Recombinant Nucleic Acid Constructs

Abstract

The present invention relates to a recombinant nucleic acid construct comprising, in a 5'→3' direction, a 5'UTR, a coding region encoding an effector molecule, and a 3'UTR, wherein the 5'UTR is selected from the group comprising the 5'UTR of a gene 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, or a derivative thereof having at least 85% nucleotide identity.

Description

[Technical field]

[0001] The present invention relates to a recombinant nucleic acid construct, a vector, preferably an expression vector, comprising the recombinant nucleic acid construct, a cell comprising the recombinant nucleic acid construct and / or said vector, a delivery vehicle comprising the recombinant nucleic acid construct, a pharmaceutical composition comprising the recombinant nucleic acid construct, a recombinant nucleic acid construct for use in a method for the treatment and / or prevention of a disease, a recombinant nucleic acid construct for use in a method for restoring cellular function of a cell, a method for the treatment and / or prevention of a disease and a method for restoring cellular function of a cell. [Background technology]

[0002] Nucleic acid molecules serve a variety of purposes in living systems. One of several purposes is to store genetic information, another is to transfer genetic information from deoxyribonucleic acid to ribosomes where such genetic information is translated into amino acid sequences. The latter process involves the use of messenger ribonucleic acid (mRNA).

[0003] In eukaryotic cells, mRNA exhibits a very basic design and typically contains, in a 5'→3' orientation, a cap structure, a 5' untranslated region (5'UTR), a coding sequence that typically begins with an AUG codon and ends with a stop codon linked to a coding sequence (CDS), a 3' untranslated region (3'UTR), and a polyA tail.

[0004] mRNA-based therapies have been proposed as treatments since the early days of biotechnology. In principle, an administered mRNA sequence can cause cells to produce a protein that can then treat a disease directly or act as a vaccine, or more indirectly, treat or ameliorate a disease by interfering with a component of a pathway in such a way that the pathway is either inhibited or stimulated. Summary of the Invention

[0005] The problem underlying the present invention is the provision of a recombinant nucleic acid construct, preferably an mRNA, that allows high expression of the coding sequence of said recombinant nucleic acid construct.

[0006] Another problem underlying the present invention is the provision of a recombinant nucleic acid construct, preferably an mRNA, which allows the expression of a coding sequence of said recombinant nucleic acid construct in endothelial cells.

[0007] These and other problems underlying the present invention are solved by the subject matter of the attached independent claims, preferred embodiments of which can be seen from the attached dependent claims.

[0008] In addition, more specifically, the problem underlying the present invention is to provide a method for the preparation of a 5'→3'-direction nucleotide sequence in a first aspect, which is also a first embodiment of the first aspect. 5'UTR, a coding region encoding an effector molecule, and 3'UTR The problem is solved by a recombinant nucleic acid construct comprising: wherein the 5'UTR is the 5'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding Ang-2 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding H3.3. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding Galectin-9 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding GADD34 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding EDN1 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding HSP70m5 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding E-selectin or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding ICAM-1 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding IL-6 or a derivative thereof having at least 85% nucleotide identity, and the 5'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, the 3'UTR is selected from the group comprising the 3'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding H3.3. or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding GADD34 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding EDN1 or a derivative thereof having at least 85% nucleotide identity and the 3'UTR of the gene encoding IL-6 or a derivative thereof having at least 85% nucleotide identity, The effector molecule is effective to restore a cellular function of the cell or to exert a therapeutic effect in or on the cell; and The recombinant nucleic acid construct differs from the wild-type mRNA encoding the effector molecule.

[0009] In a second embodiment of the first aspect, which is also an embodiment of the first embodiment of the first aspect, the 5'UTR and the 3'UTR of the recombinant construct are of different origin, preferably from different endogenous species.

[0010] In a third embodiment of the first aspect, which is also an embodiment of the first and second embodiment of the first aspect, the 5'UTR and the 3'UTR of the recombinant construct are of the same origin, preferably derived from the same endogenous gene species.

[0011] In a fourth embodiment of the first aspect which is also an embodiment of the first, second and third embodiments of the first aspect, a) the 5'UTR is the 5'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity and the 3'UTR is selected from the group comprising the 3'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity and the 3'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, or b) the 5'UTR is the 5'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity and the 3'UTR is selected from the group comprising the 3'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity and the 3'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, or c) the 5'UTR is the 5'UTR of the gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity and the 3'UTR is selected from the group comprising the 3'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity and the 3'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, or d) the 5'UTR is the 5'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is selected from the group comprising the 3'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity.

[0012] In a fifth embodiment of the first aspect which is also an embodiment of the first, second and third embodiments of the first aspect, a) the 3'UTR is the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity and the 5'UTR is selected from the group comprising the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity, or b) the 3'UTR is the 3'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR is selected from the group comprising the 5'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity; c) the 3'UTR is the 3'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity and the 5'UTR is selected from the group comprising the 5'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, or or d) the 3'UTR is the 3'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR is selected from the group comprising the 5'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity.

[0013] In a sixth aspect of the first aspect which is also an embodiment of the first, second, third, fourth and fifth aspects of the first aspect, the construct comprises A construct, wherein the 5'UTR is the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity. A construct, wherein the 5'UTR is the 5'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, A construct, wherein the 5'UTR is the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity. A construct, wherein the 5'UTR is the 5'UTR of a gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity. A construct, wherein the 5'UTR is the 5'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, A construct, wherein the 5'UTR is the 5'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity. A construct, wherein the 5'UTR is the 5'UTR of a gene encoding H3.3. or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding H3.3. or a derivative thereof having at least 85% nucleotide identity, and A construct, wherein the 5'UTR is the 5'UTR of a gene encoding galectin 9 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding galectin 9 or a derivative thereof having at least 85% nucleotide identity. The compound is selected from the group consisting of:

[0014] In a seventh aspect of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth and sixth aspect of the first aspect, the construct comprises a polyA tail, preferably at the 3' end of the recombinant nucleic acid construct.

[0015] In an eighth aspect of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth and seventh aspect of the first aspect, the construct comprises a cap structure, preferably at the 5' end of the recombinant nucleic acid construct.

[0016] In a ninth aspect of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh and eighth aspect of the first aspect, the construct comprises an IRES (internal ribosome entry site) sequence, preferably at the 5' end of the recombinant nucleic acid construct.

[0017] In a tenth aspect of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth aspects of the first aspect, the construct comprises a nucleic acid sequence encoding a signal peptide, preferably the signal peptide is in frame with the nucleic acid sequence encoding a signal peptide and is located between the 5'UTR and the coding region encoding the effector molecule.

[0018] In an eleventh embodiment of the first aspect which is also an embodiment of the tenth embodiment of the first aspect, the signal peptide allows secretion of the effector molecule.

[0019] In a twelfth embodiment of the first aspect, which is also an embodiment of the tenth and eleventh embodiments of the first aspect, the nucleotide sequence encoding the signal peptide is selected from the group comprising a nucleotide sequence encoding the signal peptide of MCP-1 or a derivative thereof having at least 85% nucleotide identity, a nucleotide sequence encoding the signal peptide of IL-6 or a derivative thereof having at least 85% nucleotide identity, a nucleotide sequence encoding the signal peptide of Ang-2 or a derivative thereof having at least 85% nucleotide identity and a nucleotide sequence encoding the signal peptide of Ang-1 or a derivative thereof having at least 85% nucleotide identity.

[0020] In a thirteenth aspect of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and twelfth aspects of the first aspect, the cell whose cellular function is to be restored and / or the cell in or on which a therapeutic effect is exerted is an endothelial cell, preferably a vascular endothelial cell, more preferably the vascular endothelial cell is a microvascular endothelial cell.

[0021] In a fourteenth aspect of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth aspects of the first aspect, the cellular function may be restored by an effector molecule having an endothelial cell anti-permeability effect, an anti-vascular leakage effect, an endothelial cell anti-apoptotic effect, or an endothelial cell anti-inflammatory effect, or an anti-stress response effect, preferably the effect is related to or associated with the Tie-2 signalling pathway, the VEGF receptor pathway, the NOTCH signalling pathway, the PI3-kinase pathway, the eNOS signalling pathway, the sirtuin-dependent metabolic and energy homeostasis pathways, the oxidative stress pathway, the shear stress response pathway, the ET-1 signalling pathway, the NO-mediated signalling pathway and the mechanochemical transduction pathway.

[0022] More specifically, the problem underlying the present invention is to provide a method for the production of a 5'→3'-directed nucleotide sequence according to a second aspect, which is also a first embodiment of the second aspect. 5' non-translated region, a coding region encoding an effector molecule, and 3' untranslated region, The present invention is solved by a recombinant nucleic acid construct for use in a method for treating and / or preventing a disease, comprising: an effector molecule that is effective for restoring cellular function of a cell, wherein the effector molecule is expressed by an endothelial cell. In one embodiment, and preferably as used herein, restoring cellular function includes and includes killing cells, more preferably killing tumor endothelial cells and / or tumor cells, respectively. In another embodiment, and preferably as used herein, restoring cellular function includes and includes neovascularization, more preferably neovascularization in the case of vascular disease, respectively.

[0023] In a second embodiment of the second aspect, which is also an embodiment of the first embodiment of the second aspect, the treatment and / or prevention of the disease involves restoration of a cellular function of the cell.

[0024] More specifically, the problem underlying the present invention is to provide a method for the preparation of a 5'→3' nucleotide sequence according to a third aspect, which is also a first embodiment of the third aspect. 5' untranslated region, a coding region encoding an effector molecule, and 3' untranslated region, The problem is solved by a recombinant nucleic acid construct for use in a method for restoring cellular function of a cell, comprising:

[0025] More specifically, the problem underlying the present invention is to provide a fourth aspect, which is also a first embodiment of the fourth aspect, in the 5'→3' direction 5' untranslated region, a coding region encoding an effector molecule, and 3' untranslated region, The problem is solved by a recombinant nucleic acid construct for use in a method for the treatment and / or prevention of a disease, comprising: an effector molecule effective to exert a therapeutic effect in or on a cell, wherein the effector molecule is expressed by an endothelial cell.

[0026] In a second embodiment of the fourth aspect, which is also an embodiment of the first embodiment of the fourth aspect, the treatment and / or prevention of the disease involves restoration of a cellular function of the cell.

[0027] More specifically, the problem underlying the present invention is to provide a method for the production of a 5'→3' oligonucleotide in a 5'→3' direction, the method being also a first embodiment of the 5' aspect. 5' untranslated region, a coding region encoding an effector molecule, and 3' untranslated region, The problem is solved by a recombinant nucleic acid construct for use in a method for restoring cellular function of a cell, comprising: an effector molecule effective to exert a therapeutic effect in or on the cell, wherein the effector molecule is expressed by an endothelial cell.

[0028] In a third embodiment of the second aspect which is also an embodiment of the first and second aspects of the second aspect, in a second embodiment of the third aspect which is also an embodiment of the first aspect of the third aspect, in a third embodiment of the fourth aspect which is also an embodiment of the first and second aspects of the fourth aspect, and in a second embodiment of the fifth aspect which is also an embodiment of the first aspect of the fifth aspect, the method wherein the effector molecule is expressed by the endothelial cell.

[0029] In the fourth embodiment of the second aspect which is also an embodiment of the first, second and third embodiment of the second aspect, in the third embodiment of the third aspect which is also an embodiment of the first and second embodiment of the third aspect, in the fourth embodiment of the fourth aspect which is also an embodiment of the first, second and third embodiment of the fourth aspect and in the third embodiment of the fifth aspect which is also an embodiment of the first and second embodiment of the fifth aspect, the endothelial cell is a vascular endothelial cell, preferably the vascular endothelial cell is a microvascular endothelial cell.

[0030] In a fifth embodiment of the second aspect which is also an embodiment of the first, second, third and fourth embodiment of the second aspect, in a fourth embodiment of the third aspect which is also an embodiment of the first, second and third embodiment of the third aspect, in a fifth embodiment of the fourth aspect which is also an embodiment of the first, second, third and fourth embodiment of the fourth aspect and in a fourth embodiment of the fifth aspect which is also an embodiment of the first, second and third embodiment of the fifth aspect, the endothelial cell is a non-dividing endothelial cell, a non-proliferating endothelial cell or a quiescent endothelial cell. In one embodiment thereof, and preferably as used herein, the endothelial cell is a vascular endothelial cell, a lymphatic endothelial cell, an endothelial stem cell (ESC) or an endothelial progenitor cell (EPC).

[0031] In the sixth embodiment of the second aspect which is also an embodiment of the first, second, third, fourth and fifth embodiment of the second aspect, in the fifth embodiment of the third aspect which is also an embodiment of the first, second, third and fourth embodiment of the third aspect, in the sixth embodiment of the fourth aspect which is also an embodiment of the first, second, third, fourth and fifth embodiment of the fourth aspect and in the fifth embodiment of the fifth aspect which is also an embodiment of the first, second, third and fourth embodiment of the fifth aspect, the endothelial cell is a senescent endothelial cell.

[0032] In a seventh aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth and sixth aspects of the second aspect, in a sixth aspect of the third aspect which is also an embodiment of the first, second, third, fourth and fifth aspects of the third aspect, in a seventh aspect of the fourth aspect which is also an embodiment of the first, second, third, fourth, fifth and sixth aspects of the fourth aspect and in a sixth aspect of the fifth aspect which is also an embodiment of the first, second, third, fourth and fifth aspects of the fifth aspect, the endothelial cells are impaired by ageing and / or show stress-related defects.

[0033] In an eighth aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth and seventh aspect of the second aspect, in a seventh aspect of the third aspect which is also an embodiment of the first, second, third, fourth, fifth and sixth aspect of the third aspect, in an eighth aspect of the fourth aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth and seventh aspect of the fourth aspect and in a seventh aspect of the fifth aspect which is also an embodiment of the first, second, third, fourth, fifth and sixth aspect of the fifth aspect, the recombinant nucleic acid construct further comprises one element selected from the group comprising a cap structure, an IRES sequence, an additional start codon providing sequence, a nucleic acid sequence encoding a signal peptide and a poly A tail.

[0034] in a ninth aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh and eighth aspects of the second aspect; in an eighth aspect of the third aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth and seventh aspects of the third aspect; in a ninth aspect of the fourth aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh and eighth aspects of the fourth aspect; and in an eighth aspect of the fifth aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth and seventh aspects of the fifth aspect, The recombinant nucleic acid construct is a) the 5' untranslated region, a coding region encoding an effector molecule, 3' untranslated region, and · PolyA tail; b) 5' untranslated region, additional start codon providing sequences, a coding region encoding an effector molecule, 3' untranslated region, and · PolyA tail; c) Cap structure, 5' untranslated region, a coding region encoding an effector molecule, and ·3' untranslated region; d) Cap structure, 5' untranslated region, additional start codon providing sequences, a coding region encoding an effector molecule, and ·3' untranslated region; e) an IRES sequence, 5' untranslated region, a coding region encoding an effector molecule, and ·3' untranslated region; f) an IRES sequence, 5' untranslated region, additional start codon providing sequences, a coding region encoding an effector molecule, and ·3' untranslated region; g) Cap structure, 5' untranslated region, a coding region encoding an effector molecule, 3' untranslated region, and · PolyA tail; h) Cap structure, 5' untranslated region, additional start codon providing sequences, a coding region encoding an effector molecule, 3' untranslated region, and · PolyA tail; i) an IRES sequence, 5' untranslated region, a coding region encoding an effector molecule, 3' untranslated region, and · PolyA tail; j) an IRES sequence, 5' untranslated region, additional start codon providing sequences, a coding region encoding an effector molecule, 3' untranslated region, and · PolyA tail; k) 5' untranslated region, a nucleic acid sequence encoding a signal peptide, a coding region encoding an effector molecule, 3' untranslated region, and · PolyA tail; l) 5' untranslated region, additional start codon providing sequences, a nucleic acid sequence encoding a signal peptide, a coding region encoding an effector molecule, 3' untranslated region, and · PolyA tail; m) Cap structure, 5' untranslated region, a nucleic acid sequence encoding a signal peptide, a coding region encoding an effector molecule, and ·3' untranslated region; Cap structure, 5' untranslated region, additional start codon providing sequences, a nucleic acid sequence encoding a signal peptide, a coding region encoding an effector molecule, and ·3' untranslated region; o) an IRES sequence, 5' untranslated region, a nucleic acid sequence encoding a signal peptide, a coding region encoding an effector molecule, and ·3' untranslated region; p) an IRES sequence, 5' untranslated region, additional start codon providing sequences, a nucleic acid sequence encoding a signal peptide, a coding region encoding an effector molecule, and ·3' untranslated region; q) Cap structure, 5' untranslated region, a nucleic acid sequence encoding a signal peptide, a coding region encoding an effector molecule, 3' untranslated region, and · PolyA tail; r) Cap structure, 5' untranslated region, additional start codon providing sequences, a nucleic acid sequence encoding a signal peptide, a coding region encoding an effector molecule, 3' untranslated region, and · PolyA tail; s) IRES sequence, 5' untranslated region, a nucleic acid sequence encoding a signal peptide, a coding region encoding an effector molecule, 3' untranslated region, and · polyA tail; and t) an IRES sequence, 5' untranslated region, additional start codon providing sequences, a nucleic acid sequence encoding a signal peptide, a coding region encoding an effector molecule, 3' untranslated region, and Poly A tail The compound is selected from the group consisting of:

[0035] In a tenth aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth aspects of the second aspect, in a ninth aspect of the third aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh and eighth aspects of the third aspect, in a tenth aspect of the fourth aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth aspects of the fourth aspect and in a ninth aspect of the fifth aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh and eighth aspects of the fifth aspect Cap structure, 5' untranslated region, a nucleic acid sequence encoding a signal peptide, a coding region encoding an effector molecule, 3' untranslated region, and Poly A tail Includes.

[0036] In an eleventh aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth aspects of the second aspect, in a tenth aspect of the third aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth aspects of the third aspect, in an eleventh aspect of the fourth aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth aspects of the fourth aspect and in a tenth aspect of the fifth aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth aspects of the fifth aspect IRES sequence, 5' untranslated region, a nucleic acid sequence encoding a signal peptide, a coding region encoding an effector molecule, 3' untranslated region, and Poly A tail Includes.

[0037] In a twelfth aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh aspects of the second aspect; in an eleventh aspect of the third aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth aspects of the third aspect; In a twelfth aspect of the fourth aspect which is also an embodiment of the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh aspects, and in an eleventh aspect of the fifth aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth aspects of the fifth aspect, the recombinant nucleic acid is Cap structure, 5' untranslated region, additional start codon providing sequences, a nucleic acid sequence encoding a signal peptide, a coding region encoding an effector molecule, 3' untranslated region, and Poly A tail Includes.

