Method for producing RNA molecule compositions

Abstract

The invention relates to a method for producing a ribonucleic acid (RNA) molecule composition comprising n different RNA molecule species, the method comprising a step of RNA in vitro transcription of a mixture of m different deoxyribonucleic acid (DNA) molecule species in a single reaction vessel in parallel, i.e. simultaneously, and a step of obtaining the RNA molecule composition. Also provided is the RNA composition provided by the inventive method and a pharmaceutical composition comprising the same as well as a pharmaceutical container. Moreover, the invention provides the RNA composition and the pharmaceutical composition for use as medicament.

Claims

1. A method for producing a ribonucleic acid (RNA) pharmaceutical composition comprising at least two different RNA molecule species, the method comprising the following steps: I) performing simultaneous RNA in vitro transcription of a mixture of at least two different deoxyribonucleic acid (DNA) molecule species in a single reaction vessel, wherein each of the at least two different DNA molecule species encode the at least two different RNA molecule species thereby generating the at least two different RNA molecule species; II) obtaining an RNA molecule composition comprising the at least two different RNA molecule species generated in step I); III) purifying the RNA from the RNA molecule composition; and IV) formulating the at least two different RNA molecule species in a pharmaceutical formulation to produce the RNA pharmaceutical composition, wherein the at least two different RNA molecules vary in length from each other by no more than 100 nucleotides and wherein the amounts of each of the at least two different RNA molecules are not more than 20% different from each other in the RNA molecule composition.

2. The method according to claim 1, further comprising prior to step a step of: 1) generating the mixture of at least two different DNA molecule species using bacterial amplification, 2) generating the mixture of at least two different DNA molecule species using polymerase chain reaction (PCR), and/or 3) generating the mixture of at least two different DNA molecule species using enzymatic amplification.

3. The method according to claim 2, wherein step 1) comprises a step of: i) transforming a bacterial cell culture with at least one single DNA plasmid species of the mixture of at least two different DNA plasmid species, wherein each DNA plasmid species encodes one of the at least two different RNA molecule species.

4. The method according to claim 2, wherein step 1) comprises a step of: i)transforming at least two single bacterial cell cultures each with a single DNA plasmid species of the at least two different DNA plasmid species, wherein the single DNA plasmid species encodes one of the at least two different RNA molecule species.

5. The method according to claim 3, further comprising a step of: ii) isolating at least one single bacterial cell clone for each DNA plasmid species of the mixture of at least two different DNA plasmid species, and iii) MHO growing each of the at least one single bacterial cell clone isolated in step ii) in a separate bacterial cell clone culture.

6. The method according to claim 4, further comprising after step i) the following steps: ii) isolating at least one single bacterial cell clone of each of the at least two single bacterial cell cultures transformed in step i), iii) growing each of the single bacterial cell clones isolated in step ii) in a separate bacterial cell culture, and iv) selecting at least one bacterial cell clone culture for each of the at least two different DNA plasmid species.

7. The method according to claim 5, further comprising: iv) determining at least one parameter of growth kinetics and/or amount of plasmid DNA of the at least one single bacterial cell clone culture, and v) selecting one bacterial cell clone culture for each of the at least two different DNA plasmid species depending on the parameter determined in step iv).

8. The method according to claim 7, wherein step iv) comprises a step of: determining a parameter of growth kinetics by measuring the optical density of the bacterial cell clone culture after a time interval, and/or determining the amount of plasmid produced per volume and time of bacterial cell culture.

9. The method according to claim 7, wherein the selected bacterial cell clone culture for each of the at least two different DNA plasmid species exhibits similar or identical growth kinetics and/or similar or identical DNA production levels.

10. The method according to claim 7, wherein step 1) further comprises a step of: inoculating and growing an amount of at least one of the one or more bacterial cell clone cultures selected for each of the at least two different DNA plasmid species in a single reaction vessel, or inoculating and growing an amount of at least one of the one or more bacterial cell clone cultures selected for each of the at least two different DNA plasmid species in one or more separate reaction vessels for each of the at least two different DNA plasmid species.

