[go: up one dir, main page]

WO2025036956A1 - Non-systemic mrna administration - Google Patents

Non-systemic mrna administration Download PDF

Info

Publication number
WO2025036956A1
WO2025036956A1 PCT/EP2024/072949 EP2024072949W WO2025036956A1 WO 2025036956 A1 WO2025036956 A1 WO 2025036956A1 EP 2024072949 W EP2024072949 W EP 2024072949W WO 2025036956 A1 WO2025036956 A1 WO 2025036956A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
mrna
administration
ionizable
mol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/072949
Other languages
French (fr)
Inventor
Carsten Rudolph
Estelle BEGUIN
Christian Dohmen
Thomas LANGENICKEL
Christian Plank
Günther HASENPUSCH
Steffen Meyer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ethris GmbH
Original Assignee
Ethris GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ethris GmbH filed Critical Ethris GmbH
Publication of WO2025036956A1 publication Critical patent/WO2025036956A1/en
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—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 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • A61K48/0075—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 delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • A61K48/0041—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 the non-active part being polymeric
    • 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
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the present invention relates to compositions designed for localized delivery within a subject's body. More particularly, the invention pertains to therapeutic compositions that remain localized to specific organs or tissues and do not exhibit systemic distribution. These compositions include specific carriers and therapeutic agents suitable for various medical applications and may be used in the prevention and/or treatment of medical conditions.
  • compositions that can deliver therapeutic agents specifically to a localized site without systemic distribution.
  • the invention provides compositions for the delivery of a therapeutic agent that does not spread into the systemic circulation. This is of the highest interest for the treatment of, inter alia, tumors and autoimmune diseases, where the drug substance may cause significant harm (“off-target”) when it is spread in the circulation.
  • compositions for localized delivery to an organ or tissue or a localized site of interest comprising: a) a therapeutic agent; b) a carrier comprising: i. an ionizable cationic lipid or lipidoid, preferably a cationic lipidoid of formula (b-l) ii. a helper lipid, and iii. optionally, a pharmaceutically acceptable excipient or diluent.
  • the present invention provides for (pharmaceutical) compositions that are particularly useful in the local administration of one or more therapeutic agent(s), as the composition (or preferably at least the therapeutic agent) remains localized at the site of administration.
  • a (pharmaceutical) composition for use in the treatment and/or prevention of a disease or disorder, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. a ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein said composition, remains localized at the site of administration and/or essentially does not exhibit systemic distribution throughout the patient's body.
  • compositions are particularly useful in the local administration of one or more therapeutic agent(s), as the composition (or preferably at least the therapeutic agent) has/shows a prolonged retention at the site of administration (as compared to a composition of the state of the art, such as a composition comprising DLin- MC3-DMA, as disclosed in US 8,158,601).
  • prolonged retention at the site of administration may cause the therapeutic agent to (largely, mainly, or even exclusively) exert its (therapeutic) effect at the site of administration, which may be desirable and can reduce off-target effect (i.e., side effect) or the required amount of therapeutic agent and/or (pharmaceutical) composition.
  • the present invention provides for a (pharmaceutical) composition for use in the treatment and/or prevention of a disease, the treatment comprising local administration of the composition, the composition comprising:
  • a carrier comprising: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) and/or diluent(s); wherein said composition has a prolonged retention at the site of administration; and/or wherein said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration.
  • the herein provided (pharmaceutical) compositions are particularly useful in the local administration I localized administration / local delivery / localized delivery or the like of one or more therapeutic agents as they may allow the skilled artisan to reduce the amount of employed/administered therapeutic agent and/or (pharmaceutical) composition, while still achieving a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
  • the present invention provides for a (pharmaceutical) composition for use in the treatment and/or prevention of a disease, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii.
  • compositions or of the therapeutic agent optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration; and/or wherein the patient has less (or fewer) side effects compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
  • the present invention further relates to a cosmetic composition having/showing local retention, prolonged retention, or the like at the site of administration, site of application, or the like.
  • the entire cosmetic composition or only parts thereof may show local retention or the like. It is herein preferred that at least the one or more active agent(s) are retained locally.
  • the present invention further provides for a cosmetic composition comprising: a) one or more active agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii.
  • said cosmetic composition is an ointment, a creme, a foam, a gel, a lotion, an aqueous liquid, or a powder, or wherein said cosmetic composition is formulated as an ointment, a creme, a foam, a gel, a lotion, an aqueous liquid, or a powder.
  • the present inventors have surprisingly found that the ionizable lipid and/or the ionizable lipidoid as comprised in the herein provided (pharmaceutical or cosmetic) compositions causes such a composition (or at least the therapeutic agents or the active agent; also collectively referred to as “agents” herein) to essentially remain localized after being administered/applied to a local site.
  • a composition or at least the therapeutic agents or the active agent; also collectively referred to as “agents” herein
  • agents also collectively referred to as “agents” herein
  • the herein provided compositions are particularly advantageous and give rise to new clinical situations, by e.g., reducing the amount of agents to be employed.
  • the present invention is thus based on the finding that various ionizable lipids/ ionizable lipidoids employed herein remain localized at the site of administration, providing a significant advancement in the field of lipid nanoparticle (LNP) technology.
  • LNP lipid nanoparticle
  • This localized or targeted retention of the ionizable lipid at the administration /injection site addresses one of the critical challenges associated with LNP-based drug delivery systems: the risk of Complement Activation-Related Pseudoallergy (CARPA).
  • CARPA Complement Activation-Related Pseudoallergy
  • CARPA is a non-lgE-mediated hypersensitivity reaction (i.e., an immune reaction that is independent of immunoglobulin E; IgE) triggered by the unintended activation of the complement system, which can occur during the systemic distribution of LNPs (see, e.g., Ferraresso et al., 2014, Molecular Therapy: Methods & Clinical Development (2024), doi: https://doi.Org/10.1016/j.omtm.2024.101314).
  • This reaction can lead to a range of adverse effects, from mild symptoms to severe anaphylaxis-like responses, posing a risk to patient safety.
  • compositions for use in the treatment and/or prevention of a disease or disorder are particularly advantageous as they reduce side effects (such as for example CARPA and/or symptoms associated with or caused by CARPA) that may occur when administering a composition not comprising the herein employed (advantageous) ionizable lipids/lipidoid (i.e., when administering a composition that does not show/have localized retention at the side of administration).
  • ionizable lipids/ionizable lipidoids in the herein employed formulations or compositions (such as pharmaceutical or cosmetic compositions) provides a targeted and localized delivery of agents (such as therapeutic agents or active agents). This not only enhances the efficacy of these agents (or drugs) by concentrating them at the desired site of administration/site of action but also reduces the likelihood of adverse immune responses (such as CARPA) that can arise from the widespread distribution of LNPs throughout the body.
  • agents such as therapeutic agents or active agents
  • the herein employed ionizable lipids/lipidoids when used in compositions (such as LNPs or pharmaceutical formulations), offer a reliable approach to mitigate the risk of undesired side effects (such as CARPA) by limiting the systemic exposure of the complement system to, e.g., LNPs.
  • This innovation enhances the safety profile of LNP-based therapies, making them more suitable for a broader range of clinical applications, particularly in patients with a (high) susceptibility to hypersensitivity reactions.
  • the present invention fulfills a long-felt and unmet need at least in the field of lipid nanoparticle (LNP) technology by providing an inventive approach for the administration of pharmaceutical agents through the localized retention of the ionizable lipid at the injection site.
  • LNP lipid nanoparticle
  • the unique capability of this invention to confine the lipid's presence to the injection site represents a significant advancement that was not previously recognized or addressed.
  • This localized retention effect is crucial for, inter alia, enhancing the safety and efficacy of LNP-based therapies, ensuring that the therapeutic agents remain concentrated at the desired site of action, thereby minimizing systemic exposure and potential side effects.
  • This innovation addresses a critical gap in the development of targeted and safe drug delivery systems.
  • Example 1 illustratively shows that intramuscular injection of a composition comprising an ionizable lipidoid in accordance with the present invention and further comprising a chemically modified RNA encoding for a luciferase gene results in the expression of said luciferase only at the local site of administration (see, for example, Figs 2 to 4).
  • hyaluronidase also referred to as Hylase herein; an enzyme that degrades hyaluronic acid in the extracellular matrix.
  • Fig 6 clearly shows that the co- administration of hyaluronidase results in increased distribution of luciferase expression which is indicative for a loss of/reduced local retention as compared to Fig 5, in which no hyaluronidase was co-administered.
  • Fig 7 and 8 i.e., the assessment of luciferase activity in excised organs from the mice shown in Figs 5 and 6, respectively
  • the herein provided compositions may interact with (bind to) / be capable of interacting with (binding to) the extracellular matrix after local administration and thereby exert the herein described effects, e.g. remain localized, essentially not exhibit systemic distribution, prolonged retention, reduced amount of composition or therapeutic agent and/or less side effects.
  • Interferons are cytokines with relevance for example in controlling inflammation, in particular in the context of viral infections.
  • the ectopic expression interferons such as human interferon lambda 1 (hlFN ⁇ 1) is of high relevance in the treatment or prevention of e.g., viral diseases.
  • the activity of hlFN ⁇ 1 (as well as many other therapeutic agents) and as such also its therapeutic effect may also depend on the tissue or organ of expression.
  • the present inventors have surprisingly found that the herein provided composition, when comprising/encapsulating an mRNA encoding for hlFN ⁇ 1 as a therapeutic agent results in the local retention of said mRNA at the site of administration (i.e., the lung in this example; see for example Fig 18). Accordingly, the present inventors have surprisingly shown the localized retention of a therapeutically relevant active agent (here, mRNA encoding hlFN ⁇ 1).
  • a therapeutically relevant active agent here, mRNA encoding hlFN ⁇ 1
  • the herein disclosed local retention effect i.e., the local retention of a composition to be administered in accordance with the present invention
  • the lipidoids of Formula (b-l) as discussed above, likely causing this local retention
  • various different helper lipids comprised in the to be administered composition and/or with compositions comprising varying concentrations of such cationic lipidoids and/or helper lipids (see, e.g., Examples 8 to 10).
  • the composition to be employed in the context of the present invention is not particularly limited, as long as it comprises a suitable therapeutic or cosmetic agent and an ionizable lipid and/or an ionizable lipidoid capable of causing local retention of said compositions.
  • Such suitable ionizable lipids and agents are further detailed herein below and the general effect of local retention as caused by such ionizable lipids and lipidoids is illustrated by exemplary and non-limiting ionizable lipids and lipidoids in the enclosed examples.
  • the local retention of a composition can be achieved across a variety of different local administration routes including, for example, intratracheal delivery such as intratracheal instillation or intratracheal microspray, subcutaneous delivery, aerosol delivery to the airways, such as upper airways or the lungs, intramuscular delivery, and nasal aerosol or nasal spray delivery (see, e.g., Examples 1 , 3. 4, and 11).
  • intratracheal delivery such as intratracheal instillation or intratracheal microspray
  • aerosol delivery to the airways such as upper airways or the lungs
  • intramuscular delivery intramuscular delivery
  • nasal aerosol or nasal spray delivery see, e.g., Examples 1 , 3. 4, and 11.
  • compositions of a state in the art such as lipid nanoparticle-based mRNA vaccines, in particular Comimaty® (comprising ALC-0315) or Spikevax® (comprising SM-102) or Onpattro® (patisiran) (comprising DLin-MC3-DMA, also referred to as “MC3” in the context of the present invention), used for the treatment of hereditary transthyretin-mediated (hATTR) amyloidosis.
  • Comimaty® comprising ALC-0315
  • Spikevax® comprising SM-102
  • Onpattro® patisiran
  • mRNA expression can be found in the plasma, liver, adrenal glands, spleen, and ovaries (see Table 1).
  • systemic movement of the intramuscular injected LNP/mRNA was observed for Spikevax®, as summarized in Table 2 below (Source 1 EMA Assessment report Comirnaty, 21 December 2020 and forSpikevax: 1 EMA Assessment report Spikevax, 11 March 2021, EMA/15689/2021 Corr.1*1 , Committee for Medicinal Products for Human Use (CHMP).
  • the intramuscular injection of state-of-the-art compositions results in the (systemic or partly systemic) distribution of the active ingredient to various organs.
  • state-of-the-art compositions in particular lipid nanoparticle compositions
  • such wide-spread distribution might cause various side effects and is thus generally undesired.
  • Table 1 Distribution of Comirnaty® after intramuscular injection
  • Table 2 Distribution of Spikevax® after intramuscular injection
  • the present invention solves the above problems with the embodiments of the invention. As mentioned above and while not wanting to be bound by any theory, it is believed that the present invention achieved advantageous results by providing formulations that effectively interact with the extracellular matrix. Destruction of the extracellular matrix with hyaluronidase (Hylase) re-establishes a systemic delivery of the delivery therapeutic agent, particularly a chemically modified mRNA. In particular, the invention relates to the embodiments as recited in following items: Item 1 .
  • a composition for local delivery to an organ or tissue or a localized site of interest comprising: a) A therapeutic agent; and a carrier comprising: b) an ionizable cationic lipid or lipidoid, and c) a helper lipid, and d) optionally a pharmaceutically acceptable excipient or diluent wherein said composition, when administered at a localized site of interest within a subject's body, remains localized and essentially does not exhibit systemic distribution throughout the subject's body.
  • Item 2 The composition of Item 1 wherein the ionizable cationic lipidoid is selected from a compound of formula (b-l):
  • variables a, b, p, m, n and R 1A to R 6A are defined as follows: a is 1 and b is an integer of 2 to 4; or a is an integer of 2 to 4 and b is 1 , p is 1 or 2, m is 1 or 2; n is 0 or 1 and m+n is s 2; and
  • R 1A to R 6A are independently of each other selected from hydrogen; CH2CH(OH)R 7A , CH(R 7A )- CH2-OH,
  • Item 3 The composition according to any one of Items 1 or 2, wherein the cationic lipidoid comprises a compound of formula (b-V) and/or formula (b-VII):
  • Item 4 The composition according to any one of Items 1 to 3, wherein the formulation or the carrier comprises a lipid or lipidoid nanoparticle.
  • Item 5. The composition according to any one of Items 1 to 4, wherein the therapeutic agent is a) an anionic therapeutical substance and/or b) a nucleic acid, preferably an RNA, more preferably an mRNA, a miRNA and/or an siRNA, most preferably an mRNA encoding one or more polypeptide(s).
  • Item 6 The composition according to Items 5, wherein the mRNA comprises one or more of the following: a) a CAP, preferably an anti-Reverse Cap Analog (ARCA) at its 5’ end, b) a 5’-untranslated region (5’-UTR) upstream of a coding sequence encoding the polypeptide(s), c) a 5’-UTR comprising before an initiation codon sequence of the mRNA, an elongated Kozak sequence: GCCACCAUG, d) a 5’-UTR comprising immediately upstream of an initiation codon sequence of the mRNA any one of the following sequences: a) GGGAGACGCCACC (SEQ ID NO:11), b) GAAGCGCCACC (SEQ ID NO: 12), c) GGGACGCCACC (SEQ ID NO: 13), d) GGGAGACTGCCACC (SEQ ID NO: 14), e) GAAGCTGCCACC (SEQ ID NO: 15), f) GGG
  • Item 7 The composition according to any one of Items 5 or 6, wherein the mRNA is a product of in-vitro transcription (IVT).
  • Item 8 The composition according to any one of Items 5 to 7, wherein the mRNA comprises a polyadenylation (poly(A)) tail downstream of an open reading frame (ORF) encoding the polypeptide.
  • poly(A) polyadenylation
  • ORF open reading frame
  • Item 9 The composition according to any one of Items 5 to 8, wherein the mRNA comprises one or more modified nucleosides.
  • Item 10 The composition according to any one of Items 5 to 9, wherein the mRNA comprises one or more modified nucleosides selected from any of the following: pseudouridine, N1-methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2- thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4- thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-iodo-uridine, 5-methoxy
  • Item 11 The composition according to any one of Items 5 to 10, wherein the mRNA comprises one or more modified nucleosides selected from any of the following, wherein the one or more modified nucleosides comprise a 1 -methylpseudouridine ( m1 ⁇ ) modification.
  • Item 12 The composition according to any one of Items 5 to 11, wherein any of the following applies: a) wherein at least 50% of the uridines in the ORF have been modified, b) wherein at least 50% of the uridines in the mRNA have been modified, c) wherein at least 50 % of the uridines in the ORF have been modified to m1 , d) wherein at least 50 % of the uridines in the mRNA have been modified to m1 ⁇ .
  • Item 13 The composition according to any one of Items 1 to 12, wherein the formulation comprises a lipid or lipidoid nanoparticle optionally comprising any of the following: a) a nucleic acid, preferably an RNA coding for a microRNA or an mRNA coding for a functional protein, or an antigen such, optionally as defined in any one of Items 6 to 12, b) a cationic lipidoid of formula (b-l), preferably a cationic lipidoid of formula (b-V) or (b- VII), most preferably a cationic lipidoid of formula (b-V), and/or c) one or more helper lipid(s), optionally selected from: c1) a phospholipid, and/or c2) a sterol, and/or c3) stealth lipid, optionally, components b), and c1-c3) are all present, optionally they are at the molar ratios of about 8.0: about 5.3: about 4.4:
  • Item 14 The composition according to Item 13, wherein the cationic lipidoid is the cationic lipidoid of formula (b-V) and optionally: a) is an R isomer of (b_V), as shown in formula (b-VI) formula (b-VI) and/or b) is present at a molar ratio % of 22 to 65%, preferably 34% to 52%, more preferably 36% to 50%, and most preferably 43.1%.
  • Item 15 The composition according to Item 13, wherein the helper lipid is a phospholipid, preferably: a) selected from phosphocholine (PC) or phosphoethanolamine (PE), most preferably phosphocholine, b) has a carbon chain length of 14 to 18, most preferably 16, and/or c) is present in a molar ratio of 10% to 45%, preferably 18% to 39%, more preferably 24% to 33%, and most preferably 28.5%.
  • PC phosphocholine
  • PE phosphoethanolamine
  • Item 16 The composition according to claim 13, wherein the helper lipid is a sterol, preferably: a) cholesterol, b) is present at a molar ratio% of 12% to 38.5%, preferably 15% to 32%, more preferably 19% to 29%, and most preferably 23.7%.
  • the helper lipid is a sterol, preferably: a) cholesterol, b) is present at a molar ratio% of 12% to 38.5%, preferably 15% to 32%, more preferably 19% to 29%, and most preferably 23.7%.
  • Item 17 The composition according to Item 13, wherein the helper lipid is a stealth lipid, preferably wherein: a) is glycerolipid-based (G) or phosphoethanolamine lipid-based (PE), b) has a carbon chain length of 14 to 18, most preferably 14, c) the PEG is from 2000 to 5000 Daton, most preferably 2000 Dalton, and/or d) is present Molar ratio %: 1.5 to 7%, preferably 3 to 6%, more preferably 4 to 5%, and most preferably 4.7%.
  • G glycerolipid-based
  • PE phosphoethanolamine lipid-based
  • Molar ratio % 1.5 to 7%, preferably 3 to 6%, more preferably 4 to 5%, and most preferably 4.7%.
  • the phospholipid of c1 is preferably a phospholipid with a carbon chain of C12 to C18, more preferably a C16 phospholipid, most preferably DPPC, and/or
  • the sterol of c2) is cholesterol, and/or
  • the stealth lipid of c3) is a PEGylated lipids, preferably a PEGylated lipid with a molar weight of the PEG chain between about 2000 to about 5000 Dalton, more preferably a PEGylated lipid with a molar weight of the PEG chain of about 2000 Dalton, most preferably the PEGylated lipid is DMG-PEG2000.
  • Item 19 The composition according to any one of Items 13 to 18, wherein said antigen is selected from the group consisting of viral antigens, bacterial antigens, a cancer or tumor associated antigen, and allergens.
  • Item 20 The composition of anyone of Items 1 to 18, wherein said localized site of interest comprises a specific tissue, organ, or anatomical region, preferably the specific tissue, organ, or anatomical region selected from the lungs, the nose, the heart, the brain, the spleen, the lymph nodes, the bones, the tendons, the skeletal muscles, joints, the stomach, the small intestine, the large intestine, the kidneys, the bladder, the breast, the testes, the ovaries, the uterus, the spleen, the thymus, the brainstem, the cerebellum, the spinal cord, the eye, the ear, the tongue, the skin and/or tumors present in said tissues, organs and anatomic regions.
  • a specific tissue, organ, or anatomical region preferably the specific tissue, organ, or anatomical region selected from the lungs, the nose, the heart, the brain, the spleen, the lymph nodes, the bones, the tendons, the skeletal muscles, joints, the stomach, the small
  • Item 21 The composition according to any of Items 1 to 20, further comprising stabilizing agents, adjuvants, or immunomodulators.
  • Item 22 The composition according to any of Items 1 to 21 , wherein said therapeutic agent or carrier is encapsulated within a hydrogel or a biocompatible matrix to enhance localized retention.
  • Item 23 The composition according to any of Items 1 to 22, for use as a medicament.
  • Item 24 The composition according to any of Items 1 to 18 for use in the prevention, treatment, or amelioration of a viral infection, preferably by immunization, more preferably by local immunization.
  • Item 25 The composition according to any one of Items 1 to 18, for use in the treatment of a disease, wherein the disease is selected from: treatment of mutations, autoimmune diseases, metabolic imbalances, neurodegenerative disorders, degenerative disorders of the joints, solid tumor diseases (e.g. soft tissue tumors, tumors of the heart, the lungs, the liver, the spleen, the kidneys, the brain, oral cavity, the intestine, the skin, the pancreas, the prostate gland, the mammaria, the ovaries, the urinary bladder, the bones (i.e.
  • osteosarkoma chondrosarkoma, Ewing sarkoma
  • tumors of pleural and periotoneal cavity lung diseases, bone fracture or lesions thereof, tendon ruptures or lesions thereof, joint infections and ligament ruptures, arthrosis, arthritis, bacterial infections, preferably infections due to Methicillin resistant Staphylococcus Aureus (MRSA), Multidrug resistant Tuberculosis, viral infection, preferably a virus infection selected from Influenza (Flu), Hepatitis A, Hepatitis B, Human Papillomavirus (HPV), Measles, Mumps, Rubella, Polio, Rabies, Varicella (Chickenpox), Shingles (Herpes Zoster), Rotavirus, Yellow Fever, Smallpox, Japanese Encephalitis, Tick-Borne Encephalitis (TBE), Dengue Fever, West Nile Virus, Chikungunya Virus, Ebola Virus, Marburg Virus, Human Immunode
  • Item 26 Use of the composition according to any one of Items 1 to 18, in the manufacture of a medicament for the prevention, treatment, or amelioration of a mutations, lung diseases, bone fracture or lesions thereof, tendon fracture or lesions thereof, bacterial infections, viral infection, an infection, preferably a virus infections, more preferably selected from Influenza (Flu), Hepatitis A, Hepatitis B, Human Papillomavirus (HPV), Measles, Mumps, Rubella, Polio, Rabies, Varicella (Chickenpox), Shingles (Herpes Zoster), Rotavirus, Yellow Fever, Smallpox, Japanese Encephalitis, Tick-Borne Encephalitis (TBE), Dengue Fever, Human Immunodeficiency Virus (HIV), COVID-19 most preferably coronavirus infection.
  • Influenza Influenza
  • Hepatitis A Hepatitis B
  • HPV Human Papillomavirus
  • Measles Measles, M
  • Item 27 A method of inducing an immune response in a subject, which comprises administering to the subject an effective amount of a composition according to any of Items 1 to 8.
  • Item 28 A method of immunizing a subject against a pathogen, which comprises administering to the subject an effective amount of an mRNA in a pharmaceutical composition according to any one of Items 1 to 18.
  • Item 29 The method of Item 28, wherein said mRNA vaccine is administered via intradermal, subcutaneous, intramuscular, or intratumoral injection.
  • Item 30 The method or composition of any of Items 28 to 29, wherein said mRNA vaccine elicits an immune response predominantly at the localized site of interest, thereby reducing the risk of systemic adverse effects.
  • Item 31 The method or composition of any of Items 28 to 31 , wherein said mRNA vaccine is designed to promote local production of antigen-specific antibodies or cellular immune responses at the localized site of interest.
  • Item 32 The method or composition of any of Items 29 to 31 , wherein said mRNA vaccine further comprises a polymeric coating or encapsulation to enhance local retention and prevent systemic dissemination.
  • Item 33 The composition or method of any of Items 1-18 or Items 28 to 32 wherein the LNP further comprises a targeting moiety or ligand that specifically binds to cells or receptors present at the localized site of interest, thereby enhancing the vaccine's specificity and efficacy.
  • Item 34 The composition or method of any of Items 1 -18 or Items 28 to 33, wherein said pharmaceutically acceptable excipient or diluent further comprises a biodegradable or bioresorbable material, facilitating gradual release and local persistence of the mRNA at the localized site of interest.
  • Item 35 The composition or method of any of Items 1 -18 or Items 28 to 34, wherein said mRNA further comprises a tissue-specific promoter and/or enhancer element to enhance the expression of the target antigen at the localized site of interest.
  • Item 36 The composition or method of any of Items 1 -18 or Items 28 to 35, wherein said mRNA is encapsulated within a biocompatible microneedle patch or implantable device, facilitating controlled and sustained release of the nucleic acid at the localized site of interest.
  • Item 37 The composition or method of any of Items 1 -18 or Items 28 to 36, wherein said mRNA further comprises a self-amplifying mRNA (saRNA) molecule, enabling enhanced protein or antigen production at the localized site of interest.
  • Item 38 The composition or method of any of Items 1-18 or Items 28 to 37, wherein the mRNA codes for an antigen.
  • the present invention further relates, inter alia, to the following items:
  • compositions for use in the treatment and/or prevention of a disease comprising:
  • a carrier comprising: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) and/or diluent(s); wherein said composition has a prolonged retention at the site of administration; and/or wherein said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration.
  • compositions for use in the treatment and/or prevention of a disease comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii.
  • compositions or of the therapeutic agent optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration; and/or wherein the patient has less side-effects compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
  • composition for use according to item 1 or 2 wherein a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration; and/or wherein the patient has less side-effects compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
  • the composition for use according to item 1 or 3 wherein said composition has a prolonged retention at the site of administration; and/or wherein said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration.
  • a cosmetic composition comprising: a) one or more active agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein said cosmetic composition is an ointment, a creme, a foam, a gel, a lotion, an aqueous liquid, or a powder, or wherein said cosmetic composition is formulated as an ointment, a creme, a foam, a gel, a lotion, an aqueous liquid, or a powder.
  • LNP lipid nanoparticle
  • LiNP lipidoid nanoparticle
  • LLCs Nanostructured Lipid Carrier
  • LDC Lipid-Drug Conjugate
  • composition for use or the cosmetic composition according to item 8 wherein said ionizable lipidoid is a compound of formula (b-l): formula (b-l), wherein a is 1 or 2 and b is an integer of 1 to 4, or a is an integer of 1 to 4 and b is 1 or 2, p is 1 or 2, m is 1 or 2, n is 0 or 1 , m+n is ⁇ 2, and
  • R 7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms comprised or contained in the compound of formula (b-l) are optionally protonated to provide a compound carrying one or more positive charges, preferably wherein the variables a, b, p, m, n and R 1A to R 6A are defined as follows: a is 1 and b is an integer of 2 to 4, or a is an integer of 2 to 4 and b is 1 , p is 1 or 2, m is 1 or 2, n is 0 or 1 , m+n is ⁇ 2, and R 1A to R 6A are independently of each other selected from hydrogen, -CH 2 -CH(OH)-R 7A ,
  • R 1A to R 6A are defined as in item 9, and wherein one or more of the nitrogen atoms comprised or contained in the compound of formula (b-ll) are optionally protonated to provide a compound carrying one or more positive charges.
  • composition for use or the cosmetic composition according any one of items 9 to 11 , wherein said ionizable lipidoid comprises or consists of a compound of formula (b- V), formula (b-XI) and/or formula (b-XII), preferably a compound of formula (b-V):
  • composition for use or the cosmetic composition according to any one of items 8 to 12, wherein said ionizable lipidoid is a compound of formula (b-V) and preferably: a) is an R isomer of the compound of formula (b-V), and/or b) is present at a molar ratio of about 22 mol% to about 65 mol%, preferably about 34 mol% to about 52 mol%, more preferably about 36 mol% to about 50 mol%, and most preferably about 43.1 mol%.
  • PC phosphocholine
  • PE phosphoethanolamine
  • PEG polyethylene glycol
  • the phospholipid is preferably a phospholipid with a carbon chain length of about 12 to about 18, more preferably phospholipid with a carbon chain length of about 16, most preferably DPPC;
  • the sterol is cholesterol; and/or c) the stealth lipid is a PEGylated lipid, preferably a PEGylated lipid with a molar mass of the PEG chain between about 2000 to about 5000 Dalton, more preferably a PEGylated lipid with a molar mass of the PEG chain of about 2000 Dalton, most preferably the PEGylated lipid is DMG-PEG2000.
  • said one or more therapeutic agent(s) is/are a) an anionic therapeutical substance and/or b) a nucleic acid, preferably an RNA, more preferably an mRNA, a miRNA, and/or an siRNA, even more preferably an mRNA, most preferably an mRNA comprising an open reading frame (ORF) encoding one or more polypeptide(s).
  • composition for use according to item 26 wherein said nucleic acid is an RNA encoding a microRNA or wherein said nucleic acid is an mRNA comprising an ORF encoding one or more polypeptides, preferably wherein said one or more polypeptide(s) are one or more functional protein(s) and/or one or more antigen(s).
  • composition for use according to any one of items 26 to 28, wherein the mRNA comprises one or more features selected from the group consisting of the following: a) a CAP, preferably an anti-Reverse Cap Analog (ARCA) at its 5’ end, b) a 5’-untranslated region (5’-UTR) upstream of the ORF encoding said one or more polypeptide(s), c) a 5'-UTR comprising an elongated Kozak sequence (GCCACCAUG; SEQ ID NO: 44) upstream of the initiation codon of the ORF, d) a 5’-UTR comprising proximately upstream of an initiation codon of the ORF any one of the following sequences: i.
  • GGGAGACGCCACC (SEQ ID NO: 11), ii.GAAGCGCCACC (SEQ ID NO: 12), iii. GGGACGCCACC (SEQ ID NO:13), iv. GGGAGACTGCCACC (SEQ ID NO:14), v.GAAGCTGCCACC (SEQ ID NO: 15), vi. GGGACTGCCACC (SEQ ID NO:16).
  • composition for use according to item 26 to 32, wherein the one or more modified nucleosides are selected from the group consisting of the following: N1 -methylpseudouridine (ml i ), pseudouridine, N1 -ethyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1- methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-iodo-uridine, 5-methoxyuridine, 2'-
  • composition for use according to item 32 or 33 wherein any one or more of the following applies: a) wherein up to 100% of the uridines comprised in the ORF have been modified, preferably wherein at least about 50 mol% of the uridines comprised in the ORF have been modified, more preferably wherein any value between 50 and 100% has been modified, even more preferably wherein 100% of the uridines have been modified; b) wherein at least about 50 mol% of the uridines comprised in the mRNA have been modified; c) wherein at least about 50 mol% of the uridines comprised in the ORF have been modified to m1 ⁇ ; d) wherein at least about 50 mol% of the uridines comprised in the mRNA have been modified to m1 ⁇ ; e) wherein about 5 mol% to about 50 mol% of the uridines comprised in the mRNA are 5-iodouridines and about 5 mol% to about 50 mol% of
  • composition for use according to any one of items 8 to 35, wherein said site of administration comprises a tissue, an organ, and/or an anatomical region, preferably said solid tissue, organ, and/or anatomical region is a solid tissue, organ and/or anatomical region, more preferably said solid tissue, organ, and/or anatomical region is selected from the group consisting of the lungs, the nose, the heart, the brain, the spleen, the lymph nodes, the bones, the tendons, the skeletal muscles, joints, the stomach, the small intestine, the large intestine, the kidneys, the bladder, the breast, the testes, the ovaries, the uterus, the spleen, the thymus, the brainstem, the cerebellum, the spinal cord, the eye, the ear, the tongue, the skin and/or tumors present in said solid tissues, organs and/or anatomical regions.
  • composition for use according to any one of items 8 to 36 further comprising one or more stabilizing agent(s), adjuvant(s), and/or immunomodulator(s).
  • a method for preventing, treating, and/or ameliorating a disease comprising administering an effective amount of the composition as defined in any one of items 1 to 6 and 8 to 38 to a subject.
  • compositions as defined in any one of items 1 to 6 and 8 to 38 in the manufacture of medicament for the prevention, treatment, and/or amelioration of a disease.
  • a method of inducing an immune response in a subject which comprises administering to said subject an effective amount of the composition as defined in or according to any one of items 1 to 6, 8 to 38, and 41 to 50.
  • a method of immunizing a subject against a pathogen which comprises administering to said subject an effective amount of an mRNA vaccine in a pharmaceutical composition, wherein said pharmaceutical composition comprises the composition as defined in or according to any one of items 1 to 6, 8 to 38, and 41 to 50.
  • mRNA vaccine further comprises a polymeric coating or encapsulation to enhance local retention and prevent systemic dissemination.
  • mRNA further comprises a self-amplifying mRNA (saRNA) molecule, enabling enhanced protein or antigen production at the site of interest.
  • composition according to any one of items 7 to 25, wherein said active ingredient is selected from the group consisting of the following: a growth factor, a peptide, an antioxidant, a retinoid, a cytokine, a siRNA, a miRNA, a mRNA, and an asRNA.
  • said active ingredient is selected from the group consisting of the following: a growth factor, a peptide, an antioxidant, a retinoid, a cytokine, a siRNA, a miRNA, a mRNA, and an asRNA.
  • a method for the amelioration of a cutaneous condition comprising the administration of the cosmetic composition according to any one of items 7 to 25, and 64.
  • a kit comprising the cosmetic composition according to any one of items 7 to 25, and 64.
  • a drug conjugate comprising an ionizable lipidoid as defined in any one of items 9 to 18 and one or more therapeutic agent(s), preferably wherein said one or more therapeutic agent(s) is/are as defined in any one of items 26 to 34.
  • compositions such as pharmaceutical compositions and cosmetic compositions
  • agents such as therapeutic agents or active agents
  • compositions this may also refer to the herein provided (pharmaceutical) composition for use as a medicament (also referred to as “pharmaceutical composition” herein) and/or to the herein provided cosmetic compositions.
  • pharmaceutical composition or accordingly a pharmaceutical composition
  • compositions may be used interchangeably.
  • formulation may be used interchangeably herein.
  • the present invention provides for a (pharmaceutical) composition for use in the treatment of a disease and/or prevention of a disease, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein one or more of the following apply:
  • a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
  • the patient has less side effects (such as a reduced risk of CARPA) compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
  • composition has a prolonged retention at the site of administration
  • said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration;
  • composition when administered to said site of administration, remains localized and essentially does not exhibit systemic distribution throughout the patient's body.
  • side effects in a patient is highly advantageous (in the context of the present invention).
  • the patient is susceptible to hypersensitivity reactions, risk group patients, and/or in pediatric patients (i.e., patients being less than about 18 years old).
  • a preferred patient in need of treatment and/or prevention of a disease
  • a patient belonging to a risk group a patient that is susceptible to hypersensitivity reactions (preferably susceptible to hypersensitivity reactions induced by pharmaceutical compositions or by the systemic or non-local distribution of pharmaceutical compositions), and/or a pediatric patient.
  • hypersensitivity reactions preferably susceptible to hypersensitivity reactions induced by pharmaceutical compositions or by the systemic or non-local distribution of pharmaceutical compositions
  • pediatric patient preferably a pediatric patient.
  • the skilled artisan is readily aware of means and methods to assess whether a patient belongs to a risk group and/or is susceptible to hypersensitivity reactions (preferably susceptible to hypersensitivity reactions induced by pharmaceutical compositions or by the systemic or non-local distribution of pharmaceutical compositions).
  • the therapeutic agent of the present invention comprises or consists of an immunomodulatory or immunoactivating polypeptide (such as an interferon, preferably interferon lambda or human interferon lambda) or a nucleic acid (such as an mRNA) encoding such an immunomodulatory or immunoactivating polypeptide (such as an interferon, preferably interferon lambda or human interferon lambda), it is particularly advantageous to limit the risk for CARPA (as potentially induced by the systemic distribution of a composition that does not comprise an ionizable lipid and/or lipidoid in accordance with the present invention), in particular in patients that are susceptible to hypersensitivity reactions.
  • an immunomodulatory or immunoactivating polypeptide such as an interferon, preferably interferon lambda or human interferon lambda
  • a nucleic acid such as an mRNA
  • Diseases to be treated and/or prevented may be any of the herein above or below detailed (in particular a diseases or disorder caused by or associated with Enterovirus, such as rhinitis as caused for example by rhinovirus).
  • a diseases or disorder caused by or associated with Enterovirus such as rhinitis as caused for example by rhinovirus.
  • the pediatric patient may be less than about 18 years old, less than about 16 years old, less than about 14 years old, less than about 12 years old, less than about 10 years old, less than about 9 years old, less than about 8 years old, less than about 7 years old, or less than about 6 years old.
  • the (pediatric) patient may be 6 month or older.
  • cosmetic compositions it is similarly advantageous to limit the distribution of said cosmetic composition or of the active agent comprised therein in order to avoid undesired side effects (such as complement activation and/or CARPA), in particular in subjects which are susceptible to hypersensitivity reactions.
  • side effects such as complement activation and/or CARPA
  • the present invention further relates to a cosmetic composition
  • a cosmetic composition comprising: a) one or more active agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii.
  • said cosmetic composition is an ointment, a creme, a foam, a gel, a lotion, an aqueous liquid, or a powder, or wherein said cosmetic composition is formulated as an ointment, a creme, a foam, a gel, a lotion, an aqueous liquid, or a powder.
  • contacting a subject/a tissue/an organ with either a pharmaceutical or cosmetic composition may be referred to as ‘‘treating a subject/a tissue/an organ”, accordingly, the terms “treat”, “treatment”, and the like may refer to both therapeutic and cosmetic application/administrations/compositions and the like.
  • At least about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of said one or more (therapeutic or active) agent(s) may be restricted to the treated tissue, organ, and/or anatomical region, as quantified by a method selected from the group consisting of qPCR, HPLC, mass spectrometry, combinations of HPLC and mass spectrometry.
  • the restriction of systemic distribution/the prolonged retention at the site of administration may preferably be quantified/assessed/determined by any suitable method, preferably a method selected from the group consisting of qPCR, HPLC, mass spectrometry, and/or combinations of HPLC and mass spectrometry.
  • means and methods to assess whether e.g., about 0.01% of the agent reaches (or is found/detected in) circulation include qPCR, HPLC, mass spectrometry, and/or combinations of HPLC and mass spectrometry.
  • agents comprising a nucleic acid such as mRNA
  • qPCR may be preferred, more preferably qPCR conducted in the enclosed examples.
  • Example 7 it was found that less than 0.01 % of the translated hlFN ⁇ 1 in the lungs reached the circulation on average approximately 2 hours after administration (between 33 minutes and 5 hours 22 minutes.
  • Example 7 illustrates how it can be assessed/detected/determined whether a composition remains localized at the site of administration and/or essentially does not exhibit systemic distribution throughout the patient's body, whether a composition has a prolonged retention at the site of administration; and/or whether the therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration, or, vice versa, whether a composition does not remain localized at the site of administration and/or essentially does exhibit systemic distribution throughout the patient's body, does not have prolonged retention at the site of administration or wherein said therapeutic agent does not exert its effect at the site of administration by prolonged retention at the site of administration.
  • composition remains localized at the site of administration” and/or “composition essentially does not exhibit systemic distribution throughout the patient's body”, “composition has a prolonged retention at the site of administration” likewise apply to and can be interchangeably used with “(therapeutic or cosmetic) agent remains localized at the site of administration” and/or “(therapeutic or cosmetic) agent essentially does not exhibit systemic distribution throughout the patient's body”, “(therapeutic or cosmetic) agent has a prolonged retention at the site of administration”, respectively.
  • a term like “(cosmetic or therapeutic) agent exerts its effect at the site of administration by prolonged retention at the site of administration” refers to “(cosmetic or therapeutic) agent exerts its effect at the site of administration by prolonged retention of the “(cosmetic or therapeutic) agent at the site of administration”.
  • composition remains localized at the site of administration” and/or if a “composition essentially does not exhibit systemic distribution throughout the patient's body”, or if a “composition has a prolonged retention at the site of administration”, e.g.
  • any suitable method preferably a method selected from the group consisting of qPCR, ligand binding assay, such as ELISA, HPLC, mass spectrometry, combinations of HPLC and mass spectrometry and/or combinations thereof.
  • compositions do not remain localized at the site of administration and/or if a composition essentially does exhibit systemic distribution throughout the patient's body, or if a composition does not have prolonged retention at the site of administration or if a (therapeutic or cosmetic) agent does not exert its effect at the site of administration by prolonged retention (of the agent or the composition) at the site of administration of the composition, e.g.
  • the percentage may be determined on basis of the amount of the translated gene product (peptide and/or protein) in the (systemic) circulation versus the amount of the translated gene product (peptide and/or protein) detected/assessed/determined at the administration site e.g. after about 10 hours after administration, after about 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or, preferably 2 hours.
  • the amount of the translated gene product (protein) detected/assessed/determined at the administration site may also be calculated based on the administered amount of the therapeutic agent (e.g. mRNA) and the thereby expected translated gene product (protein).
  • the extent to which the therapeutic agent or the proteins and/or peptides encoded by said therapeutic agent (such as an mRNA agent) remain localized at the site of administration may depend on the characteristics of said therapeutic agent, or the encoded protein and/or peptides. For example, a small peptide may be more mobile once expressed than a large protein. Thus, the expressed protein may preferably have a molecular weight of about 20 KDa or larger.
  • the determination of the relative values will take into account the respective administered (total) amount of the agent (or the (total) amount of the translated gene product (protein) in case of mRNA/DNA)). For example, if the therapeutic agent is administered in subsequent 3 doses with time interval of 2 hours, the amount of the (therapeutic or cosmetic) agent detected/assessed/determined in (systemic) circulation (or the amount the agent reaches in (systemic) circulation) may be determined e.g. 2 hours after each administration relative to the then total administered amount of the agent.
  • the quantification of therapeutic agents in e.g., different organs is exemplified in the enclosed examples. Further, the enclosed examples illustratively show that both the therapeutic agent (i.e., hlNFM mRNA; SEQ ID NO: 42) and the employed ionizable lipidoid (e.g., a compound according to formula b-V) were retained locally at the site of local administration (see for example, Figs 18 and 19).
  • the local retention of the composition for example an LiNP or components thereof, such as an ionizable lipidoid or ionizable lipid
  • the (therapeutic or active) agent is also locally retained.
  • means and methods to assess whether e.g., about 0.01% of the (ionizable) lipidoid or (ionizable) lipid reaches (or is found in) circulation include HPLC, mass spectrometry, combinations of HPLC and mass spectrometry, preferably LC-MC/MS, as detailed in the enclosed examples.
  • HPLC high-density lipoprotein
  • mass spectrometry combinations of HPLC and mass spectrometry, preferably LC-MC/MS, as detailed in the enclosed examples.
  • any suitable method preferably a method selected from the group consisting of HPLC, mass spectrometry, combinations of HPLC and mass spectrometry, more preferably LC-MC/MS.
  • compositions do not remain localized at the site of administration and/or if a composition essentially does exhibit systemic distribution throughout the patient's body, or if a composition does not have prolonged retention at the site of administration or if a (therapeutic or cosmetic) agent does not exert its effect at the site of administration by prolonged retention (of the agent or the composition) at the site of administration of the composition, e.g.
  • the lowest limit of quantification using (LLOQ) to detect the (ionizable) lipidoid shown in formula b-V (specifically) “dL_05” in serum and lung was 100 ng/mL and in liver 200 ng/mL).
  • Such a method may be performed in a subject, e.g. a (non-human) animal (like rat or mouse), for example using non-invasive imaging techniques.
  • the therapeutic use or method of treatment may comprise an (in vitro or ex vivo) step of detecting/assessing/determining whether a “composition remains localized at the site of administration” and/or if a “composition essentially does not exhibit systemic distribution throughout the patient's body”, or if a “composition has a prolonged retention at the site of administration”, and the like, as described herein above.