[0038] In a thirteenth aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and twelfth aspects of the second aspect; in a twelfth aspect of the third aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh aspects of the third aspect; In a thirteenth aspect of the fourth aspect which is also an embodiment of the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and twelfth aspects, and in a twelfth aspect of the fifth aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth and eleventh aspects of the fifth aspect, the recombinant nucleic acid is IRES sequence, 5' untranslated region, additional start codon providing sequences, a nucleic acid sequence encoding a signal peptide, a coding region encoding an effector molecule, 3' untranslated region, and Poly A tail Includes.

[0039] In a fourteenth aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth aspects of the second aspect; in a thirteenth aspect of the third aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and twelfth aspects of the third aspect; In a 14th aspect of the fourth aspect which is also an embodiment of the fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth aspects, and in a 13th aspect of the fifth aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and twelfth aspects of the fifth aspect, the further start codon providing sequence is in frame with the coding region encoding the effector molecule.

[0040] In a fifteenth aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth aspects of the second aspect; in a fourteenth aspect of the third aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth aspects of the third aspect; In a fifteenth embodiment of the fourth aspect which is also an embodiment of the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth embodiments and in a fourteenth embodiment of the fifth aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth embodiments of the fifth aspect, the 5' untranslated region is a 5' UTR.

[0041] In a sixteenth aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth and fifteenth aspects of the second aspect; in a fifteenth aspect of the third aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth aspects of the third aspect; In a 16th embodiment of the 4th aspect which is also an embodiment of the 10th, 11th, 12th, 13th, 14th and 15th embodiments, and in a 15th embodiment of the 5th aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th and 14th embodiments of the 5th aspect, the 5' untranslated region is derived from a eukaryotic organism, preferably a mammalian organism, more preferably a mammalian organism selected from the group comprising a human, a non-human primate, such as a cynomolgus monkey, a chimpanzee, a rhesus monkey, a rat and a mouse. In an alternative embodiment, the 5' untranslated region is derived from a viral gene, preferably the virus is a virus capable of infecting a eukaryotic organism.

[0042] In a seventeenth aspect of the second aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth and sixteenth aspects of the second aspect, and in a sixteenth aspect of the third aspect which is also an aspect of the fifteenth aspect, in a seventeenth aspect of the fourth aspect which is also an aspect of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth and sixteenth aspects of the fourth aspect, and in a seventeenth aspect of the fourth aspect which is also an aspect of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth aspects of the fifth aspect. In a sixteenth embodiment of the fifth aspect, which is also an embodiment of the tenth, eleventh, twelfth, thirteenth, fourteenth and fifteenth embodiments, the 5'UTR is selected from the group comprising the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding Ang-2 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding H3.3. or a derivative thereof having at least 85% nucleotide identity and the 5'UTR of a gene encoding Galectin-9 or a derivative thereof having at least 85% nucleotide identity.

[0043] In an 18th aspect of the second aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th and 17th aspects of the second aspect; in a 17th aspect of the third aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th and 16th aspects of the third aspect; In an 18th embodiment of the 4th aspect which is also an embodiment of the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th and 17th embodiments and in a 17th embodiment of the 5th aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th and 17th embodiments of the 5th aspect, the 3' untranslated region is a 3'-UTR.

[0044] In a 19th aspect of the second aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th and 18th aspects of the second aspect; in an 18th aspect of the third aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th and 17th aspects of the third aspect; In a 19th aspect of the 4th aspect which is also an embodiment of the 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th and 18th aspects, and in an 18th aspect of the 5th aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th and 15th, 16th, 17th aspects of the 5th aspect, the 3' untranslated region is derived from a eukaryotic organism, preferably a mammalian organism, more preferably a mammalian organism selected from the group comprising humans, non-human primates, such as cynomolgus monkeys, chimpanzees, rhesus monkeys, rats and mice. In an alternative embodiment, the 5' untranslated region is derived from a viral gene, preferably the virus is a virus capable of infecting eukaryotic organisms.

[0045] A 20th aspect of the second aspect which is also an aspect of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth and nineteenth aspects of the second aspect, and a 21st aspect of the second aspect which is also an aspect of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh and twelfth aspects of the third aspect in a 19th aspect of the third aspect which is also an embodiment of the 13th, 14th, 15th, 16th, 17th and 18th aspects; in a 20th aspect of the fourth aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th aspects of the fourth aspect; In a 19th embodiment of the 5th aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth and eighteenth aspects, the 3'UTR is selected from the group comprising the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity and the 3'UTR of a gene encoding H3.3. or a derivative thereof having at least 85% nucleotide identity.

[0046] In a 21st aspect of the second aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th aspects of the second aspect; in a 20th aspect of the third aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th aspects of the third aspect; In a 21st aspect of the 4th aspect which is also an aspect of the 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th aspects and in a 20th aspect of the 5th aspect which is also an aspect of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th and 19th aspects of the 5th aspect, the 5'UTR and the 3'UTR of the recombinant nucleic acid construct are of different origin, preferably from different species.

[0047] In a 22nd aspect of the second aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th and 21st aspects of the second aspect; in a 21st aspect of the third aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th aspects of the third aspect; In a 22nd aspect of the 4th aspect which is also an embodiment of the 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th and 21st aspects and in a 21st aspect of the 5th aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th aspects of the 5th aspect, the 5'UTR and the 3'UTR of the recombinant nucleic acid construct are of the same origin, preferably from the same species.

[0048] In a 23rd aspect of the second aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first and twenty-second aspects of the second aspect; in a 22nd aspect of the third aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth and twenty-first aspects of the third aspect; in a 23rd aspect of the 4th aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th, 21st and 22nd aspects of the 4th aspect, and in a 22nd aspect of the 5th aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th and 21st aspects of the 5th aspect, a) the 5'UTR is the 5'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity and the 3'UTR is selected from the group comprising the 3'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity and the 3'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, or b) the 5'UTR is the 5'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity and the 3'UTR is selected from the group comprising the 3'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity and the 3'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, or c) the 5'UTR is the 5'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity and the 3'UTR is selected from the group comprising the 3'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity and the 3'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, or d) the 5'UTR is the 5'UTR of a gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is selected from the group comprising the 3'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity.

[0049] In a 24th aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second and twenty-third aspects of the second aspect; in a 23rd aspect of the third aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first and twenty-second aspects of the third aspect; In a 24th aspect of the 4th aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, and 23rd aspects of the aspect; and in a 23rd aspect of the 5th aspect which is also an embodiment of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, and 22nd aspects of the 5th aspect, a) the 3'UTR is the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity and the 5'UTR is selected from the group comprising the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR of a gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity, and b) the 3'UTR is the 3'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR is selected from the group comprising the 5'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity; c) the 3'UTR is the 3'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity and the 5'UTR is selected from the group comprising the 5'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, or or d) the 3'UTR is the 3'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR is selected from the group comprising the 5'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity.

[0050] In a 25th aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third and twenty-fourth aspects of the second aspect, In a twenty-fourth aspect of the third aspect which is also an aspect of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second and twenty-third aspects of the third aspect, In a twenty-fifth aspect of the fourth aspect which is also an aspect of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third and twenty-fourth aspects, as well as in a twenty-fifth aspect of the fourth aspect which is also an aspect of the first, second, third, fourth, fifth, sixth, seventh, eighteenth, nineteenth, tenth, twenty-first, twenty-second, twenty-third and twenty-fourth aspects, In a 24th aspect of the fifth aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second and twenty-third aspects, the construct comprises A construct, wherein the 5'UTR is the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity. A construct, wherein the 5'UTR is the 5'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, A construct, wherein the 5'UTR is the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity. A construct, wherein the 5'UTR is the 5'UTR of a gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity. A construct, wherein the 5'UTR is the 5'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, A construct, wherein the 5'UTR is the 5'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity. A construct, wherein the 5'UTR is the 5'UTR of a gene encoding H3.3. or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding H3.3. or a derivative thereof having at least 85% nucleotide identity, or A construct, wherein the 5'UTR is the 5'UTR of a gene encoding galectin 9 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding galectin 9 or a derivative thereof having at least 85% nucleotide identity. The compound is selected from the group consisting of:

[0051] In a 26th aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth and twenty-fifth aspects of the second aspect, In a twenty-fifth aspect of the third aspect which is also an aspect of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third and twenty-fourth aspects of the third aspect, aspect, the third aspect, the fourth aspect, the fifth aspect, the sixth aspect, the seventh aspect, the eighth aspect, the ninth aspect, the tenth aspect, the eleventh aspect, the twelfth aspect, the thirteenth aspect, the fourteenth aspect, the fifteenth aspect, the sixteenth aspect, the seventeenth aspect, the eighteenth aspect, the nineteenth aspect, the twentieth aspect, the twenty-first aspect, the twenty-second aspect, and the twenty-sixth aspect of the fourth aspect which is also an aspect of the twenty-third aspect, the twenty-fourth aspect and the twenty-fifth aspect, In a 25th embodiment of the fifth aspect, which is also an embodiment of the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third and twenty-fourth embodiments, the construct comprises a polyA tail.

[0052] In a 27th aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth and twenty-sixth aspects of the second aspect, In a twenty-sixth aspect of the third aspect which is also an aspect of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth and twenty-fifth aspects of the third aspect, In the 27th aspect of the 4th aspect which is also an aspect of the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th and 26th aspects, and in the 1st aspect of the 5th aspect In a 26th embodiment of the 5th aspect, which is also an embodiment of the 2nd embodiment, the 3rd embodiment, the 4th embodiment, the 5th embodiment, the 6th embodiment, the 7th embodiment, the 8th embodiment, the 9th embodiment, the 10th embodiment, the 11th embodiment, the 12th embodiment, the 13th embodiment, the 14th embodiment, the 15th embodiment, the 16th embodiment, the 17th embodiment, the 18th embodiment, the 19th embodiment, the 20th embodiment, the 21st embodiment, the 22nd embodiment, the 23rd embodiment, the 24th embodiment and the 25th embodiment, the construct comprises a cap structure.

[0053] In a 28th aspect of the second aspect which is also an aspect of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second and twenty-third, twenty-fourth, twenty-fifth, twenty-sixth and twenty-seventh aspects of the second aspect, Aspect 27 of the third aspect which is also an aspect of aspect 2, aspect 3, aspect 4, aspect 5, aspect 6, aspect 7, aspect 8, aspect 9, aspect 10, aspect 11, aspect 12, aspect 13, aspect 14, aspect 15, aspect 16, aspect 17, aspect 18, aspect 19, aspect 20, aspect 21, aspect 22, aspect 23, aspect 24, aspect 25, and aspect 26 of the first, second, third, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, tenth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, and twenty-sixth aspects of the fourth aspect In the 28th aspect of the 4th aspect which is also an aspect of the 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th and 27th aspects, and in the 1st, 2nd, 3rd, 4th aspects of the 5th aspect, In a 27th embodiment of the 5th aspect, which is also an embodiment of the 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th and 26th embodiments, the construct comprises one or more IRES (internal ribosome entry site) sequences. In a preferred embodiment, the IRES sequences have replaced the cap structure.

[0054] In a 29th aspect of the second aspect which is also an aspect of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second and twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh and twenty-eighth aspects of the second aspect, In a twenty-eighth aspect of the third aspect which is also an aspect of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth and twenty-seventh aspects of the first, second and In a 29th aspect of the 4th aspect which is also an aspect of the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th and 28th aspects, and in the 1st, 2nd and 3rd aspects of the 5th aspect In a 28th embodiment of the fifth aspect, which is also an embodiment of the fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth and twenty-seventh embodiments, the construct comprises a nucleic acid sequence encoding a signal peptide.

[0055] In a 30th aspect of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, and twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, and twenty-ninth aspects of the second aspect, In a 29th aspect of the third aspect which is also an aspect of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh and twenty-eighth aspects of the aspect, , the third aspect, the fourth aspect, the fifth aspect, the sixth aspect, the seventh aspect, the eighth aspect, the ninth aspect, the tenth aspect, the eleventh aspect, the twelfth aspect, the thirteenth aspect, the fourteenth aspect, the fifteenth aspect, the sixteenth aspect, the seventeenth aspect, the eighteenth aspect, the nineteenth aspect, the twentieth aspect, the twenty-first aspect, the twenty-second aspect, the twenty-third aspect, the twenty-fourth aspect, the twenty-fifth aspect, the twenty-sixth aspect, the twenty-seventh aspect, the twenty-eighth aspect, and the thirtieth aspect of the fourth aspect which are also aspects of the twenty-ninth aspect, In a 29th embodiment of the fifth aspect, which is also an embodiment of the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh and twenty-eighth embodiments, the signal peptide allows secretion of the effector molecule.

[0056] In a 31st aspect of the second aspect which is also an aspect of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second and twenty-third, twenty-fourth, twenty-fifth, twenty-sixth, twenty-seventh, twenty-eighth, twenty-ninth and thirty-first aspects of the second aspect, In a 30th aspect of the third aspect which is also an aspect of aspects 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and 29 of the fourth aspect, In the 31st aspect of the 4th aspect which is also an aspect of aspect 19, aspect 20, aspect 21, aspect 22, aspect 23, aspect 24, aspect 25, aspect 26, aspect 27, aspect 28, aspect 29 and aspect 30, and in the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th and 25th aspects of the 5th aspect In a 30th embodiment of the fifth aspect, which is also an embodiment of the 26th, 27th, 28th and 29th embodiments, the nucleotide sequence encoding the signal peptide is a nucleotide sequence encoding a signal peptide of MCP-1 or a derivative thereof having at least 85% nucleotide identity, a nucleotide sequence encoding a signal peptide of IL-6 or a derivative thereof having at least 85% nucleotide identity, a nucleotide sequence encoding a signal peptide of Ang-2 or a derivative thereof having at least 85% nucleotide identity,and a nucleotide sequence encoding the signal peptide of Ang-1 or a derivative thereof having at least 85% nucleotide identity.

[0057] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the cell in which a cellular function is restored and / or in or on which a therapeutic effect is exerted is an endothelial cell, preferably a vascular endothelial cell, more preferably the vascular endothelial cell is a microvascular endothelial cell.

[0058] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the endothelial cells are non-dividing endothelial cells, non-proliferating endothelial cells or quiescent endothelial cells.

[0059] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the endothelial cell is an endothelial cell stimulated by an inflammatory stimulus.

[0060] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the endothelial cells are senescent endothelial cells.

[0061] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the endothelial cells are impaired with age and / or exhibit stress-related defects.

[0062] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the cell function may be restored by an effector molecule having an endothelial cell anti-permeability effect, an anti-vascular leakage effect, an endothelial cell anti-apoptotic effect, an endothelial cell anti-inflammatory effect, or an anti-stress response effect, in one embodiment of which the effector molecule stimulates vascular survival, inhibits regression, inhibits apoptosis, stimulates migration, stimulates remodeling, stimulates angiogenesis, stimulates lumen formation / invasion, stimulates proliferation, inhibits leukocyte adhesion, inhibits adhesion molecule expression, inhibits tissue factor expression, inhibits NFκB activity, or promotes monolayer integrity.

[0063] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the effect is related to or associated with the Tie-2 signaling pathway, the VEGF receptor pathway, the NOTCH signaling pathway, the PI3-kinase pathway, the eNOS signaling pathway, the sirtuin-dependent metabolic and energy homeostasis pathway, the oxidative stress pathway, the shear stress response pathway, the ET-1 signaling pathway, the NO-mediated signaling pathway, and the mechanochemical transduction pathway, preferably the pathway is selected from the group comprising the Ang / Tie-2 signaling pathway, the VEGF / VEGF receptor pathway and the Notch / Notch ligand pathway.

[0064] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the effector molecule is a member of a pathway selected from the group comprising the Tie-2 signaling pathway, the VEGF receptor pathway, the NOTCH signaling pathway, the PI3-kinase pathway, the eNOS signaling pathway, the sirtuin-dependent metabolic and energy homeostasis pathway, the oxidative stress pathway, the shear stress response pathway, the ET-1 signaling pathway, the NO-mediated signaling pathway, and the mechanochemical transduction pathway.

[0065] In one embodiment of the first, second, third, fourth, and fifth aspects (including any embodiment thereof), the effector molecule is selected from the group comprising Ang-1, Ang-4, COMP-Ang-1, hCOMP-Ang-1, COMP-Ang-1, COMP-ANG-2, Tie-2 receptor, Tie-1 receptor, PI3-kinase, preferably constitutively active PI3-kinase, highly active Tie-2 receptor (e.g. R849W), VE-cadherin, GRB2, GRB7, GRB14, GRB7, IQGAP1, RAC1, RAP1, DOK2, ABIN1 and ABIN2, KLF2 (Krueppel-like factor 2), alpha5beta1 integrin, CD73, Akt1, Akt2, and Akt3.

[0066] In one embodiment of the first, second, third, fourth and fifth aspect (including any embodiment thereof), the cellular function is vascular leakage and preferably the pathway is the Tie-2 pathway.

[0067] In one embodiment of the first, second, third, fourth, and fifth aspects (including any embodiment thereof), the effector molecule is selected from the group comprising VEGF, VEGF-A, VEGF-B, PDGF, bFGF, sirtuin, eNOS, RAS, and c-MYC.

[0068] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the effector molecule restores revascularization or provides revascularization, preferably vascular endothelial revascularization.

[0069] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the cell in which a cellular function is restored and / or in or on which a therapeutic effect is exerted is a non-endothelial cell.

[0070] In one embodiment of the first, second, third, fourth, and fifth aspects (including any embodiment thereof), the non-endothelial cell is selected from the group comprising an epithelial cell, an alveolar macrophage, a type I pneumocyte, a type II pneumocyte, an alveolar macrophage, a lung cell, a lung epithelial cell, a hematopoietic cell, a bone marrow cell, a bone cell, a stem cell, a mesenchymal cell, a nervous system cell, a glial cell, a neuronal cell, an astrocyte, a cell of the peripheral nervous system, a cardiac cell, an adipocyte, a vascular smooth muscle cell, a cardiac muscle cell, a skeletal muscle cell, a beta cell, a pituitary cell, a synovial lining cell, an ovarian cell, a testicular cell, a B cell, a T cell, a reticulocyte, a leukocyte, a granulocyte, a macrophage, a neutrophil, an antigen presenting cell (dendritic cell), a fibroblast, a liver cell, a tumor cell, and combinations thereof.

[0071] In one embodiment of the first, second, third, fourth, and fifth aspects (including any embodiment thereof), the effector molecule is selected from the group comprising tumor death ligands, factors involved in hematopoiesis and coagulation, stem cell factors, growth factors, and cytokines.

