Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids

Abstract

The present invention is directed to (the use of) a solution containing at least one nucleic acid (sequence) and free mannose for lyophilization, transfection and/or injection, particularly of RNA and mRNA. The inventive solution exhibits a positive effect on stabilization of the nucleic acid (sequence) during lyophilization and storage but also leads to a considerable increase of the transfection efficiency of a nucleic acid. It thus also increases in vivo expression of a protein encoded by such a nucleic acid upon increased transfection rate. The present invention is furthermore directed to a method of lyophilization using the mannose-containing solution, to pharmaceutical compositions, vaccines, kits, first and second medical uses applying such a mannose-containing solution and/or a nucleic acid (sequence) lyophilized or resuspended with such a solution.

1 . A method of using a solution, wherein the solution comprises at least one nucleic acid sequence and a free, unconjugated and non-covalently bound mannose for lyophilization, transfection and/or injection of the nucleic acid sequence, wherein the solution enhances the transfection efficiency of the nucleic acid and/or the expression of a protein encoded by the nucleic acid sequence. 
     
     
         2 . The method of  claim 1 , wherein the mannose concentration of the solution is in the range of about 0.01 to about 10% (w/w), including a concentration of about 0.01 to about 10% (w/w), a concentration of about 0.1 to about 7.5% (w/w), a concentration of about 0.5 to about 5% (w/w), a concentration of about 1 to about 4% (w/w), or a concentration of about 2 to about 4% (w/w), including a concentration of about 2.5% (w/w). 
     
     
         3 . The method of  claim 1  or  2 , wherein the mannose is selected from α-D-Mannofuranose, β-D-Mannofuranose, α-D-Mannopyranose and/or β-D-Mannopyranose. 
     
     
         4 . The method of  claim 1 , wherein the solution is present in an osmolarity in the range of about 200 mosmol/l to about 400 mosmol/l. 
     
     
         5 . The method of  claim 1 , wherein the solution additionally contains an isotonic buffer or its components wherein the isotonic buffer or its components are selected from phosphate-buffered saline (PBS), TRIS-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), standard saline citrate (SSC), HEPES-buffered saline (HBS), Grey's balanced salt solution (GBSS), or normal saline (NaCl), or hypotonic (saline) solutions with addition of glucose or dextrose. 
     
     
         6 . The method of  claim 1 , wherein the solution additionally contains lactic acid selected from free lactic acid (IUPAC systematic name: 2-hydroxypropanoic acid), including its optical isomers L-(+)-lactic acid, (S)-lactic acid, D-(−)-lactic acid or (R)-lactic acid, or its biologically active optical isomer L-(+)-lactic acid, a salt or an anion thereof, selected from sodium-lactate, potassium-lactate, or Al3+-lactate, NH 4 +-lactate, Fe-lactate, Li-lactate, Mg-lactate, Ca-lactate, Mn-lactate or Ag-lactate, or is selected from Ringer's lactate (RiLa), lactated Ringer's solution (main content sodium lactate, also termed “Hartmann's Solution” in the UK), acetated Ringer's solution, or is selected from lactate containing water or ortho-lactate-containing solutions. 
     
     
         7 . The method of  claim 1 , wherein the nucleic acid sequence is selected from DNA, including genomic DNA, single-stranded DNA molecules, double-stranded DNA molecules, coding DNA, DNA primers, DNA probes, immunostimulatory DNA, (short) DNA oligonucleotides ((short) oligodesoxyribonucleotides), or is selected from PNA (peptide nucleic acid) or is selected from RNA, including (short) RNA oligonucleotides ((short) oligoribonucleotides), a coding RNA, a messenger RNA (mRNA), an immunostimulatory RNA, a siRNA, an antisense RNA, a micro RNA or riboswitches, ribozymes aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and/or a viral RNA (vRNA). 
     
     
         8 . The method of  claim 1 , wherein the solution additionally comprises an additive selected from mannite, proteins, peptides, amino acids, alcohols, carbohydrates, metals or metal ions, surfactants, polymers or complexing agents, and/or a buffer. 
     
