WO2021094792A1 - METHOD FOR THE DEVELOPMENT OF A DELIVERY PLATFORM TO PRODUCE DELIVERABLE PTD-IVT-mRNA THERAPEUTICS - Google Patents

METHOD FOR THE DEVELOPMENT OF A DELIVERY PLATFORM TO PRODUCE DELIVERABLE PTD-IVT-mRNA THERAPEUTICS Download PDF

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WO2021094792A1
WO2021094792A1 PCT/GR2020/000059 GR2020000059W WO2021094792A1 WO 2021094792 A1 WO2021094792 A1 WO 2021094792A1 GR 2020000059 W GR2020000059 W GR 2020000059W WO 2021094792 A1 WO2021094792 A1 WO 2021094792A1
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ivt
ptd
mrna
therapeutic
pfvyli
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PCT/GR2020/000059
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French (fr)
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Lefkothea PAPADOPOULOU
Ioannis VIZIRIANAKIS
Ioannis PAPPAS
Androulla MILIOTOU
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Aristotle University Of Thessaloniki E.L.K.E.
University Of Thessaly
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Priority to EP20823912.9A priority Critical patent/EP4061393A1/en
Publication of WO2021094792A1 publication Critical patent/WO2021094792A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0091—Purification or manufacturing processes for gene therapy compositions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a method for the development of a safe delivery platform to improve health including food.
  • PTD Protein Transduction Domain
  • IVT-mRNA in vitro transcribed mRNA
  • IVT-mRNA is a synthetic mRNA that resembles endogenous mRNA and, after its successful delivery into the cell, it can be translated into the desired corresponding protein.
  • the therapeutic IVT-mRNA used in this platform can be any IVT-mRNA of interest, depending on the gene translational product that must be delivered intracellularly.
  • the platform is designed to offer numerous improvements with respect to prior delivery platforms as it successfully tackles problems of stability, transduction and translation in mRNA-therapeutics’ field.
  • the major difficulty for all IVT-mRNA therapeutics - that involves their in vivo conveyance - is their intracellular delivery.
  • the IVT-mRNA to be translated into protein, must survive in the extracellular space that contains high levels of ubiquitous RNases, reach the cells of interest, and finally, cross the cell membrane.
  • the optimum delivery system of IVT-mRNA should fulfill several functions, such as the ability to form complexes with the IVT-mRNA, to promote cellular uptake, to protect mRNA from intracellular and extracellular nuclease degradation, and to enable the release of mRNA into the cytoplasm.
  • IVT-mRNAs Conventional methods for delivering IVT-mRNAs include the production of complexes, using cationic lipid vesicles (e.g., iipofectamine or DOTAP-1 ,2-dioleoyl-3- trimethylammonium-propane) attached to the negatively charged IVT-mRNA molecules, building up the lipoplexes.
  • cationic lipid vesicles e.g., iipofectamine or DOTAP-1 ,2-dioleoyl-3- trimethylammonium-propane
  • biomaterials such as nanoparticles, viromers, protamine/IVT-mRNA complexes, microparticles, polymeric nanoparticles, self- assembled materials and biomaterial scaffolds.
  • LNPs lipid nanoparticles
  • siRNAs in vivo and promising phase III clinical trials.
  • LNPs lipid nanoparticles
  • SARS-CoV-2 lipid nanoparticles
  • LNPs increase IVT-mRNA cargo retention time in vivo and enhance IVT-mRNA cytosolic delivery.
  • LNPs face the issue of IVT-mRNA escape after endocytosis as well as they are used to accumulate in off-target organs, such as the liver, while instances of allergic reactions in human patients were observed.
  • the aim of the present invention is to remedy the above-mentioned drawbacks and shortcomings set out above by providing a solution for the needs identified above.
  • Said method is remarkable by the combination of the PTD technology with IVT-mRNA, via covalent chemical binding-conjugation, wherein said IVT-mRNA is conjugated to a said PTD, by means whereof the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is achieved, thus providing IVT-mRNA:PTD complex which are very stable; wherein said IVT-mRNAs, constituting therapeutic molecules, are submitted to an intracellular delivery through said covalently conjugation to the appropriate PTD -particularly said PFVYLI peptide-, after which said IVT-mRNA’s are directed to ribosomes and translated into corresponding targeted proteins to reveal their therapeutic effect.
  • IVT-mRNAs constituting a new generation of therapeutic molecules, after their successful intracellular delivery through the proposed disclosure of the covalent chemical reaction of the said IVT-mRNA molecules with puromycin, which is conjugated via an amide bond to the selected PTD, particularly short peptide, resp. the PFVYLI peptide notably, which are directed to the ribosomes and translated into the corresponding desired proteins, revealing their expected therapeutic effect.
  • IVT-mRNA technology The strongest advantage of IVT-mRNA technology is that mRNA molecules do not interfere with the host genome, thus no triggering oncogenic mutations take place. Furthermore, this technology is advantageous in comparison with the protein replacement therapy, by producing biotechnologically the desired recombinant proteins, especially due to the time- consuming and costly purification steps.
  • IVT-mRNA is degraded after few days in the cytoplasm through physiologic pathways and therefore does not require either inactivation or removal of the IVT-mRNA in cases of unexpected observed toxicity, in comparison with strategies via viral vectors.
  • IVT-mRNA-based therapies appear to be much safer than DNA- or viral-based therapies and they are applicable to a broad spectrum of disorders, both acute and chronic.
  • IVT-mRNAs there is no size limit for creating the desired mRNA sequence through in vitro transcription and IVT-mRNA’s production can be carried out at the desired scales with commercially available materials.
  • the use of IVT-mRNAs as therapeutics is beneficial because of its biological origin.
  • PTDs Protein Transduction Domains
  • Said method proposed according to the invention is remarkable in that a PTD is selected and in that the development of said delivery platform is accomplished by a covalent chemical reaction between said IVT-mRNA molecules with puromycin, which is conjugated via an amide bond to the selected PTD, notably the PFVYLI peptide.
  • Said method is further remarkable by the combination of the PTD technology with IVT-mRNA, via covalent chemical binding-conjugation - coupling, wherein said IVT-mRNA is conjugated to a said PTD.
  • the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is achieved, thus providing PTD-IVT-mRNA complex which are very stable.
  • Said IVT-mRNAs constituting therapeutic molecules are further submitted to an intracellular delivery through said covalent chemical reaction of the IVT-mRNA with said puromycin, which is conjugated via an amide bond to the appropriate PTD -particularly the said PFVYLI peptide-, after which said IVT-mRNA’s are directed to ribosomes and translated into corresponding targeted proteins to reveal their therapeutic effect, wherein said covalent bond reduces load loss, and enhances protection in adverse conditions during in vivo delivery or in the intracellular environment, due to the existence of RNases.
  • the method for the development of a delivery platform to produce deliverable PTD-IVT-mRNA therapeutics consists of a chemical reaction of the covalent conjugation of a therapeutic IVT-mRNA, after its polyA tail, via puromycin, which is conjugated via an amide bond to the selected PTD, such as the PFVYLI peptide, said method comprising the general steps of: i. Selection of a PTD - Dissolution of PTD ii. Puromycin as the peptide and nucleic acid linker, iii. Phosphorylation, and iv. Ligation.
  • PTDs Protein Transduction Domains
  • Conjugating of IVT-mRNA to a PTD is thus achieved thanks to the present invention and has been validated by various methods as well as by the high rates of transfection in cells.
  • the improvement lies in the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule, since the covalent bond gives an advantage in reduced “load” loss, as well as in its protection in adverse conditions during in vivo delivery or in the intracellular environment, due to the existence of RNases.
  • the PTD-IVT-mRNA complex appears to be very stable even in human plasma for long periods, as it is expected to be injected intravenously during the therapeutic administration.
  • PFVYLI peptide is selected as said PTD.
  • PTD technology employs short peptides, able to transduce almost all biological membranes, carrying intracellularly a variety of «cargos» from micro-molecules, siRNAs to macromolecules (proteins, RNAs, plasmid DNA, nanoparticles).
  • This invention thus relates to the covalently conjugat/on of a selected PTD -the PFVYLI peptide-, which is exploited as a neutral surface charge carrier for any therapeutic IVT- mRNA of interest, through the covalent chemical reaction of the said IVT-mRNA molecule with puromycin, which is conjugated via an amide bond to the selected PTD, leading to greater stability in the presence of serum, thereby increasing its functionality, increasing its tolerance to serum and reducing the variability of transfection between different cell types.
  • the PTD-IVT-mRNA disclosure demonstrates lower cytotoxicity than other delivery methods, like Lipofectamine, a commonly used cationic lipid reagent for delivering IVT- mRNAs
  • PTD-IVT-mRNA complexes exhibit high stability in low and high serum and plasma conditions compared to naked IVT-mRNA.
  • the selected Protein Transduction Domain is a 6-amino acid (6aa) peptide, the PFVYLI, which is covalently conjugated to the IVT-mRNA.
  • PFVYLI is selected as a hydrophobic peptide of six (6) amino acids, of > 95% purity and acetylated at the N-terminus (Ac-Pro-Phe-Val-Tyr-Leu-lle- COOH), notably wherein the peptide is synthesized upon order, wherein this peptide has very low water solubility, wherein it is kept in powder form at -20°C and wherein Dissolution in DMF (Dimethylformamide) occurs shortly before use.
