# US 12,565,665 B2 · Compositions and methods for controlled mRNA translation and stability

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

- Number: US 12,565,665 B2
- Document ID: US12565665B2
- Status: Granted
- Kind: B2
- Application number: 18/392,928
- Filed: 2023-12-21
- Published: 2024-08-08
- Granted: 2026-03-03
- Earliest claimed: 2022-12-21
- Assignees: Trustees of Boston University
- Applicants: Trustees of Boston University
- Inventors: Alexander Michael Marzilli; John Tuan Ngo
- Web page: https://patentgrep.com/patent/US12565665B2

## CPC

- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- A61P 35/00: Antineoplastic agents
- C07K 14/005: from viruses
- C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- 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 305/04: in cyclic amidines (3.5.4)
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2319/00: Fusion polypeptide
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2795/18022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- C12Y: ENZYMES
- 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

The technology described herein is directed to compositions, kits, systems and methods related to an engineered, inducible adenosine deaminase (iAD) enzymes, including but not limited to, an engineered inducible adenosine deaminase acting on RNA (ADAR) enzyme, which can be activated in the presence of an inducer. Also described are synthetic RNA molecules, to which the iAD can be specifically recruited to edit at least one target codon, leading to decreased or increased translation of the RNA molecules depending on the specific construct. The technology described herein is also directed to systems comprising the iAD and synthetic RNA molecule, nucleic acids and vectors encoding the iAD and synthetic RNA molecule, and methods of using such systems, nucleic acids, and vectors.

## Claims

### Claim 1

A fusion protein comprising:
(a) an N-terminal portion of a deaminase domain (DD) of an adenosine deaminase (AD-DDN), wherein the adenosine deaminase comprises Adenosine Deaminase Acting on RNA 1 (ADAR1) comprising the amino acid sequence of SEQ ID NO: 79, ADAR2 comprising the amino acid sequence of SEQ ID NO: 80, ADAR3 comprising the amino acid sequence of SEQ ID NO: 81, Adenosine Deaminase Domain Containing 1 (ADAD1) comprising the amino acid sequence of SEQ ID NO: 82, or ADAD2 comprising the amino acid sequence of SEQ ID NO: 83;
(b) a binding protein 1 (BP1) of a binding pair linked to the AD-DDN;
(c) a C-terminal portion of the DD (AD-DDC); wherein the AD-DDN and AD-DDC are split between:
(i) residues 5977 and D978 or T984 and E985 of SEQ ID NO: 79;
(ii) residues A468 and D469 of SEQ ID NO: 80;
(iii) residues 5507 and 5508 of SEQ ID NO: 81;
(iv) residues L340 and R341 of SEQ ID NO: 82;
(v) residues G357 and G358 of SEQ ID NO: 83; and
(d) a binding protein 2 (BP2) of the binding pair linked to the AD-DDC;
wherein the BP1 is capable of binding to the BP2 in the absence of an inducer, resulting in allosteric inhibition of the AD-DDN and AD-DDC,
wherein the BP1 is not capable of binding to the BP2 in the presence of the inducer, resulting in activation of the AD-DDN and AD-DDC; wherein the activation of the AD-DDN and AD-DDC comprises deaminase activity, and
wherein the BP1 and BP2 are:
(i) Bad and Bcl-xL, and the inducer of the BP1 and BP2 is A-1331852 or ABT-737;
(ii) MS1 and MCL-1, and the inducer of the BP1 and BP2 is 563845; or
(iv) a first ALFA epitope and an anti-ALFA nanobody, and the inducer of the BP1 and BP2 is a second ALFA epitope, wherein the anti-ALFA nanobody is capable of binding to the second ALFA antigen with a similar or higher affinity to the first ALFA antigen.

### Claim 2 (depends on 1)

The fusion protein of claim 1, wherein in the allosteric inhibition of the AD-DDN and AD-DDC comprises deformation of the inositol hexaphosphate (IP6) binding pocket of the AD-DDN and AD-DDC, wherein the deformation of the IP6 pocket prevents access of cofactor IP6 to the IP6 binding pocket.

