RECOMBINANT NUCLEIC ACID CONSTRUCT

Abstract

The present invention is related to a recombinant nucleic acid construct comprising in 5′->3′ direction a 5′ UTR, a coding region coding for an effector molecule, and a 3′UTR,
wherein the 5′ UTR is selected from the group comprising a 5′ UTR of a gene or a derivative thereof having a nucleotide identity of at least 85%, wherein the gene is selected from the group consisting of MCP-1, RPL12s.c., Ang-2, HSP70, H3.3., Galectin-9, GADD34, EDN1, HSP70m5, E-selectin, ICAM-1, IL-6 and vWF.

Claims

1. A recombinant nucleic acid construct comprising in 5′->3′ direction a 5′ UTR, a coding region coding for an effector molecule, and a 3′UTR, wherein the 5′ UTR is selected from the group comprising a 5′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for Ang-2 or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for HSP70 or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for H3.3. or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for Galectin-9 or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for GADD34 or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for EDN1 or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for HSP70m5 or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for E-selectin or a derivative thereof having a nucleotide identity of at least 85% a 5′ UTR of a gene coding for ICAM-1 or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for IL-6 or a derivative thereof having a nucleotide identity of at least 85% and a 5′ UTR of a gene coding for vWF or a derivative thereof having a nucleotide identity of at least 85%; wherein 3′ UTR is selected from the group comprising a 3′ UTR of a gene coding for vWF or a derivative thereof having a nucleotide identity of at least 85%, a 3′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%, a 3′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85%, a 3′ UTR of a gene coding for HSP70 or a derivative thereof having a nucleotide identity of at least 85%, a 3′ UTR of a gene coding for H3.3. or a derivative thereof having a nucleotide identity of at least 85%, a 3′ UTR of a gene coding for GADD34 or a derivative thereof having a nucleotide identity of at least 85%, a 3′ UTR of a gene coding for EDN1 or a derivative thereof having a nucleotide identity of at least 85%, and a 3′ UTR of a gene coding for IL-6 or a derivative thereof having a nucleotide identity of at least 85%, wherein the effector molecule is effective in restoring a cellular function of a cell or is effective in exercising a therapeutic effect in or on a cell, and wherein the recombinant nucleic acid construct is different from a wild type mRNA coding for the effector molecule.

2. The recombinant nucleic acid construct of claim 1, wherein a) the 5′ UTR is a 5′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%, and wherein the 3′ UTR is selected from the group comprising a 3′ UTR of a gene coding for vWF or a derivative thereof having a nucleotide identity of at least 85%, a 3′ UTR of a gene coding for HSP70 or a derivative thereof having a nucleotide identity of at least 85%, a 3′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85% and a 3′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%, or b) the 5′ UTR is a 5′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85%, and wherein the 3′ UTR is selected from the group comprising a 3′ UTR of a gene coding for vWF or a derivative thereof having a nucleotide identity of at least 85%, a 3′ UTR of a gene coding for HSP70 or a derivative thereof having a nucleotide identity of at least 85%, a 3′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85%, and a 3′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%, or c) the 5′ UTR is a 5′ UTR of a gene coding for HSP70 or a derivative thereof having a nucleotide identity of at least 85%, and wherein the 3′ UTR is selected from the group comprising a 3′ UTR of a gene coding for HSP70 or a derivative thereof having a nucleotide identity of at least 85%, a 3′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85%, a 3′ UTR of a gene coding for vWF or a derivative thereof having a nucleotide identity of at least 85% and a 3′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%, or d) the 5′ UTR is a 5′ UTR of a gene coding for ANG-2 or a derivative thereof having a nucleotide identity of at least 85%, and wherein the 3′ UTR is selected from the group comprising a 3′ UTR of a gene coding for HSP70 or a derivative thereof having a nucleotide identity of at least 85%, a 3′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85%, a 3′ UTR of a gene coding for vWF or a derivative thereof having a nucleotide identity of at least 85% and a 3′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%.

3. The recombinant nucleic acid construct of claim 1, wherein the a) 3′ UTR is a 3′ UTR of a gene coding for vWF or a derivative thereof having a nucleotide identity of at least 85% and the 5′ UTR is selected from the group comprising a 5′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for HSP70 or a derivative thereof having a nucleotide identity of at least 85% and a 5′ UTR of a gene coding for ANG-2 of a derivative thereof having a nucleotide identity of at least 85%, or b) 3′UTR is a 3′ UTR of a gene coding for HSP70 or a derivative thereof having a nucleotide identity of at least 85% and the 5′ UTR is selected from the group comprising a 5′ UTR of a gene coding for HSP70 or a derivative thereof having a nucleotide identity of at least 85% a 5′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for ANG-2 of a derivative thereof having a nucleotide identity of at least 85%, and a 5′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%, or c) 3′UTR is a 3′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85% and the 5′ UTR is selected from the group comprising a 5′ UTR of a gene coding for HSP70 or a derivative thereof having a nucleotide identity of at least 85% a 5′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for ANG-2 of a derivative thereof having a nucleotide identity of at least 85%, and a 5′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%, or d) 3′UTR is a 3′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85% and the 5′ UTR is selected from the group comprising a 5′ UTR of a gene coding for HSP70 or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85%, a 5′ UTR of a gene coding for ANG-2 of a derivative thereof having a nucleotide identity of at least 85%, and a 5′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%.

4. The recombinant nucleic acid construct of claim 1, wherein the 5′ UTR and the 3′ UTR of the recombinant construct are of different origin, and optionally are from different endogenous genes or species.

5. The recombinant nucleic acid construct of claim 1, wherein the 5′ UTR and the 3′ UTR of the recombinant construct are of the same origin, and optionally are from the same endogenous genes or species.

6. The recombinant nucleic acid construct of claim 1, wherein the construct is one selected from the group comprising a construct, wherein the 5′ UTR is a 5′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%, and the 3′ UTR is a 3′ UTR of a gene coding for vWF or a derivative thereof having a nucleotide identity of at least 85%, a construct, wherein the 5′ UTR is a 5′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85%, and the 3′ UTR is a 3′ UTR of a gene coding for vWF or a derivative thereof having a nucleotide identity of at least 85%, a construct, wherein the 5′ UTR is a 5′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%, and the 3′ UTR is a 3′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%, a construct, wherein the 5′ UTR is a 5′ UTR of a gene coding for ANG-2 or a derivative thereof having a nucleotide identity of at least 85%, and the 3′ UTR is a 3′ UTR of a gene coding for vWF or a derivative thereof having a nucleotide identity of at least 85%, a construct, wherein the 5′ UTR is a 5′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85%, and the 3′ UTR is a 3′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85%, a construct, wherein the 5′ UTR is a 5′ UTR of a gene coding for HSP70 or a derivative thereof having a nucleotide identity of at least 85%, and the 3′ UTR is a 3′ UTR of a gene coding for HSP70 or a derivative thereof having a nucleotide identity of at least 85%, a construct, wherein the 5′ UTR is a 5′ UTR of a gene coding for H3.3. or a derivative thereof having a nucleotide identity of at least 85%, and the 3′ UTR is a 3′ UTR of a gene coding for H3.3. or a derivative thereof having a nucleotide identity of at least 85%, and a construct, wherein the 5′ UTR is a 5′ UTR of a gene coding for Galectin 9 or a derivative thereof having a nucleotide identity of at least 85%, and the 3′ UTR is a 3′ UTR of a gene coding for Galectin 9 or a derivative thereof having a nucleotide identity of at least 85%, wherein optionally the construct is a construct, wherein the 5′ UTR is a 5′ UTR of a gene coding for MCP-1 or a derivative thereof having a nucleotide identity of at least 85%, and the 3′ UTR is a 3′ UTR of a gene coding for vWF or a derivative thereof having a nucleotide identity of at least 85%, or a construct, wherein the 5′ UTR is a 5′ UTR of a gene coding for RPL12s.c. or a derivative thereof having a nucleotide identity of at least 85%, and the 3′ UTR is a 3′ UTR of a gene coding for vWF or a derivative thereof having a nucleotide identity of at least 85%.

7. The recombinant nucleic acid construct of claim 1, wherein the construct comprises a poly-A tail.

8. The recombinant nucleic acid construct of claim 1, wherein the construct comprises a CAP structure.

9. The recombinant nucleic acid construct of claim 1, wherein the construct comprises a IRES (internal ribosomal entry site) sequence.

10. The recombinant nucleic acid construct of claim 1, wherein the construct comprises nucleic acid sequence coding for a signal peptide, wherein the signal peptide is in-frame with nucleic acid sequence coding for a signal peptide and is arranged between the 5′ UTR and the coding region coding for an effector molecule.

11. The recombinant nucleic acid construct of claim 10, wherein the signal peptide allows secretion of the effector molecule.

12. The recombinant nucleic acid construct of claim 10, wherein the nucleotide sequence coding for a signal peptide is selected from the group comprising a nucleotide sequence coding for a signal peptide of MCP-1 or a derivative thereof having a nucleotide identity of at least 85%, nucleotide sequence coding for a signal peptide of IL-6 or a derivative thereof having a nucleotide identity of at least 85%, a nucleotide sequence coding for a signal peptide of Ang-2 or a derivative thereof having a nucleotide identity of at least 85%, and a nucleotide sequence coding for a signal peptide of Ang-1 or a derivative thereof having a nucleotide identity of at least 85%.

13. The recombinant nucleic acid construct of claim 1, wherein the cell a cellular function of which is restored and/or the cell in or on which a therapeutic effect is exercised is an endothelial cell.

14. The recombinant nucleic acid construct of claim 1, wherein the cellular function is one which can be restored by an effector molecule having anti-permeability effect of endothelial cells, an anti-vascular leakage effect, an anti-apoptotic effect of endothelial cells or an anti-inflammatory effect of endothelial cells or an anti-stress response effect, wherein optionally the effect is linked to or associated with the TIE-2 signalling pathway, VEGF-receptor pathway, NOTCH signalling pathway, PI3-kinase pathway, eNOS signalling pathway, sirtuin-dependent metabolic and energy homeostasis pathway, oxidative stress pathway, shear stress response pathway, ET-1 signal transduction pathway, NO-mediated signal transduction pathway, and mechanochemical transduction pathway.

