Linker sequence potency assays for multiple coding nucleic acids
AssigneeBioNTech SE
InventorsDaniel Abram Rothenberg, Jens Schumacher
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Abstract
The invention provides potency assays for simultaneously analyzing at least two different nucleic acid sequences such as RNA and/or DNA sequences encoding a functional sequence such as an antigen or epitope. The potency assays of the present invention may be performed with nucleic acid sequences encoding at least two different functional sequences, including at least two different antigens or epitopes.
Claims28
A method for simultaneously analysing at least two different nucleic acid sequences, each encoding a different amino acid sequence, wherein each of the at least two different amino acid sequences comprises a different functional sequence and a different linker sequence, wherein each linker sequence is capable of being proteolytically excised from its amino acid sequence, wherein the method comprises the following steps:
- (i) providing the at least two different nucleic acid sequences;
- (ii) introducing the at least two different nucleic acid sequences into a cell;
- (iii) expressing the at least two different amino acid sequences;
- (iv) proteolytically excising the at least two different linker sequences;
- (v) determining the amounts of each of the excised linker sequences;
- (vi) using the amounts of each of the excised linker sequences as an indication for the potency of each of the nucleic acid sequences to express each of the functional sequences in a biological system.
A method for analysing the potency of nucleic acid sequences to express functional sequences in a biological system, wherein the method comprises simultaneously analysing at least two different nucleic acid sequences, each encoding a different amino acid sequence, wherein each of the at least two different amino acid sequences comprises a different functional sequence and a different linker sequence, wherein each linker sequence is capable of being proteolytically excised from its amino acid sequence, wherein the method comprises the following steps:
- (i) providing the at least two different nucleic acid sequences;
- (ii) introducing the at least two different nucleic acid sequences into a cell;
- (iii) expressing the at least two different amino acid sequences;
- (iv) proteolytically excising the at least two different linker sequences;
- (v) determining the amounts of the excised linker sequences.
A kit comprising:
- a) a first nucleic acid sequence comprising an insertion site for a first polynucleotide encoding a functional sequence, wherein the first nucleic acid sequence encodes an amino acid sequence comprising a first linker sequence, wherein the first linker sequence is flanked by proteolytic cleavage sites such that the first linker sequence is capable of being excised from its amino acid sequence, wherein the sequence of the first linker sequence is different to any other sequence that is flanked by the same proteolytic cleavage sites in its amino acid sequence or in the amino acid sequence encoded by a second nucleic acid sequence;
- and
- b) a second nucleic acid sequence comprising an insertion site for a second polynucleotide encoding a functional sequence, wherein the second nucleic acid sequence encodes an amino acid sequence comprising a second linker sequence, wherein the second linker sequence is flanked by proteolytic cleavage sites such that the second linker sequence is capable of being excised from its amino acid sequence, wherein the sequence of the second linker sequence is different to any other sequence flanked by the same proteolytic cleavage sites in its amino acid sequence or in the amino acid sequence encoded by the first nucleic acid sequence.
A method comprising simultaneously analysing the potency of at least two nucleic acid sequences to express at least two different functional sequences in a biological system, wherein each of the at least two nucleic acid sequences encodes an amino acid sequence comprising a different functional sequence and a different linker sequence, wherein each linker sequence is 6 to 30 amino acids in length and is of the general formula:
- wherein
- X is any amino acid;
- n is an integer from 5 to 29;
- Y is lysine or arginine;
- further wherein [X]n may comprise the amino acid sequence KP or RP but otherwise does not comprise lysine or arginine;
- wherein each linker sequence is preceded at its N-terminus by a lysine or arginine residue.
Dependencies are inferred from the claim text.