WO2025061863A1 - Procédé - Google Patents

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Publication number
WO2025061863A1
WO2025061863A1 PCT/EP2024/076303 EP2024076303W WO2025061863A1 WO 2025061863 A1 WO2025061863 A1 WO 2025061863A1 EP 2024076303 W EP2024076303 W EP 2024076303W WO 2025061863 A1 WO2025061863 A1 WO 2025061863A1
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Prior art keywords
rna
primer
sample
dna
species
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English (en)
Inventor
Julia SCHLERETH
Cristian DEL CAMPO
Mikaelle LE GALL
Andreas Kuhn
Andreas Czech
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Biontech SE
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Biontech SE
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8827—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving nucleic acids

Definitions

  • the present invention relates to a method for quality control analysis of an RNA sample comprising n different RNA molecule species, i.e. for an RNA mixture.
  • the method is suitable for use in quality control of RNA molecule mixtures during or following production.
  • the invention relates to a method for determining at least one quality parameter of an RNA sample by using reverse transcription and PCR, preferably droplet digital PCR.
  • the quality parameter may be the quantitative ratio of two or more RNA molecule species in an RNA sample containing RNA molecules of n RNA molecule species, or the identity of the n RNA molecule species in the RNA sample, wherein n is an integer of at least 2.
  • the quality parameter may be the integrity of an RNA sample containing RNA molecules of n RNA molecule species, wherein n is an integer of at least 1.
  • the quality parameter may be the potency of a formulated RNA sample comprising RNA molecules of interest. The method is particularly suitable for analyzing mixtures of encapsulated and/or complexed RNA molecule mixtures, in particular for liposome, lipid nanoparticle- and lipoplex-formulated RNA molecule mixtures.
  • RNA molecules represent an emerging class of drugs. RNA-based therapeutics may be used in immunotherapy, gene therapy and genetic vaccination, belonging to the most promising and quickly developing therapeutic fields of modern medicine. RNA-based therapeutics may provide highly specific and individual treatment options for the therapy of a large variety of diseases.
  • RNA mixture based treatments may include the application of polyvalent RNA mixtures that provide protection against several serotypes of a pathogen (e.g., hemagglutinin (HA) from multiple serotypes of Influenza A and B virus); RNA mixtures that provide different antigens from one pathogen (e.g., different antigens from influenza, such as HA, nucleoprotein (NP), neuraminidase (NA) etc.); RNA mixtures that provide protection against several isoforms or variants of a cancer antigen (e.g., prostate specific antigen (PSA) in the context of prostate carcinoma); RNA mixtures that provide different epitopes of an antigen; RNA mixtures that contain a cancer specific and/or patient specific mixture of cancer antigens (expressed antigens or mutated antigens); RNA mixtures that encode a variety of antibodies (e.g., antibodies that are targeted against different epitop
  • a pathogen e.g., hemagglutinin (HA)
  • the RNA molecules in the RNA (mixture) sample may be present in complexed form, i.e. in the form of at least one RNA-carrier complex.
  • the complexation or encapsulation of RNA in RNA- carrier complexes facilitates successful in vivo delivery.
  • Complexing carrier compounds used in the art typically include various types of peptides, polymers, carbohydrates, cholesterol, polyethylene glycol (PEG), lipids, phospholipids, PEGylated lipids, cationic and polycationic compounds, and combinations thereof, as well as other carrier compounds, which may be assembled into RNA-carrier complexes.
  • RNA mixture based therapeutics it is required that the n different components (n different RNA molecule species, complexed or free) of the drug product and drug substance can be characterized, in terms of presence, integrity, ratio and quantity (quality control parameter).
  • quality controls may be implemented during or following the RNA sample production, and/or during or following complexation of the RNA sample and/or as a batch release quality control.
  • RNA mixture-based therapeutics can be composed of multiple RNA species of highly similar size and sequence (e.g., polyvalent vaccines composed of multiple similar antigens) standard methods for quality control to discriminate between RNA species of similar size such as agarose gel electrophoresis or analytic HPLC are not suitable.
  • An ideal method for the quality control of RNA mixtures should be fast, robust, and cost effective allowing for the characterization of any or all of the quality control parameters selected from the group consisting of presence, quantity, and integrity of at least one RNA molecule species and ratio of at least two RNA molecule species within an RNA sample comprising n different RNA molecule species.
  • a method for RNA quality control analysis in particular in terms of cost-efficiency, robustness and the ability of the methods to discriminate highly similar RNA species in RNA samples comprising n different RNA molecule species.
  • WO20 18/211038 describes a method for quality control analysis of an RNA sample comprising n different RNA molecule species using reverse transcription and a polymerase chain reaction (PCR) based assay, wherein each of the n different RNA molecule species comprises one or more coding RNA molecules of synthetic origin, wherein n is an integer of at least 1, in some aspects at least 2, thereby determining at least one quality parameter, and wherein the PCR based assay is digital PCR (dPCR), preferably droplet digital (ddPCR).
  • dPCR digital PCR
  • ddPCR droplet digital
  • the assay may be used to determine various quality parameters, including the quantity of the RNA species present, the presence of the one or more coding RNA molecules, the integrity of the RNA molecules, and the quantitative ratio between the n RNA species.
  • the drawbacks of the method described in this publication are that it requires the introduction of artificial sequences in the RNA sequence, which are purely for analytical purposes, rather than for the functionality of the RNA. In addition,
  • the invention provides a method of determining a quality parameter of an RNA sample containing RNA molecules of n RNA molecule species, wherein n is an integer of at least 2, the quality parameter being selected from the group consisting of: i) quantitative ratio of two or more RNA molecule species of the n RNA molecule species; and ii) identity of the n RNA molecule species in the RNA sample; the method comprising the steps of: a) reverse transcription of the n RNA molecule species in the RNA sample into cDNA molecules of n DNA molecule species; and b) carrying out a polymerase chain reaction (PCR)-based assay on the resulting cDNA molecules, the PCR-based assay using a first primer set and a single second primer, wherein the first primer set comprises n primer species, wherein each primer species is capable of annealing to a first target region of only one of the n DNA molecule species in the sample, and the single second primer is capable of annealing to a
  • PCR poly
  • the quality parameter may be the quantitative ratio of two or more RNA molecule species of the n RNA molecule species. Therefore, in a second aspect, the invention provides a method of determining the quantitative ratio of two or more RNA molecule species in an RNA sample containing RNA molecules of n RNA molecule species, wherein n is an integer of at least 2, the method comprising the steps of: a) reverse transcription of the n RNA molecule species in the RNA sample into cDNA molecules of n DNA molecule species; and b) carrying out a polymerase chain reaction (PCR)-based assay on the resulting cDNA molecules, the PCR-based assay using a first primer set and a single second primer, wherein the first primer set comprises n primer species, wherein each primer species is capable of annealing to a first target region of only one of the n DNA molecule species in the sample, and the single second primer is capable of annealing to a second target region of all of the n DNA molecule species in the sample.
  • the quality parameter may also be the identity of the n RNA molecule species in the RNA sample. Therefore, in a third aspect, the invention provides a method of determining identity of n RNA molecule species in an RNA sample containing RNA molecules of n RNA molecule species, wherein n is an integer of at least 2, the method comprising the steps of: a) reverse transcription of the n RNA molecule species in the RNA sample into cDNA molecules of n DNA molecule species; and b) carrying out a polymerase chain reaction (PCR)-based assay on the resulting cDNA molecules, the PCR-based assay using a first primer set and a single second primer, wherein the first primer set comprises n primer species, wherein each primer species is capable of annealing to a first target region of only one of the n DNA molecule species in the sample, and the single second primer is capable of annealing to a second target region of all of the n DNA molecule species in the sample.
  • PCR polymerase chain reaction
  • the invention provides a method of determining integrity of an RNA sample containing RNA molecules of n RNA molecule species, wherein n is an integer of at least 1, the method comprising the steps of: a) reverse transcription of the n RNA molecules in the RNA sample into cDNA molecules of n DNA molecule species; and b) carrying out a polymerase chain reaction (PCR)-based assay on the resulting cDNA molecules, the PCR-based assay using a first primer set, a single second primer, a third primer set and a single fourth primer, wherein the first primer set comprises n primer species, wherein each primer species is capable of annealing to a first target region at the 3 ’-end region of a DNA molecule species in the sample, the single second primer is capable of annealing at the 3 ’-end region of all of the n DNA molecule species in the sample; the third primer set comprises n primer species, wherein each primer species is capable of annealing to a third target region
  • the invention provides a method of determining the potency of a formulated RNA sample comprising RNA molecules of interest, the method comprising the steps of: a) providing an RNA sample which has been isolated from cells transfected with a formulated RNA sample; b) reverse transcription of the RNA molecules in the RNA sample into cDNA molecules; c) carrying out a polymerase chain reaction (PCR)-based assay on the resulting cDNA molecules, the PCR-based assay using a first primer, a second primer, a third primer and a fourth primer, wherein the first primer and the second primer are capable of annealing to a first target region and a second target region of the cDNA molecules produced from the RNA of interest in the sample, and the third primer and the fourth primer are capable of annealing to a first target region and a second target region of the cDNA molecules derived from an endogenous RNA in the sample; and d) comparing the measured amount of the cDNA produced from the RNA
  • the PCR-based assay may use a detectable label.
  • the detectable label may be a fluorescent probe.
  • Figure 1 shows the % integrity of each RNA composing the differently degraded RNA mixtures measured with ddPCR.
  • Figure 2 shows the copy numbers (CN) measured by digital droplet polymerase chain reaction (ddPCR) measuring the potency of a ribonucleic acid (RNA) of interest compared with a housekeeping gene from total RNA isolated from Chinese Hamster Ovary (CHO) cells which were previously transfected with four different amounts of formulated RNA of interest.
  • the present inventors that carrying out the PCR method using the PCR-based assay using a first primer set and a single second primer, wherein the first primer set comprises n primer species, each primer species is capable of annealing to a first target region of only one of the n DNA molecule species in the sample (i.e. is specific to each DNA molecule species in the sample) , and the single second primer is capable of annealing to a second target region of all of the n DNA molecule species in the sample (i.e.