15. A vector comprising a nucleic acid construct of claim 1.

16. A cell comprising a nucleic acid construct of claim 1.

17. A delivery vehicle comprising a nucleic acid construct of claim 1, wherein the delivery vehicle is a cationic lipid delivery particle, wherein optionally the particle is a nanoparticle, and wherein said nanoparticle optionally the average size of the nanoparticle is from about 30 nm to about 200 nm.

18. A pharmaceutical composition comprising a nucleic acid construct of claim 1, and a pharmaceutically acceptable diluent.

19-20. (canceled)

21. A method of treating or preventing a disease in a subject, comprising administering to said subject a recombinant nucleic acid construct according to claim 1.

22. A method of restoring cellular function of a cell comprising delivering to said cell a recombinant nucleic acid construct according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0330] The present invention is further illustrated by the following FIGS., and Examples form which further features, embodiments and advantages of the present invention may be taken.

[0331] FIG. 1 is a schematic representation of conventional mRNA molecule structure.

[0332] FIG. 2 is a schematic representation of selected examples for mRNA constructs with the coding sequence with an open reading frame being the one of Nano-luciferase, namely PAN02, PAN05, PAN07, PAN08, PAN09, PAN10, PAN11, PAN12, 13, PAN28, PAN29, PAN30, PAN31, PAN32, PAN33, PAN34, PAN35, PAN36, PAN37, PAN38, PAN39, PAN40, PAN41, PAN42, PAN43, PAN44, PAN45, PAN46, PAN47, PAN48 and PAN49.

[0333] FIG. 3 is a restriction map of plasmid pcDNA3.1(−) containing construct PAN11.

[0334] FIG. 4 is a restriction digest map of plasmid pdDNA3.1(−) of FIG. 3.

[0335] FIG. 5 is an 1% agarose gel stained with EtBr showing the non-linearized (uncut, supercoiled, right lanes) and linearization product (left lanes) of pcDNA3.1(−) plasmids containing constructs PAN28 (“28”), PAN13 (“13”), PAN12 (“12”), PAN11 (“11”), PAN10 (“10”), PAN09 (“09”), PAN08 (“08”), PAN07 (“07”) and PAN05 (“05”) upon BamHI restriction.

[0336] FIG. 6 shows 1% agarose gels stained with EtBr showing PCR products of Poly-A tailing PCRs for the addition of 120 nt of Poly-A tail.

[0337] FIG. 7A shows the 5′ primers used for the 5′ UTRs of the indicated gene; the first column indicates in connection with which construct such primer is to be used, the second column indicates the origin of the 5′ UTR, and the third column indicates the position where the primer hybridizes to the nucleotide sequence.

[0338] FIG. 7B shows the 3′ primers used for the 3′ UTRs of the indicated gene; the first column indicates in connection with which construct such primer is to be used, the second column indicates the origin of the 3′ UTR, and the third column indicates the position where the primer hybridizes to the nucleotide sequence.

[0339] FIG. 8 is an image of a non-denaturing agarose gel after EtBr staining showing the in vitro mRNA transcripts of various construct.

[0340] FIGS. 9A and 9B are photographs of fluorescence microscopy 24 h post transfection of HeLa cells transfected with 0.5 μg (FIG. 9A) and 0.1 μg (FIG. 9B) of a commercially, enhanced mRNA version of the producing green fluorescent protein (TriLINK Biotechnology).

[0341] FIG. 10 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in medium, by the indicated recombinant nucleic acid constructs in HPMEC where luciferase is used as the effector molecule after 2 hours (left column), 6 hours (middle column) and 24 hours (right column) post transfection.

[0342] FIG. 11 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in whole cell lysates of HPMEC, by the indicated recombinant nucleic acid constructs in HPMEC, where luciferase is used as the effector molecule after, 6 hours (left column) and 24 hours (right column) post transfection.

[0343] FIG. 12 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in medium, by the indicated recombinant nucleic acid constructs in HUVEC where luciferase is used as the effector molecule after 2 hours (left column), 6 hours (middle column) and 24 hours (right column) post transfection.

[0344] FIG. 13 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units)] of luciferase in whole cell lysates of HUVEC, by the indicated recombinant nucleic acid constructs after lysis of HUVEC, where luciferase is used as the effector molecule after 2 hours (left column), 6 hours (middle column) and 24 hours (right column) post transfection.

[0345] FIG. 14 is a bar diagram showing expression of luciferase, indicated as RLU [(relative light units) of luciferase in medium, by the indicated recombinant nucleic acid constructs in HeLa cells where luciferase is used as the effector molecule after 2 hours (left column), 6 hours (middle column) and 24 hours (right column) post transfection.

[0346] FIG. 15A shows the 5′ UTR nucleotide sequence of the mRNA of human MCP-1 which is also referred to as Homo sapiens C—C motif chemokine ligand 2 (CCL2) (GenBank entry NM_002982.3).

[0347] FIG. 15B shows both the nucleotide sequence and the amino acid sequence of the signal peptide sequence of human MCP-1.

[0348] FIG. 15C shows the 3′ UTR nucleotide sequence of the mRNA of human MCP-1.

[0349] FIG. 16A shows the 5′ UTR nucleotide sequence of the mRNA of the 50S ribosomal protein L12, chloroplastic (LOCI 10782793), of Spinacia oleracea which is also referred to as RPL12s.c. (GenBank entry XM_021987044.1).

[0350] FIG. 16B shows the 3′ UTR from the mRNA of the 50S ribosomal protein L12, chloroplastic (LOCI 10782793), of Spinacia oleracea.

[0351] FIG. 17A shows the 5′ UTR nucleotide sequence of the mRNA of human angiopoietin 2 which is also referred to as ANGPT2 or Ang-2, transcript variant 1 (GenBank entry NM_001147.2).

[0352] FIG. 17B shows both the nucleotide sequence and the amino acid sequence of the signal sequence of the mRNA of human Ang-2.

[0353] FIG. 17C shows the 3′ UTR nucleotide sequence of the mRNA of human Ang-2.

[0354] FIG. 18A shows the 5′ UTR nucleotide sequence of the mRNA of human interleukin 6 (IL-6), transcript variant 1 (GenBank entry NM_000600.4).

[0355] FIG. 18B shows both the nucleotide sequence and the amino acid sequence of the signal sequence of the mRNA of human IL-6.

[0356] FIG. 18C shows the 3′ UTR nucleotide sequence of the mRNA of human IL-6.

[0357] FIG. 19A shows the 5′ UTR nucleotide sequence of the mRNA of human von Willebrand factor (vWF) (GenBank entry NM_000552.4).

[0358] FIG. 19B shows both the nucleotide sequence and the amino acid sequence of the signal sequence of the mRNA of von Willebrand factor (vWF).

[0359] FIG. 19C shows the 3′ UTR nucleotide sequence of the mRNA of von Willebrand factor (vWF).

[0360] FIG. 20A shows the 5′ UTR nucleotide sequence of the mRNA of human heat shock protein family A (Hsp70) member 1A, also referred to as HSPA1A (GenBank entry NM_005345.5).

[0361] FIG. 20B shows the 3′ UTR nucleotide sequence of the mRNA of human HSPA1A.

[0362] FIG. 21A shows the 5′ UTR nucleotide sequence of the mRNA of human heat shock protein family A (Hsp70) member 5, also referred to as HSPA5, (GenBank entry NM_005347.4).

[0363] FIG. 21B shows both the nucleotide sequence and the amino acid sequence of the signal sequence of the mRNA of human HSPA5.

[0364] FIG. 21B shows both the nucleotide sequence and the amino acid sequence of the signal sequence of the mRNA of human HSPA5.

[0365] FIG. 21C shows the 3′ UTR nucleotide sequence of the mRNA of human HSPA1A.

[0366] FIG. 22A shows the 5′ UTR nucleotide sequence of the mRNA of human H3 histone family member 3A (H3F3A), also referred to as H3.3, (GenBank entry NM_002107.4).

[0367] FIG. 22B shows the 3′ UTR nucleotide sequence of the mRNA of human H3.3.

[0368] FIG. 23A shows the 5′ UTR nucleotide sequence of the mRNA of human galectin-9 (LGALS9), transcript variant 1 (GenBank entry NM_009587.2).

[0369] FIG. 23B shows the 3′ UTR nucleotide sequence of the mRNA of human galectin-9.

[0370] FIG. 24A shows the basic structure and FIG. 24B shows the nucleotide sequence of recombinant nucleic acid construct PAN01.

[0371] FIG. 25A shows the basic structure and FIG. 25B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN03.

[0372] FIG. 26A shows the basic structure and FIG. 26B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN04.

[0373] FIG. 27A shows the basic structure and FIG. 27B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN06.

[0374] FIG. 28A shows the basic structure and FIG. 28B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN36.

[0375] FIG. 29A shows the basic structure and FIG. 29B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN37.

[0376] FIG. 30A shows the basic structure and FIG. 30B shows nucleotide sequence of recombinant nucleic acid construct PAN02.

[0377] FIG. 31A shows the basic structure and FIG. 31B shows nucleotide sequence of recombinant nucleic acid construct PAN05.

[0378] FIG. 32A shows the basic structure and FIG. 32B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN07.

[0379] FIG. 33A shows the basic structure and FIG. 33B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN08.

[0380] FIG. 34A shows the basic structure and FIG. 34B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN09.

[0381] FIG. 35A shows the basic structure and FIG. 35B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN10.

[0382] FIG. 36A shows the basic structure and FIG. 36B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN11.

[0383] FIG. 37A shows the basic structure and FIG. 37B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN12.

[0384] FIG. 38A shows the basic structure and FIG. 38B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN13.

[0385] FIG. 39A shows the basic structure and FIG. 39B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN28.

[0386] FIG. 40A shows the basic structure and FIG. 40B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN29.

[0387] FIG. 41A shows the basic structure and FIG. 41B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN30.

[0388] FIG. 42A shows the basic structure and FIG. 42B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN31.

[0389] FIG. 43A shows the basic structure and FIG. 43B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN32.

[0390] FIG. 44A shows the basic structure and FIG. 44B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN33.

[0391] FIG. 45A shows the basic structure and FIG. 45B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN34.

[0392] FIG. 46A shows the basic structure and FIG. 46B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN35.

[0393] FIG. 47A shows the basic structure and FIG. 47B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN38.