11. The method according to claim 10, wherein equal amounts of each bacterial cell clone culture are inoculated.

12. The method according to claim 10, wherein the amount of each bacterial cell clone culture used for inoculating is selected so that equal or similar amounts of each of the at least two different DNA plasmid species are obtained.

13. The method according to claim 1, wherein the amount of each of the at least two different RNA molecule species in the RNA molecule composition is proportional or at least 90% proportional to the amount of the corresponding DNA molecule species in the mixture of at least two different DNA molecule species.

14. The method according to claim 1, wherein the DNA sequences of the at least two different deoxyribonucleic acid (DNA) molecule species are at least 90% identical to each other.

15. The method according to claim 1, wherein the RNA sequences of the at least two different RNA molecule species are at least 90% identical to each other.

16. The method according to claim 1, wherein each of the at least two different DNA molecule species encodes for different RNA molecule species, wherein each of the at least two different RNA molecule species encodes for an antigen of different serotypes or strains of a same pathogen.

17. The method according to claim 16, wherein each of the at least two different RNA molecule species encodes for an influenza antigen.

18. The method of claim 1, wherein the at least two different RNA molecule species encode different variants of the same target peptide or protein, wherein said composition comprises the at least two different RNA molecule species in identical amounts.

19. The method according to claim 2, wherein step 1) comprises a step of: i)transforming a single bacterial cell culture with a mixture of at least two different DNA plasmid species, wherein each DNA plasmid species encodes one of the at least two different RNA molecule species.

20. The method according to claim 19, further comprising after step i) the following steps: ii) isolating at least at least two single bacterial cell clones, and iii) growing each of the at least two single bacterial cell clones isolated in step ii) in a separate bacterial cell clone culture, iv) determining the identity of the DNA plasmid species of each of the at least at least two single bacterial cell clone cultures grown in step iii), and v) selecting at least one single bacterial cell clone culture for each of the at least two different DNA plasmid species.

21. The method of claim 1, further comprising producing a RNA molecule composition comprising at least three different RNA molecule species.

22. The method of claim 21, further comprising producing a RNA molecule composition comprising at least four different RNA molecule species.

23. The method according to claim 1, wherein each of the at least two different RNA molecule species encodes for tumor antigen.

24. The method of claim 1, wherein the at least two different DNA molecule species comprise at least two different DNA plasmid species.

25. The method according to claim 24, wherein the DNA plasmid species have the same plasmid backbone.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Percent identity matrix of the pDNA sequences (FIG. 1A) and the RNA sequences (FIG. 1B). The matrix is based on sequence identity values using multiple sequence alignments (Clustal 12.1). SEQ ID NOs are indicated.

(2) FIG. 2: Overlay of growth curves (OD.sub.600) of different pDNA clones. In FIG. 2A, DH5alpha was used as a host. Three groups that show similar growth characteristics were identified (g1, g2, g3). In FIG. 2B, a CopyCutter™ strain was used as a host. All pDNA cultures exhibited similar growth characteristics. A detailed description of the experiment is provided in Example 7.

(3) FIG. 3: Pre- and main culture growth kinetics for DH5alpha (A) and CopyCutter™ (B) clones show that growth characteristics of clones are robust. A detailed description of the experiment is provided in Example 7. A: DH5alpha and B: CopyCutter™ clones

(4) FIG. 4: FIG. 4A shows a scheme of the CoStock and Colnoc co-cultivation strategies. FIG. 4B shows an overlay of the growth characteristics for different replicates (co-cultivations of 4 and 5 different clones). A detailed description of the experiment is provided in Example 8.

(5) FIG. 5: FIG. 5 shows RNA agarose gel electrophoresis of the obtained RNA molecule compositions using a pDNA mixture as template. A detailed description of the experiment is provided in Example 9.

(6) FIG. 6: Quantitative analysis of single RNA preparations, mix-4 preparations and mix-5 preparations. A detailed description of the experiment is provided in Example 9.

(7) FIG. 7: Restriction analysis of a 4-mix PCR amplified DNA mixture. M: marker lane; control: irrelevant DNA; Mix-4_A: 4-mix PCR amplified DNA mixture treated with a combination of restriction enzymes that does not digest the A product; Mix-4_B: 4-mix PCR amplified DNA mixture treated with a combination of restriction enzymes that does not digest the B product; Mix-4_C: 4-mix PCR amplified DNA mixture treated with a combination of restriction enzymes that does not digest the C product; Mix-4_D: 4-mix PCR amplified DNA mixture treated with a combination of restriction enzymes that does not digest the D product; Arrow indicates the expected band indicating that all DNA species (A, B, C, D) were present in the PCR amplified DNA mixture.

