide that is at least 95% identical to SEQ ID NO: 2, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 2 with the N-terminus beginning at amino acid 417 of SEQ ID NO: 2 or beginning at amino acid 417 of a polypeptide that is at least 95% identical to SEQ ID NO: 2; and
b) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 2 with the C-terminus ending at amino acid 421 of SEQ ID NO: 2 or ending at amino acid 421 of a polypeptide that is at least 95% identical to SEQ ID NO: 2, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 2 with the N-terminus beginning at amino acid 422 of SEQ ID NO: 2 or beginning at amino acid 422 of a polypeptide that is at least 95% identical to SEQ ID NO: 2.

### Claim 9 (depends on 1)

The system of claim 1, wherein the first (N1) and second (N2) nuclease polypeptide fragments are Cas13b endonuclease polypeptide fragments, wherein N1 and N2 are selected from the group consisting of:
a) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 3 with the C-terminus ending at amino acid 49 of SEQ ID NO: 3 or ending at amino acid 49 of a polypeptide that is at least 95% identical to SEQ ID NO: 3, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 3 with the N-terminus beginning at amino acid 50 of SEQ ID NO: 3 or beginning at amino acid 50 of a polypeptide that is at least 95% identical to SEQ ID NO: 3;
b) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 3 with the C-terminus ending at amino acid 177 of SEQ ID NO: 3 or ending at amino acid 177 of a polypeptide that is at least 95% identical to SEQ ID NO: 3, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 3 with the N-terminus beginning at amino acid 178 of SEQ ID NO: 3 or beginning at amino acid 178 of a polypeptide that is at least 95% identical to SEQ ID NO: 3;
c) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 3 with the C-terminus ending at amino acid 250 of SEQ ID NO: 3 or ending at amino acid 250 of a polypeptide that is at least 95% identical to SEQ ID NO: 3, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 3 with the N-terminus beginning at amino acid 251 of SEQ ID NO: 3 or beginning at amino acid 251 of a polypeptide that is at least 95% identical to SEQ ID NO: 3;
d) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 3 with the C-terminus ending at amino acid 431 of SEQ ID NO: 3 or ending at amino acid 431 of a polypeptide that is at least 95% identical to SEQ ID NO: 3, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 3 with the N-terminus beginning at amino acid 432 of SEQ ID NO: 3 or beginning at amino acid 432 of a polypeptide that is at least 95% identical to SEQ ID NO: 3; and
e) N1 comprises a N-terminal polypeptide fragment of SEQ ID NO: 3 with the C-terminus ending at amino acid 1065 of SEQ ID NO: 3 or ending at amino acid 1065 of a polypeptide that is at least 95% identical to SEQ ID NO: 3, and N2 comprises a C-terminal polypeptide fragment of SEQ ID NO: 3 with the N-terminus beginning at amino acid 1066 of SEQ ID NO: 3 or beginning at amino acid 1066 of a polypeptide that is at least 95% identical to SEQ ID NO: 3.

### Claim 10 (depends on 1)

The system of claim 1, wherein a split site between the first (N1) and second (N2) nuclease polypeptide fragments is in a structured region of Cas13.

### Claim 11 (depends on 1)

The system of claim 1, wherein a split site between the first (N1) and second (N2) nuclease polypeptide fragments is in an unstructured region of Cas13.

### Claim 12 (depends on 10)

The system of claim 10, wherein the split site does not comprise a conserved residue of Cas13 compared to a plurality of Cas13 orthologs.

### Claim 13 (depends on 12)

The system of claim 12, wherein the conserved residue is in a secondary structure of Cas13 or a catalytic domain of Cas13.

### Claim 14 (depends on 3)

The system of claim 3, wherein the NS3 is catalytically dead.

### Claim 15 (depends on 1)

The system of claim 1, wherein the first or second polypeptide does not comprise any protease cleavage sites.

### Claim 16 (depends on 2)

The system of claim 2, further comprising a cofactor for the repressible protease.

### Claim 17 (depends on 16)

The system of claim 16, wherein the cofactor is an HSV NS4A domain.

### Claim 18 (depends on 17)

The system of claim 17, wherein the HSV NS4A domain is adjacent and N-terminal to the repressible protease.

### Claim 19 (depends on 1)

The system of claim 1, wherein there is a lag time of 30 seconds or less in between protein complementation of the two polypeptide fragments in the absence of the repressor agent or repressor signal and the protein being in its active state.

### Claim 20 (depends on 1)

The system of claim 1, wherein the first and/or second polypeptide further comprises:
a) at least one cytosolic sequestering domain, wherein the cytosolic sequestering domain comprises a ligand binding domain (LBD), wherein, in the presence of a ligand, the sequestering of the protein to the cytosol is inhibited;
b) at least one nuclear export signal (NES); or
c) at least one nuclear localization signal (NLS).