  • compositions for use, the herein provided cosmetic compositions, the herein provided method of treatment in the context of the herein provided method of detecting/assessing/determining whether a “composition remains localized at the site of administration” and/or if a “composition essentially does not exhibit systemic distribution throughout the patient's body”, or if a “composition has a prolonged retention at the site of administration”, and the like
  • the skilled person may readily assess whether a “composition remains localized at the site of administration” and/or if a “composition essentially does not exhibit systemic distribution throughout the patient's body”, or if a “composition has a prolonged retention at the site of administration”, and the like, by comparing a composition (e.g., that may have local retention at the site of administration or the like/in accordance with the present invention) to be administered to a subject with a reference composition that does not remain localized at the site of administration and/or a reference composition that does exhibit system
  • comparing a composition with a reference composition may mean to compare the systemic distribution/the local retention of a composition with said reference compositions by means and methods detailed herein above and further illustratively demonstrated in the enclosed examples.
  • methods are not particularly limited and may comprise a method selected from the group consisting of qPCR, ligand binding assay, such as ELISA, HPLC, mass spectrometry, combinations of HPLC and mass spectrometry and/or combinations thereof.
  • a “reference composition” does not have local retention at the site of administration or the like.
  • the term “a composition that has local retention at the site of administration” or the like may also refer to “a composition that has a prolonged retention at the site of administration”, “a composition that remains localized and essentially does not exhibit systemic distribution throughout the patient's body”, “a non-systemic composition”, “a composition that does not have/exhibit systemic distribution” or the like, and vice versa.
  • a “reference composition” may also be herein referred to as a “systemic composition” (i.e., a composition that has systemic distribution/a composition that does not have localized retention at the site of administration or the like).
  • a “reference composition” in the context of the present invention does not comprise the herein employed ionizable lipids or ionizable lipidoids that confer the local retention at the site of administration.
  • a “reference composition” in the context of the present invention does not comprise an ionizable lipid or ionizable lipidoid according to e.g., any one of formulas (b-l), (b-ll), (b-V), (b-VI), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), or (b- XII).
  • Such a reference composition may for example comprise a state of the art lipid selected from e.g., DLin-MC3-DMA, ALC-0315, or SM-102 (and does not comprise an ionizable lipid or ionizable lipidoid according to any one of formulas (b-l), (b-ll), (b-V), (b-VI), (b-VII), (b-VIII), (b- IX), (b-X), (b-XI), or (b-XII)).
  • a state of the art lipid selected from e.g., DLin-MC3-DMA, ALC-0315, or SM-102 (and does not comprise an ionizable lipid or ionizable lipidoid according to any one of formulas (b-l), (b-ll), (b-V), (b-VI), (b-VII), (b-VIII), (b- IX), (b-X), (b-XI),
  • compositions or components thereof, such as the active agent or the therapeutic agent
  • agent e.g., a nucleic acid, such as an mRNA.
  • agents that are reporter polypeptides (or nucleic acids encoding such reporter polypeptides), such as luciferase (or nucleic acids encoding luciferase) to assess whether a composition has localized retention at the site of administration or the like as compared to said reference composition.
  • compositions such as a composition in accordance with the present invention/a composition comprising e.g., any one of formulas (b-l), (b-ll), (b-V), (b-VI), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), or (b-XII)
  • a reference composition e.g., comprising MC3 each comprising a reporter polypeptide (such as luciferase, as exemplified by SEQ ID NO: 46) or a nucleic acid encoding the same (as exemplified by SEQ ID NO: 45) and assess the activity of said reporter polypeptide (e.g., luciferase activity) after localized administration of the composition (in accordance with the present invention) and after localized administration (e.g., by nasal administration) of said reference composition.
  • a reporter polypeptide such as luciferase, as exemplified by SEQ ID NO:
  • the skilled person is aware that the composition and the reference composition are to be administered to two different/to two independent subjects by localized administration (in order to subsequently quantify the reporter polypeptide activity).
  • the activity of the reporter polypeptide may be assessed by means and methods employed in the enclosed examples (e.g., by IVIS imaging of the of the subjects or by IVIS imaging of excised organs of the subjects). When excising organs of the subjects, said subjects are non- human.
  • the agent i.e., the reporter polypeptide or the nucleic acid encoding the same
  • the composition (in accordance with the present invention) and the reference composition differ only in the above mentioned ionizable lipidoid).
  • reporter polypeptide activity such as reporter polypeptide activity as assessed by IVIS imaging
  • the skilled person can readily compare reporter polypeptide activity (such as reporter polypeptide activity as assessed by IVIS imaging) by comparing the resulting measurements of reporter polypeptide activity (such as the IVIS imaging measurements) in the subjects after administration of the composition and the reference composition e.g. after about 10 hours after administration, after about 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or, preferably 2 hours.
  • the skilled person may for example assess reporter polypeptide activity (such as reporter polypeptide activity as assessed by IVIS imaging) in an off-target tissue or organ.
  • An off-target tissue or organ is a tissue or organ to which the to-be-administered composition was not administered.
  • the lung when targeting the lung (i.e., when locally administering a composition to the lung of a subject) the lung is to be considered as a “target organ” (or as “target tissue”) whereas other organs (such as, e.g., the heart, the spleen, etc.) are considered as “off-target organs” (or “off-target tissue”).
  • target organ or as “target tissue”
  • off-target organs or “off-target tissue”.
  • the reference composition has/results in/exhibits about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 10 4 -fold, 10 5 -fold, etc., increase in reporter polypeptide activity as compared to a composition (in accordance with the present invention), with any values in between such ranges being also envisaged herein and with
  • Such fold-changes may be determined by dividing the measurements (such as IVIS imaging measurements) obtained from the assessment of reporter polypeptide activity in an off-target organ after localized administration of the reference composition by the measurements (such as IVIS imaging measurements) obtained from the assessment of reporter polypeptide activity in an off-target organ after localized administration of the composition (in accordance with the present invention).
  • a reference composition comprising e.g., an mRNA encoding for luciferase, as exemplified in SEQ ID NO: 46
  • the administration of a composition in accordance with the present invention; and comprising e.g., an mRNA encoding for luciferase, as exemplified in SEQ ID NO: 46
  • a composition in accordance with the present invention, and comprising e.g., an mRNA encoding for luciferase, as exemplified in SEQ ID NO: 46
  • the same off-target organ or the reporter polypeptide activity in the same off-target organ
  • the reference composition i.e. , for example that the heart (or the reporter polypeptide activity in the heart) is assessed for the composition (in accordance with the present invention) and for the reference composition) at the same or about the same time after administration of both compositions to the subjects.
  • the present invention provides for a/an (in vitro I ex vivo) method of detecting/assessing/determining whether and/or to what extend a composition remains localized at the site of administration, the method comprising the steps of:
  • composition (i) administering a composition to a target organ of a first subject via localized administration and administering a reference composition to a target organ of a second subject via localized administration, wherein each composition comprises a reporter polypeptide or a nucleic acid encoding the same;
  • reporter polypeptide activity may also apply to said method. Accordingly, instead of assessing reporter polypeptide activity, the skilled person may readily quantify the (therapeutic or active) agent by other means, such as a method selected from the group consisting of qPCR, ligand binding assay, such as ELISA, HPLC, mass spectrometry, combinations of HPLC and mass spectrometry and/or combinations thereof.
  • a method selected from the group consisting of qPCR, ligand binding assay, such as ELISA, HPLC, mass spectrometry, combinations of HPLC and mass spectrometry and/or combinations thereof such as a method selected from the group consisting of qPCR, ligand binding assay, such as ELISA, HPLC, mass spectrometry, combinations of HPLC and mass spectrometry and/or combinations thereof.
  • composition when a composition remains localized at the site of administration/in a target organ/in a target tissue, this allows the reduction of the amount of the (therapeutic or active) agent to be administered and/or allows for less side effects of the to be administered composition.
  • a method for determining a/an (reduced) amount of a (therapeutic or cosmetic) agent in a composition (in accordance with the present invention) to be administered to a target organ wherein the amount of said (therapeutic or cosmetic) agent is reduced as compared to a reference composition (e.g.
  • composition comprising MC3), preferably wherein the amount of said (therapeutic or cosmetic) agent is reduced by about 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%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,
  • a/an (reduced) amount of a side effects caused by a composition when administered to a target organ, wherein the amount of said side effects is reduced as compared to a reference composition (e.g. composition comprising MC3), preferably wherein the amount of said side effect is reduced by about 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%,
  • a reference composition e.g. composition comprising MC3
  • systemic distribution of the (therapeutic or cosmetic) agent is preferably assessed on (a) blood sample(s) of the subject to be treated.
  • a blood sample may be a whole blood sample, a plasma sample, a serum sample, or the like.
  • the restriction of the one or more (therapeutic or active) agent(s) to the treated/ tissue, organ, and/or anatomical region enables a reduction in the dose of said one or more (therapeutic or active) agent(s) to be administered by up to 20%, including any and all ranges within this limit, such as but not limited to reductions of 1-20%, 5-15%, or 10-20%.
  • the restriction of said one or more (therapeutic or active) agent(s) to the treated tissue, organ, and/or anatomical region reduces the number of administrations of said one or more (therapeutic or active) agent(s), preferably the number of administrations is reduced by about or less than about 5%, preferably by about or less than about 10%, more preferably by about or less than about 20%, even more preferably by about or less than about 30%, even more preferably by about or less than about 40%, even more preferably by about or less than about 50%, even more preferably by about or less than about 60%, even more preferably by about or less than about 70%, even more preferably by about or less than about 80%, most preferably by about or less than about 90%.
  • the restriction of said one or more (therapeutic or active) agent(s) to the treated tissue, organ, and/or anatomical region reduces toxicity caused by and/or associated with the accumulation of said one or more (therapeutic or active) agent(s) in off-target organs, preferably wherein toxicity caused by and/or associated with said one or more therapeutic agent(s) is reduced in the liver, the brain, the kidneys, the heart, and/or the spleen.
  • off-target organs/off target tissues/off-target sites or the like are organs/tissues/sites or the like that are not the (primary) target organs I target tissues/ target sites or the like; in contrast, the term (primary) target organs I target tissues/ target sites or the like in the context of the present invention refer to the organs/tissues/sites or the like to be treated using the herein provided compositions.
  • the restriction of said one or more (therapeutic or active) agent(s) to the treated tissue, organ, and/or anatomical region reduces and/or avoids off-target effects caused by and/or associated with said one or more (therapeutic or active) agent(s), preferably wherein off-target effects caused by and/or associated with said one or more (therapeutic or active) agent(s) are reduced in the liver, the brain, the kidneys, the heart, and/or the spleen.
  • the herein provided compositions locally restrict the dissemination at least of the therein comprised (therapeutic or active) agent(s), while the co-administration with a hyaluronidase surprisingly abolished this advantageous effect.
  • the herein provided (therapeutic or cosmetic) composition is preferably not to be co-administered with one or more hyaluronidase(s) and/or enzyme(s) comprising hyaluronidase activity.
  • compositions are not particularly limited as long as they are suitable for therapeutic and/or cosmetic applications and remain locally restricted/has prolonged retention at the site of administration/essentially does not exhibit systemic distribution/or the like.
  • the carrier is a lipid nanoparticle (LNP), a lipidoid nanoparticle (LiNP), a liposome, a micelle, an emulsion, an Nanostructured Lipid Carrier (NLCs), or a Lipid-Drug Conjugate (LDC), preferably an LNP or an LiNP, and/or that the agent is formulated as a lipid nanoparticle (LNP), a lipidoid nanoparticle (LiNP), a liposome, a micelle, an emulsion, a Nanostructured Lipid Carrier (NLCs), or a Lipid-Drug Conjugate (LDC), preferably as an LNP or as an LiNP.
  • LNP lipid nanoparticle
  • LiNP lipidoid nanoparticle
  • LLCs Nanostructured Lipid Carrier
  • LDC Lipid-Drug Conjugate
  • any definition relating to an LNP may also refer to an LiNP, and vice versa, with the exception that an LNP at least comprises one ionizable lipid and an LiNP comprises at least one ionizable lipidoid.
  • An aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a nucleic acid (such as an RNA or an mRNA) of the invention, a nucleic acid (such as an RNA or an mRNA) vaccine vector of the invention, or an nucleic acid (such as an RNA or an mRNA) vaccine of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • the mRNA or the mRNA vaccine of the invention can advantageously be combined in the pharmaceutical composition with further components and/or compounds which ease delivery of the mRNA to the target cells or the target tissue and/or which increase its stability.
  • RNA into liposomes or nanoparticles with suitable substances such as those described herein and, e.g. in EP3013964B1, which is incorporated herein in its entirety.
  • the mRNA or the mRNA vaccine of the invention might be formulated with liposomes, to generate lipoplexes or with subsequent generations of lipid nanocarriers, such as lipid nanoparticles (LNPs), lipidoid nanoparticles (LINPs), nanostructured lipid carriers, and/or cationic lipid-nucleic acid complexes.
  • the nucleic acid of the invention can be delivered to target cells and/or target tissues in vivo, ex vivo and/or in vitro using LNPs or LiNPs.
  • LNPs and LiNPs can be distinguished from other carriers due to their small size, their homogenous size distribution and their structure and are especially suited for immunization of a subject.
  • LNPs and LiNPs The skilled person knows methods for the production of LNPs and LiNPs.
  • the production of LNPs or LiNPs involves a combination of lipids or lipidoids, such as phospholipids, cholesterol, and other specialized lipids, which are mixed together in a solvent, such as an alcohol.
  • lipids or lipidoids such as phospholipids, cholesterol, and other specialized lipids, which are mixed together in a solvent, such as an alcohol.
  • nanoprecipitation which involves rapidly mixing the lipid solution with a non-solvent, such as a nucleic acid dissolved in water, under controlled conditions of temperature, pressure, and stirring rate.
  • a non-solvent such as a nucleic acid dissolved in water
  • the nano particles may also be further modified with various surface coatings, such as polyethylene glycol (PEG), to improve their stability and reduce their tendency to be cleared by the immune system.
  • PEG polyethylene glycol
  • compositions may further comprise one or more stabilizing agent(s), adjuvant(s), and/or immunomodulator(s).
  • stabilizing agents may be defined as anywhere herein above or below, preferably the stabilizing agent may be a triblock polymer (i.e., component (p)) as defined anywhere herein.
  • stabilizing agents such as Cholesterol, Polyethylene Glycol, poloxamer
  • may help stabilize the composition such as stabilize the lipid bilayers and improve the structural integrity of nanoparticles).
  • adjuvants may comprise for example CPG oligonucleotides, in particular in the context of herein provided vaccine compositions.
  • the LiNPs may comprise as component (a) an mRNA, an ionizable lipid or an ionizable lipidoid and optionally helper lipids as defined below.
  • the LiNPs may comprise as component (p) a triblock copolymer which contains one polypropylene oxide) block, and two poly(ethylene oxide) blocks as described above.
  • the surfactant in the context of the present invention may be a non-ionic surfactant, optionally at least one nonionic surfactant selected from the group of fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, fatty acid esters of sorbitol, ethoxylated fatty acid esters of sorbitol, fatty acid esters of glycerol, ethoxylated castor oil and ethoxylated vitamin E, preferably wherein the surfactant is selected from the list consisting of poloxamer 188 (P188), poloxamer 338 (P338), poloxamer 407 (P407), Tween-20, Tween-80, BRIJ35, tyloxapol, VitE-PEG1000, and/or Kolliphor EL.
  • the surfactant is a (tri)block copolymer of ethylene oxide and propylene oxide, more preferably a poloxamer, even more preferably poloxamer selected from the list consisting of: poloxamer 188, 338, and/or 407, most preferably poloxamer 188 (i.e., P188).
  • the nanoparticles contained in the pharmaceutical composition of the invention may comprise a mRNA coding for one or more antigen(s), wherein said one or more antigen(s) is/are selected from the group consisting of a viral antigen, a bacterial antigen, a cancer, and/or tumor associated antigen, and/or an allergen.
  • the nanoparticles in the pharmaceutical composition may further comprise an ionizable lipid or an ionizable lipidoid. It will be understood that this encompasses the possibility that the nanoparticles comprise a combination of different ionizable lipids, a combination of different ionizable lipidoids, or a combination of one or more ionizable lipids and one or more ionizable lipidoids.
  • the nanoparticles used in the context of the present invention typically comprise an mRNA (a) and as the ionizable lipid or as the ionizable lipidoid (b) a cationic lipid or cationic lipidoid, in the form of a mixture of these components.
  • Ionizable lipids and/or ionizable lipidoids that may be suitable and are, thus, envisaged in the context of the present invention are disclosed in WO 2014/207231, which is herein incorporated by reference in its entirety.
  • the pharmaceutical composition or the mRNA vaccine according to the invention optionally comprises a LiNP comprising an ionizable lipidoid of formula (b-l): wherein the variables a, b, p, m, n and R 1A to R 6A are defined as follows: a is 1 and b is an integer of 2 to 4; or a is an integer of 2 to 4 and b is 1 , p is 1 or 2, m is 1 or 2; n is 0 or 1 and m+n is ⁇ 2; and
  • R 1A to R 6A are independently of each other selected from hydrogen; -CH 2 -CH(OH)-R 7A , -CH(R 7A )-CH 2 -OH,
  • the composition according to the present invention comprises an ionizable lipidoid, wherein said ionizable lipidoid may be a compound of formula (b-l): formula (b-l), preferably wherein the variables a, b, p, m, n and R 1A to R 6A are defined as follows: a is 1 and b is an integer of 2 to 4, or a is an integer of 2 to 4 and b is 1 , p is 1 or 2, m is 1 or 2, n is 0 or 1 , m+n is ⁇ 2, and
  • R 1A to R 6A are independently of each other selected from hydrogen, -CH 2 -CH(OH)-R 7A ,
  • the composition according to the present invention comprises an ionizable lipidoid, wherein said ionizable lipidoid may be a compound of formula (b-l): formula (b-l), wherein a is 1 or 2 and b is an integer of 1 to 4, or a is an integer of 1 to 4 and b is 1 or 2, p is 1 or 2, m is 1 or 2, n is 0 or 1 , m+n is ⁇ 2, and
  • composition according to the present invention comprises an ionizable lipidoid, wherein said ionizable lipidoid may be a compound of formula (b-ll): wherein a is 1 or 2, preferably 1 , b is 1 or 2, preferably 2,
  • R 1A to R 6A are defined as herein above (e.g., as a compound of formula (b-l)), and wherein one or more of the nitrogen atoms comprised or contained in the compound of formula (b-ll) are optionally protonated to provide a compound carrying one or more positive charges.
  • R 7A may be preferably selected from C8-C16 alkyl or C8-C18 alkenyl having one C-C double bond, and more preferably from C8-C12 alkyl or C8-C12 alkenyl having one C-C double bond and most preferably from C10-C12 alkyl or C10-C12 alkenyl having one C- C double bond.
  • the Ionizable lipidoid has a structure according to formula (IVb) or (IVc): wherein a, b, and R 1 to R 6 are defined as anywhere herein above and wherein one or more of the nitrogen atoms indicated in formula (IVc) may be protonated to provide a cationic lipidoid.
  • the compound of formula (b-l) is a compound of formula (b-lb), and the ionizable lipidoid comprises or consists of a lipidoid compound of the following formula (b-lb), wherein R 1A to R 6A are defined as in formula (b-l), including preferred embodiments thereof; or a protonated form thereof wherein one or more of the nitrogen atoms indicated in formula (b- Ib) are optionally protonated to provide a compound carrying a positive charge.
  • the ionizable lipidoid comprises or consists of a lipidoid of the above formula (b-lb) or a protonated form thereof, and R 1A to R 6A are independently selected from hydrogen and -CH2-CH(OH)-R 7A , wherein R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond, provided that at least two residues among R 1A to R 6A are -CH2-CH(OH)-R 7A , more preferably at least three residues among R 1A to R 6A , and still more preferably at least four residues among R 1A to R 6A are -CH2-CH(OH)-R 7A , wherein R 7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond.
  • the mRNA vaccine, or the pharmaceutical composition according to the invention comprises a LiNP nanoparticle comprising a cationic lipidoid of formula (b-V) (also herein referred to as “dL_05” herein, with the (R)-enantiomer of the compound of formula (b-V) being herein also referred to as “dL_05(R)”) and/or formula (b-VII):
  • the mRNA vaccine, or the pharmaceutical composition according to the invention comprises a LiNP nanoparticle comprising a cationic lipidoid of formula (b-XI) and/or formula (b-XII):
  • the composition comprises an ionizable lipidoid, wherein said ionizable lipidoid comprises or consists of a compound of formula (b-VII) or a compound of formula (b-VIII), preferably a compound of formula (b-VII):
  • composition for use or the cosmetic composition as described herein wherein said ionizable lipidoid comprises or consists of a compound of formula (b-IX) or a compound of formula (b-X), preferably a compound of formula (b-X):
  • Example 10 demonstrates that a combination of a compound according to formula (b-IX) and of a compound according to formula (b-X) also causes the (desired) local retention of the to be administered composition.
  • the (carrier of the) to be administered compositions may comprise at least two ionizable lipids and/or at least two ionizable lipidoids. It is herein preferred that said at least two ionizable lipids and/or said at least two ionizable lipidoids are two ionizable lipids and/or two ionizable lipidoids.
  • Said at least two ionizable lipids and/or said at least two ionizable lipidoids may be as defined anywhere herein above. Accordingly, said at least two ionizable lipids and/or said at least two ionizable lipidoids may, for example, be a compound according to formula (b-VI), (b-V), (b-l), (b-ll), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), or (b-XII). In the context of the present invention, it is understood that said at least two ionizable lipids or said at least two ionizable lipidoids are not identical/different (e.g.
  • the (carrier of the) to be administered compositions comprises a lipidoid according to formula (b-IX) and a lipidoid according to formula (b-X).
  • the compositions to be administered in the context of the present invention may comprise an ionizable lipid or an ionizable lipidoid.
  • the ionizable lipid or the ionizable lipidoid
  • the composition comprises solely the same/identical ionizable lipid (or the same/identical ionizable lipidoid), i.e. the composition does not comprise different ionizable lipids (and/or different ionizable lipidoids).
  • the only/sole lipidoid in the composition may, for example, be a compound of formula (b-l), (b-ll), (b-V), (b-VII), (b-VIII), (b- IX), (b-X), (b-XI), or (b-XII).
  • cationic lipidoid ionizable lipidoid
  • lipidoid lipidoid
  • cationic lipid and ionizable lipid are interchangeable herein.
  • composition wherein said ionizable lipidoid is a compound of formula (b- V) and preferably: a) is an R isomer of the compound of formula (b-V), and/or b) is present at a molar ratio of about 22 mol% to about 65 mol%, preferably about 34 mol% to about 52 mol%, more preferably about 36 mol% to about 50 mol%, and most preferably about 43.1 mol%.
  • the LiNP of the pharmaceutical composition may comprise one or more helper lipid(s) as described in the following.
  • the herein described agents and reagents for delivering and/or introducing the mRNA into a target cell or a target tissue and the herein described lipids and lipidoids may be combined with one or more (e.g., two, three or four) further lipid(s) (like, for example, cholesterol, DPPC, DOPE and/or PEG-lipids (e.g. DMPE-PEG, DMG-PEG2000)).
  • helper lipids may support the desired function of the therapeutic agents and the lipidoids (support and/or increase the delivery and/or introduction of RNA into the cell or tissue and improve transfection efficiency, respectively) and function as respective “helper lipids”.
  • helper lipids are cholesterol, DPPC, DOPE and/or PEG-lipids (e.g., DMPE-PEG, DMG-PEG (e.g., DMG-PEG2000).
  • the further lipids e.g., “helper lipids” may also be part(s) of the herein disclosed complexes/particles. The skilled person is readily in the position to prepare complexes/particles in accordance with the invention.
  • helper lipids examples include a) to c): a) a phospholipid; b) a sterol; and/or c) a stealth lipid.
  • Suitable phospholipids are known in the art. Examples of such phospholipids include, inter alia, dipalmitoylphosphatidylcholine (DPPC), DMPC, DSPC, or DOPC. In the context of the present invention DPPC is a preferred phospholipid. Suitable sterols are known in the art. An exemplary, yet preferred sterol is cholesterol. Suitable stealth lipids are known in the art. Examples of such stealth lipids include, inter alia, DMG-PEG2000 and N-TETAMINE- pSar25. In the context of the present invention DMG-PEG2000 is a preferred stealth lipid.
  • helper lipids e.g., “helper lipids”
  • ratios of the cationic lipidoid(s) and the further lipids e.g. “helper lipids”.
  • ratios may be molar ratios of [1-4 : 1-5], [3-4 : 4-6], [about 4 : about 5], [about 4 : about 5.3] of cationic lipidoid(s) : further lipid(s), (the narrower ranges are preferred).
  • the cationic lipidoid may be combined with three further lipids, like DPPC, cholesterol, and DMG-PEG2000, preferably at a molar ratio of ⁇ 8.0 : ⁇ 5.3 : ⁇ 4.4 : ⁇ 0.9, respectively, or, more particularly, 8.00 : 5.29 : 4.41 : 0.88, respectively.
  • the lipidoids according to formula (b-l), (b-lb), (b- II), (b-V), (b-VI) and (b-VII) are as described above and used with helper lipids DPPC and cholesterol and PEG-lipid DMG-PEG2000 at the molar ratios 8.00:5.29:4.41:0.88 for formulating lipidoid nanoparticles.
  • the ionizable lipidoid e.g., the lipidoid according to formula b-V
  • the phospholipid lipid (e.g., DPPC) represents between 10% and 60%, more preferably between 13% and 53% of the total lipid content of the herein provided compositions.
  • sterol represents between 10% and 30%, more preferably between 12% and 29% of the total lipid content of the herein provided compositions.
  • the stealth lipid represents between 1 % and 10%, more preferably between 2% and 9% of the total lipid content of the herein provided compositions.
  • percentages of the total lipid content indicated in weight% i.e., for example 25% ionizable lipidoid means that said ionizable lipidoid accounts for 25% of the weight of a given composition).
  • the N/P ratio of the herein provided compositions or of the total lipids comprised in the herein provided compositions is between 4 and 18, more preferably between 6 and 16, most preferably 8.
  • a herein provided composition comprises about 43.1% of the (R)- enantiomer of the ionizable lipidoid of formula (b-V), about 28.5% DPPC, about 23.7% cholesterol, and about 4.7% DMG-PEG2000, with an N/P ratio of about 8.
  • a herein provided composition comprises about 34% of the (R)- enantiomer of the ionizable lipidoid of formula (b-V), about 43% DPPC, about 19% cholesterol, and about 4% DMG-PEG2000, with an N/P ratio of about 8.
  • a herein provided composition comprises about 50% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 33% DPPC, about 12% cholesterol, and about 5% DMG-PEG2000, with an N/P ratio of about 8.
  • a herein provided composition comprises about 44.4% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 29.3% DPPC, about 24.2% cholesterol, and about 2% DMG-PEG2000, with an N/P ratio of about 8.
  • a herein provided composition comprises about 25.6% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 52.3% DPPC, about 14% cholesterol, and about 8.1% DMG-PEG2000, with an N/P ratio of about 8.
  • a herein provided composition comprises about 51.5% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 13.9% DPPC, about 28.7% cholesterol, and about 5.9% DMG-PEG2000, with an N/P ratio of about 16.
  • a herein provided composition comprises about 44.4% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 29.3% DPPC, about 24.2% cholesterol, and about 2% DMG-PEG2000, with an N/P ratio of about 16.
  • a herein provided composition comprises about 22.6% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 39.2% DPPC, about 32.2% cholesterol, and about 6% DMG-PEG2000, with an N/P ratio of about 6.
  • a herein provided composition comprises about 42% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 39.2% DPPC, about 32.2% cholesterol, and about 7% DMG-PEG2000, with an N/P ratio of about 6.
  • a herein provided composition comprises about 50% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 24% DMPC, about 19% cholesterol, and about 7% N-TETAMINE-pSar25, with an N/P ratio of about 8.
  • a herein provided composition comprises about 36% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 45% DMPC, about 12% cholesterol, and about 7% N-TETAMINE-pSar25, with an N/P ratio of about 8.
  • a herein provided composition comprises about 36% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 45% DMPC, about 12% cholesterol, and about 7% DMG-PEG2000, with an N/P ratio of about 8.
  • a herein provided composition comprises about 50% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 24% DMPC, about 19% cholesterol, and about 7% N-TETAMINE-pSar25, with an N/P ratio of about 8.
  • a herein provided composition comprises about 21.6% of the ionizable lipidoid of formula (b-IX), about 21.6% of the ionizable lipidoid of formula (b-X), about 28.5% DMPC, about 23.7% cholesterol, and about 4.7% DMG-PEG2000, with an N/P ratio of about 8.
  • a herein provided composition comprises about 43.1% of the ionizable lipidoid of formula (b-IX), about 28.5% DPPC, about 23.7% cholesterol, and about 4.7% DMG-PEG2000, with an N/P ratio of about 8.
  • composition about 43.1% of the ionizable lipidoid of formula (b-XI), about 28.5% DPPC, about 23.7% cholesterol, and about 4.7% DMG-PEG2000, with an N/P ratio of about 8.
  • a herein provided composition comprises about 43.1% of the ionizable lipidoid of formula (b-XII), about 28.5% DPPC, about 23.7% cholesterol, and about 4.7% DMG-PEG2000, with an N/P ratio of about 8.
  • any other specification e.g., relating to the therapeutic or active agent detailed herein above or below may apply.
  • a/an (ionizable) lipidoid or a/an (ionizable) lipid comprised in a (pharmaceutical or cosmetic) composition in accordance with the present invention is either present as an (R)-enantiomer or as an (L)-enantiomer, the (R)- enantiomer is preferred.
  • the herein employed examples employ (R)-enantiomers of the respective ionizable lipidoids or ionizable lipids.
  • the present invention also envisages combinations (i.e., racemic mixes) of (R)-enantiomers and (L)-enantiomers.
  • the terms “(R)-enantiomer”, “R- isomer”, or the like may be used interchangeably.
  • the mRNA vaccine, or the pharmaceutical composition according to the invention comprises a LiNP comprising the following components: a) a mRNA according to the invention, b) a cationic lipidoid of formula (b-l), (b-l I), (b-lb) (b-V), (b-VI), (b-VII) or (b-VIII), and c) one or more helper lipid(s), optionally selected from: c1) DPPC, and/or c2) cholesterol, and/or c3) PEG-lipid DMG-PEG2000, optionally, components b), and c1-c3), are present, optionally component b) and c1 )-c3) are at the molar ratios of about 8.0: about 5.3: about 4.4: about 0.9, respectively, optionally, the LNP comprises a triblock copolymer which contains one polypropylene oxide) block and two poly(ethylene oxide) blocks as component (p) as defined above in vehicles.
  • compositions in accordance with the present invention may preferably comprise: a) one or more therapeutic agent(s) and/or one or more active agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein the one or more (therapeutic and/or active) agent(s) may be defined as anywhere herein above or below, wherein the carrier may be defined as anywhere herein above or below.
  • said ionizable lipid and/or said ionizable lipidoid may be defined as anywhere herein above or below.
  • said one or more helper lipid(s) may be as defined anywhere herein above or below.
  • said one or more pharmaceutically acceptable excipient(s) or diluent(s) may be defined as anywhere herein above or below.
  • composition in which the R-isomer of formula (b-V), i.e., formula (b-VI) is formulated with the lipids DPPC and cholesterol and PEG-lipid DMG-PEG2000 at the molar ratios 8.00 : 5.29 : 4.41 :0.88 is also referred herein as “Formulation I” or LF92.
  • a composition in which the lipidoid of formula (b-VI I) is formulated with the lipids DPPC and cholesterol and PEG-lipid DMG- PEG2000 at the molar ratios 8.00 : 5.29 : 4.41 : 0.88 is also referred herein as “Formulation II”.
  • the LiNPs in the pharmaceutical composition of the invention comprises Formulation I and/or Formulation II.
  • the LiNP comprises Formulation I and/or Formulation II.
  • the cationic lipidoid to mRNA ratios in the LiNP is controlled in terms of the mole ratio of nitrogen atoms of the cationic lipidoid (N) to phosphate groups in the mRNA (P) (N/P ratio).
  • the other lipid components are calculated according to the target molar lipid proportions relative to the cationic lipidoid as discussed above, and may be for example 8.00 : 5.29 : 4.41 : 0.88 for cationic lipidoid, DPPC, cholesterol and PEG-lipid DMG-PEG2000, respectively.
  • the final N/P ratio of a cationic lipidoid having formula (b-l), (b-ll), (b-lb), (b-V), (b-VI), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), and/or (b-XII) to one phosphate group of mRNA molecule is preferably 4 to 44, preferably 4 to 16, more preferably 8 nitrogen atoms of a cationic lipidoid having formula (b-l), (b-lb), (b-V), (b-VI), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), and/or (b-XII) per one phosphate group of the mRNA molecule.
  • the lipid or lipidoid nanoparticles contained in the suspension formulation and in the aerosol in accordance with the invention preferably have a Z-average diameter in the range of 10 to 500 nm, more preferably in the range of 10 to 250 nm, still more preferably 20 to 200 nm.
  • the indicated particle diameter is the hydrodynamic diameter of the particles, as determined by dynamic light scattering (DLS). Measurements are generally carried out at 25 °C.
  • the polydispersity index of the nanoparticles contained in the suspension formulation and in the aerosol in accordance with the invention is preferably in the range of 0.05 to 0.4, more preferably in the range of 0.05 to 0.2.
  • the polydispersity index can be determined by dynamic light scattering (DLS). Measurements are generally carried out at 25 °C
  • the compositions comprise a pharmaceutically acceptable carrier and/or an adjuvant.
  • the adjuvant can be alum, Freund’s complete adjuvant, a biological adjuvant or immunostimulatory oligonucleotides (such as CpG oligonucleotides).
  • compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions, and additional pharmaceutical agents are conventional.
  • Remington describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions, and additional pharmaceutical agents.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like
  • solid compositions for example, powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • an mRNA vaccine of the invention is administered intramuscularly.
  • an mRNA vaccine of the invention is administered intramuscularly, intradermally, subcutaneously by needle or by gene gun, or electroporation.
  • an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention is administered via the respiratory system.
  • the administration is in a form which allows administration to the respiratory system via inhalation, nebulization, via a spray or droplets, e.g., a nasal spray or nasal droplets.
  • the pharmaceutical composition may comprise a vehicle solution and/or a pharmaceutical acceptable carrier.
  • vehicle solution and/or the pharmaceutically acceptable carriers may include, but are not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the carrier and composition can be sterile, and the formulation suits the mode of administration.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. Any of the common pharmaceutical carriers, such as sterile saline solution or sesame oil, can be used.
  • the medium can also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like.
  • Other media that can be used with the compositions and methods provided herein are normal saline and sesame oil.
  • compositions generally comprise a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s) (i.e., also referred herein as "pharmaceutical acceptable carrier”.
  • the carrier may optionally comprise one or more pharmaceutical acceptable carrier(s).
  • the carrier in the present invention may be a lipid nanoparticle (LNP), a lipidoid nanoparticle (LiNP), a liposome, a micelle, an emulsion, a Nanostructured Lipid Carrier (NLCs), or a Lipid-Drug Conjugate (LDC), preferably an LNP or an LiNP.
  • said LNP may comprise one or more pharmaceutically acceptable excipient(s) or diluent(s).
  • the carrier i.e., for example the LNP or LiNP
  • the carrier may further be comprised in one or more pharmaceutically acceptable excipient(s) or diluent(s), which may be defined as anywhere herein above or below.
  • Such pharmaceutically acceptable excipient(s) or diluent(s) may also be herein referred to as “vehicle solution” or as “pharmaceutically acceptable carrier”.
  • vehicle solution or as “pharmaceutically acceptable carrier”.
  • said vehicle is any solution in a pharmaceutical composition in which an LNP or LiNP may be suspended.
  • the vehicle solution and/or the pharmaceutically acceptable carrier may comprise a triblock copolymer which contains one polypropylene oxide) block and two poly(ethylene oxide) blocks.
  • the triblock copolymer is an A-B-A triblock copolymer which contains one polypropylene oxide) block B of formula (p-1): wherein s is an integer of 15 to 67, preferably 20 to 40, and two polyethylene oxides) blocks A of formula (p-2): wherein r is, independently for each block, an integer of 2 to 130, preferably 50 to 100, and more preferably 60 to 90.
  • the triblock copolymer has the following structure: wherein r and t are independently of each other integers of 2 to 130, preferably 50 to 100, and more preferably 60 to 90, and s is an integer of 15 to 67, preferably 20 to 40. Most preferably, Poloxamer P188 is used as the triblock copolymer.
  • the vehicle solution and/or carrier may comprise the triblock copolymer dissolved therein.
  • the composition for intramuscular administration or for aerosol formation comprises the triblock copolymer at a concentration of 0.05 to 5 % w/v (i.e. gram per 100 mL) preferably 0.1 to 2 %, based on the total volume of the composition.
  • the vehicle solution further comprises at least one of sucrose and NaCI, more preferably sucrose and NaCI.
  • the pharmaceutical formulation in accordance with the invention can be conveniently prepared e.g. by a method including adding the triblock copolymer to a suspension comprising a vehicle solution and the lipid or lipidoid nanoparticles, or including adding the lipid or lipidoid nanoparticles to a vehicle solution comprising the triblock copolymer.
  • the (therapeutic or active) agent or carrier may be encapsulated within/comprised in a hydrogel or a biocompatible matrix.
  • the herein provided LiNP/LNP may be encapsulated within/comprised in a hydrogel or a biocompatible matrix.
  • the herein provided (pharmaceutical or cosmetic) compositions are particularly useful as they result in the local retention of the (therapeutic or active) agents comprised therein. Accordingly, in particular in the context of the herein provided pharmaceutical compositions this may be advantageous to e.g., cause expression of a therapeutically relevant mRNA at the local site of administration, and thereby for example reduced accumulation thereof in undesired off-target tissues or organs, such as the liver. Accordingly, in some embodiments the present invention provides for the means and methods for vaccination/immunization (using the herein provided composition).
  • the present invention provides methods for the generation of systemic immunization through localized expression.
  • the invention provides a mRNA vaccine comprising the cameras described above.
  • the present invention provides for a method of immunizing a subject which comprises administering to said subject an effective amount of an mRNA vaccine in a pharmaceutical composition, wherein said pharmaceutical composition comprises the composition as defined anywhere herein above or below, preferably immunizing a subject against a pathogen, against a cancer antigen, or a self- antigen.
  • an mRNA used in the invention comprises an RNA sequence of SEQ ID NO:1.
  • RNA or the complement thereof comprising a sequence of SEQ ID NO:1 , or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide identity over its entire length with the RNA sequence of SEQ ID NO:1 and which encodes an amino acid sequence of SEQ ID NO: 2.
  • RNA which encodes an amino acid sequence of SEQ ID NO:1.
  • the RNA comprises the RNA sequence of SEQ ID NO:8, SEQ ID NO: 27, SEQ ID 28, SEQ ID NO:29, or SEQ ID NO:30 or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:8, SEQ ID NO: 27, SEQ ID 28, SEQ ID NO:29, or SEQ ID NO:30 and which encodes an amino acid sequence of SEQ ID NO:1.
  • the RNA comprises an RNA sequence of SEQ ID NO:10, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:10, SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34 and which encodes an amino acid sequence of SEQ ID NO:3.
  • a further aspect of the invention is an isolated RNA which encodes an amino acid sequence of SEQ ID NO: 43.
  • the RNA comprises an RNA sequence of SEQ ID NO: 42, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:43.
  • the mRNA is a modified version of the mRNA comprising modified nucleosides.
  • the one or more modified nucleosides are 5-iodouridine and 5-iodocytidine.
  • at least 50% of the uridines in the ORF have been modified.
  • At least 50% of the uridines in the mRNA have been modified.
  • at least 50% of the uridines in the ORF have been modified to m1 ⁇ .
  • 5 to 50% of the uridine nucleotides are 5-iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine.
  • 5 to 50% of the uridine nucleotides are 2-thiouridine and 5 to 50% of the cytidine nucleotides are 5-methylcytidine.
  • advantageous immunogenic properties for example, increased antibody response and/or increased breadth of immune response
  • mRNA immunogens encoding tethered coronavirus spike protein receptor binding domains.
  • an isolated mRNA encoding a polypeptide comprising an amino acid sequence of a coronavirus spike protein receptor binding domain (RBD) linked at its C-terminal end directly, or by a linker amino acid sequence of up to 10 amino acid residues, to an amino acid sequence of a transmembrane domain.
  • RBD coronavirus spike protein receptor binding domain
  • RNA or mRNA of the invention is a product of in-vitro transcription (IVT).
  • IVT in-vitro transcription
  • nucleic acid, RNA or mRNA of the invention comprises a polyadenylation signal or a (poly(A)) tail downstream of an open reading frame (ORF) encoding the polypeptide.
  • a nucleic acid, RNA or mRNA of the invention comprises one or more modified nucleosides.
  • said modified nucleoside is selected from any of the following: pseudouridine, N1 -methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2- thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4- thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 5-iodo-uridine, 2'- O-methyl uridine, 5-methylcytidine, 5-i
  • the one or more modified nucleosides comprise a 1 -methylpseudouridine (m1 ⁇ ) modification.
  • the one or more modified nucleosides comprise at least one N1 - methylpseudouridine (N1i ) modification.
  • the one or more modified nucleosides are 5-iodouridine and 5-iodocytidine.
  • At least 50% of the uridines in the ORF have been modified.
  • At least 50% of the uridines in the ORF have been modified to m1 ⁇ .
  • 5 to 50% of the uridine nucleotides are 5-iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine.
  • 5 to 50% of the uridine nucleotides are 2-thiouridine and 5 to 50% of the cytidine nucleotides are 5-methylcytidine.
  • an mRNA vaccine vector comprising an mRNA of the invention.
  • nucleic acid, RNA or mRNA vaccine which comprises a nucleic acid, RNA or mRNA of the invention, or a nucleic acid, RNA or mRNA vaccine vector of the invention, encapsulated in a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • composition comprising a nucleic acid, RNA or mRNA of the invention, a nucleic acid, RNA or mRNA vaccine vector of the invention, or a nucleic acid, RNA or mRNA vaccine of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • nucleic acid, RNA or mRNA of the invention nucleic acid, RNA or an mRNA vaccine vector of the invention, a nucleic acid, RNA or mRNA vaccine of the invention, or a pharmaceutical composition of the invention, for use as a medicament.
  • nucleic acid an RNA or an mRNA of the invention, a nucleic acid, an RNA or an mRNA vaccine vector of the invention, a nucleic acid, an RNA or an mRNA vaccine of the invention, or a pharmaceutical composition of the invention, for use in the prevention, treatment, or amelioration of a coronavirus infection.
  • nucleic acid an RNA or an mRNA of the invention
  • nucleic acid an RNA or an mRNA vaccine vector of the invention
  • nucleic acid an RNA or an mRNA vaccine of the invention
  • pharmaceutical composition of the invention in the manufacture of a medicament for the prevention, treatment, or amelioration of a coronavirus infection.
  • a method of inducing an immune response to a coronavirus in a subject which comprises administering to the subject an effective amount of an mRNA of the invention, an mRNA vaccine vector of the invention, an mRNA vaccine of the invention, or a pharmaceutical composition of the invention.
  • a method of immunizing a subject against a coronavirus which comprises administering to the subject an effective amount of a nucleic acid, an RNA or an mRNA of the invention, an mRNA vaccine vector of the invention, a nucleic acid, an RNA or an mRNA vaccine of the invention, or a pharmaceutical composition of the invention.
  • a method of the invention comprises administering to the subject a nucleic acid, an RNA or an mRNA of the invention, a nucleic acid, an RNA or an mRNA vaccine vector of the invention, a nucleic acid, an RNA or an mRNA vaccine of the invention, or a pharmaceutical composition of the invention, as part of a prime boost regimen.
  • the coronavirus is a beta-coronavirus.
  • beta-coronavirus is a lineage B or C beta-coronavirus.
  • beta-coronavirus is a lineage B beta-coronavirus.
  • the lineage B beta-coronavirus is SARS-CoV or SARS-CoV-2.
  • the lineage C beta-coronavirus is MERS-CoV.
  • beta-coronavirus is a variant of concern (VOC).
  • VOC variant of concern
  • beta-coronavirus is a SARS-CoV-2 VOC.
  • beta-coronavirus is a SARS-CoV-2 beta, gamma, delta, or omicron VOC.
  • the subject is a human subject.
  • a method of inducing an immune response to a coronavirus in a subject which comprises administering to the subject an effective amount of an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention.
  • a method of immunizing a subject against a virus which comprises administering to the subject an effective amount of an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention.
  • An effective amount is an amount to produce an antigen-specific immune response in a subject.
  • the method comprises administering an effective amount of an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention to a subject that has previously been seroconverted with an mRNA, a vector, a pharmaceutical composition or a vaccine, coding or comprising a full-length spike protein of a coronavirus.
  • the coronavirus is a Sarbecovirus.
  • the mRNA of the invention, the vector of the invention, the pharmaceutical composition of the invention or the vaccine of the invention comprises or consist of SEQ ID NO: 4 or SEQ ID NO: 7.