  • the method is advantageous compared with the methods described in WO2018/211038 in that the experimental system is simplified compared with the by the fact that two components of each oligonucleotide set (i.e., one common primer and one common dual-labelled probe) are identical for all RNAs and only one primer of each set is RNA-specific. This reduces the amount of reagents needed, the complexity of the system and the pipetting time compared to assembling two sets each composed of RNA-specific oligonucleotides.
  • the method can also determine identity and quantitative ratio of the RNA species in parallel with determining integrity.
  • the term "about” denotes an interval of accuracy that the person of ordinary skill will understand to still ensure the technical effect of the feature in question.
  • the term typically indicates deviation from the indicated numerical value by ⁇ 10%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1%, ⁇ 0.9%, ⁇ 0.8%, ⁇ 0.7%, ⁇ 0.6%, ⁇ 0.5%, ⁇ 0.4%, ⁇ 0.3%, ⁇ 0.2%, ⁇ 0.1%, ⁇ 0.05%, and for example ⁇ 0.01%.
  • “about” indicates deviation from the indicated numerical value by ⁇ 10%.
  • “about” indicates deviation from the indicated numerical value by ⁇ 5%.
  • phrases such as "determining the amount” or “determining expression” or similar phrases with reference to an amino acid sequence refer to determining the quantity or presence of an amino acid sequence.
  • nucleic acid comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof.
  • the term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules.
  • isolated nucleic acid means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.
  • PCR polymerase chain reaction
  • RNA polymerase RNA polymerase
  • nucleoside (abbreviated herein as "N") relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.
  • the five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine.
  • the five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively.
  • thymidine is more commonly written as “dT” ("d” represents “deoxy") as it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA.
  • modified purine or pyrimidine base moieties include N7-alkyl-guanine, N6-alkyl- adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(l)-alkyl-uracil, such as N7-C1-4 alkyl-guanine, N6-C1-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5-C1-4 alkyl-uracil, and N(l)-Cl-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5- methyl-cytosine, 5-methyl-uracil, and N(l)-methyl-uracil.
  • DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA.
  • a molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule.
  • the total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).
  • DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA.
  • the cDNA may be obtained by reverse transcription of RNA.
  • a molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule.
  • the total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).
  • RNA includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA).
  • RNA refers to mRNA.
  • IVT in vitro transcription means that the transcription (i.e., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living/cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)).
  • the nucleic acids of the present invention such as one, at least two or all of the nucleic acids of the present invention, are RNA.
  • the RNA is single stranded RNA.
  • the RNA is mRNA.
  • the RNA is generated by RNA in vitro transcription.
  • the RNA comprises a 5' cap structure.
  • the RNA does not comprise modified ribonucleotides.
  • the RNA comprises modified ribonucleotides.
  • the modified ribonucleotides comprise modified uridines.
  • the modified uridines comprise Nl-methyl-pseudouri dine.
  • the nucleic acids of the present invention are DNA.
  • the DNA is present in the form of a vector.
  • the vector comprises DNA encoding an amino acid sequence comprising the amino acid sequence of a peptide or polypeptide having biological activity.
  • the vector is a DNA vector.
  • the nucleic acids of the present invention such as one, at least two or all of the nucleic acids of the present invention comprise a mixture of RNA and DNA.
  • the RNA in the mixture is single stranded RNA.
  • the RNA in the mixture is mRNA.
  • the RNA in the mixture is generated by RNA in vitro transcription.
  • the RNA in the mixture comprises a 5' cap structure.
  • the RNA in the mixture does not comprise modified ribonucleotides.
  • the RNA in the mixture comprises modified ribonucleotides.
  • the modified ribonucleotides comprise modified uridines.
  • the modified uridines comprise Nl-methyl-pseudouri dine.
  • the DNA in the mixture is present in the form of a vector.
  • the vector in the mixture comprises DNA encoding an amino acid sequence comprising the amino acid sequence of a peptide or polypeptide having biological activity.
  • the vector in the mixture is a DNA vector.
  • the nucleic acid (such as RNA and/or DNA) of the present invention which can comprise one or at least two or more nucleic acid constructs, is formulated with a delivery vehicle.
  • the nucleic acid (such as RNA and/or DNA) is formulated with one or more compounds complexing the nucleic acid (such as RNA and/or DNA).
  • the nucleic acid (such as RNA and/or DNA) is formulated as particles. In some embodiments, the nucleic acid (such as RNA and/or DNA) is formulated as lipoplex particles. In these embodiments, it is preferred that the cells are characterized by a macropinocytosis-mediated RNA uptake mechanism.
  • the nucleic acid (such as RNA and/or DNA) is formulated as lipid nanoparticles.
  • the nucleic acid (such as RNA and/or DNA) comprises a mixture of different nucleic acids (such as RNAs and/or DNAs, e.g., two or more RNAs, two or more DNAs, or one or more RNAs and one or more DNAs), wherein each nucleic acid (such as RNA and/or DNA) encodes an amino acid sequence comprising the amino acid sequence of a peptide or polypeptide having biological activity.
  • the mixture of different nucleic acids comprises nucleic acids (such as RNAs and/or DNAs, e.g., two or more RNAs, two or more DNAs, or one or more RNAs and one or more DNAs) comprises nucleic acids (such as RNAs and/or DNAs, e.g., two or more RNAs, two or more DNAs, or one or more RNAs and one or more DNAs) encoding different amino acid sequences comprising the amino acid sequence of a peptide or polypeptide having biological activity.
  • nucleic acids such as RNAs and/or DNAs, e.g., two or more RNAs, two or more DNAs, or one or more RNAs and one or more DNAs
  • the different amino acid sequences comprise the amino acid sequence of different peptides or polypeptides having biological activity.
  • the different peptides or polypeptides having biological activity comprise different antigens.
  • beta-S- ARCA(Dl) (m27,2'-OGppSpG) or m27,3’-OGppp(ml2’-O)ApG may be utilized as specific capping structure at the 5'-end of the RNA drug substances.
  • 5'-UTR sequence the 5'-UTR sequence of the human alpha-globin mRNA, optionally with an optimized ‘Kozak sequence’ to increase translational efficiency may be used.
  • Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms.
  • the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest.
  • Alphavirus-based transreplication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system).
  • Trans-replication requires the presence of both these nucleic acid molecules in a given host cell.
  • the nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.
  • the mRNA contains one or more modifications, e.g., in order to increase its stability and/or increase translation efficiency and/or decrease immunogenicity and/or decrease cytotoxicity.
  • modifications e.g., in order to increase expression of the mRNA, it may be modified within the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without altering the sequence of the expressed peptide or polypeptide.
  • Particularly preferred 5'-cap analogs are those having one or more substitutions at the bridging and non-bridging oxygen in the phosphate bridge, such as phosphorothioate modified 5'-cap analogs at the P-phosphate (such as m27,2'OG(5')ppSp(5')G (referred to as beta-S-ARCA or P-S-ARCA)), as described in WO2019/175356.
  • phosphorothioate modified 5'-cap analogs at the P-phosphate such as m27,2'OG(5')ppSp(5')G (referred to as beta-S-ARCA or P-S-ARCA)
  • Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • a cassette is disclosed in WO 2016/005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 Al may be used in the present disclosure.
  • RNA preferably mRNA which is modified by pseudouridine (replacing partially or completely, preferably completely, uridine) is referred to herein as "T-modified", whereas the term “ml'P-modified” means that the RNA (preferably mRNA) contains N(l)-methylpseudouridine (replacing partially or completely, preferably completely, uridine). Furthermore, the term “m5U-modified” means that the RNA (preferably mRNA) contains 5-methyluridine (replacing partially or completely, preferably completely, uridine).
  • the mRNA used in the present disclosure contains a combination of at least two, at least three, at least four or all five of the above-mentioned modifications, i.e., (i) incorporation of a 5'-cap structure, (ii) incorporation of a poly-A sequence, unmasking of a poly-A sequence; (iii) alteration of the 5'- and/or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and/or pseudouridine (T) or N(l)-m ethylpseudouridine (m l ) or 5-methyluridine (m5U) for uridine), and (v) codon optimization.
  • synthetic nucleotides e.g., 5-methylcytidine for cytidine and/or pseudouridine (T) or N(
  • RNA molecule species denotes at least one RNA molecule in a population of RNA molecules which do not differ in their RNA sequence and/or their sequence length. Hence, the RNA molecules within one RNA molecule species are encoded by the same template DNA. If the RNA present within the sample is a coding RNA, one RNA species may encode one target peptide or protein or variant thereof.
  • n different RNA molecule species denotes a group of n RNA molecules which may differ with respect to their RNA sequence and/or their sequence length.
  • an RNA sample comprises n different RNA molecule species and if n is 2, the RNA sample comprises RNA molecules which belong to either of the 2 RNA molecule species, i.e. have the same RNA sequence and/or their sequence length.
  • the one or more RNA molecules of the first RNA molecule species do not differ in their RNA sequence and/or their sequence length among each other but differ from the RNA sequence and/or sequence length of the one or more RNA molecules of the second RNA molecule species.
  • Each RNA molecule species comprises at least one RNA molecule, i.e. each RNA molecule species comprises one or more RNA molecules. Accordingly, if an RNA sample comprises n different RNA molecule species, the RNA sample comprises at least n RNA molecules (at least one RNA molecule per RNA molecule species). However, typically an RNA molecule species comprises a higher number of RNA molecules per RNA molecule species in one sample. In the present invention, the one or more RNA molecules of each RNA molecule species are coding RNA species of synthetic origin. If the RNA molecule species are coding RNA molecule species, each of the n different RNA molecule species preferably but not necessarily encodes one target peptide/protein or variant thereof.
  • RNA sample comprising n different RNA species which is analyzed by the method of the present invention
  • identical, similar or different amounts of each species may be present, preferably the amounts are identical or similar.
  • the number of different RNA molecule species which are present in the RNA sample are reflected by the integer n which is at least 1 and thus can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and so forth.