### Claim 3 (depends on 1)

The fusion protein of claim 1, further comprising an RNA-binding domain, wherein the RNA-binding domain (RBD) is capable of binding to a binding motif for the RBD on an RNA molecule.

### Claim 4 (depends on 3)

The fusion protein of claim 3, wherein the RNA-binding domain is selected from the group consisting of MCP, PCP, λN, and HIV tat.

### Claim 5 (depends on 1)

The fusion protein of claim 1, wherein the deaminase activity comprises:
(a) deamination of an adenosine nucleotide into an inosine nucleotide in an RNA molecule;
(b) converting at least one stop codon into at least one non-stop codon;
(c) converting at least one start codon into at least one non-start codon;
(d) converting at least one non-start codon into at least one start codon;
and/or
(d) converting at least one sense codon encoding a first amino acid into at least one mutated sense codon encoding a second amino acid.

### Claim 6 (depends on 1)

The fusion protein of claim 1, wherein in the presence of the inducer, the DD is constitutively active.

### Claim 7 (depends on 6)

The fusion protein of claim 6, wherein the constitutively active deaminase domain comprises: an E1008Q mutation in SEQ ID NO: 79 (ADAR1); an E488Q mutation in SEQ ID NO: 80 (ADAR2); or an E527Q mutation in SEQ ID NO: 81 (ADAR3).

### Claim 8 (depends on 1)

The fusion protein of claim 1, wherein the DD comprises at least one mutation in the IP6 binding pocket that decreases background deaminase activity compared to a DD without the at least one mutation: wherein the at least one mutation is in SEO ID NO: 80 (ADAR2) selected from the group consisting of: R400K, R522M, K690R, and L699G.

### Claim 9 (depends on 3)

The fusion protein of claim 3, comprising from N-terminus to C-terminus:
(a) the RNA-binding domain;
(b) the AD-DDN;
(c) the BP1;
(d) the AD-DDC; and
(e) the BP2; or
comprising from N-terminus to C-terminus
(f) the RNA-binding domain;
(g) the BP1;
(h) the AD-DDN;
(i) the AD-DDC; and
(j) the BP2.

### Claim 10 (depends on 1)

The fusion protein of claim 1, wherein the fusion protein further comprises a cleavable linker between the AD-DDC and the BP2.

### Claim 11

A fusion protein comprising:
(a) an N-terminal portion of a deaminase domain (DD) of an adenosine deaminase (AD-DDN), wherein the adenosine deaminase comprises Adenosine Deaminase Acting on RNA 1 (ADAR11comprising the amino acid sequence of SEQ ID NO: 79, ADAR2 comprising the amino acid sequence of SEQ ID NO: 80, ADAR3 comprising the amino acid sequence of SEQ ID NO: 81, Adenosine Deaminase Domain Containing 1 (ADAD1) comprising the amino acid sequence of SEQ ID NO: 82, or ADAD2 comprising the amino acid sequence of SEQ ID NO: 83;
(b) a repressible protease linked to the AD-DDN;
(c) a C-terminal portion of the DD (AD-DDC); wherein the AD-DDN and AD-DDC are split between:
(i) residues 5977 and D978 or T984 and E985 of SEQ ID NO: 79;
(ii) residues A468 and D469 of SEQ ID NO: 80;
(iii) residues 5507 and 5508 of SEQ ID NO: 81;
(iv) residues L340 and R341 of SEQ ID NO: 82;
(v) residues G357 and G358 of SEQ ID NO: 83; and
(d) a protease cleavage site linked to the AD-DDN and the AD-DDC; wherein the repressible protease is capable of binding to the protease cleavage site in the absence of an inhibitor for the repressible protease, resulting in cleavage of the protease cleavage site and inactivation of the AD-DDN and the AD-DDC,
wherein the repressible protease is not capable of binding to the protease cleavage site in the presence of the inhibitor for the repressible protease, resulting in activation of the AD-DDN and the AD-DDC; wherein the activation of the AD-DDN and AD-DDC comprises deaminase activity; and
wherein the repressible protease comprises hepatitis C virus (HCV) nonstructural protein 3 (NS3), the cleavage site is an NS3 cleavage site, and the inhibitor is selected from the group consisting of: grazoprevir, danoprevir, simeprevir, asuanprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir.