  • an mRNA of the invention a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention, for use as a medicament.
  • an mRNA of the invention a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention, for use in the prevention, treatment, or amelioration of a coronavirus infection.
  • an mRNA of the invention a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention, in the manufacture of a medicament for the prevention, treatment, or amelioration of a coronavirus infection, including long covid.
  • the coronavirus is a beta-coronavirus.
  • the beta-coronavirus is a lineage B or C beta-coronavirus.
  • beta-coronavirus is a lineage B beta -coronavirus.
  • the lineage B beta -coronavirus is SARS-CoV or SARS-CoV-2.
  • the lineage C beta -coronavirus is MERS-CoV.
  • an immune response is induced against more than one lineage B beta-coronavirus.
  • an immune response is induced against SARS-1 and SARS-2 beta-coronavirus.
  • an immune response is induced against SARS-1 and MERS beta-coronavirus.
  • an Immune response is induced against SARS-2 and MERS beta-coronavirus.
  • an immune response is induced against SARS-1, SARS-2, and MERS beta- coronavirus.
  • beta-coronavirus is a variant of concern (VOC).
  • beta-coronavirus is a SARS-CoV-2 VOC.
  • beta-coronavirus is a SARS-CoV-2 lineage B1.248 (Brazil P1 lineage) VOC.
  • beta-coronavirus is a SARS-CoV-2 lineage B1.351 (South Africa) VOC.
  • beta-coronavirus is a SARS-CoV-2 beta, gamma, or delta VOC.
  • beta-coronavirus is a SARS-CoV-2 beta VOC.
  • beta-coronavirus is a SARS-CoV-2 gamma VOC.
  • beta-coronavirus is a SARS-CoV-2 delta VOC.
  • beta-coronavirus is a SARS-CoV-2 alpha VOC.
  • beta-coronavirus is a SARS-CoV-2 omicron VOC.
  • beta-coronavirus is SARS-CoV-2 omicron BA.1.
  • the beta-coronavirus is a SARS-CoV-2 omicron BA.2. It can readily be determined whether an immune response has been induced to a beta- coronavirus using methods well-known to the skilled person. For example, a pseudotype neutralization assay as described in the example below may be used.
  • the subject is a human subject.
  • compositions comprise one or more (therapeutic or active) agent(s), and the nature of said agent(s) is not particularly limited.
  • agent(s) may comprise one or more of the following: a growth factor, a peptide, an antioxidant, a retinoid, a cytokine, a siRNA, a miRNA, a mRNA, and an asRNA.
  • said agent(s) is/are: a) an (anionic) therapeutical substance and/or b) a nucleic acid, preferably an RNA, more preferably an mRNA, a miRNA, and/or an siRNA, even more preferably an mRNA, most preferably an mRNA comprising an open reading frame (ORF) encoding one or more polypeptide(s).
  • a nucleic acid is particularly preferred herein, especially an mRNA encoding one or more polypeptide(s).
  • an mRNA comprising an open reading frame (ORF) encoding one or more polypeptide(s) may be used interchangeably herein with the term “an mRNA encoding one or more polypeptide(s)” or the like.
  • Said one or more polypeptide(s) are not particularly limited however may for example be growth factors, Copper Peptides, or Cytokines.
  • Preferred growth factors may for example be Epidermal Growth Factor (EGF) for stimulating skin growth and wound healing or Fibroblast Growth Factors (FGFs) to promote dermal fibroblasts proliferation and enhance skin elasticity.
  • Preferred peptides may be Copper Peptides which may regenerate skin tissue by stimulating collagen production or Palmitoyl Pentapeptide-4 (i.e., Matrixyl) to reduce wrinkles and improve skin texture.
  • Cytokines may preferably be Interleukins specifically modified to regulate inflammatory responses in the skin.
  • said one or more polypeptide(s) may comprise a reporter polypeptide.
  • Reporter polypeptides are not particularly limited, are well known in the art, and include for example luminescent or fluorescent proteins, such as, luciferase or green fluorescent protein (GFP), a preferred reporter polypeptide in the context of the present invention is luciferase (as exemplified in SEQ ID NO: 46).
  • the appended examples illustratively show the local retention of a luciferase reporter polypeptide encoded by an mRNA administered in accordance with the present invention.
  • therapeutically active substances such as one or more therapeutic polypeptides or such as mRNA encoding one or more therapeutic polypeptides
  • therapeutically active substances may be administered with such reporter polypeptides or with mRNA encoding such reporter polypeptides.
  • fusion polypeptides comprising a therapeutic polypeptide (covalently) coupled to a reporter polypeptide or mRNAs encoding are fusion polypeptides comprising a therapeutic polypeptide (covalently) coupled to a reporter polypeptide.
  • Means and method for the fusion/coupling/linkage of two polypeptides or coding regions encoding the same are well known in the art.
  • Such reporter peptides/proteins may particularly be useful in the herein described (in vitro I ex vivo) method of detecting/assessing/determining whether a “composition remains localized at the site of administration” and/or if a “composition essentially does not exhibit systemic distribution throughout the patient's body”, or if a “composition has a prolonged retention at the site of administration”, and the like.
  • Therapeutical substances may also be selected from antioxidants or retinoids.
  • antioxidants can preferably be selected from Vitamin C (Ascorbic acid) for photo-protection, pigmentation reduction, and collagen stimulation and Coenzyme Q10 to prevent skin aging by reducing oxidative stress.
  • An exemplary retinoid may be Retinol for accelerating skin renewal, enhancing collagen production, and reducing signs of aging.
  • such (therapeutic or cosmetic) agents are preferably nucleic acids, preferably an antisense RNA (asRNA), a non-coding RNA or a mRNA, preferably a small interfering RNA (siRNA), a microRNA (miRNA), a mRNA, most preferably an mRNA.
  • asRNA antisense RNA
  • mRNA small interfering RNA
  • miRNA microRNA
  • mRNA microRNA
  • mRNA most preferably an mRNA.
  • Said siRNA may preferably be a siRNA targeting specific mRNA involved in melanin synthesis (e.g., targeting TYR gene for skin lightening) or an siRNA targeting MMP (Matrix Metalloproteinase) mRNA to decrease collagen breakdown and reduce wrinkles.
  • siRNA targeting specific mRNA involved in melanin synthesis e.g., targeting TYR gene for skin lightening
  • MMP Microx Metalloproteinase
  • Said miRNA may preferably a miRNA-145 inhibitor (i.e., a miRNA targeting miRNA-145) to enhance collagen production by dermal fibroblasts or inhibit hypertrophic scar formation or a miRNA or a miRNA mimic that could potentially downregulate genes responsible for inflammation and aging.
  • a miRNA-145 inhibitor i.e., a miRNA targeting miRNA-145
  • a miRNA targeting miRNA-145 to enhance collagen production by dermal fibroblasts or inhibit hypertrophic scar formation
  • a miRNA or a miRNA mimic could potentially downregulate genes responsible for inflammation and aging.
  • Said asRNA may preferably comprise sequences that can inhibit the expression of genes involved in undesirable skin conditions like hyperpigmentation or excessive hair growth.
  • Said mRNA may preferably be an mRNA encoding antioxidants or growth factors that can be produced endogenously at the site of application to enhance skin appearance and health.
  • Said (one or more) mRNA (molecules) may also comprise an ORF encoding a protein, specifically a therapeutic protein, such as CFTR, Erythropoietin (EPO), Factor VIII, Factor IX, Chimeric Antigen Receptor (CAR) T-cell, Survivin (BIRC5) or a dominant-negative form thereof, P53, Vascular Endothelial Growth Factor (VEGF), Insulin, SARS-CoV-2 Spike protein, Alpha-synuclein, Dystrophin, Glucocerebrosidase (GCase), a cytokine such as lnterleukin-2 (IL-2), Interleukin-10 (IL-10), Interleukin-12 (IL-12), an interferon (including, but not limited to interferon-alpha (IFN-a
  • the above protein is a human protein.
  • CFTR may be human CFTR
  • EPO Erythropoietin
  • Factor VIII may be human Factor VIII, and so on.
  • Exemplary sequences of an mRNA (molecules) comprising an ORF encoding human interferon lambda 1 (hlFN ⁇ 1) are shown in any one of SEQ ID NOs 41 , 42 or 43.
  • SEQ ID NO. 42 is preferred herein.
  • said one or more mRNA(s) may comprise an ORF encoding human interferon lambda 1 (hlFN ⁇ 1), preferably wherein said ORF comprises a nucleic acid sequence according to SEQ ID NO: 41 ,42 or 43 or a variant thereof having about 90%/91%/92%/93%/94%/95%/96%/97%/98%/99%/100% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 ,42 and 43, respectively and wherein said variant sequence encodes functional hlFN ⁇ 1 .
  • ORF human interferon lambda 1
  • mRNA comprising an ORF encoding interferon, specifically interferon lambda, more specifically interferon lambda 1, most preferably human interferon lambda 1 (hlFN ⁇ 1 ) may be for use in the treatment or prevention of diseases or disorders, such as a viral-induced or virus-associated disorder, in particular, a viral-induced or virus-associated respiratory disorder.
  • the disease may be Chronic Obstructive Pulmonary Disease (COPD), and/or asthma.
  • COPD Chronic Obstructive Pulmonary Disease
  • the disease may be a respiratory virus infection, e.g. a seasonal and/or emerging virus infection and/or virus-driven or virus-associated exacerbation of chronic respiratory diseases, such as virus-driven or virus-associated exacerbation of asthma or COPD.
  • the virus which causes and/or is linked to said viral-induced or virus-associated respiratory disorder is selected from the group consisting of an enterovirus (such as a rhinovirus), influenza virus, parainfluenza virus, metapneumo virus, respiratory syncytial virus, adenovirus, and coronavirus.
  • the virus which causes said viral-induced respiratory disorder may be a virus which enters cells via the ACE2 receptor and may be SARS- CoV, SARS-CoV-2 or HCoV- NL63.
  • the mRNA encoding interferon described above may be used in a method of prevention of (e.g., virus-induced) rhinitis.
  • the mRNA encoding interferon has described above may be used in a method of prevention of virus infections that can reduce risk of (severe and potentially life- threatening) infections in, inter alia, immunocompromised and immunosuppressed (e.g. post- transplantation) subjects.
  • the mRNA encoding interferon may also be used in a method of prevention or mitigation of respiratory virus infections in subjects with genetic lung diseases such as PCD.
  • Preferred dosages for the administration of the nucleic acid, preferably (m)RNA is any amount between 0.01 mg and up to 60 mg of nucleic acid (mRNA), preferably between 0.05 and 30 mg.
  • a more preferred inhaled amount is between 1 mg and 20 mg, more preferably between 10 mg and 20 mg.
  • a preferred dose might be between 0.001 mg to 30 mg, more preferably between 0.01 mg to 20 mg, even more preferably between 0.05 mg to 10 mg, even more preferably between 0.05 and 5 mg, even more preferably between 0.05 and 2 mg.
  • a dose can be selected from about 0.001 mg to about 6 mg of nucleic acid, more preferably about 0.01 mg to about 2.8 mg, more preferably between 0.25 mg to 1 mg.
  • a preferred dose might be between 0.001 mg to 10 mg, more preferably between 0.01 mg to 5 mg, even more preferably between 0.05 mg to 1 mg, even more preferably between 0.1 and 1 mg.
  • Preferred dosages regimes for any route of administration include once a week, twice a week or three times a week.
  • the administration can also be a chronic administration once to three times a week, preferably once or twice a week.
  • the mRNA is to be locally administered, in particular by delivery/local administration into the respiratory system, preferably wherein said delivery/local administration into the respiratory system is inhalation and/or (through) a nasal spray or aerosol.
  • the inhalation may be Inhalation of an aerosol comprising the mRNA.
  • the present invention provides for a composition for use in the treatment and/or prevention of a disease or disorder, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises:
  • a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
  • the patient has less side effects (such as a reduced complement activation and/or reduced risk of CARPA) compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
  • composition has a prolonged retention at the site of administration
  • said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration; and/or said composition, when administered to said site of administration, remains localized and essentially does not exhibit systemic distribution throughout the patient's body.
  • the present invention provides for a composition for use in the treatment and/or prevention of a disease or disorder, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipidoid; ii.one or more helper lipid(s); and iii.
  • a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
  • the patient has less side effects (such as reduced complement activation and/or a reduced risk of CARPA) compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
  • composition has a prolonged retention at the site of administration
  • said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration;
  • the present invention provides for a composition for use in the treatment and/or prevention of a disease or disorder, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipidoid; ii.one or more helper lipid(s); and iii.
  • a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
  • the patient has less side effects (such as reduced complement activation and/or a reduced risk of CARPA) compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
  • composition has a prolonged retention at the site of administration
  • the present invention provides for a composition for use in the treatment and/or prevention of a disease or disorder, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipidoid; ii.one or more helper lipid(s); and iii.
  • a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
  • the patient has less side effects (such as reduced complement activation and/or a reduced risk of CARPA) compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
  • composition has a prolonged retention at the site of administration
  • said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration; and/or said composition, when administered to said site of administration, remains localized and essentially does not exhibit systemic distribution throughout the patient's body.
  • a nucleic acid, an RNA or mRNA of the invention may also be provided as part of a nucleic acid, an RNA or mRNA vaccine.
  • mRNA vaccines are a new form of vaccine (recently reviewed in Pardi et al., Nature Reviews Drug Discovery Volume 17, pages 261-279(2018); Wang etal., Molecular Cancer (2021) 20:33: mRNA vaccine: a potential therapeutic strategy).
  • the first mRNA vaccines to be approved for use were BNT162b2 (manufactured by Pfizer) and mRNA-1273 (manufactured by Moderna) during the COVID-19 pandemic.
  • mRNA vaccines have a unique feature of temporarily promoting the expression of antigen (typically days). The expression of the exogenous antigen is controlled by the lifetime of encoding mRNA, which is regulated by cellular degradation pathways.
  • mRNA-based vaccines trigger an immune response after the synthetic mRNA which encodes viral antigens transfects human cells.
  • the cytosolic mRNA molecules are then translated by the host’s own cellular machinery into specific viral antigens. These antigens may then be presented on the cell surface where they can be recognized by immune cells, triggering an immune response.
  • the structural elements of a vaccine vector mRNA molecule are similar to those of natural mRNA, comprising a 5’ cap, 5’ untranslated region (UTR), coding region (for example, comprising an open reading frame encoding a polypeptide of the invention), 3’ UTR, and a poly(A) tail.
  • the 5’ UTR also known as a leader sequence, transcript leader, or leader RNA
  • the 5' UTR forms complex secondary structure to regulate translation.
  • the 5' UTR begins at the transcription start site and ends one nucleotide (nt) before the initiation sequence (usually AUG) of the coding region.
  • nt nucleotide
  • AUG initiation sequence
  • the eukaryotic 5' UTR may contain a Kozak consensus sequence (ACCAUG (initiation codon underlined), which contains the initiation codon AUG.
  • An elongated Kozak sequence may be used: GCCACCAUG (initiation codon underlined).
  • a 5’IITR of an mRNA of the invention may comprise, with an initiation codon of the mRNA, a Kozak consensus sequence, or an elongated Kozak sequence.
  • a 5’UTR of an mRNA of the invention comprises immediately upstream of an initiation codon sequence anyone of the following sequences: GGGAGACGCCACC (SEQ ID NO:11), or GGGAGACUGCCACC (SEQ ID NO:14).
  • a 5’UTR of an mRNA of the invention comprises immediately upstream of an initiation codon sequence a T7, T3, SP6, or K11 polymerase binding domain, a minimal UTR and a Kozak sequence as follows: GGGAGACGCCACC (SEQ ID NO:11), GG GACGCCACC (SEQ ID NO: 12), GGGACGCCACC (SEQ ID NO:13), GGGAGACUGCCACC (SEQ ID NO: 14), GAAGCTGCCACC (SEQ ID NO:15), or GG GACTGCCACC (SEQ ID NO:16).
  • a 5' cap structure is required for efficient protein production from mRNA.
  • Various versions of 5' caps can be added during or after the transcription reaction using a vaccinia virus capping enzyme, or by incorporating synthetic cap or anti-reverse cap analogues (see Pardi et al., supra).
  • Anti-Reverse Cap Analog is a cap analog used during in vitro transcription for the generation of capped transcripts.
  • ARCA is modified in a way that ensures incorporation in the forward orientation only.
  • Anti-Reverse Cap Analog (ARCA) is a modified cap analog in which the 3' OH group (closer to m 7 G) is replaced with -OCH3:
  • the 3’ UTR may comprise a sequence for generation of a restriction site when in a vector, such as GAAUU.
  • a 3’ UTR that may be used is 3' UTR of CYBA (CCUCGCCCCGGACCUGCCCUCCCGCCAGGUGCACCCACCUGCAAUAAAUGCAGCGA AGCCGGGA, SEQ ID NO:26.
  • poly(A) tail also plays an important regulatory role in mRNA translation and stability; thus, an optimal length of poly(A) must be added to mRNA either directly from the encoding DNA template, by using poly(A) polymerase (see Pardi et al., supra) or ligation after in-vitro transcription.
  • the poly(A) may have a length of 90 A nucleotides (Ago) or more, 100 A nucleotides (A 100 ) or more, 110 A nucleotides (A 110 ) or more, 120 A nucleotides (A 120 ) or more, 130 A nucleotides (A 130 ) or more, 150 A nucleotides (A 150 ) or more, 180 A nucleotides (A 180 ) or more, 190 A nucleotides (A 190 ) or more.
  • An example of a suitable length of poly(A) tail is poly( ⁇ A 120 ).
  • the poly(A) tail may be a segmented poly(A) tail, as disclosed in WO 2020074642 A1 , which is herein incorporated by reference.
  • the segmented poly(A) may have the structure A 55-65 -S- A 55-65 wherein S is a single nucleotide selected from C, G, T or U.
  • the poly(A) have the structure: A 55-65 -N-S4-N-A 55-65 , wherein N is a nucleotide that is not adenine, and wherein S4 are four nucleotides selected from A, C, G, T or U.
  • the segmented poly(A) is a poly(A) of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40).
  • the codon usage additionally has an impact on protein translation. Replacing rare codons with frequently used synonymous codons that have abundant cognate tRNA in the cytosol is a common practice to increase protein production from mRNA. Enrichment of G:C content constitutes another form of sequence optimization that has been shown to increase steady- state mRNA levels in vitro and protein expression in vivo (see Pardi et al., supra).
  • RNA vaccines Two major types of RNA are currently studied as vaccines: non-replicating mRNA and virally derived, self-amplifying RNA. While both types of vaccines share a common structure in mRNA constructs, self-amplifying RNA vaccines contain additional sequences in the coding region for RNA replication, including RNA-dependent RNA polymerases.
  • BNT162b2 vaccine construct comprises a lipid nanoparticle (LNP) encapsulated mRNA molecule encoding trimerised full-length SARS2 S protein with a PP mutation (at residue positions 986-987).
  • the mRNA is encapsulated in 80 nm ionizable cationic lipid nanoparticles.
  • mRNA-1273 vaccine construct is also based on an LNP vector, but the synthetic mRNA encapsulated within the lipid construct encodes the full-length SARS2 S protein.
  • US Patent No. 10,702,600 B1 (ModernaTX) describes betacoronavirus mRNA vaccines, including suitable LNPs for use in such vaccines.
  • An mRNA vaccine of the invention may be formulated in a lipid nanoparticle.
  • mRNA vaccines have several advantages in comparison with conventional vaccines containing inactivated (or live attenuated) disease-causing organisms. Firstly, mRNA-based vaccines can be rapidly developed due to design flexibility and the ability of the constructs to mimic antigen structure and expression as seen in the course of a natural infection. mRNA vaccines can be developed within days or months based on sequencing information from a target virus, while conventional vaccines often take years and require a deep understanding of the target virus to make the vaccine effective and safe.
  • these novel vaccines can be rapidly produced. Due to high yields from in vitro transcription reactions, mRNA production can be rapid, inexpensive, and scalable. Thirdly, vaccine risks are low. mRNA does not contain infectious viral elements that pose risks for infection and insertional mutagenesis. Anti-vector immunity is also avoided as mRNA is the minimally immunogenic genetic vector, allowing repeated administration of the vaccine.
  • the challenge for effective application of mRNA vaccines lies in cytosolic delivery. mRNA isolates are rapidly degraded by extracellular RNases and cannot penetrate cell membranes to be transcribed in the cytosol. However, efficient in vivo delivery can be achieved by formulating mRNA into carrier molecules, allowing rapid uptake and expression in the cytoplasm.
  • lipid-, polymer-, or peptide-based delivery virus-like replicon particle, cationic nanoemulsion, naked mRNAs, and dendritic cell-based delivery (each reviewed in Wang et al., supra), cationic lipid nanoparticle (LNP) delivery is the most appealing and commonly used mRNA vaccine delivery tool.
  • LNP cationic lipid nanoparticle
  • Exogenous mRNA may be highly immunostimulatory.
  • Single-stranded RNA (ssRNA) molecules are considered a pathogen associated molecular pattern (PAMP), and are recognized by various Toll-like receptors (TLR) which elicit a pro-inflammatory reaction.
  • TLR Toll-like receptors
  • ssRNA Single-stranded RNA
  • PAMP pathogen associated molecular pattern
  • TLR Toll-like receptors
  • the U-rich sequence of mRNA is a key element to activate TLR (Wang et al., supra).
  • enzymatically synthesized mRNA preparations contain double stranded RNA (dsRNA) contaminants as aberrant products of the in vitro transcription (IVT) process.
  • dsRNA double stranded RNA
  • dsRNA is a potent PAMP, and elicits downstream reactions resulting in the inhibition of translation and the degradation of cellular mRNA and ribosomal RNA (Pardi et al., supra).
  • the mRNA may suppress antigen expression and thus reduce vaccine efficacy.
  • RNA modification may, in the context of the present invention apply to the herein above detailed mRNA vaccines and to any other RNA to be employed as or to be comprised in (therapeutic or active) agent in the context of the herein provided composition.
  • nucleoside chemical modifications include, but are not limited to, 5-methylcytidine (m5C), 5-methyluridine (m5U), N1 -methyladenosine (m1A), N6- methyladenosine (m6A), 2- thiouridine (s2U), and 5-methoxyuridine (5moU) (Wang et al., supra).
  • An RNA of the invention may comprise an mRNA.
  • An mRNA of the invention, a pharmaceutical composition, or a vector of the invention may be provided as part of an mRNA vaccine.
  • a Vector of the invention may comprise the corresponding DNA sequence encoding for the peptide (s) and or protein(s) of interest and optionally immediately upstream of an initiation codon sequence anyone of the following sequences: TAATACGACTCACTATA GGGAGACGCCACC (SEQ ID NO: 17), AATTAACCCTCACTAAA GGGAGACGCCACC (SEQ ID NO: 18), ATTTAGGTGACACTATA GAAGCGCCACC (SEQ ID NO: 19), AATTAGGGCACACTATA GGGACGCCACC (SEQ ID NO:20), TAATACGACTCACTATA GGGAGA CTGCCACC (SEQ ID NO:21), AATTAACCCTCACTAAAGGGAGA CTGCCACC (SEQ ID NO:22), ATTTAGGTGACACTATAGAAG CTGCCACC (SEQ ID NO:23), AATTAGGGCACACTATAGGGA CTGCCACC (SEQ ID NO:24), or CGCCUAGCAGUGUCCCAGCCGGGUUCGUGUCGCC (S
  • the above sequences may be placed upstream of the ATG of any of the mRNA sequences of the invention, including the full-length spike (SEQ ID NO:1).
  • An mRNA, a pharmaceutical composition, a vector, or a vaccine, of the invention may comprise one or more modified nucleosides.
  • the one or more modified nucleosides may be present in an RNA or mRNA of the invention, or in mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention.
  • at least one chemical modification is selected from pseudouridine, N1- methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2- thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine,
  • the chemical modification is in the 5-position of the uracil. In some embodiments, the chemical modification is an N1 -methylpseudouridine. In some embodiments, the chemical modification is an N1 -ethylpseudouridine.
  • an RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention may comprise one or more of the following modified nucleosides: pseudouridine (ip); N1 -methylpseudouridine ( m1 ⁇ ); 5-methylcytidine (m5C); 5- methyluridine (m5U); N1 -methyladenosine (m1A); N6-methyladenosine (m6A); 2-thiouridine (s2U); 5-methoxyuridine (5moU); 5-iodouridine; and 5-iodocytidine.ln some embodiments 100% of the uracil of the whole mRNA have a chemical modification.
  • 100% of the uracil in the open reading frame has a chemical modification.
  • a chemical modification is in the 5-position of the uracil.
  • a chemical modification is an N1-methyl pseudouridine.
  • 100% of the uracil of the mRNA have a N1-methyl pseudouridine in the 5-position of the uracil.
  • 100% of the uracil in the open reading frame have a N1 -methyl pseudouridine in the 5-position of the uracil.
  • 5 to 50% of the uridine nucleotides are
  • 5-iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine.
  • 5 to 50% of the uridine nucleotides are 5-iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine.
  • 5 to 50% of the uridine nucleotides are 2-thiouridine and 5 to 50% of the cytidine nucleotides are 5-methylcytidine.
  • RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention may contain from about 1% to about 100% modified nucleotides (or nucleosides) (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide (or nucleoside), i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%,
  • RNA ormRNA ofthe invention, or mRNAof a pharmaceutical composition, a vector, or a vaccine, of the invention comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with each 'U' replaced by m1 ⁇ .
  • RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with at least 50% of the ‘U’s replaced by m1 ⁇ .
  • the remaining ‘U’s may all be unmodified, or may comprise unmodified and one or more other modified nucleosides.
  • RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with at least 70% of the ‘U’s replaced by m.1 T ⁇ he remaining ‘U’s may all be unmodified, or may comprise unmodified and one or more other modified nucleosides.
  • RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with at least 90% of the ‘U’s replaced by m1 ⁇ .
  • the remaining ‘U’s may all be unmodified, or may comprise unmodified and one or more other modified nucleosides.
  • RNA ormRNA ofthe invention, or mRNAof a pharmaceutical composition, a vector, or a vaccine, of the invention comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with 100% of the ‘U’s replaced by m1 ⁇ .
  • mRNA vaccines of the invention may be co-administered with an immunological adjuvant, for example MF59 (Novartis), TriMix, RNActive (CureVac AG), RNAdjuvant (again reviewed in Wang et al., supra).
  • composition may be administered by any means and methods that are (routinely) applied in the art, accordingly, any suitable route of administration may be used.
  • Methods of administration, specifically of local administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, subcutaneous, submucosal, mucosal, vaginal, rectal, intranasal, inhalation or oral.
  • local administration is opposed to “systemic administration”, such as intravenous administration.
  • systemic administration such as intravenous administration.
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Routes for local administration in general include, for example, topical administration routes but also intradermal, transdermal, subcutaneous, submucosal, mucosal, aerosol delivery such as into the respiratory system including intranasal or lung delivery, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, and sublingual injections, preferably mucosal or aerosol delivery.
  • aerosol delivery refers to the (local) administration of a composition via the airway to the nasal cavities and/or the lung, and can thus herein be used interchangeably with nasal and/or “pulmonary drug delivery” or the like.
  • local site of administration refers to a (body) site suitable for local administration, such as, e.g., muscle tissue, the respiratory system (including for example the lung, or the nasal cavity).
  • a local site of administration and as mentioned above, administration to e.g., veins is to be understood as a form of systemic administration in the context of the present invention.
  • the route of administration may be adapted according to the respective needs.
  • administration routes may preferably be topical routes.
  • said cosmetic composition may preferably be formulated for topic application/topic administration.
  • the application of the cosmetic composition is preferably targeted at the skin of a subject, whereas the skin of any body part may be targeted.
  • said cosmetic composition may also be formulated for injection, preferably for intradermal, subcutaneous, submucosal, mucosal or intramuscular injection.
  • Said cosmetic composition may also be comprised in a patch, preferably wherein said patch may be a transdermal patch.
  • a transdermal patch may comprise micro-needles that facilitate the intradermal injection of the composition in accordance with the present invention, accordingly, in the context of transdermal patches the composition may preferably be formulated for injection.
  • administration routes may depend on the disease to be treated and/or prevented.
  • the herein provided pharmaceutical compositions are particularly useful in the treatment and/or prevention of a variety of diseases, accordingly, such diseases are not particularly limited.
  • Such diseases may be selected from: genetic mutations, autoimmune diseases, metabolic imbalances, neurodegenerative disorders, degenerative disorders of the joints, arthrosis, arthritis, bone fractures, non-union fractures, solid tumor diseases (including soft tissue tumors, tumors of the heart, the lungs, the liver, the spleen, the kidneys, the brain, the oral cavity, the intestine, the skin, the pancreas, the prostate gland, the mammary glands, the ovaries, the urinary bladder, the bones (including osteosarcoma, chondrosarcoma, Ewing sarcoma)), tumors of the pleural and the peritoneal cavity, diseases of the respiratory system including rhinitis and lung diseases such as asthma, viral induced asthma, COPD, including lung autoimmune diseases, ciliopathies and pulmonary alveolar proteinosis (PAP), bone fractures or lesions thereof, tendon fractures or lesions thereof, joint infections, ligament ruptures, resistant Staphylococcus Aureus (MRSA) and
  • the disease is a lung disease.
  • the lung disease is a ciliopathy, in particular primary ciliary dyskinesia (PCD).
  • PCD primary ciliary dyskinesia
  • said primary ciliary dyskinesia (PCD) is caused by a mutation in a protein or factor selected from DNAH5, DNAH11 , CCDC39, DNAI1, CCDC40, CCDC103, SPAG1, ZMYND10, ARMC4, CCDC151, DNAI2, RSPH1 , CCDC114, RSPH4A, DNAAF1 (LRRC50), DNAAF2 (KTU), LRRC6, C21orf59, CCDC65 (DRC2), CCNO, DNAAF3, DNAH1 , DNAH8, DNAL1, DRC1 (CCDC164), DYX1C1 , DNAAF5 (HEATR2), HYDIN, MCIDAS, NME8 (TXNDC3), RSPH3, RSPH9, and/or FOXJ1.
  • a ciliopathy or PCD if the disease to be treated is a ciliopathy or PCD, the skilled person is aware of suitable therapeutic agents.
  • Such agents are, inter alia, disclosed in WO 2020/165352, which is herein incorporated by reference in its entirety.
  • Particularly suitable therapeutic agents for the treatment of a ciliopathy or PCD include a nucleic acid (such as an mRNA) encoding coiled- coil domain containing 39 (CCDC39) and/or coiled-coil domain containing 40 (CCDC40).
  • nucleic acids sequences encoding CCDC39 and CCDC40 are exemplified in SEQ ID NO: 47 and 48 for CCDC39 and in SEQ ID NO: 49 and 50 for CCDC40.
  • the herein described local retention at the site of administration/the herein described non- systemic distribution of a pharmaceutical composition is also particularly advantageous in treatment of ciliopathy or PCD as it, inter alia, limits the distribution of the therapeutic agent (such as an mRNA encoding CCDC39 or CCDC40) to the site of administration (preferably the respiratory system, such as the nose, throat, larynx, trachea, bronchi, or lungs).
  • a (pharmaceutical) composition for use in the treatment of a ciliopathy or PCD, the treatment comprising local administration of the composition, preferably local administration to the respiratory system, such as the nose, throat, larynx, trachea, bronchi, or lungs, the composition comprising: a) one or more therapeutic agent(s), wherein said one or more therapeutic agent(s) comprise(s) or consist(s) of a nucleic acid (such as an mRNA) encoding CCDC39 and/or CCDC40, preferably an mRNA comprising the nucleic acid sequence as set forth in any one of SEQ ID NO: 47 to 50; and b) a carrier, wherein said carrier comprises: i.
  • an ionizable lipid and/or an ionizable lipidoid ii. optionally one or more helper lipid(s); and ill. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein one or more of the following apply:
  • a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
  • the patient has less side effects (such as a reduced risk of CARPA) compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
  • composition has a prolonged retention at the site of administration
  • said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration;
  • composition when administered to said site of administration, remains localized and essentially does not exhibit systemic distribution throughout the patient's body.
  • Any further specification or detail (such as components of the carrier or the dosage of the therapeutic agent) may be defined as anywhere herein above or below.
  • the pharmaceutical composition may be administered to one or more solid tissue(s), solid organ(s) and/or solid anatomical region(s), preferably wherein said one or more solid tissue(s), solid organ(s) and/or solid anatomical region(s) are selected from the group consisting of the lungs, the nose, the heart, the brain, the spleen, the lymph nodes, the bones, the tendons, the skeletal muscles, the joints, the stomach, the small intestine, the large intestine, the kidneys, the bladder, the breast, the testes, the ovaries, the uterus, the spleen, the thymus, the brainstem, the cerebellum, the spinal cord, the eye, the ear, the tongue, the skin and/or tumors present in said one or more solid tissue(s), solid organ(s) and/or solid anatomical region(s).
  • compositions may be administered in any suitable manner, such as with pharmaceutically acceptable carriers.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition.
  • Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with Inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • Inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid
  • Administration can be accomplished by single or multiple doses.
  • the dose administered to a subject in the context of the present disclosure should be sufficient to Induce a beneficial therapeutic response in a subject overtime, or to inhibit or prevent infection.
  • the dose required will vary from subject to subject depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the particular composition being used and its mode of administration. An appropriate dose can be determined by one of ordinary skill in the art using only routine experimentation.
  • the present disclosure includes methods comprising administering an mRNA vaccine to a subject in need thereof.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
  • the mRNA vaccine is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the mRNA vaccine may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • the effective amount of the mRNA may be as low as 20 pg, administered for example as a single dose or as two 10 pg doses. In some embodiments, the effective amount is a total dose of 20 ⁇ g-300 ⁇ g or 25 ⁇ g-300 ⁇ g.
  • the effective amount may be a total dose of 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50 ⁇ g, 55 ⁇ g, 60 ⁇ g, 65 ⁇ g, 70 ⁇ g, 75 ⁇ g, 80 ⁇ g, 85 ⁇ g, 90 ⁇ g, 95 ⁇ g, 100 ⁇ g, 110 ⁇ g, 120 ⁇ g, 130 ⁇ g, 140 ⁇ g, 150 ⁇ g, 160 ⁇ g, 170 ⁇ g, 180 ⁇ g, 190 ⁇ g, 200 ⁇ g, 250 ⁇ g, or 300 ⁇ g.
  • the effective amount is a total dose of 20 ⁇ g.
  • the effective amount is a total dose of 25 pg. In some embodiments, the effective amount is a total dose of 50 ⁇ g. In some embodiments, the effective amount is a total dose of 75 ⁇ g. In some embodiments, the effective amount is a total dose of 100 ⁇ g. In some embodiments, the effective amount is a total dose of 150 ⁇ g. In some embodiments, the effective amount is a total dose of 200 ⁇ g. In some embodiments, the effective amount is a total dose of 250 pg. In some embodiments, the effective amount is a total dose of 300 ⁇ g.
  • An mRNA vaccine described herein can be formulated into a dosage form described herein, such as an intranasal, Intratracheal, or Injectable (e.g., intravenous, intraocular, intravltreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).
  • injectable e.g., intravenous, intraocular, intravltreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous.
  • an mRNA vaccine is formulated in an effective amount to produce an antigen specific immune response in a subject.
  • the effective amount is a total dose of 1 ⁇ g to 1000 ⁇ g, 25 ⁇ g to 1000 ⁇ g, or 50 ⁇ g to 1000 ⁇ g. In some embodiments, the effective amount is a total dose of 100 ⁇ g. In some embodiments, the effective amount is a dose of 25 ⁇ g administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 100 ⁇ g administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 400 ⁇ g administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 500 ⁇ g administered to the subject a total of two times.
  • a dosage of between 10 ⁇ g/kg and 400 ⁇ g/kg of the mRNA vaccine is administered to the subject.
  • the dosage of the mRNA is 1-5 ⁇ g, 5-10 ⁇ g, 10-15 ⁇ g, 15-20 ⁇ g, 10-25 ⁇ g, 20-25 ⁇ g, 20-50 ⁇ g, 30-50 ⁇ g, 40-50 ⁇ g, 40-60 ⁇ g, 60-80 ⁇ g, 60-100 ⁇ g, 50-100 ⁇ g, 80-120 ⁇ g, 40-120 ⁇ g, 40-150 ⁇ g, 50-150 ⁇ g, 50-200 ⁇ g, 80-200 ⁇ g, 100-200 ⁇ g, 120-250 ⁇ g, 150-250 ⁇ g, 180-280 ⁇ g, 200-300 ⁇ g, 50-300 ⁇ g, 80-300 ⁇ g, 100-300 ⁇ g, 40-300 ⁇ g, 50-350 ⁇ g, 100-350 ⁇ g, 200-350 ⁇ g, 300-350 ⁇ g
  • the mRNA vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the mRNA vaccine is administered to the subject on day zero. In some embodiments, a second dose of the mRNA vaccine is administered to the subject on day twenty one.
  • a first dose of the mRNA vaccine is given as a priming step, followed by a second dose as a booster.
  • the prime-boost strategy aims to provide a stronger overall immune response.
  • the boost may be administered at least a day, at least a week, or at least two, three, four, five, six, or seven weeks, or at least two, three, four, five, or six months after the primer.
  • the boost may be administered at least three weeks after the primer.
  • sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
  • Homologs or variants of a given gene or protein will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981 ; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.
  • Sequence identity between nucleic acid sequences, or between amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same nucleotide, or amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical nucleotides or amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences.
  • Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.
  • Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include MatGat (Campanella et al., 2003, BMC Bioinformatics 4: 29;), Gap (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453), FASTA (Altschul et aL, 1990, J. Mol. Biol.
  • sequence comparisons may be undertaken using the “needle” method of the EMBOSS Pairwise Alignment Algorithms, which determines an optimum alignment (including gaps) of two sequences when considered over their entire length and provides a percentage identity score.
  • Default parameters for amino acid sequence comparisons (“Protein Molecule” option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: Blosum 62.
  • the sequence comparison may be performed over the full length of the reference sequence.
  • a polypeptide encoded by a mRNA of the invention may include one or more conservative amino acid substitutions.
  • Conservative amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original polypeptide, that is, the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
  • Examples of conservative substitutions include Ala to Ser, Arg to Lys, Asn to Gin or His, Asp to Glu, Cys to Ser, Gin to Asn, Glu to Asp, His to Asn or Gin, He to Leu or Vai, Leu to He or Vai, Lys to Arg or Gin, Met to Leu or He, Phe to Met, Leu, or Tyr, Ser to Thr, Thr to Ser, Trp to Tyr, Tyr to Trp or Phe, and Vai to He or Leu.
  • Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, serine or threonine, is substituted for (or by) a hydrophobic residue, for example, leucine, isoleucine, phenylalanine, valine or alanine; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysine, arginine, or histidine, is substituted for (or by) an electronegative residue, for example, glutamate or aspartate; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.
  • a hydrophilic residue for example, serine or threonine
  • a hydrophobic residue for example, leucine,
  • narrowly neutralizing immune response is used herein to mean an immune response elicited in a subject that is sufficient to inhibit (i.e. reduce), neutralize or prevent infection, and/or progress of infection, of a virus within the coronavirus family.
  • a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and/or progress of infection, of more than one type of beta-coronavirus (for example, SARS-CoV, and SARS-CoV-2).
  • a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and/or progress of infection, of more than one type of beta- coronavirus within the same beta-coronavirus lineage (for example, more than one type of beta-coronavirus within the subgenus Sarbecovirus, such as SARS-CoV, SARS-CoV-2, and Bat SL-CoV-WIV1).
  • a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and/or progress of infection, of coronaviruses of different beta- coronavirus lineages, such as lineage B (for example, SARS-CoV, and SARS-CoV-2) and lineage C (for example, MERS-CoV).
  • a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and/or progress of infection, of most or all different beta-coronaviruses.
  • a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and/or progress of infection, of most or all different viruses of the coronavirus family.
  • a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and/or progress of infection, of most or all variants of concern (VOCs) of SARS-CoV-2, including Beta, Gamma, Delta, Omicron (BA.1).
  • VOCs variants of concern
  • a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and/or progress of infection, of SARS-CoV, WIV16, RaTG13, SARS-CoV-2, SARS- CoV-2 Beta, SARS-CoV-2 Gamma, SARS-CoV-2 Delta, SARS-CoV-2 Omicron (BA.1).
  • the immune response may be a humoral and/or a cellular immune response.
  • a cellular immune response is a response of a cell of the immune system, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen or vaccine.
  • An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine.
  • An immune response includes, but is not limited to, an innate immune response or inflammation.
  • a polypeptide encoded by an mRNA of the invention induces a protective immune response.
  • a protective immune response refers to an immune response that protects a subject from infection or disease (i.e. prevents infection or prevents the development of disease associated with infection).
  • Methods of measuring immune responses include, for example, measuring proliferation and/or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, or antibody production.
  • a polypeptide encoded by an mRNA of the invention is able to induce the production of antibodies and/or a T-cell response in a human or non-human animal to which the mRNA has been administered (for example, expressed from an administered mRNA vaccine).
  • the present invention further relates to the (cosmetic/non-therapeutic) use of the herein above detailed cosmetic composition in the amelioration of a cutaneous condition. Accordingly, the present invention further relates to a (cosmetic/non-therapeutic) method for the amelioration of a cutaneous condition, wherein said method comprises the administration of the cosmetic composition as defined anywhere herein.
  • the active agent is selected from a growth factor, a peptide, an antioxidant, a retinoid, a cytokine, a siRNA, a miRNA, a mRNA, and an asRNA, whereas any of the listed agents may be as defined herein above.
  • the cosmetic composition may be as defined anywhere herein above.
  • the present invention further relates to a kit comprising the cosmetic composition and/or the pharmaceutical composition as detailed herein above.
  • the herein employed ionizable lipidoids (such as a lipidoid according to e.g., formula (b-V)) were surprisingly found to cause a composition they are comprised in (such as an LiNP composition comprising e.g., the lipidoid of formula (b-V)) to remain locally restricted at the site of administration following administration to a patient.
  • lipidoids may also limit systemic distribution of an agent when coupled thereto.
  • the present invention further relates to a drug conjugate comprising an ionizable lipidoid as defined herein above (preferably a compound of formula (b-l) or (b-ll), more preferably a compound of any one of formulas (b-V), (b-VII), (b-VII), (b-VIII), (b-IX), (b- X), (b-XI), or (b-XII), even more preferably a compound of formula (b-V), most preferably the (R)-enantiomer of the compound of formula (b-V)) and one or more therapeutic agent(s), preferably wherein said one or more therapeutic agent(s) is/are as defined anywhere herein above.
  • an ionizable lipidoid as defined herein above (preferably a compound of formula (b-l) or (b-ll), more preferably a compound of any one of formulas (b-V), (b-VII), (b-VII), (b-VIII), (b-IX
  • the present invention further relates to the in vitro use of an ionizable lipidoid for the restriction of the dissemination of one or more to be administered therapeutic agent(s), wherein said ionizable lipidoid is co-formulated with said one or more therapeutic agent(s), preferably wherein said ionizable lipidoid is as defined herein above (preferably a compound of formula (b-l) or (b-ll), more preferably a compound of any one of formulas (b-V), (b-VII), (b-VII), (b- VIII), (b-IX), (b-X), (b-XI), or (b-XII), even more preferably a compound of formula (b-V), most preferably the (R)-enantiomer of the compound of formula (b-V)), preferably wherein said one or more therapeutic agent(s) is/are as defined anywhere herein above.
  • a compound of formula (b-l) or (b-ll) more preferably a compound of any one
  • the present invention further relates to an (/n vitro) method for the restriction of the dissemination of one or more to be administered therapeutic agent(s), wherein said method comprises the step of co-formulating an ionizable lipidoid with said one or more therapeutic agent(s), wherein said ionizable lipidoid is as defined anywhere herein above (preferably wherein said lipidoid is a compound of formula (b-l) or (b-ll), more preferably a compound of any one of formulas (b-V), (b-VII), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), or (b-XII), even more preferably a compound of formula (b-V), most preferably the (R)-enantiomer of the compound of formula (b-V)), and wherein said therapeutic agent is as defined anywhere herein above.
  • co-formulating the lipidoid and the therapeutic agent(s) may comprise further co-formulating any component (such as, for example, a pharmaceutically acceptable excipient or diluent) of the herein above detailed compositions therewith.
  • the present invention further provides for a compound of formula (b-VII).
  • the present invention further provides for a compound of formula (b-VIII).
  • the present invention further provides for a compound of formula (b-IX).
  • the present invention further provides for a compound of formula (b-X).
  • the present invention further provides for a compound of formula (b-XI).
  • the present invention further provides for a compound of formula (b-XII).
  • terapéutica include prevention and/or treatment of any disease, disorder, or condition and includes vaccination or immunization.
  • subject means an individual.