     
         9 . A lyophilized nucleic acid, wherein the nucleic acid sequence is selected from DNA, including genomic DNA, single-stranded DNA molecules, double-stranded DNA molecules, coding DNA, DNA primers, DNA probes, immunostimulatory DNA, (short) DNA oligonucleotides ((short) oligodesoxyribonucleotides), or is selected from PNA (peptide nucleic acid) or is selected from RNA, including (short) RNA oligonucleotides ((short) oligoribonucleotides), a coding RNA, a messenger RNA (mRNA), an immunostimulatory RNA, a siRNA, an antisense RNA, a micro RNA or riboswitches, ribozymes aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and/or a viral RNA (vRNA), and wherein the sequence is obtained by lyophilizing (freeze-drying) the nucleic acid from a solution according to  claim 1 . 
     
     
         10 . The lyophilized nucleic acid sequence of  claim 9 , wherein the residual water content of the lyophilized nucleic acid sequence is reduced to a content of about 0.5% (w/w) to about 10% (w/w), including a content of about 1% (w/w) to about 5% (w/w), a content of about 2% (w/w) to about 4% (w/w), a content of about 3% (w/w), or a content of about 3% (w/w)±2% (w/w) or 3% (w/w)±1% (w/w). 
     
     
         11 . The lyophilized nucleic acid sequence of  claim 9  or  10 , wherein the relative integrity of the lyophilized nucleic acid sequence has a relative integrity of at least about 70%. 
     
     
         12 . The lyophilized nucleic acid sequence of  claim 9 , wherein the nucleic acid is RNA or an RNA, complexed with a cationic or polycationic compound. 
     
     
         13 . A method of preparing a lyophilized nucleic acid sequence as defined according to  claim 9 , comprising the following steps:
 a) optionally providing a nucleic acid sequence containing solution as defined according to  claim 1 ;   b) freezing the nucleic acid sequence containing solution, obtained according to step a);   c) drying the frozen nucleic acid sequence containing solution, obtained according to step b), via sublimation;   d) optionally floating the lyophilized nucleic acid sequence obtained according to step c) with an inert gas selected from nitrogen, or a noble gas, including helium, neon, argon, xenon, and/or krypton; and   e) optionally sealing the lyophilized nucleic acid sequence obtained according to step c) or d).   
     
     
         14 . The method of  claim 13 , wherein the freezing in step b) occurs at a temperature in the range between −20° C. and −80° C., including a range between −30° C. and −60° C., a range between −40° C. and −50° C., or a temperature of about −47° C. 
     
     
         15 . The method of  claim 13 , wherein the drying in step c) occurs in a primary drying step c1) and a secondary drying step c2), wherein the primary drying step c1) is carried out at a pressure including a pressure in a range of about 980 to about 1045 mbar, or a pressure in a range of about 0.001 mbar to about 0.2 mbar, a range of about 0.01 mbar to about 0.1 mbar, or a range of about 0.025 mbar to about 0.075 mbar, or a pressure of about 0.05 mbar and/or is carried out at a temperature of about −40° C. to about +20° C., and/or wherein the secondary drying step c2) is carried out at a temperature range of about +10° C. to about +40° C., including a range of about +25° C. to about +35° C., or a temperature of about 30° C., and/or a pressure of about 0.001 mbar to about 0.05 mbar, including a range of about 0.001 mbar to about 0.025 mbar, a range of about 0.005 mbar to about 0.015 mbar, or a pressure of about 0.01 mbar. 
     
     
         16 . At least one lyophilized nucleic acid sequence as defined according to  claim 9  for use as a medicament. 
     
     
         17 . A solution as defined according to  claim 1  or at least one lyophilized nucleic acid as defined according to  claim 9  for use in the prophylaxis, treatment and/or amelioration of diseases selected from cancer or tumor diseases, infectious diseases, including viral, bacterial or protozoological infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, including hereditary diseases, or genetic diseases, diseases which have a genetic inherited background and which are typically caused by a single gene defect, cardiovascular diseases or neuronal diseases. 
     