[0394] FIG. 48A shows the basic structure and FIG. 48B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN39.

[0395] FIG. 49A shows the basic structure and FIG. 49B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN40.

[0396] FIG. 50A shows the basic structure and FIG. 50B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN41.

[0397] FIG. 51A shows the basic structure and FIG. 51B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN42.

[0398] FIG. 52A shows the basic structure and FIG. 52B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN43.

[0399] FIG. 53A shows the basic structure and FIG. 53B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN44.

[0400] FIG. 54A shows the basic structure and FIG. 54B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN45.

[0401] FIG. 55A shows the basic structure and FIG. 55B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN46.

[0402] FIG. 56A shows the basic structure and FIG. 57B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN47.

[0403] FIG. 57A shows the basic structure and FIG. 58B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN48.

[0404] FIG. 58 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN49.

[0405] FIG. 59A shows the nucleotide sequence of mRNA of human angiopoietin 1, also referred to as ANGPT1 or Ang-1, transcript variant 1, GenBank entry NM_001146.4.

[0406] FIG. 59B shows both the nucleotide sequence and the amino acid sequence of the mRNA of human Ang-1.

[0407] FIG. 59C shows the coding sequence (CDS) of the mature peptide of mRNA of human Ang-1.

[0408] FIG. 59D shows the 3′ UTR nucleotide sequence of the mRNA of human Ang-1.

[0409] FIG. 60A shows the nucleotide sequence of mRNA of human angiopoietin 4, also referred to as ANGPT4 or Ang-4, transcript variant 1, GenBank entry NM_015985.3.

[0410] FIG. 60B shows both the nucleotide sequence and the amino acid sequence of the mRNA of human Ang-4.

[0411] FIG. 60C shows the coding sequence (CDS) of the mature peptide of mRNA of human Ang-4.

[0412] FIG. 60D shows the 3′ UTR nucleotide sequence of the mRNA of human Ang-4.

[0413] FIG. 61 shows the nucleotide sequence coding for COMP-Ang1 which is a synthetic construct.

[0414] FIG. 62 shows the nucleotide sequence coding for hCOMP-Ang1 which is a synthetic construct.

[0415] FIG. 63 shows the nucleotide sequence coding for CMP-Ang1 which is a synthetic construct.

[0416] FIG. 64 shows the nucleotide sequence coding for COMP-Ang2 which is a synthetic construct.

[0417] FIG. 65 shows the coding nucleotide sequence of mRNA of human TEK receptor tyrosine kinase (TEK), transcript variant 1, also referred to as Tie-2, GenBank entry.

[0418] FIG. 66 shows the coding nucleotide sequence of mRNA of human mutant TEK receptor tyrosine kinase (TEK), which is also referred to as TIE* having a R849W mutation.

[0419] FIG. 67A shows the basic structure and FIG. 67B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN50.

[0420] FIG. 68A shows the basic structure and FIG. 68B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN51.

[0421] FIG. 69A shows the basic structure and FIG. 69B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN52.

[0422] FIG. 70A shows the basic structure and FIG. 70B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN53.

[0423] FIG. 71A is an image of a non-denaturing agarose gel after EtBr staining showing the tail-PCR products of various construct.

[0424] FIG. 71B is an image of a non-denaturing agarose gel after EtBr staining showing the in vitro mRNA transcripts of various construct.

[0425] FIG. 72 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in medium, by the indicated recombinant nucleic acid constructs in HPMEC where luciferase is used as the effector molecule after 2 hours (left column), 6 hours (middle column) and 24 hours (right column) post transfection.

[0426] FIG. 73 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in whole cell lysates of HPMEC, by the indicated recombinant nucleic acid constructs in HPMEC, where luciferase is used as the effector molecule after 6 hours (left column) and 24 hours (right column) post transfection.

[0427] FIG. 74A (left) is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in medium, by the indicated recombinant nucleic acid constructs in HPMEC (transfected with GFP mRNA, PAN12 mRNA and PAN12* mRNA) where luciferase is used as the effector molecule after 2 hours (left column), 6 hours (middle column) and 24 hours (right column) post transfection. In PAN12* 100% of uridine and 100% cytidine nucleotides are replaced by pseudo-uridine and 5-methyl-cytidine, respectively.

[0428] FIG. 74B (right) is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in cell lysates, by the indicated recombinant nucleic acid constructs in HPMEC (transfected with GFP mRNA, PAN12 mRNA and PAN12* mRNA) where luciferase is used as the effector molecule after 6 hours (left column) and 24 hours (right column) post transfection. In PAN12* 100% of uridine and 100% cytidine nucleotides are replaced by pseudo-uridine and 5-methyl-cytidine, respectively.

[0429] FIG. 75 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in medium, by the indicated recombinant nucleic acid constructs in HPMEC where luciferase is used as the effector molecule after 1 hour, 2 hours, 4 hours and 24 hours post transfection (from left to right, with 1 hour results being shown to the utmost left side and 24 hours result being shown to the utmost right side).

[0430] FIG. 76 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in whole cell lysates of HPMEC, by the indicated recombinant nucleic acid constructs in HPMEC, where luciferase is used as the effector molecule after 1 hour, 2 hours, 4 hours and 24 hours post transfection (from left to right, with 1 hour results being shown to the utmost left side and 24 hours result being shown to the utmost right side).

[0431] FIG. 11A (left) is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in medium, by the indicated recombinant nucleic acid constructs in HPMEC (transfected with GFP mRNA, PAN12 mRNA and PAN12* mRNA) where luciferase is used as the effector molecule after 1 hour, 2 hours, 4 hours and 24 hours post transfection (from left to right, with 1 hour results being shown to the utmost left side and 24 hours result being shown to the utmost right side). In PAN12* 100% of uridine and 100% cytidine nucleotides are replaced by pseudo-uridine and 5-methyl-cytidine, respectively.

[0432] FIG. 77B (right) is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in cell lysates, by the indicated recombinant nucleic acid constructs in HPMEC (transfected with GFP mRNA, PAN12 mRNA and PAN12* mRNA) where luciferase is used as the effector molecule after 1 hour, 2 hours, 4 hours and 24 hours post transfection (from left to right, with 1 hour results being shown to the utmost left side and 24 hours result being shown to the utmost right side). In PAN12* 100% of uridine and 100% cytidine nucleotides are replaced by pseudo-uridine and 5-methyl-cytidine, respectively.

[0433] FIG. 78 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in medium, by the indicated recombinant nucleic acid constructs in HPAEC where luciferase is used as the effector molecule after 1 hour, 2 hours, 4 hours and 24 hours post transfection (from left to right, with 1 hour results being shown to the utmost left side and 24 hours result being shown to the utmost right side).

[0434] FIG. 79 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in whole cell lysates of HPAEC, by the indicated recombinant nucleic acid constructs in HPAEC, where luciferase is used as the effector molecule after 1 hour, 2 hours, 4 hours and 24 hours post transfection (from left to right, with 1 hour results being shown to the utmost left side and 24 hours result being shown to the utmost right side).

[0435] FIG. 80 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in medium, by the indicated recombinant nucleic acid constructs in HeLa cells where luciferase is used as the effector molecule after 1 hour, 2 hours, 4 hours and 24 hours post transfection (from left to right, with 1 hour results being shown to the utmost left side and 24 hours result being shown to the utmost right side).

[0436] FIG. 81 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in whole cell lysates of HeLa cells, by the indicated recombinant nucleic acid constructs in HeLA cells, where luciferase is used as the effector molecule after 1 hour, 2 hours, 4 hours and 24 hours post transfection (from left to right, with 1 hour results being shown to the utmost left side and 24 hours result being shown to the utmost right side).

[0437] FIG. 82 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in medium, by the indicated recombinant nucleic acid constructs in HPMEC where luciferase is used as the effector molecule after 2 hours, 4 hours, 6 hours and 24 hours post transfection (from left to right, with 1 hour results being shown to the utmost left side and 24 hours result being shown to the utmost right side). In constructs PAN12*, PAN28*, PAN29*, PAN34*, PAN50* and PAN51*100% of uridine and 100% cytidine nucleotides are replaced by pseudo-uridine and 5-methyl-cytidine, respectively.

[0438] FIG. 83 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in whole cell lysates of HPMEC, by the indicated recombinant nucleic acid constructs in HPMEC, where luciferase is used as the effector molecule after 2 hours, 4 hours, 6 hours and 24 hours post transfection (from left to right, with 1 hour results being shown to the utmost left side and 24 hours result being shown to the utmost right side). In constructs PAN12*, PAN28*, PAN29*, PAN34*, PAN50* and PAN51*100% of uridine and 100% cytidine nucleotides are replaced by pseudo-uridine and 5-methyl-cytidine, respectively.

[0439] FIG. 84 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in medium, by the indicated recombinant nucleic acid constructs in HPMEC where luciferase is used as the effector molecule after 2 hours (left column), 4 hours (middle column) and 6 hours (right column) post transfection. In construct PAN02*, 100% of uridine and 100% cytidine nucleotides are replaced by pseudo-uridine and 5-methyl-cytidine, respectively; and in construct PAN51 w/o CAP the mRNA sequence is the one of construct PAN51, but without any 5′-capping structure.

[0440] FIG. 85 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in whole cell lysates of HPMEC, by the indicated recombinant nucleic acid constructs in HPMEC, where luciferase is used as the effector molecule after 2 hours (left column) 4 hours (middle column) and 6 hours (right column) post transfection. In construct PAN02*, 100% of uridine and 100% cytidine nucleotides are replaced by pseudo-uridine and 5-methyl-cytidine, respectively; and in construct PAN51 w/o CAP the mRNA sequence is the one of construct PAN51, but without any 5′-capping structure.

[0441] FIG. 86A shows the basic structure and FIG. 86B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN54.

[0442] FIG. 87A shows the basic structure and FIG. 87B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN55.

[0443] FIG. 88A shows the basic structure and FIG. 88B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN56.

[0444] FIG. 89A shows the basic structure and FIG. 89B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN57.

[0445] FIG. 90A shows the basic structure and FIG. 90B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN58.

[0446] FIG. 91A shows the basic structure and FIG. 91B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN59.