  • a subject in the sense of the invention can be a mammal, preferably a human.
  • the singular forms “a,” “an”, and “the” include the plural referents unless the context clearly indicates otherwise.
  • the terms “include”, “such as”, and “the like” are intended to convey inclusion without limitation, unless otherwise specifically indicated.
  • the term “or” is generally employed in its usual sense including “and/or” unless the content clearly dictates otherwise.
  • the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.
  • the term “comprising” also specifically includes embodiments “consisting of and “consisting essentially of the recited elements, unless specifically indicated otherwise.
  • FIG. 1 Bioluminescence image of excised organs. Animals were treated by intratracheal administration of 1 ⁇ g luciferase chemically modified mRNA encapsulated in Formulation I (i.e., Formulation I formulated Luciferase). D-Luciferin substrate was applied to animals by intraperitoneal and intranasal application before they were euthanized 4 hours after application. Luciferase activity was measured in explanted organs using a Lumlna XR In Vivo Imaging system (Perkin Elmer, USA). In this context Luciferase activity was only detected in the lungs but not In the liver or the spleen.
  • Formulation I Formulation I formulated Luciferase
  • Luciferase radiance is measured as p/sec/cm 2 /sr, indicating the number of photons per second per square centimeter per steradian. Accordingly, the scale represents radiance in p/sec/cm2/sr.
  • FIGS. 2 and 3 BALB/c laboratory mice were short time anesthetized through inhalation of Isoflurane (2-3 %).
  • Formulation I formulated Luciferase coding modified-RNA (i.e., Formulation I) was injected at a dose level of 20 ⁇ g/20 ⁇ L into the cranial tibial muscle of both hind limbs using 0.3 mL Insulin injection syringes (BD, Germany). Luciferase activity was measured 1 and 4 days (Fig 2 and 3, respectively) subsequently under full anesthesia and following the application of 3 mg D-Luciferin/100 ⁇ L PBS which was applied intraperitoneally.
  • Formulation I formulated Luciferase coding modified-RNA (i.e., Formulation I) was injected at a dose level of 20 ⁇ g/20 ⁇ L into the cranial tibial muscle of both hind limbs using 0.3 mL Insulin injection syringes (BD, Germany). Luciferas
  • Luciferase activity was measured using a Lumina XR In Vivo Imaging System (Perkin Elmer, USA). Local Luciferase activity was measured using a defined Region-Of-lnterest (ROI) which was drawn over the injection site. A ROI over the chest region served as internal control. Scale represents radiance in p/sec/cm2/sr.
  • ROI Region-Of-lnterest
  • Results showed expression of Luciferase at a comparable level In both hind limbs as soon as 1 day ( Figure 2) following application of the test item. Luciferase activity remained stable for at least 4 days ( Figure 3) following application. No Luciferase activity was observed in any other region of the body, indicating that there was no systemic distribution of the test item.
  • FIG. 4 Intramuscular injection using Formulation I formulated Firefly Luciferase with chemically modified RNA.
  • BALB/c laboratory mice were short time anesthetized through inhalation of Isoflurane (2-3 %).
  • Formulation I formulated Luciferase coding chemically modified RNA was injected at a dose level of 20 ⁇ g/20 ⁇ L into the cranial tibial muscle of both hind limbs using 0.3 mL Insulin injection syringes (BD, Germany). Luciferase activity was measured 1 and 4 days subsequently under full anesthesia and following the application of 3 mg D-Luciferin/100 ⁇ L PBS which was applied intraperitoneally.
  • Luciferase activity was measured using a Lumina XR In Vivo Imaging System (Perkin Elmer, USA). Local Luciferase activity was measured using a defined Region-Of-lnterest (ROI) which was drawn over the injection site. A ROI over the chest region served as internal control. Scale represents radiance in p/sec/cm2/sr.
  • ROI Region-Of-lnterest
  • Results showed expression of Luciferase at a comparable level in both hind limbs as soon as 1 day following application of the test item. Luciferase activity remained stable for 4 days following application. No Luciferase activity was observed in any other region of the body, indicating that there was no systemic distribution of the test item.
  • Figure 5 and 6 BALB/c laboratory mice were short time anesthetized through inhalation of Isoflurane (2-3 %).
  • Formulation I formulated Luciferase coding chemically modified RNA was injected at a dose level of 20 ⁇ g either alone (diluted in 300 ⁇ L 0.9% NaCI) ( Figure 5) or combined with hyaluronidase (Hylase "Dessau" 150 I.U. diluted in 300 ⁇ L 0.9% NaCI which was prepared according to the manufacturer's instructions) ( Figure 6) subcutaneously at the back of the animal using 0.3 mL Insulin injection syringes (BD, Germany).
  • Hyaluronidase is an enzyme which facilitates degradation of the extracellular matrix, and which is typically used in dermatology for the aesthetic treatment of scar tissue. Due to its capacity for the degradation of the extracellular matrix hyaluronidase enables the locally applied Formulation I formulated Luciferase chemically modified RNA to spread into the systemic circulation.
  • the Luciferase activity was measured 6 hours after application of the test item under full anesthesia and following the application of 3 mg D-Luciferin/100 ⁇ L PBS which was applied intraperitoneally. Luciferase activity was measured using a Lumina XR In Vivo Imaging System (Perkin Elmer, USA). Local Luciferase activity was measured using a defined Region- Of-lnterest (ROI) which was drawn over the injection site. Scale represents radiance in p/sec/cm2/sr.
  • Figure 7 and 8 Four hours following application of formulated Luciferase mRNA as Formulation I either diluted in 0.9% NaCI or hyaluronidase solution animals underwent necropsy. Subsequent ex vivo in vivo imaging using the Lumina XR In Vivo Imaging System (Perkin Elmer, USA) showed Luciferase activity explanted liver and spleen of animals being treated with hyaluronidase dissolved Formulation I (see organs at top and very left of Figure 8; spleen and liver, respectively). In contrast to this, no activity in liver and spleen was observed following treatment using Formulation I which was dissolved in 0.9% NaCI only (see organs at top and very left of Figure 7). Independent from the dilution agent, no activity was observed in the heart, the right kidney, and the lungs. Scale represents radiance in p/sec/cm2/sr.
  • Figure 9 Effect of phospholipid length.
  • the figure shows the effect of phospholipid length on spike protein of SARS-Cov2 expression level measured as the area under the curve of antibody production for antibodies targeting the expressed protein (coronavirus spike protein receptor binding domain (RBD)).
  • the tested phospholipid lengths were 14, 16 and 18 carbon atoms. Each point represents an individual mouse.
  • PE Phosphoethanolamine.
  • PC Phosphocholine (see Table 5).
  • the dot diameter corresponds to the value of the area under the curve (AUC).
  • FIG 10 immunization effect of phospholipid type: PC (Phosphocholine) or PE (Phosphoethanolamine).
  • PC Phosphocholine
  • PE Phosphoethanolamine
  • the chart illustrates how different types of phospholipids impact the immunization level against the expressed SARS-CoV-2 spike protein. This impact is quantified by measuring the average area under the curve (AUC) for antibodies targeting the expressed protein, 36 days post-immunization. The tested phospholipid lengths were 14, 16 and 18 carbon atoms. Each dot in the figure represents data from an individual mouse.
  • PE Phosphoethanolamine.
  • PC Phosphocholine.
  • the dot diameter corresponds to the value of the area under the curve (AUC).
  • Figure 11 Effect of stealth PEG length (14 to 18 C PEG 2000 or 5000 Da).
  • the figure presents the impact of stealth PEG length on the expression of the SARS-CoV-2 spike protein, quantified by the area under the curve (AUC) of antibodies targeting the protein 36 days after the initial immunization in mice. Additionally, the effect of stealth lipid chain lengths — comprising 14, 16, or 18 carbon atoms — was evaluated.
  • Each dot in the figure represents data from an individual mouse.
  • G Dimyristoylglycerin-polyethylenglycol
  • PE Dipalmitoylglycerin- polyethylenglycol Phosphoethanolamine.
  • the dot diameter corresponds to the value of the area under the curve (AUC).
  • Figure 12 Effect of stealth PEG %.
  • the figure presents the impact of the % of PEG-lipid on the expression of the SARS-CoV-2 spike protein, quantified by the area under the curve (AUC) of antibodies targeting the protein 36 days after the initial immunization in mice.
  • G Dimyristoylglycerin-polyethylenglycol-2000 or Dimyristoylglycerin-polyethylenglycol-5000
  • PE Dimyristoylphosphoethanolamin-polyethylenglycol-2000 or Dimyristoylphosphoethanolamin- polyethylenglycol-5000 (see Table 5).
  • Figure 13 Effect of sterol percentage.
  • the figure presents the impact of the % of sterol lipid on the expression/immunization against the SARS-CoV-2 spike protein, quantified by the area under the curve (AUC) of antibodies targeting the RBD of the spike protein 36 days after the initial immunization in mice.
  • Figure 14 effect cationic lipidoid %.
  • the figure presents the impact of the amount of cationic lipidoid in % on the expression of/immunization against SARS-CoV-2 spike protein, quantified as the area under the curve (AUC) of antibodies targeting the RBD of the spike protein 36 days after the initial immunization in mice.
  • AUC area under the curve
  • Figure 15 Effect of N/P ratio.
  • the figure presents the impact of the ratio of nitrogen to phosphorous atoms on the expression of the SARS-CoV-2 spike protein, quantified by the area under the curve (AUC) of antibodies targeting the RBD of the spike protein 36 days after the initial immunization in mice.
  • AUC area under the curve
  • Figure 16 hlFN ⁇ 1 -mRNA levels in lungs homogenates following single inhaled administration measured by qPCR.
  • A copies of hlFN ⁇ 1 mRNA in lung homogenates 24 hours after administration of 0.024 mg/m 2 of hlFN ⁇ 1-LiNP;
  • B copies of hlFN ⁇ 1 mRNA in lung homogenates 24 hours after administration of 0.048 mg/m 2 of hlFN ⁇ 1-LiNP,
  • C copies of hlFN ⁇ 1 mRNA in lung homogenates 24 hours after administration of 0.096 mg/m 2 of hlFN ⁇ 1- LiNP;
  • D copies of hlFN ⁇ 1 mRNA in lung homogenates 24 to 144 hours after administration of 0.024 mg/m 2 of hlFN ⁇ 1-LiNP.
  • hlFN ⁇ 1-mRNA was quantified in lung homogenates by RT- qPCR.
  • Control is a non-translatable mRNA-LiNP. Calculation of mRNA concentrations was done using a standard curve. Each dot represents one animal. Mean values and standard deviation are shown. “Vehicle” does not contain any LiNP.
  • FIG 17 Biodistribution of hlFN ⁇ 1-mRNA in lungs homogenates 24h after single (1x) and repeated (4x) inhaled administration in rats.
  • hlFN ⁇ 1 LNP-mRNA was measured in serum and organ homogenates by RT-qPCR 24 h after single or repeated inhaled administration of test product hlFN ⁇ 1 LNP.
  • Vehicle group was derived from study WP2_3_14 (see Table 10). Values were quantified by interpolating a standard curve of mRNA in spiked-in neat matrix of untreated animals. Signals in vehicle-treated animals are considered background levels of the assay. Each dot represents one animal. Columns and error bars represent mean values and standard deviation. “Vehicle” does not contain any LiNP.
  • Figure 18 Biodistribution of hlFN ⁇ 1-mRNA in lungs, serum, and key organs 24h after single (1x) (Fig 18 A) and multiple (3x) (Fig 18 B) administration in rats.
  • Test product hlFN ⁇ 1 LNP- mRNA was measured in serum and organ homogenates by RT-qPCR 24 h after single or repeated inhaled administration of test product hlFN ⁇ 1 LNP.
  • Vehicle group was derived from study WP2_3_14. Values were quantified by interpolating a standard curve of mRNA in spiked- in neat matrix of untreated animals. Each dot represents one animal. Columns and error bars represent mean values and standard deviation. No mRNA could be detected in the serum, liver, brain, heart, spleen, and kidney after single or repeated administration.
  • "Vehicle” does not contain any LiNP.
  • FIG 19 Biodistribution of the ionizable lipidoid dL_05 in lungs, serum and key organs following single(1x) and multiple (3x) inhaled administration in rats.
  • the lipid fractions of serum and organ lysates were analyzed by a validated LC-MS/MS method for the lipidoid dL_05 following single or repeated inhaled administration of test product hlFN ⁇ 1 LNP. Values were quantified by interpolating a standard curve of dL_05 spiked in neat matrix of untreated animals. The values of all matrices, except for lungs, are below the limit of detection. Each dot represents one animal. Columns and error bars represent mean values and standard deviation. “Vehicle” does not contain any LiNP.
  • Figure 20 hlFN ⁇ 1 protein concentrations in lung homogenates following single dose administration by nasal sniffing of test product hlFN ⁇ 1 LNP in Formulation I in mice.
  • Fig 20 A shows results after administration of hlFN ⁇ 1 LNP at 0.020 mg/m 2 .
  • Fig 20 B shows results after administration of hlFN ⁇ 1 LNP at 0.056 mg/m 2 indicating that hlFN ⁇ 1 protein was produced dose-dependently in lung homogenates at 5 h after administration.
  • Lungs of animals necropsied at later time points (24 to 96 h) showed no evidence of hlFN ⁇ 1 protein concentration above LLOQ. Mean and standard deviation of three animals is shown. “Vehicle” does not contain any LiNP.
  • Administration resulted in highest hlFN ⁇ 1 protein concentration measured at 6 h post administration which returned to baseline at 48 h. Error bars indicate mean values and standard deviation.
  • Figure 22 hlFN ⁇ 1 protein levels in lung homogenate and serum of rats following single and repeated inhaled administration of test product hlFN ⁇ 1 LiNP in the GLP Toxicology study. Serum and lung homogenate were analyzed with a validated hlFN ⁇ 1 ECL assay. Measured values are reported per lung tissue mass or per mL serum. Each dot represents one animal, error bars indicate mean values and standard deviation. hlFN ⁇ 1 concentrations in all vehicle- treated animals were BLQ for both matrices. hlFN ⁇ 1was detected in both lung and serum, but at much lower levels in the serum. hlFN ⁇ 1 was detected in both lung and serum, but at much lower levels in the serum. These results confirm the localized expression after administration to the lungs. “Vehicle” does not contain any LINP.
  • Figure 23 IVIS imaging of BALB/c mice (left column) and ex vivo lungs, heart, liver, spleen and right kidney of the same animals (top to bottom, right column), 6 hours after intratracheal instillation of 50 ⁇ L (0.06 mg Luciferase mRNA/mL) for carrier formulations with different ratios for the same components as Formulation I (dL_05(R), DPPC, Cholesterol, DMG-PEG2000).
  • FIG. 25 IVIS imaging of BALB/c mice (left col.) and ex vivo lungs, heart, liver, spleen and right kidney of the same animals (right coL), 6 hours after intratracheal instillation of 50 ⁇ L (0.06 mg Luciferase mRNA/mL) for formulations with dL_05(R) (formula b-VI) and Cholesterol but different helper and stealth lipid, as well as different lipid-to-lipid ratios.
  • A Formulation M02-102-006
  • B Formulation M02-103-003
  • C Formulation M02-002-003
  • D Formulation M02-109-006.
  • Scale represents radiance in p/sec cm2/sr. All tested formulations result in a localized luciferase signal in the lungs of BALB/c mice. No expression could be detected in other tissues, such as explanted hearts, livers, spleen, and right kidneys. This is indicative of a localized retention of the administered formulations at the site of administration (i.e., in the lungs).
  • Figure 26 IVIS imaging of BALB/c mice (left col.) and ex vivo heart, lungs, right kidney, liver, and spleen (right coL), 6 hours after intratracheal instillation of 50 ⁇ L (0.06 mg of Luciferase mRNA/mL) for a formulation (Formulation M03-076-3) comprising a combination of two ethyl-propyl-ethyl lipidoids different to dL_05(R) (used in Formulation I), and also different helper lipids, as well as different lipid-to-lipid ratios.
  • A experiment with three mice
  • B experiment with two mice.
  • Formulation M03-076-3 comprises lipidoids LG2C (formula b-IX) and LE1 D (formula b-X). Scale represents radiance in p/sec/cm2/sr. All tested formulations result in a localized luciferase signal in the lungs of BALB/c mice. No expression could be detected in other tissues, such as explanted hearts, livers, spleen, and right kidneys. This is indicative of a localized retention of the administered formulations at the site of administration (i.e., in the lungs).
  • Figure 27 IVIS imaging of BALB/c mice (left) and corresponding organs (lungs, liver, spleen), 6 hours after intratracheal microspray of 50 ⁇ L (0.2 mg of Luciferase mRNA/mL) for formulations with EPE-based dL but different than dL_05(R), otherwise same lipids and lipid- to-lipid molar ratios as Formulation I.
  • A Formulation LF110
  • B Formulation LF181
  • C Formulation LF53.
  • the figure scale represents radiance, measured in photons per second per square centimeter per steradian (p/sec/cm 2 /sr).
  • Figure 28 IVIS imaging of C57BL6&JRj female adult mice, 4 hours after intranasal delivery showing localized expression in the nasal airways when administered with 0.15 mg/mL (A), 0.5 mg/mL (B), 1.5 mg/mL (C) modified mRNA coding for luciferase formulated with Formulation I (i.e., comprising dL_05(R)).
  • Left images show color image and right images show the same mice in grey scale. Circles indicate radiance values measured in regions of interest (ROIs). Scale represents radiance in p/sec/cm2/sr.
  • composition I (herein also referred to as Formulation I or LF92)).
  • composition I (herein also referred to as Formulation I or LF92)
  • the only difference between this tool product and the therapeutic hlFN ⁇ 1 LNP test drug is the mRNA of Examples 3 to 7 is the coding sequence of hlFN ⁇ 1 , whereas in Example 1 the mRNA encodes luciferase, an easily measurable reporter protein.
  • Intra tracheal administration of mRNA formulated in Composition I results in efficient delivery of the mRNA to the respiratory tissue followed by translation of the encoded target gene (Luciferase).
  • test composition I (herein also referred to as Formulation I or LF92) comprising mRNA encoding luciferase was tested in vivo via intratracheal instillation in mice (1 ⁇ g/50 ⁇ L]
  • luciferase signals were recorded 6 hours post- administration, following exposure of the organs to luciferin, and bioluminescence was quantified using an in vivo imaging system (IVIS) as shown in Fig 1.
  • IVIS in vivo imaging system
  • Test Item Formulation I with chemically modified mRNA coding for luciferase and modified during in-vitro transcription to comprise 30 % I5U/ 3% I5C at a final concentration of 0.02 mg/mL.
  • mice of the C57BL/6J strain were obtained from Charles River Laboratories for use in this study.
  • Formulation I consists of the excipient mix dL_05(R) (i.e., the (R)-enantiomer of a compound according to formula (b-V) in the context of the present invention), DPPC, Cholesterol and DMG-PEG200 at a molar mixing ratio of 8: 5.29: 4.41 :0.88 at an N/P ratio of 8 (referring to the ratio of nitrogen atoms presents in the lipids to the amount of phosphorous atoms present in the therapeutic agent, preferably a nucleic acid, being packed; a lipid/lipidoid having a high N/P ratio, such as an N/P ratio of 8, allows to pack the same amount of RNA in an LiNP/LNP as compared to a lipid/lipidoid having a lower N/P ratio).
  • dL_05(R) i.e., the (R)-enantiomer of a compound according to formula (b-V) in the context of the present invention
  • the mRNA sequences encoding the sequences luciferase were synthesized by in vitro transcription (IVT) from linearized plasmid DNA templates using modified nucleotides to generate partial modified mRNAs.
  • IVT was performed for 120 min at 37°C using T7-RNA polymerase including co-transcriptional capping via Anti-Reverse Cap Analog (ARCA), followed by digestion of the template using DNAse I.
  • mRNAs were dephosphorylated for 15 min at 37°C using alkaline phosphates and enzymatically polyadenylated for 10-30 min at 37°C using PolyA polymerase to produce a Poly A tail of approximately 120 nucleotides.
  • mRNAs were stored at -80°C until LNP encapsulation.
  • Each mRNA was LNP encapsulated via nanoprecipitation (NanoAssemblr lgnite+, Precision Nanosystems, Vancouver) by microfluidic mixing of mRNA in citrate buffer (pH 4.5) with ionizable-, structural-, helper- and polyethylene glycol (PEG) lipids in ethanol, followed by buffer exchange and concentration via tangential flow filtration.
  • mRNA LNPs were filtered through a 0.2pm membrane and stored at -20°C until use.
  • mice were set under full anesthesia through intraperitoneal injection of Fentanyl/Midazolam/Medetomidin (0.05/5.0/0.5 mg/kg bw).
  • D-Luciferin (3 mg/100 ⁇ L PBS) was applied by intraperitoneal injection and intranasally using the “sniffing” method (1.5 mg/5 ⁇ L PBS).
  • mice were killed by cervical dislocation, immediately after blood withdrawal.
  • Blood samples were centrifuged at 2.000 x g for 5 min at 4°C. The abdominal cavity was opened in the median axis. A careful cut was made in the diaphragm, which led to atmospheric pressure in the thoracal cavity and immediate collapse of the lungs.
  • the aim of this task was to investigate whether mRNA encapsulated in Formulation I is able to transfect cells of the respiratory tissue after intratracheal administration and whether said formulation remains local to the administered organ or tissue.
  • Results demonstrate that a clear signal of the reporter protein (Luciferase) encoded by the mRNA was detectable after treatment with a dose of 1 ⁇ g per animal. While a clear signal was detectable in the lung no protein could be detected in liver and spleen ( Figure 1 ). This confirms that Formulation I (M01-001) surprisingly remains localized to the administered organ.
  • Formulation I (M01-001) efficiently deliver mRNA to respiratory cells resulting in translation into the target protein.
  • the formulation remains local, and no expression is detectable in the liver or spleen.
  • Luciferase coding chemically modified mRNA was injected at a dose level of 20 ⁇ g/20 ⁇ L into the cranial tibial muscle of both hind limbs using 0.3 mL Insulin injection syringes (BD, Germany). Luciferase activity was measured 1 and 4 days subsequently under full anesthesia and following the application of 3 mg D-Luciferin/100 ⁇ L PBS which was applied intraperitoneally. Luciferase activity was measured using a Lumina XR In Vivo Imaging System (Perkin Elmer, USA). Local Luciferase activity was measured using a defined Region-Of-lnterest (ROI) which was drawn over the injection site. A ROI over the chest region served as internal control.
  • ROI Region-Of-lnterest
  • Luciferase mRNA (20 ⁇ g) formulated as Formulation I was dissolved either in 300 ⁇ L physiological saline (0.9% NaCI) or in 300 ⁇ L hyaluronidase solution (Hylase “Dessau” 150 LU., HWI Pharma Services GmbH, Germany) and was subcutaneously injected in the cervical back of female BALB/c mice which were short time anesthetized by Isofluran inhalation. Luciferase activity was measured 4 hours following application using a Lumina XR In Vivo Imaging System (Perkin Elmer, USA).
  • Luciferase activity was observed for both, following application of saline dissolved and following application of hyaluronidase dissolved LF92 (Formulation I) LUC mRNA. However, Luciferase activity was substantially higher (2-fold) following application of hyaluronidase dissolved LF92 (Formulation I) LUC mRNA compared to mRNA dissolved in saline. Subsequent to in vivo measurement of Luciferase activity ( Figures 5 and 6), the animals were euthanized, and organs were explanted for ex vivo measurement of Luciferase activity.
  • Figure 7 there is no detectable luciferase signal in any of the organs.
  • the values correspond to background signal and the image corresponds to the visible light image taken from the organs.
  • Figure 8 there is a significant signal coming from the luciferase. In order to detect that signal, the camera was set to the highest sensitivity. This causes the generation of a pixelated image.
  • the comparison of figures 7 and 8 shows that after intramuscular administration together with Hylase a significative luciferase expression in at least the liver and spleen, which is indicative of reduced localized retention (or increased systemic distribution) of the mRNA encoding the luciferase.
  • Fig 9 summarizes the effect of Effect of phospholipid length.
  • Fig 10 summarizes the effect of phospholipid type: PC (Phosphocholine) or PE (Phosphoethanolamine).
  • Fig 11 summarizes the effect of stealth PEG length (14 to 18 C PEG 2000 or 5000 Da).
  • the figure presents the impact of stealth PEG length on the expression of the SARS-CoV-2 spike protein, quantified by the area under the curve (AUG) of antibodies targeting the protein 36 days after the initial immunization in mice. Additionally, the effect of stealth lipid chain lengths — comprising 14, 16, or 18 carbon atoms — was evaluated. All tested stealth lipids provided excellent immunization effect. Especially the use of 14 C PEG 2000 Da provided the best results (i.e. , the highest immunization level). Fig 12 also highlights the effects of using G (glycerol DMG PEG) or PE (DMG PEG 2000phosphoethanolamine) as shown in Fig 11. Both provided good immunization levels. Dot size is indicative of immunization AUG after 36 days.
  • G glycerol DMG PEG
  • PE DMG PEG 2000phosphoethanolamine
  • LINPs were complexed with mRNA encoding for the spike protein of SARS-Cov2 (Wuhan variant) and injected intramuscular at day 1 and day 21. Animals subjected to treatment were evaluated for spike protein receptor binding domain (RBD)-specific antibodies following the first and second immunizations and for the cytokine expression profile of splenocytes using ELISpot analysis
  • M01-001 is depicted in Figures 9 to 15 as an open circle (O).
  • M04-001 comprises the same LNP components as M01-001 (also referred to as Formulation I or LF92 herein above) and further comprises a poloxamer (P188); M04-001 is depicted as a cross (+).
  • P188 poloxamer
  • M04-001 is depicted as a cross (+).
  • the addition of Poloxamer provided a higher immunogenicity.
  • Fig 13, Fig 14 and Fig 15 show that a range of cholesterol, dL_05(R) and different N/P, respectively, can be used to induce high immunization level, recorded as AUG of antibodies targeting the protein, 36 days after the initial immunization.
  • Carriers were mixed using a NanoAssemblrTM benchtop system as described in Example 1 .
  • Lipid mixes were prepared according to Table 3 and Table 4.
  • Downstream processing was performed via dialysis in Slide-A-Lyzer MINI dialysis devices. The devices were prepared according to the manual of the manufacturer. 2 mL of formulation were transferred into the sample reservoir and dialyzed against water by shaking on an orbital shaker at 300 rpm. After 1.5 - 2 h the dialysis buffer was replaced by fresh water and the formulations were dialyzed overnight (14 - 18 h).
  • SARS-CoV-2 RBD-specific antibodies were quantified with ELISA.
  • a 384- well plate was coated over night at 4 °C with 2 ⁇ g/mL recombinant SARS-CoV-2 spike protein RBD (Sino Biological, 40592-V08B), diluted in CBB buffer, using 20 ⁇ L per well.
  • the plate was washed 3x with PBST using a microplate washer.
  • the plate was blocked 1 h at RT and 600 rpm with 50 ⁇ L 1 % casein blocking solution (ThermoFisher Scientific, Cat. Nr. 37528) per well.
  • the plate was washed 3x with PBST using a microplate washer.
  • An anti-RBD IgG standard curve ranging from 0.06 -1,000 ng/mL was prepared in PBST using mouse anti-SARS-CoV-2 RBD antibody (R&D Systems, Cat. Nr. MAB105808-100). Standard curve and samples (Serum was applied in serial 1 :4-dilution starting at 1 :50) were applied to the plate using 20 ⁇ L per well and incubated for 1 h at RT and 600 rpm. The plate was washed 3x with PBST using a microplate washer. The detection antibody (Abeam, ab205719) was diluted 1 :2000 in PBST, applied to the plate using 20 ⁇ L per well and incubated for 1 h at RT and 600 rpm.
  • the plate was washed 3x with PBST using a microplate washer. Lastly, 20 ⁇ L TMB was added to each well and the plate was incubated for 1.5 min at RT and 600 rpm. The reaction was stopped using 10 ⁇ L H2SO4 per well. Absorption is measured at 450/650 nm using a microplate reader. To obtain IgG concentrations in samples, the results were interpolated using the standard curve.
  • the lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) of the assay are 0.5 ng/mL and 100 ng/mL, respectively. Serum MRD is 1 :800. In addition to quantification of antibody levels, also reciprocal titers were determined.
  • Titers are defined as highest measured concentrations that obtains results > limit of detection (LOD).
  • LOD of the assay is defined as 3*Mean OD of pure PBST.
  • AUC area under the curve
  • Table 5 discloses the standard names of helper lipids and stealth lipids used in all formulations. Abbreviations for Examples 4-6
  • Example 4 Local delivery of Formulation l-based LiNPs carrying an mRNA encoding a therapeutic protein - human interferon lambda 1 (hlFN ⁇ 1 ).
  • a frozen LiNP Formulation I suspension at a concentration of 2.5 mg/mL comprising a modified messenger RNA (mRNA) encoding human interferon lambda 1 (hlFN ⁇ 1), referred in the following as the test drug product hlFN ⁇ 1 LNP was used to test the localized delivery of a therapeutic polypeptide of interest.
  • mRNA messenger RNA
  • hlFN ⁇ 1 LNP modified messenger RNA
  • Interferons are potent cytokines that play a key role as first line defense in viral infections by controlling inflammation and directly inducing anti-viral mechanisms.
  • type III interferons such as interferon lambda 1 (IFN ⁇ 1) are critically important for viral defense directly at the respiratory epithelium, the common entry site for respiratory viruses.
  • IFN ⁇ 1 interferon lambda 1
  • the advantages of localized expression of a therapeutic protein such as IFN ⁇ 1 is that side effects caused by systemic activation of downstream targets can be avoided.
  • the mRNA coding for a human codon optimized Interferon Lambda 1 according to SEQ ID NO: 41 with an average length of 788 nucleotides was manufactured by in-vitro transcription (IVT) from a linearized plasmid DNA template using a T7 RNA polymerase using in the mix 25% of modified bases 5-methylcytosine and 75% cytidine and 25% 2-thiouridine and 75% uridine to generate a partially modified mRNA.
  • IVTT in-vitro transcription
  • modified nucleosides in the mRNA reduces the affinity and recognition of mRNA by intracellular like toll-like receptors (TLRs), retinoid-inducible gene 1 (RIG-1), and others and substantially decrease activation of the innate immune system in vitro and in vivo and concomitantly increase the stability of the mRNA, allowing for prolonged, high-level cellular translation.
  • the LiNPs comprise the lipidoid component (dL_05 (R)) and excipients optimized for aerosol delivery of LNP-formulated mRNA.
  • the complete structure of the mRNA includes a 5’ Cap, a 5’ minimal UTR, a human codon-optimized IFN ⁇ 1 coding sequence, and a poly(A) tail (SEQ ID NO: 42).
  • the poly(A) might be an encoded poly A or might be added enzymatically. In the tested mRNA, the poly(A) was added enzymatically. 4.3. Generation of the hlFN ⁇ 1 Test LiNP
  • the drug product tested was a preservative-free, sterile dispersion of mRNA encoding hlFN ⁇ 1 formulated in Formulation I lipidoid nanoparticles (LiNP) in an aqueous cryoprotectant solution.
  • the lipidoid nanoparticles were generated by mixing the mRNA with a solution of one lipidoid and three lipid excipients (lipidoid dL_05(R), and the lipids DPPC [1 ,2-dipalmitoyl-sn-glycero- 3-phosphocholine], cholesterol, and DMG-PEG 2000 [1 ,2-Dimyristoyl-rac-glycero-3- methyl polyoxyethylene glycol 2000]).
  • the excipients associate with the mRNA, protect it from degradation and aid its delivery to the target cells in the respiratory epithelium.
  • Downstream processing after mixing lipids and mRNA included buffer exchange and concentration.
  • the drug was aseptically filled at a nominal filling volume of 1 .0 mL with an mRNA concentration of 2.50 ⁇ 0.50 mg/mL and stored frozen at -20 ⁇ 5°C. The drug was characterized as a white to off-white suspension.
  • the delivered dose in inhalation toxicology studies was calculated using the recommendations of the Association of Inhalation Toxicologists considering aerosol concentration, respiratory minute volume and the inhalation duration (Alexander et al. 2008).
  • the resulting nasal and lung surface exposures are dependent on the droplet size of the aerosol.
  • MMAD mass median aerodynamic diameter
  • hlFN ⁇ 1 LNP-mRNA drug substance
  • cDNA synthesis was performed with Transcriptor First Strand cDNA Synthesis Kit (Roche®) using Oligo(dT) priming. qPCR was run with customized TaqMan Primer/Probe using the TaqManTM Fast Advanced Master Mix (Thermo Fisher Scientific).
  • hlFN ⁇ 1 LNP- mRNA was extracted from rat tissues using QIAGEN RNeasy Mini Kit (QIAGEN) and from serum using miRNeasy Serum/Plasma Advanced Kit (QIAGEN).
  • Rat tissue and serum RNA extracts were quantified by a 2-step reverse-transcription RT-qPCR using the Roche Transcriptor High Fidelity cDNA synthesis Kit with Oligo(dT) priming followed by qPCR using TaqManTM Environmental Master Mix (2.0) (Thermo Fisher Scientific). mRNA extraction and RT-qPCR for the GLP study were validated methods.
  • hlFN ⁇ 1 LNP-mRNA In the non-GLP Dose Range Finding (DRF) study involving Wistar Han rats, the highest concentrations of hlFN ⁇ 1 LNP-mRNA from the test product were detected in lung homogenates at 6 hours post single inhaled administration (the earliest measurement after drug administration). Lung levels at 24 hours were lower than at 6 hours but remained consistent up to 48 hours. Beyond 48 hours, hlFN ⁇ 1 LNP-mRNA levels in the lung further decreased (see Fig 16D). Notably, 24 hours post-administration, hlFN ⁇ 1 LNP-mRNA levels in the lungs showed a dose-dependent increase, aligning with the dose-dependent rise in hlFN ⁇ 1 protein concentrations in the lung (see Fig 21A-E).
  • Test product hlFN ⁇ 1 LNP-mRNA was measured in serum and homogenates of major perfused organs by RT-qPCR within 2h following single inhaled administration of test product hlFN ⁇ 1 LNP. Values were quantified by interpolating a standard curve of mRNA in spiked-in neat matrix of untreated animals. Signals in vehicle-treated animals are considered background levels of the assay. Each dot represents one animal. Columns and error bars represent mean values and standard deviation. The hlFN ⁇ 1 coding mRNA from the test product was not detected in the serum, and its liver copy numbers matched the background levels of vehicle- treated animals (Fig 17).
  • dL_05 defined ionizable lipidoid 05
  • dL_05 defined ionizable lipidoid 05
  • Detection of LNPs using dL_05 is particularly suitable as a specific biomarker for the LNP biodistribution as no endogenous levels are expected.
  • the other lipid components of the LNP are cholesterol (occurs endogenously), DPPC and DMG-PEG.
  • DPPC is naturally occurring and the main phospholipid of pulmonary surfactant, while detection of DMG-PEG in humans is potentially biased by the exposure to PEG from other sources as it is a widely used excipient in many pharmaceutical and cosmetical products.
  • LLOQ the lowest limit of quantification using (LLOQ) to detect dL_05 in serum and lung was 100 ng/mL and in liver 200 ng/ml.
  • Pulmonary hlFN ⁇ 1 production following local delivery of hlFN ⁇ 1 LNP to the lungs was confirmed in several studies in mice and rats by measuring hlFN ⁇ 1 in lung homogenates.
  • test product hlFN ⁇ 1 LNP was well tolerated following administration to the lungs of mice and resulted in dose-dependent pulmonary production of hlFN ⁇ 1 at the 5 h time (Fig 20A and Fig 20B). Protein levels returned to background level from 24 h onwards.
  • hlFN ⁇ 1 protein was produced dose-dependently in lung homogenates at 5 h after administration. Lungs of animals necropsied at later time points (24 to 96 h) showed no evidence of hlFN ⁇ 1 protein concentration above lower limit of quantification (LLOQ). Mean and standard deviation of three animals is shown.
  • Wistar Han rats received test product hlFN ⁇ 1 LNP via nose-only inhalation at lung exposures of 0.012, 0.024, 0.048, 0.096 mg/m 2 or formulated untranslatable control-mRNA at a lung exposure of 0.024 mg/m 2 (Fig 21 A to 21 E).
  • a control group receiving only a vehicle solution containing all components of the drug product formulation was included. Animals of all dose groups were euthanized 24h post administration. In the 0.024 mg/m 2 dose group satellite animals were also sacrificed at 6, 24, 48, 72, 96 and 144 h post administration.
  • test product hlFN ⁇ 1 LNP Single-dose inhalation administration of test product hlFN ⁇ 1 LNP in rats led to dose- and time-dependent hlFN ⁇ 1 production in the lungs at 24 h, whereas hlFN ⁇ 1 protein concentrations in animals treated with untranslatable control-mRNA and vehicle were below the lower limit of quantification.
  • Highest hlFN ⁇ 1 protein levels were measured at 6 h post administration and returned to baseline at 48 hours following administration of test product hlFN ⁇ 1 LNP at a dose of 0.024 mg/m 2 (Fig 19B). No hlFN ⁇ 1 was detected in serum.
  • the doses are specified as surface exposure [mg/m 2 ] in lung or nose for better comparability of studies conducted in different species following different routes of administration.
  • the surface exposures were calculated based on deposited hlFN ⁇ 1 LNP following nasal sniffing or inhalation and the respective surface area in the animal models (Fernandes, C. A., and R. Vanbever. 2009. 'Preclinical models for pulmonary drug delivery', Expert Opin Drug Deliv, 6: 1231-45; Gizurarson, S. 1990. Animal models for intranasal drug delivery studies. A review article', Acta Pharm Nord, 2: 105-22). Principles of calculation of surfaces exposures and extrapolation across species are further detailed in Section 7.1.
  • hlFN ⁇ 1 LNP was administered via nose- only inhalation. This included a single-dose group that received the highest dose once, and a vehicle group that underwent three administrations of the vehicle solution, which contains all components of the drug product formulation. Additionally, satellite animals designated for pharmacokinetic (PK) analysis were euthanized immediately after plethysmography on the day of their last administration, which was on day 1 for the single-dose group. hlFN ⁇ 1 protein levels in lung homogenates and serum were measured using a validated ECL assay.
  • test product hlFN ⁇ 1 LNP was administered to Wistar Han rats at doses that result in a nasal surface exposure of 3, 11 and 44 mg/m 2 , respectively.
  • Nasal administration was as a single droplet of 13 ⁇ L per nostril (max. volume retained in the nasal cavity).
  • Single dose administration all dose levels of hlFN ⁇ 1 LNP, vehicle, and control-mRNA and rat IFN ⁇ 1 -encoding mRNAat 0.075 mg/kg bw).
  • Vehicle is the buffer in which the drug product hlFN ⁇ 1 LNP is diluted after purification and contains 10 % Sucrose w/v, 1% P188w/v, 50 mM NaCI, and water for Injection.
  • Control-mRNA is a mRNA in which the ATGs have been scrambled and thus does not express any protein.
  • Repeated dose administration (dose level 0.075 mg/kg bw; day 1 , 3, 5, 7).
  • Control groups received untranslatable control-mRNA or mRNA coding for rat IFN ⁇ at a dose that results in a nasal surface exposure of 11 mg/m 2 . Animals were sacrificed one day after the last administration. The two highest dose groups also included animals that were sacrificed 5 days after the last administration.
  • test product hlFN ⁇ 1 LNP received four administrations of test product hlFN ⁇ 1 LNP at a dose resulting in 11 mg/m 2 nasal surface exposure. Animals of this group were sacrificed 1 day after the last exposure. No hlFN ⁇ 1 protein was measured in serum and lungs confirming the absence of systemic bioavailability of hlFN ⁇ 1 protein encoded by test product hlFN ⁇ 1 LNP upon nasal administration.
  • Example 1 50 ⁇ L of LINPs formulations comprising 0.06 mg of chemically modified mRNA coding for luciferase mRNA per mL were administered to five BALB/c mice by intratracheal instillation of encapsulated formulation.
  • D-Luciferin substrate was applied to animals by intraperitoneal and intranasal application before they were euthanized 6 hours after application. Luciferase activity was measured in explanted organs using a Lumina XR In vivo Imaging system (Perkin Elmer, USA).
  • mice Five mice were sacrificed six hours after intratracheal instillation of 50 ⁇ L (0.06 mg Luciferase mRNA/mL) for formulations with dL_05(R) and cholesterol but different helper and stealth lipid , as well as different lipid-to-lipid ratios. Results are show in Fig 25 (A-D): IVIS imaging of BALB/c mice (left col.) and ex vivo lungs, heart, liver, spleen, and right kidney of the same animals (right col.), (A): Formulation M02-102-006, (B) Formulation M02-103-003, (C) Formulation M02-002-003, and (D) Formulation M02-109-006. Scale represents radiance in p/sec/cm2/sr.
  • a test formulation comprising a combination of LG2C and LE1D was formulated with mRNA coding for luciferase and administered as shown in Table 14 to five BALC mice by instillation as previously described in Example 8 and 9.
  • LG2C N 1 ,N 17 -Didecyl-4,7,11,14-tetrakis(3-(decylamino)-3-oxopropyl)-4,7,11,74- tetraazaheptadecanediamide
  • LE1D (13R,27R)-15, 18,22-Tris((R)-2-hydroxytetradecyl)-15,18,22,25- tetraazanonatriacontane-13,27-diol Result Results are summarized for three mice in Fig 26 (A and B): showing localized expression of luciferase in BALB/c mice (left col.) and after ex vivo analysis of heart, lungs, right kidney, liver, and sple
  • Figure 26 (A) shows an experiment with three mice and Fig 26 (B) an experiment with two mice.
  • Formulation M03-076-3 comprises lipidoids DL_F (LG2C) (formula b-IX) and LE1D (formula b-X). Scale represents radiance in p/sec/cm2/sr.
  • lipidoids DL_F (formula b-IX above), DL_L (formula b-XI) and DL_N (formula b-XI I) were formulated as summarized in Table 15 below. The results after I VIS in mice and their excised organs are shown in Fig 27 (A), (B) and (C).
  • Test formulation The test formulation was formulated with mRNA coding for luciferase and administered as shown in Table 15 to three BALC mice by instillation as previously described in Example 8 and 9.
  • the tested lipidoid comprises amide bonds in its lipid side chains that are potentially biodegradable which is thought to be advantageous regarding long-term tolerability after administration to a subject.
  • This study aimed at exploring a library of lipidoid structures varying the substitution degree of alkyl chains per oligo amine as well as the alkyl chain length. For this purpose, complexes were formed with modified RNA generated in vitro in the presence of 25% m5C and 25% S2U, and encoding for firefly luciferase.
  • test mRNA 50 ⁇ L of the test item was applied to mice using an intratracheal spray application device (MicroSprayer 1A device (PennCentury, USA) under Isofluran inhalation anesthesia.
  • the efficiency of delivery was quantified after 6 h via measurement of reporter protein level (as quantified by a luciferase luminescence signal) in the lung.
  • mice were anesthetized through intraperitoneal injection of medetomidine/midazolam/fentanyl (0.15/2/0.005 mg/kg bw). 50 ⁇ L D-Luciferin (1.5 mg dissolved in PBS) were applied intranasally and 100 ⁇ L (3 mg) intraperitoneally. Bioluminescence Imaging was conducted using an MS Lumina XR in vivo imaging system (Perkin Elmer, USA). After in vivo imaging mice were killed by cervical dislocation. The left kidney artery was dissected, and the circulation was flushed through injection of 5 mL PBS through the right heart ventricle. Lung, liver and spleen were placed on a petri dish and were as well imaged ex vivo.
  • the mRNA coded for Firefly luciferase was generated in vitro in the presence of 25% s2U and 25% m5C.
  • Figure 28 shows MS imaging of C57BL6&JRj female adult mice, 4 hours after intranasal delivery showing localized expression in the nasal airways when administered with 0.15 mg/mL (A), 0.5 mg/mL (B), 1.5 mg/mL (C) modified mRNA coding for luciferase formulated with Formulation I (i.e. , comprising dL_05(R)).
  • Left images show color image and right images show the same mice in grey scale. Circles indicate radiance values measured in regions of interest (ROIs). Scale represents radiance in p/sec/cm2/sr.
  • Formulations LF110, LF181 and LF53, each comprising an ethyl-propyl-ethyl lipidoid different to dL_05(R), showed, surprisingly, a localized expression of luciferase.
  • the results further support that the lipidoids of the invention (as well as compositions comprising the same) in general have the capacity to remain localized to the tissue to which they are administered.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Otolaryngology (AREA)
  • Physics & Mathematics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Dispersion Chemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Pulmonology (AREA)
  • Neurosurgery (AREA)
  • Dermatology (AREA)
  • Nanotechnology (AREA)
  • Optics & Photonics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates to compositions designed for localized delivery within a subject's body. More particularly, the invention pertains to therapeutic compositions that remain localized to specific organs or tissues and do not exhibit systemic distribution. These compositions include specific carriers and therapeutic agents suitable for various medical applications.

