US 12,565,665 B2Granted

Compositions and methods for controlled mRNA translation and stability

AssigneeTrustees of Boston University

InventorsAlexander Michael Marzilli, John Tuan Ngo

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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.

Claims17

Dependent claims

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.

26 more elements

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.

24 more elements

Dependencies are inferred from the claim text.

Facts

Application: 18/392,928Filed 21 Dec 2023 · us provisional application
Priority: 21 Dec 2022
Published: US 2024/0263193 A18 Aug 2024
Granted: 3 Mar 2026
Term ends: Not estimatedThe source records don’t support an estimate.

Classification16 codes

C12N 15/87
Recombinant DNA-technology: introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
A61P 35/00
Antineoplastic agents
C07K 14/005
Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof: from viruses
C12N 9/78
Hydrolases: acting on carbon to nitrogen bonds other than peptide bonds

Family

Application63/434,275Us provisional application · filed 21 Dec 2022
US 2024/0263193 A1Pre-grant publication · published 8 Aug 2024
US 12,565,665 B2This patent

From the US filing history. Foreign family members aren’t listed.

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