  • n is an integer of at least 2 or at least 3, or in the range of 1 to 200, or 2 to 200, more preferably of 2 to 150, even more preferably of 2 to 100, and most preferably of 2 to 50.
  • the n different RNA molecule species may have a similar length, as the detection and discrimination of the n different RNA species is not dependent on differences in the length.
  • the length of the n different RNA species within the sample may differ by not more than 10% or 8%, preferably not more than 7% or 6%, more preferably by not more than 5% and most preferably by not more than 4%.
  • the RNA sequences of the one or more coding RNA molecules of each of the n different RNA molecule species may be at least 80% identical to each other, which considers the identity of the whole RNA molecule sequence and not only the target sequence.
  • Integrity of the one or more coding RNA molecules of an or at least two RNA molecule species describes whether the complete target RNA sequence is present in the sample of in vitro produced RNA. Low integrity could be due to, amongst others, degradation, cleavage, incorrect or incomplete chemical synthesis, incorrect base pairing, integration of modified nucleotides or the modification of already integrated nucleotides, lack of or incomplete capping, lack of or incomplete polyadenylation, or incomplete transcription.
  • the primers used in the methods of the present invention may target the 3'- untranslated region (3'-UTR) or the 5'-untranslated region (3'-UTR).
  • 3'-UTR refers to a part of the RNA molecule, which is located 3' (i.e. "downstream") of a coding sequence and which is not translated into protein.
  • a 3'-UTR is the part of an mRNA which is located between the protein coding region (coding sequence (CDS)) and the 3' terminus of the mRNA.
  • CDS protein coding region
  • the term 3'-UTR may also comprise elements, which are not encoded in the template, from which an RNA is transcribed, but which are added after transcription during maturation, e.g. a poly(A) sequence (or poly(A) 'tail').
  • a 3'-UTR of the mRNA is not translated into an amino acid sequence.
  • the 3'-UTR sequence is generally encoded by the DNA template, which is transcribed into the corresponding mRNA during the gene expression process.
  • a 3'-UTR corresponds to the sequence of a mature mRNA, which is located between the stop codon of the protein coding region, preferably immediately 3' to the stop codon of the protein coding region, and the poly(A) sequence of the mRNA.
  • a 5'-untranslated region is typically understood to be a particular section of messenger RNA (mRNA). It is located 5' of the coding sequence of the mRNA. Typically, the 5'-UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the coding sequence.
  • the 5'-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites.
  • the 5'-UTR may be post-transcriptionally modified, for example by addition of a 5' cap structure. In the context of the present invention, the term “5'-UTR" typically refers to the sequence of an mRNA, which is located between the 5' cap structure and the start codon.
  • the 5'-UTR is the sequence, which extends from a nucleotide located 3' to the 5' cap structure, preferably from the nucleotide located immediately 3' to the 5' cap structure, to a nucleotide located 5' to the start codon of the coding sequence, preferably to the nucleotide located immediately 5' to the start codon of the coding sequence.
  • the methods of the invention may involve the preparation of the sample including RNA. This method may include one or more purification steps.
  • purification or “purifying” is understood to mean that the desired RNA in a sample is separated and/or isolated from impurities, intermediates, by-products and/or reaction components present therein or that the impurities, intermediates, by-products and/or reaction components are at least depleted from the sample comprising the RNA.
  • Non-limiting examples of undesired constituents of RNA-containing samples which therefore need to be depleted may comprise degraded fragments or fragments which have arisen as a result of premature termination of transcription, or also excessively long transcripts if plasmids are not completely linearized.
  • intermediates may be depleted from the sample such as e.g. template DNA (for RNA IVT).
  • reaction components such as enzymes, proteins, bacterial DNA and RNA, small molecules such as spermidine, buffer components etc. may have to be depleted from the RNA sample.
  • impurities such as organic solvents, nucleotides, nucleosides or other small molecules may be separated.
  • Each of the methods of the invention comprises the step of reverse transcription of the n RNA molecule species in the RNA sample into cDNA molecules of n DNA molecule species.
  • RNA i.e. one or more coding RNA molecules of at least two RNA molecule species of the n different RNA molecule species, is typically incubated with the enzyme reverse transcriptase, deoxynucleotides (dNTPs), and at least one suitable primer for a time and under conditions sufficient for cDNA synthesis to occur, e.g. incubation for thirty minutes to one hour at a temperature of about 37 °C to 42 °C.
  • dNTPs deoxynucleotides
  • the primer(s) used for reverse transcription may be random so that any RNA molecule, e.g. one or more coding RNA molecules of all n different RNA molecule species, present in a sample may be reverse transcribed into cDNA or may be targetspecific so that only the target RNA, e.g. one or more coding RNA molecules of at least two RNA molecule species of the n different RNA molecule species, are reverse transcribed into the corresponding cDNA.
  • the methods of the invention further comprise carrying out a polymerase chain reaction (PCR)-based assay on the cDNA molecules resulting from reverse transcription of the RNA.
  • PCR polymerase chain reaction
  • PCR based assay encompasses any assay that employs a PCR reaction.
  • the PCR assay is quantitative Polymerase chain reaction (qPCR).
  • the PCR assay is digital PCR (dPCR).
  • the PCR assay is droplet digital PCR (ddPCR).
  • PCR polymerase chain reaction
  • the DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a PCR template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis.
  • DNA oligonucleotides also called DNA primers
  • the vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample through a defined series of temperature steps. In the first step, the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting. In the second step, the temperature is lowered and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA.
  • RT-qPCR An RT-qPCR assay involves a first step of reverse transcription and a second step of quantitative PCR as described above. The reverse transcription reaction and the quantitative PCR reaction may be performed separately so that in a first reaction the RNA is reverse transcribed into cDNA and in a second reaction the cDNA is transferred into a new reaction mixture for the quantitative PCR.
  • the reverse transcription reaction and the quantitative PCR reaction may be performed in one step so that the reaction mixture comprises both the components of the reverse transcription reaction and the components of the quantitative PCR.
  • the PCR process generally consists of a series of temperature changes, known as thermal cycles, with each cycle commonly consisting of at least two, optionally three or four, discrete temperature steps.
  • the cycling is often preceded by a single temperature step at a very high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage.
  • the temperatures used and the length of time they are applied in each cycle depend on a variety of parameters, including the enzyme used for DNA synthesis, the concentration of bivalent ions and dNTPs in the reaction, and the melting point of the primers.
  • the individual steps common to most PCR methods are as follows.
  • the first step is the heating of the reaction chamber in order to activate the DNA polymerase.
  • the initialization step takes place at a temperature between 90-100 °C. In one embodiment, the initialization step takes place at a temperature between 94-96 °C. In one embodiment, the initialization step takes place at a temperature of 95 °C. In one embodiment, the initialization step lasts 1 to 20 minutes. In one embodiment, the initialization step lasts 5 to 15 minutes. In one embodiment, the initialization step lasts 8 to 12 minutes. In one embodiment, the initialization step lasts 10 minutes. In one embodiment, the initialization step is omitted.
  • the second step known as the denaturation step, allows the separation of the nucleic acid's double chain yielding two single-stranded DNA molecules.
  • the denaturation step takes place at a temperature between 90- 100 °C. In one embodiment, the denaturation step takes place at a temperature between 92-97 °C. In one embodiment, the denaturation step takes place at a temperature of 94 °C. In one embodiment, the denaturation step lasts 1 second to 1 minute. In one embodiment, the denaturation step lasts 20 to 40 seconds. In one embodiment, the denaturation step lasts 30 seconds.
  • the third step allows the binding of the primers with each of the single- stranded DNA templates, and the subsequent polymerisation carried out by the DNA polymerase.
  • the temperature of the annealing and extension step must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i.e., the primer should bind only to a perfectly complementary part of the strand, and nowhere else. If the temperature is too low, the primer may bind imperfectly. If it is too high, the primer may not bind at all.
  • a typical annealing temperature is about 3-5°C below the melting point of the primers used. Stable hydrogen bonds between complementary bases are formed only when the primer sequence very closely matches the template sequence.
  • the polymerase binds to the primer-template hybrid and begins DNA formation.
  • the temperature at which the annealing and extension step is carried out depends on the RNA under investigation and the primers used. In one embodiment, the annealing and extension step takes place at a temperature between 55-65 °C. In one embodiment, the annealing and extension step takes place at a temperature between 55-65 °C.
  • the annealing and extension step takes place at a temperature of 63 °C. In one embodiment, the annealing and extension step lasts 10 seconds to 5 minutes. In one embodiment, the annealing and extension step lasts 20 seconds to 2 minutes. In one embodiment, the annealing and extension step lasts 40 to 80 seconds. In one embodiment, the annealing and extension step lasts 60 seconds.
  • the denaturation and annealing / extension steps may be repeated to the number of times necessary to allow the DNA to be sufficiently amplified.
  • the denaturation annealing and polymerisation steps are repeated 10 to 100 times.
  • the denaturation annealing and polymerisation steps are repeated 20 to 60 times.
  • the denaturation annealing and polymerisation steps are repeated 30 to 50 times.
  • the denaturation annealing and polymerisation steps are repeated 35 to 45 times.
  • the denaturation annealing and polymerisation steps are repeated 40 times.
  • the PCR procedure includes an enzyme inactivation step subsequent to completion of the cycles of denaturation and annealing / extension steps. In one embodiment, the enzyme inactivation step is omitted.
  • the enzyme inactivation step takes place at a temperature between 97-99 °C. In one embodiment, the enzyme inactivation step takes place at a temperature of 98 °C.
  • the enzyme inactivation step lasts 1 to 20 minutes. In one embodiment, the enzyme inactivation step lasts 5 to 15 minutes. In one embodiment, the enzyme inactivation step lasts 8 to 12 minutes. In one embodiment, the enzyme inactivation step lasts 10 minutes.
  • the enzyme inactivation step lasts 10 seconds to 5 minutes. In one embodiment, the enzyme inactivation step lasts 20 seconds to 2 minutes. In one embodiment, the enzyme inactivation step lasts 40 to 80 seconds. In one embodiment, the enzyme inactivation step lasts 60 seconds.