Description

Non-Systemic mRNA administration
The present invention relates to compositions designed for localized delivery within a subject's body. More particularly, the invention pertains to therapeutic compositions that remain localized to specific organs or tissues and do not exhibit systemic distribution. These compositions include specific carriers and therapeutic agents suitable for various medical applications and may be used in the prevention and/or treatment of medical conditions.
Background of the Invention
Localized delivery of therapeutic agents to specific tissues, organs, or sites of interest is desirable for treating localized diseases and conditions. Systemic administration of therapeutic agents can lead to unwanted side effects and off-target effects. Therefore, there is a need for compositions that can deliver therapeutic agents specifically to a localized site without systemic distribution.
Summary of the Invention
The invention provides compositions for the delivery of a therapeutic agent that does not spread into the systemic circulation. This is of the highest interest for the treatment of, inter alia, tumors and autoimmune diseases, where the drug substance may cause significant harm ("off-target") when it is spread in the circulation.
The invention provides compositions for localized delivery to an organ or tissue or a localized site of interest, comprising: a) a therapeutic agent; b) a carrier comprising: i. an ionizable cationic lipid or lipidoid, preferably a cationic lipidoid of formula (b-l) ii. a helper lipid, and iii. optionally, a pharmaceutically acceptable excipient or diluent.
Accordingly, the present invention provides for (pharmaceutical) compositions that are particularly useful in the local administration of one or more therapeutic agent(s), as the composition (or preferably at least the therapeutic agent) remains localized at the site of administration. Accordingly, the present invention provides for a (pharmaceutical) composition for use in the treatment and/or prevention of a disease or disorder, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. a ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein said composition, remains localized at the site of administration and/or essentially does not exhibit systemic distribution throughout the patient's body.
The herein provided (pharmaceutical) compositions are particularly useful in the local administration of one or more therapeutic agent(s), as the composition (or preferably at least the therapeutic agent) has/shows a prolonged retention at the site of administration (as compared to a composition of the state of the art, such as a composition comprising DLin- MC3-DMA, as disclosed in US 8,158,601).
In the context of the present invention, prolonged retention at the site of administration may cause the therapeutic agent to (largely, mainly, or even exclusively) exert its (therapeutic) effect at the site of administration, which may be desirable and can reduce off-target effect (i.e., side effect) or the required amount of therapeutic agent and/or (pharmaceutical) composition.
Accordingly, the present invention provides for a (pharmaceutical) composition for use in the treatment and/or prevention of a disease, the treatment comprising local administration of the composition, the composition comprising:
(a) one or more therapeutic agent(s); and
(b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) and/or diluent(s); wherein said composition has a prolonged retention at the site of administration; and/or wherein said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration.
As already mentioned herein above, the herein provided (pharmaceutical) compositions are particularly useful in the local administration I localized administration / local delivery / localized delivery or the like of one or more therapeutic agents as they may allow the skilled artisan to reduce the amount of employed/administered therapeutic agent and/or (pharmaceutical) composition, while still achieving a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration. In the context of the present invention, it may be desirable to reduce the amount of therapeutic agents and/or of the (pharmaceutical) composition, in order to limit potential side effects thereof.
Accordingly, the present invention provides for a (pharmaceutical) composition for use in the treatment and/or prevention of a disease, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration; and/or wherein the patient has less (or fewer) side effects compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
The present invention further relates to a cosmetic composition having/showing local retention, prolonged retention, or the like at the site of administration, site of application, or the like. In this context, the entire cosmetic composition or only parts thereof may show local retention or the like. It is herein preferred that at least the one or more active agent(s) are retained locally. Accordingly, the present invention further provides for a cosmetic composition comprising: a) one or more active agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein said cosmetic composition is an ointment, a creme, a foam, a gel, a lotion, an aqueous liquid, or a powder, or wherein said cosmetic composition is formulated as an ointment, a creme, a foam, a gel, a lotion, an aqueous liquid, or a powder.
The present inventors have surprisingly found that the ionizable lipid and/or the ionizable lipidoid as comprised in the herein provided (pharmaceutical or cosmetic) compositions causes such a composition (or at least the therapeutic agents or the active agent; also collectively referred to as “agents” herein) to essentially remain localized after being administered/applied to a local site. This could not have been foreseen and as already mentioned above, has drastic and advantageous effects, inter alia, on the amount of composition to be administered, potential side effects, and the like. Thus, the herein provided compositions are particularly advantageous and give rise to new clinical situations, by e.g., reducing the amount of agents to be employed.
The present invention is thus based on the finding that various ionizable lipids/ ionizable lipidoids employed herein remain localized at the site of administration, providing a significant advancement in the field of lipid nanoparticle (LNP) technology. This localized or targeted retention of the ionizable lipid at the administration /injection site, inter alia, addresses one of the critical challenges associated with LNP-based drug delivery systems: the risk of Complement Activation-Related Pseudoallergy (CARPA).
CARPA is a non-lgE-mediated hypersensitivity reaction (i.e., an immune reaction that is independent of immunoglobulin E; IgE) triggered by the unintended activation of the complement system, which can occur during the systemic distribution of LNPs (see, e.g., Ferraresso et al., 2014, Molecular Therapy: Methods & Clinical Development (2024), doi: https://doi.Org/10.1016/j.omtm.2024.101314). This reaction can lead to a range of adverse effects, from mild symptoms to severe anaphylaxis-like responses, posing a risk to patient safety. By ensuring that the herein employed ionizable lipids/ionizable lipidoids and the associated LNPs/the associated compositions remain at the injection site, the exposure of the complement system to these particles is significantly reduced, thereby minimizing the risk of systemic complement activation and the subsequent development of CARPA. Accordingly, the herein provided compositions for use in the treatment and/or prevention of a disease or disorder are particularly advantageous as they reduce side effects (such as for example CARPA and/or symptoms associated with or caused by CARPA) that may occur when administering a composition not comprising the herein employed (advantageous) ionizable lipids/lipidoid (i.e., when administering a composition that does not show/have localized retention at the side of administration).
The use of the ionizable lipids/ionizable lipidoids in the herein employed formulations or compositions (such as pharmaceutical or cosmetic compositions) provides a targeted and localized delivery of agents (such as therapeutic agents or active agents). This not only enhances the efficacy of these agents (or drugs) by concentrating them at the desired site of administration/site of action but also reduces the likelihood of adverse immune responses (such as CARPA) that can arise from the widespread distribution of LNPs throughout the body.
In summary, the herein employed ionizable lipids/lipidoids, when used in compositions (such as LNPs or pharmaceutical formulations), offer a reliable approach to mitigate the risk of undesired side effects (such as CARPA) by limiting the systemic exposure of the complement system to, e.g., LNPs. This innovation, inter alia, enhances the safety profile of LNP-based therapies, making them more suitable for a broader range of clinical applications, particularly in patients with a (high) susceptibility to hypersensitivity reactions.
The present invention fulfills a long-felt and unmet need at least in the field of lipid nanoparticle (LNP) technology by providing an inventive approach for the administration of pharmaceutical agents through the localized retention of the ionizable lipid at the injection site. The unique capability of this invention to confine the lipid's presence to the injection site represents a significant advancement that was not previously recognized or addressed. This localized retention effect is crucial for, inter alia, enhancing the safety and efficacy of LNP-based therapies, ensuring that the therapeutic agents remain concentrated at the desired site of action, thereby minimizing systemic exposure and potential side effects. This innovation addresses a critical gap in the development of targeted and safe drug delivery systems.
The enclosed examples illustratively detail the local retention of therapeutic agents/active agents/active ingredients. In particular, Example 1 illustratively shows that intramuscular injection of a composition comprising an ionizable lipidoid in accordance with the present invention and further comprising a chemically modified RNA encoding for a luciferase gene results in the expression of said luciferase only at the local site of administration (see, for example, Figs 2 to 4). The present inventors considered that the herein provided compositions interacted with the extracellular matrix, and thus, co-administered a composition according to the present invention with hyaluronidase (also referred to as Hylase herein; an enzyme that degrades hyaluronic acid in the extracellular matrix). Fig 6 clearly shows that the co- administration of hyaluronidase results in increased distribution of luciferase expression which is indicative for a loss of/reduced local retention as compared to Fig 5, in which no hyaluronidase was co-administered. The comparison of Fig 7 and 8 (i.e., the assessment of luciferase activity in excised organs from the mice shown in Figs 5 and 6, respectively) further corroborates these results and demonstrates that the herein provided compositions likely interact with the extracellular matrix and are thereby locally retained. Without wishing to be bound by theory, the herein provided compositions may interact with (bind to) / be capable of interacting with (binding to) the extracellular matrix after local administration and thereby exert the herein described effects, e.g. remain localized, essentially not exhibit systemic distribution, prolonged retention, reduced amount of composition or therapeutic agent and/or less side effects.
As is detailed in enclosed Example 4, Interferons are cytokines with relevance for example in controlling inflammation, in particular in the context of viral infections. Thus, the ectopic expression interferons such as human interferon lambda 1 (hlFNλ 1) is of high relevance in the treatment or prevention of e.g., viral diseases. However, the activity of hlFNλ 1 (as well as many other therapeutic agents) and as such also its therapeutic effect may also depend on the tissue or organ of expression. The present inventors have surprisingly found that the herein provided composition, when comprising/encapsulating an mRNA encoding for hlFNλ1 as a therapeutic agent results in the local retention of said mRNA at the site of administration (i.e., the lung in this example; see for example Fig 18). Accordingly, the present inventors have surprisingly shown the localized retention of a therapeutically relevant active agent (here, mRNA encoding hlFNλ1).
Furthermore, it was surprisingly found that the herein disclosed local retention effect (i.e., the local retention of a composition to be administered in accordance with the present invention) can be achieved with the lipidoids of Formula (b-l) (as discussed above, likely causing this local retention), with various different helper lipids comprised in the to be administered composition, and/or with compositions comprising varying concentrations of such cationic lipidoids and/or helper lipids (see, e.g., Examples 8 to 10). This indicates that the composition to be employed in the context of the present invention is not particularly limited, as long as it comprises a suitable therapeutic or cosmetic agent and an ionizable lipid and/or an ionizable lipidoid capable of causing local retention of said compositions. Such suitable ionizable lipids and agents are further detailed herein below and the general effect of local retention as caused by such ionizable lipids and lipidoids is illustrated by exemplary and non-limiting ionizable lipids and lipidoids in the enclosed examples.
Further, it was illustratively demonstrated that the local retention of a composition can be achieved across a variety of different local administration routes including, for example, intratracheal delivery such as intratracheal instillation or intratracheal microspray, subcutaneous delivery, aerosol delivery to the airways, such as upper airways or the lungs, intramuscular delivery, and nasal aerosol or nasal spray delivery (see, e.g., Examples 1 , 3. 4, and 11). This suggests that the type of local administration is not particular limiting in the context of the present invention as long as an interaction of the to be administered composition (in particular of the ionizable lipid and/or ionizable lipidoid) with the extracellular matrix can occur.
This localized expression is highly surprising when compared to the known localization of a composition of a state in the art, such as lipid nanoparticle-based mRNA vaccines, in particular Comimaty® (comprising ALC-0315) or Spikevax® (comprising SM-102) or Onpattro® (patisiran) (comprising DLin-MC3-DMA, also referred to as “MC3” in the context of the present invention), used for the treatment of hereditary transthyretin-mediated (hATTR) amyloidosis. For example, after intramuscular injection of 50 micrograms of mRNA formulated as Comirnaty (i.e., formulated using ALC-0315), mRNA expression can be found in the plasma, liver, adrenal glands, spleen, and ovaries (see Table 1). Similarly, systemic movement of the intramuscular injected LNP/mRNA was observed for Spikevax®, as summarized in Table 2 below (Source 1 EMA Assessment report Comirnaty, 21 December 2020 and forSpikevax: 1 EMA Assessment report Spikevax, 11 March 2021, EMA/15689/2021 Corr.1*1 , Committee for Medicinal Products for Human Use (CHMP). Accordingly, the intramuscular injection of state-of-the-art compositions (in particular lipid nanoparticle compositions) results in the (systemic or partly systemic) distribution of the active ingredient to various organs. As mentioned above, such wide-spread distribution might cause various side effects and is thus generally undesired.
Table 1 - Distribution of Comirnaty® after intramuscular injection
Figure imgf000008_0001
Table 2 - Distribution of Spikevax® after intramuscular injection
Figure imgf000009_0001
It is thus necessary to find new delivery systems that are spatially limited to the tissues or organs wherein expression was intended.
The present invention solves the above problems with the embodiments of the invention. As mentioned above and while not wanting to be bound by any theory, it is believed that the present invention achieved advantageous results by providing formulations that effectively interact with the extracellular matrix. Destruction of the extracellular matrix with hyaluronidase (Hylase) re-establishes a systemic delivery of the delivery therapeutic agent, particularly a chemically modified mRNA. In particular, the invention relates to the embodiments as recited in following items: Item 1 . A composition for local delivery to an organ or tissue or a localized site of interest, comprising: a) A therapeutic agent; and a carrier comprising: b) an ionizable cationic lipid or lipidoid, and c) a helper lipid, and d) optionally a pharmaceutically acceptable excipient or diluent wherein said composition, when administered at a localized site of interest within a subject's body, remains localized and essentially does not exhibit systemic distribution throughout the subject's body.
Item 2. The composition of Item 1 wherein the ionizable cationic lipidoid is selected from a compound of formula (b-l):
Figure imgf000010_0001
Preferably wherein the variables a, b, p, m, n and R1A to R6A are defined as follows: a is 1 and b is an integer of 2 to 4; or a is an integer of 2 to 4 and b is 1 , p is 1 or 2, m is 1 or 2; n is 0 or 1 and m+n is s 2; and
R1A to R6A are independently of each other selected from hydrogen; CH2CH(OH)R7A, CH(R7A)- CH2-OH,
-CH2-CH2-(C=O)-O-R7A, or CH2R7A; wherein R7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; -C(NH)-NH2; a poly(ethylene glycol) chain; and a receptor ligand; provided that at least two residues among R1A to R6A are a group -CH2-CH(OH)-R7A, -CH(R7A)-CH2OH, -CH2CH2(C=O)-O-R7, - CH2CH2(C=O)-NH-R7A or -CH2R7 wherein R7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms contained in the compound of formula (b-l) are optionally protonated to provide a compound carrying a positive charge.
Item 3. The composition according to any one of Items 1 or 2, wherein the cationic lipidoid comprises a compound of formula (b-V) and/or formula (b-VII):
Figure imgf000011_0001
Item 4. The composition according to any one of Items 1 to 3, wherein the formulation or the carrier comprises a lipid or lipidoid nanoparticle. Item 5. The composition according to any one of Items 1 to 4, wherein the therapeutic agent is a) an anionic therapeutical substance and/or b) a nucleic acid, preferably an RNA, more preferably an mRNA, a miRNA and/or an siRNA, most preferably an mRNA encoding one or more polypeptide(s).
Item 6. The composition according to Items 5, wherein the mRNA comprises one or more of the following: a) a CAP, preferably an anti-Reverse Cap Analog (ARCA) at its 5’ end, b) a 5’-untranslated region (5’-UTR) upstream of a coding sequence encoding the polypeptide(s), c) a 5’-UTR comprising before an initiation codon sequence of the mRNA, an elongated Kozak sequence: GCCACCAUG, d) a 5’-UTR comprising immediately upstream of an initiation codon sequence of the mRNA any one of the following sequences: a) GGGAGACGCCACC (SEQ ID NO:11), b) GAAGCGCCACC (SEQ ID NO: 12), c) GGGACGCCACC (SEQ ID NO: 13), d) GGGAGACTGCCACC (SEQ ID NO: 14), e) GAAGCTGCCACC (SEQ ID NO: 15), f) GGGACTGCCACC (SEQ ID NO: 16). e) a 3’-untranslated region (3’-UTR) downstream of a coding sequence encoding the polypeptide(s), and/or d) a 3’-UTR sequence selected from: a) GAAUU, or b)CCTCGCCCCGGACCTGCCCTCCCGCCAGGTGCACCCACCTGCAATAAATGCAGCGA AGO CGGGA (SEQ ID NO:26).
Item 7. The composition according to any one of Items 5 or 6, wherein the mRNA is a product of in-vitro transcription (IVT).
Item 8. The composition according to any one of Items 5 to 7, wherein the mRNA comprises a polyadenylation (poly(A)) tail downstream of an open reading frame (ORF) encoding the polypeptide.
Item 9. The composition according to any one of Items 5 to 8, wherein the mRNA comprises one or more modified nucleosides.
Item 10. The composition according to any one of Items 5 to 9, wherein the mRNA comprises one or more modified nucleosides selected from any of the following: pseudouridine, N1-methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2- thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4- thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-iodo-uridine, 5-methoxyuridine, 2'- O-methyl uridine, 5-iodocytidine, 5-methylcytosine, 5-methylcytidine, N1 -methyladenosine, /V6-methyladenosine.
Item 11. The composition according to any one of Items 5 to 10, wherein the mRNA comprises one or more modified nucleosides selected from any of the following, wherein the one or more modified nucleosides comprise a 1 -methylpseudouridine ( m1ψ ) modification.
Item 12. The composition according to any one of Items 5 to 11, wherein any of the following applies: a) wherein at least 50% of the uridines in the ORF have been modified, b) wherein at least 50% of the uridines in the mRNA have been modified, c) wherein at least 50 % of the uridines in the ORF have been modified to m1 , d) wherein at least 50 % of the uridines in the mRNA have been modified to m1ψ . e) wherein 5 to 50% of the uridine nucleotides are 5-iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine, f) wherein 5 to 50% of the uridine nucleotides are 2-thiouridine and 5 to 50% of the cytidine nucleotides are 5-methylcytidine.
Item 13. The composition according to any one of Items 1 to 12, wherein the formulation comprises a lipid or lipidoid nanoparticle optionally comprising any of the following: a) a nucleic acid, preferably an RNA coding for a microRNA or an mRNA coding for a functional protein, or an antigen such, optionally as defined in any one of Items 6 to 12, b) a cationic lipidoid of formula (b-l), preferably a cationic lipidoid of formula (b-V) or (b- VII), most preferably a cationic lipidoid of formula (b-V), and/or c) one or more helper lipid(s), optionally selected from: c1) a phospholipid, and/or c2) a sterol, and/or c3) stealth lipid, optionally, components b), and c1-c3) are all present, optionally they are at the molar ratios of about 8.0: about 5.3: about 4.4: about 0.9, respectively, optionally, a triblock copolymer which contains one polypropylene oxide) block, and two poly(ethylene oxide) blocks is comprised in the LNP or LiNP as component (p) or is present in a vehicle or in an aqueous phase of the composition.
Item 14. The composition according to Item 13, wherein the cationic lipidoid is the cationic lipidoid of formula (b-V) and optionally: a) is an R isomer of (b_V), as shown in formula (b-VI) formula (b-VI)
Figure imgf000014_0001
and/or b) is present at a molar ratio % of 22 to 65%, preferably 34% to 52%, more preferably 36% to 50%, and most preferably 43.1%.
Item 15. The composition according to Item 13, wherein the helper lipid is a phospholipid, preferably: a) selected from phosphocholine (PC) or phosphoethanolamine (PE), most preferably phosphocholine, b) has a carbon chain length of 14 to 18, most preferably 16, and/or c) is present in a molar ratio of 10% to 45%, preferably 18% to 39%, more preferably 24% to 33%, and most preferably 28.5%.
Item 16. The composition according to claim 13, wherein the helper lipid is a sterol, preferably: a) cholesterol, b) is present at a molar ratio% of 12% to 38.5%, preferably 15% to 32%, more preferably 19% to 29%, and most preferably 23.7%.
Item 17. The composition according to Item 13, wherein the helper lipid is a stealth lipid, preferably wherein: a) is glycerolipid-based (G) or phosphoethanolamine lipid-based (PE), b) has a carbon chain length of 14 to 18, most preferably 14, c) the PEG is from 2000 to 5000 Daton, most preferably 2000 Dalton, and/or d) is present Molar ratio %: 1.5 to 7%, preferably 3 to 6%, more preferably 4 to 5%, and most preferably 4.7%.
Item 18. The composition according to Items 13, wherein
(i) the phospholipid of c1 ) is preferably a phospholipid with a carbon chain of C12 to C18, more preferably a C16 phospholipid, most preferably DPPC, and/or
(ii) the sterol of c2) is cholesterol, and/or
(iii) the stealth lipid of c3) is a PEGylated lipids, preferably a PEGylated lipid with a molar weight of the PEG chain between about 2000 to about 5000 Dalton, more preferably a PEGylated lipid with a molar weight of the PEG chain of about 2000 Dalton, most preferably the PEGylated lipid is DMG-PEG2000.
Item 19. The composition according to any one of Items 13 to 18, wherein said antigen is selected from the group consisting of viral antigens, bacterial antigens, a cancer or tumor associated antigen, and allergens.
Item 20. The composition of anyone of Items 1 to 18, wherein said localized site of interest comprises a specific tissue, organ, or anatomical region, preferably the specific tissue, organ, or anatomical region selected from the lungs, the nose, the heart, the brain, the spleen, the lymph nodes, the bones, the tendons, the skeletal muscles, joints, the stomach, the small intestine, the large intestine, the kidneys, the bladder, the breast, the testes, the ovaries, the uterus, the spleen, the thymus, the brainstem, the cerebellum, the spinal cord, the eye, the ear, the tongue, the skin and/or tumors present in said tissues, organs and anatomic regions.
Item 21. The composition according to any of Items 1 to 20, further comprising stabilizing agents, adjuvants, or immunomodulators. Item 22. The composition according to any of Items 1 to 21 , wherein said therapeutic agent or carrier is encapsulated within a hydrogel or a biocompatible matrix to enhance localized retention.
Item 23. The composition according to any of Items 1 to 22, for use as a medicament.
Item 24 The composition according to any of Items 1 to 18 for use in the prevention, treatment, or amelioration of a viral infection, preferably by immunization, more preferably by local immunization.
Item 25. The composition according to any one of Items 1 to 18, for use in the treatment of a disease, wherein the disease is selected from: treatment of mutations, autoimmune diseases, metabolic imbalances, neurodegenerative disorders, degenerative disorders of the joints, solid tumor diseases (e.g. soft tissue tumors, tumors of the heart, the lungs, the liver, the spleen, the kidneys, the brain, oral cavity, the intestine, the skin, the pancreas, the prostate gland, the mammaria, the ovaries, the urinary bladder, the bones (i.e. osteosarkoma, chondrosarkoma, Ewing sarkoma)), tumors of pleural and periotoneal cavity, lung diseases, bone fracture or lesions thereof, tendon ruptures or lesions thereof, joint infections and ligament ruptures, arthrosis, arthritis, bacterial infections, preferably infections due to Methicillin resistant Staphylococcus Aureus (MRSA), Multidrug resistant Tuberculosis, viral infection, preferably a virus infection selected from Influenza (Flu), Hepatitis A, Hepatitis B, Human Papillomavirus (HPV), Measles, Mumps, Rubella, Polio, Rabies, Varicella (Chickenpox), Shingles (Herpes Zoster), Rotavirus, Yellow Fever, Smallpox, Japanese Encephalitis, Tick-Borne Encephalitis (TBE), Dengue Fever, West Nile Virus, Chikungunya Virus, Ebola Virus, Marburg Virus, Human Immunodeficiency Virus (HIV), COVID-19 most preferably coronavirus infection.
Item 26. Use of the composition according to any one of Items 1 to 18, in the manufacture of a medicament for the prevention, treatment, or amelioration of a mutations, lung diseases, bone fracture or lesions thereof, tendon fracture or lesions thereof, bacterial infections, viral infection, an infection, preferably a virus infections, more preferably selected from Influenza (Flu), Hepatitis A, Hepatitis B, Human Papillomavirus (HPV), Measles, Mumps, Rubella, Polio, Rabies, Varicella (Chickenpox), Shingles (Herpes Zoster), Rotavirus, Yellow Fever, Smallpox, Japanese Encephalitis, Tick-Borne Encephalitis (TBE), Dengue Fever, Human Immunodeficiency Virus (HIV), COVID-19 most preferably coronavirus infection.
Item 27. A method of inducing an immune response in a subject, which comprises administering to the subject an effective amount of a composition according to any of Items 1 to 8. Item 28. A method of immunizing a subject against a pathogen, which comprises administering to the subject an effective amount of an mRNA in a pharmaceutical composition according to any one of Items 1 to 18.
Item 29. The method of Item 28, wherein said mRNA vaccine is administered via intradermal, subcutaneous, intramuscular, or intratumoral injection.
Item 30. The method or composition of any of Items 28 to 29, wherein said mRNA vaccine elicits an immune response predominantly at the localized site of interest, thereby reducing the risk of systemic adverse effects.
Item 31. The method or composition of any of Items 28 to 31 , wherein said mRNA vaccine is designed to promote local production of antigen-specific antibodies or cellular immune responses at the localized site of interest.
Item 32. The method or composition of any of Items 29 to 31 , wherein said mRNA vaccine further comprises a polymeric coating or encapsulation to enhance local retention and prevent systemic dissemination.
Item 33. The composition or method of any of Items 1-18 or Items 28 to 32 wherein the LNP further comprises a targeting moiety or ligand that specifically binds to cells or receptors present at the localized site of interest, thereby enhancing the vaccine's specificity and efficacy.
Item 34. The composition or method of any of Items 1 -18 or Items 28 to 33, wherein said pharmaceutically acceptable excipient or diluent further comprises a biodegradable or bioresorbable material, facilitating gradual release and local persistence of the mRNA at the localized site of interest.
Item 35. The composition or method of any of Items 1 -18 or Items 28 to 34, wherein said mRNA further comprises a tissue-specific promoter and/or enhancer element to enhance the expression of the target antigen at the localized site of interest.
Item 36. The composition or method of any of Items 1 -18 or Items 28 to 35, wherein said mRNA is encapsulated within a biocompatible microneedle patch or implantable device, facilitating controlled and sustained release of the nucleic acid at the localized site of interest.
Item 37. The composition or method of any of Items 1 -18 or Items 28 to 36, wherein said mRNA further comprises a self-amplifying mRNA (saRNA) molecule, enabling enhanced protein or antigen production at the localized site of interest. Item 38. The composition or method of any of Items 1-18 or Items 28 to 37, wherein the mRNA codes for an antigen.
The present invention further relates, inter alia, to the following items:
1. A composition for use in the treatment and/or prevention of a disease or disorder, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein said composition, remains localized at the site of administration and/or essentially does not exhibit systemic distribution throughout the patient's body.
2. A composition for use in the treatment and/or prevention of a disease, the treatment comprising local administration of the composition, the composition comprising:
(a) one or more therapeutic agent(s); and
(b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) and/or diluent(s); wherein said composition has a prolonged retention at the site of administration; and/or wherein said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration.
3. A composition for use in the treatment and/or prevention of a disease, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration; and/or wherein the patient has less side-effects compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration. The composition for use according to item 1 or 2, wherein a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration; and/or wherein the patient has less side-effects compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration. The composition for use according to item 1 or 3, wherein said composition has a prolonged retention at the site of administration; and/or wherein said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration. The composition for use according to item 2 or 3, wherein said composition, when administered to said site of administration, remains localized and essentially does not exhibit systemic distribution throughout the patient's body. A cosmetic composition comprising: a) one or more active agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein said cosmetic composition is an ointment, a creme, a foam, a gel, a lotion, an aqueous liquid, or a powder, or wherein said cosmetic composition is formulated as an ointment, a creme, a foam, a gel, a lotion, an aqueous liquid, or a powder. The composition for use according to any one of items 1 to 6, or the cosmetic composition according to item 7, wherein the carrier is a lipid nanoparticle (LNP), a lipidoid nanoparticle (LiNP), a liposome, a micelle, an emulsion, a Nanostructured Lipid Carrier (NLCs), or a Lipid-Drug Conjugate (LDC), preferably an LNP or an LiNP, and/or wherein the agent is formulated as a lipid nanoparticle (LNP), a lipidoid nanoparticle (LiNP), a liposome, a micelle, an emulsion, a Nanostructured Lipid Carrier (NLCs), or a Lipid-Drug Conjugate (LDC), preferably as an LNP or as an LiNP. The composition for use or the cosmetic composition according to item 8, wherein said ionizable lipidoid is a compound of formula (b-l):
Figure imgf000020_0001
formula (b-l), wherein a is 1 or 2 and b is an integer of 1 to 4, or a is an integer of 1 to 4 and b is 1 or 2, p is 1 or 2, m is 1 or 2, n is 0 or 1 , m+n is ≥ 2, and
R1A to R6A are independently of each other selected from hydrogen, -CH2-CH(OH)-R7A, -CH(R7A)-CH2-OH, -CH2-CH2-C(=O)-O-R7A, -CH2-CH2-C(=O)-NH-R7A, and -CH2-R7A, wherein R7A is selected from C3-C18 alkyl, C3-C18 alkenyl having one C-C double bond, a protecting group for an amino group, -C(NH)-NH2, a polyethylene glycol) chain, and a receptor ligand; and wherein at least two residues among R1A to R6A are a group selected from -CH2-CH(OH)-R7A, -CH(R7A)-CH2OH, -CH2-CH2-C(=O)-O-R7A,
-CH2-CH2-C(=O)-NH-R7A, and -CH2R7A, wherein R7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms comprised or contained in the compound of formula (b-l) are optionally protonated to provide a compound carrying one or more positive charges, preferably wherein the variables a, b, p, m, n and R1A to R6A are defined as follows: a is 1 and b is an integer of 2 to 4, or a is an integer of 2 to 4 and b is 1 , p is 1 or 2, m is 1 or 2, n is 0 or 1 , m+n is ≥ 2, and R1A to R6A are independently of each other selected from hydrogen, -CH2-CH(OH)-R7A,
-CH(R7A)-CH2-OH, -CH2-CH2-C(=O)-O-R7A, -CH2CH2C(=O)-NH-R7A, and -CH2-R7A, wherein R7A is selected from C3-C18 alkyl, C3-C18 alkenyl having one C-C double bond, a protecting group for an amino group, -C(NH)-NH2, a polyethylene glycol) chain, and a receptor ligand; and wherein at least two residues among R1A to R6A are a group selected from -CH2- CH(OH)-R7A, -CH(R7A)-CH2OH, -CH2CH2-C(=O)-O-R7A, -CH2CH2-C(=O)-NH- R7A, and -CH2R7A, wherein R7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms comprised or contained in the compound of formula (b-l) are optionally protonated to provide a compound carrying one or more positive charges. The composition for use or the cosmetic composition according to item 8 or 9, wherein said ionizable lipidoid is a compound of formula (b-ll):
Figure imgf000021_0001
formula (b-ll), wherein a is 1 or 2, preferably 1 , b is 1 or 2, preferably 2,
R1A to R6A are defined as in item 9, and wherein one or more of the nitrogen atoms comprised or contained in the compound of formula (b-ll) are optionally protonated to provide a compound carrying one or more positive charges. The composition for use or the cosmetic composition according to any one of items 8 to 10, wherein R1A to R6A are independently of each other selected from hydrogen, -CH2-CH(OH)-R7A, -CH2-CH2-C(=O)-O-R7A, -CH2CH2-C(=O)-NH-R7A, wherein R7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond, and wherein at least three, preferably at least four of R1A to R6A are selected from -CH2-CH(OH)-R7A, -CH2-CH2-C(=O)-O-R7A, and -CH2-CH2-C(=O)-NH-R7A, wherein R7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond.
12. The composition for use or the cosmetic composition according any one of items 9 to 11 , wherein said ionizable lipidoid comprises or consists of a compound of formula (b- V), formula (b-XI) and/or formula (b-XII), preferably a compound of formula (b-V):
Figure imgf000022_0001
13. The composition for use or the cosmetic composition according to any one of items 8 to 12, wherein said ionizable lipidoid comprises or consists of a compound of formula (b-IX) and/or a compound of formula (b-X), preferably a compound of formula (b-X):
Figure imgf000023_0001
The composition for use or the cosmetic composition according to any one of items 8 to 13, wherein said carrier comprises at least two ionizable lipids and/or at least two ionizable lipidoids. The composition for use or the cosmetic composition according to item 14, wherein said at least two ionizable lipids and/or said at least two ionizable lipidoids are as defined in any one of items 9 to 14. The composition for use or the cosmetic composition according to item 15, wherein said carrier comprises a lipidoid according to formula (b-IX) and a lipidoid according to formula (b-X). The composition for use or the cosmetic composition according to any one of items 8 to 11, wherein said ionizable lipidoid comprises or consists of a compound of formula (b-VII) or a compound of formula (b-VIII), preferably a compound of formula (b-VII):
Figure imgf000024_0001
18. The composition for use or the cosmetic composition according to any one of items 8 to 12, wherein said ionizable lipidoid is a compound of formula (b-V) and preferably: a) is an R isomer of the compound of formula (b-V), and/or b) is present at a molar ratio of about 22 mol% to about 65 mol%, preferably about 34 mol% to about 52 mol%, more preferably about 36 mol% to about 50 mol%, and most preferably about 43.1 mol%.
19. The composition for use or the cosmetic composition according to any one of items 8 to 18, wherein said one or more helper lipid(s) are selected from the group consisting of a) to c): a) a phospholipid; b) a sterol; and/or c) a stealth lipid.
20. The composition for use or the cosmetic composition according to item 19, wherein said composition comprises said ionizable lipid and/or said ionizable lipidoid, said phospholipid, said sterol, and said stealth lipid, preferably at a molar ratio of about 8.0 : about 5.3 : about 4.4 : about 0.9,
21. The composition for use or the cosmetic composition according to item 19 or 20, wherein said phospholipid: a) is selected from phosphocholine (PC) or phosphoethanolamine (PE), preferably PC; b) has a carbon chain length of about 14 to about 18, most preferably about 16; and/or c) is present in a molar ratio of about 10 mol% to about 45 mol%, preferably about 18 mol% to about 39 mol%, more preferably about 24 mol% to about 33 mol%, and most preferably about 28.5 mol%.
22. The composition for use or the cosmetic composition according to any one of items 19 to 21, wherein said sterol: a) is cholesterol; and/or b) is present at a molar ratio of about 12 mol% to about 38.5 mol%, preferably about 15 mol% to about 32 mol%, more preferably about 19 mol% to about 29 mol%, and most preferably about 23.7 mol%.
23. The composition for use or the cosmetic composition according to any one of items 19 to 22, wherein said stealth lipid: a) is glycerolipid-based or PE lipid-based; b) has a carbon chain length of about 14 to about 18, most preferably about 14; c) comprises polyethylene glycol (PEG), and wherein said PEG has a molar mass of about 2000 to about 5000 Daton, most preferably about 2000 Dalton; and/or d) is present molar ratio of about 1.5 mol% to about 7 mol%, preferably about 3 mol% to about 6 mol%, more preferably about 4 mol% to about 5 mol%, and most preferably about 4.7 mol%.
24. The composition for use or the cosmetic composition according to any one of items 19 to 23, wherein: a) the phospholipid is preferably a phospholipid with a carbon chain length of about 12 to about 18, more preferably phospholipid with a carbon chain length of about 16, most preferably DPPC; b) the sterol is cholesterol; and/or c) the stealth lipid is a PEGylated lipid, preferably a PEGylated lipid with a molar mass of the PEG chain between about 2000 to about 5000 Dalton, more preferably a PEGylated lipid with a molar mass of the PEG chain of about 2000 Dalton, most preferably the PEGylated lipid is DMG-PEG2000.
25. The composition for use or the cosmetic composition according to any one of items 8 to 24, wherein said composition further comprises a triblock copolymer as component (p) preferably wherein said triblock copolymer comprises about one polypropylene oxide) block and about two polyethylene oxide) blocks. The composition for use according to any one of items 8 to 25, wherein said one or more therapeutic agent(s) is/are a) an anionic therapeutical substance and/or b) a nucleic acid, preferably an RNA, more preferably an mRNA, a miRNA, and/or an siRNA, even more preferably an mRNA, most preferably an mRNA comprising an open reading frame (ORF) encoding one or more polypeptide(s). The composition for use according to item 26, wherein said nucleic acid is an RNA encoding a microRNA or wherein said nucleic acid is an mRNA comprising an ORF encoding one or more polypeptides, preferably wherein said one or more polypeptide(s) are one or more functional protein(s) and/or one or more antigen(s). The composition for use according to item 27, wherein said one or more antigen(s) is/are selected from the group consisting of a viral antigen, a bacterial antigen, a cancer, and/or tumor associated antigen, and/or an allergen. The composition for use according to any one of items 26 to 28, wherein the mRNA comprises one or more features selected from the group consisting of the following: a) a CAP, preferably an anti-Reverse Cap Analog (ARCA) at its 5’ end, b) a 5’-untranslated region (5’-UTR) upstream of the ORF encoding said one or more polypeptide(s), c) a 5'-UTR comprising an elongated Kozak sequence (GCCACCAUG; SEQ ID NO: 44) upstream of the initiation codon of the ORF, d) a 5’-UTR comprising proximately upstream of an initiation codon of the ORF any one of the following sequences: i. GGGAGACGCCACC (SEQ ID NO: 11), ii.GAAGCGCCACC (SEQ ID NO: 12), iii. GGGACGCCACC (SEQ ID NO:13), iv. GGGAGACTGCCACC (SEQ ID NO:14), v.GAAGCTGCCACC (SEQ ID NO: 15), vi. GGGACTGCCACC (SEQ ID NO:16). e) a 3’-untranslated region (3’-UTR) downstream of the ORF encoding said one or more polypeptide(s), and f) a 3’-UTR sequence downstream of the ORF encoding said one or more polypeptide(s) selected from: i. GAAUU, and ii.
Figure imgf000026_0001
30. The composition for use according to any one of items 26 to 29, wherein the mRNA is a product of in-vitro transcription (IVT).
31. The composition for use according to any one of items 26 to 30, wherein the mRNA comprises a polyadenylation signal or a (poly(A)) tail downstream of the ORF encoding said one or more polypeptide(s).
32. The composition for use according to any one of items 26 to 31, wherein the mRNA comprises one or more modified nucleosides.
33. The composition for use according to item 26 to 32, wherein the one or more modified nucleosides are selected from the group consisting of the following: N1 -methylpseudouridine (ml i ), pseudouridine, N1 -ethyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1- methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-iodo-uridine, 5-methoxyuridine, 2'-O-methyl uridine, 5- iodocytidine, 5-methylcytosine, 5-methylcytidine, N1 -methyladenosine, and N6- methyladenosine, preferably N1 -methylpseudouridine.
34. The composition for use according to item 32 or 33, wherein any one or more of the following applies: a) wherein up to 100% of the uridines comprised in the ORF have been modified, preferably wherein at least about 50 mol% of the uridines comprised in the ORF have been modified, more preferably wherein any value between 50 and 100% has been modified, even more preferably wherein 100% of the uridines have been modified; b) wherein at least about 50 mol% of the uridines comprised in the mRNA have been modified; c) wherein at least about 50 mol% of the uridines comprised in the ORF have been modified to m1ψ ; d) wherein at least about 50 mol% of the uridines comprised in the mRNA have been modified to m1ψ ; e) wherein about 5 mol% to about 50 mol% of the uridines comprised in the mRNA are 5-iodouridines and about 5 mol% to about 50 mol% of the cytidines comprised in the mRNA are 5-iodocytidines; and/or f) wherein about 0.5 mol% to about 50 mol% of the uridines comprised in the mRNA are 2-thiouridine, preferably about 1 mol% to about 50 mol%, more preferably 1 mol% to 5 ml% of the uridines are 2-thiouridine, and of the cytidines comprised in the mRNA 0.5 mol% to about 50 mol% are 5-methylcytidines.
35. The composition for use according to any one of items 8 to 34, wherein the composition is to be administered to a patient in need thereof.
36. The composition for use according to any one of items 8 to 35, wherein said site of administration comprises a tissue, an organ, and/or an anatomical region, preferably said solid tissue, organ, and/or anatomical region is a solid tissue, organ and/or anatomical region, more preferably said solid tissue, organ, and/or anatomical region is selected from the group consisting of the lungs, the nose, the heart, the brain, the spleen, the lymph nodes, the bones, the tendons, the skeletal muscles, joints, the stomach, the small intestine, the large intestine, the kidneys, the bladder, the breast, the testes, the ovaries, the uterus, the spleen, the thymus, the brainstem, the cerebellum, the spinal cord, the eye, the ear, the tongue, the skin and/or tumors present in said solid tissues, organs and/or anatomical regions.
37. The composition for use according to any one of items 8 to 36, further comprising one or more stabilizing agent(s), adjuvant(s), and/or immunomodulator(s).
38. The composition for use according to any one of items 8 to 37, wherein said therapeutic agent or carrier is encapsulated within a hydrogel or a biocompatible matrix.
39. A method for preventing, treating, and/or ameliorating a disease, wherein the method comprises administering an effective amount of the composition as defined in any one of items 1 to 6 and 8 to 38 to a subject.
40. Use of a composition as defined in any one of items 1 to 6 and 8 to 38 in the manufacture of medicament for the prevention, treatment, and/or amelioration of a disease.
41. The composition for use according to any one of items 1 to 6 and 8 to 38, the method of treatment according to item 39, or the use of the composition according to item 40, wherein the prevention of said disease comprises prevention by immunization, even more preferably in the prevention by local immunization. The composition for use according to any one of items 1 to 6, 8 to 38, and 41 , the method of treatment according to item 39 or 41 , or the use of the composition according to item 40 or 41 , wherein said disease is selected from: genetic mutations, autoimmune diseases, metabolic imbalances, neurodegenerative disorders, degenerative disorders of the joints, arthrosis, arthritis, bone fractures, non-union fractures, solid tumor diseases (including soft tissue tumors, tumors of the heart, the lungs, the liver, the spleen, the kidneys, the brain, the oral cavity, the intestine, the skin, the pancreas, the prostate gland, the mammary glands, the ovaries, the urinary bladder, the bones (including osteosarcoma, chondrosarcoma, Ewing sarcoma)), tumors of the pleural and the peritoneal cavity, diseases of the respiratory system including rhinitis and lung diseases such as asthma, viral induced asthma, COPD, including lung autoimmune diseases and ciliopathies, bone fractures or lesions thereof, tendon fractures or lesions thereof, joint infections, ligament ruptures, resistant Staphylococcus Aureus (MRSA) and/or Multidrug resistant Tuberculosis), viral infections, preferably a viral infection, more preferably a viral infection selected from enterovirus, rhinovirus, Influenza (Flu), respiratory syncytial virus (RSV) Hepatitis A, Hepatitis B, Hepatitis C, Human Papillomavirus (HPV), Measles, Mumps, Rubella, Polio, Rabies, Varicella (Chickenpox), Shingles (Herpes Zoster), Rotavirus, Yellow Fever, Smallpox, Japanese Encephalitis, Tick-Borne Encephalitis (TBE), Dengue Fever, West Nile Virus, Chikungunya Virus, Ebola Virus, Marburg Virus, Human Immunodeficiency Virus (HIV), a coronavirus infection (including COVID-19), most preferably a coronavirus infection. The composition for use according to any one of items 1 to 6, 8 to 38, 41 , and 42, the method of treatment according to any one of items 39, 41, and 42, or the use of the composition according to any one of items 40 to 42, wherein said composition is to be administered to one or more solid tissue(s), solid organ(s) and/or solid anatomical region(s), preferably wherein said one or more solid tissue(s), solid organ(s) and/or solid anatomical region(s) are selected from the group consisting of the lungs, the nose, the heart, the brain, the spleen, the lymph nodes, the bones, the tendons, the skeletal muscles, the joints, the stomach, the small intestine, the large intestine, the kidneys, the bladder, the breast, the testes, the ovaries, the uterus, the spleen, the thymus, the brainstem, the cerebellum, the spinal cord, the eye, the ear, the tongue, the skin and/or tumors present in said one or more solid tissue(s), solid organ(s) and/or solid anatomical region(s). The composition for use according to any one of items 1 to 6, 8 to 38, and 41 to 43, the method of treatment according to any one of items 39, and 41 to 43, or the use of the composition according to any one of items 40 to 43, wherein at least about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of said one or more therapeutic agent(s) are restricted to the treated tissue, organ, and/or anatomical region, as quantified by a method selected from the group consisting of qPCR, HPLC, mass spectrometry, combinations of HPLC and mass spectrometry.
45. The composition for use according to any one of items 1 to 6, 8 to 38, and 41 to 44, the method of treatment according to any one of items 39, and 41 to 44, or the use of the composition according to any one of items 40 to 44, wherein said restriction of the one or more therapeutic agent(s) to the treated tissue, organ, and/or anatomical region enables a reduction in the dose of said one or more therapeutic agent(s) to be administered by up to 20%, including any and all ranges within this limit, such as but not limited to reductions of 1-20%, 5-15%, or 10-20%.