[0072] In one embodiment of the first, second, third, fourth, and fifth aspects (including any embodiment thereof), the tumor death ligand is selected from the group including IL-12, a member of the TNF gene superfamily of death ligand proteins, such as Apo2L / TRAIL, IL-13, IL-10, IL-8, IL-2, interferon beta, secreted frizzled related protein (SFRP) 1, SFRP2, SFRP3, SFRP4, and SFRP5.

[0073] In one embodiment of the first, second, third, fourth, and fifth aspects (including any embodiment thereof), the factor involved in hematopoiesis and coagulation is selected from the group comprising erythropoietin, factor VII, factor VIII, factor IX, and heparan-N-sulfatase.

[0074] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the stem cell factor is selected from the group comprising Oct4, Sox2, Klf4 and c-Myc.

[0075] In one embodiment of the first, second, third, fourth, and fifth aspects (including any embodiment thereof), the growth factors and cytokines are selected from the group consisting of adrenomedullin, angiopoietin family, autocrine motility stimulating factor, bone morphogenetic proteins, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), macrophage colony stimulating factor (m-CSF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), epidermal growth factor (EGF), ephrin, erythrocyte stimulating factor (ESF), and / or erythrocyte-specific cytokines (EGF). Also called endothelial cell line (EPO), fibroblast growth factor family (FGF1-23), somatotropin, GDNF ligand family (e.g. glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin and artemin), growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor-1 and insulin-like growth factor-2 (IGF-1, IGF-2), keratinocyte growth factor (KGF), migration stimulating factor (MSF), hepatocyte growth factor-like protein (HGFLP) Macrophage-stimulating protein (MSP), myostatin (GDF-8), neuregulin 1-4 (NRG1-4), neurotrophins (brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS) - an anti-apoptotic survival factor, T cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor family (VEGF), Wnt signaling pathway signaling glycoproteins (WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-α, interferon-β,Interleukin 2 (IL-2), Interleukin 11 (IL-11), Interferon-γ, IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL- 17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, and IL-36. ,

[0076] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the disease is characterized by or caused by vascular leakage, preferably leakage of vascular endothelial cells, more preferably leakage of microvascular endothelial cells.

[0077] In one embodiment of the first, second, third, fourth, and fifth aspects (including any embodiment thereof), the subject suffering from the disease exhibits inflammation in the lungs.

[0078] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the disease is selected from the group comprising pneumonia, sepsis and trauma.

[0079] In one embodiment of the first, second, third, fourth, and fifth aspects (including any embodiment thereof), the subject suffering from the disease exhibits acute respiratory distress syndrome (ARDS).

[0080] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the disease is pneumonia, preferably pneumonia selected from the group comprising severe community-acquired pneumonia (sCAP), community-acquired pneumonia (CAP), hospital-acquired pneumonia (HAP).

[0081] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the disease is characterised by or exhibits lung damage.

[0082] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the lung injury is immediate lung damage.

[0083] In one embodiment of the first, second, third, fourth, and fifth aspects (including any embodiment thereof), the direct lung injury results from inhalation of toxic gases, toxic pulmonary edema, viral or bacterial lung infection, aspiration of gastric contents, aspiration of fresh water, aspiration of salt water, pulmonary contusion, fat embolism, amniotic fluid embolism, and inhalation of hyperbaric oxygen.

[0084] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the lung injury is indirect lung injury.

[0085] In one embodiment of the first, second, third, fourth, and fifth aspects (including any embodiment thereof), the indirect lung injury results from sepsis, bacteremia, endotoxemia, severe trauma, multiple trauma including shock, burns, pancreatitis, falciparum malaria, drugs and immunosuppression, chronic alcohol abuse, chronic lung disease, low serum pH.

[0086] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the disease or effect is vascular regeneration or life extension, in which case preferably the effector molecule is selected from the group comprising VEGF, VEGF-A, VEGF-B, PDGF, bFGF, Sirtuin (Sirt1), PPAR-γ, AMPK, eNOS, RAS, c-MYC, FOXO3, PI3 kinase*, Akt, Akt* (* indicates fusions or truncations to generate hyperactive and constitutively active derivatives of the kinase), adenosine A1 receptor, adenosine A2A receptor, adenosine A2B receptor, telomerase, Yamanaka factors (i.e. Oct4, sox2, klf4, c-My).

[0087] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the effector molecule is an anti-apoptotic factor, preferably an anti-apoptotic factor that protects normal endothelium, i.e. non-diseased endothelium, and the anti-apoptotic factor is preferably selected from the group comprising PTEN, survivin, IAP, cIAP2 and XIAP, neuroglobin (binds cytochrome c), the pro-survival proteins Bcl-2, Bcl-xl, Bcl-w, mcl-1 A1, NR-13, BHRF1, LMW5-HL, ORF16, KS-Bcl-2, E1b-19K and P53.

[0088] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the effector molecule is an apoptotic factor, preferably an apoptotic factor that targets the tumor endothelium, and the apoptotic factor is preferably selected from the group comprising PTEN, survivin, IAP, cIAP2 and XIAP, neuroglobin (binds cytochrome c), pro-survival proteins Bcl-2, Bcl-xl, Bcl-w, mcl-1 A1, NR-13, BHRF1, LMW5-HL, ORF16, KS-BCl-2, E1b-19K, P53.

[0089] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the effector molecule is a tumor death ligand, preferably the tumor death ligand is pro-apoptotic or anti-angiogenic, more preferably the tumor death ligand is a member of the TNF gene superfamily of death ligand proteins, such as TNF-α, Fas-L, Apo2L / TRAIL, IL-12, IL-13, IL-10, IL-8, IL-2, the secreted frizzled related protein (SFRP) family (SFRP1, 2, 3, 4, 5), ELTD1, TGF-β, TNF-a, IL-6, P The inhibitor is selected from the group including TEN, p53 and other tumor suppressor genes with pro-apoptotic function, thrombospondin C, TIMP-1, TIMP-2, interferon-alpha, interferon-beta, interferon-gamma, Ang-1 and derivatives with Tie-2 agonist function, cytochrome c, BH3-only proteins, the pro-apoptotic Bcl-2 proteins Bad, Bid, Bax, Bak and Bim, Apaf1, pro-caspase-8, pro-caspase-9, pro-caspase-10, caspase 2, caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, and caspase 10.

[0090] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the effector molecule is anti-inflammatory, preferably the effector molecule is selected from the group comprising IκB and derivatives with mutated phosphorylation sites, PI3 kinase and Akt kinase and hyperactive forms.

[0091] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the effector molecule is active in blood coagulation and / or hematopoiesis, preferably the effector molecule is selected from the group comprising erythropoietin, Factor VII, Factor VIII, Factor IX and heparan-N-sulfatase.

[0092] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the effector molecule is a gene-editing nuclease such as Cas9.

[0093] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the effector molecule encodes a therapeutic antibody or a decoy.

[0094] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the effector molecule is effective as a tumour vaccine, preferably the effector molecule is an antigen which induces an immune response against tumour cells.

[0095] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the effector molecule is a secreted protein.

[0096] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the effector molecule is effective as an autocrine factor.

[0097] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the effector molecule is effective as a paracrine factor.

[0098] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the recombinant nucleic acid construct consists of ribonucleotides.

[0099] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the recombinant nucleic acid construct consists of deoxyribonucleotides.

[0100] In one embodiment of the first, second, third, fourth and fifth aspects (including any embodiment thereof), the recombinant nucleic acid construct consists of ribonucleotides and deoxyribonucleotides.

[0101] More particularly, the problem underlying the present invention is solved in a sixth aspect, which is also a first aspect of the sixth aspect, by a vector comprising a recombinant nucleic acid construct of the first aspect (including any aspect thereof) and / or a recombinant nucleic acid construct as described in relation to any aspect, such as described in relation to any aspect thereof, preferably any aspect of the second, third, fourth and fifth aspect.

[0102] In a second embodiment of the sixth aspect which is also an embodiment of the first embodiment of the sixth aspect the vector is an expression vector.

[0103] In a third embodiment of the sixth aspect which is also an embodiment of the second embodiment of the sixth aspect, the vector allows expression of the recombinant nucleic acid construct in a cell, preferably an endothelial cell, more preferably the vector is a plasmid or a virus.

[0104] More particularly, the problem underlying the present invention is solved in a seventh aspect, which is also a first aspect of the seventh aspect, by a cell comprising a recombinant nucleic acid construct of the first, second, third, fourth and fifth aspect (including any aspect thereof) and / or a vector of the seventh aspect (including any aspect thereof).

[0105] In a second embodiment of the seventh aspect which is also an embodiment of the first embodiment of the seventh aspect the cell is an endothelial cell or a mesenchymal stem cell.

[0106] In a third embodiment of the seventh aspect, which is also an embodiment of the first and second embodiments of the seventh aspect, the cell is a recombinant cell.

[0107] In a fourth embodiment of the seventh aspect, which is also an embodiment of the first, second and third embodiment of the seventh aspect, the cell is an isolated cell.

[0108] More specifically, the problem underlying the present invention is solved in an eighth aspect, which is also a first aspect of the eighth aspect, by a delivery vehicle which is a cationic lipid delivery particle comprising a recombinant nucleic acid construct of the first, second, third, fourth and fifth aspects (including any aspects thereof).

[0109] In a second embodiment of the eighth aspect which is also an embodiment of the first embodiment of the eighth aspect the particles are nanoparticles.

[0110] In a third embodiment of the eighth aspect, which is also an embodiment of the first and second embodiments of the eighth aspect, the average size of the nanoparticles is from about 30 nm to about 200 nm, preferably from about 30 nm to about 140 nm, and more preferably from about 30 nm to about 60 nm.

[0111] More particularly, the problem underlying the present invention is solved in a ninth aspect, which is also a first aspect of the ninth aspect, by a composition comprising a recombinant nucleic acid construct of the first, second, third, fourth and fifth aspects (including any embodiment thereof), a vector of the sixth aspect (including any embodiment thereof), a cell of the seventh aspect (including any embodiment thereof) and / or a delivery vehicle of the eighth aspect (including any embodiment thereof). In a preferred embodiment, the composition is a pharmaceutical composition comprising a pharma- ceutically acceptable excipient.

[0112] More specifically, the problem underlying the present invention is also solved in a tenth aspect by the use of a recombinant nucleic acid construct of the first, second, third, fourth and fifth aspects (including any embodiment thereof), a vector of the sixth aspect (including any embodiment thereof), a cell of the seventh aspect (including any embodiment thereof), a cell of the seventh aspect (including any embodiment thereof) and / or a delivery vehicle of the eighth aspect (including any embodiment thereof) in the manufacture of a medicament. In one embodiment thereof the medicament is for the treatment of a disease, preferably for the treatment of a disease as described herein in connection with the second, third, fourth and fifth aspects (including any embodiment thereof).

[0113] More specifically, the problem underlying the present invention is also solved in an eleventh aspect by a method for treating a subject, preferably a human subject, comprising administering to the subject a therapeutically effective amount of a recombinant nucleic acid construct of the first, second, third, fourth and fifth aspects (including any embodiment thereof), a vector of the sixth aspect (including any embodiment thereof), a cell of the seventh aspect (including any embodiment thereof), a delivery vehicle of the eighth aspect (including any embodiment thereof) and / or a composition of the ninth aspect (including any embodiment thereof). In one embodiment thereof, the method is a method for the treatment of a disease, preferably a method for the treatment of a disease as described herein in connection with the second, third, fourth and fifth aspects (including any embodiment thereof).

[0114] More specifically, the problem underlying the present invention is also solved in a twelfth aspect by a method for vaccinating a subject, preferably a human subject, comprising administering to the subject a recombinant nucleic acid construct of the first, second, third, fourth and fifth aspects (including any of their embodiments), a vector of the sixth aspect (including any of its embodiments), a cell of the seventh aspect (including any of its embodiments), a delivery vehicle of the eighth aspect (including any of its embodiments) and / or a composition of the ninth aspect (including any of its embodiments) in an amount effective to induce an immune response, preferably a desired immune response, in the subject. In one embodiment thereof, the method is a method for the treatment of a disease, preferably a method for the treatment of a disease as described herein in connection with the second, third, fourth and fifth aspects (including any of their embodiments). In a preferred embodiment, the effector molecule is capable or suitable for eliciting such an immune response.

[0115] In one embodiment, and preferably as used herein, a heterologous construct or a heterologous recombinant construct is a construct that does not exist in a wild-type biological system, such as a virus, a cell, a tissue, an organ or an organism. More preferably, a heterologous construct or a recombinant construct is one in which at least one element selected from the group consisting of a 5' untranslated region, a 3' untranslated region, a cap structure, a signal sequence and a polyA tail is not combined with the coding region of an effector. Preferably, the 5' untranslated region is a 5' UTR and the 3' untranslated region is a 3' UTR.

[0116] In one embodiment, and as preferably used herein, a method of therapy or a method for the treatment and / or prevention of a disease is a therapy.

[0117] In one embodiment of all aspects of the invention, the coding region encoding the effector molecule is expressed by an endothelial cell. In a preferred embodiment thereof, the endothelial cell expressing the effector molecule is an endothelial cell whose function is impaired. In an alternative preferred embodiment, the endothelial cell expressing the effector molecule is an endothelial cell whose function is not impaired, in which case the effector molecule is exported or secreted by the effector molecule-producing endothelial cell and exerts its effect on any other cells whose function is impaired, typically on any other endothelial cells whose function is impaired. In a preferred embodiment, the impaired function of the cell is a function of the cell that is restored in accordance with various aspects of the invention.

[0118] Preferably, as used herein, a derivative of a sequence exhibits the same functionality as the parent sequence, which is preferably a wild-type sequence. In a preferred embodiment, the sequence of the derivative and the sequence of the parent have a similar functionality to each other by at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least It is within the scope of the present invention that the derivative is a truncated version of the parent sequence. Such truncation may be at the 5' end, at the 3' end, or at both the 5' and 3' ends. When referring to the parent sequence in terms of identity, such identity preferably refers to the subsequent stretch of nucleotides shared by both the derivative and the parent sequence, with the sequence of the parent sequence serving as the reference sequence.

[0119] In one embodiment, and as preferably used herein, a coding region encoding an effector molecule comprises or consists of a nucleic acid sequence encoding said effector molecule.

[0120] In one embodiment, and preferably as used herein, a 5' untranslated region is a 5' untranslated sequence.

[0121] In one embodiment, and preferably as used herein, a 3' untranslated region is a 5' untranslated sequence.

[0122] In one embodiment, and preferably as used herein, the indication of any source of any one of the elements contained in the recombinant nucleic acid construct of the present invention shall be understood as a substitute for an alternative description of the nucleotide sequence. Such nucleotide sequence is preferably of the respective wild type sequence, more preferably of the respective human wild type sequence. It will be understood by those skilled in the art that species other than human may also provide such wild type sequences. And, in one embodiment of the present invention, such wild type sequences of such species other than human may be used in the practice of the present invention. It will be understood by those skilled in the art that such wild type sequences may be obtained from public databases such as GenBank. Wild type sequences may be modified over time, typically to correct sequencing errors. In one embodiment of the present invention, the sequence is as described in said public databases such as GenBank as of the filing date of this application. To the extent that various respective wild type sequences exist, any such wild type sequences shall be encompassed by the present invention. In another embodiment, when there are two or more wild type sequences and the two or more wild type sequences are contributed by different human groups, e.g., by different geographically defined human groups and / or by different genetically defined human groups, the wild type sequence that is predominant in the most widespread of the different human groups may be considered the wild type sequence.

[0123] In one embodiment of the invention, and preferably as used herein, when reference is made to a derivative of the 5'UTR of a gene indicating that the derivative has at least 85% nucleotide identity, the identity is with reference to or based on the 5'UTR of that gene.

[0124] In one embodiment of the invention, and preferably as used herein, when reference is made to a derivative of the 3'UTR of a gene indicating that the derivative has at least 85% nucleotide identity, the identity is with reference to or based on the 3'UTR of that gene.

[0125] In one aspect, and preferably as used herein, a signal peptide is a peptide that directly or indirectly mediates the transport or secretion into the extracellular space of a polypeptide or protein to which it is fused or attached, after which the signal sequence is preferably removed from the polypeptide and protein, respectively.

[0126] In one embodiment of the present invention, the recombinant nucleic acid construct is a recombinant nucleic acid construct disclosed in the figures, more specifically, a recombinant nucleic acid construct disclosed in Figure 40 (PAN29), Figure 90 (PAN58), Figure 86 (PAN54), Figure 67 (PAN50), Figure 45 (PAN34), Figure 44 (PAN33), Figure 39 (PAN28), Figure 37 (PAN12), Figure 88 (PAN56).

[0127] In one embodiment of the present invention, and as preferably used herein, a decoy is a protein encoded by an mRNA that binds to a ligand and neutralizes its signaling activity.

[0128] In one embodiment, and as preferably used herein, a poly-A tail is a nucleotide sequence that includes or consists of multiple As covalently linked to each other, such covalent bonds preferably being phosphodiester bonds. In one embodiment thereof, the poly-A tail includes from about 20 to about 240 A (i.e., adenosine phosphate) nucleotides, preferably from about 60 to about 120 As, more preferably from about 100 to about 140 As, and most preferably about 120 As.

[0129] PolyA tails are believed to stabilize natural messengers and synthetic sense RNA. Therefore, in one embodiment, a long polyA tail can be added to an mRNA molecule to make the RNA more stable. PolyA tails can be added using various techniques recognized in the art. For example, polyA polymerase can be used to add long polyA tails to synthetic or in vitro transcribed RNA (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode a long polyA tail. In addition, polyA tails can be added by transcription directly from PCR products. PolyA can be ligated to the 3' end of sense RNA using RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Edition, edited by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)). In one embodiment, the length of the poly-A tail is at least about 90, 200, 300, 400 nucleotides, or at least 500 nucleotides. In one embodiment, the length of the poly-A tail is adjusted to control the stability of the modified sense mRNA of the present invention, and therefore the transcription of the protein. For example, since the length of the poly-A tail can affect the half-life of the sense mRNA molecule, the length of the poly-A tail can be adjusted to change the level of resistance of the mRNA to nucleases, thereby controlling the time course of protein expression in cells. In one embodiment, the stabilized nucleic acid molecules are sufficiently resistant to degradation in vivo (e.g., by nucleases) that they can be delivered to target cells without a delivery vehicle. In yet another embodiment, the nucleic acid molecules can be purified using HPLC to remove double-stranded RNA contaminants, thereby reducing the activation of the innate immune system (see, e.g., Kariko et al., Nucleic Acids Res. 2011 Nov;39(21):e142).