(8) FIG. 8: FIG. 8 shows RNA agarose gel electrophoresis of the obtained RNA molecule compositions using a PCR amplified DNA mixture as a template. A detailed description of the experiment is provided in Example 10.

(9) FIG. 9: NGS analysis of the PCR amplified DNA mixture and the RNA composition (4-mix) obtained using the PCR amplified DNA mixture as a template. A detailed description of the experiment is provided in Example 10.

EXAMPLES

(10) The following examples are intended to illustrate the invention in a further way. They are merely illustrative and not intended to limit the subject matter of the invention.

Example 1: Preparation of DNA Encoding HA Proteins of Several Serotypes

(11) For the present examples, DNA sequences encoding different heamagglutinin proteins, a glycoprotein found on the surface of influenza viruses (Influenza A and Influenza B), were generated. For the present examples, several HA proteins of various serotypes were used (see Table 1 below). The DNA sequences were prepared by modifying the wild type encoding DNA sequence by introducing a GC-optimized sequence for stabilization. Sequences were introduced into the same vector backbone, a pUC19 derived vector and modified to comprise a 5′-UTR derived from the 32L4 ribosomal protein (32L4 TOP 5′-UTR) and a 3′-UTR derived from albumin, a histone-stem-loop structure, and a stretch of 64 adenosines at the 3′-terminal end. The respective plasmid DNA sequences as well as the corresponding RNA sequences are provided in the sequence protocol (SEQ ID NOs: 1-14 (RNA sequences) and SEQ ID NOs: 15-28 (plasmid DNA sequences). The generated sequences show high sequence similarity (Sequence identity matrix of plasmid DNA sequences and expected RNA sequences provided in FIG. 1). The obtained plasmid DNA constructs were transformed and propagated in bacteria (Escherichia coli) and glycerol stocks were prepared using common protocols known in the art.

(12) TABLE-US-00001 TABLE 1 HA-constructs used in the experiment GC SEQ ID SEQ ID Length Length content NO NO of of of of the of RNA pDNA HA protein description pDNA RNA RNA 1 15 H1N1 Influenza A virus 3952 1915 60.78 (Puerto Rico/1934) 2 16 H1N1 Influenza A virus 3955 1918 60.84 (Netherlands/2009) 3 17 H1N1 Influenza A virus 3955 1918 61.00 (California/2009) 4 18 H5N1 Influenza A virus 3949 1912 60.25 (NIBRG-14) 5 19 H5N1 Influenza A virus 3961 1924 60.34 (Vietnam 2004) 6 20 H5N1 Influenza A virus 3961 1924 60.60 (Bavaria/2006) 7 21 H1N1 Influenza A virus 3952 1915 60.78 (Brisbane/2007) 8 22 H3N2 Influenza A virus 3955 1918 60.74 (Uruguay/2007) 9 23 H3N2 Influenza A virus 3955 1918 61.52 (Hongkong/1968) 10 24 H2N2 Influenza A virus 3943 1906 61.12 (Japan/1957) 11 25 H7N7 Influenza A virus 3946 1909 62.28 (Bratislava/1979) 12 26 H1N1 Influenza A virus 3955 1918 60.85 (Netherlands2009)-wBB (California2009) 13 27 HA Influenza B virus 4012 1975 63.39 (Brisbane 2008) 14 28 H1N1 Influenza A virus 3955 1918 61.00 (California2009)-wBB (Netherlands2009)

Example 2: Screening of the Growth Behavior of Individual Clones

(13) The goal of this experiment is to evaluate the individual growth and production behavior of bacteria cultures bearing plasmids obtained in Example 1. This analysis is necessary to identify uniformly growing clones from HA plasmid DNAs (see Table 1) which guarantees that all plasmid DNA variants are produced in similar amounts (see Example 3).

