Granted Patent B2

US 12,612,654 · App. 16/077,366 · Granted Apr 28, 2026

Inventors: Aniela Wochner (Tübingen, DE); Tilmann Roos (Kusterdingen, DE); Fabian Johannes Eber (Tübingen, DE); Philipp Hofmann (Stuttgart, DE)

Assignee: CureVac SE

C12Q1/6806C12Q2531/113C12Q2565/518

Abstract

Claims (22)

1 . A method of analyzing a sample comprising at least two different RNA species, each having a sequence comprising a target sequence that is a distinctive protein coding sequence and a distinctive PCR tag in a 3′ untranslated region (UTR), said at least two different RNA species prepared by RNA in vitro transcription, the method comprising determining the presence, integrity and/or quantity of each of the RNA species present in said sample using RT-qPCR that comprises the following steps:

a) contacting said sample with at least one primer for reverse transcription under conditions sufficient for reverse transcription, thereby providing a sample containing cDNA;

b) contacting the sample containing cDNA with different sets of PCR primers under conditions sufficient for PCR amplification of the cDNA, wherein each set of PCR primers includes one primer capable of binding to the distinctive PCR tag of the cDNA corresponding to one RNA species within the sample, but not to the cDNA corresponding to other RNA species within the sample, thereby producing amplified DNA corresponding to the at least two different RNA species; and

c) quantifying the amplified DNA corresponding to the at least two different RNA species,

wherein said at least two different RNA species are at least 200 nucleotides in length and: (1) have a length that is no more than 10% different from one another; and (2) 3′ UTR sequences that are at least 80% identical to one another.

2 . The method according to claim 1 , wherein the distinctive PCR tag is a synthetic sequence.

3 . The method according to claim 1 , wherein the amplified DNA is quantified using a fluorescent dye.

4 . The method according to claim 3 , wherein a different fluorescent dye is used for each amplified DNA corresponding to one RNA species.

5 . The method according to claim 1 , wherein the at least one primer for reverse transcription is complimentary to a sequence in the 3′UTR of said different RNA species.

6 . The method according to claim 1 , wherein the different RNA species comprise at least four three different RNA species.

7 . The method according to claim 6 , wherein the three different RNA species encode different influenza HA antigens.

8 . The method according to claim 7 , wherein the three different RNA species encode different influenza HA antigens from influenza A and influenza B.

9 . The method according to claim 8 , wherein the three different RNA species encode H1 and H3 antigens from influenza A and a HA antigen from influenza B.

10 . The method according to claim 1 , wherein said at least two different RNA species have a length that is no more than 5% different from one another.

11 . The method according to claim 1 , wherein said at least two different RNA species are at least 600 nucleotides in length.

12 . The method of claim 1 , wherein the distinctive PCR tag is 70-100 nucleotides in length.

13 . The method of claim 1 , wherein the at least two RNA species each comprises, from 5′ to 3′, a 5′ Cap, the distinctive protein coding sequence, the 3′ UTR, and a poly(A) sequence.

14 . The method of claim 1 , wherein the poly(A) is 60 to 250 adenosine nucleotides in length.

15 . The method of claim 14 , wherein the at least one primer for reverse transcription is an oligo (dT) primer.

16 . The method of claim 14 , wherein the 3′ UTR of each of the at least two RNA species comprise a sequence derived from a β-globin gene.

17 . The method of claim 14 , wherein the 3′ UTR of each of the at least two RNA species are at least 85% identical to one another.

18 . The method of claim 17 , wherein the 3′ UTR of each of the at least two RNA species are at least 90% identical to one another.

Assignments (2)

Continuity (1)

References Cited (224)