[0130] In one embodiment, and as preferably used herein, a cap structure is a nucleotide sequence that forms a cap structure known to those skilled in the art. In eukaryotic cells, the cap structure consists of a m7G(5')ppp(5')G5'-5'-triphosphate bond structure. The first two ribose sugars at the 5' end of the mRNA can be methylated at the 2' position. This is known as CAP-1 (when the first of the two ribose sugars is methylated) and CAP-2 structure (when the second of the two ribose sugars is methylated). The cap structure can be incorporated during mRNA transcription or can be incorporated post-transcriptionally using a capping enzyme and a 2'-O-methyltransferase enzyme with S-adenosylmethionine (SAM) as the methyl donor. In addition, phosphatase treatment can be used to remove the immunostimulatory 5' phosphate group.

[0131] In a preferred embodiment of the recombinant nucleic acid construct of any aspect (including any embodiment thereof), the recombinant nucleic acid construct is an mRNA.

[0132] In one embodiment of all aspects of the invention (including any embodiment thereof), the 5'UTR of the gene encoding MCP-1 comprises the nucleotide sequence of SEQ ID NO:20.

[0133] In one embodiment of all aspects of the invention (including any embodiment thereof), the 3'UTR of the gene encoding MCP-1 comprises the nucleotide sequence of SEQ ID NO:23.

[0134] In one embodiment of all aspects of the invention (including any embodiment thereof), the 5'UTR of the gene encoding RPL12s.c. comprises the nucleotide sequence of SEQ ID NO:24.

[0135] In one embodiment of all aspects of the invention (including any embodiment thereof), the 3'UTR of the gene encoding RPL12s.c. comprises the nucleotide sequence of SEQ ID NO:25.

[0136] In one embodiment of all aspects of the invention (including any embodiments thereof), the 5'UTR of the gene encoding Ang-2 comprises the nucleotide sequence of SEQ ID NO:26.

[0137] In one embodiment of all aspects of the invention (including any embodiment thereof), the 3'UTR of the gene encoding Ang-2 comprises the nucleotide sequence of SEQ ID NO:29.

[0138] In one embodiment of all aspects of the invention (including any embodiment thereof), the 5'UTR of the gene encoding HSP70 comprises the nucleotide sequence of SEQ ID NO:38 or SEQ ID NO:40.

[0139] In one embodiment of all aspects of the invention (including any embodiment thereof), the 3'UTR of the gene encoding HSP70 comprises the nucleotide sequence of SEQ ID NO:39 or SEQ ID NO:43.

[0140] In one embodiment of all aspects of the invention (including any embodiment thereof), the 5'UTR of the gene encoding H3.3. comprises the nucleotide sequence of SEQ ID NO:44.

[0141] In one embodiment of all aspects of the invention (including any embodiment thereof), the 3'UTR of the gene encoding H3.3. comprises the nucleotide sequence of SEQ ID NO:45.

[0142] In one embodiment of all aspects of the invention (including any embodiment thereof), the 5'UTR of the gene encoding galectin-9 (LHALS9) comprises the nucleotide sequence of SEQ ID NO:46.

[0143] In one embodiment of all aspects of the invention (including any embodiment thereof), the 3'UTR of the gene encoding galectin-9 (LHALS9) comprises the nucleotide sequence of SEQ ID NO:47.

[0144] In one embodiment of all aspects of the invention (including any embodiment thereof), the 5'UTR of the gene encoding IL-6 comprises the nucleotide sequence of SEQ ID NO:30.

[0145] In one embodiment of all aspects of the invention (including any embodiment thereof), the 3'UTR of the gene encoding IL-6 comprises the nucleotide sequence of SEQ ID NO:33.

[0146] In one embodiment of all aspects of the invention (including any embodiment thereof), the 5'UTR of the gene encoding vWF comprises the nucleotide sequence of SEQ ID NO:34.

[0147] In one embodiment of all aspects of the invention (including any embodiment thereof), the 3'UTR of the gene encoding vWF comprises the nucleotide sequence of SEQ ID NO:37.

[0148] In one embodiment of all aspects of the invention (including any embodiment thereof), the 5'UTR of the gene encoding Ang-1 comprises the nucleotide sequence of SEQ ID NO:83.

[0149] In one embodiment of all aspects of the invention (including any embodiment thereof), the 3'UTR of the gene encoding Ang-1 comprises the nucleotide sequence of SEQ ID NO:88.

[0150] In one embodiment of all aspects of the invention (including any embodiment thereof), the 5'UTR of the gene encoding Ang-4 comprises the nucleotide sequence of SEQ ID NO:89.

[0151] In one embodiment of all aspects of the invention (including any embodiment thereof), the 3'UTR of the gene encoding Ang-4 comprises the nucleotide sequence of SEQ ID NO:94.

[0152] In one embodiment of all aspects of the invention (including any embodiment thereof), the 5'UTR of construct PAN57 comprises the nucleotide sequence of SEQ ID NO:120.

[0153] In one embodiment of all aspects of the invention (including any embodiment thereof), the 3'UTR of construct PAN57 comprises the nucleotide sequence of SEQ ID NO:121.

[0154] In one embodiment of all aspects of the present invention (including any embodiments thereof), the various combinations of 5'UTRs and 3'UTRs realized in any specific construct disclosed herein are combinations of 5'UTRs and 3'UTRs that may be realized in a recombinant nucleic acid construct independent of any other components, and in particular independent of any coding sequence encoding an effector molecule contained in a recombinant nucleic acid construct of the present invention.

[0155] In one embodiment of all aspects of the present invention (including any of its embodiments), and preferably as used herein, endogenous gene refers to a gene contained in any cell described herein.Thus, a recombinant nucleic acid construct derived from an endogenous gene with different 5'UTR and 3'UTR comprises a 5'UTR derived from a first gene of a cell and a 3'UTR derived from a second gene of a cell, for example, the 5'UTR derived from a gene encoding MCP-1 or Ang-2, and the 3'UTR derived from a gene encoding vWF.The cells may be the same or different cells, and / or the cells may be the same or different types of cells.Preferably, the cells are cells of human origin.

[0156] In a preferred embodiment of the recombinant nucleic acid construct of any aspect (including any embodiment thereof), the recombinant nucleic acid construct is a recombinant nucleic acid molecule.

[0157] It is within the scope of the present invention, as defined by any aspect, including any of its embodiments, that the nucleotides forming the recombinant nucleic acid molecule may be modified. Suitable modifications include changing one or more nucleotides of a codon to code for the same amino acid, but to a codon that is more stable than the codon found in the wild-type version of the nucleic acid. For example, an inverse relationship between RNA stability and the abundance of cytidine (C) and / or uridine (U) residues has been demonstrated, and RNA lacking C and U residues has been found to be stable against most RNases (Heidenreich, et al. J Biol Chem 269, 2131-8 (1994)). In some embodiments, the number of C and / or U residues in the mRNA sequence is reduced. In another embodiment, the number of C and / or U residues is reduced by substituting a codon that codes for a particular amino acid for another codon that codes for the same or a related amino acid. Possible modifications to the mRNA nucleic acid of the present invention also include the incorporation of pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, and 5'-methylcytidine. The incorporation of such modified nucleotides into the recombinant nucleic acid construct, preferably into the mRNA of the present invention, can increase stability and translational capacity, as well as reduce immunogenicity in vivo. (See, for example, Kariko, K., et al., Molecular Therapy 16(11):1833-1840(2008)). Substitutions and modifications to the recombinant nucleic acid construct of the present invention, particularly at the level of individual nucleotides of the recombinant nucleic acid construct, can be performed by methods readily apparent to those skilled in the art.

[0158] The constraints on reducing the number of C and U residues in a sequence are probably greater in the coding regions of an mRNA than in untranslated regions (i.e., it probably would not be possible to remove all of the C and U residues present in a message while still retaining the ability of the message to code for the desired amino acid sequence). However, due to the degeneracy of the genetic code, there is the opportunity to reduce the number of C and / or U residues present in a sequence while still maintaining the same coding capacity (i.e., depending on which amino acid a codon codes for, there may be several different possibilities for modifying an RNA sequence). For example, codons for Gly can be changed to GGA or GGG instead of GGU or GGC.

[0159] The term modification also encompasses, for example, the incorporation of non-nucleotide bonds or modified nucleotides into the nucleic acid sequences of the invention (e.g., modification of either or both of the 3' and 5' ends of an mRNA molecule encoding a functional protein or enzyme). Such modifications include the addition of bases to the nucleic acid sequence (e.g., incorporation of a polyA tail or a longer polyA tail), alteration of the 3' or 5' UTR, complexation of the nucleic acid with an agent (e.g., a protein or a complementary nucleic acid molecule), and incorporation of elements that alter the structure of the nucleic acid molecule (e.g., elements that form secondary structures).

[0160] The delivery vehicle of the eighth aspect is preferably based on a composition of one or more suitable lipids (e.g., liposomes) that are combined with a recombinant nucleic acid construct of the invention to collectively result in a cationic-lipid-nucleic acid particle (cLNP). Such cLNPs can be used to functionally introduce nucleic acids, particularly mRNA, into endothelial cells of the mammalian vasculature.

[0161] Lipid-nucleic acid nanoparticles (LNPs), formed by complexing nucleic acids, such as the recombinant nucleic acid constructs of the invention, with cationic lipids in combination with other lipid components, such as zwitterionic phospholipids, cholesterol, and PEGylated lipids, have already been used to prevent RNA degradation in plasma and facilitate functional cellular uptake of RNA.

[0162] However, in the context of therapy, especially considering parenteral therapeutic applications, the overall charge of lipid-nucleic acid nanoparticles, characterized by zeta potential, is crucial in directing the organ distribution of nanoparticles. Those skilled in the art will understand that overall neutrally charged LNPs are taken up almost exclusively by organs of the reticuloendothelial system, especially the liver and spleen. On the other hand, those skilled in the art also know that overall positively charged lipid nanoparticles are particularly useful for functionally delivering pharmaceutical payloads into cells of the mammalian vasculature.

[0163] It will also be appreciated by those skilled in the art that free nucleic acids, particularly mRNA, when applied intravenously, are immunostimulatory.

[0164] Therefore, in consideration of parenteral therapeutic applications, complete and stable complex formation of nucleic acid inside lipid nanoparticles is considered to be essential to prevent undesirable immunogenic side reactions.Furthermore, it is known to those skilled in the art that the size of lipid-nucleic acid nanoparticles is extremely important in suppressing the reticuloendothelial clearance of the particles.

[0165] It has recently been described that highly potent, limit-sized siRNA LNPs (average particle size approx. 30 nm) can be prepared using a microfluidic mixing process based on a staggered herringbone micromixer (SHM) device (Belliveau et al.; Molecular Therapy-Nucleic Acids (2012) 1(8)).

[0166] Due to the millisecond mixing of lipids and nucleic acids, such as the recombinant nucleic acid constructs of the present invention, on the nanoliter scale, the resulting particles are characterized by extremely tight packing of nucleic acids within a dense solid particle core, whereas those skilled in the art will appreciate that macrofluidic mixing processes using T-connectors generally result in larger nanoparticles with more heterogeneous multilamellar morphology.

[0167] In one aspect, the invention provides novel positively charged lipid-nucleic acid nanoparticles comprising a nucleic acid, such as a recombinant nucleic acid construct of the invention, for functional delivery of the recombinant nucleic acid construct of the invention, particularly mRNA, into cells of the mammalian vasculature, particularly endothelial cells of the pulmonary vasculature.

[0168] In one embodiment of the various aspects of the present invention (including any embodiment thereof), a cationic lipid selected from the group comprising β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleyl-amide, L-arginyl-β-alanine-N-palmityl-N-oleyl-amide, DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl chloride), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt)), or DC-cholesterol is combined with a neutral lipid and an additional shielding lipid component having a polyethylene glycol (PEG) modification. The neutral lipid can be a zwitterionic phospholipid selected from the group including diphytanoyl-PE (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-lauroyl-sn-glycero-3-phosphoethanolamine), DMPE, POPE, DSPE, or an uncharged sterol lipid selected from the group including cholesterol and stigmasterol. The PEGylated lipid component is selected from the group including methoxy PEG-DSPE, methoxy PEG-DLG, methoxy PEG-DMG, methoxy PEG-DPG, methoxy PEG-DSG, methoxy PEG-c-DMA, methoxy PEG-C8-ceramide, methoxy PEG-C16-ceramide. In a particular embodiment, the chain length of the PEG chain corresponds to a molecular weight in the range of 750 Da to 5000 Da, preferably in the range of 1500 Da to 3000 Da.

[0169] In another embodiment of the present invention, the molar ratio of lipids in the lipid mixture is in the range of 20-80 mol % cationic lipid, 10-70 mol % neutral lipid, and 1-10 mol % PEGylated lipid, preferably in the range of 35-65 mol % cationic lipid, 35-65 mol % neutral lipid, and 1-5 mol % PEGylated lipid (total lipid content is taken as 100%).

[0170] In a preferred embodiment, the cationic lipid is β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleyl-amide or L-arginyl-β-alanine-N-palmityl-N-oleyl-amide, the neutral lipid is phospholipid diphytanoyl-PE, and the PEGylated lipid is methoxyPEG2000-DSPE. The molar ratio is 50 mol% cationic lipid, 49 mol% diphytanoyl-PE, and 1 mol% mPEG2000-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](sodium salt)).

[0171] In yet another embodiment, the mixture of lipids described above is dissolved in a water-miscible solvent selected from the group comprising ethanol, acetone, 1-butanol, 2-butanol, tert.-butanol, 3-methyl-1-butanol, 2-methyl-1-propanol, 1-propanol, 2-propanol, dimethylsulfoxide, preferably the group comprising ethanol, tert.-butanol, and 1-butanol.

[0172] The dissolved lipid mixture is then rapidly mixed with a nucleic acid in an aqueous medium, such as a lysate of a recombinant nucleic acid construct of the invention, resulting in the formation of lipid-nucleic acid nanoparticles (LNPs). In a preferred embodiment of the invention, mixing is performed using a microfluidic mixing device, in particular a staggered herringbone mixing chamber, such as a NanoAssemblr™ (Precision NanoSystems Inc., Vancouver, Canada) device.

[0173] In yet another embodiment, the mass ratio of total lipids to nucleic acids of the nucleic acids, such as the recombinant nucleic acid constructs of the present invention, is in the range of 5 to 60, preferably in the range of 15 to 40, and the volume mixing ratio of the aqueous nucleic acid solution to the total lipid organic solution is in the range of 1:1 to 6:1, preferably in the range of 2:1 to 4:1. The mixing flow rate of the solutions is in the range of 5 ml / min to 25 ml / min, preferably in the range of 10 ml / min to 20 ml / min.

[0174] In certain embodiments of the present invention, the organic solvent of the resulting mixture is removed after the mixing step by dialysis or by tangential flow filtration using an ultrafiltration membrane, where the ultrafiltration membrane can be either a hollow fiber membrane or a flat-screen membrane. The pore size of the ultrafiltration membrane corresponds to a molecular weight cut-off in the range of 1500 Da to 500.000 Da, preferably in the range of 1500 Da to 100.000 Da, more preferably in the range of 1500 Da to 30.000 Da. It is also possible to concentrate the LNPs to the desired concentration within the tangential flow filtration step.

[0175] The average particle size of the resulting LNPs can be measured using dynamic light scattering (DLS) techniques and is preferably in the range of 15 nm to 400 nm, more preferably in the range of 25 nm to 200 nm, and even more preferably the particles have a size in the range of 25 nm to 100 nm. The overall particle charge of the LNPs is positive and the zeta potential of the particles is preferably in the range of +5 mV to +60 mV, more preferably in the range of +25 mV to +60 mV.

[0176] In certain embodiments, the delivery vehicle of the present invention is a liposomal delivery vehicle, such as a lipid nanoparticle or lipidoid nanoparticle. In one embodiment, the delivery vehicle can be selected and / or prepared to optimize the delivery of the recombinant nucleic acid construct of the present invention to the target cell. For example, when the target cell is an endothelial cell, the properties of the delivery vehicle (e.g., size, charge and / or pH) can be optimized to effectively deliver such delivery vehicle to the target cell, reduce immune clearance, and / or enhance retention in the target cell. Alternatively, when the target cell is in the central nervous system (e.g., when the mRNA, such as the recombinant nucleic acid construct of the present invention, administered for the treatment of neurodegenerative disease, can specifically target brain or spinal cord tissue), the selection and preparation of the delivery vehicle must take into account the penetration of the blood-brain barrier and retention in the blood-brain barrier and / or the use of alternative means to directly deliver such delivery vehicle to such target cell. In one embodiment, the compositions of the invention may be combined with an agent that facilitates the transfer of exogenous mRNA (e.g., an agent that enhances the transfer of exogenous mRNA into target cells by disrupting or improving the permeability of the blood-brain barrier).

[0177] Liposomes (e.g., liposomal lipid nanoparticles) are known to be particularly suitable for their use in vivo as vehicles for the introduction of diagnostic or therapeutic compounds (Lasic, Trends Biotechnol, 16:307-321, 1998; Drummond et al, Pharmacol.Rev., 51:691-743, 1999), and are usually characterized as microscopic vesicles with an internal aqueous space separated from the outside medium by one or more bilayer membranes. The bilayer membrane of liposomes is typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that contain spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol, 16:307-321, 1998). The bilayer membrane of liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). Such liposomes may also be used as the lipid moiety in a delivery vehicle of the present invention comprising a recombinant nucleic acid construct of the present invention.

[0178] It should be appreciated that any feature of a recombinant nucleic acid construct disclosed in relation to a particular aspect of the invention is also an embodiment of a recombinant nucleic acid construct of any other aspect of the invention, including any embodiments thereof. More specifically, any embodiment of a recombinant nucleic acid construct disclosed in relation to the first aspect is also an embodiment of a recombinant nucleic acid construct of the second, third and fourth aspects. Similarly, any embodiment of a recombinant nucleic acid construct disclosed in relation to the second, third and fourth aspects is also an embodiment of a recombinant nucleic acid construct of the first aspect.

[0179] It is generally accepted in the art that UTRs flanking a coding region do not interfere with the coding region, and more specifically that UTRs can, in principle, be exchanged without interfering with expression of the coding region (see, for example, WO 2017 / 100551 A1, Trepotec et al.; Tissue Engineering Part A, Vol 0 Nr ja (Apr. 2018) (https: / / doi.org / 10.1089 / ten.TEA.2017.0485)).

