Methods and compositions to spread protein cargoes across multi-nucleated cells
Inventors
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Assignees
University of Florida Research Foundation Inc
MemberUniversity of MiamiUniversity of MiamiThe University of Miami, established in 1925 and based in Coral Gables, Florida, is a private research university recognized for its comprehensive academic offerings, robust research infrastructure, and strong interdisciplinary focus. Home to more than 19,000 students and with more than 400 acres of campuses across the Miami region, its mission encompasses education, research, innovation, and community service. The institution supports clinical, biomedical, marine, and atmospheric research initiatives, delivers diverse undergraduate and graduate programs, and maintains numerous research centers and institutes dedicated to scientific, medical, and societal advancements.
The University of Miami, established in 1925 and based in Coral Gables, Florida, is a private research university recognized for its comprehensive academic offerings, robust research infrastructure, and strong interdisciplinary focus. Home to more than 19,000 students and with more than 400 acres of campuses across the Miami region, its mission encompasses education, research, innovation, and community service. The institution supports clinical, biomedical, marine, and atmospheric research initiatives, delivers diverse undergraduate and graduate programs, and maintains numerous research centers and institutes dedicated to scientific, medical, and societal advancements.
Publication Number
US-12662517-B2
Publication Date
2026-06-23
Expiration Date
Abstract
Methods and compositions useful for delivering a protein to a plurality of nuclei in a cell. Fusion proteins comprising one or more signals useful for promoting delivery of the proteins to a plurality of nuclei in a multinucleate cell. Methods comprising administering isolated nucleic acids to a multinucleate cell, wherein the isolated nucleic acid comprises a sequence encoding a fusion protein, the fusion protein comprising, a protein of interest fused to at least the following migration signals: (a) at least one nuclear export signal (NFS); and (b) at least one nuclear localization signal (NLS) and/or at least one nucleolar localization signal (NoLS). Fusion proteins comprising: (a) a protein of interest; and (b) at least the following migration signals: (i) a nuclear export signal (NFS); (ii) a nuclear localization signal (NLS); and (iii) a nucleolar localization signal (NoLS). Methods of making and using the same. Methods of delivering recombinant genes and/or fusion proteins to multinucleate cells.
Core Innovation
The invention relates to methods comprising administering an isolated nucleic acid to a multinucleate cell. The isolated nucleic acid comprises a sequence encoding a fusion protein in which a protein of interest is fused to at least one nuclear export signal (NES) and at least one nuclear localization signal (NLS) and/or at least one nucleolar localization signal (NoLS). The protein of interest comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 13.
The described fusion-protein migration-signal arrangement supports movement between subcellular compartments in multinucleate cells by combining nuclear export signals with nuclear localization signals and/or nucleolar localization signals. Administering the nucleic acid provides the fusion protein program through expression of the encoded fusion protein in the multinucleate cell.
Compositions and embodiments include an isolated nucleic acid flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs) and optional structural refinements for the fusion protein, including linker-mediated attachment and positioning of migration signals at either the N-terminus or C-terminus of the protein of interest. The document also associates the approach with therapeutic target use cases including DUX4 and Facioscapulohumeral dystrophy, including a dominant-negative DUX4 variant.
Claims Coverage
The independent claim is directed to a method of administering an isolated nucleic acid to a multinucleate cell encoding a nuclear-migrating fusion protein that includes both NES and NLS/NoLS, with a sequence-identity constraint relative to SEQ ID NO: 13.
Administering an isolated nucleic acid encoding an NES and NLS/NoLS fusion protein to multinucleate cells
A method comprising administering an isolated nucleic acid to a multinucleate cell, wherein the isolated nucleic acid comprises a sequence encoding a fusion protein, the fusion protein comprising a protein of interest fused to at least one nuclear export signal (NES) and at least one nuclear localization signal (NLS) and/or at least one nucleolar localization signal (NoLS), wherein the protein of interest comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 13.
Across the dependent claim refinements described in the provided material, the core inventive concept is maintained while narrowing or specifying the fusion construct and delivery cassette features, including linker attachment, migration-signal positioning at the N-terminus or C-terminus, identity thresholds of migration signals relative to specified SEQ IDs, and AAV ITR flanking. The disease-context limitation is optionally introduced via a disorder such as Facioscapulohumeral dystrophy.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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