  • the temperatures and the timings used for each cycle depend on a wide variety of parameters, such as: the enzyme used to synthesize the DNA, the concentration of divalent ions and deoxyribonucleotides (dNTPs) in the reaction and the bonding temperature of the primers.
  • the type of quantitative PCR technique used depends on the DNA sequence in the samples, the technique can either use non-specific fluorochromes or hybridization probes.
  • the PCR methods used in the present invention employ a detectable label.
  • a detectable label is a detectable compound which is attached directly or indirectly to another molecule. Within the method of the present invention the detectable label is attached to a single-stranded nucleic acid molecule.
  • the skilled person knows methods for attaching labels to nucleic acid molecules. Specific, nonlimiting examples of labels include fluorescent probes and fluorogenic moieties, chromogenic moieties, haptens, affinity tags and radioactive isotopes.
  • the label can be detectable directly (e.g. optically) or indirectly (e.g. by interaction with one or more molecules which are in turn detectable).
  • fluorescent probes are used.
  • a fluorescent probe also known as a fluorescent label or a fluorescent tag
  • a fluorescent probe is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid.
  • the fluorescent probe carries one label. In one embodiment, the fluorescent probe carries multiple labels (such as two, three, or four labels).
  • the fluorescent probe is labelled with a fluorophore.
  • a fluorophore is a fluorescent substance which can re-emit light upon light excitation. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically.
  • fluorophores examples include [6-amino-9-[2-carboxy-4-[5-(2,5-dioxopyrrol-l-yl)pentyl- carbamoyl]phenyl]-4,5-disulfoxanthen-3-ylidene]azanium (Alexa 488), 6- carboxyfluorescein (FAM), 4-(2,7-difluoro-3-hydroxy-6-oxoxanthen-9-yl)benzene- 1,3 -dicarboxylic acid (Oregon Green), [6-amino-9-(2,5-dicarboxyphenyl)xanthen-3- ylidene]azanium (Rhodamine Green), 6-[(7-nitro-2, l,3-benzoxadiazol-4-yl)amino]- Hexanoic acid (NBD-X), tetrachlorofluorescein (TET), [9-[6-(2,
  • the fluorescent probe is also labelled with a quencher.
  • a quencher is a substance which decreases the fluorescent intensity of a given substance.
  • quenchers include 2-[N- (2 -hydroxy ethyl)-4-[[2-methoxy-5-methyl-4-[(4-methyl-2-nitrophenyl)diazenyl]- phenyl]diazenyl]anilino]ethanol (Black Hole Quencher 1, BHQ1), ZEN quencher (available from IDT), Iowa black fluorescein quencher (IBFQ) (available from IDT) 4-N-methyl-N-(4'-nitro-2'-chloroazobenzen-4-yl)-aminobutanamido- 1 - (2-0- dimethoxytrityloxymethyl)-pyrrolidin-4-yl-succinoyl long chain alkylamino-CPG (ECLIPSE quencher),
  • the fluorophore is 5'-hexachlorofluorescein and the quencher is Black Hole Quencher 1. In one embodiment, the fluorophore is 6-carboxyfluorescein and the quencher is Black Hole Quencher 1. Both the fluorophore and the quencher are commercially available, for example from Eurofins Genomics.
  • the method of detecting the double-stranded nucleic acid molecules produced in the PCR based assay of the invention depends on the detectable label attached to the nucleic acid probe. For example, if the nucleic acid probe is labelled with a radioactive isotope, the double-stranded nucleic acid molecules are detected by autoradiography. If the nucleic acid probe is labelled with a fluorescent probe, the double-stranded nucleic acid molecules are detected by fluorescence spectroscopy.
  • the PCR is digital PCR (dPCR).
  • the dPCR- based assay uses a detectable label.
  • the detectable label is a fluorescent probe.
  • dPCR digital PCR
  • respective PCR reactions are partitioned into multiple smaller reactions so that individual nucleic acid molecules within the sample are localized and amplified in many separate regions.
  • Micro well plates, capillaries, oil emulsion (droplets), and arrays of miniaturized chambers with nucleic acid binding surfaces can be used to separate the RNA sample in multiple small reactions per reaction vessel.
  • the samples are analyzed for fluorescence signals with a binary readout of "0" (no signal) or "1" (signal).
  • 0 no signal
  • 1 signal
  • dPCR is not dependent on the number of amplification cycles to determine the initial sample amount, and thus eliminates the reliance on uncertain exponential data to quantify target nucleic acids. Therefore, dPCR allows absolute quantification of nucleic acids.
  • Suitable commercial systems that may be used to perform dPCR comprise chip-based QuantStudioTM 3D digital PCR System (Thermo Fisher, Waltham, MA, USA), Rain Drop PlusTM system (RainDance Technologies, Lexington, MA, USA), or QX200TM, AutoDGTM, and Droplet DigitalTM PCR System (BioRad Laboratories, Hercules, CA, USA).
  • Chip-based digital PCR measures absolute quantities by counting nucleic acid molecules partitioned in independent reaction wells.
  • Commercially available systems for chip-based dPCR comprise QuantStudioTM 3D digital PCR System (Thermo Fisher, Waltham, MA, USA).
  • a PCR reaction is divided into around 20,000 independent reaction wells on a chip that either contain or not contain template.
  • the sealed chip is then subjected to PCR amplification.
  • Each well containing template is leading to PCR positive signals (positive well) and each well that is not containing template is leading to negative PCR signals (negative wells). Positive and negative wells are counted allowing quantitation of template concentration using Poisson distribution algorithm.
  • the PCR is droplet digital PCR (ddPCR).
  • the ddPCR-based assay uses a detectable label.
  • the detectable label is a fluorescent probe.
  • Droplet digital PCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined water-in-oil droplet partitions.
  • Commercially available systems for ddPCR comprise Droplet DigitalTM PCR System (BioRad Laboratories, Hercules, CA, USA) or Rain Drop PlusTM system (RainDance Technologies, Lexington, MA, USA).
  • a PCR reaction is divided into around 20,000 droplets that either contain or not contain template leading to PCR positive and negative droplets that are counted allowing quantitation of template concentration using Poisson distribution algorithm.
  • the droplets are generated using a droplet generator.
  • ddPCR enables more precise absolute quantification as compared to classic relative quantitation which is limited due to the doubling during each cycle.
  • ddPCR has several advantages over conventional methods (e.g. qPCR) because no standard curve is needed for quantification, it is a more robust method (even sub-optimal primer pairs that lead to false positive or false negative signals will be eventually give a correct concentration due to the Poisson distribution algorithm), and enables precise (diagnostic) quantification (Resolution of standard qPCR: 0.5 cycles (+/-50%) vs. 10% for ddPCR (using acoustic pipetting 1.5% accuracy possible).
  • qPCR qPCR
  • the method according to the present invention is suitable for determining at least one quality parameter.
  • quality parameter comprises any parameter of the RNA sample which is related to a property of the RNA sample and typically obtained for quality control during or following production.
  • quality parameters are quantity of the one or more coding RNA molecules of at least two RNA molecule species, integrity of the one or more coding RNA molecules of at least two RNA molecule species and quantitative ratio between the one or more coding RNA molecules of at least two RNA molecule species.
  • the quality parameter is useful to determine, e.g., whether an RNA sample comprises all required n different RNA molecule species, the quantitative ratio between at least two of the n different RNA molecule species, whether the RNA molecules of the n different RNA molecule species are present in intact form (integrity) and to determine the amount of each RNA molecule species in the RNA sample.
  • an RNA sample can be analyzed and rated according to regulatory requirements as necessary for marketing approval of a medicinal product.
  • the quality parameter measured is the quantitative ratio of two or more RNA molecule species in an RNA sample.
  • the quantitative ratio may be between the one or more coding RNA molecules of at least two RNA molecule species:
  • the quantitative ratio of the one or more coding RNA molecules of at least two RNA molecule species may be determined by determining the quantity of the one or more coding RNA molecules of at least two RNA molecule species and evaluating the quantitative ratio of both or more quantities. Since the method allows determining the quantity of the one or more coding RNA molecules of all RNA molecule species, it is possible to determine the quantitative ratio between all RNA molecule species.
  • the invention provides a method of determining the quantitative ratio of two or more RNA molecule species in an RNA sample containing RNA molecules of n RNA molecule species, wherein n is an integer of at least 2, the method comprising the steps of: a) reverse transcription of the n RNA molecule species in the RNA sample into cDNA molecules of n DNA molecule species; and b) carrying out a polymerase chain reaction (PCR)-based assay on the resulting cDNA molecules, the PCR-based assay using a first primer set and a single second primer, wherein the first primer set comprises n primer species, wherein each primer species is capable of annealing to a first target region of only one of the n DNA molecule species in the sample, and the single second primer is capable of annealing to a second target region of all of the n DNA molecule species in the sample.
  • PCR polymerase chain reaction
  • the first target region is within the coding sequence of the DNA molecule. In one embodiment, the first target region spans the coding sequence and the untranslated region of the DNA molecule.
  • the first target region is at the 3 ’-end region of the DNA molecule. In one embodiment, the first target region is within the coding sequence at the 3 ’-end region of the DNA molecule. In one embodiment, the first target region spans the coding sequence and the 3 ’-untranslated region (as defined herein) of the DNA molecule. In one embodiment, the first target region is at the 5 ’-end region of the DNA molecule. In one embodiment, the first target region is within the coding sequence at the 5’-end region of the DNA molecule. In one embodiment, the first target region spans the coding sequence and the 5 ’-untranslated region (as defined herein) of the DNA molecule.
  • the second target region is within the coding sequence of the DNA molecule. In one embodiment, the second target region spans the coding sequence and the untranslated region of the DNA molecule. In one embodiment, the second target region is within the untranslated region of the DNA molecule.
  • the second target region is at the 3 ’-end region of the DNA molecule. In one embodiment, the second target region is within the coding sequence at the 3 ’-end region of the DNA molecule. In one embodiment, the second target region spans the coding sequence and the 3 ’-untranslated region (as defined herein) of the DNA molecule. In one embodiment, the second target region is at the 5 ’-end region of the DNA molecule. In one embodiment, the second target region is within the coding sequence at the 5 ’-end region of the DNA molecule. In one embodiment, the second target region spans the coding sequence and the 5 ’-untranslated region (as defined herein) of the DNA molecule.