  • DMF Diamethylformamide
  • Puromycin (Puromycin dihydrochloride) binds to the N-terminus of PFVYLI with EDC.HCI [N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride], for the conjugation via an amide bond to the PFVYLI; and/or in said third step Phosphorylation of the puro-PFVYLI product is carried out with T4 PNK, and/or in said fourth step Ligation of the phosphorylated puro-PFVYLI product is carried out with the selected, therapeutic IVT-mRNA, with T4 RNA ligase. steps in a specific and determinate order:
  • Phosphorylation of the puro-PFVYLI complex is then carried out using 1 mI of T4 PNK, 4 mI of T4 PNK buffer, 17 mI FLO DEPC-treated and 10 mI of DMF.
  • the phosphorylation reaction is carried out at 37°C, for 1 hour and 40 minutes, followed by inactivation of the enzyme for 20 minutes, at 65°C.
  • IVT-mRNA For 1 mg of PFVYLI peptide is estimated to require 18 nM of the respective therapeutic IVT-mRNA.
  • the molecular weight of IVT-mRNA can be calculated using any bioinformatics tool. For this, one enters the sequence of the desired IVT-mRNA, and with the molecular weight being calculated, the concentration to be added to the reaction is found.
  • the successful chemical covalent conjugation of PFVYLI to IVT-mRNA can be evaluated by electrophoresis on 8 M urea/6% PAGE after ethidium bromide staining (Miyamoto-Sato et al. , 2003).
  • This method can also be termed a “retardation assay” since binding to the PFVYLI PTD peptide clearly shows the delayed transposition of IVT-mRNA into the polyacrylamide gel in comparison with naked IVT-mRNA.
  • a Protein Transduction Domain (PTD), preferably hydrophobic, is selected without a free amino group; more preferably wherein the selected PTD has a purity > 95% and is acetylated at the N-terminus; even more preferably wherein the selected PTD is synthesized upon order; in particular wherein PFVYLI is selected as the PTD, of six (6) amino acids (Ac-Pro-Phe-Val-Tyr-Leu- lle-COOH).
  • PTD Protein Transduction Domain
  • said PTD is dissolved in an appropriate solvent, depending on the chemical characteristics of the PTD; in particular wherein PFVYLI is selected as the PTD, and is synthesized, wherein it has a very low water solubility, and it is thus kept in powder form at -20°C and, just prior use, is dissolved in an organic solvent, like DMA (Dimethylacetamide) or DMSO (Dimethyl sulfoxide), preferably DMF (Dimethylformamide).
  • an organic solvent like DMA (Dimethylacetamide) or DMSO (Dimethyl sulfoxide), preferably DMF (Dimethylformamide).
  • puromycin for said Coupling or dissolution step of the PTD and puromycin with EDC.HCI, puromycin, particularly Puromycin dihydrochloride, is used as linker, which is dissolved preferably in DMF, whereas other nucleosides are possibly used as linkers; still more particularly wherein the EDC.FICI [N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride] is dissolved, preferably in DEPC (Diethyl pyrocarbonate)-treated distilled FI2O; wherein
  • Puromycin is the linker between the PTD and the IVT-mRNA, which are added in subsequent step below.
  • the puro-PTD product is phosphorylated by the enzyme T4 Polynucleotide Kinase (T4 PNK); in particular wherein the puro-PTD product is preferably phosphorylated by using 10 units of T4 PNK, 1X of T4 PNK buffer, 10 mI of the PTD’s solvent and DEPC-treated distilled Ft 0 up to 40 mI; more particularly wherein the reaction for the phosphorylation is carried out at 37°C, for 1 hour and 40 minutes, followed by inactivation of the enzyme T4 PNK, preferably for 20 minutes, at 65°C, which produces the phosphorylated puro-PTD product.
  • T4 PNK T4 Polynucleotide Kinase
  • the phosphorylated puro-PTD product is incubated with RNase Inhibitor, preferably 60 units, for 15 minutes, at 37°C, thereby removing any RNases from the mixture of the above reaction and thus protecting the IVT-mRNA, which are added at the next step.
  • RNase Inhibitor preferably 60 units, for 15 minutes, at 37°C
  • the above phosphorylated puro-PTD product (from 1 mg of PTD) is ligated with the selected - produced therapeutic IVT- mRNA by using the enzyme T4 RNA ligase; wherein particularly for the ligation reaction, the phosphorylated puro-PTD product is incubated with 15 units of T4 RNA ligase, 1X of T4 RNA ligase buffer, 18 nM IVT-mRNA and up to 60 pi with DEPC-treated H2O; preferably wherein the ligation reaction is carried out at 16°C, overnight and then, the enzyme T4 RNA ligase is inactivated, more preferably at 70°C for 10 minutes.
  • the chemical covalent conjugation of the PTD to IVT- mRNA is evaluated by gel electrophoresis, notably wherein this method is qualified as a retardation assay, in that binding to the PTD peptide shows a delayed transposition of IVT- mRNA into the gel in comparison with naked IVT-mRNA reference; preferably wherein the electrophoresis is carried out on denaturing 8M urea / 6% polyacrylamide gel, after ethidium bromide staining ; or wherein alternatively, agarose gel is used for the assessment of the generation of IVT-mRNA:PTD complexes, such as simple gel electrophoresis or denaturing agarose gels, containing formaldehyde or glyoxal/DMSO.
  • this method is qualified as a retardation assay, in that binding to the PTD peptide shows a delayed transposition of IVT- mRNA into the gel in comparison with naked IVT-mRNA reference; preferably wherein the electrophoresis is
  • the invention also relates to the use of Protein Transduction Domains (PTDs) technology as a transduction delivery platform for therapeutic IVT-mRNAs, wherein said delivery platform is accomplished by a covalent chemical reaction between said IVT-mRNA molecules and a selected transduction peptide, notably the PFVYLI peptide, including the combination of the PTD technology with IVT-mRNA, via covalent chemical binding- conjugation, wherein said IVT-mRNA is conjugated to a PTD, by means whereof a IVT- mRNA: PTD complex is produced which is very stable, wherein the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is being enhanced; wherein said IVT-mRNAs, constituting a selected generation of therapeutic molecules, are submitted to an effective intracellular delivery through said covalently conjugation to the appropriate PTD, particularly the said PFVYLI peptide, after which said IVT-mRNAs are directed to the ribosomes and translated into the corresponding targeted proteins thereby revealing
  • IVT-mRNAs as therapeutics is beneficial because of its biological origin.
  • a delivery platform produces deliverable PTD-IVT-mRNA therapeutics, wherein a neutral surface carrier is provided for delivering a therapeutic IVT-mRNA to its target site with a set of predetermined limitations being removed.
  • This invention achieves high rates of transfection in cellular models, even increase of seven (7) folds of the translation levels of the desired protein, increasing the Mean Fluorescent Intensity (MFI) three times (3X) as well, in comparison with the control cells.
  • MFI Mean Fluorescent Intensity
  • this invention relates to a method and its use for the development of a delivery platform to produce deliverable PTD-IVT-mRNA therapeutics, wherein Protein Transduction Domains (PTDs) technology is used as a transduction delivery platform for therapeutic IVT-mRNAs and the development of said delivery platform is accomplished by a covalent chemical reaction between said IVT-mRNA molecules and a selected transduction peptide PFVYLI.
  • PTDs Protein Transduction Domains
  • this invention also relates to a delivery platform and its use to produce deliverable PTD-IVT-mRNA therapeutics, wherein Protein Transduction Domains (PTDs) technology is used as a transduction delivery platform for therapeutic IVT-mRNAs.
  • PTDs Protein Transduction Domains
  • the development of said delivery platform is accomplished by a covalent chemical reaction between said IVT- mRNA molecules and a selected transduction peptide PFVYLI. It is remarkable by the combination of the PTD technology with IVT-mRNA, via covalent chemical binding - conjugation, wherein said IVT-mRNA is conjugated to a PTD, by means whereof the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is achieved, thus providing IVT-mRNA:PTD complex which are very stable. Said IVT-mRNAs constituting therapeutic molecules, are delivered intracellularly through said covalently conjugation to the appropriate PTD, whereas said IVT-mRNA’s are directed to ribosomes and translated into corresponding targeted
  • the proposed technology is fast and low cost, while it has the advantage of:
  • the PTD-IVT-mRNA complexes proposed according to the invention have the potential to:
  • (b) be transferred to the membranes, in the context of cellular therapies, such as the CAR (chimeric antigen receptor) cancer immunotherapy.
