SELECTABLE MARKER PROTEINS, EXPRESSION VECTORS, ENGINEERED CELLS AND EXTRACELLULAR VESICLES FOR THE PRODUCTION OF VIRUS-LIKE PARTICLES FOR THERAPEUTIC AND PROPHYLACTIC APPLICATIONS
The present invention relates to engineered selectable marker proteins for recombinant protein expression, as well as novel expression vector designs for achieving high-level recombinant protein expression, and cells transfected therewith as a platform technology for producing extracellular vesicle-based therapeutic or prophylactic compositions, wherein one or more recombinant proteins of interest are displayed on the surface of the extracellular vesicles. As an example, the present invention relates to a virus-like article composition comprising such extracellular vesicles displaying one or more antigens configured to induce immune responses against SARS-CoV-2.
1 . A non-naturally occurring selectable marker (SM) protein, wherein the SM protein comprises a destabilization domain (DD) operably connected to a SM protein, thereby providing a non-naturally occurring SM protein.
2 . The non-naturally occurring SM protein according to claim 1 , wherein said SM protein is an SM protein that functions in a mammalian cell.
3 . The non-naturally occurring SM protein according to claim 1 , wherein said DD is appended to the N-terminus of said SM protein, the C-terminus of said SM protein, or both the N-terminus and the C-terminus of said SM protein.
4 . The non-naturally occurring SM protein according to claim 1 , wherein said DD is appended to the N-terminus of said SM protein.
5 . The non-naturally occurring SM protein according to claim 1 , wherein said SM protein is a dominant SM protein.
6 . The non-naturally occurring SM protein according to claim 1 , wherein said non-naturally occurring SM protein confers resistance to zeocin, puromycin, hygromycin, G418, and/or blasticidin.
7 . The non-naturally occurring SM protein according to claim 6 , wherein said SM protein that confers resistance to zeocin is BleoR, wherein said SM protein that confers resistance to puromycin is PuroR; wherein said SM protein that confers resistance to hygromycin is HygR; wherein said SM protein that confers resistance to G418 is NeoR; and/or wherein said SM protein for mammalian cells that confers resistance to blasticidin is BsdR.
8 . The non-naturally occurring SM protein according to claim 1 , wherein said DD is derived from the human estrogen receptor (ER50), thereby providing a SM protein operably connected to the ER50(DD).
9 . The non-naturally occurring SM protein according to claim 8 , wherein said SM protein operably connected to the ER50(DD) is BleoR operably connected to the ER50(DD), i.e., ER50BleoR; PuroR operatively connected to the ER50(DD), i.e., ER50PuroR; HygR operatively connected to the ER50(DD), i.e., ER50HygR; NeoR operatively connected to the ER50(DD), i.e., ER50NeoR; or BsdR operatively connected to the ER50(DD), i.e., ER50BsdR.
10 . The non-naturally occurring SM protein according to claim 1 , wherein said DD is derived from the Escherichia coli dihydrofolate reductase (ecDHFR), thereby providing an SM protein operatively connected to the ecDHFR(DD).
11 . The non-naturally occurring SM protein according to claim 10 , wherein said SM operatively linked to the ecDHFR(DD) is BleoR operatively linked to the ecDHFR(DD), i.e., ecDHFRBleoR; PuroR operatively linked to the ecDHFR(DD), i.e., ecDHFRPuroR; HygR operatively linked to the ecDHFR(DD), i.e., ecDHFRHygR; NeoR operatively linked to the ecDHFR(DD), i.e., ecDHFRNeoR; or BsdR operatively linked to the ecDHFR(DD), i.e., ecDHFRBsdR.
12 . The non-naturally occurring SM protein according to claim 1 , wherein the engineered SM protein further comprises an altered amino acid sequence resulting from a frameshift mutation within a nucleotide sequence that encodes the last about 10, 20, 30, 40, or 50 amino acids at the 3′ end of the DD-tagged SM.
13 . An isolated nucleic acid, the nucleotide sequence of which encodes the engineered SM protein according to claim 1 .
14 . An expression vector comprising a nucleic acid, the nucleotide sequence of which encodes a selectable marker (SM) protein, optionally an unstable and/or degraded SM protein, and an operably linked recombinant protein of interest (POI), wherein the nucleic acid is operably linked to an expression control sequence.
15 - 16 . (canceled)
17 . The expression vector according to claim 14 , wherein said nucleic acid comprises an open reading frame (ORF) that encodes (a) the POI, followed by (b) a self-cleaving peptide which can induce ribosomal skipping during translation, and (c) the SM protein.
18 - 28 . (canceled)
29 . A cell comprising the expression vector of claim 14 .
30 . A cultured cell line comprising the expression vector of claim 14 , wherein the cells in the cultured cell line are selected by culturing in a selection-containing media.
31 - 35 . (canceled)
36 . A method of making extracellular vesicles (EVs) comprising culturing a cell line of claim 30 , wherein the cell line produces EVs comprising one or more of the POIs, and isolating the EVs produced.
37 . The method according to claim 36 , wherein said EVs are exosomes or microvesicles.
38 - 43 . (canceled)
44 . An expression vector comprising:
(a) a nucleic acid the nucleotide sequence of which encodes a transposon comprised of inverted terminal repeats (ITR-L and ITR-R elements) that define the left and right ends of the transposon;
(b) one or more genes of interest (GOIs) encoding one or more proteins of interest (POIs) inserted between the ITR-L and ITR-R elements, wherein the GOIs are operably linked to expression control sequences; and
(c) a nucleic acid the nucleotide sequence of which encodes a transposase enzyme, wherein the nucleic acid is located outside the transposon, and wherein the nucleic acid is operably linked to an expression control sequence.
45 - 133 . (canceled)