  • the PCR is digital PCR (dPCR).
  • the dPCR- based assay uses a detectable label.
  • the detectable label is a fluorescent probe.
  • the PCR is droplet digital PCR (ddPCR).
  • the ddPCR-based assay uses a detectable label.
  • the detectable label is a fluorescent probe.
  • the quality parameter measured is the identity of n RNA molecule species in an RNA sample.
  • the invention provides a method of determining identity of n RNA molecule species in an RNA sample containing RNA molecules of n RNA molecule species, wherein n is an integer of at least 2, the method comprising the steps of a) reverse transcription of the n RNA molecule species in the RNA sample into cDNA molecules of n DNA molecule species; and b) carrying out a polymerase chain reaction (PCR)-based assay on the resulting cDNA molecules, the PCR-based assay using a first primer set and a single second primer, wherein the first primer set comprises n primer species, wherein each primer species is capable of annealing to a first target region of only one of the n DNA molecule species in the sample, and the single second primer is capable of annealing to a second target region of all of the n DNA molecule species in the sample.
  • PCR polymerase chain reaction
  • the first target region is within the coding sequence of the DNA molecule. In one embodiment, the first target region spans the coding sequence and the untranslated region of the DNA molecule.
  • the first target region is at the 3 ’-end region of the DNA molecule. In one embodiment, the first target region is within the coding sequence at the 3 ’-end region of the DNA molecule. In one embodiment, the first target region includes a part of the coding sequence and a part of the 3 ’-untranslated region (as defined herein) of the DNA molecule. In one embodiment, the first target region is at the 5’-end region of the DNA molecule. In one embodiment, the first target region is within the coding sequence at the 5 ’-end region of the DNA molecule. In one embodiment, the first target region includes a part of the coding sequence and a part of the 5 ’-untranslated region (as defined herein) of the DNA molecule.
  • the second target region is within the coding sequence of the DNA molecule. In one embodiment, the second target region includes a part of the coding sequence and a part of the untranslated region of the DNA molecule. In one embodiment, the second target region is within the untranslated region of the DNA molecule
  • the second target region is at the 3 ’-end region of the DNA molecule. In one embodiment, the second target region is within the coding sequence at the 3 ’-end region of the DNA molecule. In one embodiment, the second target region includes a part of the coding sequence and a part of the 3 ’-untranslated region (as defined herein) of the DNA molecule. In one embodiment, the second target region is at the 5’-end region of the DNA molecule. In one embodiment, the second target region is within the coding sequence at the 5 ’-end region of the DNA molecule. In one embodiment, the second target region includes a part of the coding sequence and a part of the 5 ’-untranslated region (as defined herein) of the DNA molecule.
  • the PCR is digital PCR (dPCR).
  • the dPCR- based assay uses a detectable label.
  • the detectable label is a fluorescent probe.
  • the PCR is droplet digital PCR (ddPCR).
  • the ddPCR-based assay uses a detectable label.
  • the detectable label is a fluorescent probe.
  • the quality parameter measured is the integrity (as defined herein) of n RNA molecule species in an RNA sample. Therefore, in one aspect, the invention provides a method of determining integrity of an RNA sample containing RNA molecules of n RNA molecule species, wherein n is an integer of at least 1, the method comprising the steps of a) reverse transcription of the n RNA molecules in the RNA sample into cDNA molecules of n DNA molecule species; and b) carrying out a polymerase chain reaction (PCR)-based assay on the resulting cDNA molecules, the PCR-based assay using a first primer set, a single second primer, a third primer set and a single fourth primer, wherein the first primer set comprises n primer species, wherein each primer species is capable of annealing to a first target region at the 3 ’-end region of a DNA molecule species in the sample, the single second primer is capable of annealing to a second target region at the 3’- end region of all of the n DNA
  • the first target region is within the coding sequence of the DNA molecule. In one embodiment, the first target region includes a part of the coding sequence and a part of the untranslated region of the DNA molecule.
  • the first target region is at the 3 ’-end region of the DNA molecule. In one embodiment, the first target region is within the coding sequence at the 3 ’-end region of the DNA molecule. In one embodiment, the first target region includes a part of the coding sequence and a part of the 3 ’-untranslated region (as defined herein) of the DNA molecule. In one embodiment, the first target region is at the 5’-end region of the DNA molecule. In one embodiment, the first target region is within the coding sequence at the 5 ’-end region of the DNA molecule. In one embodiment, the first target region includes a part of the coding sequence and a part of the 5 ’-untranslated region (as defined herein) of the DNA molecule.
  • the second target region is within the coding sequence of the DNA molecule. In one embodiment, the second target region includes a part of the coding sequence and a part of the untranslated region of the DNA molecule. In one embodiment, the second target region is within the untranslated region of the DNA molecule.
  • the second target region is at the 3 ’-end region of the DNA molecule. In one embodiment, the second target region is within the coding sequence at the 3 ’-end region of the DNA molecule. In one embodiment, the second target region includes a part of the coding sequence and a part of the 3 ’-untranslated region (as defined herein) of the DNA molecule. In one embodiment, the second target region is at the 5’-end region of the DNA molecule. In one embodiment, the second target region is within the coding sequence at the 5 ’-end region of the DNA molecule. In one embodiment, the second target region includes a part of the coding sequence and a part of the 5 ’-untranslated region (as defined herein) of the DNA molecule. In one embodiment, the third target region is within the coding sequence of the DNA molecule. In one embodiment, the third target region includes a part of the coding sequence and a part of the untranslated region of the DNA molecule.
  • the third target region is at the 3 ’-end region of the DNA molecule. In one embodiment, the third target region is within the coding sequence at the 3 ’-end region of the DNA molecule. In one embodiment, the third target region includes a part of the coding sequence and a part of the 3 ’-untranslated region (as defined herein) of the DNA molecule. In one embodiment, the third target region is at the 5’-end region of the DNA molecule. In one embodiment, the third target region is within the coding sequence at the 3 ’-end region of the DNA molecule. In one embodiment, the third target region includes a part of the coding sequence and a part of the 5 ’-untranslated region (as defined herein) of the DNA molecule.
  • the fourth target region is within the coding sequence of the DNA molecule. In one embodiment, the fourth target region includes a part of the coding sequence and a part of the untranslated region of the DNA molecule. In one embodiment, the fourth target region is within the untranslated region of the DNA molecule.
  • the fourth target region is at the 3 ’-end region of the DNA molecule. In one embodiment, the fourth target region is within the coding sequence at the 3 ’-end region of the DNA molecule. In one embodiment, the fourth target region includes a part of the coding sequence and a part of the 3 ’-untranslated region (as defined herein) of the DNA molecule. In one embodiment, the fourth target region is at the 5 ’-end region of the DNA molecule. In one embodiment, the fourth target region is within the coding sequence at the 5 ’-end region of the DNA molecule. In one embodiment, the fourth target region includes a part of the coding sequence and a part of the 5 ’-untranslated region (as defined herein) of the DNA molecule.
  • the PCR is digital PCR (dPCR). In one embodiment, the dPCR- based assay uses a detectable label. In one embodiment, the detectable label is a fluorescent probe. In one embodiment, the PCR is droplet digital PCR (ddPCR). In one embodiment, the ddPCR-based assay uses a detectable label. In one embodiment, the detectable label is a fluorescent probe.
  • the quality parameter measured is the potency of a formulated RNA sample comprising RNA molecules of interest.
  • the invention provides a method of determining the potency of a formulated RNA sample comprising RNA molecules of interest, the method comprising the steps of: a) providing an RNA sample which has been isolated from cells transfected with a formulated RNA sample; b) reverse transcription of the RNA molecules in the RNA sample into cDNA molecules; c) carrying out a polymerase chain reaction (PCR)-based assay on the resulting cDNA molecules, the PCR-based assay using a first primer, a second primer, a third primer and a fourth primer, wherein the first primer and the second primer are capable of annealing to a first target region and a second target region of the cDNA molecules produced from the RNA of interest in the sample, and the third primer and the fourth primer are capable of annealing to a first target region and a second target region of the cDNA molecules derived from an endogenous
  • PCR
  • the first target region is within the coding sequence of the cDNA molecule. In one embodiment, the first target region includes a part of the coding sequence and a part of the untranslated region of the cDNA molecule.
  • the first target region is at the 3 ’-end region of the cDNA molecule. In one embodiment, the first target region is within the coding sequence at the 3 ’-end region of the cDNA molecule. In one embodiment, the first target region includes a part of the coding sequence and a part of the 3 ’-untranslated region (as defined herein) of the cDNA molecule. In one embodiment, the first target region is at the 5’-end region of the cDNA molecule. In one embodiment, the first target region is within the coding sequence at the 3 ’-end region of the cDNA molecule. In one embodiment, the first target region includes a part of the coding sequence and a part of the 5 ’-untranslated region (as defined herein) of the cDNA molecule.
  • the second target region is within the coding sequence of the cDNA molecule. In one embodiment, the second target region includes a part of the coding sequence and a part of the untranslated region of the cDNA molecule.
  • the second target region is at the 3 ’-end region of the cDNA molecule. In one embodiment, the second target region is within the coding sequence at the 3 ’-end region of the cDNA molecule. In one embodiment, the second target region includes a part of the coding sequence and a part of the 3 ’-untranslated region (as defined herein) of the cDNA molecule. In one embodiment, the second target region is at the 5 ’-end region of the cDNA molecule. In one embodiment, the second target region is within the coding sequence at the 5 ’-end region of the cDNA molecule. In one embodiment, the second target region includes a part of the coding sequence and a part of the 5 ’-untranslated region (as defined herein) of the cDNA molecule.
  • the PCR is digital PCR (dPCR).
  • the dPCR- based assay uses a detectable label.
  • the detectable label is a fluorescent probe.
  • the PCR is droplet digital PCR (ddPCR).
  • the ddPCR-based assay uses a detectable label.