46. The composition for use according to any one of items 1 to 6, 8 to 38, and 41 to 45, the method of treatment according to any one of items 39, and 41 to 45, or the use of the composition according to any one of items 40 to 45, wherein said restriction of said one or more therapeutic agent(s) to the treated tissue, organ, and/or anatomical region reduces the number of administrations of said one or more therapeutic agent(s), preferably the number of administrations is reduced by about or less than about 25%.
47. The composition for use according to any one of items 1 to 6, 8 to 38, and 41 to 46, the method of treatment according to any one of items 39, and 41 to 46, or the use of the composition according to any one of items 40 to 46, wherein said restriction of said one or more therapeutic agent(s) to the treated tissue, organ, and/or anatomical region reduces toxicity caused by and/or associated with the accumulation of said one or more therapeutic agents in off-target organs, preferably wherein toxicity caused by and/or associated with said one or more therapeutic agent(s) is reduced in the liver, the brain, the kidneys, the heart, and/or the spleen.
48. The composition for use according to any one of items 1 to 6, 8 to 38, and 41 to 47, the method of treatment according to any one of items 39, and 41 to 47, or the use of the composition according to any one of items 40 to 47, wherein said restriction of said one or more therapeutic agent(s) to the treated tissue, organ, and/or anatomical region reduces and/or avoids off-target effects caused by and/or associated with said one or more therapeutic agent(s), preferably wherein off-target effects caused by and/or associated with said one or more therapeutic agent(s) are reduced in the liver, the brain, the kidneys, the heart, and/or the spleen.
49. The composition for use according to any one of items 1 to 6, 8 to 38, and 41 to 48, the method of treatment according to any one of items 39, and 41 to 48, or the use of the composition according to any one of items 40 to 48, wherein said composition is not to be co-administered with one or more hyaluronidase(s) and/or enzyme(s) comprising hyaluronidase activity.
50. The composition for use according to any one of items 1 to 6, 8 to 38, and 41 to 49, the method of treatment according to any one of items 39, and 41 to 49, or the use of the composition according to any one of items 40 to 49, wherein the subject to be treated is a mammal, preferably a human.
51. The composition for use according to any one of items 1 to 6, 8 to 38, and 41 to 50, the method of treatment according to any one of items 39, and 41 to 50, or the use of the composition according to any one of items 40 to 50, wherein said composition is to be administered via intradermal, subcutaneous, intramuscular, or intratumoral injection, aerosol delivery such as into the respiratory system including intranasal or lung delivery, or topic application.
52. A method of inducing an immune response in a subject, which comprises administering to said subject an effective amount of the composition as defined in or according to any one of items 1 to 6, 8 to 38, and 41 to 50.
53. A method of immunizing a subject against a pathogen, which comprises administering to said subject an effective amount of an mRNA vaccine in a pharmaceutical composition, wherein said pharmaceutical composition comprises the composition as defined in or according to any one of items 1 to 6, 8 to 38, and 41 to 50.
54. The method according to item 53, wherein said mRNA vaccine is administered via intradermal, subcutaneous, intramuscular, or intratumoral injection.
55. The method according to item 53 or 54, wherein said mRNA vaccine elicits an immune response predominantly at the site of administration, thereby reducing the risk of systemic adverse effects.
56. The method according to any one of items 53 to 55, wherein said mRNA vaccine is designed to promote local production of antigen-specific antibodies or cellular immune responses at the site of administration.
57. The method according to any one of items 53 to 56, wherein said mRNA vaccine further comprises a polymeric coating or encapsulation to enhance local retention and prevent systemic dissemination. The composition for use according to any one of items 8 to 38, and 41 to 51 , or the method according to any one of items 53 to 57, wherein said carrier further comprises a targeting moiety or ligand that specifically binds to cells or receptors present at the site of interest, thereby enhancing the specificity and efficacy of said therapeutic agent. The composition for use according to any one of items 8 to 38, 41 to 51 , and 58, or the method according to any one of items 53 to 58, wherein said pharmaceutically acceptable excipient or diluent further comprises a biodegradable or bioresorbable material, facilitating gradual release and local persistence of said therapeutic agent at the site of interest. The composition for use according to any one of items 8 to 38, 41 to 51 , 58, and 59, or the method according to any one of items 53 to 59, wherein said mRNA further comprises a tissue-specific promoter and/or enhancer element to enhance the expression of the antigen at the site of interest. The composition for use according to any one of items 8 to 38, 41 to 51 , and 58 to 60, or the method according to any one of items 53 to 60, wherein said therapeutic agent is encapsulated within a biocompatible microneedle patch or implantable device, facilitating controlled and/or sustained release of said therapeutic agent at the site of interest. The composition for use according to any one of items 8 to 38, 41 to 51 , and 58 to 61 , or the method according to any one of items 53 to 61 , wherein said mRNA further comprises a self-amplifying mRNA (saRNA) molecule, enabling enhanced protein or antigen production at the site of interest. The composition for use according to any one of items 8 to 38, 41 to 51 , and 58 to 62, or the method according to any one of items 53 to 62, wherein said one or more mRNA molecules comprise an ORF encoding CFTR, Erythropoietin (EPO), Factor VIII, Factor IX, Chimeric Antigen Receptor (CAR) T-cell, Survivin (BIRC5) or a dominant-negative form thereof, P53, Vascular Endothelial Growth Factor (VEGF), Insulin, SARS-CoV-2 Spike protein, Alpha-synuclein, Dystrophin, Glucocerebrosidase (GCase), a cytokine such as lnterleukin-2 (IL-2), Interleukin-10 (IL-10), lnterieukin-12 (IL-12), an interferon, Interferon-alpha (IFN-α), Interferon-beta (IFN-β), Interferon-gamma (IFN-γ), interferon lambda (IFN ), such as interferon lambda 1 (IFN-λ1 , also known as IL-29), IFN-λ2 (also known as IL-28A), IFN-λ3 (also known as IL-28B), and/or IFN-λ4, human interferon lambda 1 (hlFNλ1), Tumor Necrosis Factor-alpha (TNF-α), Granulocyte-macrophage colony-stimulating factor (GM-CSF), a primary ciliary dyskinesia protein or factor such as DNAH5, DNAH11, CCDC39, DNAI1 , CCDC40, CCDC103, SPAG1, ZMYND10, ARMC4, CCDC151 , DNAI2, RSPH1 , CCDC114, RSPH4A, DNAAF1 (LRRC50), DNAAF2 (KTU), LRRC6, C21orf59, CCDC65 (DRC2), CCNO, DNAAF3, DNAH1 , DNAH8, DNAL1 , DRC1 (CCDC164), DYX1C1 , DNAAF5 (HEATR2), HYDIN, MCIDAS, NME8 (TXNDC3), RSPH3, RSPH9, or FOXJ1, preferably wherein said one or more mRNA molecules comprise an ORF encoding interferon lambda 1 (IFNλ1), more preferably human interferon lambda 1 (hlFNλ1)
64. The cosmetic composition according to any one of items 7 to 25, wherein said active ingredient is selected from the group consisting of the following: a growth factor, a peptide, an antioxidant, a retinoid, a cytokine, a siRNA, a miRNA, a mRNA, and an asRNA.
65. Use of the cosmetic composition according to any one of items 7 to 25, and 64, in the amelioration of cutaneous condition.
66. A method for the amelioration of a cutaneous condition, wherein said method comprises the administration of the cosmetic composition according to any one of items 7 to 25, and 64.
67. A kit comprising the cosmetic composition according to any one of items 7 to 25, and 64.
68. A drug conjugate comprising an ionizable lipidoid as defined in any one of items 9 to 18 and one or more therapeutic agent(s), preferably wherein said one or more therapeutic agent(s) is/are as defined in any one of items 26 to 34.
69. In vitro use of an ionizable lipidoid for the restriction of the dissemination of one or more to be administered therapeutic agent(s), wherein said ionizable lipidoid is co-formulated with said one or more therapeutic agent (s), preferably wherein said ionizable lipidoid is as defined in any one of items 9 to 18, preferably wherein said one or more therapeutic agent(s) is/are as defined in any one of items 26 to 34.
70. An (in vitro) method for the restriction of the dissemination of one or more to be administered therapeutic agent(s), wherein said method comprises the step of co- formulating an ionizable lipidoid with said one or more therapeutic agent(s), wherein said ionizable lipidoid is as defined in any one of items 9 to 18. 71. The drug conjugate according to item 68, the in vitro use according to item 69, or the in vitro method according to item 70, wherein said ionizable lipidoid comprises a compound of formula (b-l), more preferably a compound of formula (b-V) or formula (b- VII), even more preferably a compound of formula (b-V).
As mentioned above, the present invention provides compositions (such as pharmaceutical compositions and cosmetic compositions) and their uses in the local delivery of agents (such as therapeutic agents or active agents). Accordingly, in the context of the present invention, when referring to a “composition” this may also refer to the herein provided (pharmaceutical) composition for use as a medicament (also referred to as “pharmaceutical composition” herein) and/or to the herein provided cosmetic compositions. Generally, all definitions and specifications relating to a "composition” (or accordingly a pharmaceutical composition) may apply to all such compositions (i.e., to pharmaceutical compositions and cosmetic compositions) herein. Herein, “pharmaceutical composition” and “therapeutic composition” may be used interchangeably. Further, the terms “composition” and “formulation” may be used interchangeably herein.
Accordingly, the present invention provides for a (pharmaceutical) composition for use in the treatment of a disease and/or prevention of a disease, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein one or more of the following apply:
- a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
- the patient has less side effects (such as a reduced risk of CARPA) compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
- said composition has a prolonged retention at the site of administration;
- said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration; and/or
- said composition, when administered to said site of administration, remains localized and essentially does not exhibit systemic distribution throughout the patient's body. As mentioned above, the reduction of side effects in a patient (as compared to a composition that does not have prolonged retention at the site of administration), such as a reduction of the risk for CARPA, is highly advantageous (in the context of the present invention). Particularly, if the patient is susceptible to hypersensitivity reactions, risk group patients, and/or in pediatric patients (i.e., patients being less than about 18 years old). Accordingly, in the context of the herein provided composition for use, a preferred patient (in need of treatment and/or prevention of a disease) is a patient belonging to a risk group, a patient that is susceptible to hypersensitivity reactions (preferably susceptible to hypersensitivity reactions induced by pharmaceutical compositions or by the systemic or non-local distribution of pharmaceutical compositions), and/or a pediatric patient. The skilled artisan is readily aware of means and methods to assess whether a patient belongs to a risk group and/or is susceptible to hypersensitivity reactions (preferably susceptible to hypersensitivity reactions induced by pharmaceutical compositions or by the systemic or non-local distribution of pharmaceutical compositions). When the therapeutic agent of the present invention comprises or consists of an immunomodulatory or immunoactivating polypeptide (such as an interferon, preferably interferon lambda or human interferon lambda) ora nucleic acid (such as an mRNA) encoding such an immunomodulatory or immunoactivating polypeptide (such as an interferon, preferably interferon lambda or human interferon lambda), it is particularly advantageous to limit the risk for CARPA (as potentially induced by the systemic distribution of a composition that does not comprise an ionizable lipid and/or lipidoid in accordance with the present invention), in particular in patients that are susceptible to hypersensitivity reactions. Diseases to be treated and/or prevented may be any of the herein above or below detailed (in particular a diseases or disorder caused by or associated with Enterovirus, such as rhinitis as caused for example by rhinovirus). In particular, for the treatment of asthma (such as the treatment of asthma using a nucleic acid encoding human interferon lambda, as also detailed herein and exemplified in SEQ ID NO: 46) the pediatric patient may be less than about 18 years old, less than about 16 years old, less than about 14 years old, less than about 12 years old, less than about 10 years old, less than about 9 years old, less than about 8 years old, less than about 7 years old, or less than about 6 years old. In particular for the treatment of PCD, the (pediatric) patient may be 6 month or older.
In the context of the herein provided cosmetic compositions, it is similarly advantageous to limit the distribution of said cosmetic composition or of the active agent comprised therein in order to avoid undesired side effects (such as complement activation and/or CARPA), in particular in subjects which are susceptible to hypersensitivity reactions.
As mentioned above, the present invention further relates to a cosmetic composition comprising: a) one or more active agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein said cosmetic composition is an ointment, a creme, a foam, a gel, a lotion, an aqueous liquid, or a powder, or wherein said cosmetic composition is formulated as an ointment, a creme, a foam, a gel, a lotion, an aqueous liquid, or a powder.
In the context of the present invention, contacting a subject/a tissue/an organ with either a pharmaceutical or cosmetic composition may be referred to as ‘‘treating a subject/a tissue/an organ”, accordingly, the terms “treat”, “treatment”, and the like may refer to both therapeutic and cosmetic application/administrations/compositions and the like.
Accordingly, in the context of the present invention, at least about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of said one or more (therapeutic or active) agent(s) may be restricted to the treated tissue, organ, and/or anatomical region, as quantified by a method selected from the group consisting of qPCR, HPLC, mass spectrometry, combinations of HPLC and mass spectrometry.
Accordingly, in the context of the present invention, the restriction of systemic distribution/the prolonged retention at the site of administration may preferably be quantified/assessed/determined by any suitable method, preferably a method selected from the group consisting of qPCR, HPLC, mass spectrometry, and/or combinations of HPLC and mass spectrometry. It is herein preferred that only about 10%, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 %, or less, preferably about 1 % or less, more preferably about 0.9 %, 0.8 %, 0.7 %, 0.6 %, 0.5 %, 0.4 %, 0.3 %, 0.2 %, 0.1% or less, even more preferably about 0.09 %, 0.08 %, 0.07 %, 0.06 %, 0.05 %, 0.04 %, 0.03 %, 0.02 %, 0.01% or less, even more preferably less than about 0.01% of the (amount of the ) (therapeutic or cosmetic) agent reaches/can be detected/assessed/determined in (systemic) circulation after local administration of the composition (such as intradermal, subcutaneous, mucous administration, submucous, intramuscular, or intratumoral injection, aerosol delivery, or topic application), e.g. after about 10 hours after administration, after about 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or, preferably 2 hours.
As mentioned above, means and methods to assess whether e.g., about 0.01% of the agent reaches (or is found/detected in) circulation include qPCR, HPLC, mass spectrometry, and/or combinations of HPLC and mass spectrometry. For agents comprising a nucleic acid (such as mRNA) qPCR may be preferred, more preferably qPCR conducted in the enclosed examples. In Example 7 it was found that less than 0.01 % of the translated hlFNλ1 in the lungs reached the circulation on average approximately 2 hours after administration (between 33 minutes and 5 hours 22 minutes. Thus, inter alia, Example 7 illustrates how it can be assessed/detected/determined whether a composition remains localized at the site of administration and/or essentially does not exhibit systemic distribution throughout the patient's body, whether a composition has a prolonged retention at the site of administration; and/or whether the therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration, or, vice versa, whether a composition does not remain localized at the site of administration and/or essentially does exhibit systemic distribution throughout the patient's body, does not have prolonged retention at the site of administration or wherein said therapeutic agent does not exert its effect at the site of administration by prolonged retention at the site of administration.
It is anticipated herein that the (therapeutic or cosmetic) agent is released following administration of the composition, inter alia, to exert its (therapeutic or cosmetic) effect. Thus, terms like “composition remains localized at the site of administration” and/or “composition essentially does not exhibit systemic distribution throughout the patient's body”, “composition has a prolonged retention at the site of administration” likewise apply to and can be interchangeably used with “(therapeutic or cosmetic) agent remains localized at the site of administration” and/or “(therapeutic or cosmetic) agent essentially does not exhibit systemic distribution throughout the patient's body”, “(therapeutic or cosmetic) agent has a prolonged retention at the site of administration”, respectively. Specifically, a term like “(cosmetic or therapeutic) agent exerts its effect at the site of administration by prolonged retention at the site of administration” refers to “(cosmetic or therapeutic) agent exerts its effect at the site of administration by prolonged retention of the “(cosmetic or therapeutic) agent at the site of administration”.
In other words, if a “composition remains localized at the site of administration” and/or if a “composition essentially does not exhibit systemic distribution throughout the patient's body”, or if a “composition has a prolonged retention at the site of administration”, e.g. about 10%, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 %, or less, preferably about 1% or less, more preferably about 0.9 %, 0.8 %, 0.7 %, 0.6 %, 0.5 %, 0.4 %, 0.3 %, 0.2 %, 0.1% or less, even more preferably about 0.09 %, 0.08 %, 0.07 %, 0.06 %, 0.05 %, 0.04 %, 0.03 %, 0.02 %, 0.01% or less, even more preferably less than about 0.01% of the (amount of the) (therapeutic or cosmetic) agent reaches/can be detected/assessed/determined in (systemic) circulation after local administration of the composition (such as intradermal, subcutaneous, submucosal, intramuscular, or intratumoral injection, mucosal delivery, aerosol delivery, or topic application), e.g. after about 10 hours after administration, after about 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or, preferably 2 hours, e.g. as quantified/assessed/determined by any suitable method, preferably a method selected from the group consisting of qPCR, ligand binding assay, such as ELISA, HPLC, mass spectrometry, combinations of HPLC and mass spectrometry and/or combinations thereof.
Vice versa, if a composition does not remain localized at the site of administration and/or if a composition essentially does exhibit systemic distribution throughout the patient's body, or if a composition does not have prolonged retention at the site of administration or if a (therapeutic or cosmetic) agent does not exert its effect at the site of administration by prolonged retention (of the agent or the composition) at the site of administration of the composition, e.g. about 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10%, or more, preferably about 1% or more, more preferably about 0.1%, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9 %, or more, even more preferably about 0.01%, 0.02 %, 0.03 %, 0.04 %, 0.05 %, 0.06 %, 0.07 %, 0.08 %, 0.09 %, or more, even more preferably more than about 0.01% of the (amount of the) (therapeutic or cosmetic) agent reaches/can be detected/assessed/determined in (systemic) circulation after local administration of the composition (such as intradermal, subcutaneous, submucosal, intramuscular, or intratumoral injection, mucosal delivery, aerosol delivery, or topic application), e.g. after about, 10 hours after administration, after about 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or, preferably 2 hours, e.g. as quantified/assessed/determined by any suitable method, preferably a method selected from the group consisting of qPCR, HPLC, mass spectrometry, combinations of HPLC and mass spectrometry.
The relative values indicated above (e.g. about 10%, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 %, or less, preferably about 1% or less, more preferably about 0.9 %, 0.8 %, 0.7 %, 0.6 %, 0.5 %, 0.4 %, 0.3 %, 0.2 %, 0.1% or less, even more preferably about 0.09 %, 0.08 %, 0.07 %, 0.06 %, 0.05 %, 0.04 %, 0.03 %, 0.02 %, 0.01% or less, even more preferably less than about 0.01% of the (amount of the) (therapeutic or cosmetic) agent for the compositions of the invention and/or about 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10%, or more, preferably about 1% or more, more preferably about 0.1%, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9 %, or more, even more preferably about 0.01%, 0.02 %, 0.03 %, 0.04 %, 0.05 %, 0.06 %, 0.07 %, 0.08 %, 0.09 %, or more, even more preferably more than about 0.01% of the (amount of the) (therapeutic or cosmetic) agent for reference compositions) refer to the amount of the (therapeutic or cosmetic) agent detected/assessed/determined in (systemic) circulation (or to the amount the agent reaches (systemic) circulation) relative to the total administered amount of the agent).
In the case of e.g. mRNA or DNA as therapeutic agent the percentage may be determined on basis of the amount of the translated gene product (peptide and/or protein) in the (systemic) circulation versus the amount of the translated gene product (peptide and/or protein) detected/assessed/determined at the administration site e.g. after about 10 hours after administration, after about 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or, preferably 2 hours. The amount of the translated gene product (protein) detected/assessed/determined at the administration site may also be calculated based on the administered amount of the therapeutic agent (e.g. mRNA) and the thereby expected translated gene product (protein).
In an embodiment of the invention, the extent to which the therapeutic agent or the proteins and/or peptides encoded by said therapeutic agent (such as an mRNA agent) remain localized at the site of administration may depend on the characteristics of said therapeutic agent, or the encoded protein and/or peptides. For example, a small peptide may be more mobile once expressed than a large protein. Thus, the expressed protein may preferably have a molecular weight of about 20 KDa or larger.
If the therapeutic agent is administered in multiple doses (e.g. in subsequently administered 2, 3, or 4 doses) the determination of the relative values will take into account the respective administered (total) amount of the agent (or the (total) amount of the translated gene product (protein) in case of mRNA/DNA)). For example, if the therapeutic agent is administered in subsequent 3 doses with time interval of 2 hours, the amount of the (therapeutic or cosmetic) agent detected/assessed/determined in (systemic) circulation (or the amount the agent reaches in (systemic) circulation) may be determined e.g. 2 hours after each administration relative to the then total administered amount of the agent.
The quantification of therapeutic agents in e.g., different organs is exemplified in the enclosed examples. Further, the enclosed examples illustratively show that both the therapeutic agent (i.e., hlNFM mRNA; SEQ ID NO: 42) and the employed ionizable lipidoid (e.g., a compound according to formula b-V) were retained locally at the site of local administration (see for example, Figs 18 and 19). The local retention of the composition (for example an LiNP or components thereof, such as an ionizable lipidoid or ionizable lipid) may indicate that the (therapeutic or active) agent is also locally retained. Accordingly, it is herein preferred that only about 10%, preferably about 1%, more preferably about 0.1%, even more preferably about 0.01%, even more preferably less than about 0.01% of the (ionizable) lipidoid or (ionizable) lipid reaches (systemic) circulation after about two hours of local administration (such as intradermal, subcutaneous, submucosal, intramuscular, or intratumoral injection, mucosal delivery, aerosol delivery, or topic application). As mentioned above, means and methods to assess whether e.g., about 0.01% of the (ionizable) lipidoid or (ionizable) lipid reaches (or is found in) circulation include HPLC, mass spectrometry, combinations of HPLC and mass spectrometry, preferably LC-MC/MS, as detailed in the enclosed examples. In other words, if a “composition remains localized at the site of administration” and/or if a “composition essentially does not exhibit systemic distribution throughout the patient's body”, or if a “composition has a prolonged retention at the site of administration”, e.g. about 10%, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 %, or less, preferably about 1% or less, more preferably about 0.9 %, 0.8 %, 0.7 %, 0.6 %, 0.5 %, 0.4 %, 0.3 %, 0.2 %, 0.1 % or less, even more preferably about 0.09 %, 0.08 %, 0.07 %, 0.06 %, 0.05 %, 0.04 %, 0.03 %, 0.02 %, 0.01% or less, even more preferably less than about 0.01%, more preferably below detection limit, of the (amount of the) ((ionizable) lipidoid or (ionizable) lipid reaches/can be detected/assessed/determined in (systemic) circulation after local administration of the composition (such as intradermal, subcutaneous, submucosal, intramuscular, or intratumoral injection, mucosal delivery, aerosol delivery, or topic application), e.g. after about 10 hours after administration, after about 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or, preferably 2 hours, e.g. as quantified/assessed/determined by any suitable method, preferably a method selected from the group consisting of HPLC, mass spectrometry, combinations of HPLC and mass spectrometry, more preferably LC-MC/MS.
Vice versa, if a composition does not remain localized at the site of administration and/or if a composition essentially does exhibit systemic distribution throughout the patient's body, or if a composition does not have prolonged retention at the site of administration or if a (therapeutic or cosmetic) agent does not exert its effect at the site of administration by prolonged retention (of the agent or the composition) at the site of administration of the composition, e.g. about 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10%, or more, preferably about 1% or more, more preferably about 0.1%, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 0.6 %, 0.7 %, 0.8 %, 0.9 %, or more, even more preferably about 0.01%, 0.02 %, 0.03 %, 0.04 %, 0.05 %, 0.06 %, 0.07 %, 0.08 %, 0.09 %, or more, even more preferably more than about 0.01% of the (amount of the) (ionizable) lipidoid or (ionizable) lipid reaches/can be detected/assessed/determined in (systemic) circulation after local administration of the composition (such as intradermal, subcutaneous, intramuscular, or intratumoral injection, aerosol delivery, or topic application), e.g. after about 10 hours after administration, after about 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or, preferably 2 hours, e.g. as quantified/assessed/determined by any suitable method, preferably a method selected from the group consisting of HPLC, mass spectrometry, combinations of HPLC and mass spectrometry, more preferably LC-MC/MS.
For example, when using HPLC-MS/MS, the lowest limit of quantification using (LLOQ) to detect the (ionizable) lipidoid shown in formula b-V (specifically) “dL_05” in serum and lung was 100 ng/mL and in liver 200 ng/mL).
Herein provided is a/an (in vitro /ex vivo) method of detecting/assessing/determining whether a “composition remains localized at the site of administration” and/or if a “composition essentially does not exhibit systemic distribution throughout the patient's body”, or if a ‘‘composition has a prolonged retention at the site of administration”, and the like, as described herein above. Such a method may be performed in a subject, e.g. a (non-human) animal (like rat or mouse), for example using non-invasive imaging techniques. The therapeutic use or method of treatment may comprise an (in vitro or ex vivo) step of detecting/assessing/determining whether a “composition remains localized at the site of administration” and/or if a “composition essentially does not exhibit systemic distribution throughout the patient's body”, or if a “composition has a prolonged retention at the site of administration”, and the like, as described herein above.
In the context of the present invention (such as in the context of the herein provided compositions for use, the herein provided cosmetic compositions, the herein provided method of treatment, in the context of the herein provided method of detecting/assessing/determining whether a “composition remains localized at the site of administration” and/or if a "composition essentially does not exhibit systemic distribution throughout the patient's body”, or if a “composition has a prolonged retention at the site of administration”, and the like), the skilled person may readily assess whether a “composition remains localized at the site of administration” and/or if a “composition essentially does not exhibit systemic distribution throughout the patient's body”, or if a “composition has a prolonged retention at the site of administration”, and the like, by comparing a composition (e.g., that may have local retention at the site of administration or the like/in accordance with the present invention) to be administered to a subject with a reference composition that does not remain localized at the site of administration and/or a reference composition that does exhibit systemic distribution throughout the patient's body, or a reference composition that has a prolonged retention at the site of administration, or the like. In this context “comparing a composition with a reference composition” may mean to compare the systemic distribution/the local retention of a composition with said reference compositions by means and methods detailed herein above and further illustratively demonstrated in the enclosed examples. As mentioned above, such methods are not particularly limited and may comprise a method selected from the group consisting of qPCR, ligand binding assay, such as ELISA, HPLC, mass spectrometry, combinations of HPLC and mass spectrometry and/or combinations thereof.
In the context of the present invention, a “reference composition” does not have local retention at the site of administration or the like. The term “a composition that has local retention at the site of administration” or the like may also refer to “a composition that has a prolonged retention at the site of administration”, “a composition that remains localized and essentially does not exhibit systemic distribution throughout the patient's body”, “a non-systemic composition”, “a composition that does not have/exhibit systemic distribution” or the like, and vice versa. Accordingly, a “reference composition” may also be herein referred to as a “systemic composition” (i.e., a composition that has systemic distribution/a composition that does not have localized retention at the site of administration or the like). It is illustratively shown in the enclosed examples that the local retention at the site of administration or the like is conferred by the employed ionizable lipid or ionizable lipidoid (that may interact with the extracellular matrix). Accordingly, a “reference composition” in the context of the present invention does not comprise the herein employed ionizable lipids or ionizable lipidoids that confer the local retention at the site of administration. Accordingly, a “reference composition” in the context of the present invention does not comprise an ionizable lipid or ionizable lipidoid according to e.g., any one of formulas (b-l), (b-ll), (b-V), (b-VI), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), or (b- XII). Such a reference composition may for example comprise a state of the art lipid selected from e.g., DLin-MC3-DMA, ALC-0315, or SM-102 (and does not comprise an ionizable lipid or ionizable lipidoid according to any one of formulas (b-l), (b-ll), (b-V), (b-VI), (b-VII), (b-VIII), (b- IX), (b-X), (b-XI), or (b-XII)).
The enclosed examples illustratively demonstrate that the local retention of a composition (or components thereof, such as the active agent or the therapeutic agent) does not depend on the active agent or the therapeutic agent, in particular when said agent is, e.g., a nucleic acid, such as an mRNA. Accordingly, the person skilled in the art can readily employ agents that are reporter polypeptides (or nucleic acids encoding such reporter polypeptides), such as luciferase (or nucleic acids encoding luciferase) to assess whether a composition has localized retention at the site of administration or the like as compared to said reference composition. Accordingly, the person skilled in the art may compare a composition (such as a composition in accordance with the present invention/a composition comprising e.g., any one of formulas (b-l), (b-ll), (b-V), (b-VI), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), or (b-XII)) and a reference composition (e.g., comprising MC3) each comprising a reporter polypeptide (such as luciferase, as exemplified by SEQ ID NO: 46) or a nucleic acid encoding the same (as exemplified by SEQ ID NO: 45) and assess the activity of said reporter polypeptide (e.g., luciferase activity) after localized administration of the composition (in accordance with the present invention) and after localized administration (e.g., by nasal administration) of said reference composition. In this context, the skilled person is aware that the composition and the reference composition are to be administered to two different/to two independent subjects by localized administration (in order to subsequently quantify the reporter polypeptide activity). The activity of the reporter polypeptide may be assessed by means and methods employed in the enclosed examples (e.g., by IVIS imaging of the of the subjects or by IVIS imaging of excised organs of the subjects). When excising organs of the subjects, said subjects are non- human. The skilled person is aware that the agent (i.e., the reporter polypeptide or the nucleic acid encoding the same) needs to be the same/identical in the composition (in accordance with the present invention) and in the reference composition. Preferably, the composition (in accordance with the present invention) and the reference composition differ only in the above mentioned ionizable lipidoid).
The skilled person can readily compare reporter polypeptide activity (such as reporter polypeptide activity as assessed by IVIS imaging) by comparing the resulting measurements of reporter polypeptide activity (such as the IVIS imaging measurements) in the subjects after administration of the composition and the reference composition e.g. after about 10 hours after administration, after about 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or, preferably 2 hours. The skilled person may for example assess reporter polypeptide activity (such as reporter polypeptide activity as assessed by IVIS imaging) in an off-target tissue or organ. An off-target tissue or organ is a tissue or organ to which the to-be-administered composition was not administered. For example, when targeting the lung (i.e., when locally administering a composition to the lung of a subject) the lung is to be considered as a “target organ” (or as “target tissue”) whereas other organs (such as, e.g., the heart, the spleen, etc.) are considered as “off-target organs” (or “off-target tissue”). It is herein preferred that the reference composition has/results in/exhibits about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 104-fold, 105-fold, etc., increase in reporter polypeptide activity as compared to a composition (in accordance with the present invention), with any values in between such ranges being also envisaged herein and with increasing fold-changes being preferred. Such fold-changes (e.g., a 10-fold increase in reporter polypeptide activity) may be determined by dividing the measurements (such as IVIS imaging measurements) obtained from the assessment of reporter polypeptide activity in an off-target organ after localized administration of the reference composition by the measurements (such as IVIS imaging measurements) obtained from the assessment of reporter polypeptide activity in an off-target organ after localized administration of the composition (in accordance with the present invention). For example, when the administration of a reference composition (comprising e.g., an mRNA encoding for luciferase, as exemplified in SEQ ID NO: 46) to a (local) target organ results in an IVIS imaging measurement of 106 p/s/cm2/ sr and the administration of a composition (in accordance with the present invention; and comprising e.g., an mRNA encoding for luciferase, as exemplified in SEQ ID NO: 46) to a (local) target organ results in an IVIS imaging measurement of 105 p/s/cm2/ sr, the skilled person is aware that this would result in a fold change of about 10 (i.e., in an about 10-fold increase of the reporter polypeptide activity/a 10-fold increase in localized retention at the target organ) when dividing the measurements for the reference composition by the measurements for the composition (in accordance with the present) (i.e., by dividing 106 p/s/cm2/ sr by 105 p/s/cm2/ sr). For example, a 10-fold increase in localized retention at the target organ corresponds to a 10-fold decrease in systemic distribution.
The skilled person is aware that the same off-target organ (or the reporter polypeptide activity in the same off-target organ) needs to be assessed for the composition (in accordance with the present invention) and for the reference composition (i.e. , for example that the heart (or the reporter polypeptide activity in the heart) is assessed for the composition (in accordance with the present invention) and for the reference composition) at the same or about the same time after administration of both compositions to the subjects.
Accordingly, the present invention provides for a/an (in vitro I ex vivo) method of detecting/assessing/determining whether and/or to what extend a composition remains localized at the site of administration, the method comprising the steps of:
(i) administering a composition to a target organ of a first subject via localized administration and administering a reference composition to a target organ of a second subject via localized administration, wherein each composition comprises a reporter polypeptide or a nucleic acid encoding the same;
(ii) assessing reporter polypeptide activity in said off-target organs from said two subjects; and
(iii) comparing the reporter polypeptide activity in said off-target organs from said two subjects thereby determining whether and/or to what extent a composition remains localized at the site of administration (as compared to said reference composition).
Any other specification and detail mentioned in the sections herein above may also apply to said method. Accordingly, instead of assessing reporter polypeptide activity, the skilled person may readily quantify the (therapeutic or active) agent by other means, such as a method selected from the group consisting of qPCR, ligand binding assay, such as ELISA, HPLC, mass spectrometry, combinations of HPLC and mass spectrometry and/or combinations thereof.
The skilled person is further able to employ such a method in the context of the herein provided therapeutic compositions, cosmetic compositions, methods of treatment and the like in order to assess whether and/or to what extend such a composition remains localized.
As mentioned above, when a composition remains localized at the site of administration/in a target organ/in a target tissue, this allows the reduction of the amount of the (therapeutic or active) agent to be administered and/or allows for less side effects of the to be administered composition.
Accordingly, further herein provided is a method for determining a/an (reduced) amount of a (therapeutic or cosmetic) agent in a composition (in accordance with the present invention) to be administered to a target organ, wherein the amount of said (therapeutic or cosmetic) agent is reduced as compared to a reference composition (e.g. composition comprising MC3), preferably wherein the amount of said (therapeutic or cosmetic) agent is reduced by about 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%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,
36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,
68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, (with any values in between such percentages being also envisaged herein and with increasing percentages being preferred) as compared to the amount of (therapeutic or cosmetic) agent comprised in said reference composition, and preferably wherein the (reduced) amount of therapeutic agent has essentially the same therapeutic effect as the amount of the therapeutic agent in the reference composition.
The skilled person is further able to employ such a method in the context of the herein provided therapeutic compositions, cosmetic compositions, methods of treatment and the like in order to assess whether and/or to what extend a reduced amount of agent can be employed, preferably while still obtaining the same therapeutic/cosmetic effect. Any other specification and detail mentioned in the sections herein above may also apply to said method.
Accordingly, further herein provided is a method for determining a/an (reduced) amount of a side effects caused by a composition (in accordance with the present invention) when administered to a target organ, wherein the amount of said side effects is reduced as compared to a reference composition (e.g. composition comprising MC3), preferably wherein the amount of said side effect is reduced by about 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%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,
44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,
60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, (with any values in between such percentages being also envisaged herein and with increasing percentages being preferred) as compared to the side effects caused by said reference composition, and preferably wherein the to be administered composition (in accordance with the present invention) has essentially the same therapeutic effect as the amount of the therapeutic agent in the reference composition.
The skilled person is further able to employ such a method in the context of the herein provided therapeutic compositions, cosmetic compositions, methods of treatment and the like in order to assess whether and/or to what extend side effects can be reduced, preferably while still obtaining the same therapeutic/cosmetic effect. Any other specification and detail mentioned in the sections herein above may also apply to said method.
In the context of the present invention, systemic distribution of the (therapeutic or cosmetic) agent is preferably assessed on (a) blood sample(s) of the subject to be treated. In this context, a blood sample may be a whole blood sample, a plasma sample, a serum sample, or the like.
Accordingly, the restriction of the one or more (therapeutic or active) agent(s) to the treated/ tissue, organ, and/or anatomical region enables a reduction in the dose of said one or more (therapeutic or active) agent(s) to be administered by up to 20%, including any and all ranges within this limit, such as but not limited to reductions of 1-20%, 5-15%, or 10-20%.
Accordingly, the restriction of said one or more (therapeutic or active) agent(s) to the treated tissue, organ, and/or anatomical region reduces the number of administrations of said one or more (therapeutic or active) agent(s), preferably the number of administrations is reduced by about or less than about 5%, preferably by about or less than about 10%, more preferably by about or less than about 20%, even more preferably by about or less than about 30%, even more preferably by about or less than about 40%, even more preferably by about or less than about 50%, even more preferably by about or less than about 60%, even more preferably by about or less than about 70%, even more preferably by about or less than about 80%, most preferably by about or less than about 90%.
Accordingly, the restriction of said one or more (therapeutic or active) agent(s) to the treated tissue, organ, and/or anatomical region reduces toxicity caused by and/or associated with the accumulation of said one or more (therapeutic or active) agent(s) in off-target organs, preferably wherein toxicity caused by and/or associated with said one or more therapeutic agent(s) is reduced in the liver, the brain, the kidneys, the heart, and/or the spleen. In the context of the present invention, off-target organs/off target tissues/off-target sites or the like are organs/tissues/sites or the like that are not the (primary) target organs I target tissues/ target sites or the like; in contrast, the term (primary) target organs I target tissues/ target sites or the like in the context of the present invention refer to the organs/tissues/sites or the like to be treated using the herein provided compositions.
Accordingly, the restriction of said one or more (therapeutic or active) agent(s) to the treated tissue, organ, and/or anatomical region reduces and/or avoids off-target effects caused by and/or associated with said one or more (therapeutic or active) agent(s), preferably wherein off-target effects caused by and/or associated with said one or more (therapeutic or active) agent(s) are reduced in the liver, the brain, the kidneys, the heart, and/or the spleen. As mentioned herein above, and as illustratively shown in e.g., Figs 7 and 8, the herein provided compositions locally restrict the dissemination at least of the therein comprised (therapeutic or active) agent(s), while the co-administration with a hyaluronidase surprisingly abolished this advantageous effect. Accordingly, the herein provided (therapeutic or cosmetic) composition is preferably not to be co-administered with one or more hyaluronidase(s) and/or enzyme(s) comprising hyaluronidase activity.
The herein provided compositions are not particularly limited as long as they are suitable for therapeutic and/or cosmetic applications and remain locally restricted/has prolonged retention at the site of administration/essentially does not exhibit systemic distribution/or the like.
It may however be preferred herein that the carrier is a lipid nanoparticle (LNP), a lipidoid nanoparticle (LiNP), a liposome, a micelle, an emulsion, an Nanostructured Lipid Carrier (NLCs), or a Lipid-Drug Conjugate (LDC), preferably an LNP or an LiNP, and/or that the agent is formulated as a lipid nanoparticle (LNP), a lipidoid nanoparticle (LiNP), a liposome, a micelle, an emulsion, a Nanostructured Lipid Carrier (NLCs), or a Lipid-Drug Conjugate (LDC), preferably as an LNP or as an LiNP. Accordingly, in the context of the present invention, when referring to a nanoparticle/a particle/a complex/a LiNP/a LNP/ or the like herein, this refers to specific embodiments of the carrier comprised in the herein provided composition. Generally, any definition relating to an LNP may also refer to an LiNP, and vice versa, with the exception that an LNP at least comprises one ionizable lipid and an LiNP comprises at least one ionizable lipidoid.
An aspect of the invention relates to a pharmaceutical composition comprising a nucleic acid (such as an RNA or an mRNA) of the invention, a nucleic acid (such as an RNA or an mRNA) vaccine vector of the invention, or an nucleic acid (such as an RNA or an mRNA) vaccine of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent.
The mRNA or the mRNA vaccine of the invention can advantageously be combined in the pharmaceutical composition with further components and/or compounds which ease delivery of the mRNA to the target cells or the target tissue and/or which increase its stability. One possibility in this regard is the formation of the RNA into liposomes or nanoparticles with suitable substances such as those described herein and, e.g. in EP3013964B1, which is incorporated herein in its entirety. In particular, the mRNA or the mRNA vaccine of the invention might be formulated with liposomes, to generate lipoplexes or with subsequent generations of lipid nanocarriers, such as lipid nanoparticles (LNPs), lipidoid nanoparticles (LINPs), nanostructured lipid carriers, and/or cationic lipid-nucleic acid complexes. In some embodiments, the nucleic acid of the invention can be delivered to target cells and/or target tissues in vivo, ex vivo and/or in vitro using LNPs or LiNPs. LNPs and LiNPs can be distinguished from other carriers due to their small size, their homogenous size distribution and their structure and are especially suited for immunization of a subject. The skilled person knows methods for the production of LNPs and LiNPs. The production of LNPs or LiNPs involves a combination of lipids or lipidoids, such as phospholipids, cholesterol, and other specialized lipids, which are mixed together in a solvent, such as an alcohol. This mixture is then subjected to a process called nanoprecipitation, which involves rapidly mixing the lipid solution with a non-solvent, such as a nucleic acid dissolved in water, under controlled conditions of temperature, pressure, and stirring rate. During this process, the lipids self-assemble into complex nanoscale structures, which trap and protect the therapeutic nucleic acids of the invention inside. The nano particles may also be further modified with various surface coatings, such as polyethylene glycol (PEG), to improve their stability and reduce their tendency to be cleared by the immune system.
The herein provided compositions may further comprise one or more stabilizing agent(s), adjuvant(s), and/or immunomodulator(s). Such stabilizing agents may be defined as anywhere herein above or below, preferably the stabilizing agent may be a triblock polymer (i.e., component (p)) as defined anywhere herein. Generally, stabilizing agents (such as Cholesterol, Polyethylene Glycol, poloxamer) may help stabilize the composition (such as stabilize the lipid bilayers and improve the structural integrity of nanoparticles).
In the context of the present invention, adjuvants may comprise for example CPG oligonucleotides, in particular in the context of herein provided vaccine compositions.
The LiNPs may comprise as component (a) an mRNA, an ionizable lipid or an ionizable lipidoid and optionally helper lipids as defined below. Optionally, the LiNPs may comprise as component (p) a triblock copolymer which contains one polypropylene oxide) block, and two poly(ethylene oxide) blocks as described above.
The surfactant in the context of the present invention may be a non-ionic surfactant, optionally at least one nonionic surfactant selected from the group of fatty alcohol ethoxylates, fatty acid ethoxylates, block copolymers of ethylene oxide and propylene oxide, alkylphenol ethoxylates or oligomers of alkylphenol ethoxylates, fatty acid esters of sorbitol, ethoxylated fatty acid esters of sorbitol, fatty acid esters of glycerol, ethoxylated castor oil and ethoxylated vitamin E, preferably wherein the surfactant is selected from the list consisting of poloxamer 188 (P188), poloxamer 338 (P338), poloxamer 407 (P407), Tween-20, Tween-80, BRIJ35, tyloxapol, VitE-PEG1000, and/or Kolliphor EL. Preferably, the surfactant is a (tri)block copolymer of ethylene oxide and propylene oxide, more preferably a poloxamer, even more preferably poloxamer selected from the list consisting of: poloxamer 188, 338, and/or 407, most preferably poloxamer 188 (i.e., P188).
As component (a), the nanoparticles contained in the pharmaceutical composition of the invention, for example in the form of a formulation for intramuscular delivery or for aerosol delivery, may comprise a mRNA coding for one or more antigen(s), wherein said one or more antigen(s) is/are selected from the group consisting of a viral antigen, a bacterial antigen, a cancer, and/or tumor associated antigen, and/or an allergen.
The nanoparticles in the pharmaceutical composition may further comprise an ionizable lipid or an ionizable lipidoid. It will be understood that this encompasses the possibility that the nanoparticles comprise a combination of different ionizable lipids, a combination of different ionizable lipidoids, or a combination of one or more ionizable lipids and one or more ionizable lipidoids. The nanoparticles used in the context of the present invention typically comprise an mRNA (a) and as the ionizable lipid or as the ionizable lipidoid (b) a cationic lipid or cationic lipidoid, in the form of a mixture of these components.
Ionizable lipids and/or ionizable lipidoids that may be suitable and are, thus, envisaged in the context of the present invention are disclosed in WO 2014/207231, which is herein incorporated by reference in its entirety.