[0447] FIG. 92 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in medium, by the indicated recombinant nucleic acid constructs in HPMEC, where luciferase is used as the effector molecule after 4 hours (left column). Additional 4-hours luciferase activity values are assessed 24 hours (middle column) and 48 hours (right column) post transfection, after washing of the cells and supplementing the cells with fresh medium.

[0448] FIG. 93 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in medium, by the indicated recombinant nucleic acid constructs in HEK293 cells, where luciferase is used as the effector molecule after 3 hours (left column). An additional 3-hours luciferase activity value is assessed 24 hours (right column) post transfection, after washing of the cells and supplementing the cells with fresh medium. The human embryonic kidney 293 cell line (HEK293) is grown in EMEM (EBSS)+2 mM Glutamine+1% Non Essential Amino Acids (NEAA)+10% FCS culture medium. Subculture Routine is to split sub-confluent cultures (70-80%) 1:2 to 1:6 i.e. seeding at 2-5×10,000 cells/cm.sup.2 using 0.25% trypsin or trypsin/EDTA; 5% CO.sub.2; 37° C. mRNA transfection are performed with the Lipofectamine™ MessengerMAX™ Transfection Reagent (Invitrogen™) according to the manufactures protocol.

[0449] FIG. 94 is a bar diagram showing expression of luciferase, indicated as RLU (relative light units) of luciferase in medium, by the indicated recombinant nucleic acid constructs in HeLa cells, where luciferase is used as the effector molecule after 3 hours (left column). An additional 3-hours luciferase activity value is assessed 24 hours (right column) post transfection, after washing of the cells and supplementing the cells with fresh medium.

[0450] FIG. 95A shows the basic structure and FIG. 95B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN60.

[0451] FIG. 96A shows the basic structure and FIG. 96B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN61.

[0452] FIG. 97A shows the basic structure and FIG. 97B shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN66.

[0453] FIG. 98 shows Western Blot analysis of protein lysates from HPMEC cells transfected with indicated PAN mRNA constructs. Cells were transfected with 1 μg the mRNAs and harvested after 6 hours. Total cell lysates were separated by SDS-PAGE and analyzed by immunoblot using anti-Ang1 antibody. M, Marker; UT, untreated cells, rec. Ang1, recombinant hAng1.

[0454] FIG. 99 shows Agarose gels of mRNAs after in vitro transcription. 2 μg of indicated mRNAs were separated by agarose gel electrophoresis and visualized by ethidium bromide staining and UV illumination.

[0455] FIG. 100 shows the sequences of: a) the 5′UTR of PAN57; b) the 3′UTR of PAN57; c) the 5′PCR-Primer for PAN57; d) the 3′PCR-Primer for PAN57 and e) the 5′PCR-Primer for PAN55, PAN56.

DETAILED DESCRIPTION

[0456] FIG. 1 is a schematic representation of conventional mRNA molecule structure. Eukaryotic including mammalian mRNAs consist of a cap region, a 5′ untranslated region (UTR), the coding sequence (CDS) with an open reading frame (ORF) starting with a consensus Kozak sequence for optimal translation initiation and in case of secreted proteins followed by an signal peptide leader sequences, a 3′ UTR, and a poly A-tail (>120 nt) at the 3′ end.

[0457] FIG. 2 is a schematic representation of selected examples for mRNA constructs with the coding sequence with an open reading frame being the one of Nano-luciferase. It is within the present invention that for each and any of the indicated mRNA constructs the coding region comprising a sequence with an open reading frame for Nano-luciferase may be replaced by a coding regions comprising a sequence coding for an effector molecule, particularly by a coding region comprising a sequence coding for an effector molecule disclosed herein; in a preferred embodiment thereof, the effector molecule is Ang1 or COMP-Ang1. In accordance with the present invention, the recombinant nucleic acid constructs are designed as follows:

[0458] Recombinant nucleic acid construct PAN02 comprises as the 5′ non-translated region the 5′ UTR of Ang1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of Ang1, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of Ang1.

[0459] Recombinant nucleic acid construct PAN05 comprises as the 5′ non-translated region the 5′ UTR of Ang2, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of Ang2, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0460] Recombinant nucleic acid construct PAN07 comprises as the 5′ non-translated region the 5′ UTR of Galectin-9, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of Ang2, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0461] Recombinant nucleic acid construct PAN08 comprises as the 5′ non-translated region the 5′ UTR of Hsp70, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of Ang2, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of HSP70.

[0462] Recombinant nucleic acid construct PAN09 comprises as the 5′ non-translated region the 5′ UTR of H3.3, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of Ang2, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of H3.3.

[0463] Recombinant nucleic acid construct PAN10 comprises as the 5′ non-translated region the 5′ UTR of RPL12s.c., as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of Ang2, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of RPL12s.c. This construct additionally comprises another start codon preceding the start codon of the coding region for Nano-luciferase in-frame.

[0464] Recombinant nucleic acid construct PAN11 comprises as the 5′ non-translated region the 5′ UTR of GADD34, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of Ang2, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of GADD34.

[0465] Recombinant nucleic acid construct PAN12 comprises as the 5′ non-translated region the 5′ UTR of MCP-1 as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of MCP-1.

[0466] Recombinant nucleic acid construct PAN13 comprises as the 5′ non-translated region the 5′ UTR of EDN1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of EDN1, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of EDN1.

[0467] Recombinant nucleic acid construct PAN28 comprises as the 5′ non-translated region the 5′ UTR of Ang2, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of IL-6, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of vWF.

[0468] Recombinant nucleic acid construct PAN29 comprises as the 5′ non-translated region the 5′ UTR of MCP-1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0469] Recombinant nucleic acid construct PAN30 comprises as the 5′ non-translated region the 5′ UTR of MCP-1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of HSP70.

[0470] Recombinant nucleic acid construct PAN31 comprises as the 5′ non-translated region the 5′ UTR of MCP-1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of IL-6, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of MCP-1.

[0471] Recombinant nucleic acid construct PAN32 comprises as the 5′ non-translated region the 5′ UTR of MCP-1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of Ang2, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of MCP-1.

[0472] Recombinant nucleic acid construct PAN33 comprises as the 5′ non-translated region the 5′ UTR of MCP-1, no nucleic acid sequence coding for a signal peptide, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of MCP-1.

[0473] Recombinant nucleic acid construct PAN34 comprises as the 5′ non-translated region the 5′ UTR of Hsp70, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of MCP-1.

[0474] Recombinant nucleic acid construct PAN35 comprises as the 5′ non-translated region the 5′ UTR of RPL12 s.c. (spinach chloroplast), as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of MCP-1.

[0475] Recombinant nucleic acid construct PAN36 comprises as the 5′ non-translated region the 5′ UTR of MCP-1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Ang1, and as the 3′ non-translated region the 3′ UTR of MCP-1.

[0476] Recombinant nucleic acid construct PAN37 comprises as the 5′ non-translated region the 5′ UTR of MCP-1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for hCOMP-Ang1, and as the 3′ non-translated region the 3′ UTR of MCP-1.

[0477] Recombinant nucleic acid construct PAN38 comprises as the 5′ non-translated region the 5′ UTR of Ang2, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0478] Recombinant nucleic acid construct PAN39 comprises as the 5′ non-translated region the 5′ UTR of Ang2* (Ang2 5′-UTR with deleted upstream ATGs), as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of IL-6, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0479] Recombinant nucleic acid construct PAN40 comprises as the 5′ non-translated region the 5′ UTR of Ang2, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of Gaussia luciferase, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0480] Recombinant nucleic acid construct PAN41 comprises as the 5′ non-translated region the 5′ UTR of RPL12 s.c., as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1 with an additional upstream ATG for translational start, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of RPL12 s.c. This construct additionally comprises another start codon preceding the start codon of the coding region for Nano-luciferase in-frame.

[0481] Recombinant nucleic acid construct PAN42 comprises as the 5′ non-translated region the 5′ UTR of RPL12 s.c., as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of RPL12 s.c.

[0482] Recombinant nucleic acid construct PAN43 comprises as the 5′ non-translated region the 5′ UTR of RPL12 s.c., as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of Ang2, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of RPL12 s.c.

[0483] Recombinant nucleic acid construct PAN44 comprises as the 5′ non-translated region the 5′ UTR of Hsp70, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of Hsp70.

[0484] Recombinant nucleic acid construct PAN45 comprises as the 5′ non-translated region the 5′ UTR of Hsp70m5, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0485] Recombinant nucleic acid construct PAN46 comprises as the 5′ non-translated region the 5′ UTR of E-selectin, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of IL-6, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0486] Recombinant nucleic acid construct PAN47 comprises as the 5′ non-translated region the 5′ UTR of ICAM1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of IL-6, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0487] Recombinant nucleic acid construct PAN48 comprises as the 5′ non-translated region the 5′ UTR of IL-6, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of IL-6, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of IL-6).

[0488] Recombinant nucleic acid construct PAN49 comprises as the 5′ non-translated region the 5′ UTR of von Willebrand Factor (vWF), as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of von Willebrand Factor (vWF), as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0489] FIG. 3 shows a restriction map of a plasmid pcDNA3.1(−) used as an illustrative example for a plasmid for the expression of an exemplary recombinant nucleic acid construct of the present invention, whereby such exemplary recombinant nucleic acid construct is construct PAN 11; restriction sites XhoI/HindIII are shown.

[0490] FIG. 4 shows a restriction digestion map of the PAN11 expressing pcDNA3.1− plasmid of FIG. 3; the insert represents PAN11 sequence with engineered 5′ and 3′ UTR, signal sequence and the coding region coding for Nano-Luciferase.

[0491] FIG. 5 is an 1% agarose gel stained with EtBr showing the non-linearized (uncut, supercoiled, right lanes) and linearization product (left lanes) of pcDNA3.1(−) plasmids upon BamHI restriction of the constructs PAN28 (“28”), PAN13 (“13”), PAN12 (“12”), PAN11 (“11”), PAN10 (“10”), PAN09 (“09”), PAN08 (“08”), PAN07 (“07”) and PAN05 (“05”).

[0492] FIG. 6 shows 1% agarose gels stained with EtBr showing PCR products of Poly-A tailing PCRs for the addition of 120 nt of Poly-A tail by 120 nt long poly-T 3′ primer flanking the different recombinant nucleic acid constructs. On top are the optimized PCR conditions for the indicated recombinant nucleic acid constructs. For example, for Pan02 the conditions are as follows: denaturation for 2 minutes at 94° C. 33 cycles of 30 seconds at 96° C., 15 seconds at 55° C. and 4 minutes at 72° C. Final extension for 8 minutes at 72° C.