### Claim 12 (depends on 1)

A system for modulating RNA translation comprising:
(a) the fusion protein of claim 1
(b) a RNA molecule comprising:
(I)
(a) a double-stranded region comprising:
(i) at least one target codon; and
(ii) an RNA binding motif capable of being bound by an RNA-binding domain; and
(b) at least one open reading frame, wherein the at least one open reading frame is operatively linked to the double-stranded region; or
(II)
(a) a first open reading frame;
(b) a double-stranded region comprising:
(i) at least one target codon; and
(ii) an RNA binding motif capable of being bound by an RNA-binding domain; and
(c) a second open reading frame, wherein the second open reading frame is operatively linked to the double-stranded region; or
(III)
(a) an open reading frame:
(b) a double-stranded region comprising:
(i) at least one target codon; and
(ii) an RNA binding motif capable of being bound by an RNA-binding domain; and
(c) a poly-A tail.

### Claim 13 (depends on 12)

The system of claim 12, further comprising an inducer of the BP1 and BP2.

### Claim 14 (depends on 12)

The system of claim 12, wherein the BP1 and BP2 of the fusion protein are capable of binding to each other in the absence of an inducer and reduce or prevent:
(a) the formation of the inositol hexaphosphate (IP6) binding pocket of the BP1 and BP2;
(b) deaminase activity of the AD-DDN and AD-DDC;
(c) conversion by the deaminase domain of the at least one target codon of the RNA molecule into at least one inosine-comprising codon;
(d) conversion by the deaminase domain of the at least one stop codon of the RNA molecule into at least one non-stop codon;
(e) conversion by the deaminase domain of the at least one start codon of the RNA molecule into at least one non-start codon;
(f) conversion by the deaminase domain of the at least one non-start codon of the RNA molecule into at least one start codon; and/or
(g) conversion by the deaminase domain of the at least one sense codon of the RNA molecule encoding for a first amino acid into at least one mutated codon encoding for a second amino acid.

### Claim 15 (depends on 12)

The system of claim 12 wherein the BP1 and BP2 of the fusion protein are not capable of binding to each other in the presence of an inducer, allowing for:
(a) the formation of the inositol hexaphosphate (IP6) binding pocket of the BP1 and BP2;
(b) deaminase activity of the AD-DDN and AD-DDC;
(c) conversion by the deaminase domain of the at least one target codon of the RNA molecule into at least one inosine-comprising codon;
(d) conversion by the deaminase domain of the at least one stop codon of the RNA molecule into at least one non-stop codon;
(e) conversion by the deaminase domain of the at least one start codon of the RNA molecule into at least one non-start codon;
(f) conversion by the deaminase domain of the at least one non-start codon of the RNA molecule into at least one start codon; and/or
(g) conversion by the deaminase domain of the at least one sense codon of the RNA molecule encoding for a first amino acid into at least one mutated sense codon encoding for a second amino acid.

### Claim 16 (depends on 15)

The system of claim 15, wherein conversion by the deaminase domain of at least one target codon in the RNA molecule into at least one inosine-comprising codon increases or decreases:
(a) translation of the reading frame of the RNA molecule;
(b) translation of the second reading frame of the RNA molecule;
(c) degradation of the RNA molecule; and/or
(d) translation of a variant polypeptide encoded by the RNA molecule.

### Claim 17 (depends on 11)

The fusion protein of claim 11, wherein the allosteric inhibition of the first and second portions of the DD comprises deformation of the inositol hexaphosphate (IP6) binding pocket of first and second portions of the DD, wherein the deformation of the IP6 pocket prevents access of cofactor IP6 to the IP6 binding pocket.