The pharmaceutical composition or the mRNA vaccine according to the invention optionally comprises a LiNP comprising an ionizable lipidoid of formula (b-l):
Figure imgf000049_0001
wherein the variables a, b, p, m, n and R1A to R6A are defined as follows: a is 1 and b is an integer of 2 to 4; or a is an integer of 2 to 4 and b is 1 , p is 1 or 2, m is 1 or 2; n is 0 or 1 and m+n is ≥ 2; and
R1A to R6A are independently of each other selected from hydrogen; -CH2-CH(OH)-R7A, -CH(R7A)-CH2-OH,
-CH2-CH2-C(=O)-O-R7A, or -CH2-R7A; wherein R7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; -C(NH)-NH2; a poly(ethylene glycol) chain; and a receptor ligand; provided that at least two residues among R1A to R6A are a group -CH2-CH(OH)-R7A, -CH(R7A)-CH2OH, -CH2CH2-C(=O)-O-R7, -CH2CH2- C(=O)-NH-R7A or -CH2R7 wherein R7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms contained in the compound of formula (b-l) are optionally protonated to provide a compound carrying a positive charge.
Accordingly, the composition according to the present invention, comprises an ionizable lipidoid, wherein said ionizable lipidoid may be a compound of formula (b-l):
Figure imgf000050_0001
formula (b-l), preferably wherein the variables a, b, p, m, n and R1A to R6A are defined as follows: a is 1 and b is an integer of 2 to 4, or a is an integer of 2 to 4 and b is 1 , p is 1 or 2, m is 1 or 2, n is 0 or 1 , m+n is ≥ 2, and
R1A to R6A are independently of each other selected from hydrogen, -CH2-CH(OH)-R7A,
-CH(R7A)-CH2-OH, -CH2-CH2-C(=O)-O-R7A, -CH2CH2-C(=O)-NH-R7A, or -CH2-R7A, wherein R7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond, a protecting group for an amino group, -C(NH)-NH2, a polyethylene glycol) chain, and a receptor ligand; wherein at least two residues among R1A to R6A are a group selected from -CH2- CH(OH)-R7A, -CH(R7A)-CH2OH, -CH2CH2-C(=O)-O-R7A, -CH2CH2-C(=O)-NH- R7A, or -CH2R7A, wherein R7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms comprised or contained in the compound of formula (b-l) are optionally protonated to provide a compound carrying one or more positive charges.
Accordingly, the composition according to the present invention, comprises an ionizable lipidoid, wherein said ionizable lipidoid may be a compound of formula (b-l):
Figure imgf000051_0001
formula (b-l), wherein a is 1 or 2 and b is an integer of 1 to 4, or a is an integer of 1 to 4 and b is 1 or 2, p is 1 or 2, m is 1 or 2, n is 0 or 1 , m+n is ≥ 2, and
R1A to R6A are independently of each other selected from hydrogen, -CH2-CH(OH)-R7A, -CH(R7A)-CH2-OH, -CH2-CH2-C(=O)-O-R7A, -CH2-CH2-C(=O)-NH-R7A, and -CH2-R7A, wherein R7A is selected from C3-C18 alkyl, C3-C18 alkenyl having one C-C double bond, a protecting group for an amino group, -C(NH)-NH2, a polyethylene glycol) chain, and a receptor ligand; and wherein at least two residues among R1A to R6A are a group selected from -CH2-CH(OH)-R7A, -CH(R7A)-CH2OH, -CH2-CH2-C(=O)-O-R7A, -CH2-CH2-C(=O)-NH-R7A, and -CH2R7A, wherein R7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms comprised or contained in the compound of formula (b-l) are optionally protonated to provide a compound carrying one or more positive charges, preferably wherein the variables a, b, p, m, n and R1A to R6A are defined as follows: a is 1 and b is an integer of 2 to 4, or a is an integer of 2 to 4 and b is 1 , p is 1 or 2, m is 1 or 2, n is 0 or 1 , m+n is ≥ 2, and R1A to R6A are independently of each other selected from hydrogen, -CH2-CH(OH)-R7A,
-CH(R7A)-CH2-OH, -CH2-CH2-C(=O)-O-R7A, -CH2CH2C(=O)-NH-R7A, and -CH2-R7A, wherein R7A is selected from C3-C18 alkyl, C3-C18 alkenyl having one C-C double bond, a protecting group for an amino group, -C(NH)-NH2, a polyethylene glycol) chain, and a receptor ligand; and wherein at least two residues among R1A to R6A are a group selected from -CH2- CH(OH)-R7A, -CH(R7A)-CH2OH, -CH2CH2-C(=O)-O-R7A, -CH2CH2-C(=O)-NH- R7A, and -CH2R7A, wherein R7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms comprised or contained in the compound of formula (b-l) are optionally protonated to provide a compound carrying one or more positive charges. The composition according to the present invention, comprises an ionizable lipidoid, wherein said ionizable lipidoid may be a compound of formula (b-ll):
Figure imgf000052_0001
wherein a is 1 or 2, preferably 1 , b is 1 or 2, preferably 2,
R1A to R6A are defined as herein above (e.g., as a compound of formula (b-l)), and wherein one or more of the nitrogen atoms comprised or contained in the compound of formula (b-ll) are optionally protonated to provide a compound carrying one or more positive charges.
In this context, R7A may be preferably selected from C8-C16 alkyl or C8-C18 alkenyl having one C-C double bond, and more preferably from C8-C12 alkyl or C8-C12 alkenyl having one C-C double bond and most preferably from C10-C12 alkyl or C10-C12 alkenyl having one C- C double bond.
Moreover, It Is generally preferred herein that the Ionizable lipidoid has a structure according to formula (IVb) or (IVc):
Figure imgf000052_0002
wherein a, b, and R1 to R6 are defined as anywhere herein above and wherein one or more of the nitrogen atoms indicated in formula (IVc) may be protonated to provide a cationic lipidoid.
Optionally, the cationic lipidoid formula (b-l) comprises at least two residues among R1A to R6A, optionally at least three residues among R1A to R6A, or at least four residues among R1A to R6A are a group selected from -CH2-CH(OH)-R7A, -CH(R7A)-CH2-OH, -CH2-CH2-C(=O)-O-R7A, -CH2-CH2-C(=O)-NH-R7A and -CH2-R7A, wherein R7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond.
Optionally, the ionizable lipidoid comprised in the herein provided composition may be an ionizable lipidoid according to formula (b-l), wherein R1A to R6* are independently of each other selected from -CH2-CH(OH)-R7A, -CH2-CH2-C(=O)-O-R7A, -CH2CH2-C(=O)-NH-R7A, wherein R7A is defined as herein above or below.
Accordingly, the ionizable lipidoid comprised in the herein provided composition may be an ionizable lipidoid according to formula (b-l), wherein R1A to R6A may be independently of each other selected from hydrogen, -CH2-CH(OH)-R7A, -CH2-CH2-C(=O)-O-R7A, -CH2CH2-C(=O)- NH-R7A, wherein R7A may be selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond, and wherein at least three, preferably at least four of R1A to R6A may be selected from -CH2-CH(OH)-R7A, -CH2-CH2-C(=O)-O-R7A, and -CH2-CH2-C(=O)-NH-R7A, wherein R7A may be selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond.
In accordance with an optional embodiment, the compound of formula (b-l) is a compound of formula (b-lb), and the ionizable lipidoid comprises or consists of a lipidoid compound of the following formula (b-lb),
Figure imgf000053_0001
wherein R1A to R6A are defined as in formula (b-l), including preferred embodiments thereof; or a protonated form thereof wherein one or more of the nitrogen atoms indicated in formula (b- Ib) are optionally protonated to provide a compound carrying a positive charge.
Thus, in a accordance with a particularly preferred embodiment, the ionizable lipidoid comprises or consists of a lipidoid of the above formula (b-lb) or a protonated form thereof, and R1A to R6A are independently selected from hydrogen and -CH2-CH(OH)-R7A, wherein R7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond, provided that at least two residues among R1A to R6A are -CH2-CH(OH)-R7A, more preferably at least three residues among R1A to R6A, and still more preferably at least four residues among R1A to R6A are -CH2-CH(OH)-R7A, wherein R7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond.
In the context of the present invention formula (b-l) and formula (b-1) are interchangeable.
In certain embodiments, the mRNA vaccine, or the pharmaceutical composition according to the invention comprises a LiNP nanoparticle comprising a cationic lipidoid of formula (b-V) (also herein referred to as “dL_05” herein, with the (R)-enantiomer of the compound of formula (b-V) being herein also referred to as “dL_05(R)”) and/or formula (b-VII):
Figure imgf000054_0001
In certain embodiments, the mRNA vaccine, or the pharmaceutical composition according to the invention comprises a LiNP nanoparticle comprising a cationic lipidoid of formula (b-XI) and/or formula (b-XII):
Figure imgf000055_0001
The present inventors have surprisingly found that certain ionizable lipidoids to be employed in the context of the present invention are particularly biodegradable, which may be particularly useful. Accordingly, in certain embodiments, the composition comprises an ionizable lipidoid, wherein said ionizable lipidoid comprises or consists of a compound of formula (b-VII) or a compound of formula (b-VIII), preferably a compound of formula (b-VII):
Figure imgf000055_0002
Figure imgf000056_0001
The composition for use or the cosmetic composition as described herein wherein said ionizable lipidoid comprises or consists of a compound of formula (b-IX) or a compound of formula (b-X), preferably a compound of formula (b-X):
Figure imgf000056_0002
As discussed above and as shown in appended Example 10, the present inventors have surprisingly found that also combinations of different ionizable lipidoids may be employed in the context ofthe present invention. In particular, Example 10 demonstrates that a combination of a compound according to formula (b-IX) and of a compound according to formula (b-X) also causes the (desired) local retention of the to be administered composition. Accordingly, in the context of the present invention, the (carrier of the) to be administered compositions may comprise at least two ionizable lipids and/or at least two ionizable lipidoids. It is herein preferred that said at least two ionizable lipids and/or said at least two ionizable lipidoids are two ionizable lipids and/or two ionizable lipidoids.
Said at least two ionizable lipids and/or said at least two ionizable lipidoids may be as defined anywhere herein above. Accordingly, said at least two ionizable lipids and/or said at least two ionizable lipidoids may, for example, be a compound according to formula (b-VI), (b-V), (b-l), (b-ll), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), or (b-XII). In the context of the present invention, it is understood that said at least two ionizable lipids or said at least two ionizable lipidoids are not identical/different (e.g. compound according to formula (b-IX) and of a compound according to formula (b-X)). In a preferred embodiment, the (carrier of the) to be administered compositions comprises a lipidoid according to formula (b-IX) and a lipidoid according to formula (b-X).
In one aspect, the compositions to be administered in the context of the present invention may comprise an ionizable lipid or an ionizable lipidoid. In this context it is preferred that the ionizable lipid (or the ionizable lipidoid) are identical/not different. In other words, it may be preferred that the composition comprises solely the same/identical ionizable lipid (or the same/identical ionizable lipidoid), i.e. the composition does not comprise different ionizable lipids (and/or different ionizable lipidoids). For example, the only/sole lipidoid in the composition may, for example, be a compound of formula (b-l), (b-ll), (b-V), (b-VII), (b-VIII), (b- IX), (b-X), (b-XI), or (b-XII).
In the context of the present invention and without being bound be theory, the terms “cationic lipidoid”, “ionizable lipidoid”, and “lipidoid” are interchangeable. Similarly, the term “cationic lipid” and “ionizable lipid” are interchangeable herein.
The herein provided composition, wherein said ionizable lipidoid is a compound of formula (b- V) and preferably: a) is an R isomer of the compound of formula (b-V), and/or b) is present at a molar ratio of about 22 mol% to about 65 mol%, preferably about 34 mol% to about 52 mol%, more preferably about 36 mol% to about 50 mol%, and most preferably about 43.1 mol%.
The LiNP of the pharmaceutical composition may comprise one or more helper lipid(s) as described in the following. In particular, the herein described agents and reagents for delivering and/or introducing the mRNA into a target cell or a target tissue and the herein described lipids and lipidoids may be combined with one or more (e.g., two, three or four) further lipid(s) (like, for example, cholesterol, DPPC, DOPE and/or PEG-lipids (e.g. DMPE-PEG, DMG-PEG2000)). These further lipids may support the desired function of the therapeutic agents and the lipidoids (support and/or increase the delivery and/or introduction of RNA into the cell or tissue and improve transfection efficiency, respectively) and function as respective “helper lipids”. Particular examples of such “helper lipids” are cholesterol, DPPC, DOPE and/or PEG-lipids (e.g., DMPE-PEG, DMG-PEG (e.g., DMG-PEG2000). The further lipids (e.g., “helper lipids”) may also be part(s) of the herein disclosed complexes/particles. The skilled person is readily in the position to prepare complexes/particles in accordance with the invention. Examples of further lipids (e.g., “helper lipids”) are also known in the art. In the context of the present invention, such helper lipids may preferably be selected from the group consisting of a) to c): a) a phospholipid; b) a sterol; and/or c) a stealth lipid.
Suitable phospholipids are known in the art. Examples of such phospholipids include, inter alia, dipalmitoylphosphatidylcholine (DPPC), DMPC, DSPC, or DOPC. In the context of the present invention DPPC is a preferred phospholipid. Suitable sterols are known in the art. An exemplary, yet preferred sterol is cholesterol. Suitable stealth lipids are known in the art. Examples of such stealth lipids include, inter alia, DMG-PEG2000 and N-TETAMINE- pSar25. In the context of the present invention DMG-PEG2000 is a preferred stealth lipid. The skilled person is readily in the position to choose suitable further lipids (e.g., “helper lipids”) and ratios of the cationic lipidoid(s) and the further lipids (e.g. “helper lipids”). Such ratios may be molar ratios of [1-4 : 1-5], [3-4 : 4-6], [about 4 : about 5], [about 4 : about 5.3] of cationic lipidoid(s) : further lipid(s), (the narrower ranges are preferred). For example, the cationic lipidoid may be combined with three further lipids, like DPPC, cholesterol, and DMG-PEG2000, preferably at a molar ratio of ~8.0 : ~5.3 : ~4.4 : ~0.9, respectively, or, more particularly, 8.00 : 5.29 : 4.41 : 0.88, respectively. Preferably, the lipidoids according to formula (b-l), (b-lb), (b- II), (b-V), (b-VI) and (b-VII) are as described above and used with helper lipids DPPC and cholesterol and PEG-lipid DMG-PEG2000 at the molar ratios 8.00:5.29:4.41:0.88 for formulating lipidoid nanoparticles. In preferred embodiments, the ionizable lipidoid (e.g., the lipidoid according to formula b-V) represents between 20% to 60%, more preferably between 25% and 52% of the total lipid content of the herein provided compositions. In preferred embodiments, the phospholipid lipid (e.g., DPPC) represents between 10% and 60%, more preferably between 13% and 53% of the total lipid content of the herein provided compositions. In preferred embodiment sterol represents between 10% and 30%, more preferably between 12% and 29% of the total lipid content of the herein provided compositions. In preferred embodiment the stealth lipid represents between 1 % and 10%, more preferably between 2% and 9% of the total lipid content of the herein provided compositions. In the context of the present invention, percentages of the total lipid content indicated in weight% (i.e., for example 25% ionizable lipidoid means that said ionizable lipidoid accounts for 25% of the weight of a given composition). In preferred embodiments the N/P ratio of the herein provided compositions or of the total lipids comprised in the herein provided compositions is between 4 and 18, more preferably between 6 and 16, most preferably 8.
In a preferred embodiment, a herein provided composition comprises about 43.1% of the (R)- enantiomer of the ionizable lipidoid of formula (b-V), about 28.5% DPPC, about 23.7% cholesterol, and about 4.7% DMG-PEG2000, with an N/P ratio of about 8.
In a preferred embodiment, a herein provided composition comprises about 34% of the (R)- enantiomer of the ionizable lipidoid of formula (b-V), about 43% DPPC, about 19% cholesterol, and about 4% DMG-PEG2000, with an N/P ratio of about 8.
In another preferred embodiment, a herein provided composition comprises about 50% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 33% DPPC, about 12% cholesterol, and about 5% DMG-PEG2000, with an N/P ratio of about 8.
In another preferred embodiment, a herein provided composition comprises about 44.4% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 29.3% DPPC, about 24.2% cholesterol, and about 2% DMG-PEG2000, with an N/P ratio of about 8.
In another preferred embodiment, a herein provided composition comprises about 25.6% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 52.3% DPPC, about 14% cholesterol, and about 8.1% DMG-PEG2000, with an N/P ratio of about 8.
In another preferred embodiment, a herein provided composition comprises about 51.5% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 13.9% DPPC, about 28.7% cholesterol, and about 5.9% DMG-PEG2000, with an N/P ratio of about 16.
In another preferred embodiment, a herein provided composition comprises about 44.4% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 29.3% DPPC, about 24.2% cholesterol, and about 2% DMG-PEG2000, with an N/P ratio of about 16.
In another preferred embodiment, a herein provided composition comprises about 22.6% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 39.2% DPPC, about 32.2% cholesterol, and about 6% DMG-PEG2000, with an N/P ratio of about 6.
In another preferred embodiment, a herein provided composition comprises about 42% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 39.2% DPPC, about 32.2% cholesterol, and about 7% DMG-PEG2000, with an N/P ratio of about 6.
In another preferred embodiment, a herein provided composition comprises about 50% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 24% DMPC, about 19% cholesterol, and about 7% N-TETAMINE-pSar25, with an N/P ratio of about 8.
In another preferred embodiment, a herein provided composition comprises about 36% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 45% DMPC, about 12% cholesterol, and about 7% N-TETAMINE-pSar25, with an N/P ratio of about 8.
In another preferred embodiment, a herein provided composition comprises about 36% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 45% DMPC, about 12% cholesterol, and about 7% DMG-PEG2000, with an N/P ratio of about 8.
In another preferred embodiment, a herein provided composition comprises about 50% of the (R)-enantiomer of the ionizable lipidoid of formula (b-V), about 24% DMPC, about 19% cholesterol, and about 7% N-TETAMINE-pSar25, with an N/P ratio of about 8.
In another preferred embodiment, a herein provided composition comprises about 21.6% of the ionizable lipidoid of formula (b-IX), about 21.6% of the ionizable lipidoid of formula (b-X), about 28.5% DMPC, about 23.7% cholesterol, and about 4.7% DMG-PEG2000, with an N/P ratio of about 8.
In another preferred embodiment, a herein provided composition comprises about 43.1% of the ionizable lipidoid of formula (b-IX), about 28.5% DPPC, about 23.7% cholesterol, and about 4.7% DMG-PEG2000, with an N/P ratio of about 8.
In another preferred embodiment, a herein provided composition about 43.1% of the ionizable lipidoid of formula (b-XI), about 28.5% DPPC, about 23.7% cholesterol, and about 4.7% DMG-PEG2000, with an N/P ratio of about 8.
In another preferred embodiment, a herein provided composition comprises about 43.1% of the ionizable lipidoid of formula (b-XII), about 28.5% DPPC, about 23.7% cholesterol, and about 4.7% DMG-PEG2000, with an N/P ratio of about 8. In the context of the herein above detailed compositions any other specification (e.g., relating to the therapeutic or active agent) detailed herein above or below may apply.
If not further specified, it is herein generally preferred that a/an (ionizable) lipidoid or a/an (ionizable) lipid comprised in a (pharmaceutical or cosmetic) composition in accordance with the present invention is either present as an (R)-enantiomer or as an (L)-enantiomer, the (R)- enantiomer is preferred. Accordingly, if not further specified, the herein employed examples employ (R)-enantiomers of the respective ionizable lipidoids or ionizable lipids. However, the present invention also envisages combinations (i.e., racemic mixes) of (R)-enantiomers and (L)-enantiomers. In the context of the present invention, the terms “(R)-enantiomer”, “R- isomer”, or the like may be used interchangeably.
In some embodiments, the mRNA vaccine, or the pharmaceutical composition according to the invention comprises a LiNP comprising the following components: a) a mRNA according to the invention, b) a cationic lipidoid of formula (b-l), (b-l I), (b-lb) (b-V), (b-VI), (b-VII) or (b-VIII), and c) one or more helper lipid(s), optionally selected from: c1) DPPC, and/or c2) cholesterol, and/or c3) PEG-lipid DMG-PEG2000, optionally, components b), and c1-c3), are present, optionally component b) and c1 )-c3) are at the molar ratios of about 8.0: about 5.3: about 4.4: about 0.9, respectively, optionally, the LNP comprises a triblock copolymer which contains one polypropylene oxide) block and two poly(ethylene oxide) blocks as component (p) as defined above in vehicles.
Accordingly, the compositions in accordance with the present invention may preferably comprise: a) one or more therapeutic agent(s) and/or one or more active agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein the one or more (therapeutic and/or active) agent(s) may be defined as anywhere herein above or below, wherein the carrier may be defined as anywhere herein above or below. Accordingly, said ionizable lipid and/or said ionizable lipidoid may be defined as anywhere herein above or below. Accordingly, said one or more helper lipid(s) may be as defined anywhere herein above or below. Accordingly, said one or more pharmaceutically acceptable excipient(s) or diluent(s) may be defined as anywhere herein above or below.
A composition in which the R-isomer of formula (b-V), i.e., formula (b-VI) is formulated with the lipids DPPC and cholesterol and PEG-lipid DMG-PEG2000 at the molar ratios 8.00 : 5.29 : 4.41 :0.88 is also referred herein as “Formulation I” or LF92. A composition in which the lipidoid of formula (b-VI I) is formulated with the lipids DPPC and cholesterol and PEG-lipid DMG- PEG2000 at the molar ratios 8.00 : 5.29 : 4.41 : 0.88 is also referred herein as “Formulation II”. In some embodiments the LiNPs in the pharmaceutical composition of the invention comprises Formulation I and/or Formulation II. In some embodiments, the LiNP comprises Formulation I and/or Formulation II.
The cationic lipidoid to mRNA ratios in the LiNP is controlled in terms of the mole ratio of nitrogen atoms of the cationic lipidoid (N) to phosphate groups in the mRNA (P) (N/P ratio). The other lipid components are calculated according to the target molar lipid proportions relative to the cationic lipidoid as discussed above, and may be for example 8.00 : 5.29 : 4.41 : 0.88 for cationic lipidoid, DPPC, cholesterol and PEG-lipid DMG-PEG2000, respectively. In some embodiments, the final N/P ratio of a cationic lipidoid having formula (b-l), (b-ll), (b-lb), (b-V), (b-VI), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), and/or (b-XII) to one phosphate group of mRNA molecule, is preferably 4 to 44, preferably 4 to 16, more preferably 8 nitrogen atoms of a cationic lipidoid having formula (b-l), (b-lb), (b-V), (b-VI), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), and/or (b-XII) per one phosphate group of the mRNA molecule.
The lipid or lipidoid nanoparticles contained in the suspension formulation and in the aerosol in accordance with the invention preferably have a Z-average diameter in the range of 10 to 500 nm, more preferably in the range of 10 to 250 nm, still more preferably 20 to 200 nm. The indicated particle diameter is the hydrodynamic diameter of the particles, as determined by dynamic light scattering (DLS). Measurements are generally carried out at 25 °C.
The polydispersity index of the nanoparticles contained in the suspension formulation and in the aerosol in accordance with the invention is preferably in the range of 0.05 to 0.4, more preferably in the range of 0.05 to 0.2. The polydispersity index can be determined by dynamic light scattering (DLS). Measurements are generally carried out at 25 °C In some embodiments, the compositions comprise a pharmaceutically acceptable carrier and/or an adjuvant. For example, the adjuvant can be alum, Freund’s complete adjuvant, a biological adjuvant or immunostimulatory oligonucleotides (such as CpG oligonucleotides).
The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions, and additional pharmaceutical agents.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Optionally an mRNA vaccine of the invention is administered intramuscularly.
Optionally an mRNA vaccine of the invention is administered intramuscularly, intradermally, subcutaneously by needle or by gene gun, or electroporation.
Optionally, an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention is administered via the respiratory system. In some embodiments the administration is in a form which allows administration to the respiratory system via inhalation, nebulization, via a spray or droplets, e.g., a nasal spray or nasal droplets.
The pharmaceutical composition may comprise a vehicle solution and/or a pharmaceutical acceptable carrier. The vehicle solution and/or the pharmaceutically acceptable carriers may include, but are not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile, and the formulation suits the mode of administration. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. Any of the common pharmaceutical carriers, such as sterile saline solution or sesame oil, can be used. The medium can also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like. Other media that can be used with the compositions and methods provided herein are normal saline and sesame oil.
In the context of the present invention the term “pharmaceutical acceptable carrier” may be used synonymously with the term “pharmaceutically acceptable excipient or diluent". Accordingly, the herein provided compositions generally comprise a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s) (i.e., also referred herein as "pharmaceutical acceptable carrier”. Accordingly, in the context of the present invention the carrier may optionally comprise one or more pharmaceutical acceptable carrier(s). As mentioned above, the carrier in the present invention may be a lipid nanoparticle (LNP), a lipidoid nanoparticle (LiNP), a liposome, a micelle, an emulsion, a Nanostructured Lipid Carrier (NLCs), or a Lipid-Drug Conjugate (LDC), preferably an LNP or an LiNP. Accordingly, for example said LNP may comprise one or more pharmaceutically acceptable excipient(s) or diluent(s). In the context of the present invention the carrier (i.e., for example the LNP or LiNP) may further be comprised in one or more pharmaceutically acceptable excipient(s) or diluent(s), which may be defined as anywhere herein above or below. Such pharmaceutically acceptable excipient(s) or diluent(s) may also be herein referred to as “vehicle solution” or as “pharmaceutically acceptable carrier”. In particular, said vehicle is any solution in a pharmaceutical composition in which an LNP or LiNP may be suspended.
The vehicle solution and/or the pharmaceutically acceptable carrier may comprise a triblock copolymer which contains one polypropylene oxide) block and two poly(ethylene oxide) blocks. Preferably, the triblock copolymer is an A-B-A triblock copolymer which contains one polypropylene oxide) block B of formula (p-1):
Figure imgf000065_0001
wherein s is an integer of 15 to 67, preferably 20 to 40, and two polyethylene oxides) blocks A of formula (p-2):
Figure imgf000065_0002
wherein r is, independently for each block, an integer of 2 to 130, preferably 50 to 100, and more preferably 60 to 90. More preferably, the triblock copolymer has the following structure:
Figure imgf000065_0003
wherein r and t are independently of each other integers of 2 to 130, preferably 50 to 100, and more preferably 60 to 90, and s is an integer of 15 to 67, preferably 20 to 40. Most preferably, Poloxamer P188 is used as the triblock copolymer.
The vehicle solution and/or carrier may comprise the triblock copolymer dissolved therein. However, as will be appreciated by the skilled reader, this does not exclude the possibility that a certain amount of the copolymer molecules is adsorbed to the lipid or lipidoid nanoparticles which are contained in the composition and will be considered component (p) of the LNPs/LiNPs. Preferably, the composition for intramuscular administration or for aerosol formation comprises the triblock copolymer at a concentration of 0.05 to 5 % w/v (i.e. gram per 100 mL) preferably 0.1 to 2 %, based on the total volume of the composition. In addition to the triblock copolymer, other excipients may be present in the vehicle solution. Preferably, the vehicle solution further comprises at least one of sucrose and NaCI, more preferably sucrose and NaCI.
The pharmaceutical formulation in accordance with the invention can be conveniently prepared e.g. by a method including adding the triblock copolymer to a suspension comprising a vehicle solution and the lipid or lipidoid nanoparticles, or including adding the lipid or lipidoid nanoparticles to a vehicle solution comprising the triblock copolymer.
In the context of the present invention, the (therapeutic or active) agent or carrier may be encapsulated within/comprised in a hydrogel or a biocompatible matrix. Accordingly, the herein provided LiNP/LNP may be encapsulated within/comprised in a hydrogel or a biocompatible matrix.
As mentioned above, the herein provided (pharmaceutical or cosmetic) compositions are particularly useful as they result in the local retention of the (therapeutic or active) agents comprised therein. Accordingly, in particular in the context of the herein provided pharmaceutical compositions this may be advantageous to e.g., cause expression of a therapeutically relevant mRNA at the local site of administration, and thereby for example reduced accumulation thereof in undesired off-target tissues or organs, such as the liver. Accordingly, in some embodiments the present invention provides for the means and methods for vaccination/immunization (using the herein provided composition).
Accordingly, the present invention, inter alia, provides methods for the generation of systemic immunization through localized expression. In particular, the invention provides a mRNA vaccine comprising the cameras described above. Accordingly, the present invention provides for a method of immunizing a subject which comprises administering to said subject an effective amount of an mRNA vaccine in a pharmaceutical composition, wherein said pharmaceutical composition comprises the composition as defined anywhere herein above or below, preferably immunizing a subject against a pathogen, against a cancer antigen, or a self- antigen.
Optionally an mRNA used in the invention comprises an RNA sequence of SEQ ID NO:1.
There is also provided according to the invention an isolated RNA or the complement thereof comprising a sequence of SEQ ID NO:1 , or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide identity over its entire length with the RNA sequence of SEQ ID NO:1 and which encodes an amino acid sequence of SEQ ID NO: 2.
There is also provided according to the invention an isolated RNA which encodes an amino acid sequence of SEQ ID NO:1. Optionally, the RNA comprises the RNA sequence of SEQ ID NO:8, SEQ ID NO: 27, SEQ ID 28, SEQ ID NO:29, or SEQ ID NO:30 or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:8, SEQ ID NO: 27, SEQ ID 28, SEQ ID NO:29, or SEQ ID NO:30 and which encodes an amino acid sequence of SEQ ID NO:1.
There is also provided according to the invention an isolated RNA which encodes an amino acid sequence of SEQ ID NO:3. Optionally, the RNA comprises an RNA sequence of SEQ ID NO:10, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:10, SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34 and which encodes an amino acid sequence of SEQ ID NO:3.
A further aspect of the invention is an isolated RNA which encodes an amino acid sequence of SEQ ID NO: 43. Optionally, the RNA comprises an RNA sequence of SEQ ID NO: 42, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:43. Optionally, the mRNA is a modified version of the mRNA comprising modified nucleosides. Optionally the one or more modified nucleosides are 5-iodouridine and 5-iodocytidine. Optionally at least 50% of the uridines in the ORF have been modified. Optionally at least 50% of the uridines in the mRNA have been modified. Optionally at least 50% of the uridines in the ORF have been modified to m1ψ . Optionally, 5 to 50% of the uridine nucleotides are 5-iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine. Optionally, 5 to 50% of the uridine nucleotides are 2-thiouridine and 5 to 50% of the cytidine nucleotides are 5-methylcytidine.
We have appreciated that advantageous immunogenic properties (for example, increased antibody response and/or increased breadth of immune response) may also be provided with mRNA immunogens encoding tethered coronavirus spike protein receptor binding domains.
According to the invention there is also provided an isolated mRNA encoding a polypeptide comprising an amino acid sequence of a coronavirus spike protein receptor binding domain (RBD) linked at its C-terminal end directly, or by a linker amino acid sequence of up to 10 amino acid residues, to an amino acid sequence of a transmembrane domain.
Optionally a nucleic acid, RNA or mRNA of the invention is a product of in-vitro transcription (IVT).
Optionally a nucleic acid, RNA or mRNA of the invention comprises a polyadenylation signal or a (poly(A)) tail downstream of an open reading frame (ORF) encoding the polypeptide. Optionally a nucleic acid, RNA or mRNA of the invention comprises one or more modified nucleosides.
Optionally said modified nucleoside is selected from any of the following: pseudouridine, N1 -methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2- thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4- thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 5-iodo-uridine, 2'- O-methyl uridine, 5-methylcytidine, 5-iodo-cytidine, N1 -methyladenosine, N6- methyladenosine.
Optionally the one or more modified nucleosides comprise a 1 -methylpseudouridine (m1ψ ) modification. Optionally the one or more modified nucleosides comprise at least one N1 - methylpseudouridine (N1i ) modification.
Optionally the one or more modified nucleosides are 5-iodouridine and 5-iodocytidine.
Optionally at least 50% of the uridines in the ORF have been modified.
Optionally at least 50% of the uridines in the ORF have been modified to m1ψ .
Optionally, 5 to 50% of the uridine nucleotides are 5-iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine. Optionally, 5 to 50% of the uridine nucleotides are 2-thiouridine and 5 to 50% of the cytidine nucleotides are 5-methylcytidine.
There is also provided according to the invention an mRNA vaccine vector comprising an mRNA of the invention.
There is also provided according to the invention a nucleic acid, RNA or mRNA vaccine, which comprises a nucleic acid, RNA or mRNA of the invention, or a nucleic acid, RNA or mRNA vaccine vector of the invention, encapsulated in a lipid nanoparticle (LNP).
There is further provided according to the invention a pharmaceutical composition comprising a nucleic acid, RNA or mRNA of the invention, a nucleic acid, RNA or mRNA vaccine vector of the invention, or a nucleic acid, RNA or mRNA vaccine of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent.
There is also provided according to the invention a nucleic acid, RNA or mRNA of the invention, nucleic acid, RNA or an mRNA vaccine vector of the invention, a nucleic acid, RNA or mRNA vaccine of the invention, or a pharmaceutical composition of the invention, for use as a medicament.
There is further provided according to the invention a nucleic acid, an RNA or an mRNA of the invention, a nucleic acid, an RNA or an mRNA vaccine vector of the invention, a nucleic acid, an RNA or an mRNA vaccine of the invention, or a pharmaceutical composition of the invention, for use in the prevention, treatment, or amelioration of a coronavirus infection.
There is also provided according to the invention use of a nucleic acid, an RNA or an mRNA of the invention, a nucleic acid, an RNA or an mRNA vaccine vector of the invention, a nucleic acid, an RNA or an mRNA vaccine of the invention, or a pharmaceutical composition of the invention, in the manufacture of a medicament for the prevention, treatment, or amelioration of a coronavirus infection.
There is also provided according to the invention a method of inducing an immune response to a coronavirus in a subject, which comprises administering to the subject an effective amount of an mRNA of the invention, an mRNA vaccine vector of the invention, an mRNA vaccine of the invention, or a pharmaceutical composition of the invention.
There is also provided according to the invention a method of immunizing a subject against a coronavirus, which comprises administering to the subject an effective amount of a nucleic acid, an RNA or an mRNA of the invention, an mRNA vaccine vector of the invention, a nucleic acid, an RNA or an mRNA vaccine of the invention, or a pharmaceutical composition of the invention.
Optionally a method of the invention comprises administering to the subject a nucleic acid, an RNA or an mRNA of the invention, a nucleic acid, an RNA or an mRNA vaccine vector of the invention, a nucleic acid, an RNA or an mRNA vaccine of the invention, or a pharmaceutical composition of the invention, as part of a prime boost regimen.
Optionally the coronavirus is a beta-coronavirus.
Optionally the beta-coronavirus is a lineage B or C beta-coronavirus.
Optionally the beta-coronavirus is a lineage B beta-coronavirus.
Optionally the lineage B beta-coronavirus is SARS-CoV or SARS-CoV-2.
Optionally the lineage C beta-coronavirus is MERS-CoV.
Optionally the beta-coronavirus is a variant of concern (VOC). Optionally the beta-coronavirus is a SARS-CoV-2 VOC.
Optionally the beta-coronavirus is a SARS-CoV-2 beta, gamma, delta, or omicron VOC.
Optionally the subject is a human subject.
There is also provided according to the invention a method of inducing an immune response to a coronavirus in a subject, which comprises administering to the subject an effective amount of an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention.
There is also provided according to the invention a method of immunizing a subject against a virus, which comprises administering to the subject an effective amount of an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention.
An effective amount is an amount to produce an antigen-specific immune response in a subject.
Optionally, the method comprises administering an effective amount of an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention to a subject that has previously been seroconverted with an mRNA, a vector, a pharmaceutical composition or a vaccine, coding or comprising a full-length spike protein of a coronavirus. Optionally, the coronavirus is a Sarbecovirus. Optionally, the mRNA of the invention, the vector of the invention, the pharmaceutical composition of the invention or the vaccine of the invention comprises or consist of SEQ ID NO: 4 or SEQ ID NO: 7.
There is further provided according to the invention an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention, for use as a medicament.
There is further provided according to the invention an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention, for use in the prevention, treatment, or amelioration of a coronavirus infection.
There is also provided according to the invention use of an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention, in the manufacture of a medicament for the prevention, treatment, or amelioration of a coronavirus infection, including long covid.
Optionally the coronavirus is a beta-coronavirus. Optionally the beta-coronavirus is a lineage B or C beta-coronavirus.
Optionally the beta-coronavirus is a lineage B beta -coronavirus.
Optionally the lineage B beta -coronavirus is SARS-CoV or SARS-CoV-2.
Optionally the lineage C beta -coronavirus is MERS-CoV.
Optionally an immune response is induced against more than one lineage B beta-coronavirus.
Optionally an immune response is induced against SARS-1 and SARS-2 beta-coronavirus.
Optionally an immune response is induced against SARS-1 and MERS beta-coronavirus.
Optionally an Immune response is induced against SARS-2 and MERS beta-coronavirus.
Optionally an immune response is induced against SARS-1, SARS-2, and MERS beta- coronavirus.
Optionally the beta-coronavirus is a variant of concern (VOC).
Optionally the beta-coronavirus is a SARS-CoV-2 VOC.
Optionally the beta-coronavirus is a SARS-CoV-2 lineage B1.248 (Brazil P1 lineage) VOC.
Optionally the beta-coronavirus is a SARS-CoV-2 lineage B1.351 (South Africa) VOC.
Optionally the beta-coronavirus is a SARS-CoV-2 beta, gamma, or delta VOC.
Optionally the beta-coronavirus is a SARS-CoV-2 beta VOC.
Optionally the beta-coronavirus is a SARS-CoV-2 gamma VOC.
Optionally the beta-coronavirus is a SARS-CoV-2 delta VOC.
Optionally the beta-coronavirus is a SARS-CoV-2 alpha VOC.
Optionally the beta-coronavirus is a SARS-CoV-2 omicron VOC.
Optionally the beta-coronavirus is SARS-CoV-2 omicron BA.1.
Optionally the beta-coronavirus is a SARS-CoV-2 omicron BA.2. It can readily be determined whether an immune response has been induced to a beta- coronavirus using methods well-known to the skilled person. For example, a pseudotype neutralization assay as described in the example below may be used.
Optionally the subject is a human subject.
As mentioned above, the herein provided compositions comprise one or more (therapeutic or active) agent(s), and the nature of said agent(s) is not particularly limited. Such agent(s) may comprise one or more of the following: a growth factor, a peptide, an antioxidant, a retinoid, a cytokine, a siRNA, a miRNA, a mRNA, and an asRNA.
It is however, preferred that said agent(s) is/are: a) an (anionic) therapeutical substance and/or b) a nucleic acid, preferably an RNA, more preferably an mRNA, a miRNA, and/or an siRNA, even more preferably an mRNA, most preferably an mRNA comprising an open reading frame (ORF) encoding one or more polypeptide(s). A nucleic acid is particularly preferred herein, especially an mRNA encoding one or more polypeptide(s). In the context of the present invention, the terms “an mRNA comprising an open reading frame (ORF) encoding one or more polypeptide(s)” may be used interchangeably herein with the term “an mRNA encoding one or more polypeptide(s)” or the like.
Said one or more polypeptide(s) (encoded by said mRNA) are not particularly limited however may for example be growth factors, Copper Peptides, or Cytokines. Preferred growth factors may for example be Epidermal Growth Factor (EGF) for stimulating skin growth and wound healing or Fibroblast Growth Factors (FGFs) to promote dermal fibroblasts proliferation and enhance skin elasticity. Preferred peptides may be Copper Peptides which may regenerate skin tissue by stimulating collagen production or Palmitoyl Pentapeptide-4 (i.e., Matrixyl) to reduce wrinkles and improve skin texture. In this context, Cytokines may preferably be Interleukins specifically modified to regulate inflammatory responses in the skin.
In one embodiment of the present invention, said one or more polypeptide(s) (encoded by said mRNA) may comprise a reporter polypeptide. Reporter polypeptides are not particularly limited, are well known in the art, and include for example luminescent or fluorescent proteins, such as, luciferase or green fluorescent protein (GFP), a preferred reporter polypeptide in the context of the present invention is luciferase (as exemplified in SEQ ID NO: 46). The appended examples illustratively show the local retention of a luciferase reporter polypeptide encoded by an mRNA administered in accordance with the present invention. It is further herein envisaged that therapeutically active substances (such as one or more therapeutic polypeptides or such as mRNA encoding one or more therapeutic polypeptides) may be administered with such reporter polypeptides or with mRNA encoding such reporter polypeptides. Further herein envisaged are fusion polypeptides comprising a therapeutic polypeptide (covalently) coupled to a reporter polypeptide or mRNAs encoding are fusion polypeptides comprising a therapeutic polypeptide (covalently) coupled to a reporter polypeptide. Means and method for the fusion/coupling/linkage of two polypeptides or coding regions encoding the same are well known in the art. Such reporter peptides/proteins may particularly be useful in the herein described (in vitro I ex vivo) method of detecting/assessing/determining whether a “composition remains localized at the site of administration” and/or if a “composition essentially does not exhibit systemic distribution throughout the patient's body”, or if a “composition has a prolonged retention at the site of administration”, and the like.
Therapeutical substances (i.e., therapeutic agents or active agents) may also be selected from antioxidants or retinoids. Such antioxidants can preferably be selected from Vitamin C (Ascorbic acid) for photo-protection, pigmentation reduction, and collagen stimulation and Coenzyme Q10 to prevent skin aging by reducing oxidative stress. An exemplary retinoid may be Retinol for accelerating skin renewal, enhancing collagen production, and reducing signs of aging.
As mentioned above, such (therapeutic or cosmetic) agents are preferably nucleic acids, preferably an antisense RNA (asRNA), a non-coding RNA or a mRNA, preferably a small interfering RNA (siRNA), a microRNA (miRNA), a mRNA, most preferably an mRNA.
Said siRNA may preferably be a siRNA targeting specific mRNA involved in melanin synthesis (e.g., targeting TYR gene for skin lightening) or an siRNA targeting MMP (Matrix Metalloproteinase) mRNA to decrease collagen breakdown and reduce wrinkles.
Said miRNA may preferably a miRNA-145 inhibitor (i.e., a miRNA targeting miRNA-145) to enhance collagen production by dermal fibroblasts or inhibit hypertrophic scar formation or a miRNA or a miRNA mimic that could potentially downregulate genes responsible for inflammation and aging.
Said asRNA may preferably comprise sequences that can inhibit the expression of genes involved in undesirable skin conditions like hyperpigmentation or excessive hair growth.
Said mRNA may preferably be an mRNA encoding antioxidants or growth factors that can be produced endogenously at the site of application to enhance skin appearance and health. Said (one or more) mRNA (molecules) may also comprise an ORF encoding a protein, specifically a therapeutic protein, such as CFTR, Erythropoietin (EPO), Factor VIII, Factor IX, Chimeric Antigen Receptor (CAR) T-cell, Survivin (BIRC5) or a dominant-negative form thereof, P53, Vascular Endothelial Growth Factor (VEGF), Insulin, SARS-CoV-2 Spike protein, Alpha-synuclein, Dystrophin, Glucocerebrosidase (GCase), a cytokine such as lnterleukin-2 (IL-2), Interleukin-10 (IL-10), Interleukin-12 (IL-12), an interferon (including, but not limited to interferon-alpha (IFN-a), interferon-beta (IFN-β), interferon-gamma (IFN-y), and/or interferon lambda (IFNλ), such as interferon lambda 1 (IFN-λ1 , also known as IL-29; (preferably human interferon lambda 1 (hlFNλ1), IFN-λ2 (also known as IL-28A), IFN-λ3 (also known as IL-28B), and/or IFN-λ4), Tumor Necrosis Factor-alpha (TNF-α), Granulocyte-macrophage colony- stimulating factor (GM-CSF), a primary ciliary dyskinesia (PCD) protein or factor such as DNAH5, DNAH11 , CCDC39, DNAI1, CCDC40, CCDC103, SPAG1, ZMYND10, ARMC4, CCDC151, DNAI2, RSPH1 , CCDC114, RSPH4A, DNAAF1 (LRRC50), DNAAF2 (KTU), LRRC6, C21orf59, CCDC65 (DRC2), CCNO, DNAAF3, DNAH1 , DNAH8, DNAL1, DRC1 (CCDC164), DYX1C1 , DNAAF5 (HEATR2), HYDIN, MCIDAS, NME8 (TXNDC3), RSPH3, RSPH9, or FOXJ1. Preferably said (one or more) mRNA (molecules) may comprise an ORF encoding interferon lambda 1 (IFNλ1), more preferably human interferon lambda 1 (hlFNλ1).
Preferably, the above protein, specifically the therapeutic protein, is a human protein. For example, CFTR may be human CFTR, Erythropoietin (EPO) may be human EPO, Factor VIII may be human Factor VIII, and so on.