[0493] FIG. 8 is an image of a 1% non-denaturing agarose gel stained with EtBr showing the in vitro transcribed mRNAs of constructs PAN28 (“28”), PAN13 (“13”), PAN12 (“12”), PAN11 (“11”), PAN10 (“10”), PAN09 (“09”), PAN08 (“08”), PAN07 (“07”) and PAN05 (“05”). M: is a lane with a high range RiboRuler RNA ladder. From this FIG. 8 may be taken that the transcribed mRNAs were intact and, more specifically, were not degraded.

[0494] FIG. 24 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN01. PAN1 is an embodiment of the present invention comprising as the 5′ non-translated region the 5′ UTR of Ang1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of Ang1, as the coding region coding for an effector molecule the coding region coding for Ang1, and as the 3′ non-translated region the 3′ UTR of Ang1.

[0495] FIG. 25 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN03. PAN03 is an embodiment of the present invention comprising as the 5′ non-translated region the 5′ UTR of Ang1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of Ang1, as the coding region coding for an effector molecule the coding region coding for COMP-Ang1, and as the 3′ non-translated region the 3′ UTR of Ang1.

[0496] FIG. 26 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN04. PAN04 is an embodiment of the present invention comprising as the 5′ non-translated region the 5′ UTR of Ang2, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of Ang2, as the coding region coding for an effector molecule the coding region coding for Ang1, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0497] FIG. 27 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN06. PAN06 is an embodiment of the present invention comprising as the 5′ non-translated region the 5′ UTR of Ang2, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of Ang2, as the coding region coding for an effector molecule the coding region coding for COMP-Ang1, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0498] FIG. 28 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN36. PAN36 is an embodiment of the present invention comprising as the 5′ non-translated region the 5′ UTR of MCP-1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Ang1, and as the 3′ non-translated region the 3′ UTR of MCP-1.

[0499] FIG. 67 shows recombinant nucleic acid construct PAN50 which is another embodiment of an mRNA of the present invention comprising as the 5′ non-translated region the 5′ UTR of MCP-1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of IL-6, as the coding region coding for an effector molecule the coding region coding for Nano luciferase, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0500] FIG. 68 shows recombinant nucleic acid construct PAN51 which is another embodiment of an mRNA of the present invention comprising as the 5′ non-translated region the 5′ UTR of HSP70, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of IL-6, as the coding region coding for an effector molecule the coding region coding for Nano luciferase (NLuc), and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0501] FIG. 69 shows recombinant nucleic acid construct PAN52 which is another embodiment of an mRNA of the present invention comprising as the 5′ non-translated region the 5′ UTR of MCP-1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Ang1, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0502] FIG. 70 shows recombinant nucleic acid construct PAN53 which is another embodiment of an mRNA of the present invention comprising as the 5′ non-translated region the 5′ UTR of MCP-1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for hCOMP-Ang1, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0503] FIG. 71A is an image of a 1% non-denaturing agarose gel stained with EtBr showing the tail-PCR products of constructs PAN35 (“35”), PA34 (“34”), PAN33 (“33”), PAN32 (“32”), PAN31 (“31”), PAN30 (“30”), PAN29 (“29”) and PAN12 (“12”). M: is a lane with a high range RiboRuler RNA ladder. From this FIG. 71A may be taken that the transcribed mRNAs were intact and, more specifically, were not degraded.

[0504] FIG. 71B is an image of a 1% non-denaturing agarose gel stained with EtBr showing the in vitro mRNA transcripts of constructs PAN35 (“35”), PA34 (“34”), PAN33 (“33”), PAN32 (“32”), PAN31 (“31”), PAN30 (“30”), PAN29 (“29”) and PAN12 (“12”). M: is a lane with a high range RiboRuler RNA ladder. From this FIG. 71B may be taken that the transcribed mRNAs were intact and, more specifically, were not degraded.

[0505] FIG. 86 shows recombinant nucleic acid construct PAN54 which is another embodiment of an mRNA of the present invention comprising as the 5′ non-translated region the 5′ UTR of RPL12s.c. as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Nano-Luciferase (NLuc), and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0506] FIG. 87 shows recombinant nucleic acid construct PAN55 which is another embodiment of an mRNA of the present invention comprising as the 5′ non-translated region the 5′ UTR of vWF, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Nano Luciferase (NLuc), and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0507] FIG. 88 shows recombinant nucleic acid construct PAN56 which is another embodiment of an mRNA of the present invention comprising as the 5′ non-translated region the 5′ UTR of vWF, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of vWF, as the coding region coding for an effector molecule the coding region coding for Nano Luciferase (NLuc), and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0508] FIG. 89 shows recombinant nucleic acid construct PAN57 which is another embodiment of an mRNA of the present invention comprising as the 5′ non-translated region a synthetic nucleic acid sequence as described in Jiang, Lei et al. (2018). Systemic messenger RNA as an etiological treatment for acute intermittent porphyria. Nature Medicine. 24, 1899-2909 (2018), as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Nano Luciferase (NLuc), and as the 3′ non-translated region the 3′ UTR a nucleic acid sequence as described in Jiang, Lei et al. (2018). Systemic messenger RNA as an etiological treatment for acute intermittent porphyria. Nature Medicine. 24; 1899-1909 (2018). The 5′- and 3′ sequences are shown underlined and in bold.

[0509] FIG. 90 shows recombinant nucleic acid construct PAN58 which is another embodiment of an mRNA of the present invention comprising as the 5′ non-translated region the 5′ UTR of MCP-1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for Nano Luciferase (NLuc), and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF). In addition, PAN58 comprises a consensus Kozak sequence (GCCACC) in the six bases upstream of the start AUG.

[0510] FIG. 91 shows recombinant nucleic acid construct PAN59 which is another embodiment of an mRNA of the present invention comprising as the 5′ non-translated region the 5′ UTR of MCP-1, as the nucleic acid sequence coding for a signal peptide the nucleic acid sequence coding for a signal peptide of MCP-1, as the coding region coding for an effector molecule the coding region coding for CMP-Ang1, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0511] FIG. 95 shows recombinant nucleic acid construct PAN60 which is another embodiment of an mRNA of the present invention comprising as the 5′ non-translated region the 5′ UTR of MCP-1, as the coding region coding for an effector molecule the coding region coding for wild type (wt) Tie2, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0512] FIG. 96 shows recombinant nucleic acid construct PAN61 which is another embodiment of an mRNA of the present invention comprising as the 5′ non-translated region the 5′ UTR of MCP-1, as the coding region coding for an effector molecule the coding region coding for Tie2 mutant R849W, and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0513] FIG. 97 shows recombinant nucleic acid construct PAN66 which is another embodiment of an mRNA of the present invention comprising as the 5′ non-translated region the 5′ UTR of MCP-1, as the coding region coding for an effector molecule the coding region coding for wild type PIK3CA which is, according to GenBank Homo sapiens phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) with the respective reference number being 006218.4., and as the 3′ non-translated region the 3′ UTR of von Willebrand Factor (vWF).

[0514] The SEQ ID NOs: of the sequence listing are related to the instant disclosure as summarized in Table 1.

TABLE-US-00001 TABLE 1 Seq ID Subject to No: FIG. Further information 1   7A 5′Primer 2   7A 5′Primer 3   7A 5′Primer 4   7A 5′Primer 5   7A 5′Primer 6   7A 5′Primer 7   7A 5′Primer 8   7A 5′Primer 9   7A 5′Primer 10   7A 5′Primer 11   7B 3′Primer 12   7B 3′Primer 13   7B 3′Primer 14   7B 3′Primer 15   7B 3′Primer 16   7B 3′Primer 17   7B 3′Primer 18   7B 3′Primer 19   7B 3′Primer 20   15A 5′UTR MCP1 21   15B SP (signal peptide) nucleotide sequence of MCP1 22   15B SP amino acid sequence of MCP1 23   15C 3′UTR of MCP1 24   16A 5′UTR of RPL12 25   16B 3′UTR of RPL12 26   17A 5′UTR of Ang2 27   17B SP (signal peptide) nucleotide sequence of Ang2 28   17B SP (signal peptide) amino acid sequence of Ang2 29   17C 3′UTR of Ang2 30   18A 5′UTR of IL6 31   18B SP (signal peptide) nucleotide sequence of IL6 32   18B SP (signal peptide) amino acid sequence of IL6 33   18C 3′UTR of IL6 34   19A 5′UTR of vWF 35   19B SP(signal peptide) nucleotide sequence of vWF 36   19B SP (signal peptide) amino aicd sequence of vWF 37   19C 3′UTR of vWF 38   20A 5′UTR of HSP70 A1 39   20B 3′UTR of HSP70 A1 40   21A 5′UTR of HSP70 A5 41   21B SP (signal peptide) nucleotide sequence of HSP70 A5 42   21B SP (signal peptide) amino acid sequence of HSP70 A5 43   21C 3′UTR of HSP70 A5 44   22A 5′UTR of H3.3 45   22B 3′UTR of H3.3 46   23A 5′UTR of LGALS9 47   23B 3′UTR of LGALS9 48 24 Construct PAN01 49 25 Construct PAN03 50 26 Construct PAN04 51 27 Construct PAN06 52 28 Construct PAN36 53 29 Construct PAN37 54 30 Construct PAN02 55 31 Construct PAN05 56 32 Construct PAN07 57 33 Construct PAN08 58 34 Construct PAN09 59 35 Construct PAN10 60 36 Construct PAN11 61 37 Construct PAN12 62 38 Construct PAN13 63 39 Construct PAN28 64 40 Construct PAN29 65 41 Construct PAN30 66 42 Construct PAN31 67 43 Construct PAN32 68 44 Construct PAN33 69 45 Construct PAN34 70 46 Construct PAN35 71 47 Construct PAN38 72 48 Construct PAN39 73 49 Construct PAN40 74 50 Construct PAN41 75 51 Construct PAN42 76 52 Construct PAN43 77 53 Construct PAN44 78 54 Construct PAN45 79 55 Construct PAN46 80 56 Construct 81 57 Construct PAN48 82 58 Construct PAN49 83   59A 5′UTR of Ang1 84   59B SP (signal peptide) nucleotide sequence of Angl 85   59B SP (signal peptide) amino acid sequence of Ang1 86   59C CDS of Ang1 (mature peptide + stop codon) 87 — Amino acid sequence derived from SEQ ID No. 86 88   59D 3′UTR of Ang1 89   60A 5′UTR of Ang4 90   60B SP (signal peptide) nucleotide sequence of Ang4 91   60B SP (signal peptide) amino acid sequecne of Ang4 92   60C CDS of Ang4 (mature peptide + stop codon) 93 — Amino acid sequence derived from SEQ ID No. 92 94   60D 3′UTR of Ang4 95 61 COMP-Ang1 CDS (mature pep + stop codon) (rat) 96 — Amino acid sequence derived from SEQ ID No. 95 97 62 hCOMP-Ang1 CDS (mature pep + stop codon) (human) 98 — Amino acid sequence derived from SEQ ID No. 97 99 63 CMP-Ang1 CDS (mat peptide + stop codon) 100 64 COMP-Ang2 CDS (mat peptide + stop codon) 101 65 Tie2 CDS (start + mat pep + stop codon) 102 — Amino acid sequence derived from SEQ ID No. 101 103 66 Tie2* CDS (R849W)(start + mat pep + stop codon) 104 — Amino acid sequence derived from SEQ ID No. 103 105 67 Construct PAN50 106 68 Construct PAN51 107 69 Construct PAN52 108 70 Construct PAN53 109 86 Construct PAN54 110 87 Construct PAN55 111 88 Construct PAN56 112 89 Construct PAN57 (Mod) 113 90 Construct PAN58 114 91 Construct PAN59 115 95 Construct PAN60 (Tie2 wt) 116 96 Construct PAN61 (Tie2 R849W) 117 97 Construct PAN66 (PIK3CA wt) 118 Construct CDS (start + mat pep + stop codon) PIK3CA 119 CDS (mature peptide + stop cod) Nluc 120 100a 5′UTR of (PAN57) 121 100b 3′UTR of (PAN57) 122   7B 3′Primer for constructs PAN01-PAN03 123 100c 5′Primer for construct PAN57 124 100d 3′Primer for construct PAN57 125 100e 5′Primer for construct PAN55 (FIG. 87) and PAN56 (FIG. 88)