  • the detectable label is a fluorescent probe.
  • the endogenous RNA is RNA expressed from a housekeeping gene in the sample.
  • the endogenous RNA is RNA expressed from a GAPDH gene in the sample.
  • the formulated RNA composition comprises two or more different RNA molecule species of interest and the PCR-based assay uses two or more different primer pairs which are each specific for one of the RNA molecule species of interest.
  • steps a) to d) are repeated for further RNA samples which have been isolated from cells transfected with formulated RNA compositions comprising the RNA molecule(s) of interest to determine a potency level for each formulated RNA composition.
  • carrying out the method establishes an expected potency level for a particular formulated RNA composition comprising a RNA of interest, the expected potency level being defined as a reference potency level.
  • the method is carried out on a further formulated RNA composition comprising a RNA of interest and comparing it with the reference potency level for the formulated RNA composition.
  • the method further comprises the following step z) prior to step a): z) isolating/purifying RNA from cells which have been transfected with the formulated RNA sample.
  • the method further comprises the following step y) prior to step z): y) transfecting the cell with the formulated RNA composition.
  • the cells are transfected using RNA which is complexed with at least one carrier compound, thereby forming at least one RNA-carrier complex.
  • the at least one carrier compound is a member selected from the group consisting of lipids, phospholipids, PEGylated lipids, cationic and polycationic compounds, and combinations thereof.
  • At least one RNA-carrier complex is selected from the group consisting of a liposome, a lipid nanoparticle, a lipoplex, and a mixture thereof.
  • RNA of the invention may be present in particles comprising (i) the RNA, and (ii) at least one cationic or cationically ionizable compound such as a polymer or lipid complexing the RNA. Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acid are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles.
  • RNA containing particles have been described previously to be suitable for delivery of RNA in particulate form (cf., e.g., Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60).
  • nanoparticle encapsulation of RNA physically protects RNA from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.
  • the term "particle” relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds.
  • the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids).
  • amphiphilic substance means that the substance possesses both hydrophilic and lipophilic properties.
  • the envelope may also comprise additional substances (e.g., additional lipids) which do not have to be amphiphilic.
  • the particle may be a monolamellar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids) optionally in combination with additional substances (e.g., additional lipids) which do not have to be amphiphilic.
  • the term "particle” relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term “particle” includes nanoparticles.
  • a lipoplex is obtainable from mixing two aqueous phases, namely a phase comprising nucleic acid (such as RNA and/or DNA) and a phase comprising a dispersion of lipids.
  • the lipid phase comprises liposomes.
  • liposomes comprise unilamellar or multilamellar phospholipid bilayers enclosing an aqueous core (also referred to herein as an aqueous lumen). They may be prepared from materials possessing polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups.
  • cationic lipids employed in formulating liposomes designed for the delivery of nucleic acids are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked to a hydrocarbon chain or cholesterol derivative via glycerol.
  • a lipid nanoparticle is obtainable from direct mixing of nucleic acid (such as RNA and/or DNA) in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol.
  • nucleic acid such as RNA and/or DNA
  • lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.
  • the particles (e.g., LNPs and LPXs) described herein have a size (such as a diameter) in the range of about 10 to about 2000 nm, such as at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and/or at most 1900 nm (e.g., at most about 1900 nm, at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm) and/
  • the particles (e.g., LNPs and LPXs) described herein have an average diameter that in some embodiments ranges from about 50 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 300 nm, from about 50 nm to about 250 nm, from about 50 nm to about 200 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 800 nm, from about 100 nm to about 700 nm, from about 100 nm to about 600 nm, from about 100 nm to about 500 nm, from about 100 nm to about 450
  • the particles described herein are nanoparticles.
  • nanoparticle relates to a nano-sized particle comprising nucleic acid (especially mRNA) as described herein and at least one cationic or cationically ionizable lipid, wherein all three external dimensions of the particle are in the nanoscale, z.e., at least about 1 nm and below about 1000 nm.
  • the size of a particle is its diameter.
  • Nucleic acid particles described herein may exhibit a poly dispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05.
  • PDI poly dispersity index
  • the nucleic acid particles can exhibit a poly dispersity index in a range of about 0.01 to about 0.4 or about 0.1 to about 0.3.
  • the N/P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the nucleic acid. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged.
  • the N/P ratio where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N/P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, RNA is considered to be completely bound to nanoparticles.
  • Nucleic acid particles (especially RNA particles such as mRNA particles) described herein can be prepared using a wide range of methods that may involve obtaining a colloid from at least one cationic or cationically ionizable lipid and mixing the colloid with nucleic acid to obtain nucleic acid particles.
  • the term "colloid” as used herein relates to a type of homogeneous mixture in which dispersed particles do not settle out.
  • the insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers.
  • the mixture may be termed a colloid or a colloidal suspension. Sometimes the term “colloid” only refers to the particles in the mixture and not the entire suspension.
  • colloids comprising at least one cationic or cationically ionizable lipid
  • methods are applicable herein that are conventionally used for preparing liposomal vesicles and are appropriately adapted.
  • the most commonly used methods for preparing liposomal vesicles share the following fundamental stages: (i) lipids dissolution in organic solvents, (ii) drying of the resultant solution, and (iii) hydration of dried lipid (using various aqueous media).
  • lipids are firstly dissolved in a suitable organic solvent, and dried down to yield a thin film at the bottom of the flask.
  • the obtained lipid film is hydrated using an appropriate aqueous medium to produce a liposomal dispersion.
  • an additional downsizing step may be included.
  • Reverse phase evaporation is an alternative method to the film hydration for preparing liposomal vesicles that involves formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. A brief sonication of this mixture is required for system homogenization. The removal of the organic phase under reduced pressure yields a milky gel that turns subsequently into a liposomal suspension.
  • colloidal liposome dispersion is, in some embodiments, formed as follows: an ethanol solution comprising lipids, such as cationic or cationically ionizable lipids like DOTMA and/or DODMA and additional lipids, is injected into an aqueous solution under stirring.
  • lipids such as cationic or cationically ionizable lipids like DOTMA and/or DODMA and additional lipids
  • the nucleic acid (such as RNA and/or DNA, especially mRNA) lipoplex particles described herein are obtainable without a step of extrusion.
  • extruding refers to the creation of particles having a fixed, cross-sectional profile. In particular, it refers to the downsizing of a particle, whereby the particle is forced through filters with defined pores.
  • LNPs comprise four components: ionizable cationic lipids, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer conjugated lipid.
  • LNPs may be prepared by mixing lipids dissolved in ethanol rapidly with nucleic acid (such as RNA and/or DNA) in an aqueous buffer. While nucleic acid (such as RNA and/or DNA) particles described herein may comprise polymer conjugated lipids such as PEG lipids, provided herein are also nucleic acid (such as RNA and/or DNA) particles which do not comprise polymer conjugated lipids such as PEG lipids.
  • the LNPs comprising nucleic acid (such as RNA and/or DNA) and at least one cationic or cationically ionizable lipid described herein are prepared by (a) preparing a nucleic acid (such as RNA and/or DNA) solution containing water and a buffering system; (b) preparing an ethanolic solution comprising the cationic or cationically ionizable lipid and, if present, one or more additional lipids; and (c) mixing the nucleic acid (such as RNA and/or DNA) solution prepared under (a) with the ethanolic solution prepared under (b), thereby preparing the formulation comprising LNPs. After step (c) one or more steps selected from diluting and filtrating, such as tangential flow filtrating, can follow.
  • diluting and filtrating such as tangential flow filtrating
  • the LNPs comprising nucleic acid (such as RNA and/or DNA) and at least one cationic or cationically ionizable lipid described herein are prepared by (a’) preparing liposomes or a colloidal preparation of the cationic or cationically ionizable lipid and, if present, one or more additional lipids in an aqueous phase; and (b’) preparing a nucleic acid (such as RNA and/or DNA) solution containing water and a buffering system; and (c’) mixing the liposomes or colloidal preparation prepared under (a’) with the nucleic acid (such as RNA and/or DNA) solution prepared under (b’). After step (c’) one or more steps selected from diluting and filtrating, such as tangential flow filtrating, can follow.
  • diluting and filtrating such as tangential flow filtrating
  • the present disclosure describes particles comprising nucleic acid (such as RNA and/or DNA, especially mRNA) and at least one cationic or cationically ionizable lipid which associates with the nucleic acid (such as RNA and/or DNA) to form nucleic acid (such as RNA and/or DNA) particles and compositions comprising such particles.
  • the nucleic acid (such as RNA and/or DNA) particles may comprise nucleic acid (such as RNA and/or DNA) which is complexed in different forms by non- covalent interactions to the particle.
  • the particles described herein are not viral particles, in particular infectious viral particles, z.e., they are not able to virally infect cells.
  • Suitable cationic or cationically ionizable lipids are those that form nucleic acid particles and are included by the term “particle forming components” or “particle forming agents”.
  • the term “particle forming components” or “particle forming agents” relates to any components which associate with nucleic acid to form nucleic acid particles. Such components include any component which can be part of nucleic acid particles.
  • nucleic acid particles (such as RNA and/or DNA particles, especially mRNA particles) comprise more than one type of nucleic acid (such as RNA and/or DNA) molecules, where the molecular parameters of the nucleic acid (such as RNA and/or DNA) molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping (only RNA), coding regions or other features.
  • each nucleic acid (such as RNA and/or DNA) species is separately formulated as an individual particulate formulation.
  • each individual particulate formulation will comprise one nucleic acid (such as RNA and/or DNA) species.
  • the individual particulate formulations may be present as separate entities, e.g. in separate containers.
  • Such formulations are obtainable by providing each nucleic acid (such as RNA and/or DNA) species separately (typically each in the form of a nucleic acid (such as RNA and/or DNA)-containing solution) together with a particle-forming agent, thereby allowing the formation of particles.
  • Respective particles will contain exclusively the specific nucleic acid (such as RNA and/or DNA) species that is being provided when the particles are formed (individual particulate formulations).
  • a composition such as a pharmaceutical composition comprises more than one individual particle formulation.
