Method for producing RNA molecule compositions

#This application is a national phase application under 35 U.S.C. § 371 of International Application No. PCT/EP2016/082487, filed Dec. 22, 2016, which claims benefit of International Application No. PCT/EP2015/081000, filed Dec. 22, 2015, the entire contents of each of which are hereby incorporated by reference.

#The sequence listing disclosed herein is included in a text file having the name “CRVCP0203US.TXT,” created on Dec. 3, 2018, having a size of 111101 bytes.

#The present invention is directed to a method for producing a ribonucleic acid (RNA) molecule composition comprising n different RNA molecule species which are derived from m different deoxyribonucleic acid (DNA) molecule species comprising RNA in vitro transcription of a mixture of m different DNA molecule species in a single reaction vessel, wherein each of the m different DNA molecule species encodes one or more of the n different RNA molecule species thereby generating the n different RNA molecule species, and obtaining the RNA molecule composition comprising n different RNA molecule species, wherein n is an integer of at least 2, and wherein m is an integer of at least 1. The present invention also provides the RNA molecule composition produced by the above method as well as a pharmaceutical composition and pharmaceutical container comprising the RNA molecule composition. In a further aspect, the inventive RNA molecule composition and the pharmaceutical composition are useful as medicament, such as in the treatment or prophylaxis of a disease selected from the group consisting of genetic diseases, allergies, autoimmune diseases, infectious diseases, neoplasms, cancer, and tumor-related diseases, inflammatory diseases, diseases of the blood and blood-forming organs, endocrine, nutritional and metabolic diseases, diseases of the nervous system, diseases of the circulatory system, diseases of the respiratory system, diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue, and diseases of the genitourinary system, independently if they are inherited or acquired, and combinations thereof. Also provided is the use of the RNA molecule composition as immunotherapeutic agent, gene-therapeutic agent or as vaccine.

#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.

#FIG. 2: Overlay of growth curves (OD600) 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.

#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

#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.

#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.

#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)