  • CAR chimeric antigen receptor
  • Another approach to targeted cancer treatment is also the systemic administration of IVT-mRNA, encoding a suicide gene [such as herpes simplex virus 1 -thymidine kinase (HSV1-tk) or other cancers-related viruses];
  • RNA molecules such as shRNAs - miRNAs for gene silencing in case of therapeutic or diagnostic purposes;
  • cell reprogramming for the immediate induction of cell differentiation, the reprogramming of somatic cells into induced pluripotent stem cells (e.g. using IVT-mRNA of various transcription factors such as Oct4, Sox2, Klf4 and cMyc), the directed (trans-) differentiation of cells into desired cell types (e.g. from fibroblasts to myocytes or hepatocytes) or and for overexpression of receptors or paracrine factors (e.g. VEGFa) in mesenchymal stem cells to improve homing behaviour;
  • IVT-mRNA of various transcription factors such as Oct4, Sox2, Klf4 and cMyc
  • desired cell types e.g. from fibroblasts to myocytes or hepatocytes
  • desired cell types e.g. from fibroblasts to myocytes or hepatocytes
  • paracrine factors e.g. VEGFa
  • Fig. 1 represents the main embodiment of the invention, which is the chemical reaction of the covalent conjugation of the PTD (such as the PFVYLI peptide) to a therapeutic IVT- mRNA, through the covalent chemical reaction of the said IVT-mRNA molecule with puromycin, which is conjugated via an amide bond to the selected PTD.
  • the PTD such as the PFVYLI peptide
  • Fig. 2, resp. 3 is a simplified, resp. schematized representation of a previous variant thereof, i.e. of a chemical reaction according to the of the invention.
  • this invention relates to a method the general steps whereof comprise according to a preferred embodiment as notably represented in Fig. 1 :
  • PFVYLI is a hydrophobic peptide of six (6) amino acids, of > 95% purity and acetylated at the N-terminus (Ac-Pro-Phe-Val-Tyr-Leu-lle-COOFI).
  • the peptide is synthesized upon order. This peptide has very low water solubility, therefore it is kept in powder form at -20°C and solubilized in DMF (Dimethylformamide) shortly before use.
  • Puromycin Puromycin (Puromycin dihydrochloride) will bind to the N-terminus of PFVYLI with EDC.HCI [N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride] for the conjugation via an amide bond to the PFVYLI;
  • PTDs Protein Transduction Domains
  • the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is a decisive achievement, since the covalent bond gives an advantage in reduced “load” loss, and in its protection in adverse conditions during in vivo delivery or in the intracellular environment, due to the existence of RNases.
  • the PTD-IVT-mRNA complex appears to be very stable even in human plasma for long periods, as it is expected to be injected intravenously during the therapeutic administration.
  • PTD Protein Transduction Domain
  • the selected PTD is preferably of > 95% purity and is acetylated at the N-terminus.
  • the selected PTD is preferably synthesized upon order.
  • PFVYLI is selected as the PTD, of six (6) amino acids (Ac-Pro-Phe-Val-Tyr- Leu-lle-COOH).
  • the PTD is dissolved in the appropriate solvent, depending on the chemical characteristics of the PTD.
  • the selected PTD. the PFVYLI is synthesized. It has very low water solubility, therefore it is kept in powder form at -20°C and, just prior use, is dissolved in DMF (Dimethylformamide). Any organic solvent, like DMA (Dimethylacetamide) or DMSO (Dimethyl sulfoxide), can be used instead of DMF.
  • DMF Dimethylformamide
  • linker can be used puromycin (in particular, Puromycin dihydrochloride) which is dissolved (preferably in DMF)
  • puromycin in particular, Puromycin dihydrochloride
  • other nucleosides could also be used as linkers.
  • EDC.HCI N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride
  • DEPC Diethyl pyrocarbonate
  • Puromycin is the linker between the PTD and the IVT-mRNA, which will be added in step 6
  • the puro-PTD product is phosphorylated by the enzyme T4 Polynucleotide Kinase (T4 PNK).
  • the puro-PTD product is preferably phosphorylated by using 10 units of T4 PNK, 1X of T4 PNK buffer, 10 mI of the PTD’s solvent and DEPC-treated distilled H20 up to 40 mI.
  • the reaction for the phosphorylation is carried out at 37°C, for 1 hour and 40 minutes, followed by inactivation of the enzyme T4 PNK, preferably for 20 minutes, at 65°C, in order to produce the phosphorylated puro-PTD product
  • the phosphorylated puro-PTD product is incubated with RNase Inhibitor, preferably 60 units, for 15 minutes, at 37°C, in order to remove any RNases from the mixture of the above reaction and to protect the IVT-mRNA, which will be added at the next step.
  • RNase Inhibitor preferably 60 units, for 15 minutes, at 37°C
  • the above phosphorylated puro-PTD product (from 1 mg of PTD) is ligated with the selected - produced therapeutic IVT-mRNA by using the enzyme T4 RNA ligase.
  • the phosphorylated puro-PTD product is incubated with 15 units of T4 RNA ligase, 1X of T4 RNA ligase buffer, 18 nM IVT-mRNA and up to 60 mI with DEPC-treated H20.
  • RNA ligase is inactivated, preferably at 70°C for 10 minutes.
  • the successful chemical covalent conjugation of the PTD to IVT-mRNA can be evaluated by gel electrophoresis.
  • This method can also be termed a “band-shift assay”, since binding to the PTD peptide clearly shows the delayed transposition of IVT-mRNA into the gel in comparison with to naked IVT-mRNA.
  • the electrophoresis is carried out preferably on denaturing 8 M urea/6% polyacrylamide gel, after ethidium bromide staining (Miyamoto-Sato et al., 2003).
  • agarose gel can be used for the assessment of the successful generation of PTD-IVT-mRNA complexes (simple gel electrophoresis or denaturing agarose gels, containing formaldehyde or glyoxal/DMSO).
  • the proposed technology is fast and low cost, while it has the advantage of the safety, since there is no integration into the host genome, as well as the transient nature of IVT-mRNA; further enhancing the stability of the therapeutic IVT-mRNAs; also the efficacy of intracellular transduction; as well as the increase of the expression levels of the corresponding therapeutic protein.
  • IVT-mRNA:PTD complexes have the potential to: (a) replace (i) corresponding absent intracellular proteins, even ones localised in organelles, in monogenic-metabolic diseases, as protein replacement therapy, as well as (ii) systemically secreted therapeutic molecules; (b) to be transferred to the membranes, in the context of cellular therapies, such as the CAR (chimeric antigen receptor) cancer immunotherapy.
  • CAR chimeric antigen receptor
  • IVT-mRNA encoding a suicide gene [such as herpes simplex virus 1 -thymidine kinase (HSV1-tk) or other cancers- related viruses]; (c) to be secreted in the context of the development of personalized vaccines against specific types of cancer, against the identified neo-antigens, in the context crucial role in the body's inflammatory-allergic reactions in the context of prophylactic vaccines; (e) to encode cytokines and growth factors in a number of somatic and stem cells; (f) to contribute to intracellular delivery of non-protein-coded RNA molecules, such as shRNAs - miRNAs for gene silencing in case of therapeutic or diagnostic purposes;
  • a suicide gene such as herpes simplex virus 1 -thymidine kinase (HSV1-tk) or other cancers- related viruses
  • Intracellular delivery of these nucleases will have several advantages, such as transient expression with efficient in vivo and in vitro translocation, non-genomic integration, potentially low off-target phenomena and high efficiency of gene editing.

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Abstract

This invention relates to a method for the production of a delivery platform to produce deliverable PTD-IVT-mRNA therapeutics, wherein Protein Transduction Domains (PTDs) technology is used as a transduction delivery platform for therapeutic IVT-mRNAs and the development of said delivery platform is accomplished by a covalent chemical reaction between said IVT-mRNA molecules and a selected transduction peptide PFVYLI, Said method is remarkable by the combination of the PTD technology with IVT-mRNA, via covalent chemical binding-conjugation, wherein said IVT-mRNA is conjugated to a PTD, by means whereof the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is achieved, thus providing very stable IVT-mRNA:PTD complex. Said IVT-mRNAs constituting therapeutic molecules, are submitted to an intracellular delivery through said covalently conjugation to the appropriate PTD, after which said IVT-mRNA's are directed to ribosomes and translated into corresponding targeted proteins to reveal their therapeutic effect. Delivery platform produced with said method and its use.

Description

METHOD FOR THE DEVELOPMENT OF A DELIVERY PLATFORM TO PRODUCE DELIVERABLE PTD-IVT- mRNA THERAPEUTICS
Field of the invention
The present invention relates to a method for the development of a safe delivery platform to improve health including food.
Background of the invention
An improved delivery platform is being developed by the exploitation of the Protein Transduction Domain (PTD) Technology, for the intracellular transduction of therapeutic in vitro transcribed (IVT)-mRNAs and the subsequent expression of the desired protein.
In this field of the invention, in vitro transcribed mRNA (IVT-mRNA) is a synthetic mRNA that resembles endogenous mRNA and, after its successful delivery into the cell, it can be translated into the desired corresponding protein.
The therapeutic IVT-mRNA used in this platform can be any IVT-mRNA of interest, depending on the gene translational product that must be delivered intracellularly.
The platform is designed to offer numerous improvements with respect to prior delivery platforms as it successfully tackles problems of stability, transduction and translation in mRNA-therapeutics’ field.
The major difficulty for all IVT-mRNA therapeutics - that involves their in vivo conveyance - is their intracellular delivery.
What is needed, therefore, is an improved method for the safe intracellular delivery of the therapeutic IVT-mRNAs, in order to be translated into the desired protein.