(14) 2.1. Pre-Cultivation from Glycerol Stocks

(15) For each bacterial clone, 1 ml LB medium (containing 100 μg/ml ampicillin) is inoculated with the respective glycerol stock and incubated for 16 h at 37° C. in a shaking incubator. Following that, 10 μl of the individual bacterial culture is transferred to solid LB medium (supplemented with 100 μg/ml ampicillin) and incubated for 16 h at 37° C. to obtain single discrete colonies. Single discrete colonies from each plate are taken to inoculate 1 ml of liquid LB medium (containing 100 μg/ml ampicillin) for pre-cultivation prior to the screening main culture. In this way, first growth synchronization is achieved.

(16) 2.2. Screening of Growth Performance in Microtiter Plate

(17) After determination of pre-cultures' biomass concentrations by optical density measurement in a plate reader, the respective volume of each clone is transferred to inoculate 1 mL of liquid TB medium (containing 100 μg/ml ampicillin) to a uniform initial cell density of 0.1. The growth of the individual clones is monitored in a special microtiter plate with a transparent bottom using online measurements (scattered light and dissolved oxygen tension (DOT) measurement). The recorded online signals allow for a detailed determination of growth kinetics such as lag phase duration, growth rate, and final biomass formation. After cultivation, plasmid titer quantification and next generation sequencing is performed. Uniformly growing clones are identified and used to generate glycerol stocks. Subsequently, those clones are used for large-scale plasmid DNA production.

(18) 2.3. Screening of RNA In Vitro Transcription Performance of Individual Clones

(19) The DNA plasmids are enzymatically linearized using EcoRI and transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture under respective buffer conditions. To assess the transcription efficiency of individual clones over time, samples taken at different time points are analyzed quantitatively.

(20) 2.4. In Vitro and In Vivo Characterization of Individual mRNA Constructs

(21) Expression analysis of individual mRNAs (in vitro translation, in-vitro expression and analysis by western blot, FACS, and ELISA. Individual analysis of antigenicity of antigens

Example 3: Large-Scale Plasmid DNA Production

(22) From each HA antigen (see Table 1), one uniformly growing pre-selected clone is taken (glycerol stocks) to inoculate a heterologous pre-culture (containing clones from each antigen) in shake flasks (200 ml LB medium containing 100 μg/ml ampicillin) for 16 h at 37° C. 100 ml of that pre-culture is taken to inoculate a production-scale fermenter (Eppendorf BioFlo415, volume 15 liter).

(23) To obtain optimal bacteria growth, feeding solution (LB medium comprising ampicillin (100 μg/ml) with 2% glucose) is constantly fed into the fermenter tank. During fermentation, standard parameters are precisely regulated and continuously monitored (e.g., pH: 7.0, temperature: 37° C.). The cell density is controlled by photometric determination at 600 nm. The fermentation procedure is stopped after 20 hours of incubation time. The bacterial culture is centrifuged down at 6000 g for 15 minutes at room temperature, the supernatant is discarded and the cell pellet used for plasmid DNA isolation.

(24) Since all clones show the same growth and production behavior, all plasmid DNA species are potentially produced in similar amounts.

Example 4: Plasmid DNA Preparation and Quality Controls

(25) The obtained bacterial cell pellet (see example 3) is used for plasmid preparation, using a commercially available endotoxin free plasmid DNA giga-preparation kit (Macherey Nagel). After purification, the plasmid DNA mixture is analyzed regarding its identity and quantity via next generation sequencing (NGS), qPCR or restriction mapping in order to confirm the presence of each individual antigen encoding plasmid in the respective amounts.

(26) Additionally, plasmid DNA content and purity are determined via UV absorption and anion exchange chromatography.

Example 5: RNA In Vitro Transcription

(27) The DNA plasmid mixture is enzymatically linearized using EcoRI and transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture under respective buffer conditions. The obtained mRNA mixture is purified using PureMessenger® (CureVac, Tübingen, Germany; WO 2008/077592 A1) and used for in vitro and in vivo experiments.

