EP-4735054-A2 - IMPROVED MULTICISTRONIC SYSTEMS AND USES THEREOF

EP4735054A2EP 4735054 A2EP4735054 A2EP 4735054A2EP-4735054-A2

Abstract

Provided herein are optimized 2A ribosome skipping elements and polynucleotide sequences encoding such optimized 2A ribosome skipping elements, as well as multicistronic systems, constructs, vectors, and engineered cells utilizing the same for expressing multiple polypeptides from a single transcript.

Inventors

COTTMAN, Rebecca Tayler

Assignees

Senti Biosciences, Inc.

Dates

Publication Date: 20260506
Application Date: 20240628

Claims (9)

1. An engineered polynucleotide comprising a first sequence encoding for a first polypeptide and a second sequence encoding for a second polypeptide, wherein the first sequence and the second sequence are operably linked via a linker polynucleotide sequence encoding a 2A ribosome skipping element comprising at least a portion of a P2A element and a T2A element.
2. The engineered polynucleotide of claim 1, comprising a sequence according to formula, oriented from 5’ to 3’: Pl - L - P2 wherein Pl comprises the first sequence, L comprises the linker polynucleotide sequence encoding the 2A ribosome skipping element, and P2 comprises the second sequence.
3. The engineered polynucleotide of any one of the preceding claims, wherein the 2A ribosome skipping element comprises the amino acid sequence ATNFSLLKQAGDVEENPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 269).
4. A vector comprising the engineered polynucleotide of any one of the preceding claims.
5. An engineered cell comprising the engineered polynucleotide or vector of any one of the preceding claims.
6. A pharmaceutical composition comprising the engineered polynucleotide or vector of any one of the preceding claims and a pharmaceutically acceptable carrier.
7. A pharmaceutical composition comprising the engineered cell of claim 5 and a pharmaceutically acceptable carrier.
8. A method, comprising administering to a subject a pharmaceutical composition of any one of the preceding claims.
9. A method of producing an engineered cell, comprising introducing an engineered polynucleotide or vector of any one of the preceding claims into a cell.

Description

IMPROVED MULTICISTRONIC SYSTEMS AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/511,459, filed on June 30, 2023, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML file, created on Month XX, 20XX, is named XXXXX.xml, and is X, XXX, XXX bytes in size. BACKGROUND [0003] Multicistronic expression systems can employ 2A ribosome skipping elements to generate multiple separated polypeptides from a single transcript. However, conventional 2A ribosome skipping elements can result in incomplete separation of polypeptides due to inefficient ribosome skipping. SUMMARY [0004] Provided herein are optimized 2 A ribosome skipping elements and polynucleotide sequences encoding such optimized 2A ribosome skipping elements. [0005] In some embodiments, a 2A ribosome skipping element comprises at least a functional portion of a P2A ribosome skipping element and at least a functional portion of a T2A ribosome skipping element. The 2A ribosome skipping element may comprise P2A ribosome skipping element and a T2A ribosome skipping element, e.g., a P2A-T2A sequence. In some embodiments, the 2A ribosome skipping element comprises the amino acid sequence ATNFSLLKQAGDVEENPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 269). [0006] Also provided herein is an engineered polynucleotide comprising a first sequence encoding for a first polypeptide and a second sequence encoding for a second polypeptide, wherein the first sequence and the second sequence are operably linked via a linker polynucleotide sequence encoding a 2A ribosome skipping element comprising at least a portion of a P2A element and a T2A element. In some embodiments, the engineered polynucleotide comprises a sequence according to formula, oriented from 5’ to 3’: Pl - L - P2 wherein Pl comprises the first sequence, L comprises the linker polynucleotide sequence encoding the 2A ribosome skipping element, and P2 comprises the second sequence. [0007] In some embodiments, the 2A ribosome skipping element comprises the amino acid sequence ATNFSLLKQAGDVEENPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 269). [0008] Also provided herein are vectors comprising engineered polynucleotides described herein, engineered cells comprising engineered polynucleotides or vectors described herein, pharmaceutical compositions comprising engineered polynucleotides, vectors, or cells described herein, and methods of preparing or using such cells. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS [0009] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description, and accompanying drawings. [0010] FIGs. 1A-1E show results of an assay to evaluate different 2A linkers between different pay load components in a multicistronic system. DETAILED DESCRIPTION [0011] The present disclosure generally relates to modified degron polypeptides with increased sensitivity to pomalidomide. Also provided are inducibly degradable proteins, inducible cell death systems, and activation-conditional control polypeptides employing modified degron polypeptides and methods and/or use thereof as described herein. [0012] Terms used in the claims and specification are defined as set forth below unless otherwise specified. [0013] The term “ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a cancer disease state, including prophylaxis, lessening in the severity or progression, remission, or cure thereof. [0014] The term “zTz situ” refers to processes that occur in a living cell growing separate from a living organism, e.g., growing in tissue culture. [0015] The term “zzz vivo” refers to processes that occur in a living organism. [0016] The term “mammal” as used herein includes both humans and non-humans, and includes, but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines. [0017] The term “percent identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm e.g., any of those described herein (e.g., BLASTP and BLASTN or other algorithms available to persons of skill), or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. [0018] For sequence comparison, typically o