The IVT-mRNA, to be translated into protein, must survive in the extracellular space that contains high levels of ubiquitous RNases, reach the cells of interest, and finally, cross the cell membrane.
The optimum delivery system of IVT-mRNA should fulfill several functions, such as the ability to form complexes with the IVT-mRNA, to promote cellular uptake, to protect mRNA from intracellular and extracellular nuclease degradation, and to enable the release of mRNA into the cytoplasm.
Prior art
Conventional methods for delivering IVT-mRNAs include the production of complexes, using cationic lipid vesicles (e.g., iipofectamine or DOTAP-1 ,2-dioleoyl-3- trimethylammonium-propane) attached to the negatively charged IVT-mRNA molecules, building up the lipoplexes. In addition, various biomaterials are used, such as nanoparticles, viromers, protamine/IVT-mRNA complexes, microparticles, polymeric nanoparticles, self- assembled materials and biomaterial scaffolds.
A promising delivery platform for IVT-mRNA delivery consists of lipid nanoparticles (LNPs) and it has recently succeeded in delivering siRNAs in vivo and promising phase III clinical trials. LNP based IVT-mRNAs are developed as a therapeutic approach for a range of diseases, including multiple types of cancer, as well as Zika, Ebola, HCV, influenza viruses and recently for SARS-CoV-2. LNPs increase IVT-mRNA cargo retention time in vivo and enhance IVT-mRNA cytosolic delivery. However, LNPs face the issue of IVT-mRNA escape after endocytosis as well as they are used to accumulate in off-target organs, such as the liver, while instances of allergic reactions in human patients were observed.
Overall, the presence of serum has been well documented to regulate the efficacy of transfection of «lipoplexes» and «polyplexes». In addition, the size of lipoplexes, the surface charge density, the colloidal stability and the modified uptake mechanisms (usually clathrin- and caveolae-mediated endocytosis) have been suggested to play an important role and raise practical issues for in vivo applications.
There is a need to develop a new, neutral surface carrier without the above-mentioned limitations for delivering a therapeutic IVT-mRNA to its target site.
Aim of the invention
The aim of the present invention is to remedy the above-mentioned drawbacks and shortcomings set out above by providing a solution for the needs identified above.
Summary of the invention To solve this problem, there is thus primarily proposed according to the present invention a method for the production of a safe delivery platform to produce deliverable PTD-IVT-mRNA therapeutics, by the exploitation of the Protein Transduction Domain (PTDs) Technology, which is used as a transduction delivery platform for therapeutic IVT-mRNAs for the intracellular transduction of therapeutic in vitro transcribed (IVT)-mRNAs and the subsequent expression of the desired protein, wherein the development of said delivery platform is accomplished by a covalent chemical reaction between said IVT-mRNA molecules and a selected transduction peptide, notably the PFVYLI peptide. Said method is remarkable by the combination of the PTD technology with IVT-mRNA, via covalent chemical binding-conjugation, wherein said IVT-mRNA is conjugated to a said PTD, by means whereof the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is achieved, thus providing IVT-mRNA:PTD complex which are very stable; wherein said IVT-mRNAs, constituting therapeutic molecules, are submitted to an intracellular delivery through said covalently conjugation to the appropriate PTD -particularly said PFVYLI peptide-, after which said IVT-mRNA’s are directed to ribosomes and translated into corresponding targeted proteins to reveal their therapeutic effect. Said covalent bond reduces load loss and enhances protection in adverse conditions during in vivo delivery or in the intracellular environment, due to the existence of RNases. IVT-mRNAs, constituting a new generation of therapeutic molecules, after their successful intracellular delivery through the proposed disclosure of the covalent chemical reaction of the said IVT-mRNA molecules with puromycin, which is conjugated via an amide bond to the selected PTD, particularly short peptide, resp. the PFVYLI peptide notably, which are directed to the ribosomes and translated into the corresponding desired proteins, revealing their expected therapeutic effect.
The strongest advantage of IVT-mRNA technology is that mRNA molecules do not interfere with the host genome, thus no triggering oncogenic mutations take place. Furthermore, this technology is advantageous in comparison with the protein replacement therapy, by producing biotechnologically the desired recombinant proteins, especially due to the time- consuming and costly purification steps.
IVT-mRNA is degraded after few days in the cytoplasm through physiologic pathways and therefore does not require either inactivation or removal of the IVT-mRNA in cases of unexpected observed toxicity, in comparison with strategies via viral vectors.
It can be characterized as a “hit-and-run” system, which leaves no ultimate genetic residue in recipient cells. In principle, IVT-mRNA-based therapies appear to be much safer than DNA- or viral-based therapies and they are applicable to a broad spectrum of disorders, both acute and chronic.
In theory, there is no size limit for creating the desired mRNA sequence through in vitro transcription and IVT-mRNA’s production can be carried out at the desired scales with commercially available materials. In addition, the use of IVT-mRNAs as therapeutics is beneficial because of its biological origin.
There is further proposed according to a main embodiment of the invention a method for the development of a delivery platform to produce deliverable PTD-IVT-mRNA therapeutics, wherein said Protein Transduction Domains (PTDs) technology is used as a transduction delivery platform for therapeutic IVT-mRNAs. Said method proposed according to the invention is remarkable in that a PTD is selected and in that the development of said delivery platform is accomplished by a covalent chemical reaction between said IVT-mRNA molecules with puromycin, which is conjugated via an amide bond to the selected PTD, notably the PFVYLI peptide. Said method is further remarkable by the combination of the PTD technology with IVT-mRNA, via covalent chemical binding-conjugation - coupling, wherein said IVT-mRNA is conjugated to a said PTD. By means thereof, the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is achieved, thus providing PTD-IVT-mRNA complex which are very stable. Said IVT-mRNAs constituting therapeutic molecules, are further submitted to an intracellular delivery through said covalent chemical reaction of the IVT-mRNA with said puromycin, which is conjugated via an amide bond to the appropriate PTD -particularly the said PFVYLI peptide-, after which said IVT-mRNA’s are directed to ribosomes and translated into corresponding targeted proteins to reveal their therapeutic effect, wherein said covalent bond reduces load loss, and enhances protection in adverse conditions during in vivo delivery or in the intracellular environment, due to the existence of RNases.
According to a further embodiment of the invention, the method for the development of a delivery platform to produce deliverable PTD-IVT-mRNA therapeutics, consists of a chemical reaction of the covalent conjugation of a therapeutic IVT-mRNA, after its polyA tail, via puromycin, which is conjugated via an amide bond to the selected PTD, such as the PFVYLI peptide, said method comprising the general steps of: i. Selection of a PTD - Dissolution of PTD ii. Puromycin as the peptide and nucleic acid linker, iii. Phosphorylation, and iv. Ligation. One of the highlights of the clue of the present invention is to employ the Protein Transduction Domains (PTDs) technology as a transduction delivery platform for therapeutic IVT-mRNAs, mediated by the said puromycin molecule as a linker, which is conjugated via an amide bond to the selected PTD and which constitutes a main feature provided thanks to the present invention which is to be considered as a substantial progress over the known prior art.
Indeed, the development of this delivery platform is accomplished by a novel covalent chemical reaction of the therapeutic IVT-mRNAs with puromycin, which is conjugated via an amide bond to the selected PTD, the PFVYLI peptide, giving serious advantages over the known transfection methods.
Besides, the combination of the PTD technology with IVT-mRNA, mediated by the puromycin molecule as a linker, which is conjugated via an amide bond to the selected PTD, for the covalent chemical reaction with the IVT-mRNAs, has not been mentioned in the known literature so far.
Conjugating of IVT-mRNA to a PTD is thus achieved thanks to the present invention and has been validated by various methods as well as by the high rates of transfection in cells.
The improvement lies in the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule, since the covalent bond gives an advantage in reduced “load” loss, as well as in its protection in adverse conditions during in vivo delivery or in the intracellular environment, due to the existence of RNases.
According to the results achieved thanks to the present invention, the PTD-IVT-mRNA complex appears to be very stable even in human plasma for long periods, as it is expected to be injected intravenously during the therapeutic administration.
According to a preferred embodiment of the method according to invention, PFVYLI peptide is selected as said PTD.
PTD technology employs short peptides, able to transduce almost all biological membranes, carrying intracellularly a variety of «cargos» from micro-molecules, siRNAs to macromolecules (proteins, RNAs, plasmid DNA, nanoparticles). This invention thus relates to the covalently conjugat/on of a selected PTD -the PFVYLI peptide-, which is exploited as a neutral surface charge carrier for any therapeutic IVT- mRNA of interest, through the covalent chemical reaction of the said IVT-mRNA molecule with puromycin, which is conjugated via an amide bond to the selected PTD, leading to greater stability in the presence of serum, thereby increasing its functionality, increasing its tolerance to serum and reducing the variability of transfection between different cell types.
In fact, the PTD-IVT-mRNA disclosure demonstrates lower cytotoxicity than other delivery methods, like Lipofectamine, a commonly used cationic lipid reagent for delivering IVT- mRNAs
PTD-IVT-mRNA complexes exhibit high stability in low and high serum and plasma conditions compared to naked IVT-mRNA.
According to a further preferred embodiment of the invention, the selected Protein Transduction Domain is a 6-amino acid (6aa) peptide, the PFVYLI, which is covalently conjugated to the IVT-mRNA.