[0180] [The present invention 1001] In the 5' to 3' direction 5'UTR, a coding region encoding an effector molecule, and 3'UTR A recombinant nucleic acid construct comprising: The 5'UTR is the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding Ang-2 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding H3.3. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding Galectin-9 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding GADD34 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding EDN1 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding HSP70m5 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding E-selectin or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding ICAM-1 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding IL-6 or a derivative thereof having at least 85% nucleotide identity, and the 5'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, the 3'UTR is selected from the group comprising the 3'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding H3.3. or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding GADD34 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding EDN1 or a derivative thereof having at least 85% nucleotide identity and the 3'UTR of the gene encoding IL-6 or a derivative thereof having at least 85% nucleotide identity, the effector molecule is effective to restore a cellular function of the cell or to exert a therapeutic effect in or on the cell; and the recombinant nucleic acid construct is distinct from the wild-type mRNA encoding the effector molecule; The recombinant nucleic acid construct. [The present invention 1002] a) said 5'UTR is the 5'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity and said 3'UTR is selected from the group comprising: the 3'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity and the 3'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, or b) said 5'UTR is the 5'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity and said 3'UTR is selected from the group comprising the 3'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity and the 3'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, or c) said 5'UTR is the 5'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity and said 3'UTR is selected from the group comprising: the 3'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity and the 3'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, or d) said 5'UTR is the 5'UTR of the gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity, and said 3'UTR is selected from the group comprising: the 3'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 3'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, 1001. A recombinant nucleic acid construct of the present invention. [The present invention 1003] a) the 3'UTR is the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity and the 5'UTR is selected from the group comprising the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR of a gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity, or b) the 3'UTR is the 3'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR is selected from the group comprising the 5'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of a gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, or c) the 3'UTR is the 3'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity and the 5'UTR is selected from the group comprising the 5'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity and the 5'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, or d) the 3'UTR is the 3'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, and the 5'UTR is selected from the group comprising the 5'UTR of the gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, the 5'UTR of the gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity, and the 5'UTR of the gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, 1001. A recombinant nucleic acid construct of the present invention. [The present invention 1004] The recombinant nucleic acid construct of any of claims 1001 to 1003, wherein the 5'UTR and the 3'UTR of the recombinant construct are of different origin, preferably derived from different endogenous genes or endogenous species. [The present invention 1005] The recombinant nucleic acid construct of any of claims 1001 to 1003, wherein the 5'UTR and the 3'UTR of the recombinant construct are of the same origin, preferably derived from the same endogenous gene or endogenous species. [The present invention 1006] A construct, wherein the 5'UTR is the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity. A construct, wherein the 5'UTR is the 5'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, A construct, wherein the 5'UTR is the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity. A construct, wherein the 5'UTR is the 5'UTR of a gene encoding ANG-2 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity. A construct, wherein the 5'UTR is the 5'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, A construct, wherein the 5'UTR is the 5'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding HSP70 or a derivative thereof having at least 85% nucleotide identity. A construct, wherein the 5'UTR is the 5'UTR of a gene encoding H3.3. or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding H3.3. or a derivative thereof having at least 85% nucleotide identity, and A construct, wherein the 5'UTR is the 5'UTR of a gene encoding galectin 9 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding galectin 9 or a derivative thereof having at least 85% nucleotide identity. is selected from the group comprising: Preferably, A construct, wherein the 5'UTR is the 5'UTR of a gene encoding MCP-1 or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of a gene encoding vWF or a derivative thereof having at least 85% nucleotide identity, or A construct, wherein the 5'UTR is the 5'UTR of the gene encoding RPL12s.c. or a derivative thereof having at least 85% nucleotide identity, and the 3'UTR is the 3'UTR of the gene encoding vWF or a derivative thereof having at least 85% nucleotide identity. That is, A recombinant nucleic acid construct according to any one of claims 1001 to 1005. [The present invention 1007] The recombinant nucleic acid construct of any one of claims 1001 to 1006, preferably comprising a poly A tail at the 3' end of said recombinant nucleic acid construct. [The present invention 1008] The recombinant nucleic acid construct of any one of claims 1001 to 1007, preferably comprising a cap structure at the 5' end of the recombinant nucleic acid construct. [The present invention 1009] The recombinant nucleic acid construct of any of claims 1001 to 1008, which preferably comprises an IRES (internal ribosome entry site) sequence at the 5' end of the recombinant nucleic acid construct. [The present invention 1010] The recombinant nucleic acid construct of any of claims 1001 to 1009, wherein the construct comprises a nucleic acid sequence encoding a signal peptide, preferably the signal peptide is in frame with a nucleic acid sequence encoding one signal peptide, and is positioned between the 5'UTR and the coding region encoding an effector molecule. [The present invention 1011] 10. The recombinant nucleic acid construct of the present invention, wherein said signal peptide allows for secretion of said effector molecule. [The present invention 1012] The recombinant nucleic acid construct for use according to the invention 1010 or 1011, wherein the nucleotide sequence encoding the signal peptide is selected from the group comprising a nucleotide sequence encoding the signal peptide of MCP-1 or a derivative thereof having at least 85% nucleotide identity, a nucleotide sequence encoding the signal peptide of IL-6 or a derivative thereof having at least 85% nucleotide identity, a nucleotide sequence encoding the signal peptide of Ang-2 or a derivative thereof having at least 85% nucleotide identity, and a nucleotide sequence encoding the signal peptide of Ang-1 or a derivative thereof having at least 85% nucleotide identity. [The present invention 1013] The recombinant nucleic acid construct of any of the present inventions 1001 to 1012, wherein the cell whose cellular function is restored and / or the cell in or on which a therapeutic effect is exerted is an endothelial cell, preferably a vascular endothelial cell, more preferably the vascular endothelial cell is a microvascular endothelial cell. [The present invention 1014] The recombinant nucleic acid construct of any of the present inventions 1001 to 1013, wherein the cellular function can be restored by an effector molecule having an anti-permeability effect on endothelial cells, an anti-vascular leakage effect, an anti-apoptotic effect on endothelial cells, or an anti-inflammatory effect on endothelial cells, or an anti-stress response effect, preferably the effect is related or associated with the TIE-2 signaling pathway, the VEGF receptor pathway, the NOTCH signaling pathway, the PI3-kinase pathway, the eNOS signaling pathway, the Sirtuin-dependent metabolic pathway and the energy homeostasis pathway, the oxidative stress pathway, the shear stress response pathway, the ET-1 signaling pathway, the NO-mediated signaling pathway, and the mechanochemical transduction pathway. [The present invention 1015] A vector comprising any one of the nucleic acid constructs of the present inventions 1001 to 1014. [The present invention 1016] A cell comprising the nucleic acid construct of any one of the present inventions 1001 to 1014 and / or the vector of the present invention 1015. [The present invention 1017] A delivery vehicle comprising any one of the nucleic acid constructs of the present inventions 1001 to 1014, which is a cationic lipid delivery particle, preferably the particle is a nanoparticle, more preferably the average size of the nanoparticles is about 30 nm to about 200 nm, preferably about 30 nm to about 140 nm, more preferably about 30 nm to about 60 nm. [The present invention 1018] A pharmaceutical composition comprising any one of the nucleic acid constructs of the present invention 1001 to 1014, the vector of the present invention 1015, the cell of the present invention 1016, and / or the delivery vehicle of the present invention 1017, and a pharma- ceutically acceptable diluent. [The present invention 1019] The recombinant nucleic acid construct of any of claims 1001 to 1014 for use in a method for the treatment and / or prevention of a disease. [The present invention 1020] The recombinant nucleic acid construct of any of claims 1001 to 1014 for use in a method for restoring a cellular function of a cell. The present invention is further illustrated by the following figures and examples from which further features, aspects and advantages of the present invention can be taken. [Brief description of the drawings]

[0181] [Figure 1] Schematic diagram of a typical mRNA molecule structure. [Figure 2-1] FIG. 1 is a schematic diagram of selected examples of mRNA constructs, namely PAN02, PAN05, PAN07, PAN08, and PAN09, whose coding sequence has an open reading frame that of Nano-luciferase. [Figure 2-2] FIG. 1 is a schematic diagram of selected examples of mRNA constructs, namely PAN10, PAN11, PAN12, 13, and PAN28, whose coding sequence has an open reading frame that of nano-luciferase. [Figure 2-3] FIG. 1 is a schematic diagram of selected examples of mRNA constructs, namely PAN29, PAN30, PAN31, PAN32, PAN33, PAN34, PAN35, PAN36, and PAN37, having a coding sequence whose open reading frame is that of nano-luciferase. [Figure 2-4] FIG. 1 is a schematic diagram of selected examples of mRNA constructs, namely PAN38, PAN39, PAN40, PAN41, PAN42, PAN43, and PAN44, whose coding sequence has an open reading frame of nano-luciferase. [Figure 2-5] FIG. 1 is a schematic diagram of selected examples of mRNA constructs, namely PAN45, PAN46, PAN47, PAN48, and PAN49, whose coding sequence has an open reading frame that of nano-luciferase. [Diagram 3]1 is a restriction map of plasmid pcDNA3.1(-) containing construct PAN11. [Figure 4] FIG. 4 is a restriction digest map of the plasmid pdDNA3.1(-) of FIG. 3. [Diagram 5] 1 is an EtBr stained 1% agarose gel showing the non-linearized (uncut, supercoiled, right lane) and linearized products after BamHI restriction digestion (left lane) 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"). [Figure 6] 1 shows an EtBr stained 1% agarose gel showing the PCR products of Poly-A tailing PCR to add a 120 nt poly-A tail. [Figure 7A] Shown are the 5' primers used for the 5'UTR of the indicated genes: the first column indicates which construct the primer is used in conjunction with, the second column indicates the origin of the 5'UTR, and the third column indicates the position to which the primer hybridizes to the nucleotide sequence. [Figure 7B] Shown are the 3' primers used for the 3'UTR of the indicated genes: the first column indicates which construct the primer is used in conjunction with, the second column indicates the origin of the 3'UTR, and the third column indicates the position to which the primer hybridizes to the nucleotide sequence. [Figure 8] 13 is an image of a non-denaturing agarose gel after EtBr staining showing in vitro mRNA transcripts of various constructs. [Figure 9]These are photographs taken under a fluorescent microscope 24 hours after transfection of HeLa cells transfected with 0.5 μg (left) and 0.1 μg (right) of a commercially available enhanced mRNA version of the producing green fluorescent protein (TriLINK Biotechnology). [Figure 10] 1 is a bar graph showing luciferase expression, indicated as RLU (relative light units) of luciferase in the medium, by the indicated recombinant nucleic acid constructs in HPMECs, where luciferase was used as an effector molecule, 2 hours (left column), 6 hours (middle column), and 24 hours (right column) after transfection. [Figure 11] 1 is a bar graph showing luciferase expression, shown as RLU (relative light units) of luciferase in whole cell lysates of HPMECs, by the indicated recombinant nucleic acid constructs in HPMECs, where luciferase was used as an effector molecule, 6 hours (left column) and 24 hours (right column) after transfection. [Figure 12] 1 is a bar graph showing luciferase expression, shown as RLU (relative light units) of luciferase in the medium, by the indicated recombinant nucleic acid constructs in HUVECs, where luciferase was used as an effector molecule, 2 hours (left column), 6 hours (middle column), and 24 hours (right column) after transfection. [Figure 13] 1 is a bar graph showing luciferase expression, shown as RLU (relative light units) of luciferase in whole cell lysates of HUVECs, by the indicated recombinant nucleic acid constructs after lysis of HUVECs, in which luciferase was used as an effector molecule, 2 hours (left column), 6 hours (middle column), and 24 hours (right column) after transfection. [Figure 14]1 is a bar graph showing luciferase expression, indicated as RLU (relative light units) of luciferase in the medium, by the indicated recombinant nucleic acid constructs in HeLa cells, where luciferase was used as an effector molecule, 2 hours (left column), 6 hours (middle column), and 24 hours (right column) after transfection. [Figure 15A] FIG. 1 depicts the 5′UTR nucleotide sequence of the mRNA for human MCP-1 (GenBank entry NM_002982.3), also known as homo sapiens CC motif chemokine ligand 2 (CCL2). [Figure 15B] Both the nucleotide and amino acid sequences of the signal peptide sequence of human MCP-1 are presented. [Figure 15C] 1 shows the 3'UTR nucleotide sequence of human MCP-1 mRNA. [Figure 16A] FIG. 1 depicts the 5'UTR nucleotide sequence of the mRNA for 50S ribosomal protein L12, chloroplast (LOC110782793) from spinach (Spinacia oleracea), also called RPL12s.c. (GenBank entry XM_021987044.1). [Figure 16B] FIG. 1 depicts the 3′UTR derived from the mRNA of spinach 50S ribosomal protein L12, chloroplast (LOC110782793). [Figure 17A] FIG. 1 depicts the 5′UTR nucleotide sequence of the mRNA for human angiopoietin 2 transcript variant 1, also known as ANGPT2 or Ang-2 (GenBank entry NM_001147.2). [Figure 17B] Both the nucleotide and amino acid sequences of the signal sequence of human Ang-2 mRNA are presented. [Figure 17C-1] 1 shows the 3'UTR nucleotide sequence of human Ang-2 mRNA. [Figure 17C-2] 1 shows the 3'UTR nucleotide sequence of human Ang-2 mRNA. [Figure 18A]FIG. 1 depicts the 5′UTR nucleotide sequence of the mRNA of human interleukin 6 (IL-6) transcript variant 1 (GenBank entry NM_000600.4). [Figure 18B] Both the nucleotide and amino acid sequences of the signal sequence of human IL-6 mRNA are presented. [Figure 18C] 1 shows the 3'UTR nucleotide sequence of human IL-6 mRNA. [Figure 19A] FIG. 1 depicts the 5′UTR nucleotide sequence of the mRNA for human von Willebrand factor (vWF) (GenBank entry NM_000552.4). [Figure 19B] 1 shows both the nucleotide and amino acid sequences of the von Willebrand factor (vWF) mRNA signal sequence. [Figure 19C] Figure 2 shows the 3'UTR nucleotide sequence of von Willebrand factor (vWF) mRNA. [Figure 20] Figure 20A depicts the 5'UTR nucleotide sequence of the mRNA for human heat shock protein family A (Hsp70) member 1A, also known as HSPA1A (GenBank entry NM_005345.5). Figure 20B depicts the 3'UTR nucleotide sequence of the mRNA for human HSPA1A. [Figure 21A] FIG. 1 depicts the 5′UTR nucleotide sequence of the mRNA for human heat shock protein family A (Hsp70) member 5, also known as HSPA5 (GenBank entry NM_005347.4). [Figure 21B] Figure 21B shows both the nucleotide and amino acid sequences of the signal sequence of human HSPA5 mRNA. [Figure 21C] 1 shows the 3'UTR nucleotide sequence of human HSPA1A mRNA. [Figure 22]Figure 22A depicts the 5'UTR nucleotide sequence of the mRNA for human H3 histone family member 3A (H3F3A) (GenBank entry NM_002107.4), also referred to as H3.3. Figure 22B depicts the 3'UTR nucleotide sequence of the mRNA for human H3.3. [Figure 23] Figure 23A depicts the 5'UTR nucleotide sequence of the mRNA of human galectin-9 (LGALS9) transcript variant 1 (GenBank entry NM_009587.2). Figure 23B depicts the 3'UTR nucleotide sequence of the mRNA of human galectin-9. [Figure 24-1] 1 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN01. [Figure 24-2] 1 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN01. [Figure 24-3] 1 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN01. [Figure 25-1] 1 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN03. [Figure 25-2] 1 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN03. [Figure 26] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN04. [Figure 27] 1 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN06. [Figure 28-1] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN36. [Figure 28-2] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN36. [Figure 29] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN37. [Figure 30-1] 1 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN02. [Figure 30-2]1 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN02. [Diagram 31] 1 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN05. [Diagram 32] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN07. [Diagram 33] 1 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN08. [Diagram 34] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN09. [Diagram 35] 1 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN10. [Diagram 36] 1 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN11. [Figure 37] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN12. [Figure 38] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN13. [Figure 39] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN28. [Diagram 40] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN29. [Diagram 41] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN30. [Diagram 42] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN31. [Diagram 43] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN32. [Diagram 44] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN33. [Diagram 45] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN34. [Diagram 46]1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN35. [Figure 47] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN38. [Figure 48] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN39. [Figure 49] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN40. [Figure 50] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN41. [Figure 51] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN42. [Figure 52] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN43. [Figure 53] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN44. [Figure 54] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN45. [Figure 55] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN46. [Figure 56] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN47. [Figure 57] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN48. [Figure 58] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN49. [Figure 59A] FIG. 1 depicts the nucleotide sequence of the mRNA for human angiopoietin 1 transcript variant 1, also known as ANGPT1 or Ang-1, GenBank entry NM_001146.4. [Figure 59B] Both the nucleotide and amino acid sequences of human Ang-1 mRNA are presented. [Figure 59C] 1 shows the mature peptide coding sequence (CDS) of human Ang-1 mRNA. [Fig. 59D] 1 shows the 3'UTR nucleotide sequence of human Ang-1 mRNA. [Figure 60A] FIG. 1 depicts the nucleotide sequence of the mRNA for human angiopoietin 4 transcript variant 1, also known as ANGPT4 or Ang-4, GenBank entry NM_015985.3. [Figure 60B] Both the nucleotide and amino acid sequences of human Ang-4 mRNA are presented. [Figure 60C] 1 shows the mature peptide coding sequence (CDS) of human Ang-4 mRNA. [Figure 60D-1] 1 shows the 3'UTR nucleotide sequence of human Ang-4 mRNA. [Figure 60D-2] 1 shows the 3'UTR nucleotide sequence of human Ang-4 mRNA. [Figure 61] 1 depicts the nucleotide sequence encoding the synthetic construct, COMP-Ang1. [Figure 62] 1 depicts the nucleotide sequence encoding the synthetic construct, hCOMP-Ang1. [Figure 63] 1 depicts the nucleotide sequence encoding the synthetic construct, CMP-Ang1. [Figure 64] 1 depicts the nucleotide sequence encoding the synthetic construct, COMP-Ang2. [Figure 65-1] FIG. 1 depicts the coding nucleotide sequence of the mRNA for human TEK receptor tyrosine kinase (TEK) transcript variant 1, also known as Tie-2, GenBank entry. [Figure 65-2] FIG. 1 depicts the coding nucleotide sequence of the mRNA for human TEK receptor tyrosine kinase (TEK) transcript variant 1, also known as Tie-2, GenBank entry. [Figure 66-1] 1 depicts the coding nucleotide sequence of the mRNA for human mutant TEK receptor tyrosine kinase (TEK), also called TIE*, which has the R849W mutation. [Figure 66-2]1 depicts the coding nucleotide sequence of the mRNA for human mutant TEK receptor tyrosine kinase (TEK), also called TIE*, which has the R849W mutation. [Figure 67] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN50. [Figure 68] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN51. [Figure 69] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN52. [Figure 70] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN53. [Figure 71] Figure 71A is an image of a non-denaturing agarose gel after EtBr staining showing the tail-PCR products of the various constructs. Figure 71B is an image of a non-denaturing agarose gel after EtBr staining showing the in vitro mRNA transcription products of the various constructs. [Figure 72] 1 is a bar graph showing luciferase expression, indicated as RLU (relative light units) of luciferase in the medium, by the indicated recombinant nucleic acid constructs in HPMECs, where luciferase was used as an effector molecule, 2 hours (left column), 6 hours (middle column), and 24 hours (right column) after transfection. [Figure 73] 1 is a bar graph showing luciferase expression, shown as RLU (relative light units) of luciferase in whole cell lysates of HPMECs, by the indicated recombinant nucleic acid constructs in HPMECs, where luciferase was used as an effector molecule, 6 hours (left column) and 24 hours (right column) after transfection. [Figure 74]Figure 74A (left) is a bar graph showing luciferase expression, expressed as RLU (relative light units) of luciferase in the medium, by the indicated recombinant nucleic acid constructs in HPMEC (transfected with GFP mRNA, PAN12 mRNA and PAN12* mRNA) 2 hours (left column), 6 hours (middle column) and 24 hours (right column) after transfection, where luciferase was used as an effector molecule. In PAN12*, 100% of uridine nucleotides and 100% of cytidine nucleotides are replaced by pseudo-uridine and 5-methyl-cytidine, respectively. Figure 74B (right) is a bar graph showing luciferase expression, expressed 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) 6 hours (left column) and 24 hours (right column) after transfection, where luciferase was used as an effector molecule. In PAN12*, 100% of uridine nucleotides and 100% of cytidine nucleotides are replaced by pseudo-uridine and 5-methyl-cytidine, respectively. [Figure 75] 1 is a bar graph showing luciferase expression, indicated as RLU (relative light units) of luciferase in the medium, by the indicated recombinant nucleic acid constructs in HPMECs where luciferase was used as an effector molecule, at 1 hour, 2 hours, 4 hours, and 24 hours after transfection (from left to right, 1 hour results are shown on the far left and 24 hours results are shown on the far right). [Figure 76] 1 is a bar graph showing luciferase expression, shown as RLU (relative light units) of luciferase in whole cell lysates of HPMECs, by the indicated recombinant nucleic acid constructs in HPMECs, where luciferase was used as an effector molecule, at 1 hour, 2 hours, 4 hours, and 24 hours after transfection (from left to right, 1 hour results are shown on the far left and 24 hours results are shown on the far right). [Figure 77] Figure 77A (left) is a bar graph showing luciferase expression, expressed as RLU (relative light units) of luciferase in the medium, by the indicated recombinant nucleic acid constructs in HPMEC (transfected with GFP mRNA, PAN12 mRNA, and PAN12* mRNA) at 1 hour, 2 hours, 4 hours, and 24 hours after transfection (from left to right, 1 hour results are shown on the far left, and 24 hours results are shown on the far right), where luciferase was used as an effector molecule. In PAN12*, 100% of the uridine nucleotides and 100% of the cytidine nucleotides are replaced by pseudo-uridine and 5-methyl-cytidine, respectively. Figure 77B (right) is a bar graph showing luciferase expression, expressed 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) at 1 hour, 2 hours, 4 hours, and 24 hours after transfection (from left to right, 1 hour results are shown on the far left, and 24 hours results are shown on the far right), where luciferase was used as an effector molecule. In PAN12*, 100% of the uridine nucleotides and 100% of the cytidine nucleotides are replaced by pseudo-uridine and 5-methyl-cytidine, respectively. [Figure 78] 1 is a bar graph showing luciferase expression, indicated as RLU (relative light units) of luciferase in the medium, by the indicated recombinant nucleic acid constructs in HPAECs where luciferase was used as an effector molecule, at 1 hour, 2 hours, 4 hours, and 24 hours after transfection (from left to right, 1 hour results are shown on the far left and 24 hours results are shown on the far right). [Figure 79]1 is a bar graph showing luciferase expression, shown as RLU (relative light units) of luciferase in whole cell lysates of HPAECs, by the indicated recombinant nucleic acid constructs in HPAECs, where luciferase was used as an effector molecule, at 1 hour, 2 hours, 4 hours, and 24 hours after transfection (from left to right, 1 hour results are shown on the far left and 24 hours results are shown on the far right). [Figure 80] FIG. 1 is a bar graph showing luciferase expression, indicated as RLU (relative light units) of luciferase in the medium, by the indicated recombinant nucleic acid constructs in HeLa cells, where luciferase was used as an effector molecule, at 1 hour, 2 hours, 4 hours, and 24 hours after transfection (from left to right, 1 hour results are shown on the far left and 24 hours results are shown on the far right). [Figure 81] 1 is a bar graph showing luciferase expression, 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 was used as an effector molecule, at 1 hour, 2 hours, 4 hours, and 24 hours after transfection (from left to right, 1 hour results are shown on the far left, and 24 hours results are shown on the far right). [Figure 82] 1 is a bar graph showing luciferase expression, expressed as RLU (relative light units) of luciferase in the medium, from the indicated recombinant nucleic acid constructs in HPMECs where luciferase was used as an effector molecule, at 2, 4, 6, and 24 hours after transfection (from left to right, 1 hour results are shown on the far left and 24 hour results are shown on the far right). In constructs PAN12*, PAN28*, PAN29*, PAN34*, PAN50*, and PAN51*, 100% of the uridine nucleotides and 100% of the cytidine nucleotides are replaced with pseudouridine and 5-methyl-cytidine, respectively. [Figure 83]1 is a bar graph showing luciferase expression, expressed as RLU (relative light units) of luciferase in whole cell lysates of HPMECs, by the indicated recombinant nucleic acid constructs in HPMECs where luciferase was used as an effector molecule, at 2, 4, 6 and 24 hours after transfection (from left to right, 1 hour results are shown on the far left and 24 hour results are shown on the far right). In constructs PAN12*, PAN28*, PAN29*, PAN34*, PAN50* and PAN51*, 100% of the uridine nucleotides and 100% of the cytidine nucleotides are replaced with pseudouridine and 5-methyl-cytidine, respectively. [Figure 84] Figure 1 is a bar graph showing expression of luciferase, shown as RLU (relative light units) of luciferase in the medium, from the indicated recombinant nucleic acid constructs in HPMECs where luciferase was used as an effector molecule, 2 hours (left column), 4 hours (middle column), and 6 hours (right column) after transfection. In construct PAN02*, 100% of uridine nucleotides and 100% of cytidine nucleotides are replaced with pseudouridine and 5-methyl-cytidine, respectively, and in uncapped construct PAN51, the mRNA sequence is that of construct PAN51 but does not have any 5'-capping structure. [Figure 85] 1 is a bar graph showing expression of luciferase, shown as RLU (relative light units) of luciferase in whole cell lysates of HPMECs by the indicated recombinant nucleic acid constructs in HPMECs where luciferase was used as an effector molecule, 2 hours (left column), 4 hours (middle column), and 6 hours (right column) after transfection. In construct PAN02*, 100% of uridine nucleotides and 100% of cytidine nucleotides are replaced with pseudouridine and 5-methyl-cytidine, respectively, and in uncapped construct PAN51, the mRNA sequence is that of construct PAN51 but does not have any 5'-capping structure. [Figure 86] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN54. [Figure 87] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN55. [Figure 88] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN56. [Figure 89] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN57. [Figure 90] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN58. [Figure 91] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN59. [Figure 92] 1 is a bar graph showing luciferase expression, indicated as RLU (relative light units) of luciferase in the medium, by the indicated recombinant nucleic acid constructs in HPMECs where luciferase was used as the effector molecule after 4 hours (left column). 24 hours (middle column) and 48 hours (right column) after transfection, the cells are washed and replenished with fresh medium before assessing luciferase activity values ​​for an additional 4 hours. [Figure 93]Bar graphs showing luciferase expression, indicated as RLU (relative light units) of luciferase in the medium, by the indicated recombinant nucleic acid constructs in HEK293 cells, where luciferase was used as the effector molecule, after 3 hours (left column). 24 hours after transfection (right column), the cells are washed and replenished with fresh medium before luciferase activity values ​​are assessed for another 3 hours. 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 subconfluent cultures (70-80%) 1:2-1:6, i.e., seeding at 2-5 × 10,000 cells / cm2 using 0.25% trypsin or trypsin / EDTA, 5% CO2, 37 °C. mRNA transfection is performed with Lipofectamine™ MessengerMAX™ Transfection Reagent (Invitrogen™) according to the manufacturer's protocol. [Figure 94] Bar graphs depicting luciferase expression, expressed as RLU (relative light units) of luciferase in the medium, by the indicated recombinant nucleic acid constructs in HeLa cells, where luciferase was used as an effector molecule, after 3 hours (left column). 24 hours after transfection (right column), the cells are washed and replenished with fresh medium before luciferase activity values ​​are assessed for an additional 3 hours. [Figure 95-1] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN60. [Figure 95-2] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN60. [Figure 96-1] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN61. [Figure 96-2] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN61. [Figure 97-1] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN66. [Figure 97-2] 1 shows the basic structure and nucleotide sequence of the recombinant nucleic acid construct PAN66. [Figure 98] Western blot analysis of protein lysates from HPMEC cells transfected with the indicated PAN mRNA constructs. Cells were transfected with 1 μg of mRNA and harvested 6 h later. Total cell lysates were separated by SDS-PAGE and analyzed by immunoblot with anti-Ang1 antibody. M, marker; UT, untreated cells; rec.Ang1, recombinant hAng1. [Figure 99] Figure 1 shows an agarose gel of in vitro transcribed mRNA. 2 μg of the indicated mRNA was separated by agarose gel electrophoresis and visualized by ethidium bromide staining and UV illumination. [Figure 100] a) The sequences of 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 primers for PAN55 and PAN56. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0182] Figure 1 is a schematic diagram of a typical mRNA molecule structure. Eukaryotic, including mammalian, mRNAs consist of a cap region, a 5' untranslated region (UTR), a coding sequence (CDS) with an open reading frame (ORF) beginning with a consensus Kozak sequence for optimal translation initiation and followed by a signal peptide leader sequence in the case of secreted proteins, a 3'UTR, and a polyA-tail (>120 nt) at the 3' end.

