Methods and compositions to spread protein cargoes across multi-nucleated cells
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.
1 . 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 the following migration signals:
(a) at least one nuclear export signal (NES); and
(b) 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.
2 . The method of claim 1 , wherein the fusion protein further comprises at least one additional migration signal, wherein the additional migration signal may be identical or distinct from the migration signals used in claim 1 , or wherein the fusion protein further comprises at least two additional migration signals, wherein the additional migration signals may be identical or distinct from the migration signals used in claim 1 .
3 . The method of claim 1 , wherein the isolated nucleic acid encodes a fusion protein wherein at least one of the migration signals is positioned at the C-terminus of the protein of interest or at the N-terminus of the protein of interest.
4 . The method of claim 1 , wherein the protein of interest carries RNA.
5 . The method of claim 1 , wherein the protein of interest is used to treat Facioscapulohumeral dystrophy.
6 . The method of claim 1 , wherein the isolated nucleic acid is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs).
7 . The method of claim 1 , wherein the multinucleate cell is a skeletal muscle cell.
8 . The method of claim 1 , wherein the multinucleate cell is a liver cell.
9 . The method of claim 1 , wherein the nucleic acid is administered to a subject, optionally wherein the subject is mammalian.
10 . The method of claim 1 , wherein the subject has a disorder, optionally wherein the disorder is Facioscapulohumeral dystrophy.
11 . The method of claim 1 , wherein at least one of the migration signals is linked to the protein of interest via a linker.
12 . The method of claim 1 , wherein at least one of the migration signals comprises an amino acid sequence with at least 95% identity to any one of SEQ ID NO: 1-6 or 14-570.
13 . The method of claim 1 , wherein at least one of the migration signals comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 3.
14 . The method of claim 1 , wherein at least one of the migration signals comprises an amino acid sequence of SEQ ID NO: 1.
15 . The method of claim 1 , wherein at least one of the migration signals comprises an amino acid sequence of SEQ ID NO: 3.