According to a more detailed development of said further embodiment of the invention, as to said first Selection step, PFVYLI is selected as a hydrophobic peptide of six (6) amino acids, of > 95% purity and acetylated at the N-terminus (Ac-Pro-Phe-Val-Tyr-Leu-lle- COOH), notably wherein the peptide is synthesized upon order, wherein this peptide has very low water solubility, wherein it is kept in powder form at -20°C and wherein Dissolution in DMF (Dimethylformamide) occurs shortly before use.
According to another more detailed development of said further embodiment of the invention, as to said second step of Puromycin as the peptide and nucleic acid linker, Puromycin (Puromycin dihydrochloride) binds to the N-terminus of PFVYLI with EDC.HCI [N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride], for the conjugation via an amide bond to the PFVYLI; and/or in said third step Phosphorylation of the puro-PFVYLI product is carried out with T4 PNK, and/or in said fourth step Ligation of the phosphorylated puro-PFVYLI product is carried out with the selected, therapeutic IVT-mRNA, with T4 RNA ligase.
Figure imgf000008_0001
steps in a specific and determinate order:
Analytical steps of covalent conjugating of IVT-mRNA to the PTD (PFVYLI) peptide: 1. EDC.HCI (0,242 mg EDC.HCI dissolved in 200 mI H20 = 6,3 mM) 4 mI are mixed with 2 mI of Puromycin [6,92 mg, dissolved in 1 ml DMF = 12,68 mM] and 1 mg PFVYLI in 2 mI DMF and left at room temperature for 2 hours, for the conjugation of puromycin to the PFVYLI, via an amide bond.
2. Phosphorylation of the puro-PFVYLI complex is then carried out using 1 mI of T4 PNK, 4 mI of T4 PNK buffer, 17 mI FLO DEPC-treated and 10 mI of DMF. The phosphorylation reaction is carried out at 37°C, for 1 hour and 40 minutes, followed by inactivation of the enzyme for 20 minutes, at 65°C.
3. Incubate the complex with 1 ,5 mI of RNase Inhibitor, for 15 minutes, at 37°C.
4. For 1 mg of PFVYLI peptide is estimated to require 18 nM of the respective therapeutic IVT-mRNA. The molecular weight of IVT-mRNA can be calculated using any bioinformatics tool. For this, one enters the sequence of the desired IVT-mRNA, and with the molecular weight being calculated, the concentration to be added to the reaction is found.
An example is set out hereafter Supposing the X sequence. According to a tool:
1 M -> 291056 g 18 nm -^5,240 pg.
Thus, for the reaction with 1mg of PFVYLI peptide, 5,24 pg of IVT-mRNA are required.
5. Finally, add 1 ,5 mI of T4 RNA ligase, 6 mI of T4 RNA ligase buffer, 18 nM IVT-mRNA and add up to 60 mI with DEPC-treated distilled H20. The ligation reaction is carried out at 16°C overnight and the enzyme is inactivated at 70°C for 10 minutes.
According to a more specific embodiment of the invention, the successful chemical covalent conjugation of PFVYLI to IVT-mRNA can be evaluated by electrophoresis on 8 M urea/6% PAGE after ethidium bromide staining (Miyamoto-Sato et al. , 2003). This method can also be termed a “retardation assay” since binding to the PFVYLI PTD peptide clearly shows the delayed transposition of IVT-mRNA into the polyacrylamide gel in comparison with naked IVT-mRNA.
According to a particular embodiment of the invention, for said selection step of a PTD, a Protein Transduction Domain (PTD), preferably hydrophobic, is selected without a free amino group; more preferably wherein the selected PTD has a purity > 95% and is acetylated at the N-terminus; even more preferably wherein the selected PTD is synthesized upon order; in particular wherein PFVYLI is selected as the PTD, of six (6) amino acids (Ac-Pro-Phe-Val-Tyr-Leu- lle-COOH). According to a more particular embodiment of the invention, for said dissolving step, said PTD is dissolved in an appropriate solvent, depending on the chemical characteristics of the PTD; in particular wherein PFVYLI is selected as the PTD, and is synthesized, wherein it has a very low water solubility, and it is thus kept in powder form at -20°C and, just prior use, is dissolved in an organic solvent, like DMA (Dimethylacetamide) or DMSO (Dimethyl sulfoxide), preferably DMF (Dimethylformamide).
According to an even more particular embodiment of the invention, for said Coupling or dissolution step of the PTD and puromycin with EDC.HCI, puromycin, particularly Puromycin dihydrochloride, is used as linker, which is dissolved preferably in DMF, whereas other nucleosides are possibly used as linkers; still more particularly wherein the EDC.FICI [N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride] is dissolved, preferably in DEPC (Diethyl pyrocarbonate)-treated distilled FI2O; wherein
1 mg of the particular PTD (dissolved with 2 pi in DMF) is incubated at room temperature, for 2 hours, with both
• 2 pi of the above solution of puromycin [12,68 mM] and
• 4 pi of the above solution of EDC.FICI [6,3 mM] thus producing the puro-PTD product; wherein
Puromycin is the linker between the PTD and the IVT-mRNA, which are added in subsequent step below.
According to a yet more particular embodiment of the invention, for said Phosphorylation step of the puro-PTD product, the puro-PTD product is phosphorylated by the enzyme T4 Polynucleotide Kinase (T4 PNK); in particular wherein the puro-PTD product is preferably phosphorylated by using 10 units of T4 PNK, 1X of T4 PNK buffer, 10 mI of the PTD’s solvent and DEPC-treated distilled Ft 0 up to 40 mI; more particularly wherein the reaction for the phosphorylation is carried out at 37°C, for 1 hour and 40 minutes, followed by inactivation of the enzyme T4 PNK, preferably for 20 minutes, at 65°C, which produces the phosphorylated puro-PTD product.
According to a still more particular embodiment of the invention, for the Inhibition step of RNases, the phosphorylated puro-PTD product is incubated with RNase Inhibitor, preferably 60 units, for 15 minutes, at 37°C, thereby removing any RNases from the mixture of the above reaction and thus protecting the IVT-mRNA, which are added at the next step. According to an even more particular embodiment of the invention, for the Ligation step of the phosphorylated puro-PTD product with the IVT-mRNA, the above phosphorylated puro- PTD product (from 1 mg of PTD) is ligated with the selected - produced therapeutic IVT- mRNA by using the enzyme T4 RNA ligase; wherein particularly for the ligation reaction, the phosphorylated puro-PTD product is incubated with 15 units of T4 RNA ligase, 1X of T4 RNA ligase buffer, 18 nM IVT-mRNA and up to 60 pi with DEPC-treated H2O; preferably wherein the ligation reaction is carried out at 16°C, overnight and then, the enzyme T4 RNA ligase is inactivated, more preferably at 70°C for 10 minutes.
According to a very particular embodiment of the invention, for the Evaluation of the outcome of this production method, the chemical covalent conjugation of the PTD to IVT- mRNA is evaluated by gel electrophoresis, notably wherein this method is qualified as a retardation assay, in that binding to the PTD peptide shows a delayed transposition of IVT- mRNA into the gel in comparison with naked IVT-mRNA reference; preferably wherein the electrophoresis is carried out on denaturing 8M urea / 6% polyacrylamide gel, after ethidium bromide staining ; or wherein alternatively, agarose gel is used for the assessment of the generation of IVT-mRNA:PTD complexes, such as simple gel electrophoresis or denaturing agarose gels, containing formaldehyde or glyoxal/DMSO.
According to a quite particular embodiment of the invention, it has been validated notably by the high rates of transfection in cells.
The invention also relates to the use of Protein Transduction Domains (PTDs) technology as a transduction delivery platform for therapeutic IVT-mRNAs, wherein said delivery platform is accomplished by a covalent chemical reaction between said IVT-mRNA molecules and a selected transduction peptide, notably the PFVYLI peptide, including the combination of the PTD technology with IVT-mRNA, via covalent chemical binding- conjugation, wherein said IVT-mRNA is conjugated to a PTD, by means whereof a IVT- mRNA: PTD complex is produced which is very stable, wherein the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is being enhanced; wherein said IVT-mRNAs, constituting a selected generation of therapeutic molecules, are submitted to an effective intracellular delivery through said covalently conjugation to the appropriate PTD, particularly the said PFVYLI peptide, after which said IVT-mRNAs are directed to the ribosomes and translated into the corresponding targeted proteins thereby revealing a therapeutic effect. Indeed, the use of IVT-mRNAs as therapeutics is beneficial because of its biological origin. In a primary variant of the use of IVT-mRNA based therapy with biological origin with the method of intracellular delivery of the therapeutic IVT-mRNA’s being translated into a targeted protein, a delivery platform produces deliverable PTD-IVT-mRNA therapeutics, wherein a neutral surface carrier is provided for delivering a therapeutic IVT-mRNA to its target site with a set of predetermined limitations being removed.
This invention achieves high rates of transfection in cellular models, even increase of seven (7) folds of the translation levels of the desired protein, increasing the Mean Fluorescent Intensity (MFI) three times (3X) as well, in comparison with the control cells.