  • Respective pharmaceutical compositions are referred to as mixed particulate formulations.
  • Mixed particulate formulations according to the invention are obtainable by forming, separately, individual particulate formulations, followed by a step of mixing of the individual particulate formulations. By the step of mixing, a formulation comprising a mixed population of nucleic acid (such as RNA and/or DNA)-containing particles is obtainable. Individual particulate populations may be together in one container, comprising a mixed population of individual particulate formulations.
  • nucleic acid (such as RNA and/or DNA) species of the pharmaceutical composition are formulated together as a combined particulate formulation.
  • Such formulations are obtainable by providing a combined formulation (typically combined solution) of all nucleic acid (such as RNA and/or DNA) species together with a particle-forming agent, thereby allowing the formation of particles.
  • a combined particulate formulation will typically comprise particles which comprise more than one nucleic acid (such as RNA and/or DNA) species.
  • different nucleic acid (such as RNA and/or DNA) species are typically present together in a single particle.
  • polymers are commonly used materials for nanoparticle-based delivery.
  • cationic polymers are used to electrostatically condense the negatively charged nucleic acid into nanoparticles.
  • These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture.
  • Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic polymers herein.
  • some investigators have synthesized polymers specifically for nucleic acid delivery. Poly(P-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability.
  • Such synthetic polymers are also suitable as cationic polymers herein.
  • a "polymer,” as used herein, is given its ordinary meaning, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds.
  • the repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer.
  • the polymer is biologically derived, i.e., a biopolymer such as a protein.
  • additional moieties can also be present in the polymer, for example targeting moieties.
  • the polymer is said to be a "copolymer.” It is to be understood that the polymer being employed herein can be a copolymer.
  • the repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc.
  • Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.
  • the polymer is biocompatible.
  • Biocompatible polymers are polymers that typically do not result in significant cell death at moderate concentrations.
  • the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body.
  • polymer may be protamine or polyalkyleneimine.
  • protamine refers to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish).
  • protamine refers to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.
  • protamine as used herein is meant to comprise any protamine amino acid sequence obtained or derived from natural or biological sources including fragments thereof and multimeric forms of said amino acid sequence or fragment thereof as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.
  • the polyalkyleneimine comprises polyethylenimine and/or polypropylenimine, preferably polyethyleneimine.
  • a preferred polyalkyleneimine is polyethyleneimine (PEI).
  • the average molecular weight of PEI is preferably 0.75-10 2 to 10 7 Da, preferably 1000 to 10 5 Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.
  • linear polyalkyleneimine such as linear polyethyleneimine (PEI).
  • Cationic polymers contemplated for use herein include any cationic polymers which are able to electrostatically bind nucleic acid.
  • cationic polymers contemplated for use herein include any cationic polymers with which nucleic acid can be associated, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.
  • Particles described herein may also comprise polymers other than cationic polymers, i.e., non-cationic polymers and/or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers
  • lipid and "lipid-like material” are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to selfassemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment.
  • Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s).
  • the hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.
  • hydrophobic refers to any a molecule, moiety or group which is substantially immiscible or insoluble in aqueous solution.
  • hydrophobic group includes hydrocarbons having at least 6 carbon atoms.
  • the hydrophobic group can have functional groups (e.g., ether, ester, halide, etc.) and atoms other than carbon and hydrogen as long as the group satisfies the condition of being substantially immiscible or insoluble in aqueous solution.
  • hydrocarbon includes alkyl, alkenyl, or alkynyl as defined herein.
  • hydrocarbon groups can also include a cyclic (alkyl, alkenyl or alkynyl) group or an aryl group, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.
  • alkyl refers to a saturated linear or branched monovalent hydrocarbon moiety which may have six to thirty, typically six to twenty, often six to eighteen carbon atoms.
  • exemplary nonpolar alkyl groups include, but are not limited to, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, and the like.
  • alkenyl refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon double bond in which the total carbon atoms may be six to thirty, typically six to twenty often six to eighteen.
  • compositions for storing nucleic acid (such as RNA and/or DNA) particles such as for freezing nucleic acid (such as RNA and/or DNA) particles comprise low sodium chloride concentrations, or comprises a low ionic strength.
  • the sodium chloride is at a concentration from 0 mM to about 50 mM, from 0 mM to about 40 mM, or from about 10 mM to about 50 mM.
  • compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in “a pharmaceutically acceptable preparation".
  • pharmaceutically acceptable refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.
  • the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intranodally, intramuscularly, intratumorally, or peri turn orally.
  • the pharmaceutical composition is formulated for local administration or systemic administration.
  • Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration.
  • parenteral administration refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection.
  • the pharmaceutical compositions are formulated for systemic administration.
  • the systemic administration is by intravenous administration.
  • compositions comprising nucleic acids described herein, optionally formulated in particles, may be used in the therapeutic or prophylactic treatment of various diseases, in particular diseases in which provision of a peptide or polypeptide to a subject results in a therapeutic or prophylactic effect.
  • provision of an antigen or epitope which is derived from a virus may be useful in the treatment of a viral disease caused by said virus.
  • Provision of a tumor antigen or epitope may be useful in the treatment of a cancer disease wherein cancer cells express said tumor antigen.
  • Provision of a functional protein or enzyme may be useful in the treatment of genetic disorder characterized by a dysfunctional protein, for example in lysosomal storage diseases (e.g. Mucopolysaccharidoses) or factor deficiencies.
  • Provision of a cytokine or a cytokine-fusion may be useful to modulate tumor microenvironment.
  • treatment relates to the management and care of a subject for the purpose of combating a condition such as a disease.
  • the term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the therapeutically effective compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications.
  • prophylactic treatment or “preventive treatment” relate to any treatment that is intended to prevent a disease from occurring in an individual.
  • the terms “prophylactic treatment” or “preventive treatment” are used herein interchangeably.
  • the terms "individual” and “subject” are used herein interchangeably. They refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate), or any other non-mammal-animal, including birds (chicken), fish or any other animal species that can be afflicted with or is susceptible to a disease (e.g., cancer, infectious diseases) but may or may not have the disease, or may have a need for prophylactic intervention such as vaccination, or may have a need for interventions such as by protein replacement.
  • the individual is a human being.
  • Nucleic acid in particular RNA, having potency according to assays described herein, may be administered to a subject for delivering the nucleic acid to cells of the subject.
  • Nucleic acid in particular RNA, having potency according to assays described herein, may be administered to a subject for delivering a therapeutic or prophylactic peptide or polypeptide (e.g., a pharmaceutically active peptide or polypeptide) to the subject, wherein the nucleic acid encodes a therapeutic or prophylactic peptide or polypeptide.
  • a therapeutic or prophylactic peptide or polypeptide e.g., a pharmaceutically active peptide or polypeptide
  • Nucleic acid in particular RNA, having potency according to assays described herein, may be administered to a subject for treating or preventing a disease in a subject, wherein the nucleic acid encodes a therapeutic or prophylactic peptide or polypeptide and wherein delivering the therapeutic or prophylactic peptide or polypeptide to the subject is beneficial in treating or preventing the disease.
  • the subject is a mammal. In some embodiments, the mammal is a human.
  • the aim is to induce an immune response by providing a vaccine.
  • a person skilled in the art will know that one of the principles of immunotherapy and vaccination is based on the fact that an immunoprotective reaction to a disease is produced by immunizing a subject with an antigen or an epitope, which is immunologically relevant with respect to the disease to be treated. Accordingly, nucleic acids described herein are applicable for inducing or enhancing an immune response. Nucleic acids described herein are thus useful in a prophylactic and/or therapeutic treatment of a disease involving an antigen or epitope.
  • the aim is to treat cancer by vaccination. In some embodiments of the disclosure, the aim is to provide protection against an infectious disease by vaccination.
  • the aim is to provide secreted therapeutic proteins, such as antibodies, bispecific antibodies, cytokines, cytokine fusion proteins, enzymes, to a subject, in particular a subject in need thereof.
  • secreted therapeutic proteins such as antibodies, bispecific antibodies, cytokines, cytokine fusion proteins, enzymes
  • the aim is to provide a protein replacement therapy, such as production of erythropoietin, Factor VII, Von Willebrand factor, P- galactosidase, Alpha-N-acetylglucosaminidase, to a subject, in particular a subject in need thereof.
  • a protein replacement therapy such as production of erythropoietin, Factor VII, Von Willebrand factor, P- galactosidase, Alpha-N-acetylglucosaminidase
  • the aim is to modulate/reprogram immune cells in the blood.
  • the aim is to provide one or more cytokines or cytokine fusions which modulate tumor microenvironment to a subject, in particular a subject in need thereof.
  • the aim is to provide one or more cytokines or cytokine fusions which have anti -turn or activity to a subject, in particular a subject in need thereof.
  • RNA synthesis was performed according to the manufacturer’s protocol using the SuperScript IV First-Strand synthesis kit (Invitrogen).
  • the RNA mixture samples were diluted to 5 ng/pL.
  • 5 ng of RNA mixture, 1 pL of 10 pM cDNA primer, and 1 pL of 10 mM dNTPs were combined, and the volume was adjusted to 13.5 pL with H2O.
  • the cDNA primer anneals in the 3’UTR, partially covering the poly(A)-tail.
  • a master mix containing RNA sample, primers, dNTP, and water was made and divided into four tubes to measure the sample in triplicate and one negative control.
  • RNA was denatured at 80°C for 5 min, snap-cooled on ice for more than 1 min, and 4 pL of 5* SuperScript IV buffer, 1 pL of 40 U/pL RNase inhibitor, 1 pL of 0.1 M DTT, and 0.5 pL of 200 U/pL SuperScript IV reverse transcriptase were added.
  • 0.5 pL of water were added instead of the reverse transcriptase.
  • Samples were incubated in a PCR cycler with the following program: 55°C for 10 min, 80°C for 10 min, hold at 4°C. Afterwards, 0.5 pL RNase H (2 U/pL) was added and the sample incubated for 20 min at 37°C.
  • the cDNA samples were either stored at -20°C in DNA low binding tubes or directly processed for ddPCR.