[0183] Figure 2 is a schematic diagram of selected examples of mRNA constructs with a coding sequence whose open reading frame is that of nano-luciferase. For any of the mRNA constructs shown, it is within the scope of the present invention that the coding region comprising a sequence with an open reading frame for nano-luciferase may be replaced with a coding region comprising a sequence encoding an effector molecule, in particular with a coding region comprising a sequence encoding an effector molecule as disclosed herein, in a preferred embodiment of which the effector molecule is Ang1 or COMP-Ang1. According to the present invention, a recombinant nucleic acid construct is designed as follows:

[0184] The recombinant nucleic acid construct PAN02 includes the 5'UTR of Ang1 as the 5'untranslated region, a nucleic acid sequence encoding the signal peptide of Ang1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nano luciferase as the coding region encoding the effector molecule, and the 3'UTR of Ang1 as the 3'untranslated region.

[0185] The recombinant nucleic acid construct PAN05 includes the 5'UTR of Ang2 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of Ang2 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0186] The recombinant nucleic acid construct PAN07 includes the 5'UTR of galectin-9 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of Ang2 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0187] The recombinant nucleic acid construct PAN08 comprises the 5'UTR of Hsp70 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of Ang2 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of HSP70 as the 3' untranslated region.

[0188] The recombinant nucleic acid construct PAN09 comprises the 5'UTR of H3.3 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of Ang2 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of H3.3 as the 3' untranslated region.

[0189] The recombinant nucleic acid construct PAN10 comprises the 5'UTR of RPL12s.c. as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of Ang2 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of RPL12s.c. as the 3' untranslated region. This construct further comprises another start codon in frame before the start codon of the coding region of nano-luciferase.

[0190] The recombinant nucleic acid construct PAN11 comprises the 5'UTR of GADD34 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of Ang2 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of GADD34 as the 3' untranslated region.

[0191] The recombinant nucleic acid construct PAN12 comprises the 5'UTR of MCP-1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of MCP-1 as the 3' untranslated region.

[0192] The recombinant nucleic acid construct PAN13 comprises the 5'UTR of EDN1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of EDN1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of EDN1 as the 3' untranslated region.

[0193] The recombinant nucleic acid construct PAN28 comprises the 5'UTR of Ang2 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of IL-6 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of vWF as the 3' untranslated region.

[0194] The recombinant nucleic acid construct PAN29 includes the 5'UTR of MCP-1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0195] The recombinant nucleic acid construct PAN30 comprises the 5'UTR of MCP-1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of HSP70 as the 3' untranslated region.

[0196] The recombinant nucleic acid construct PAN31 comprises the 5'UTR of MCP-1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of IL-6 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of MCP-1 as the 3' untranslated region.

[0197] The recombinant nucleic acid construct PAN32 comprises the 5'UTR of MCP-1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of Ang2 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of MCP-1 as the 3' untranslated region.

[0198] The recombinant nucleic acid construct PAN33 comprises the 5'UTR of MCP-1 as the 5' untranslated region, does not comprise a nucleic acid sequence encoding a signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of MCP-1 as the 3' untranslated region.

[0199] The recombinant nucleic acid construct PAN34 comprises the 5'UTR of Hsp70 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of MCP-1 as the 3' untranslated region.

[0200] The recombinant nucleic acid construct PAN35 comprises the 5'UTR of RPL12s.c. (spinach chloroplast) as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of MCP-1 as the 3' untranslated region.

[0201] The recombinant nucleic acid construct PAN36 comprises the 5'UTR of MCP-1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding Ang1 as the coding region encoding the effector molecule, and the 3'UTR of MCP-1 as the 3' untranslated region.

[0202] The recombinant nucleic acid construct PAN37 includes the 5'UTR of MCP-1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding hCOMP-Ang1 as the coding region encoding the effector molecule, and the 3'UTR of MCP-1 as the 3' untranslated region.

[0203] The recombinant nucleic acid construct PAN38 includes the 5' UTR of Ang2 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3' UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0204] The recombinant nucleic acid construct PAN39 contains the 5'UTR of Ang2* (Ang2 5'-UTR with the upstream ATG deleted) as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of IL-6 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0205] The recombinant nucleic acid construct PAN40 includes the 5'UTR of Ang2 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of Gaussia luciferase as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0206] The recombinant nucleic acid construct PAN41 includes the 5'UTR of RPL12s.c. as a 5'untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 with an ATG added upstream for translation initiation as a nucleic acid sequence encoding a signal peptide, a coding region encoding nanoluciferase as a coding region encoding an effector molecule, and the 3'UTR of RPL12s.c. as a 3'untranslated region. This construct further includes another in-frame start codon before the start codon of the coding region of nanoluciferase.

[0207] The recombinant nucleic acid construct PAN42 comprises the 5'UTR of RPL12s.c. as the 5'untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of RPL12s.c. as the 3'untranslated region.

[0208] The recombinant nucleic acid construct PAN43 includes the 5'UTR of RPL12s.c. as the 5'untranslated region, a nucleic acid sequence encoding the signal peptide of Ang2 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of RPL12s.c. as the 3'untranslated region.

[0209] The recombinant nucleic acid construct PAN44 comprises the 5'UTR of Hsp70 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of Hsp70 as the 3' untranslated region.

[0210] The recombinant nucleic acid construct PAN45 includes the 5' UTR of Hsp70m5 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3' UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0211] The recombinant nucleic acid construct PAN46 includes the 5' UTR of E-selectin as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of IL-6 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3' UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0212] The recombinant nucleic acid construct PAN47 includes the 5'UTR of ICAM1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of IL-6 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0213] The recombinant nucleic acid construct PAN48 comprises the 5'UTR of IL-6 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of IL-6 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of IL-6 as the 3' untranslated region.

[0214] The recombinant nucleic acid construct PAN49 includes the 5'UTR of von Willebrand factor (vWF) as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of von Willebrand factor (vWF) as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3'UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0215] 3 shows a restriction map of the plasmid pcDNA3.1(-) used as an example for a plasmid to express an exemplary recombinant nucleic acid construct of the present invention, the construct PAN11, with the restriction sites XhoI / HindIII shown.

[0216] Figure 4 shows the restriction digestion map of the PAN11 expressing pcDNA3.1-plasmid of Figure 3. The insert represents the PAN11 sequence with engineered 5'UTR and 3'UTR, signal sequence, and coding region encoding nano-luciferase.

[0217] FIG. 5 is an EtBr stained 1% agarose gel showing the non-linearized (uncut, supercoiled, right lane) and linearized products after BamHI restriction digestion (left lane) of the pcDNA3.1(-) plasmids of constructs PAN28 ("28"), PAN13 ("13"), PAN12 ("12"), PAN11 ("11"), PAN10 ("10"), PAN09 ("09"), PAN08 ("08"), PAN07 ("07"), and PAN05 ("05").

[0218] Figure 6 depicts an EtBr stained 1% agarose gel showing the PCR products of polyA tailing PCR to add 120 nt polyA tails with 120 nt long poly-T 3' primers flanking various recombinant nucleic acid constructs. At the top, the optimized PCR conditions are shown for the indicated recombinant nucleic acid constructs. For example, for Pan02, the conditions are: denaturation at 94°C for 2 min, 33 cycles of 96°C for 30 s, 55°C for 15 s and 72°C for 4 min, with a final extension at 72°C for 8 min.

[0219] FIG. 8 is an image of an EtBr stained 1% non-denaturing agarose gel showing 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 the high range RiboRuler RNA ladder lane. From this FIG. 8 it can be seen that the transcribed mRNAs were intact, more specifically, not degraded.

[0220] 24 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN01. PAN1 is an embodiment of the present invention, which comprises the 5' UTR of Ang1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of Ang1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding Ang1 as the coding region encoding the effector molecule, and the 3' UTR of Ang1 as the 3' untranslated region.

[0221] 25 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN03. PAN03 is an embodiment of the present invention, which includes the 5' UTR of Ang1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of Ang1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding COMP-Ang1 as the coding region encoding the effector molecule, and the 3' UTR of Ang1 as the 3' untranslated region.

[0222] 26 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN04. ​​PAN04 is an embodiment of the present invention, which includes the 5' UTR of Ang2 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of Ang2 as the nucleic acid sequence encoding the signal peptide, a coding region encoding Ang1 as the coding region encoding the effector molecule, and the 3' UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0223] 27 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN06. PAN06 is an embodiment of the present invention, which includes the 5' UTR of Ang2 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of Ang2 as the nucleic acid sequence encoding the signal peptide, a coding region encoding COMP-Ang1 as the coding region encoding the effector molecule, and the 3' UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0224] 28 shows the basic structure and nucleotide sequence of recombinant nucleic acid construct PAN36. PAN36 is an embodiment of the present invention, which comprises the 5'UTR of MCP-1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding Ang1 as the coding region encoding the effector molecule, and the 3'UTR of MCP-1 as the 3' untranslated region.