Furthermore, subcellular targeted delivery of the produced protein, seems to be achieved even in organelles, like mitochondria of the cell-model used.
To summarize, this invention relates to a method and its use for the development of a delivery platform to produce deliverable PTD-IVT-mRNA therapeutics, wherein Protein Transduction Domains (PTDs) technology is used as a transduction delivery platform for therapeutic IVT-mRNAs and the development of said delivery platform is accomplished by a covalent chemical reaction between said IVT-mRNA molecules and a selected transduction peptide PFVYLI. It is remarkable by the combination of the PTD technology with IVT-mRNA, via covalent chemical binding - conjugation, wherein said IVT-mRNA is conjugated to a PTD, by means whereof the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is achieved, thus providing IVT- mRNA:PTD complex which are very stable; wherein said IVT-mRNAs, constituting a therapeutic molecules, are submitted to an intracellular delivery through said covalently conjugation to the appropriate PTD , after which said IVT-mRNA’s are directed to ribosomes and translated into corresponding targeted proteins to reveal their therapeutic effect.
At last, this invention also relates to a delivery platform and its use to produce deliverable PTD-IVT-mRNA therapeutics, wherein Protein Transduction Domains (PTDs) technology is used as a transduction delivery platform for therapeutic IVT-mRNAs. The development of said delivery platform is accomplished by a covalent chemical reaction between said IVT- mRNA molecules and a selected transduction peptide PFVYLI. It is remarkable by the combination of the PTD technology with IVT-mRNA, via covalent chemical binding - conjugation, wherein said IVT-mRNA is conjugated to a PTD, by means whereof the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is achieved, thus providing IVT-mRNA:PTD complex which are very stable. Said IVT-mRNAs constituting therapeutic molecules, are delivered intracellularly through said covalently conjugation to the appropriate PTD, whereas said IVT-mRNA’s are directed to ribosomes and translated into corresponding targeted proteins to reveal their therapeutic effect.
In conclusion, the proposed technology is fast and low cost, while it has the advantage of:
• the safety, since there is no integration into the host genome, as well as the transient nature of IVT-mRNA;
• enhancing the stability of the therapeutic IVT-rmRNAs;
• the efficacy of intracellular transduction; and
• the increase of the expression levels of the corresponding therapeutic protein.
The PTD-IVT-mRNA complexes proposed according to the invention have the potential to:
(a) replace (i) corresponding absent intracellular proteins, even ones localised in organelles, in monogenic - metabolic diseases, as protein replacement therapy, as well as (ii) systemically secreted therapeutic molecules;
(b) be transferred to the membranes, in the context of cellular therapies, such as the CAR (chimeric antigen receptor) cancer immunotherapy. Another approach to targeted cancer treatment is also the systemic administration of IVT-mRNA, encoding a suicide gene [such as herpes simplex virus 1 -thymidine kinase (HSV1-tk) or other cancers-related viruses];
(c) be secreted in the context of the development of personalized vaccines against specific types of cancer, against the identified neo-antigens, in the context of personalized medicine;
(d) encode antibodies secreted to neutralize cytokines that play crucial role in the body's inflammatory - allergic reactions in the context of prophylactic vaccines;
(e) encode cytokines and growth factors in a number of somatic and stem cells;
(f) contribute to intracellular delivery of non-protein-coded RNA molecules, such as shRNAs - miRNAs for gene silencing in case of therapeutic or diagnostic purposes;
(g) cell reprogramming for the immediate induction of cell differentiation, the reprogramming of somatic cells into induced pluripotent stem cells (e.g. using IVT-mRNA of various transcription factors such as Oct4, Sox2, Klf4 and cMyc), the directed (trans-) differentiation of cells into desired cell types (e.g. from fibroblasts to myocytes or hepatocytes) or and for overexpression of receptors or paracrine factors (e.g. VEGFa) in mesenchymal stem cells to improve homing behaviour;
(h) gene editing therapies for the intracellular delivery of programmable nucleases (ZFN, TALEN, and Cas9) to the target tissue or cell. Intracellular delivery of these nucleases, as PTD-IVT-mRNA complexes, will have several advantages, such as transient expression with efficient in vivo and in vitro translocation, non-genomic integration, potentially low off- target phenomena and high efficiency of gene editing. Further details and particulars will appear from the following description of a way of carrying out the invention, and of some exemplary embodiments of the method according to the invention and the uses thereof, with the use of examples, explaining the application of the invention more in detail based on the appended drawings.
Brief description of the drawings
Fig. 1 represents the main embodiment of the invention, which is the chemical reaction of the covalent conjugation of the PTD (such as the PFVYLI peptide) to a therapeutic IVT- mRNA, through the covalent chemical reaction of the said IVT-mRNA molecule with puromycin, which is conjugated via an amide bond to the selected PTD.
Fig. 2, resp. 3 is a simplified, resp. schematized representation of a previous variant thereof, i.e. of a chemical reaction according to the of the invention.
Description
In general, this invention relates to a method the general steps whereof comprise according to a preferred embodiment as notably represented in Fig. 1 :
1 . Selection of a hydrophobic PTD - Dissolution of PTD:
PFVYLI is a hydrophobic peptide of six (6) amino acids, of > 95% purity and acetylated at the N-terminus (Ac-Pro-Phe-Val-Tyr-Leu-lle-COOFI). The peptide is synthesized upon order. This peptide has very low water solubility, therefore it is kept in powder form at -20°C and solubilized in DMF (Dimethylformamide) shortly before use.
2. Puromycin as the peptide and nucleic acid linker:
Puromycin (Puromycin dihydrochloride) will bind to the N-terminus of PFVYLI with EDC.HCI [N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride] for the conjugation via an amide bond to the PFVYLI;
3. Phosphorylation of the puro-PFVYLI product, with T4 PNK;
4. Ligation of the phosphorylated puro-PFVYLI product with the selected, therapeutic IVT-mRNA, with T4 RNA ligase.
A substantial highlight of the development of the present invention is to apply the Protein Transduction Domains (PTDs) technology as a transduction delivery platform for therapeutic IVT-mRNAs, which is mediated by the puromycin molecule as a linker. The latter is conjugated via an amide bond to the selected PTD and it constitutes an essential feature. The development of this delivery platform is accomplished by a novel covalent chemical reaction of the therapeutic IVT-mRNAs with puromycin, which is conjugated via an amide bond to the selected PTD, the PFVYLI peptide, giving striking advantages compared to the known transfection methods.
Besides, the combination of the PTD technology with IVT-mRNA, mediated by the puromycin molecule as a linker, which is conjugated via an amide bond to the selected PTD, for the covalent chemical reaction with the IVT-mRNAs, constituting a significant progress.
Conjugating of IVT-mRNA to a PTD has thus presently been achieved and it was validated by various methods as well as by the high rates of transfection in cells.
The stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is a decisive achievement, since the covalent bond gives an advantage in reduced “load” loss, and in its protection in adverse conditions during in vivo delivery or in the intracellular environment, due to the existence of RNases.
According to the results achieved herewith, the PTD-IVT-mRNA complex appears to be very stable even in human plasma for long periods, as it is expected to be injected intravenously during the therapeutic administration.
A description of an exemplary embodiment is set out hereafter in more details based upon said primary method to produce deliverable PTD-IVT-mRNA therapeutics shown in Fig. 2 with its various successive steps as follows:
1. As to the Selection step of a PTD,
Any Protein Transduction Domain (PTD), preferably hydrophobic, without a free amino group, can be selected.
The selected PTD is preferably of > 95% purity and is acetylated at the N-terminus.
The selected PTD is preferably synthesized upon order.
In particular, PFVYLI is selected as the PTD, of six (6) amino acids (Ac-Pro-Phe-Val-Tyr- Leu-lle-COOH).
2. As to step Dissolution of PTD,
The PTD is dissolved in the appropriate solvent, depending on the chemical characteristics of the PTD.
The selected PTD. the PFVYLI, is synthesized. It has very low water solubility, therefore it is kept in powder form at -20°C and, just prior use, is dissolved in DMF (Dimethylformamide). Any organic solvent, like DMA (Dimethylacetamide) or DMSO (Dimethyl sulfoxide), can be used instead of DMF.
3. As to Coupling step of the PTD and purormycin with EDC.HCI, via an amide bond,
As linker can be used puromycin (in particular, Puromycin dihydrochloride) which is dissolved (preferably in DMF) Instead of puromycin, other nucleosides could also be used as linkers.
The EDC.HCI [N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride] is dissolved, preferable in DEPC (Diethyl pyrocarbonate)-treated distilled H2O.
1 mg of the particular PTD (dissolved with 2 pi in DMF) is incubated at room temperature, for 2 hours, with both
• 2 pi of the above solution of puromycin [12.68 mM] and
• 4 mI of the above solution of EDC.HCI [6.3 mM] in order to produce the puro-PTD product;
Puromycin is the linker between the PTD and the IVT-mRNA, which will be added in step 6
4. As to the Phosphorylation step of the puro-PTD product,
The puro-PTD product is phosphorylated by the enzyme T4 Polynucleotide Kinase (T4 PNK).
In particular, the puro-PTD product is preferably phosphorylated by using 10 units of T4 PNK, 1X of T4 PNK buffer, 10 mI of the PTD’s solvent and DEPC-treated distilled H20 up to 40 mI.