  • Denaturation step 2 30s at 94°C,
  • QuantaSoft is generally able to automatically distinguish and define positive and negative populations. In the rare case that this was not possible, the threshold was set manually. The software automatically calculates the CN/pL. For RNA identity confirmation, the sample must show a minimum of 300 CN/pL and the negative control a maximum of 10 CN/pL.
  • each oligonucleotide set was tested against each single RNA composing the sample RNA mixture.
  • the ddPCR generated positive droplets only if the RNA-specific forward primer was combined with the target RNA (e.g., when the RNA A was amplified with the RNA A forward primer). In all other forward primer/RNA combinations, only negative droplets were observed.
  • RNA synthesis was performed according to the manufacturer’s protocol using the SuperScript IV First-Strand synthesis kit (Invitrogen).
  • the RNA mixture samples were diluted to 5 ng/pL.
  • 5 ng of RNA mixture, 1 pL of 10 pM cDNA primer, and 1 pL of 10 mM dNTPs were combined, and the volume was adjusted to 13.5 pL with FEO.
  • the cDNA primer anneals in the 3’UTR, partially covering the poly(A)-tail.
  • a master mix containing RNA sample, primers, dNTP, and water was made and divided into four tubes to measure the sample in triplicate and one negative control.
  • RNA was denatured at 80°C for 5 min, snap-cooled on ice for more than 1 min, and 4 pL of 5* SuperScript IV buffer, 1 pL of 40 U/pL RNase inhibitor, 1 pL of 0.1 M DTT, and 0.5 pL of 200 U/pL SuperScript IV reverse transcriptase were added.
  • 0.5 pL of water were added instead of the reverse transcriptase.
  • Samples were incubated in a PCR cycler with the following program: 55°C for 10 min, 80°C for 10 min, hold at 4°C. Afterwards, 0.5 pL RNase H (2 U/pL) was added and the sample incubated for 20 min at 37°C.
  • the cDNA samples were either stored at -20°C in DNA low binding tubes or directly processed for ddPCR.
  • Droplet digital PCR was conducted on a QX200/C1000 system (Bio-Rad) according to the manufacturer’s instructions.
  • the cDNA was always freshly diluted to a concentration of -1000 CN/pL.
  • 5.5 pL of cDNA, 11 pL of 2x ddPCR SuperMix (Bio-Rad), 0.25 pM dual-labelled HEX-BHQ1 -probe, 0.9 pM of RNA-specific forward primer, and 0.9 pM of common reverse primer were combined in a final volume of 22 pL.
  • Each sample was measure in triplicate, together with one negative control per sample. After oil droplet generation, the samples were incubated in the Cl 000 thermocycler (Bio-Rad) and the following thermal program was executed:
  • Activation step 1) 600s at 95°C, Denaturation step 2) 30s at 94°C, Annealing and extension step 3) 60s at 63°C, Enzyme inactivation step 4) 600s at 98°C. Step 2 and 3 were repeated 40 times. After PCR completion, the droplet fluorescence was read with the QX200 droplet reader (Bio-Rad).
  • RNA ratio calculation the CN/pL were converted into mass/volume concentration (g/pL) according to the following formula: g (CN MW pL “ ⁇ pL / * 6.022 * 10 23 where MW is the molecular weight of the RNAs. Afterwards, the ratios (in %) were calculated with the following formulas:
  • RNA B, C or D concentration of the respective RNA must be given in the numerator.
  • the %recovery spanned between 89.3% and 119.4%.
  • the ratio of the four RNAs is calculated as the amount of one RNA in solution with respect to the other three; that is, if the amount of one RNA is lower, the amount of the other three will increase in a complementary fashion.
  • delta was also evaluated, where delta is the mathematical distance of the measurement for both the RNAs in one mixture from the target value.
  • Table 2 report the mean and standard deviation (SD) of the triplicates as well as the %recovery (mean of measured values theoretical value x 100) and delta (the mathematical distance of the measurement from the theoretical value). Compare the “Theoretical RNA ratio” column with the “Mean” to verify that the measured ratio reflects the theoretical value with minimal deviation.
  • RNA synthesis was performed according to the manufacturer’s protocol using the SuperScript IV First-Strand synthesis kit (Invitrogen).
  • the RNA mixture samples were diluted to 5 ng/pL.
  • 5 ng of RNA mixture, 1 pL of 10 pM cDNA primer, and 1 pL of 10 mM dNTPs were combined, and the volume was adjusted to 13.5 pL with H2O.
  • the cDNA primer anneals in the 3’UTR, partially covering the poly(A)-tail.
  • a master mix containing RNA sample, primers, dNTP, and water was made and divided into four tubes to measure the sample in triplicate and one negative control.
  • RNA was denatured at 80°C for 5 min, snap-cooled on ice for more than 1 min, and 4 pL of 5* SuperScript IV buffer, 1 pL of 40 U/pL RNase inhibitor, 1 pL of 0.1 M DTT, and 0.5 pL of 200 U/pL SuperScript IV reverse transcriptase were added.
  • 0.5 pL of water were added instead of the reverse transcriptase.
  • Samples were incubated in a PCR cycler with the following program: 55°C for 10 min, 80°C for 10 min, hold at 4°C. Afterwards, 0.5 pL RNase H (2 U/pL) was added and the sample incubated for 20 min at 37°C.
  • the cDNA samples were either stored at -20°C in DNA low binding tubes or directly processed for ddPCR.
  • Droplet digital PCR was conducted on a QX200/C1000 system (Bio-Rad) according to the manufacturer’s instructions.
  • the cDNA was always freshly diluted to a concentration of -1000 CN/pL.
  • 5.5 pL of cDNA, 11 pL of 2x ddPCR SuperMix (Bio-Rad), 0.25 pM dual-labelled HEX-BHQ1 -probes, 0.9 pM of RNA-specific primers, and 0.9 pM of common reverse primers were combined in a final volume of 22 pL.
  • the reaction mix includes two oligonucleotides sets, one targeting the 5 ’-end and the other the 3 ’-end of the RNA.
  • the 5 ’-end oligonucleotide set is composed of one common forward primer, one RNA-specific reverse primer, and one common FAM-BHQ1 dual -labelled probe.
  • the 3 ’-end oligonucleotide set is composed of one RNA-specific forward primer, one common reverse primer, and one common HEX-BHQ1 dual-labelled probe. Each sample was measure in triplicate, together with one negative control per sample.
  • Step 2 and 3 were repeated 40 times. After PCR completion, the droplet fluorescence was read with the QX200 droplet reader (Bio-Rad).
  • the linkage describes the CN/droplet concentration of 5'- and 3 '-end double-positive droplets, once normalized for the possibility of random co-partitioning of single- and double-positive templates.
  • the linkage automatically calculated by the ddPCR software and converted in CN/pL is divided by the total number of 3 '-end (HEX) CN/pL to obtain the %linkage. This number correlates with the RNA integrity. 100
  • each RNA was individually degraded to different integrity levels by incubation at 90°C for different time spans. The RNAs from the same degradation time point were then mixed and measured with ddPCR. The method successfully detected the decrease in RNA integrity over time for all the RNAs.
  • Figure 1 shows the integrity of each RNA composing the differently degraded RNA mixtures was measured with ddPCR. The longer the RNA was degraded at 90°C, the lower the integrity measured with ddPCR.
  • the 3’-end CN/pL information is quantified. This is the same information needed for the assessment of RNA ratio and RNA identity.
  • the method presented here can measure RNA identity, RNA ratio, and RNA integrity in parallel.
  • the expermental system is simplified by the fact that 2 components of each oligonucleotide set (i.e., one common primer and one common dual-labelled probe) are identical for all RNAs and only one primer of each set is RNA-specific. This reduces the amount of reagents needed, the complexity of the system and the pipetting time compared to assembling two sets each composed of RNA-specific oligonucleotides.
  • RNA Total ribonucleic acid
  • CHO Chinese Hamster Ovary
  • Total RNA was isolated using a commercial kit (RNeasy Micro kit, Qiagen) and reverse transcribed (SuperScript IV, Invitrogen) into complementary deoxyribonucleic acid (cDNA) with the help of an oligo(dT) primer (Oligo (dT)20, part of the Super Script IV kit from Invitrogen).
  • cDNA was diluted and analyzed by digital droplet polymerase chain reaction (ddPCR) using two sets of specific primers and fluorescently labelled probe against RNA of interest and housekeeping RNA. Probes were labelled with hexachlorofluorescein (HEX) and 6-carboxyfluorescein (FAM) and carried a Black Hole Quencher 1 (BHQ1).
  • PCR cycling included initial activation for 600 s at 95°C and 40 cycles of denaturation for 30 s at 94°C and annealing / extension for 60 s at 61 °C. Subsequently, a final enzyme inactivation for 60 s at 98°C followed.
  • Figure 2 shows that copy numbers (CN) measured by digital droplet polymerase chain reaction (ddPCR) for a ribonucleic acid (RNA) of interest and a housekeeping gene from total RNA isolated from Chinese Hamster Ovary (CHO) cells which were previously transfected with four different amounts of formulated RNA of interest.
  • ddPCR digital droplet polymerase chain reaction

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Abstract

L'invention concerne un procédé de détermination d'au moins un paramètre de qualité d'un échantillon d'ARN à l'aide d'une transcription inverse et d'une PCR, de préférence une PCR numérique de gouttelettes. Le paramètre de qualité peut être (a) le rapport quantitatif d'au moins deux espèces de molécule d'ARN dans un échantillon d'ARN contenant des molécules d'ARN de n espèces de molécule d'ARN ; ou (b) l'identité de l'espèce de molécule d'ARN n dans l'échantillon d'ARN, n étant un nombre entier d'au moins 2 ; ou (c) l'intégrité d'un échantillon d'ARN contenant des molécules d'ARN d'espèces de molécules d'ARN n, n étant un nombre entier d'au moins 1 ; ou (d) la puissance d'un échantillon d'ARN formulé comprenant des molécules d'ARN d'intérêt.
PCT/EP2024/076303 2023-09-20 2024-09-19 Procédé Pending WO2025061863A1 (fr)

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