Example 1: Materials and Methods

[0515] Plasmid Template Generation by Gene Synthesis and Cloning

[0516] Sequences encoding Nluc reporter protein, Ang-1 protein and derivatives thereof (e.g. COMP-Ang-1, CMP-Ang-1) with different signal peptides were flanked by different heterologous 5′ and 3′ UTRs and were designed and produced by gene synthesis by BioCat (Heidelberg) and cloned (Xho1-BamHI) into pcDNA3.1− (Thermo Fisher).

[0517] Sequences encoding wt Tie-2 and sequences with the specified Tie-2 mutation in the coding region flanked by different heterologous 5′ and 3′ UTRs were designed (see FIGS. 95 and 96) and produced by gene synthesis by BioCat (Heidelberg) and cloned (Xho1-BamHI) into pcDNA3.1− (Thermo Fisher).

[0518] In Vitro Transcription from PCR Products to Generate Polyadenylated mRNAs

[0519] In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed. A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2.sup.+ ions. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed. In order to generate DNA templates with a poly-(A) tail a tail-PCR using 5′ primers containing T7 RNA polymerase sequences and 3′ primers with a 120 nt Poly-T sequence were synthesized and purified (BioSpring, Frankfurt).

[0520] The Linear DNA templates for in vitro transcription were generated by PCR from linearized plasmid. Plasmids were digested with BamHI that cut once 3-terminal in the vector backbone to produce a linearized vector that can be used as the template for the poly-(A) tail PCR. 5 μg plasmid are digested for 2 h at 37° C. with 5 U BamHI restriction enzyme in 50 μl and 1×HF Buffer. Small Aliquots of digested mix were analyzed by gel electrophoresis to check for complete digestion of the plasmid (see, for example, FIG. 5 for constructs PAN28 (“28”), PAN13 (“13”), PAN12 (“12”), PAN11 (“11”), PAN10 (“10”), PAN09 (“09”), PAN08 (“08”), PAN07 (“07”) and PAN05 (“05”)). The restriction enzyme is heat-inactivated by incubating at 80° C. for 20 min and the digested plasmid is purified by a PCR purification column following the manufacturer's protocol (NucleoSpin Gel and PCR clean-up, Macherey-Nagel). The linearized plasmid can be stored at −20° C. for several months and can be used for PCR template generation. The purpose of linearization was to eliminate circular templates that could potentially generate run-on transcripts during the IVT reaction.

[0521] Addition of poly-(A) tail by PCR was performed by using Hot StarHiFidelity polymerase (Qiagen) or Taq DNA Polymerase (Roche) following the manufacturer's protocol. For this purpose, Adapter primers containing T7 promoter sequences and 3′ Poly A tail sequences (120 nt) were used to amplify linear DNA-templates. Each of the adapter primers had overlap sequences which fused regulatory sequences necessary for translation and transcription to the gene of interest (see, for example, FIGS. 7a and 7b). A typical 50 μl PCR reaction contained final concentrations of 200 μM (of each) dNTP, 0.5 μM for each primer, 50 ng linearized plasmid DNA, 1×PCR reaction buffer (1.5 mM MgCl2) and 1.25 U Taq DNA Polymerase per reaction (0.25 μl of 5 U/μl). The specific PCR cycle programs or thermal profiles were adjusted according to the Tm of the primer pairs, according to the expected length of the PCR product and according to the used thermal cycler. The quality of the PCR products was checked by analyzing an aliquot by gel electrophoresis and the reactions are purified by commercial PCR purification kits ((NucleoSpin Gel and PCR clean-up, Macherey-Nagel).

[0522] In Vitro Transcription Reaction

[0523] Addition of a 5′ end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability, efficient translation, nuclear transport and splicing. The process involved addition of a 7-methylguanosine cap at the 5′ triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs such as ARCA (Jena Bioscience) or CleanCap (Trilink Biotechnologies) or EZ-Cap (ApexBio). In the enzymatic method, the mRNA may be capped using the vaccinia virus mRNA capping enzyme (NEB) as per manufacturer's protocol. The enzyme adds on a 7-methylguanosine cap at the 5′ end of the RNA using GTP and S-adenosyl methionine as donors (cap-0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications. In addition, a 2′-O-Methyltransferase can be used to introduce cap-1 and cap-2 structures.

[0524] In the example described below, the T7 High Yield Transcription Kit (Thermo Scientific) was used to synthesize capped RNA transcripts (PAN 01-XX) using cap analogs co-transriptionally.

[0525] The DNA template for the transcription reaction was a linearized plasmid or a PCR product. For the capped RNA the reaction at room temperature was set up in the following order:

TABLE-US-00002 Component Volume (μl) Final Nuclease free water* To 20 5X Reaction Buffer 4 1X ATP (100 mM) 2 10 mM CTP (100 mM) or 2 10 mM 5′-Methylcytidine-5′-Triphophate (100 mM) UTP(100 mM) or 2 10 mM Pseudouridine-5′-Triphosphate (100 mM) or 5-Methoxy uridine-5′-Triphosphate (100 mM) N1-Methyl-pseudouridine (100 mM) GTP (30 mM) 2  3 mM 3′-0-Me-m.sup.7G(5′)ppp(5′)G cap analog 2 10 mM (ARCA; AntiReverse Cap Analog) (100 mM) Template DNA* X 1 μg T7 RNA Polymerase Mix 2 Total 20 

[0526] 1 μg DNA was added and the total reaction volume to 20 μl was obtained by adding nuclease free water. The amount of water to be added varied based on the concentration of the template DNA. The reactions at were well mixed by vortexing and incubated at 37° C. for 2 hours in a dry air incubator. The transcription reactions were treated with DNase I to remove the DNA template before proceeding with purification as follows: [0527] 1. 70 μl nuclease free water was added to the transcription reactions followed by 10 μl of DNase I reaction buffer. [0528] 2. 2 μl DNase I were added to the reactions. [0529] 3. The reaction was incubated at 37° C. for 15 minutes.
Column Purification of Capped mRNA [0530] 1. The capped RNA was purified using the MEGAclearKit as per the manufacturer's instructions (any spin column based RNA purification kit may be used). [0531] 2. The RNA was quantified using a NanoDrop Spectrophotometer. [0532] 3. As per the manufacturer's instructions, RNA sample quality was assessed using the Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer or by native 1% agarose gel electrophoresis and ethidium bromide staining (see, FIG. 8).

[0533] mRNA Transfection of HeLa Cells, HEK293 Cells, HPMEC Cells, HPAEC Cells and HUVEC Cells

[0534] HeLa cells, were grown in DMEM complete medium. The human embryonic kidney 293 cell line (HEK293) is grown in EMEM (EBSS)+2 mM Glutamine+1% Non Essential Amino Acids (NEAA)+10% FCS culture medium.

[0535] The human embryonic kidney 293 cell line (HEK293) is grown in EMEM (EBSS)+2 mM Glutamine+1% Non Essential Amino Acids (NEAA)+10% FCS culture medium. Subculture Routine is to split sub-confluent cultures (70-80%) 1:2 to 1:6 i.e. seeding at 2-5×10,000 cells/cm.sup.2 using 0.25% trypsin or trypsin/EDTA; 5% CO.sub.2; 37° C. mRNA transfection are performed with the Lipofectamine™ MessengerMAX™ Transfection Reagent (Invitrogen™) according to the manufactures protocol.

[0536] Primary Human Pulmonary Microvascular Endothelial Cells (HPMEC) and Primary Human Pulmonary Artery Endothelial Cells (HPAEC) are isolated from human pulmonary arteries are most appropriate for studying human lung diseases and are isolated from the lung from a single donor. Since lung tissue contains blood and lymphatic capillaries, HPMEC comprise Blood and Lymphatic Microvascular Endothelial Cells.

[0537] The cell type of HPMEC's are characterized by immunofluorescent staining. They stain positive for CD31 and von Willebrand factor and negative for smooth muscle alpha-actin.