Example 6: Formulation of a Polyvalent HA Vaccine

(28) 6.1. Formulation with Protamine

(29) The mRNA mixture is furthermore complexed with protamine prior to use in in vivo vaccination. The mRNA formulation consists of a mixture of 50% free mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1 (according to WO/2010/037539). First, mRNA is complexed with protamine by addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes were stably generated, free mRNA is added, and the final concentration of the vaccine is adjusted with Ringer's lactate solution.

(30) 6.2. LNP Encapsulation

(31) A lipid nanoparticle (LNP)-encapsulated mRNA mixture is prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. LNPs are prepared as follows. Cationic lipid, DSPC, cholesterol and PEG-lipid are solubilized in ethanol. Briefly, the mRNA mixture is diluted to a total concentration of 0.05 mg/mL in 50 mM citrate buffer, pH 4. Syringe pumps are used to mix the ethanolic lipid solution with the mRNA mixture at a ratio of about 1:6 to 1:2 (vol/vol). The ethanol is then removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 μm pore sterile filter. Lipid nanoparticle particle diameter size is determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK).

Example 7: Cultivation and Characterization of Growth Behaviour of Different pDNA Strains

(32) 12 different HA pDNA constructs (obtained according to Example 1) were propagated in DH5alpha and CopyCutter™ E. coli strains and characterized for their growth behavior.

(33) 7.1 Re-Transformation of pDNA in Escherichia coli:

(34) For the re-transformation of pDNA (obtained according to Example 1) harboring influenza antigen coding sequences (SEQ ID NOs: 1-11, 13), 123 μl SOC medium was prepared and maintained at 37° C. 25 μl competent cells (DH5alpha and CopyCutter™) were thawed on ice, mixed, and 2 μL of the respective pDNA were added. After incubation on ice for 30 min, cells were heat shocked for 30 s at 42° C. Then, cells were cooled on ice for 2 min. After adding 123 μL SOC medium, cells were incubated for 37° C. for 60 min and 500 rpm. Afterwards, cells were plated on 1.5% agarose LB APS ampicillin [100 mg/L] and incubated over night at 37° C. to allow growth of discrete colonies.

(35) 7.2 Characterization of Growth Behavior of Obtained Clones:

(36) For screening of growth characteristics of clones, 48 multi-well BOH flower plates with optodes for pH and DO were used. The cultivation and characterization of growth was performed with main cultures on DH5alpha and CopyCutter™ hosts. For inoculation of the pre-culture, two discrete colonies per transformation were picked and transferred into separate wells in 1.2 mL LB APS ampicillin [100 mg/L]. The 48 well flower plate was sealed with a gas permeable sealing foil with evaporation reduction and cultivated in a Biolector® (m2p-labs) microbioreactor (Conditions: 37° C.; 1200 rpm shaking frequency; 20.95% 02; humidity of 85%; Well readout: cycle time 10 min; biomass gain of 25; pO.sub.2 gain of 38; pH gain of 22). The plate was incubated until an OD.sub.600 between 0.4 and 0.7 was reached. Main-cultures were inoculated with pre-cultures to a final OD.sub.600 of 0.05-0.1 in 1.1 ml LB APS ampicillin [100 mg/L], CopyCutter™ clones were additionally treated with induction solution. The cultivation was continued with the same cultivation settings, until all clones reached the stationary phase. Cells were harvested for pDNA preparation. After characterization of DH5α and CopyCutter™ clones, glycerol stocks of all clones with a final OD600 of 1.0 were prepared for further experiments. FIG. 2 shows the growth characteristics of each individual main culture for DH5alpha and CopyCutter™ strains. FIG. 2 shows a comparison of the growth kinetics of pre- and main-culture for two different clones per pDNA construct for DH5alpha and CopyCutter™ strains.

(37) 7.3 Results:

(38) The results of FIG. 2 show that several analyzed clones show similar growth characteristics, both for DH5alpha strain and CopyCutter™ strain. For example, DH5alpha clones can be grouped in three characteristic groups that may potentially be cultured together (g1, g2, g3). CopyCutter™ clones show a more balanced and homogeneous growth characteristics. These clones may potentially be grouped and cultured together. The data suggests that it is feasible to generate a co-culture of clones for producing a pDNA template mixture for RNA in vitro transcription which would dramatically economize and streamline the RNA production process.