[0225] FIG. 67 shows a recombinant nucleic acid construct PAN50, which is another embodiment of the mRNA of the present invention, comprising the 5' UTR of MCP-1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of IL-6 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase as the coding region encoding the effector molecule, and the 3' UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0226] Figure 68 shows recombinant nucleic acid construct PAN51, which is another embodiment of the mRNA of the present invention, comprising the 5' UTR of HSP70 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of IL-6 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase (NLuc) as the coding region encoding the effector molecule, and the 3' UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0227] Figure 69 shows recombinant nucleic acid construct PAN52, which is another embodiment of the mRNA of the present invention, comprising the 5' UTR of MCP-1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding Ang1 as the coding region encoding the effector molecule, and the 3' UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0228] Figure 70 shows recombinant nucleic acid construct PAN53, which is another embodiment of the mRNA of the present invention, comprising the 5' UTR of MCP-1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding hCOMP-Ang1 as the coding region encoding the effector molecule, and the 3' UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0229] FIG. 71A is an EtBr stained 1% non-denaturing agarose gel image showing tail-PCR products of constructs PAN35 ("35"), PA34 ("34"), PAN33 ("33"), PAN32 ("32"), PAN31 ("31"), PAN30 ("30"), PAN29 ("29"), and PAN12 ("12"). M: is the high range RiboRuler RNA ladder lane. From this FIG. 71A, it can be seen that the transcribed mRNA was intact, more specifically, not degraded.

[0230] FIG. 71B is an image of an EtBr stained 1% non-denaturing agarose gel showing in vitro mRNA transcription products of constructs PAN35 ("35"), PA34 ("34"), PAN33 ("33"), PAN32 ("32"), PAN31 ("31"), PAN30 ("30"), PAN29 ("29"), and PAN12 ("12"). M: is the high range RiboRuler RNA ladder lane. From this FIG. 71B, it can be seen that the transcribed mRNA was intact, more specifically, not degraded.

[0231] FIG. 86 shows a recombinant nucleic acid construct PAN54, which is another embodiment of the mRNA of the present invention, comprising the 5' UTR of RPL12s.c. as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nano-luciferase (NLuc) as the coding region encoding the effector molecule, and the 3' UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0232] FIG. 87 shows recombinant nucleic acid construct PAN55, which is another embodiment of the mRNA of the present invention, comprising the 5' UTR of vWF as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase (NLuc) as the coding region encoding the effector molecule, and the 3' UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0233] Figure 88 shows a recombinant nucleic acid construct PAN56, which is another embodiment of the mRNA of the present invention, comprising the 5'UTR of vWF as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of vWF as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase (NLuc) as the coding region encoding the effector molecule, and the 3'UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0234] FIG. 89 shows a recombinant nucleic acid construct PAN57, which is another embodiment of the mRNA of the present invention, comprising 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 a 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as a nucleic acid sequence encoding a signal peptide, a coding region encoding nanoluciferase (NLuc) as a coding region encoding an effector molecule, and a 3' UTR of the 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) as a 3' untranslated region. The 5' and 3' sequences are underlined and bolded.

[0235] Figure 90 shows a recombinant nucleic acid construct PAN58, which is another embodiment of the mRNA of the present invention, including the 5'UTR of MCP-1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding nanoluciferase (NLuc) as the coding region encoding the effector molecule, and the 3'UTR of von Willebrand factor (vWF) as the 3' untranslated region. In addition, PAN58 includes a consensus Kozak sequence (GCCACC) 6 bases upstream of the initiating AUG.

[0236] Figure 91 shows recombinant nucleic acid construct PAN59, which is another embodiment of the mRNA of the present invention, comprising the 5' UTR of MCP-1 as the 5' untranslated region, a nucleic acid sequence encoding the signal peptide of MCP-1 as the nucleic acid sequence encoding the signal peptide, a coding region encoding CMP-Ang1 as the coding region encoding the effector molecule, and the 3' UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0237] Figure 95 shows recombinant nucleic acid construct PAN60, which is another embodiment of the mRNA of the present invention, comprising the 5' UTR of MCP-1 as the 5' untranslated region, a coding region encoding wild-type (wt) Tie2 as the coding region encoding the effector molecule, and the 3' UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0238] Figure 96 shows recombinant nucleic acid construct PAN61, which is another embodiment of the mRNA of the present invention, comprising the 5' UTR of MCP-1 as the 5' untranslated region, a coding region encoding Tie2 mutant R849W as the coding region encoding the effector molecule, and the 3' UTR of von Willebrand factor (vWF) as the 3' untranslated region.

[0239] FIG. 97 shows a recombinant nucleic acid construct PAN66, which is another embodiment of the mRNA of the present invention, comprising the 5'UTR of MCP-1 as the 5'untranslated region, a coding region encoding wild-type PIK3CA, which is human phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) according to GenBank and has the respective reference number 006218.4, as a coding region encoding an effector molecule, and the 3'UTR of von Willebrand factor (vWF) as the 3'untranslated region.

[0240] The SEQ ID NOs of the sequence listing are relevant to this disclosure, as summarized in Table 1.

[0241] [Table 1] TIFF0007663494000002.tif248163TIFF0007663494000003.tif228163 EXAMPLES

[0242] Example 1: Materials and Methods Generation of plasmid templates by gene synthesis and gene cloning We designed sequences encoding the Nluc reporter protein, Ang-1 protein and its derivatives (e.g., COMP-Ang-1, CMP-Ang-1) with various signal peptides flanked by various heterologous 5′UTRs and heterologous 3′UTRs. They were produced by gene synthesis by BioCat (Heidelberg) and cloned (Xho1-BamHI) into pcDNA3.1- (Thermo Fisher).

[0243] Sequences encoding wt Tie-2 and sequences carrying the specified Tie-2 mutations in the coding region flanked by various heterologous 5'UTRs and 3'UTRs were designed (see Figures 95 and 96), which were generated by gene synthesis by BioCat (Heidelberg) and cloned (Xho1-BamHI) into pcDNA3.1- (Thermo Fisher).

[0244] In vitro transcription from PCR products to generate polyadenylated mRNA 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 template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor, and Mg2. +The buffer consists of a buffer containing ions. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have a T7 promoter sequence upstream of the gene to be transcribed. To generate DNA templates with poly(A) tails, tail-PCR was performed using a 5' primer containing a T7 RNA polymerase sequence and a 3' primer with a 120 nt poly-T sequence synthesized and purified (BioSpring, Frankfurt).

[0245] Linear DNA templates for in vitro transcription were generated by PCR from linearized plasmids. Plasmids were digested with BamHI, which cuts once at the 3′ end in the vector backbone, generating linearized vectors that can be used as templates for poly-(A) tail PCR. 5 μg of plasmid is digested with 5 U of BamHI restriction enzyme in 50 μl and 1×HF buffer at 37° C. for 2 hours. Complete digestion of the plasmid is checked by analyzing a small portion of the digested mixture by gel electrophoresis (see, e.g., 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 enzymes are heat-inactivated by incubation at 80°C for 20 min, and the digested plasmid is purified by PCR purification columns according to 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 used to generate PCR templates. The purpose of linearization was to eliminate circular templates that could potentially generate run-on transcripts during the IVT reaction.

[0246] Addition of poly(A) tails by PCR was performed using Hot Star HiFidelity polymerase (Qiagen) or Taq DNA polymerase (Roche) according to the manufacturer's protocol. For this purpose, linear DNA templates were amplified using an adapter primer containing a T7 promoter sequence and an adapter primer containing a 3' polyA tail sequence (120 nt). Each adapter primer had an overlapping sequence fusing regulatory sequences required for translation and transcription to the gene of interest (see e.g. Figures 7a and 7b). A typical 50 μl PCR reaction contained 200 μM (each) dNTPs, 0.5 μM for each primer, 50 ng linearized plasmid DNA, 1× PCR reaction buffer (1.5 mM MgCl2) and 1.25 U of Taq DNA polymerase per reaction (0.25 μl at 5 U / μl). The specific PCR cycle program or temperature profile was adjusted according to the Tm of the primer pairs, according to the expected length of the PCR products, and according to the thermal cycler used. The quality of the PCR products was checked by analyzing an aliquot by gel electrophoresis. The reactions were purified by a commercial PCR purification kit (NucleoSpin Gel and PCR clean-up, Macherey-Nagel).

[0247] In vitro transcription reaction The addition of 5'-end cap structures to RNA is an important process in eukaryotes. It is essential for RNA stability, efficient translation, nuclear transport and splicing. This process involved the addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be performed 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, mRNA can be capped using vaccinia virus mRNA capping enzyme (NEB) following the manufacturer's protocol. This enzyme uses GTP and S-adenosylmethionine as donors to add a 7-methylguanosine cap to the 5' end of the RNA (cap-0 structure). Both methods give functionally active capped RNA suitable for transfection or other applications. In addition, 2'-O-methyltransferase can be used to introduce cap-1 and cap-2 structures.

[0248] In the examples described below, the T7 High Yield Transcription Kit (Thermo Scientific) was used to co-transcriptionally synthesize capped RNA transcripts (PAN 01-XX) with cap analogs.

[0249] The DNA templates for the transcription reactions were linearized plasmids or PCR products. For capped RNA, reactions were set up at room temperature in the following order: TIFF0007663494000004.tif97170

[0250] 1 μg of DNA was added and the total reaction volume was brought to 20 μl by adding nuclease-free water. The amount of water to add varies based on the concentration of the template DNA. The reaction was mixed well by vortexing and incubated at 37° C. in a dry air incubator for 2 hours. Before proceeding with purification, the transcription reaction was treated with DNase I to remove the DNA template as follows: 1. 70 μl of nuclease free water was added to the transcription reaction, followed by 10 μl of DNase I reaction buffer. 2. 2 μl of DNase I was added to the reaction. 3. The reaction was incubated at 37°C for 15 minutes.

[0251] Column purification of capped mRNA 1. Capped RNA was purified using MEGAclearKit according to manufacturer's instructions (any spin column based RNA purification kit can be used). 2. RNA was quantified using a NanoDrop spectrophotometer. 3. The quality of the RNA samples was assessed using the Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer according to the manufacturer's instructions or by non-denaturing 1% agarose gel electrophoresis and ethidium bromide staining (see FIG. 8).

[0252] mRNA transfection of HeLa, HEK293, HPMEC, HPAEC, and HUVEC cells HeLa cells were grown in DMEM complete medium. 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.

[0253] 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 involves splitting subconfluent cultures (70-80%) 1:2-1:6, i.e., at 2-5 × 10,000 cells / cm using 0.25% trypsin or trypsin / EDTA, 5% CO2, 37 °C. 2 mRNA transfection is performed with Lipofectamine™ MessengerMAX™ Transfection Reagent (Invitrogen™) according to the manufacturer's protocol.

[0254] Primary human pulmonary microvascular endothelial cells (HPMEC) and primary human pulmonary artery endothelial cells (HPAEC) are isolated from human pulmonary arteries and are best suited for the study of human lung disease, as they are isolated from the lungs of a single donor. Because lung tissue contains blood capillaries and lymphatic capillaries, HPMEC contain microvascular and lymphatic endothelial cells.

[0255] The HPMEC cell types were characterized by immunofluorescence staining: they stained positively for CD31 and von Willebrand factor and negatively for smooth muscle α-actin.

[0256] HUVEC, HPAEC, and HPMEC cells were grown in endothelial cell growth medium as recommended by PromoCell (Heidelberg, Germany), which is a basal medium supplemented with fetal bovine 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. Experiments using HUVEC, HPAEC, and HPMEC cells are performed with cells at less than passage 8.

[0257] RNA transfections, as well as mRNA transfections, were performed using Lipofectamin MessengerMAX (Invitrogen, Carlsbad, CA; Thermo Fisher Scientific). For mRNA transfections in a 24-well plate format, 1 × 10 cells were added 24 h prior to transfection to reach a confluence of 70–90 immediately prior to transfection. 5 Cells / well were seeded. In a single well, 1.5 μl Lipofectamine MessengerMAX reagent was added to 25 μl OptMEM (Invitrogen, Carlsbad, CA; Thermo Fisher Scientific), vortexed, and incubated at room temperature for 10 min. In another well, 500 ng or 1 μg mRNA was added to 25 μl OptiMEM and vortexed. After incubation, diluted mRNA was added to diluted Lipofectamin, mixed well, incubated at room temperature for 5 min, and added dropwise to the cells in the presence of 0.5 ml complete medium for Hela and HEK293. For HUVEC, HPAEC, and HPMEC, the endothelial cell growth medium was replaced and the cells were washed with OptiMEM, after which the Lipofectamin MessengerMAxX mRNA complex solution was added to the wells containing 0.5 ml OptiMEM pre-warmed to 37°C. The cells were incubated at 37°C for 2 h, and the OptiMEM medium was replaced with complete endothelial cell growth medium (PromoCell, Heidelberg, Germany).

[0258] Cell lysates or serum from transfected cells 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-diaminopropionic acid-N-palmityl-N-oleyl-amide in combination with neutral co-lipids and PEGylated co-lipids were used for in vitro transfection. The co-lipid was diphytanoyl-PE. The PEGylated lipid was methoxy PEG2000-DSPE.

[0259] Successful introduction of the mRNA into the host cell was monitored using various known methods, for example, using a fluorescent marker, such as green fluorescent protein (GFP), such as a reporter enzyme (e.g., a luciferase derivative). Alternatively, transfection of the modified mRNA could be determined by measuring the protein expression level of the target polypeptide, for example, by Western blotting or immunocytochemistry or ELISA.

[0260] Fully modified mRNA with 5-methylcytidine and / or pseudouridine (or 5-methoxyuridine or N1-methylpseudouridine) is transfected into HUVEC or HPMEC (human pulmonary microvascular endothelial cells, PromoCell, Heidelberg) using Lipofectamin MessengerMAX (Invitrogen, Carlsbad, CA; Thermo Fisher Scientific). At various time points post-transfection (30", 1 h, 2 h, 4 h, 6 h), cell lysates are harvested and subjected to ELISA or immunoblot assays (Western) to determine protein expression.

[0261] Nano-Luc luciferase activity reporter measurement Time-dependent quantitative detection of Nano-Glo-Luciferase expression (Promega) was performed by analyzing samples from tissue culture supernatants for secreted forms according to the reporter assay technical manual of the Nano-Glo Luciferase Assay System (Promega). Briefly, 5–20 μl of serum or lysate was diluted in a final volume of 100 μl H2O and mixed with an equal volume of reconstituted Nano-Glo Luciferase Assay Reagent in a 96-well GloMax 96 microplate. After at least 3 min of incubation at room temperature, luminescence was measured in an appropriate luminometer.

[0262] Immunoblot detection of Tie-2 and derivatives For immunoblot detection of Tie-2 and derivatives, protein lysates are loaded onto a NuPage SDS-PAGE system (chamber and power supply) (both from Life Technologies, Grand Island, NY) using 1.5 mm ready-to-use Bis-Tris gels and a 4-12% acrylamide gradient with MOPS buffer as running aid. Each lysate sample is prepared in a final volume of 40 μl. The samples contain 25 μg of protein lysate in various volumes, RIPA buffer to a volume of 26 μl, 4 μl of 10× reducing agent and 10 μl of 4×SDS loading buffer (both from Life Technologies, Grand Island, NY). Samples are heated at 95° C. for 5 min and loaded onto the gel. Standard settings are chosen by the manufacturer and are 200 V, 120 mA, and 25 W max. The run time is 60 min, until the running dye reaches the bottom of the gel.

[0263] After the run is completed, the plastic case is cracked and the gel inside is transferred to a pre-prepared nitrocellulose membrane kit and power supply (iBLOT; LifeTechnologies, Grand Island, NY). Using default settings, the protein lysate is transferred from the gel to the membrane with high Ampere electricity.

[0264] After transfer, the membrane is incubated for 15 min in 5% BSA in IX TBS, then for another 15 min in 5% BSA in IX TBS + 0.1% Tween. Primary or phospho-specific antibodies against human Tie-2 protein (P-Tie-2, P-Akt, see below) are applied at 1:500-1:2000 dilution in 3 ml of 5% BSA in IX TBS solution for 3 h at room temperature with gentle shaking on an orbital shaker. The membrane is washed three times with IX TBS / 0.1% Tween, 5 min each time with gentle shaking. The secondary antibody (goat anti-rabbit HRP conjugate; Abeam, Cambridge, MA) is conjugated to horseradish peroxidase and binds to the antibody of the primary antibody. The secondary antibody is diluted 1:1000-1:5000 in 5% BSA in IX TBS and incubated for 3 hours at room temperature. After the incubation period, wash the membrane three times with IX TBS / 0.1% Tween for 5 minutes each time with gentle agitation. Develop the membrane in 5 ml of Pierce West Pico Chemiluminescent Substrate (Thermo Fisher, Rockford, IL) as directed.

[0265] Example 2: Expression of recombinant nucleic acid constructs expressing luciferase, Ang-1, or Ang-1 derivatives in HPMEC, HUVEC, HPAEC, and HeLa cells mRNA transfection of HeLa, HUVEC, HPAEC, and HPMEC cells Primary human pulmonary microvascular endothelial cells (HPMEC) are best suited for the study of human lung diseases and are isolated from the lungs of a single donor. As lung tissue contains blood capillaries and lymphatic capillaries, HPMEC contain microvascular and lymphatic endothelial cells. Cells were routinely analyzed by immunofluorescence staining. They stain positive for CD31 and von Willebrand factor and negative for smooth muscle α-actin. HeLa, HUVEC, HPMEC and HPAEC (human pulmonary artery endothelial cells) cells were grown in DMEM complete medium or endothelial cell growth medium (PromoCell, Heidelberg, Germany, Cat. No. C-22020), respectively. Endothelial cell growth medium was basal medium supplemented with fetal bovine serum (0.05 ml / ml), Endothelial Cell Growth Supplement (bovine hypothalamic extract; 0.004 ml), heparin 90 μg / ml, and hydrocortisone 1 μg / ml. Experiments using HUVEC and HPMEC cells were performed with cells less than passage 8.

[0266] mRNA transfections were performed using Lipofectamin MessengerMAX (Invitrogen, Carlsbad, CA; Thermo Fisher Scientific). For mRNA transfections in a 24-well plate format, 1 × 10 cells were added 24 h prior to transfection to reach a confluence of 70–90% immediately prior to transfection. 5Cells / well were seeded. In a single well, 1.5 μl Lipofectamine MessengerMAX reagent was added to 25 μl OptiMEM, vortexed, and incubated at room temperature for 10 min. In another well, 500 ng or 1 μg mRNA was added to 25 μl OptiMEM and vortexed. After incubation, diluted mRNA was added to diluted Lipofectamin, mixed well, incubated at room temperature for 5 min, and for HeLa, added dropwise to the cells in the presence of 0.5 ml DMEM complete medium. For HUVEC, HPMEC, and HPAEC, the endothelial cell growth medium was replaced and the cells were washed with OptiMEM, after which the Lipofectamin MessengerMAX mRNA complex solution was added to the wells containing 0.5 ml OptiMEM pre-warmed to 37°C. Cells were incubated for 2 h at 37 °C, 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-diaminopropionic acid-N-palmityl-N-oleyl-amide in combination with neutral and PEGylated co-lipids can be used for in vitro transfection.