The reaction for the phosphorylation is carried out at 37°C, for 1 hour and 40 minutes, followed by inactivation of the enzyme T4 PNK, preferably for 20 minutes, at 65°C, in order to produce the phosphorylated puro-PTD product
5. As to the Inhibition step of RNases,
The phosphorylated puro-PTD product is incubated with RNase Inhibitor, preferably 60 units, for 15 minutes, at 37°C, in order to remove any RNases from the mixture of the above reaction and to protect the IVT-mRNA, which will be added at the next step.
6. As to the Ligation step of the phosphorylated puro-PTD product with the IVT-mRNA,
The above phosphorylated puro-PTD product (from 1 mg of PTD) is ligated with the selected - produced therapeutic IVT-mRNA by using the enzyme T4 RNA ligase.
In particular, for the ligation reaction, the phosphorylated puro-PTD product is incubated with 15 units of T4 RNA ligase, 1X of T4 RNA ligase buffer, 18 nM IVT-mRNA and up to 60 mI with DEPC-treated H20.
Figure imgf000016_0001
RNA ligase is inactivated, preferably at 70°C for 10 minutes.
7. As to the Evaluation step of the successful outcome of the proposed innovative method, The successful chemical covalent conjugation of the PTD to IVT-mRNA can be evaluated by gel electrophoresis. This method can also be termed a “band-shift assay”, since binding to the PTD peptide clearly shows the delayed transposition of IVT-mRNA into the gel in comparison with to naked IVT-mRNA.
The electrophoresis is carried out preferably on denaturing 8 M urea/6% polyacrylamide gel, after ethidium bromide staining (Miyamoto-Sato et al., 2003).
Alternatively, agarose gel can be used for the assessment of the successful generation of PTD-IVT-mRNA complexes (simple gel electrophoresis or denaturing agarose gels, containing formaldehyde or glyoxal/DMSO).
High rates of transfection in cellular models are achieved, even doubling of the translation levels of the desired protein, increasing the Mean Fluorescent Intensity (MFI) three times (3X) in comparison with the control cells.
Furthermore, subcellular targeted delivery of the produced protein, seems to be achieved even in organelles, like mitochondria of the cell-model used.
In conclusion, the proposed technology is fast and low cost, while it has the advantage of the safety, since there is no integration into the host genome, as well as the transient nature of IVT-mRNA; further enhancing the stability of the therapeutic IVT-mRNAs; also the efficacy of intracellular transduction; as well as the increase of the expression levels of the corresponding therapeutic protein.
The obtained IVT-mRNA:PTD complexes have the potential to: (a) replace (i) corresponding absent intracellular proteins, even ones localised in organelles, in monogenic-metabolic diseases, as protein replacement therapy, as well as (ii) systemically secreted therapeutic molecules; (b) to be transferred to the membranes, in the context of cellular therapies, such as the CAR (chimeric antigen receptor) cancer immunotherapy. Another approach to targeted cancer treatment is also the systemic administration of IVT-mRNA, encoding a suicide gene [such as herpes simplex virus 1 -thymidine kinase (HSV1-tk) or other cancers- related viruses]; (c) to be secreted in the context of the development of personalized vaccines against specific types of cancer, against the identified neo-antigens, in the context
Figure imgf000017_0001
crucial role in the body's inflammatory-allergic reactions in the context of prophylactic vaccines; (e) to encode cytokines and growth factors in a number of somatic and stem cells; (f) to contribute to intracellular delivery of non-protein-coded RNA molecules, such as shRNAs - miRNAs for gene silencing in case of therapeutic or diagnostic purposes;
(g) to cell reprogramming for the immediate induction of cell differentiation, the reprogramming of somatic cells into induced pluripotent stem cells (e.g. using IVT-mRNA of various transcription factors such as Oct4, Sox2, Klf4 and cMyc), the directed (trans-) differentiation of cells into desired cell types (e.g. from fibroblasts to myocytes or hepatocytes) or and for overexpression of receptors or paracrine factors (e.g. VEGFa) in mesenchymal stem cells to improve homing behaviour; and further (h) to gene editing therapies for the intracellular delivery of programmable nucleases (ZFN, TALEN, and Cas9) to the target tissue or cell. Intracellular delivery of these nucleases, as IVT-mRNA:PTD complexes, will have several advantages, such as transient expression with efficient in vivo and in vitro translocation, non-genomic integration, potentially low off-target phenomena and high efficiency of gene editing.

Claims

1. Method for the development of a delivery platform to produce deliverable PTD-IVT- mRNA therapeutics, wherein Protein Transduction Domains (PTDs) technology is used as a transduction delivery platform for therapeutic IVT-mRNAs, wherein for said delivery platform there is accomplished a covalent chemical reaction between said IVT-mRNA molecules and a selected transduction peptide, notably PFVYLI peptide, wherein said method is characterised by the combination of the PTD technology with IVT-mRNA, via covalent chemical binding - conjugation, wherein said IVT-mRNA is conjugated to a said PTD, by means whereof the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is stabilized, thereby also stabilizing IVT-mRNA:PTD complex; wherein said IVT-mRNAs constituting a therapeutic molecule, are submitted to an intracellular delivery through said covalently conjugation to the appropriate PTD -particularly the said PFVYLI peptide-, after which said IVT-mRNA’s are directed to ribosomes and translated into corresponding targeted proteins to reveal their therapeutic effect.
2, Method for the development of a delivery platform to produce deliverable PTD-IVT- mRNA therapeutics, particularly according to claim 1 , wherein Protein Transduction Domains (PTDs) technology is used as a transduction delivery platform for therapeutic IVT- mRNAs, characterized in that a PTD is selected and in that said delivery platform is produced by a covalent chemical reaction between said IVT-mRNA molecules with puromycin, which is conjugated via an amide bond to the selected PTD, wherein said PTD technology is combined with IVT-mRNA, via covalent chemical binding - conjugation - coupling, wherein said IVT-mRNA is conjugated to a said PTD, by means whereof the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is achieved, thus providing PTD-IVT-mRNA complex which are very stable; wherein said IVT-mRNAs, constituting therapeutic molecules, are submitted to an intracellular delivery through said covalent chemical reaction of the IVT-mRNA with puromycin, which is conjugated via an amide bond to the appropriate PTD, after which said IVT-mRNA’s are directed to ribosomes and translated into corresponding targeted proteins to reveal their therapeutic effect, wherein said covalent bond reduces load loss, and enhances protection in adverse conditions during in vivo delivery or in the intracellular environment, due to the existence of RNases.
3. Method for developing a delivery platform to produce deliverable PTD-IVT-mRNA therapeutics, particularly according to claim 1 or 2, characterized in that said method consists of a chemical reaction of the covalent conjugation of a therapeutic IVT-mRNA, after its polyA tail, via puromycin, which is conjugated via an amide bond to the selected PTD, which comprises the general steps of: i. Selection of a PTD - Dissolution of PTD ii. Puromycin as the peptide and nucleic acid linker, iii. Phosphorylation, and iv. Ligation.
4. Method according to one of the claims 1 to 3, characterised in that PFVYLI peptide is selected as said PTD.
5. Method according to one of the claims 3 or 4, characterized in that in said first Selection step, PFVYLI is selected as a hydrophobic peptide of six amino acids, having > 95% purity and being acetylated at the N-terminus (Ac-Pro-Phe-Val-Tyr-Leu-lle-COOH).
6. Method according to claim 4 or 5, characterized in that the peptide is synthesized upon order, wherein this peptide has very low water solubility, wherein it is kept in powder form at about -20°C and wherein Dissolution in DMF (Dimethylformamide) occurs shortly before use.
7. Method according to one of the claims 3 to 6, characterised in that in said second step (ii) with Puromycin as the peptide and nucleic acid linker, Puromycin (Puromycin dihydrochloride) binds to the N-terminus of PFVYLI with EDC.HCI [N-(3- Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride], after which conjugation is carried out via an amide bond to the PFVYLI.
8. Method according to one of the claims 3 to 7, characterised in that in said third step (iii) Phosphorylation of the puro-PFVYLI product is carried out with T4 PNK.
9. Method according to one of the claims 3 to 8, characterised in that in said fourth step Ligation of the phosphorylated puro-PFVYLI product is carried out with the selected, therapeutic IVT-mRNA, with T4 RNA ligase.