[0538] HUVEC, HPAEC and HPMEC cells were grown in Endothelial Cell Growth Medium as recommended from PromoCell (Heidelberg Germany) respectively. The Endothelial Cell Growth Medium is basal Medium supplemented with Fetal Calf Serum (0.05 ml/ml), Endothelial Cell Growth Supplement (bovine hypothalamic extract; 0.004 ml ml), Heparin 90 μg/ml and Hydrocortisone 1 μg/ml. The experiments with HUVEC, HPAEC and HPMEC cells are performed on cells with passage numbers below 8.

[0539] RNA transfections and more mRNA transfections were carried out using Lipofectamin MessengerMAX (Invitrogen, Carlsbad, Calif.; Thermo Fisher Scientific). For mRNA transfection in 24-well plate format 1×10.sup.5 cells/well were seeded 24 h before transfection to reach 70-90 confluence immediately before transfection. For one single well 1.5 μl Lipofectamine MessengerMAX Reagent was added to 25 μl OptiMEM (Invitrogen, Carlsbad, Calif.; Thermo Fisher Scientific), vortexed and incubated for 10 min at RT. In a second well 500 ng or 1 μg mRNA were added to 25 μl OptiMEM, and vortexed. After incubation diluted mRNA was added to diluted Lipofectamin, mixed well, incubated for 5 min at RT and added drop wise to cells in the presence of 0.5 ml of complete medium for Hela and HEK293. For HUVEC, HPAEC and HPMEC the Endothelial Cell Growth medium was replaced and the cells washed with OptiMEM and then the Lipofectamin MessengerMAxX mRNA complex solution added to the well with 0.5 ml OptiMEM prewarmed to 37° C. Cells were incubated at 37° C. for 2 h and the OptiMEM medium replaced with complete Endothelial Cell Growth Medium (PromoCell, Heidelberg Germany).

[0540] Cell Lysates from Transfected cells or serum were analyzed at the indicated time points. Alternatively, mRNA-LNPs based on the cationic lipids L-Arginyl-β-alanine-N-palmityl-N-oleyl-amide or β-(L-Arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide in combination with neutral and PEGylated co-lipids were used for in vitro transfection. The co-lipids were Diphytanoyl-PE and the PEGylated lipid is methoxyPEG2000-DSPE

[0541] Successful introduction of mRNA into host cells was monitored using various known methods, such as a fluorescent marker, such as Green Fluorescent Protein (GFP), such as reporter enzymes (e.g. luciferase derivatives). Alternatively, transfection of a modified mRNA could also be determined by measuring the protein expression level of the target polypeptide by e.g., Western Blotting or immunocytochemistry or ELISA.

[0542] Fully modified mRNAs with 5-methylcytidine and/or pseudouridine (or 5-methoxyuridine or N1-methylpseudouridine) are transfected into HUVEC or HPMEC (Human Pulmonary Microvascular Endothelial Cells, PromoCell, Heidelberg) using Lipofectamin MessengerMAX (Invitrogen, Carlsbad, Calif.; Thermo Fisher Scientific). The cell lysates are harvested and run by ELISA (or on immunoblot assays (western) at different time points (30″, 1 h, 2 h, 4 h, 6 h hours) after transfection to determine the protein expression.

[0543] Nano-Luc Luciferase Activity Reporter Measurement

[0544] A time dependent quantitative detection of Nano-Glo-Luciferase expression (Promega) was performed for secreted versions by analyzing samples from the tissue culture supernatant according to the reporter assay technical manual Nano-Glo Luciferase Assay System (Promega). In brief, 5-20 μl of serum or lysate were diluted in 100 μl H.sub.2O final volume and equal volume of reconstituted Nano-Glo luciferase assay reagent was combined in a 96 well GloMax 96 Microplate. After at least 3 min incubation at RT luminescence were measured in an appropriate luminometer.

[0545] Immunoblot Detection of Tie-2 and Derivates

[0546] For immunoblot detection of Tie-2 and derivatives protein lysates are loaded on NuPage SDS-PAGE system (chambers and power supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12% acrylamide gradient with MOPS-buffer as running aid (all Life Technologies, Grand Island, N.Y.). Each lysate sample is prepared to 40 μl final volume. This sample contains 25 μg protein lysate in variable volume, RIPA buffer to make up volume to 26 μl, 4 μl of 10× reducing agent and 10 μl 4×SDS loading buffer (both from Life Technologies, Grand Island, N.Y.). Samples are heated at 95° C. for 5 min and loaded on the gel. Standard settings are chosen by the manufacturer, 200V, 120 mA and max. 25 W. Run time is 60 min, but no longer than running dye reaching the lower end of the gel.

[0547] After the run is terminated, the plastic case is cracked and the encased gel transferred to a ready-to-use nitrocellulose membrane kit and power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Using default settings, the protein lysate is transferred by high Ampere electricity from the gel to the membrane.

[0548] After the transfer, the membranes are incubated in 5% BSA in IX TBS for 15 minutes then in 5% BSA in 1×TBS+0.1% Tween for another 15 minutes. Primary antibodies against human Tie-2 proteins or Phospho-specific Antibodies (P-Tie-2, P-Akt, see below) are applied in 3 ml of 5% BSA in 1×TBS solution at a 1:500 to 1:2000 dilution for 3 hours at room temperature and gentle agitation on an orbital shaker. Membranes are washed 3 times with 1×TBS/0.1% Tween, 5 minutes each time with gentle agitation. The secondary antibody (Goat anti-rabbit HRP conjugate; Abeam, Cambridge, Mass.) is conjugated to horse radish peroxidase and binds to the primary antibody antibodies. The secondary antibody is diluted from 1:1000 to 1:5000 in 5% BSA in IX TBS and incubated for 3 hrs at RT. At the end of incubation time, the membranes are washed 3 times with IX TBS/0.1% Tween, 5 minutes each time with gentle agitation. The membranes are developed in 5 ml Pierce WestPico Chemiluminescent Subtrate (Thermo Fisher, Rockford, Ill.) as directed.

Example 2: Expression of Luciferase, Ang-1 or Ang-1-Derivatives Expressing Recombinant Nucleic Acid Constructs in HPMEC, HUVEC, HPAEC and HeLa Cells

[0549] mRNA Transfection of HeLa, HUVEC, HPAEC and HPMEC Cells

[0550] Primary Human Pulmonary Microvascular Endothelial Cells (HPMEC) are most appropriate for studying human lung diseases and are isolated from the lung from a single donor. Since lung tissue contains blood and lymphatic capillaries, HPMEC comprise Blood and Lymphatic Microvascular Endothelial Cells. The cells were routinely analyzed by immunofluorescent staining: they stain positive for CD31 and von Willebrand factor and negative for smooth muscle alpha-actin. HeLa cells, HUVEC, HPMEC and HPAEC (human pulmonary artery endothelial cells) cells, respectively, were grown in DMEM complete medium or Endothelial Cell Growth Medium (PromoCell, Heidelberg Germany, CatNo.: C-22020) respectively. The Endothelial Cell Growth Medium was basal Medium supplemented with Fetal Calf Serum (0.05 ml/ml), Endothelial Cell Growth Supplement (bovine hypothalamic extract; 0.004 ml ml), Heparin 90 μg/ml and Hydrocortisone 1 μg/ml. The experiments with HUVEC and HPMEC cells were performed on cells with passage numbers below 8.

[0551] mRNA transfections were carried out using Lipofectamin MessengerMAX (Invitrogen, Carlsbad, Calif.; Thermo Fisher Scientific). For mRNA transfection in 24-well plate format 1×10.sup.5 cells/well were seeded 24 h before transfection to reach 70-90 confluence immediate before transfection. For one single well 1.5 μl Lipofectamine MessengerMAX Reagent was added to 25 μl OptiMEM, vortex and incubated for 10 min at RT. In a second well add 500 ng or 1 μg mRNA were added to 25 μl OptiMEM, vortex. After incubation diluted mRNA was added to diluted Lipofectamin, mixed well, incubated for 5 min at RT and added dropwise to cells in the presence of 0.5 ml of DMEM complete medium for HeLa. For HUVEC, HPMEC and HPAEC the Endothelial Cell Growth medium was replaced and the cells washed with OptiMEM and then added the Lipofectamin MessengerMAX mRNA complex solution to the well with 0.5 ml OptiMEM prewarmed to 37° C. Cells were incubated at 37° C. for 2 h and the OptiMEM medium was replaced with complete Endothelial Cell Growth Medium (PromoCell, Heidelberg Germany) and transfected cells or serum were analyzed at the indicated time points. Alternatively, mRNA-LNPs based on the cationic lipids L-Arginyl-β-alanine-N-palmityl-N-oleyl-amide or β-(L-Arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide in combination with neutral and PEGylated co-lipids can be used for in vitro transfection.

[0552] Successful introduction of mRNA into host cells can be monitored using various known methods, such as a fluorescent marker, such as Green Fluorescent Protein (GFP), such as reporter enzymes (e.g. luciferase derivatives) or transfection of a modified mRNA can also be determined by measuring the protein expression level of the target polypeptide by e.g., Western Blotting or immunocytochemistry or ELISA.

[0553] Nano-Luc Luciferase Activity Reporter Measurement

[0554] A time dependent quantitative detection of Nano-Glo-Luciferase expression (Promega) was performed for secreted versions by analyzing samples from the tissue culture supernatant according to the reporter assay technical manual Nano-Glo Luciferase Assay System (Promega). In brief, 5-20 μl of serum or lysate were diluted in 100 μl H.sub.2O final volume and equal volume of reconstituted Nano-Glo luciferase assay reagent was combined in a 96 well GloMax 96 Microplate. After at least 3 min incubation at RT luminescence could be measured in an appropriate luminometer.

[0555] Detection of Ang-1, COM-Ang-1, CMP-Ang-1 Protein in Cell Lysates and Supernatant

[0556] A time dependent quantitative detection of secreted Ang-1 and secreted COMP-Ang-1 or COMP-Ang-2 expression is performed by analyzing samples from the supernatant tissue culture by ELISA (Human Angiopoietin-1 Quantikine ELISA Kit (DANG10, R&D systems) or by Western blot according to the technical manual.

