Rotavirus vectors for heterologous gene delivery
AssigneeMerck Sharp & Dohme Corp.
InventorsDai Wang, Jiajie Wei
Life of this patent
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Abstract
Rotavirus vectors encoding in their genome a heterologous gene, and nucleic acid constructs encoding such rotavirus vectors. The rotavirus vector genome may include a rotavirus non-structural protein, a 2A peptide downstream of the rotavirus non-structural protein, and a heterologous protein downstream of the 2A peptide. The heterologous gene may be, for example, a SARS-CoV-2 spike protein or a fragment thereof, or an RSV F protein or a fragment thereof.
Claims43
An isolated nucleic acid molecule comprising:
- a promoter sequence;
- and
- a cDNA molecule encoding:
- a rotavirus non-structural protein;
- a 2A peptide downstream of the rotavirus non-structural protein;
- and a heterologous protein downstream of the 2A peptide.
A recombinant rotavirus comprising in its genome a cDNA sequence encoding a 2A peptide downstream of NSP1, NSP3, or NSP5, and a heterologous gene downstream of the 2A peptide.
A method for rescuing recombinant rotavirus, the method comprising:
- a) transfecting cells with
- i) eleven individual rotavirus genomic segment plasmids (RGSP), each RGSP having a promoter and encoding one of a single rotavirus protein VP1, VP2, VP3, VP4, NSP1, VP6, NSP3, NSP2, VP7, NSP4, or NSP5, wherein one or more of the plasmids encoding NSP1, NSP3, and NSP5 protein includes a sequence encoding a 2A protein that is downstream of the NSP protein and a sequence encoding a heterologous protein that is downstream of the sequence encoding the 2A protein, and
- ii) five individual helper plasmids (HPs), each HP having a promoter and encoding one of a fusogenic Fusion-Associated Small Transmembrane (FAST) protein, RNA capping enzyme DIR, RNA capping enzyme D12L, NSP2 protein, or NSP5 protein;
- b) maintaining the transfected cells in conditions suitable for the production of recombinant rotavirus;
- and
- c) harvesting the resulting recombinant rotavirus.
Dependencies are inferred from the claim text.