Selectable marker proteins, expression vectors, engineered cells and extracellular vesicles for the production of virus-like particles for therapeutic and prophylactic applications

Abstract

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)