(39) The results in FIG. 3 show that the growth characteristics of different clones that carry the same pDNA constructs are comparable to each other, suggesting that the characterization of the growth behavior is robust and therefore suitable to assess and predict the growth behavior also for a setting of a co-cultivation.

(40) Summarizing the above, the data shows that co-culturing of groups of clones is applicable in (industrial) production of RNA mixtures.

Example 8: Co-Cultivation and Characterization of Different Clone Mixes

(41) Based on the results of Example 7, CopyCutter™ clones with similar growth characteristics were selected and used to co-cultivate different influenza antigen clones and to produce a pDNA template mixture. In one setup, four different pDNAs (“4-mix”) were selected for co-culturing (A; B; C; D), in another setup, five different pDNAs (“5-mix”) were selected for co-culturing (A; B; C; D; E); the selected constructs show high similarity in sequence length, GC content and sequence similarity on the RNA level (see Table 2 and Table 3).

(42) TABLE-US-00002 TABLE 2 Overview of the 4-mix and 5-mix selected for co-culturing GC content RNA SEQ ID SEQ ID NO Antigen pDNA length in % NO RNA pDNA 4-mix A 3955 60.84 2 16 B 3961 60.34 5 19 C 3955 60.74 8 22 D 3943 61.12 10 24 5-mix A 3955 60.84 2 16 B 3961 60.34 5 19 C 3955 60.74 8 22 D 3943 61.12 10 24 E 4012 63.39 13 27

(43) TABLE-US-00003 TABLE 3 Sequence identity matrix (in %) of 4-mix and 5-mix sequences on the pDNA level E C A B D E 100 82.27 81.90 82.11 82.33 C 82.27 100 84.38 84.37 84.29 A 81.90 84.38 100 88.76 89.03 B 82.11 84.37 88.76 100 90.91 D 82.33 84.29 89.03 90.91 100

(44) Two concepts of co-cultivation were tested: the co-stock (“CoStock”) and the co-inocula (“Colnoc”) strategies (see FIG. 4A). In the CoStock strategy, the glycerol stocks of the respective clones were mixed in equimolar ratios and used as inoculum for a pre-culture (1.2 mL LB APS ampicillin [100 mg/L]). For a mixture of 4 clones, 15 μL from each glycerol stock (OD.sub.600=1) was used (60 μL in total). For a mixture of 5 clones, 12.5 μL from each glycerol stock (OD.sub.600=1) was used (60 μL in total). After the pre-cultures reached an OD.sub.600 of 10, the pre-cultures were used as inocula for a main-culture. In the CoInoc strategy, 60 μL of each individual glycerol stock (OD.sub.600=1) was used as inoculum for individual pre-cultures (1.2 mL LB APS ampicillin [100 mg/L]). Cultivation of each clone was performed separately in a pre-culture until the individual cultures reached an OD.sub.600 of 10. A mixture of said pre-cultures was used for the inoculation of a main-culture.

(45) The main cultures in both strategies were grown to a concentration of OD.sub.600 0.2. To each main-culture, the CopyCutter™ induction solution was added. When the respective cultures reached the late log-phase after approximately 7 h, cells were harvested. Afterwards, the pDNA was extracted, measured with spectrophotometry, and quantitatively analyzed (restriction analysis, sequencing, quantitative PCR). Growth characteristics of the respective co-culture replicates (CoStock strategy and CoInoc strategy with several replicates) were monitored as outlined in Example 7. The results are shown in FIG. 4B.

(46) Results:

(47) FIG. 4 shows that major differences for the respective tested setups (Colnoc strategy, CoStock strategy, 4-mix, and 5-mix) could not be observed, suggesting that the overall growth characteristics of the respective co-cultures were comparable to each other. In that experiment, the CoStock culture replicates showed a very homogeneous growth behavior with very small inter-sample variation.

(48) Summarizing the above, the data shows that co-culturing of several different clones works in a robust and reproducible way. Therefore, the generation of a pDNA mixture for RNA in vitro transcription can be obtained by bacterial amplification in a co-culture which streamlines and improves the production process of RNA mixture based therapeutics.