     
         18 . A pharmaceutical composition comprising at least one lyophilized nucleic acid as defined according to  claim 9  and optionally a pharmaceutically acceptable carrier and/or vehicle. 
     
     
         19 . The pharmaceutical composition of  claim 18 , wherein the pharmaceutical composition is a vaccine. 
     
     
         20 . A kit of parts, comprising in one or more parts of the kit at least one lyophilized nucleic acid as defined according to  claim 9 , and optionally in one or more parts of the kit additives as defined according to  claim 8 , and in one or more parts of the kit water, a liquid and/or a buffer or solution as defined according to  claim 1 , and optionally technical instructions with information on the administration and dosage of the lyophilized nucleic acid. 
     
     
         21 . A solution comprising at least one nucleic acid sequence and a free, unconjugated and non-covalently bound mannose for lyophilization, transfection and/or injection of the nucleic acid sequence, wherein the solution enhances the transfection efficiency of the nucleic acid and/or the expression of a protein encoded by the nucleic acid sequence. 
     
     
         22 . The solution of  claim 21 , wherein the mannose concentration of the solution is in the range of about 0.01 to about 10% (w/w), including a concentration of about 0.01 to about 10% (w/w), a concentration of about 0.1 to about 7.5% (w/w), a concentration of about 0.5 to about 5% (w/w), a concentration of about 1 to about 4% (w/w), or a concentration of about 2 to about 4% (w/w), including a concentration of about 2.5% (w/w). 
     
     
         23 . The solution of  claim 21  or  22 , wherein the mannose is selected from α-D-Mannofuranose, β-D-Mannofuranose, α-D-Mannopyranose and/or β-D-Mannopyranose. 
     
     
         24 . The solution of  claim 21 , wherein the solution is present in an osmolarity in the range of about 200 mosmol/l to about 400 mosmol/l. 
     
     
         25 . The solution of  claim 21 , wherein the solution additionally contains an isotonic buffer or its components wherein the isotonic buffer or its components are selected from phosphate-buffered saline (PBS), TRIS-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), standard saline citrate (SSC), HEPES-buffered saline (HBS), Grey's balanced salt solution (GBSS), or normal saline (NaCl), or hypotonic (saline) solutions with addition of glucose or dextrose. 
     
     
         26 . The solution of  claim 21 , wherein the solution additionally contains lactic acid selected from free lactic acid (IUPAC systematic name: 2-hydroxypropanoic acid), including its optical isomers L-(+)-lactic acid, (S)-lactic acid, D-(−)-lactic acid or (R)-lactic acid, or its biologically active optical isomer L-(+)-lactic acid, a salt or an anion thereof, selected from sodium-lactate, potassium-lactate, or Al3+-lactate, NH 4 +-lactate, Fe-lactate, Li-lactate, Mg-lactate, Ca-lactate, Mn-lactate or Ag-lactate, or is selected from Ringer's lactate (RiLa), lactated Ringer's solution (main content sodium lactate, also termed “Hartmann's Solution” in the UK), acetated Ringer's solution, or is selected from lactate containing water or ortho-lactate-containing solutions. 
     
     
         27 . The solution of  claim 21 , wherein the nucleic acid sequence is selected from DNA, including genomic DNA, single-stranded DNA molecules, double-stranded DNA molecules, coding DNA, DNA primers, DNA probes, immunostimulatory DNA, (short) DNA oligonucleotides ((short) oligodesoxyribonucleotides), or is selected from PNA (peptide nucleic acid) or is selected from RNA, including (short) RNA oligonucleotides ((short) oligoribonucleotides), a coding RNA, a messenger RNA (mRNA), an immunostimulatory RNA, a siRNA, an antisense RNA, a micro RNA or riboswitches, ribozymes aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and/or a viral RNA (vRNA). 
     
     
         28 . The solution of  claim 21 , wherein the solution additionally comprises an additive selected from mannite, proteins, peptides, amino acids, alcohols, carbohydrates, metals or metal ions, surfactants, polymers or complexing agents, and/or a buffer.