[0557] 500 ng of COMP-Ang-1 (mRNA sequence shown in SEQ ID NO: PAN05) with poly A tail of approximately 120 nucleotides not shown in sequence; 5′ cap) fully modified with 5-methylcytidine and pseudouridine (COMP-Ang-1, 5 mC/pU), fully modified with 5-methylcytidine and Nl-methyl-pseudouridine (COMP-Ang-1, 5mC/NlmpU) or unmodified (COMP-Ang-, unmod) is transfected into HUVEC or HPMEC (Human Pulmonary Microvascular Endothelial Cells, PromoCell, Heidelberg) using Lipofectamin MessengerMAX (Invitrogen, Carlsbad, Calif.; Thermo Fisher Scientific). The supernatant is harvested and run by ELISA 4 hours after transfection to determine the protein expression and cytokine induction.

[0558] For immunoblot detection of Ang-1 and derivatives protein lysates were loaded on NuPage SDS-PAGE system (chambers and power supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12% acrylamide gradient with MOPS-buffer as running aid (all Life Technologies, Grand Island, N.Y.). Each lysate sample was prepared to 40 ul final volume. This sample contained 25 ug protein lysate in variable volume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducing agent and 10 μl 4×SDS loading buffer (both from Life Technologies, Grand Island, N.Y.). Samples were heated at 95° C. for 5 min and loaded on the gel. Standard settings were chosen by the manufacturer, 200V, 120 mA and max. 25 W. Run time was 60 min, but no longer than running dye reaching the lower end of the gel.

[0559] After the run is terminated, the plastic case is cracked and the encased gel transferred to a ready-to-use nitrocellulose membrane kit and power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Using default settings, the protein lysate is transferred by high Ampere electricity from the gel to the membrane.

[0560] After the transfer, the membranes were incubated in 5% BSA in 1×TBS for 15 minutes then in 5% BSA in IX TBS+0.1% Tween for another 15 minutes. Primary antibodies (Ang-1 ab183701 Abeam, Cambridge, UK) against human Ang-1 proteins are applied in 3 ml of 5% BSA in IX TBS solution at a 1:5000 dilution overnight at 4° C. and gentle agitation on an orbital shaker. Membranes are washed 3 times with IX TBS/0.1% Tween, 5 minutes each time with gentle agitation. The secondary antibody (Goat anti-rabbit HRP conjugate; Abeam, Cambridge, Mass.) is conjugated to horse radish peroxidase and binds to the primary antibody antibodies. The secondary antibody is diluted of 1:10000 in 5% BSA in IX TBS and incubated for 1 hr at RT. At the end of incubation time, the membranes are washed 3 times with IX TBS/0.1% Tween, 5 minutes each time with gentle agitation. The membranes are developed in 5 ml Pierce WestPico Chemiluminescent Subtrate (Thermo Fisher, Rockford, Ill.) as directed. The Western Blot detects protein around the expected size of 70 kd for human Ang-1, and of 37 kDa for COMP-Ang-1 and CMP-Ang1. For reference, recombinant human Ang-1 protein was purchased from R&D Systems (biotechne).

[0561] Results

[0562] Protein expression data presented in the FIGS. 10 to 14, 72 to 85 and 92 to 94 underline that combinations of different regulatory nucleotide sequences flanking a coding region can significantly change the protein expression levels. Such change is further impacted by modification of the nucleotides forming the constructs as exemplified by replacing 100% of uridine and 100% cytidine nucleotides by pseudo-uridine and 5-methyl-cytidine (the constructs marked with “*” in FIG. 74 and FIGS. 77 to 85. Changes are not only observed in a particular cell type but also across different cell types. For example, PAN02 (wild-type Ang-1 mRNA) shows the lowest Luciferase activity in all cell lines (FIGS. 10 to 14). All other constructs show enhanced protein expressions throughout all cell types. Therefore, the approach of using regulatory sequences from different genes can increase protein expression activity. To this end, replacing the endogenous Ang-1 sequences have shown superior effects on the protein expression of the reporter.

[0563] Worth noting, even the same regulatory sequences showed distinct protein expression activity in different cell types (FIGS. 75 to 81. These data further support our approach that the use of specific regulatory sequences can influence the activity of protein expression in a context-specific, i.e. cell-type specific cellular environment.

[0564] Furthermore, we have shown that our data are also concerning a different aspect i.e. secretion. For example, the regulatory sequences used in construct PAN12 show cell-type specific (HPMEC) expression in whole cell lysates but also in medium (supernatant) (FIGS. 10-14). This indicated that these sequences are not only optimal for protein expression (translation) but also secretion. In contrast, construct PAN28 shows expression in all cell types more or less equally. The regulatory sequences used in construct PAN29 (and PAN58), namely the 5′-UTR and the signal peptide sequence of MCP-1 in combination with the 3′-UTR of vWF, were found to be particularly useful for highly efficient protein expression in human (microvascular) pulmonary endothelial cells (see e.g. FIGS. 72 and 75). The regulatory sequences used in construct PAN54, namely the 5′-UTR of spinach chloroplast RPL12, the signal peptide sequence of MCP-1 and the 3′-UTR of vWF, were found to be equally efficient for protein expression and secretion as shown in FIG. 92. As shown in FIG. 98 the regulatory sequences of PAN29 are also functional using three different ORFs, namely wt Ang-1 in PAN52; hCOMP-Ang-1 in PAN53 and CMP-Ang-1 in PAN59 in directing expression in primary human pulmonary microendothelial cells (HPMEC). In contrast the mRNA construct containing the endogenous 5′- and 3′-UTRs of human Ang-1 (PAN01) is not translated (FIG. 98). FIG. 99 shows that all four mRNA constructs display comparable quality (integrity and purity).

Example 3: Tie-2 Pathway Activation after Transfection of Modified mRNAs Encoding the Wildtype Tie-2 (PAN60) or the Activating Tie-2 Mutations (e.g. R849W (PAN61))

[0565] Modified mRNAs containing heterologous 5′ and 3′ UTRs are generated by in vitro transcription of PCR templates as described previously in example 1 above. Different concentration of this mRNA encoding the activating Tie-2 mutations (e.g. R849W) are transfected in different cell lines (HeLa, primary endothelial cells such as HUVECs or HPMEC or HPAEC) next to mRNAs encoding the wild-type Tie-2 using Lipofectamin MessengerMAX (Invitrogen, Carlsbad, Calif.; Thermo Fisher Scientific). For qualitative Western blot cell lysates at different time points post transfection of mRNAs (1-24 h) are probed with anti-phosphotyrosine antibody (R&D systems) to evaluate Tie-2 phosphorylation (pTyr, 140 kD). Blots are stripped and re-probed with anti-Tie-2 antibody (R&D systems) to detect total Tie-2. For quantitative measurement two different ELIS As are performed measuring human Tie-2 or Phospho-Tie-2 using the human Tie-2 DuoSet ELISA kit or human-Phospho-Tie-2 DuoSet IC ELISA according to the manufactures (e.g. both ELISA-Kits from R&D systems) protocol.

[0566] Transfection of Tie-2 mutation (PAN61, R849W) encoding mRNAs increase the presence of Phospho-Tie-2 in comparison to cell lysates derived from cells transfected with mRNAs encoding wildtype Tie-2 mRNA or in comparison to non-transfected cells. This increase in Tie-2 phosphorylation is observed in the presence or absence of co-stimulation with recombinant human Ang-1 ligand and can be observed in different cell lines. To confirm the activation of the Tie-2 signaling pathway and to demonstrate a biological relevant functional downstream signal transduction by expressing the Tie-2 (R849W) derivate a phosphorylation of Akt is demonstrated in a time course experiment. These experiments indicate that mRNAs encoding for the Tie-2 activating mutation (R849W) are functional and stronger in activating the Tie-2 signalling pathway than wt Tie-2 encoding mRNAs. These hyperactivation of the pathway can be demonstrated in the absence or presence of Tie-2 ligands (e.g. Ang-1) in human endothelial and/or non-endothelial cells.

Example 4: mRNA Formulation in Cationic LNPs for In Vivo Applications by Intravenous Administration

[0567] For in vivo experiments mRNA-LNPs are prepared in a formulation process with β-(L-Arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide as cationic lipid. Alternatively, the cationic lipid L-Arginyl-β-alanine-N-palmityl-N-oleyl-amide can be used in an identical procedure to prepare mRNA-LNPs.

[0568] mRNA-LNP formulations are prepared using a modified procedure of a method described for siRNA (Chen, S., Tam, Y. Y., Lin, P. J., Sung, M. M., Tam, Y. K., and Cullis, P. R. (2016), Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA. J. Control. Release 235, 236-244. & (ii) patent application US20170121712).

[0569] Briefly, lipids are dissolved in ethanol at appropriate molar ratios (e.g. 50:49:1 β-(L-arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide: DPyPE: mPEG2000-DSPE). The lipid mixture is combined with an isotonic Sucrose solution of mRNA at a volume ratio of 2:1 (aqueous:ethanol) using a microfluidic mixer (NanoAssemblr®; Precision Nanosystems, Vancouver, BC) and flow rates of 18 ml/min. Similarly, LNP formulations can be obtained using citrate or acetate buffered mRNA solutions (pH 3-4) and a slightly differing mixing ratio of 3:1 (v/v; aqueous:ethanol) and flow rates of 12 ml/min.

[0570] After the mixing process, the formulations are dialyzed against 10 mM HEPES or TRIS buffered isotonic Sucrose solution using 3.5 K MWCO Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher Scientific) for at least 18 hours at 4° C. Instead of Sucrose, other sugars like Trehalose or Glucose can be equally used within the formulation process.

[0571] Subsequently, the formulations are tested for particle size (Zetasizer Nano ZS instrument (Malvern Instruments Ltd, Malvern, UK), RNA encapsulation (Quant-iT RiboGreen RNA Assay Kit following manufacturer's (Thermo Fisher Scientific) protocol), and endotoxin and are found to be between 30 to 100 nm in size with a Zeta-potential of >25 mV, display greater than 90% mRNA encapsulation and <1 EU/ml of endotoxin.

[0572] mRNA-LNP formulations are stored at −80° C. at a concentration of RNA of 0.3 μg/μl and an RNA to total lipid ratio of 0.03-0.05 (wt/wt) until further in vitro or in vivo use.

[0573] The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.