Example 9: RNA In Vitro Transcription Using pDNA Template Mixtures

(49) The aim of the experiment was to show that an mRNA mixture can be generated in one reaction by RNA in vitro transcription using a pDNA cocktail as a template. In the present example, a 4-mix and 5-mix RNA mixture was produced (see Table 2 and 3).

(50) 9.1 Generation of pDNA Template Mixtures:

(51) First, the pDNAs of mix-4 and mix-5 (see Table 2) were separately linearized (200 μg pDNA each) using 60 μL EcoRI (10 U/μL) enzyme in the respective digestion buffer. The reactions were incubated for 4-5 h at 37° C. Linearized pDNAs were recovered using isopropanol precipitation. The obtained linearized pDNA samples were re-dissolved in WFI and analyzed for completeness of linearization using agarose gel electrophoresis. The linearized pDNA templates were used to generate pDNA mix-4 and mix-5 mixtures (0.09 μg/μL linearized pDNA each).

(52) 9.2 RNA In Vitro Transcription Using pDNA Template Mixtures:

(53) RNA in vitro transcription was performed with the respective mix-4 and mix-5 pDNA mixtures (25 μg/mL DNA in total) in the presence of a sequence-optimized NTP-mix (13.45 mM) comprising cap analog (4×GTP), 2500 U/mL T7 Polymerase, 24 mM MgCl.sub.2, 5 U/mL Pyrophosphatase (PPase), and 0.2 U/μL Ribolock in Tris-HCl transcription buffer. The reactions were incubated at 37° C. After 90 minutes incubation time, Tris-HCl transcription buffer and NTPs were added (26.9 mM final NTP concentration) and incubated at 37° C. for additional 5 h. Afterwards, DNA template was removed using a DNasel digest. The digestion reaction was stopped with 25 mM EDTA and samples were subjected to LiCl precipitation. Precipitated RNA was re-dissolved in WFI. Following that the RNA was analyzed using RNA agarose gel electrophoresis (see FIG. 5). The composition of the RNA mixture was quantitatively and qualitatively analyzed (see FIG. 6). Moreover the obtained RNA composition is chraracterized using NGS and qPCR. The obtained mRNA mixture may further be formulated according to Example 6.

(54) 9.3 Results:

(55) RNA agarose gel electrophoresis (see FIG. 5A) showed that RNA was produced in comparable amounts for the control reaction (single RNA preparation), the mix-4 and mix-5 reactions. Defined bands were visible for the standard, mix-4 and mix-5 between in the expected size. Moreover, side-products could not be determined and no variation in band intensity in the respective duplicates could be observed. The data demonstrates that it is feasible to generate a RNA mixture in one RNA in vitro transcription reaction using a pDNA mixture as template. In addition the data demonstrates that the RNA in vitro reaction yields the expected product in a robust, clean and reproducible manner.

(56) FIG. 6 shows that RNA in vitro transcription on a pDNA template mixture yields amounts of RNA comparable to those obtained from single RNA preparations. Moreover, the data shows that there is very little inter-sample variation in the mix-4 and mix-5 RNA preparations suggesting that the process works in a robust and reproducible way.

(57) Summarizing the above, the data shows that RNA in vitro transcription on a pDNA mixture works in a robust and reproducible way. Moreover, the obtained RNA mixture displays the same quality attributes than single RNA preparations. Therefore, the inventive RNA in vitro transcription procedure streamlines and economize the production process of RNA mixture based therapeutics.

Example 10: Production of Template Cocktails Using Preparative PCR with Subsequent RNA In Vitro Transcription

(58) The aim of the experiment was to evaluate whether PCR on a DNA mixture is suitable to generate a DNA template mixture for RNA in vitro transcription.

(59) 10.1 Generation of PCR-Amplified DNA Template Mixtures:

(60) As PCR template, the 4-mix pDNA mixture was used (see Example 9.1). The final concentrations of all components in WFI were 1×KAPA HiFi HotStart ReadyMix, 1 ng 4-mix DNA mixture, 1 M betaine, 0.3 μM T7 forward primer, and 0.3 μM reverse primer. The PCR was performed using a commercially available Thermocycler. The obtained PCR product mixture was purified using Agencourt® AMPure® XP-Kit (according to the manufacturer's instructions) and analyzed with restriction analysis on the 4-mix PCR product to reveal that each product was amplified to a similar extend (see FIG. 7). In addition, the PCR product was analyzed using NGS (FIG. 9).