[0267] Successful introduction of the mRNA into the host cell can be monitored using various known methods, for example using a fluorescent marker, such as green fluorescent protein (GFP), such as a reporter enzyme (e.g. a luciferase derivative), or transfection of the modified mRNA can be determined by measuring the protein expression level of the target polypeptide, for example by Western blotting or immunocytochemistry or ELISA.

[0268] Nano-Luc luciferase activity reporter measurement Time-dependent quantitative detection of Nano-Glo-Luciferase expression (Promega) for secreted forms was performed by analyzing samples from tissue culture supernatants according to the reporter assay technical manual of the Nano-Glo Luciferase Assay System (Promega). Briefly, 5–20 μl of serum or lysate was diluted in a final volume of 100 μl H2O and mixed with an equal volume of reconstituted Nano-Glo Luciferase Assay Reagent in a 96-well GloMax 96 microplate. After at least 3 min of incubation at room temperature, luminescence could be measured in an appropriate luminometer.

[0269] Detection of Ang-1, COM-Ang-1, and CMP-Ang-1 proteins in cell lysates and cell supernatants Time-dependent quantitative detection of secreted Ang-1 expression and secreted COMP-Ang-1 or secreted COMP-Ang-2 expression is performed by analyzing samples from tissue culture supernatants by ELISA (Human Angiopoietin-1 Quantikine ELISA Kit (DANG10, R&D systems)) or Western blot according to the technical manual.

[0270] 500 ng of COMP-Ang-1 (mRNA sequence shown in SEQ ID NO:PAN05) fully modified with 5-methylcytidine and pseudouridine (COMP-Ang-1,5mC / pU), fully modified with 5-methylcytidine and Nl-methyl-pseudouridine (COMP-Ang-1,5mC / NlmpU), or unmodified (COMP-Ang-, unmod) with a polyA tail of approximately 120 nucleotides not shown in the sequence is transfected into HUVEC or HPMEC (human pulmonary microvascular endothelial cells, PromoCell, Heidelberg) using Lipofectamin MessengerMAX (Invitrogen, Carlsbad, CA; Thermo Fisher Scientific). Supernatants are harvested 4 hours after transfection and subjected to ELISA to determine protein expression and cytokine induction.

[0271] For immunoblot detection of Ang-1 and derivatives, protein lysates were loaded onto a NuPage SDS-PAGE system (chamber and power supply) (both from Life Technologies, Grand Island, NY) using 1.5 mm pre-prepared Bis-Tris gels and 4-12% acrylamide gradients with MOPS buffer as the running aid. Each lysate sample was prepared to a final volume of 40ul. The samples contained 25ug of protein lysate in various volumes, RIPA buffer to a volume of 26ul, 4ul of 10x reducing agent and 10ul of 4x SDS loading buffer (both from Life Technologies, Grand Island, NY). Samples were heated at 95°C for 5 minutes and loaded onto the gel. Standard settings were chosen by the manufacturer and were 200V, 120mA, and 25W max. The run time was 60 minutes, until the running dye reached the bottom of the gel.

[0272] After the run is completed, the plastic case is cracked and the gel inside is transferred to a pre-prepared nitrocellulose membrane kit and power supply (iBLOT; LifeTechnologies, Grand Island, NY). Using default settings, the protein lysate is transferred from the gel to the membrane by high electrical current.

[0273] After transfer, the membrane is incubated in 5% BSA in IX TBS for 15 minutes, then in 5% BSA in IX TBS + 0.1% Tween for another 15 minutes. A primary antibody against human Ang-1 protein (Ang-1 ab183701 Abcam, Cambridge, UK) is applied at 1:5000 dilution in 3 ml of 5% BSA in IX TBS solution overnight at 4°C with gentle shaking on an orbital shaker. The membrane is washed three times with IX TBS / 0.1% Tween, 5 minutes each time with gentle shaking. The secondary antibody (goat anti-rabbit HRP conjugate; Abeam, Cambridge, MA) is conjugated to horseradish peroxidase and binds to the primary antibody. The secondary antibody is diluted 1:10000 in 5% BSA in IX TBS and incubated at room temperature for 1 hour. After the incubation period, the membrane is washed three times with IX TBS / 0.1% Tween, 5 min each time with gentle shaking. The membrane is developed in 5 ml of Pierce West Pico chemiluminescent substrate (Thermo Fisher, Rockford, IL) as indicated. Western blots detect proteins at approximately the expected size of 70 kDa for human Ang-1 and 37 kDa for COMP-Ang-1 and CMP-Ang1. For reference, recombinant human Ang-1 protein was purchased from R&D Systems (biotechne).

[0274] result The protein expression data presented in Figures 10-14, 72-85, and 92-94 make it clear that the combination of various regulatory nucleotide sequences flanking the coding region can significantly alter protein expression levels. Such alterations are further driven by the modification of the nucleotides forming the construct, as exemplified by replacing 100% of the uridine and 100% of the cytidine nucleotides with pseudouridine and 5-methyl-cytidine (constructs marked with "*" in Figures 74 and 77-85). The alterations are not only observed in specific cell types, but also between different cell types. For example, PAN02 (wild type Ang-1 mRNA) shows the lowest luciferase activity in any cell line (Figures 10-14). All other constructs show enhanced protein expression across all cell types. Thus, approaches using regulatory sequences from different genes can increase protein expression activity. To this end, replacing the endogenous Ang-1 sequence showed a superior effect on protein expression of the reporter.

[0275] Notably, the same regulatory sequence exhibited different protein expression activity in different cell types (Figures 75-81). These data further support our approach that the use of specific regulatory sequences can affect protein expression activity in a context-specific, i.e., cell type-specific, cellular environment.

[0276] Furthermore, we have found that our data also relate to a different aspect, namely secretion. For example, the regulatory sequences used in construct PAN12 show cell type specific (HPMEC) expression in whole cell lysates as well as in the culture medium (supernatant) (Figures 10-14). This shows that these sequences are not only optimal for protein expression (translation) but also optimal for secretion. In contrast, construct PAN28 shows a more or less equal expression in all cell types. The regulatory sequences used in constructs PAN29 (and PAN58), namely the 5'-UTR and signal peptide sequences of MCP-1 in combination with the 3'-UTR of vWF, proved to be particularly useful for highly efficient protein expression in human (microvascular) pulmonary endothelial cells (see, for example, Figures 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 in protein expression and secretion, as shown in Figure 92. As shown in Figure 98, the regulatory sequences of PAN29 are functional in directing expression in primary human pulmonary microendothelial cells (HPMECs) using three different ORFs, namely wt Ang-1 in PAN52, hCOMP-Ang-1 in PAN53 and CMP-Ang-1 in PAN59. In contrast, the mRNA construct (PAN01) containing the endogenous 5'-UTR and 3'-UTR of human Ang-1 is not translated (Figure 98). Figure 99 shows that all four mRNA constructs exhibit comparable quality (integrity and purity).

[0277] Example 3: Activation of the Tie-2 pathway after transfection with modified mRNA encoding wild-type Tie-2 (PAN60) or an activating Tie-2 mutation (e.g., R849W) (PAN61) Modified mRNAs containing heterologous 5'UTRs and heterologous 3'UTRs are generated by in vitro transcription of PCR templates as already described in Example 1 above. This mRNA encoding an activating Tie-2 mutation (e.g. R849W) as well as wild-type Tie-2 is transfected at various concentrations into various cell lines (primary endothelial cells such as HeLa, HUVEC or HPMEC or HPAEC) using Lipofectamin MessengerMAX (Invitrogen, Carlsbad, CA; Thermo Fisher Scientific.). For quantitative Western blots, cell lysates at various time points (1-24 hours) after transfection of the mRNA are probed with an anti-phosphotyrosine antibody (R&D systems) to assess Tie-2 phosphorylation (pTyr, 140 kD). Total Tie-2 is detected by stripping the blots and reprobing with an anti-Tie-2 antibody (R&D systems). For quantitative measurements, two different ELISAs are performed to measure human Tie-2 or phospho-Tie-2 using the Human Tie-2 DuoSet ELISA Kit or the Human-Phospho-Tie-2 DuoSet IC ELISA according to the manufacturer's protocol (e.g., both ELISA kits from R&D systems).

[0278] Transfection of mRNA encoding a Tie-2 mutation (PAN61, R849W) increases the presence of phospho-Tie-2 compared to cell lysates derived from cells transfected with mRNA encoding wild-type Tie-2 mRNA or compared to non-transfected cells. This increase in Tie-2 phosphorylation is observed in the presence or absence of costimulation with recombinant human Ang-1 ligand and can be observed in different cell lines. To confirm activation of the Tie-2 signaling pathway and demonstrate biologically relevant and functional downstream signaling by expressing the Tie-2 (R849W) derivative, phosphorylation of Akt is demonstrated in time-course experiments. These experiments show that mRNA encoding a Tie-2 activating mutation (R849W) is functional and more potent than mRNA encoding wt Tie-2 in activating the Tie-2 signaling pathway. These hyperactivations of this pathway can be demonstrated in human endothelial and / or non-endothelial cells in the absence or presence of Tie-2 ligands (eg Ang-1).

[0279] Example 4: mRNA formulation in cationic LNPs for in vivo application by intravenous administration For in vivo experiments, mRNA-LNPs are prepared using a formulation process with β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleyl-amide as the cationic lipid. Alternatively, mRNA-LNPs can be prepared using the same procedure with the cationic lipid L-arginyl-β-alanine-N-palmityl-N-oleyl-amide.

[0280] The mRNA-LNP formulations are prepared using a modification of a method described for siRNA (Chen, S., Tam, YY, Lin, PJ, Sung, MM, Tam, YK, and Cullis, PR (2016), Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA. J. Control. Release 235, 236-244 and (ii) published patent application US20170121712).

[0281] Briefly, lipids are dissolved in ethanol in an appropriate molar ratio (e.g., 50:49:1 β-(L-arginyl)-L-2,3-diaminopropionic acid-N-palmityl-N-oleyl-amide:DPyPE:mPEG2000-DSPE). The lipid mixture is mixed with an isotonic sucrose solution of mRNA in a volume ratio of 2:1 (aqueous phase:ethanol) using a microfluidic mixer (NanoAssemblr®; Precision Nanosystems, Vancouver, BC) at a flow rate of 18 ml / min. Similarly, LNP formulations can be obtained using a slightly different mixing ratio of 3:1 (v / v; aqueous phase:ethanol) with a citrate- or acetate-buffered mRNA solution (pH 3-4) and a flow rate of 12 ml / min.

[0282] After the mixing process, the formulation is dialyzed against 10 mM HEPES-buffered or TRIS-buffered isotonic sucrose solution using a 3.5K MWCO Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific) for at least 18 hours at 4° C. Other sugars such as trehalose or glucose can be used in place of sucrose equally well in the formulation process.

[0283] The formulations are then 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 30–100 nm in size, have a ζ potential of >25 mV, exhibit >90% mRNA encapsulation and <1 EU / ml endotoxin.

[0284] The mRNA-LNP formulations are stored at -80 °C until further in vitro or in vivo use, with an RNA concentration of approximately 0.3 µg / µl and an RNA to total lipid ratio of approximately 0.03-0.05.

[0285] The features of the invention disclosed in the specification, the claims and / or the drawings may be important for realizing the invention in diverse forms thereof, both separately and in any combination thereof.

Claims

1. In the 5' to 3' direction 5'UTR, a coding region encoding an effector molecule, and ・3'UTR A recombinant nucleic acid construct comprising: the 5'UTR being (i) the 5'UTR of the gene encoding MCP-1 or (ii) a derivative of the 5'UTR of the gene encoding MCP-1, said derivative having at least 90% nucleotide identity with said 5'UTR and the same function as said 5'UTR; the 3'UTR is (i) the 3'UTR of the gene encoding vWF or (ii) a derivative of the 3'UTR of the gene encoding vWF, which has at least 90% nucleotide identity with the 3'UTR and the same function as the 3'UTR; the effector molecule is effective to restore a cellular function of the cell or to exert a therapeutic effect in or on the cell; and the recombinant nucleic acid construct is distinct from the wild-type mRNA encoding the effector molecule; The recombinant nucleic acid construct.

2. The recombinant nucleic acid construct of claim 1, wherein the 5'UTR and the 3'UTR of the recombinant nucleic acid construct are derived from different species.

3. The recombinant nucleic acid construct of claim 1, wherein the 5'UTR and the 3'UTR of the recombinant nucleic acid construct are derived from the same species.

4. 4. The recombinant nucleic acid construct of claim 1, comprising a polyA tail at the 3' end of the recombinant nucleic acid construct.

5. The recombinant nucleic acid construct of any one of claims 1 to 4, wherein the recombinant nucleic acid construct comprises a cap structure.

6. The recombinant nucleic acid construct of claim 5, comprising a cap structure at the 5' end of the recombinant nucleic acid construct.

7. The recombinant nucleic acid construct according to any one of claims 1 to 6, wherein the recombinant nucleic acid construct comprises an IRES (internal ribosome entry site) sequence.

8. The recombinant nucleic acid construct of claim 7, comprising an IRES (internal ribosome entry site) sequence at the 5' end of the recombinant nucleic acid construct.

9. 9. The recombinant nucleic acid construct according to any one of claims 1 to 8, wherein the recombinant nucleic acid construct comprises a nucleic acid sequence encoding a signal peptide.

10. The recombinant nucleic acid construct described in claim 9, wherein the signal peptide is in frame with the nucleic acid sequence encoding the effector molecule and is positioned between the 5'UTR and the coding region encoding the effector molecule.

11. 11. The recombinant nucleic acid construct of claim 9 or 10, wherein said signal peptide allows secretion of said effector molecule.

12. The nucleotide sequence encoding the signal peptide is (i) a nucleotide sequence encoding the signal peptide of MCP-1; (ii) a derivative of a nucleotide sequence encoding a signal peptide of MCP-1, which has at least 90% nucleotide identity with the nucleotide sequence encoding said signal peptide and has the same function as said signal peptide; (iii) a nucleotide sequence encoding a signal peptide of IL-6; (iv) a derivative of a nucleotide sequence encoding a signal peptide of IL-6, which has at least 90% nucleotide identity with the nucleotide sequence encoding the signal peptide and has the same function as the signal peptide; (v) a nucleotide sequence encoding the signal peptide of Ang-2; (vi) a derivative of a nucleotide sequence encoding a signal peptide of Ang-2, which has at least 90% nucleotide identity with the nucleotide sequence encoding the signal peptide and has the same function as the signal peptide; (vii) a nucleotide sequence encoding the signal peptide of Ang-1, and (viii) A derivative of a nucleotide sequence encoding a signal peptide of Ang-1, the derivative having at least 90% nucleotide identity with the nucleotide sequence encoding the signal peptide and the same function as the signal peptide.

12. The recombinant nucleic acid construct of any one of claims 9 to 11, selected from the group consisting of:

13. 13. The recombinant nucleic acid construct of any one of claims 1 to 12, wherein the cell in which a cellular function is restored and / or in or on which a therapeutic effect is exerted is an endothelial cell.

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

15. The recombinant nucleic acid construct of claim 14, wherein the vascular endothelial cells are microvascular endothelial cells.

16. 16. The recombinant nucleic acid construct of any one of claims 1 to 15, wherein the cellular function can be restored by an effector molecule having an endothelial cell anti-permeability effect, an anti-vascular leakage effect, an endothelial cell anti-apoptotic effect, an endothelial cell anti-inflammatory effect, or an endothelial cell anti-stress response effect.

17. The recombinant nucleic acid construct of claim 16, wherein the effect is related to the TIE-2 signaling pathway, the VEGF receptor pathway, the NOTCH signaling pathway, the PI3-kinase pathway, the eNOS signaling pathway, the sirtuin-dependent metabolic pathway, the energy homeostasis pathway, the oxidative stress pathway, the ET-1 signaling pathway, or the NO-mediated signaling pathway.

18. A recombinant nucleic acid construct according to any one of claims 1 to 17, capable of expressing the effector molecule and / or suitable for expressing the effector molecule.

19. 19. The recombinant nucleic acid construct of any one of claims 1 to 18, which is an mRNA.

20. 19. The recombinant nucleic acid construct of any one of claims 1 to 18, which consists of ribonucleotides, deoxyribonucleotides, or a combination thereof.

21. A vector comprising the recombinant nucleic acid construct of any one of claims 1 to 20.

22. A cell comprising a recombinant nucleic acid construct according to any one of claims 1 to 20 and / or a vector according to claim 21.

23. 21. A delivery vehicle comprising one or more lipids and the recombinant nucleic acid construct of any one of claims 1 to 20, wherein the one or more lipids and the recombinant nucleic acid construct collectively form a cationic lipid particle.

24. The delivery vehicle of claim 23, wherein the particle is a nanoparticle.

25. The delivery vehicle of claim 24, wherein the average size of the nanoparticles is from about 30 nm to about 200 nm.

26. A pharmaceutical composition comprising a recombinant nucleic acid construct according to any one of claims 1 to 20, a vector according to claim 21, a cell according to claim 22, and / or a delivery vehicle according to any one of claims 23 to 25, and a pharma- ceutically acceptable diluent.

27. A pharmaceutical composition comprising a recombinant nucleic acid construct according to any one of claims 1 to 20 for the treatment and / or prevention of a disease.

28. 21. A pharmaceutical composition comprising the recombinant nucleic acid construct of any one of claims 1 to 20 for restoring a cellular function of a cell, wherein the cell is an endothelial cell or a non-endothelial cell.

29. 29. The pharmaceutical composition of claim 28, wherein the non-endothelial cells are selected from the group consisting of epithelial cells, alveolar macrophages, type I pneumocytes, type II pneumocytes, alveolar macrophages, lung cells, lung epithelial cells, hematopoietic cells, bone marrow cells, bone cells, stem cells, mesenchymal cells, nervous system cells, glial cells, neuronal cells, astrocytes, cells of the peripheral nervous system, cardiac cells, adipocytes, vascular smooth muscle cells, cardiac muscle cells, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, B cells, T cells, reticulocytes, leukocytes, granulocytes, macrophages, neutrophils, antigen presenting cells (dendritic cells), fibroblasts, liver cells, tumor cells, and combinations thereof.

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