Recombinant polypeptides for membrane fusion and uses thereof

Inventors

Duncan, Roy • Clancy, Eileen Kathryn • Lewis, John • De Antueno, Roberto Justo • Nesbitt, Rae-Lynn

Assignees

Entos Pharmaceuticals Inc

Abstract

Disclosed is a recombinant polypeptide for facilitating membrane fusion. The recombinant polypeptide having a sequence with at least 80% sequence identity with the ectodomain of p14 fusion-associated small transmembrane (FAST) protein and having a functional myristoylation motif, a transmembrane domain from a FAST protein and a sequence with at least 80% sequence identity with the endodomain of p15 FAST protein. A targeting ligand can be added to the recombinant polypeptide for selective fusion. The recombinant polypeptide can be included in the membrane of a liposome, or the like, to facilitate the delivery of bioactive compounds, such as siRNA, or the recombinant polypeptide can be mixed with a lipid carrier and added to cultured cells to induce cell-cell fusion and heterokaryon formation.

Core Innovation

The invention relates to a recombinant polypeptide for facilitating membrane fusion that comprises an ectodomain sequence comprising the sequence of SEQ ID NO: 19, a p14 fusion-associated small transmembrane (FAST) protein transmembrane domain connected to the ectodomain sequence, and an endodomain sequence connected to the transmembrane domain. The endodomain comprises a hydrophobic patch having the sequence of SEQ ID NO: 8.

The FAST transmembrane region is described as having defined structural requirements, including a length of 23 aa with hydrophobic β-branched residues, tri-serine residues, and specific glycine positions. The endodomain hydrophobic patch is described as a hydrophobic patch in the p15 endodomain region, including p15 hydrophobic patch in p14 chimeras, and functional myristoylation motif is described as part of the design context.

The invention further describes targeting via a ligand, notably bombesin, and incorporation into liposomes/proteoliposomes for selective membrane fusion and intracellular delivery of payloads. Reported results are described as supporting modular enhancement of syncytia and pore formation, including roles of the p15 endodomain and FAST protein transmembrane residues such as glycine/proline and tri-serine.

Claims Coverage

The document provides one independent claim and dependent claims refining at least three core inventive elements: the ectodomain defined by SEQ ID NO: 19 or SEQ ID NO: 2, the FAST protein transmembrane domain with motif constraints, and the p15 endodomain hydrophobic patch defined by SEQ ID NO: 8, with further refinements to liposome/proteoliposome formulations and an optional targeting ligand.

Overall, the claim coverage centers on a membrane-fusion facilitating recombinant polypeptide defined by a specific ectodomain, a p14 FAST transmembrane domain, and an endodomain hydrophobic patch defined by SEQ ID NO: 8, with dependent refinements adding ectodomain identity or sequence constraints, transmembrane motif exclusion, and liposome embodiments that can include a targeting ligand such as bombesin.

Stated Advantages

Documented Applications