10. Method according to one of the preceding claims, particularly any of claims 4 to 9, characterized in that it comprises the following steps in a specific and determinate order as specified for the following analytical steps of covalent conjugating of IVT-mRNA to the PTD (PFVYLI) peptide consisting of
- EDC.HCI (0,242 mg EDC.HCI dissolved in 200 pi H20 = 6,3 mM) 4 pi are mixed with 2 pi of Puromycin [6.92 mg, dissolved in 1 ml DMF = 12,68 mM] and 1 mg PFVYLI in 2 pi DMF and left at room temperature for 2 hours;
- Phosphorylation of the puro-PFVYLI complex is then carried out using 1 pi of T4 PNK, 4 pi of T4 PNK buffer, 17 pi H20 DEPC-treated and 10 pi of DMF, wherein the phosphorylation reaction is carried out at 37°C, for 1 hour and 40 minutes, followed by inactivation of the enzyme for 20 minutes, at 65°C;
- the complex is incubated with 1 ,5pl of RNase Inhibitor, for 15 minutes, at 37°C;
- for 1 mg of PFVYLI peptide is estimated to require 18 nM of the respective therapeutic IVT-mRNA, wherein the molecular weight of IVT-mRNA is calculated using a bioinformatics tool as identified, for which the sequence of the desired IVT-mRNA is entered, and wherein the molecular weight is calculated thereby yielding the concentration to be added to the reaction; and
- finally, 1 ,5 pi of T4 RNA ligase, 6 pi of T4 RNA ligase buffer, 18 nM IVT-mRNA are added and up to 60 pi with DEPC-treated distilled H20 is added, wherein the ligation reaction is carried out at 16°C overnight and the enzyme is inactivated at 70°C for 10 minutes.
11. Method according to the preceding claim 10, characterized in that said chemical covalent conjugation of PFVYLI to IVT-mRNA, wherein covalent chemical reaction between said IVT-mRNA molecules and puromycin, which is conjugated via an amide bond to the selected PTD, is evaluated by electrophoresis on 8 M urea/6% PAGE after ethidium bromide staining, wherein this method acts as a so-called “band-shift assay”, whereas binding to the PFVYLI PTD peptide causes a delayed transposition of IVT-mRNA into the polyacrylamide gel in comparison with a preset reference.
12. Method according to one of the claims 3 to 11 , characterized in that for said Selection step (i), a Protein Transduction Domain (PTD), preferably hydrophobic, is selected without any free amino group.
13. Method according to the preceding claim 12, characterised in that said PTD is hydrophobic.
14. Method according to one of both preceding claims 12 or 13, characterized in that the selected PTD has a purity > 95% and is acetylated at the N-terminus.
15. Method according to one of the claims 12 to 14, characterized in that the selected PTD is synthesized upon order; in particular wherein PFVYLI is selected as the PTD, of six amino acids (Ac-Pro-Phe-Val-Tyr-Leu-lle-COOH).
16. Method according to one of the claims 3 to 15, characterized in that for said Dissolution step (i), said PTD is dissolved in an appropriate solvent, depending on the chemical characteristics of the PTD.
17. Method according to one of the preceding claims, particularly claim 16, characterised in that said selected PFVYLI is synthesized, wherein it has a very low water solubility, and it is kept in powder form at about -20°C and, just prior use, is dissolved in an organic solvent, like DMA (Dimethylacetamide) or DMSO (Dimethyl sulfoxide), preferably DMF (Dimethylformamide).
18. Method according to one of the preceding claims, particularly any of the claims 10 to 17, characterized in that for said Coupling step of the PTD and puromycin with EDC.HCI, puromycin, particularly Puromycin dihydrochloride, is used as linker, wherein puromycin is conjugated via an amide bond to the selected PTD, which is dissolved preferably in DMF, wherein the EDC.HCI [N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride] is dissolved, preferably in DEPC (Diethyl pyrocarbonate)-treated distilled H20; in particular wherein 1 mg of the particular PTD -dissolved with 2 pi in DMF- is incubated at room temperature, for 2 hours, with both
« 2 pi of the above solution of puromycin [1 ,.68 mM] and • 4 mI of the above solution of EDC.HCI [6,3 mM] by means whereof the puro-PTD product is produced; wherein
Puromycin is the linker between the PTD and the IVT-mRNA, which are added in a subsequent step.
19. Method according to one of the preceding claims, particularly any of the claims 10 to 17 or 18, characterized in that other nucleosides are used as linkers.
20. Method according to one of the claims 3 to 19, characterized in that for said Phosphorylation step (iii) of the puro-PTD product, said puro-PTD product is phosphorylated by the enzyme T4 Polynucleotide Kinase.
21. Method according to the preceding claim 20, characterized in that said puro-PTD product is phosphorylated preferably by using 10 units of T4 PNK, 1X of T4 PNK buffer, 10 pi of the PTD’s solvent and DEPC-treated distilled H2O up to 40 mI.
22. Method according to one of both preceding claims 20 or 21 , characterized in that the reaction for the phosphorylation is carried out at 37°C, for 1 hour and 40 minutes, followed by inactivation of the enzyme T4 PNK, preferably for 20 minutes, at 65°C, by which the phosphorylated puro-PTD product is produced.
23. Method according to one of the claims 20 to 22, particularly claim 22, characterized in that for the Inhibition step of RNases, the phosphorylated puro-PTD product is incubated with RNase Inhibitor, preferably 60 units, for 15 minutes, at 37°C, thereby removing any RNases from the mixture of the above reaction and thus protecting the IVT-mRNA, which are added at a next step.
24. Method according to one of the claims 3 to 23, particularly any of the claims 20 to 23, characterized in that for said Ligation step (iv) of the phosphorylated puro-PTD product with the IVT-mRNA, the above phosphorylated puro-PTD product (from 1 mg of PTD) is ligated with the selected - produced therapeutic IVT-mRNA by using the enzyme T4 RNA ligase; wherein particularly for the ligation reaction, the phosphorylated puro-PTD product is incubated with 15 units of T4 RNA ligase, 1X of T4 RNA ligase buffer, 18 nM IVT-mRNA and up to 60 mI with DEPC-treated H2O; preferably wherein the ligation reaction is carried out at 16°C, overnight and then, the enzyme T4 RNA ligase is inactivated, more preferably at 70°C for 10 minutes.
25. Method according to one of the preceding claims, characterized in that the outcome of this production method is evaluated, wherein the chemical covalent conjugation of the PTD to IVT-mRNA is evaluated by gel electrophoresis, notably wherein this method is qualified as a so-called “band-shift assay”, in that binding to the PTD peptide shows a delayed transposition of IVT-mRNA into the gel in comparison with naked IVT-mRNA reference.
26. Method according to the preceding claim 25, characterized in that the electrophoresis is carried out on denaturing 8 M urea/6% polyacrylamide gel, after ethidium bromide staining; or wherein alternatively, agarose gel is used for the assessment of the generation of PTD-IVT-mRNA complexes, such as simple gel electrophoresis or denaturing agarose gels, containing formaldehyde or glyoxal/DMSO.
27. Method according to one of the preceding claims, characterized in that it is validated by the high rates of transfection in cells.
28. Delivery platform produced according to a method as defined in any of the preceding claims for deliverable PTD-IVT-mRNA therapeutics, wherein Protein Transduction Domains (PTDs) technology is used as a transduction delivery platform for therapeutic IVT-mRNAs, wherein said delivery platform is characterised by a covalent chemical reaction between said IVT-mRNA molecules and a selected transduction peptide, wherein it includes the combination of the PTD technology with IVT-mRNA, via covalent chemical binding- conjugation, wherein said IVT-mRNA is conjugated to a said PTD, having the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule, thus providing very stable IVT-mRNA:PTD complex; wherein said IVT-mRNAs are delivered intracellularly through said covalent conjugation to the appropriate PTD, wherein said IVT- mRNA’s are directed to ribosomes and translated into corresponding targeted proteins, revealing their therapeutic effect.
29. Delivery platform producing deliverable PTD-IVT-mRNA therapeutics, particularly according to the preceding claim 28, including Protein Transduction Domains (PTDs) technology as a transduction delivery platform for therapeutic IVT-mRNAs, characterized in that it includes a selected PTD and a covalent chemical reaction between said IVT-mRNA molecules with puromycin, which is conjugated via an amide bond to the selected PTD, PFVYLI peptide, wherein it further includes a combination of the PTD technology with IVT- mRNA, via covalent chemical binding - conjugation - coupling, wherein said IVT-mRNA is conjugated to a said PTD, thus including the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule, thereby providing a very stable PTD- IVT-mRNA complex; wherein said IVT-mRNAs therapeutic molecules include an intracellular delivery through said covalent chemical reaction of the IVT-mRNA with puromycin, which is conjugated via an amide bond to the appropriate PTD, wherein said IVT-mRNA’s are directed to ribosomes and translated into corresponding targeted proteins thereby revealing their therapeutic effect.
30. Use of Protein Transduction Domains (PTDs) technology as a transduction delivery platform for therapeutic IVT-mRNAs, wherein said delivery platform is accomplished by a covalent chemical reaction between said IVT-mRNA molecules with puromycin, which is conjugated via an amide bond to the selected PTD, especially the PFVYLI peptide accomplished by a covalent chemical reaction between said IVT-mRNA molecules and a selected transduction peptide, notably said PFVYLI peptide, including the combination of the PTD technology with IVT-mRNA, via covalent chemical reaction of the said IVT-mRNAs with puromycin, which is conjugated via an amide bond to the PTD, by means whereof the stability of the connection between the PTD transporter and the therapeutic IVT-mRNA molecule is increased, thereby generating a PTD-IVT-mRNA complex which is very stable; wherein said IVT-mRNAs, constituting a selected generation of therapeutic molecules, are submitted to an effective intracellular delivery through said covalently conjugation to said selected PTD, after which said IVT-mRNAs are directed to the ribosomes and translated into the corresponding targeted proteins, thereby generating a therapeutic effect.
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