(61) 10.2 RNA In Vitro Transcription Using PCR-Amplified DNA Template Mixtures:

(62) The obtained purified 4-mix PCR amplified DNA mixture was used in RNA in vitro transcription as described in Example 9.2. The composition of the produced RNA mixture was quantitatively and qualitatively analyzed using RNA agarose gelelectrophoresis (see FIG. 8) and NGS (see FIG. 9). Moreover the obtained RNA molecule composition is chraracterized using qPCR. The obtained mRNA mixture may further be formulated according to Example 6.

(63) 10.3 Results:

(64) FIG. 7 shows that PCR is suitable to amplify and generate a DNA mixture that can be used as a template for the production of an RNA mixture via RNA in vitro transcription. Each PCR product was present in the obtained 4-mix PCR amplified DNA mixture.

(65) RNA AGE (see FIG. 8) showed that RNA was produced in comparable amounts for the control reaction (control RNA), and mix-4 reactions. Defined bands were visible for the control and the mix-4 in the expected size. Moreover, side-products could not be determined and no variation in band intensity in the respective duplicates could be observed. The data demonstrates that it is feasible to generate a RNA mixture via RNA in vitro transcription in one reaction using a PCR amplified DNA mixture as template. In addition, the data demonstrates that the RNA in vitro reaction yields the expected product in a robust, clean and reproducible manner.

(66) Next generation sequencing (see FIG. 9) showed that simultaneous PCR amplification of a DNA template mixture yielded DNA in almost equal amounts. In addition, using the PCR-amplified DNA mixture as a template for simultaneous RNA in vitro transcription, a homogeneous RNA mixture (4-mix) was generated (25:27:25:22) that almost matched the theoretically expected ratio of 1:1:1:1. Notably, the NGS results also demonstrate the linearity of the process, meaning that the ratio of the PCR-generated mixture matched the ratio of the final RNA mixture.

(67) Therefore, the results show that the inventive method is suitable to generate RNA mixtures also in other ratios, depending on the application or purpose.

(68) Summarizing the above, the data shows that RNA in vitro transcription on a PCR amplified DNA mixture works in a robust and reproducible way. Moreover, the obtained RNA mixture displays the same quality attributes than single RNA preparations. Therefore, the inventive RNA in vitro transcription procedure streamlines and economize the production process of RNA mixture based therapeutics.

Example 11: Production of Template Cocktails Using On-Chip PCR with Subsequent RNA In Vitro Transcription

(69) A chip harboring a mixture of synthetic, immobilized DNA is used as a template for preparative PCR (DNA chip obtained from TWIST bioscience). The preparative PCR is performed essentially according to Example 10. The obtained PCR product is purified and used for RNA in vitro transcription to generate a mixture of RNA (essentially performed according to Example 10) and subjected to quantitative and qualitative measurements (e.g., RNA AGE, RT-qPCR, NGS, and Spectrometry). Following that, a purification step (e.g. PureMessenger®; WO2008077592) and, optionally, a formulation step is performed (e.g., protamine complexation, LNP encapsulation).

Example 12: Production of Template Cocktails Using dbDNA Templates with Subsequent RNA In Vitro Transcription

(70) An in vitro cell free process for amplifying a DNA template and converting the amplified DNA into closed linear “doggybone” DNAs (dbDNA) is carried out to generate a DNA mixture for subsequent RNA in vitro transcription. Rolling circle DNA template amplification and generation of dbDNA is performed according to WO 2010/086626. The obtained dbDNA templates are individually linearized using an appropriate restriction enzyme (e.g., EcoRI), purified, and mixed to generate a linearized template mixture (e.g., mix-4, mix-5; e.g. see Table 2). The linearized template mixture is used for RNA in vitro transcription (essentially performed according to Example 9) and subjected to quantitative and qualitative measurements (e.g., RNA AGE, RT-qPCR, NGS, and Spectrometry). Following that, a purification step (e.g. PureMessenger®; WO2008077592) and, optionally, a formulation step is performed (e.g., protamine complexation, LNP encapsulation).