Exemplary sequences of an mRNA (molecules) comprising an ORF encoding human interferon lambda 1 (hlFNλ1) are shown in any one of SEQ ID NOs 41 , 42 or 43. SEQ ID NO. 42 is preferred herein. In accordance with the above, said one or more mRNA(s) may comprise an ORF encoding human interferon lambda 1 (hlFNλ1), preferably wherein said ORF comprises a nucleic acid sequence according to SEQ ID NO: 41 ,42 or 43 or a variant thereof having about 90%/91%/92%/93%/94%/95%/96%/97%/98%/99%/100% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 ,42 and 43, respectively and wherein said variant sequence encodes functional hlFNλ1 . mRNA (molecules) comprising an ORF encoding interferon, specifically interferon lambda, more specifically interferon lambda 1, most preferably human interferon lambda 1 (hlFNλ1 ) may be for use in the treatment or prevention of diseases or disorders, such as a viral-induced or virus-associated disorder, in particular, a viral-induced or virus-associated respiratory disorder. The disease may be Chronic Obstructive Pulmonary Disease (COPD), and/or asthma. The disease may be a respiratory virus infection, e.g. a seasonal and/or emerging virus infection and/or virus-driven or virus-associated exacerbation of chronic respiratory diseases, such as virus-driven or virus-associated exacerbation of asthma or COPD. The virus which causes and/or is linked to said viral-induced or virus-associated respiratory disorder is selected from the group consisting of an enterovirus (such as a rhinovirus), influenza virus, parainfluenza virus, metapneumo virus, respiratory syncytial virus, adenovirus, and coronavirus. The virus which causes said viral-induced respiratory disorder may be a virus which enters cells via the ACE2 receptor and may be SARS- CoV, SARS-CoV-2 or HCoV- NL63.
The mRNA encoding interferon described above may be used in a method of prevention of (e.g., virus-induced) rhinitis. The mRNA encoding interferon has described above may be used in a method of prevention of virus infections that can reduce risk of (severe and potentially life- threatening) infections in, inter alia, immunocompromised and immunosuppressed (e.g. post- transplantation) subjects. The mRNA encoding interferon may also be used in a method of prevention or mitigation of respiratory virus infections in subjects with genetic lung diseases such as PCD.
Preferred dosages for the administration of the nucleic acid, preferably (m)RNA, is any amount between 0.01 mg and up to 60 mg of nucleic acid (mRNA), preferably between 0.05 and 30 mg. For conditions that require a therapeutic protein that is not a cytokine, such as a protein replacement treatment in PCD, a more preferred inhaled amount is between 1 mg and 20 mg, more preferably between 10 mg and 20 mg.
For conditions that require an inhaled cytokine administration such as interferon lambda-1, a preferred dose might be between 0.001 mg to 30 mg, more preferably between 0.01 mg to 20 mg, even more preferably between 0.05 mg to 10 mg, even more preferably between 0.05 and 5 mg, even more preferably between 0.05 and 2 mg.
In the case of nasal administration, for conditions that require a therapeutic protein that is not a cytokine, such a protein replacement treatment in PCD, a dose can be selected from about 0.001 mg to about 6 mg of nucleic acid, more preferably about 0.01 mg to about 2.8 mg, more preferably between 0.25 mg to 1 mg.
For conditions that require a nasal cytokine administration such as administration of interferon lambda-1, a preferred dose might be between 0.001 mg to 10 mg, more preferably between 0.01 mg to 5 mg, even more preferably between 0.05 mg to 1 mg, even more preferably between 0.1 and 1 mg.
Preferred dosages regimes for any route of administration, preferably nasal or lung administration, include once a week, twice a week or three times a week. The administration can also be a chronic administration once to three times a week, preferably once or twice a week.
In this context, it is preferred that the mRNA is to be locally administered, in particular by delivery/local administration into the respiratory system, preferably wherein said delivery/local administration into the respiratory system is inhalation and/or (through) a nasal spray or aerosol. The inhalation may be Inhalation of an aerosol comprising the mRNA.
Accordingly, the present invention provides for a composition for use in the treatment and/or prevention of a disease or disorder, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises:
I. an ionizable lipidoid; ii. one or more helper lipid(s); and iii. one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein said one or more therapeutic agent(s) are one or more mRNA(s), wherein said ionizable lipidoid is a lipidoid according to formula (b-l) or (b-ll), preferably a lipidoid according to any one of formulas (b-V), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), or (b-XII), more preferably according to formula (b-V), even more preferably the (R)-enantiomer of a compound according to formula (b-V), preferably wherein said disease or disorder is a diseases or disorder of the respiratory tract, more preferably a diseases or disorder caused by or associated with Enterovirus (such as rhinitis as caused for example by rhinovirus), preferably wherein the composition is to be administered by nasal spray delivery, and wherein one or more of the following apply:
- a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
- the patient has less side effects (such as a reduced complement activation and/or reduced risk of CARPA) compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
- said composition has a prolonged retention at the site of administration;
- said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration; and/or said composition, when administered to said site of administration, remains localized and essentially does not exhibit systemic distribution throughout the patient's body.
Accordingly, the present invention provides for a composition for use in the treatment and/or prevention of a disease or disorder, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipidoid; ii.one or more helper lipid(s); and iii. one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein said one or more therapeutic agent(s) are one or more mRNA(s), wherein said ionizable lipidoid is a lipidoid according to formula (b-l) or (b-ll), preferably a lipidoid according to any one of formulas (b-V), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), or (b-XII), more preferably according to formula (b-V), even more preferably the (R)- enantiomer of a compound according to formula (b-V), preferably wherein said disease or disorder is a diseases or disorder of the respiratory tract, more preferably a diseases or disorder caused by or associated with Enterovirus (such as rhinitis as caused for example by rhinovirus), preferably wherein the composition is to be administered by nasal spray delivery, wherein said one or more helper lipid(s) are a phospholipid, a sterol, and/or a stealth lipid, preferably wherein said phospholipid is phosphocholine (PC), preferably wherein said sterol is cholesterol, preferably wherein said stealth lipid is glycerolipid-based or phosphoethanolaminelipid- based, and wherein one or more of the following apply:
- a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
- the patient has less side effects (such as reduced complement activation and/or a reduced risk of CARPA) compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
- said composition has a prolonged retention at the site of administration;
- said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration; and/or
- said composition, when administered to said site of administration, remains localized and essentially does not exhibit systemic distribution throughout the patient's body. Accordingly, the present invention provides for a composition for use in the treatment and/or prevention of a disease or disorder, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipidoid; ii.one or more helper lipid(s); and iii. one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein said one or more therapeutic agent(s) are one or more mRNA(s), and wherein said one or more mRNA(s) comprise an ORF encoding human interferon lambda 1 (hlFNλ1), preferably wherein said ORF comprises a nucleic acid sequence according to SEQ ID NO: 42 or a variant thereof having about 90%/91%/92%/93%/94%/95%/96%/97%/98%/99%/100% sequence identity to the nucleic acid sequence of SEQ ID NO: 42 and wherein said variant sequence encodes functional hlFNλ1, wherein said ionizable lipidoid is a lipidoid according to formula (b-l) or (b-ll), preferably a lipidoid according to any one of formulas (b-V), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), or (b-XII), more preferably according to formula (b-V), even more preferably the (R)- enantiomer of a compound according to formula (b-V), preferably wherein said disease or disorder is a diseases or disorder of the respiratory tract, more preferably a diseases or disorder caused by or associated with Enterovirus (such as rhinitis as caused for example by rhinovirus), preferably wherein the composition is to be administered by nasal spray delivery, and wherein one or more of the following apply:
- a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
- the patient has less side effects (such as reduced complement activation and/or a reduced risk of CARPA) compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
- said composition has a prolonged retention at the site of administration;
- said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration; and/or said composition, when administered to said site of administration, remains localized and essentially does not exhibit systemic distribution throughout the patient's body. Accordingly, the present invention provides for a composition for use in the treatment and/or prevention of a disease or disorder, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipidoid; ii.one or more helper lipid(s); and iii. one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein said one or more therapeutic agent(s) are one or more mRNA(s), and wherein said one or more mRNA(s) comprise an ORF encoding human interferon lambda 1 (hlFNλ1), preferably wherein said ORF comprises a nucleic acid sequence according to SEQ ID NO: 42 or a variant thereof having about 90%/91%/92%/93%/94%/95%/96%/97%/98%/99%/100% sequence identity to the nucleic acid sequence of SEQ ID NO: 42 and wherein said variant sequence encodes functional hlFNλ1, wherein said ionizable lipidoid is a lipidoid according to formula (b-l) or (b-ll), preferably a lipidoid according to any one of formulas (b-V), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), or (b-XII), more preferably according to formula (b-V), even more preferably the (R)- enantiomer of a compound according to formula (b-V), preferably wherein said disease or disorder is a diseases or disorder of the respiratory tract, more preferably a diseases or disorder caused by or associated with Enterovirus (such as rhinitis as caused for example by rhinovirus), preferably wherein the composition is to be administered by nasal spray delivery, wherein said one or more helper lipid(s) are a phospholipid, a sterol, and/or a stealth lipid, preferably wherein said phospholipid is phosphocholine (PC), preferably wherein said sterol is cholesterol, preferably wherein said stealth lipid is glycerolipid-based or phosphoethanolaminelipid- based, and wherein one or more of the following apply:
- a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
- the patient has less side effects (such as reduced complement activation and/or a reduced risk of CARPA) compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
- said composition has a prolonged retention at the site of administration;
- said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration; and/or said composition, when administered to said site of administration, remains localized and essentially does not exhibit systemic distribution throughout the patient's body.
A nucleic acid, an RNA or mRNA of the invention may also be provided as part of a nucleic acid, an RNA or mRNA vaccine.
Messenger RNA (mRNA) vaccines are a new form of vaccine (recently reviewed in Pardi et al., Nature Reviews Drug Discovery Volume 17, pages 261-279(2018); Wang etal., Molecular Cancer (2021) 20:33: mRNA vaccine: a potential therapeutic strategy). The first mRNA vaccines to be approved for use were BNT162b2 (manufactured by Pfizer) and mRNA-1273 (manufactured by Moderna) during the COVID-19 pandemic. mRNA vaccines have a unique feature of temporarily promoting the expression of antigen (typically days). The expression of the exogenous antigen is controlled by the lifetime of encoding mRNA, which is regulated by cellular degradation pathways. While this transient nature of protein expression requires repeated administration for the treatment of genetic diseases and cancers, it is extremely beneficial for vaccines, where prime or prime-boost vaccination is sufficient to develop highly specific adaptive immunity without any exposure to the contagion. mRNA-based vaccines trigger an immune response after the synthetic mRNA which encodes viral antigens transfects human cells. The cytosolic mRNA molecules are then translated by the host’s own cellular machinery into specific viral antigens. These antigens may then be presented on the cell surface where they can be recognized by immune cells, triggering an immune response.
The structural elements of a vaccine vector mRNA molecule are similar to those of natural mRNA, comprising a 5’ cap, 5’ untranslated region (UTR), coding region (for example, comprising an open reading frame encoding a polypeptide of the invention), 3’ UTR, and a poly(A) tail. The 5’ UTR (also known as a leader sequence, transcript leader, or leader RNA) is the region of an mRNA that is directly upstream from the initiation codon. This region is important for the regulation of translation of a transcript. In many organisms, the 5' UTR forms complex secondary structure to regulate translation. The 5' UTR begins at the transcription start site and ends one nucleotide (nt) before the initiation sequence (usually AUG) of the coding region. In eukaryotes, the length of the 5' UTR tends to be anywhere from 100 to several thousand nucleotides long. The differing sizes are likely due to the complexity of the eukaryotic regulation which the 5' UTR holds as well as the larger pre-initiation complex that must form to begin translation. The eukaryotic 5' UTR may contain a Kozak consensus sequence (ACCAUG (initiation codon underlined), which contains the initiation codon AUG. An elongated Kozak sequence may be used: GCCACCAUG (initiation codon underlined). The 5’ and 3' UTR elements flanking the coding sequence profoundly influence the stability and translation of mRNA, both of which are critical concerns for vaccines. These regulatory sequences can be derived from viral or eukaryotic genes and greatly increase the half-life and expression of therapeutic mRNAs. For example, a 5’IITR of an mRNA of the invention may comprise, with an initiation codon of the mRNA, a Kozak consensus sequence, or an elongated Kozak sequence. Optionally a 5’UTR of an mRNA of the invention comprises immediately upstream of an initiation codon sequence anyone of the following sequences: GGGAGACGCCACC (SEQ ID NO:11), or GGGAGACUGCCACC (SEQ ID NO:14).
Optionally, a 5’UTR of an mRNA of the invention comprises immediately upstream of an initiation codon sequence a T7, T3, SP6, or K11 polymerase binding domain, a minimal UTR and a Kozak sequence as follows: GGGAGACGCCACC (SEQ ID NO:11), GG GACGCCACC (SEQ ID NO: 12), GGGACGCCACC (SEQ ID NO:13), GGGAGACUGCCACC (SEQ ID NO: 14), GAAGCTGCCACC (SEQ ID NO:15), or GG GACTGCCACC (SEQ ID NO:16).
A 5' cap structure is required for efficient protein production from mRNA. Various versions of 5' caps can be added during or after the transcription reaction using a vaccinia virus capping enzyme, or by incorporating synthetic cap or anti-reverse cap analogues (see Pardi et al., supra). Anti-Reverse Cap Analog (ARCA) is a cap analog used during in vitro transcription for the generation of capped transcripts. ARCA is modified in a way that ensures incorporation in the forward orientation only. Anti-Reverse Cap Analog (ARCA) is a modified cap analog in which the 3' OH group (closer to m7G) is replaced with -OCH3:
Figure imgf000081_0001
Conventional Cap Analog: R=H, m7G(5’)pppG;
ARCA: R=CH3, 3’-0-Me-m7G(5’)pppG
Because of this substitution, the RNA polymerase can only initiate transcription with the remaining hydroxyl group thus forcing ARCA incorporation in the forward orientation. As a result, unlike transcripts synthesized with conventional cap analogs, 100% of the transcripts synthesized with ARCA at the 5' end are translatable leading to a strong stimulatory effect on translation. The 3’ UTR may comprise a sequence for generation of a restriction site when in a vector, such as GAAUU. Alternatively, a 3’ UTR that may be used is 3' UTR of CYBA (CCUCGCCCCGGACCUGCCCUCCCGCCAGGUGCACCCACCUGCAAUAAAUGCAGCGA AGCCGGGA, SEQ ID NO:26.
The poly(A) tail also plays an important regulatory role in mRNA translation and stability; thus, an optimal length of poly(A) must be added to mRNA either directly from the encoding DNA template, by using poly(A) polymerase (see Pardi et al., supra) or ligation after in-vitro transcription. The poly(A) may have a length of 90 A nucleotides (Ago) or more, 100 A nucleotides (A100) or more, 110 A nucleotides (A110) or more, 120 A nucleotides (A120) or more, 130 A nucleotides (A130) or more, 150 A nucleotides (A150) or more, 180 A nucleotides (A180) or more, 190 A nucleotides (A190) or more. An example of a suitable length of poly(A) tail is poly(~A120). The poly(A) tail may be a segmented poly(A) tail, as disclosed in WO 2020074642 A1 , which is herein incorporated by reference. Optionally the segmented poly(A) may have the structure A55-65-S- A55-65 wherein S is a single nucleotide selected from C, G, T or U. Optionally the poly(A) have the structure: A55-65-N-S4-N-A55-65, wherein N is a nucleotide that is not adenine, and wherein S4 are four nucleotides selected from A, C, G, T or U. Optionally, the segmented poly(A) is a poly(A) of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40).
The codon usage additionally has an impact on protein translation. Replacing rare codons with frequently used synonymous codons that have abundant cognate tRNA in the cytosol is a common practice to increase protein production from mRNA. Enrichment of G:C content constitutes another form of sequence optimization that has been shown to increase steady- state mRNA levels in vitro and protein expression in vivo (see Pardi et al., supra).
Two major types of RNA are currently studied as vaccines: non-replicating mRNA and virally derived, self-amplifying RNA. While both types of vaccines share a common structure in mRNA constructs, self-amplifying RNA vaccines contain additional sequences in the coding region for RNA replication, including RNA-dependent RNA polymerases.
BNT162b2 vaccine construct comprises a lipid nanoparticle (LNP) encapsulated mRNA molecule encoding trimerised full-length SARS2 S protein with a PP mutation (at residue positions 986-987). The mRNA is encapsulated in 80 nm ionizable cationic lipid nanoparticles. mRNA-1273 vaccine construct is also based on an LNP vector, but the synthetic mRNA encapsulated within the lipid construct encodes the full-length SARS2 S protein.
US Patent No. 10,702,600 B1 (ModernaTX) describes betacoronavirus mRNA vaccines, including suitable LNPs for use in such vaccines. An mRNA vaccine of the invention may be formulated in a lipid nanoparticle. mRNA vaccines have several advantages in comparison with conventional vaccines containing inactivated (or live attenuated) disease-causing organisms. Firstly, mRNA-based vaccines can be rapidly developed due to design flexibility and the ability of the constructs to mimic antigen structure and expression as seen in the course of a natural infection. mRNA vaccines can be developed within days or months based on sequencing information from a target virus, while conventional vaccines often take years and require a deep understanding of the target virus to make the vaccine effective and safe. Secondly, these novel vaccines can be rapidly produced. Due to high yields from in vitro transcription reactions, mRNA production can be rapid, inexpensive, and scalable. Thirdly, vaccine risks are low. mRNA does not contain infectious viral elements that pose risks for infection and insertional mutagenesis. Anti-vector immunity is also avoided as mRNA is the minimally immunogenic genetic vector, allowing repeated administration of the vaccine. The challenge for effective application of mRNA vaccines lies in cytosolic delivery. mRNA isolates are rapidly degraded by extracellular RNases and cannot penetrate cell membranes to be transcribed in the cytosol. However, efficient in vivo delivery can be achieved by formulating mRNA into carrier molecules, allowing rapid uptake and expression in the cytoplasm. To date, numerous delivery methods have been developed including lipid-, polymer-, or peptide-based delivery, virus-like replicon particle, cationic nanoemulsion, naked mRNAs, and dendritic cell-based delivery (each reviewed in Wang et al., supra), cationic lipid nanoparticle (LNP) delivery is the most appealing and commonly used mRNA vaccine delivery tool.
Exogenous mRNA may be highly immunostimulatory. Single-stranded RNA (ssRNA) molecules are considered a pathogen associated molecular pattern (PAMP), and are recognized by various Toll-like receptors (TLR) which elicit a pro-inflammatory reaction. Although a strong cellular and humoral immune response is desirable in response to vaccination, the innate immune reaction elicited by exogenous mRNA may cause undesirable side effects in the subject. The U-rich sequence of mRNA is a key element to activate TLR (Wang et al., supra). Additionally, enzymatically synthesized mRNA preparations contain double stranded RNA (dsRNA) contaminants as aberrant products of the in vitro transcription (IVT) process. dsRNA is a potent PAMP, and elicits downstream reactions resulting in the inhibition of translation and the degradation of cellular mRNA and ribosomal RNA (Pardi et al., supra). Thus, the mRNA may suppress antigen expression and thus reduce vaccine efficacy.
Studies over the past decade have shown that the immunostimulatory effect of mRNA can be shaped by the purification of IVT mRNA, the introduction of modified nucleosides, complexing the mRNA with various carrier molecules (Pardi et al., supra), adding poly(A) tails or optimizing mRNA with GC-rich sequence (Wang et al., supra). Chemical modification of uridine is a common approach to minimize the immunogenicity of foreign mRNA. Incorporation of pseudouridine (ip) and N1 - methylpseudouridine ( m1ψ ) to IVT mRNA prevents TLR activation and other innate immune sensors, thus reducing pro-inflammatory signaling in response to the exogenous mRNA. Such nucleoside modification also suppresses recognition of dsRNA species (Pardi et al., supra) and can reduce innate immune sensing of exogenous mRNA translation (Hou et al. Nature Reviews Materials, 2021).
Any above and below mentioned RNA modification may, in the context of the present invention apply to the herein above detailed mRNA vaccines and to any other RNA to be employed as or to be comprised in (therapeutic or active) agent in the context of the herein provided composition.
Other nucleoside chemical modifications include, but are not limited to, 5-methylcytidine (m5C), 5-methyluridine (m5U), N1 -methyladenosine (m1A), N6- methyladenosine (m6A), 2- thiouridine (s2U), and 5-methoxyuridine (5moU) (Wang et al., supra).
An RNA of the invention may comprise an mRNA.
An mRNA of the invention, a pharmaceutical composition, or a vector of the invention may be provided as part of an mRNA vaccine.
A Vector of the invention may comprise the corresponding DNA sequence encoding for the peptide (s) and or protein(s) of interest and optionally immediately upstream of an initiation codon sequence anyone of the following sequences: TAATACGACTCACTATA GGGAGACGCCACC (SEQ ID NO: 17), AATTAACCCTCACTAAA GGGAGACGCCACC (SEQ ID NO: 18), ATTTAGGTGACACTATA GAAGCGCCACC (SEQ ID NO: 19), AATTAGGGCACACTATA GGGACGCCACC (SEQ ID NO:20), TAATACGACTCACTATA GGGAGA CTGCCACC (SEQ ID NO:21), AATTAACCCTCACTAAAGGGAGA CTGCCACC (SEQ ID NO:22), ATTTAGGTGACACTATAGAAG CTGCCACC (SEQ ID NO:23), AATTAGGGCACACTATAGGGA CTGCCACC (SEQ ID NO:24), or CGCGCCUAGCAGUGUCCCAGCCGGGUUCGUGUCGCC (SEQ ID NO:25). The above sequences may be placed upstream of the ATG of any of the mRNA sequences of the invention, including the full-length spike (SEQ ID NO:1). An mRNA, a pharmaceutical composition, a vector, or a vaccine, of the invention may comprise one or more modified nucleosides.
The one or more modified nucleosides may be present in an RNA or mRNA of the invention, or in mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention. Optionally, at least one chemical modification is selected from pseudouridine, N1- methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2- thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine,
4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4- thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 5-lodo-uridine, and 2'-O-methyl uridine. In some embodiments, the chemical modification is in the 5-position of the uracil. In some embodiments, the chemical modification is an N1 -methylpseudouridine. In some embodiments, the chemical modification is an N1 -ethylpseudouridine.
For example, an RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention, may comprise one or more of the following modified nucleosides: pseudouridine (ip); N1 -methylpseudouridine ( m1ψ ); 5-methylcytidine (m5C); 5- methyluridine (m5U); N1 -methyladenosine (m1A); N6-methyladenosine (m6A); 2-thiouridine (s2U); 5-methoxyuridine (5moU); 5-iodouridine; and 5-iodocytidine.ln some embodiments 100% of the uracil of the whole mRNA have a chemical modification. In some embodiments, 100% of the uracil in the open reading frame has a chemical modification. In some embodiments, a chemical modification is in the 5-position of the uracil. In some embodiments, a chemical modification is an N1-methyl pseudouridine. In some embodiments, 100% of the uracil of the mRNA have a N1-methyl pseudouridine in the 5-position of the uracil. In some embodiments, 100% of the uracil in the open reading frame have a N1 -methyl pseudouridine in the 5-position of the uracil. In some embodiments, 5 to 50% of the uridine nucleotides are
5-iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine. In some embodiments, 5 to 50% of the uridine nucleotides are 5-iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine. In some embodiments, 5 to 50% of the uridine nucleotides are 2-thiouridine and 5 to 50% of the cytidine nucleotides are 5-methylcytidine.
RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention, may contain from about 1% to about 100% modified nucleotides (or nucleosides) (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide (or nucleoside), i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). Any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.
Optionally RNA ormRNA ofthe invention, or mRNAof a pharmaceutical composition, a vector, or a vaccine, of the invention, comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with each 'U' replaced by m1ψ .
Optionally RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention, comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with at least 50% of the ‘U’s replaced by m1ψ . The remaining ‘U’s may all be unmodified, or may comprise unmodified and one or more other modified nucleosides.
Optionally RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention, comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with at least 70% of the ‘U’s replaced by m.1 Tψhe remaining ‘U’s may all be unmodified, or may comprise unmodified and one or more other modified nucleosides.
Optionally RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention, comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with at least 90% of the ‘U’s replaced by m1ψ . The remaining ‘U’s may all be unmodified, or may comprise unmodified and one or more other modified nucleosides.
Optionally RNA ormRNA ofthe invention, or mRNAof a pharmaceutical composition, a vector, or a vaccine, of the invention, comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with 100% of the ‘U’s replaced by m1ψ . mRNA vaccines of the invention may be co-administered with an immunological adjuvant, for example MF59 (Novartis), TriMix, RNActive (CureVac AG), RNAdjuvant (again reviewed in Wang et al., supra).
The herein provided composition may be administered by any means and methods that are (routinely) applied in the art, accordingly, any suitable route of administration may be used. Methods of administration, specifically of local administration, include, but are not limited to, intradermal, intramuscular, intraperitoneal, subcutaneous, submucosal, mucosal, vaginal, rectal, intranasal, inhalation or oral. The terms “local administration” is opposed to “systemic administration”, such as intravenous administration. Thus, the here envisaged local administration does not comprise “systemic administration”, such as intravenous administration. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Routes for local administration in general include, for example, topical administration routes but also intradermal, transdermal, subcutaneous, submucosal, mucosal, aerosol delivery such as into the respiratory system including intranasal or lung delivery, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, and sublingual injections, preferably mucosal or aerosol delivery. As used herein, “aerosol delivery” refers to the (local) administration of a composition via the airway to the nasal cavities and/or the lung, and can thus herein be used interchangeably with nasal and/or “pulmonary drug delivery” or the like. Similarly, the term “local site of administration” or “local site” herein refers to a (body) site suitable for local administration, such as, e.g., muscle tissue, the respiratory system (including for example the lung, or the nasal cavity). As opposed to “a local site of administration” and as mentioned above, administration to e.g., veins is to be understood as a form of systemic administration in the context of the present invention.
The route of administration may be adapted according to the respective needs. In the context of the herein provided cosmetic compositions administration routes may preferably be topical routes. Accordingly, said cosmetic composition may preferably be formulated for topic application/topic administration. Generally, the application of the cosmetic composition is preferably targeted at the skin of a subject, whereas the skin of any body part may be targeted.
In the context of the present invention, said cosmetic composition may also be formulated for injection, preferably for intradermal, subcutaneous, submucosal, mucosal or intramuscular injection.
Said cosmetic composition may also be comprised in a patch, preferably wherein said patch may be a transdermal patch. In the context of the present invention, a transdermal patch may comprise micro-needles that facilitate the intradermal injection of the composition in accordance with the present invention, accordingly, in the context of transdermal patches the composition may preferably be formulated for injection. In the context of the pharmaceutical compositions, administration routes may depend on the disease to be treated and/or prevented. In general, the herein provided pharmaceutical compositions are particularly useful in the treatment and/or prevention of a variety of diseases, accordingly, such diseases are not particularly limited. Such diseases may be selected from: genetic mutations, autoimmune diseases, metabolic imbalances, neurodegenerative disorders, degenerative disorders of the joints, arthrosis, arthritis, bone fractures, non-union fractures, solid tumor diseases (including soft tissue tumors, tumors of the heart, the lungs, the liver, the spleen, the kidneys, the brain, the oral cavity, the intestine, the skin, the pancreas, the prostate gland, the mammary glands, the ovaries, the urinary bladder, the bones (including osteosarcoma, chondrosarcoma, Ewing sarcoma)), tumors of the pleural and the peritoneal cavity, diseases of the respiratory system including rhinitis and lung diseases such as asthma, viral induced asthma, COPD, including lung autoimmune diseases, ciliopathies and pulmonary alveolar proteinosis (PAP), bone fractures or lesions thereof, tendon fractures or lesions thereof, joint infections, ligament ruptures, resistant Staphylococcus Aureus (MRSA) and/or Multidrug resistant Tuberculosis), viral infections, preferably a viral infection, more preferably a viral infection selected from enterovirus, rhinovirus, Influenza (Flu), respiratory syncytial virus (RSV) Hepatitis A, Hepatitis B, Hepatitis C, Human Papillomavirus (HPV), Measles, Mumps, Rubella, Polio, Rabies, Varicella (Chickenpox), Shingles (Herpes Zoster), Rotavirus, Yellow Fever, Smallpox, Japanese Encephalitis, Tick-Borne Encephalitis (TBE), Dengue Fever, West Nile Virus, Chikungunya Virus, Ebola Virus, Marburg Virus, Human Immunodeficiency Virus (HIV), a coronavirus infection (including COVID-19), rhinovirus, influenza virus, parainfluenza virus, metapneumo virus, respiratory syncytial virus, adenovirus, more preferably rhinovirus, influenza virus, parainfluenza virus, metapneumo virus, respiratory syncytial virus, adenovirus, Hepatitis C and coronavirus, even more preferably a coronavirus infection, influenza, Hepatitis C, most preferably a coronavirus infection.
Preferably, the disease is a lung disease. In some embodiments the lung disease is a ciliopathy, in particular primary ciliary dyskinesia (PCD). In preferred embodiments, said primary ciliary dyskinesia (PCD) is caused by a mutation in a protein or factor selected from DNAH5, DNAH11 , CCDC39, DNAI1, CCDC40, CCDC103, SPAG1, ZMYND10, ARMC4, CCDC151, DNAI2, RSPH1 , CCDC114, RSPH4A, DNAAF1 (LRRC50), DNAAF2 (KTU), LRRC6, C21orf59, CCDC65 (DRC2), CCNO, DNAAF3, DNAH1 , DNAH8, DNAL1, DRC1 (CCDC164), DYX1C1 , DNAAF5 (HEATR2), HYDIN, MCIDAS, NME8 (TXNDC3), RSPH3, RSPH9, and/or FOXJ1.
In the context of the herein provided pharmaceutical compositions and methods of treatment and the like, if the disease to be treated is a ciliopathy or PCD, the skilled person is aware of suitable therapeutic agents. Such agents are, inter alia, disclosed in WO 2020/165352, which is herein incorporated by reference in its entirety. Particularly suitable therapeutic agents for the treatment of a ciliopathy or PCD include a nucleic acid (such as an mRNA) encoding coiled- coil domain containing 39 (CCDC39) and/or coiled-coil domain containing 40 (CCDC40). Particularly preferred nucleic acids sequences encoding CCDC39 and CCDC40 are exemplified in SEQ ID NO: 47 and 48 for CCDC39 and in SEQ ID NO: 49 and 50 for CCDC40. The herein described local retention at the site of administration/the herein described non- systemic distribution of a pharmaceutical composition is also particularly advantageous in treatment of ciliopathy or PCD as it, inter alia, limits the distribution of the therapeutic agent (such as an mRNA encoding CCDC39 or CCDC40) to the site of administration (preferably the respiratory system, such as the nose, throat, larynx, trachea, bronchi, or lungs).
Accordingly, in one preferred embodiment of the present invention, herein provided is a (pharmaceutical) composition for use in the treatment of a ciliopathy or PCD, the treatment comprising local administration of the composition, preferably local administration to the respiratory system, such as the nose, throat, larynx, trachea, bronchi, or lungs, the composition comprising: a) one or more therapeutic agent(s), wherein said one or more therapeutic agent(s) comprise(s) or consist(s) of a nucleic acid (such as an mRNA) encoding CCDC39 and/or CCDC40, preferably an mRNA comprising the nucleic acid sequence as set forth in any one of SEQ ID NO: 47 to 50; and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and ill. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein one or more of the following apply:
- a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
- the patient has less side effects (such as a reduced risk of CARPA) compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration;
- said composition has a prolonged retention at the site of administration;
- said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration; and/or
- said composition, when administered to said site of administration, remains localized and essentially does not exhibit systemic distribution throughout the patient's body. Any further specification or detail (such as components of the carrier or the dosage of the therapeutic agent) may be defined as anywhere herein above or below.
Depending on the disease to be treated and/or prevented the pharmaceutical composition may be administered to one or more solid tissue(s), solid organ(s) and/or solid anatomical region(s), preferably wherein said one or more solid tissue(s), solid organ(s) and/or solid anatomical region(s) are selected from the group consisting of the lungs, the nose, the heart, the brain, the spleen, the lymph nodes, the bones, the tendons, the skeletal muscles, the joints, the stomach, the small intestine, the large intestine, the kidneys, the bladder, the breast, the testes, the ovaries, the uterus, the spleen, the thymus, the brainstem, the cerebellum, the spinal cord, the eye, the ear, the tongue, the skin and/or tumors present in said one or more solid tissue(s), solid organ(s) and/or solid anatomical region(s).
Compositions may be administered in any suitable manner, such as with pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with Inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. Administration can be accomplished by single or multiple doses. The dose administered to a subject in the context of the present disclosure should be sufficient to Induce a beneficial therapeutic response in a subject overtime, or to inhibit or prevent infection. The dose required will vary from subject to subject depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the particular composition being used and its mode of administration. An appropriate dose can be determined by one of ordinary skill in the art using only routine experimentation.
The present disclosure includes methods comprising administering an mRNA vaccine to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
The mRNA vaccine is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the mRNA vaccine may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
The effective amount of the mRNA, as provided herein, may be as low as 20 pg, administered for example as a single dose or as two 10 pg doses. In some embodiments, the effective amount is a total dose of 20 μg-300 μg or 25 μg-300 μg. For example, the effective amount may be a total dose of 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 250 μg, or 300 μg. In some embodiments, the effective amount is a total dose of 20 μg. In some embodiments, the effective amount is a total dose of 25 pg. In some embodiments, the effective amount is a total dose of 50 μg. In some embodiments, the effective amount is a total dose of 75 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a total dose of 150 μg. In some embodiments, the effective amount is a total dose of 200 μg. In some embodiments, the effective amount is a total dose of 250 pg. In some embodiments, the effective amount is a total dose of 300 μg. An mRNA vaccine described herein can be formulated into a dosage form described herein, such as an intranasal, Intratracheal, or Injectable (e.g., intravenous, intraocular, intravltreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).
Optionally, an mRNA vaccine is formulated in an effective amount to produce an antigen specific immune response in a subject.
In some embodiments, the effective amount is a total dose of 1 μg to 1000 μg, 25 μg to 1000 μg, or 50 μg to 1000 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a dose of 25 μg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 100 μg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 400 μg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 500 μg administered to the subject a total of two times.
Optionally a dosage of between 10 μg/kg and 400 μg/kg of the mRNA vaccine is administered to the subject. In some embodiments the dosage of the mRNA is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200 μg, 100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg, 80-300 μg, 100-300 μg, 40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg, 300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100- 400 μg, 200-400 μg, or 300-400 μg per dose.
In some embodiments, the mRNA vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the mRNA vaccine is administered to the subject on day zero. In some embodiments, a second dose of the mRNA vaccine is administered to the subject on day twenty one.
In a strategy called “prime-boost”, a first dose of the mRNA vaccine is given as a priming step, followed by a second dose as a booster. The prime-boost strategy aims to provide a stronger overall immune response. The boost may be administered at least a day, at least a week, or at least two, three, four, five, six, or seven weeks, or at least two, three, four, five, or six months after the primer. For example, the boost may be administered at least three weeks after the primer.
The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a given gene or protein will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981 ; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237- 244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids’ Research 16:10881-10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119-129, 1994. The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al., J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.
Sequence identity between nucleic acid sequences, or between amino acid sequences, can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same nucleotide, or amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical nucleotides or amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.
Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include MatGat (Campanella et al., 2003, BMC Bioinformatics 4: 29;), Gap (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453), FASTA (Altschul et aL, 1990, J. Mol. Biol. 215: 403-410;), Clustal W 2.0 and X 2.0 (Larkin et al., 2007, Bioinformatics 23: 2947-2948) and EMBOSS Pairwise Alignment Algorithms (Needleman & Wunsch, 1970, supra; Kruskal, 1983, In: Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Sankoff & Kruskal (eds), pp 1-44, Addison Wesley). All programs may be run using default parameters.
For example, sequence comparisons may be undertaken using the “needle” method of the EMBOSS Pairwise Alignment Algorithms, which determines an optimum alignment (including gaps) of two sequences when considered over their entire length and provides a percentage identity score. Default parameters for amino acid sequence comparisons (“Protein Molecule” option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: Blosum 62.
The sequence comparison may be performed over the full length of the reference sequence.
A polypeptide encoded by a mRNA of the invention may include one or more conservative amino acid substitutions. Conservative amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original polypeptide, that is, the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. Examples of conservative substitutions include Ala to Ser, Arg to Lys, Asn to Gin or His, Asp to Glu, Cys to Ser, Gin to Asn, Glu to Asp, His to Asn or Gin, He to Leu or Vai, Leu to He or Vai, Lys to Arg or Gin, Met to Leu or He, Phe to Met, Leu, or Tyr, Ser to Thr, Thr to Ser, Trp to Tyr, Tyr to Trp or Phe, and Vai to He or Leu. Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, serine or threonine, is substituted for (or by) a hydrophobic residue, for example, leucine, isoleucine, phenylalanine, valine or alanine; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysine, arginine, or histidine, is substituted for (or by) an electronegative residue, for example, glutamate or aspartate; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.
The term “broadly neutralizing immune response” is used herein to mean an immune response elicited in a subject that is sufficient to inhibit (i.e. reduce), neutralize or prevent infection, and/or progress of infection, of a virus within the coronavirus family. Optionally a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and/or progress of infection, of more than one type of beta-coronavirus (for example, SARS-CoV, and SARS-CoV-2). Optionally a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and/or progress of infection, of more than one type of beta- coronavirus within the same beta-coronavirus lineage (for example, more than one type of beta-coronavirus within the subgenus Sarbecovirus, such as SARS-CoV, SARS-CoV-2, and Bat SL-CoV-WIV1). Optionally a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and/or progress of infection, of coronaviruses of different beta- coronavirus lineages, such as lineage B (for example, SARS-CoV, and SARS-CoV-2) and lineage C (for example, MERS-CoV). Optionally a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and/or progress of infection, of most or all different beta-coronaviruses. Optionally a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and/or progress of infection, of most or all different viruses of the coronavirus family. Optionally a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and/or progress of infection, of most or all variants of concern (VOCs) of SARS-CoV-2, including Beta, Gamma, Delta, Omicron (BA.1). Optionally a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and/or progress of infection, of SARS-CoV, WIV16, RaTG13, SARS-CoV-2, SARS- CoV-2 Beta, SARS-CoV-2 Gamma, SARS-CoV-2 Delta, SARS-CoV-2 Omicron (BA.1).
The immune response may be a humoral and/or a cellular immune response. A cellular immune response is a response of a cell of the immune system, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen or vaccine. An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate immune response or inflammation.
Optionally a polypeptide encoded by an mRNA of the invention induces a protective immune response. A protective immune response refers to an immune response that protects a subject from infection or disease (i.e. prevents infection or prevents the development of disease associated with infection). Methods of measuring immune responses are well known in the art and include, for example, measuring proliferation and/or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, or antibody production.
Optionally a polypeptide encoded by an mRNA of the invention is able to induce the production of antibodies and/or a T-cell response in a human or non-human animal to which the mRNA has been administered (for example, expressed from an administered mRNA vaccine).
The present invention further relates to the (cosmetic/non-therapeutic) use of the herein above detailed cosmetic composition in the amelioration of a cutaneous condition. Accordingly, the present invention further relates to a (cosmetic/non-therapeutic) method for the amelioration of a cutaneous condition, wherein said method comprises the administration of the cosmetic composition as defined anywhere herein.
In the context the herein provided (cosmetic/non-therapeutic) use and method, it is particularly preferred that the active agent is selected from a growth factor, a peptide, an antioxidant, a retinoid, a cytokine, a siRNA, a miRNA, a mRNA, and an asRNA, whereas any of the listed agents may be as defined herein above. In this context, the cosmetic composition may be as defined anywhere herein above.
The present invention further relates to a kit comprising the cosmetic composition and/or the pharmaceutical composition as detailed herein above.
As is detailed in the enclosed examples and mentioned above, the herein employed ionizable lipidoids (such as a lipidoid according to e.g., formula (b-V)) were surprisingly found to cause a composition they are comprised in (such as an LiNP composition comprising e.g., the lipidoid of formula (b-V)) to remain locally restricted at the site of administration following administration to a patient. Thus, it is conceivable that such lipidoids may also limit systemic distribution of an agent when coupled thereto.
Accordingly, in the context of the present invention it is envisaged that said ionizable lipidoids may be coupled/linked/fused/conjugated to an active agent and/or a therapeutic agent (such as a drug). Accordingly, the present invention further relates to a drug conjugate comprising an ionizable lipidoid as defined herein above (preferably a compound of formula (b-l) or (b-ll), more preferably a compound of any one of formulas (b-V), (b-VII), (b-VII), (b-VIII), (b-IX), (b- X), (b-XI), or (b-XII), even more preferably a compound of formula (b-V), most preferably the (R)-enantiomer of the compound of formula (b-V)) and one or more therapeutic agent(s), preferably wherein said one or more therapeutic agent(s) is/are as defined anywhere herein above.
The present invention further relates to the in vitro use of an ionizable lipidoid for the restriction of the dissemination of one or more to be administered therapeutic agent(s), wherein said ionizable lipidoid is co-formulated with said one or more therapeutic agent(s), preferably wherein said ionizable lipidoid is as defined herein above (preferably a compound of formula (b-l) or (b-ll), more preferably a compound of any one of formulas (b-V), (b-VII), (b-VII), (b- VIII), (b-IX), (b-X), (b-XI), or (b-XII), even more preferably a compound of formula (b-V), most preferably the (R)-enantiomer of the compound of formula (b-V)), preferably wherein said one or more therapeutic agent(s) is/are as defined anywhere herein above.
Accordingly, the present invention further relates to an (/n vitro) method for the restriction of the dissemination of one or more to be administered therapeutic agent(s), wherein said method comprises the step of co-formulating an ionizable lipidoid with said one or more therapeutic agent(s), wherein said ionizable lipidoid is as defined anywhere herein above (preferably wherein said lipidoid is a compound of formula (b-l) or (b-ll), more preferably a compound of any one of formulas (b-V), (b-VII), (b-VII), (b-VIII), (b-IX), (b-X), (b-XI), or (b-XII), even more preferably a compound of formula (b-V), most preferably the (R)-enantiomer of the compound of formula (b-V)), and wherein said therapeutic agent is as defined anywhere herein above. In the context of said use or method, co-formulating the lipidoid and the therapeutic agent(s) may comprise further co-formulating any component (such as, for example, a pharmaceutically acceptable excipient or diluent) of the herein above detailed compositions therewith.
The present invention further provides for a compound of formula (b-VII). The present invention further provides for a compound of formula (b-VIII). The present invention further provides for a compound of formula (b-IX). The present invention further provides for a compound of formula (b-X). The present invention further provides for a compound of formula (b-XI). The present invention further provides for a compound of formula (b-XII).
Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.
As used herein, the terms “therapeutic” or “therapy” include prevention and/or treatment of any disease, disorder, or condition and includes vaccination or immunization.
As used herein “subject” means an individual. In general, a subject in the sense of the invention can be a mammal, preferably a human.
The disclosures in context of the methods described herein are disclosed as the corresponding uses mutatis mutandis. The disclosures in context of the uses described herein are disclosed as corresponding methods mutatis mutandis.
Concentrations, amounts, and other numerical data may be expressed or presented herein in a “range” format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of "150 mg to 600 mg" should be interpreted to include not only the explicitly recited values of 150 mg to 600 mg, but to also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 150, 160, 170, 180, 190, ..., 580, 590, 600 mg and sub-ranges such as from 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc. This same principle applies to ranges reciting only one numerical value. Furthermore, such an interpretation should apply regardless of the breadth of the range, or the characteristics being described.
The term “about" when used in connection with a numerical value is meant to encompass numerical values within a range having a lower limit that is 10% smaller than the indicated numerical value and having an upper limit that is 10% larger than the indicated numerical value.
In the context of the present invention, a number of individual elements, characterizing features, techniques and/or steps are disclosed. It is readily recognized that each of these has benefits not only individually when considered or used alone, but also when considered and used in combination with one another. Accordingly, to avoid exceedingly repetitious and redundant passages, this description has refrained from reiterating every possible combination and permutation. Nevertheless, whether expressly recited or not, it is understood that such combinations are entirely within the scope of the presently disclosed subject matter.
As used herein, the singular forms “a,” “an”, and “the” include the plural referents unless the context clearly indicates otherwise. The terms “include”, “such as”, and “the like” are intended to convey inclusion without limitation, unless otherwise specifically indicated. As used herein, the term “or” is generally employed in its usual sense including “and/or” unless the content clearly dictates otherwise. The term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements. As used herein, the term “comprising” also specifically includes embodiments “consisting of and “consisting essentially of the recited elements, unless specifically indicated otherwise.
All publications, patent applications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference(s) should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document is authoritative. Embodiments of the invention are described below, by way of example only, with reference to the accompanying drawings in which:
Figure 1 Bioluminescence image of excised organs. Animals were treated by intratracheal administration of 1μg luciferase chemically modified mRNA encapsulated in Formulation I (i.e., Formulation I formulated Luciferase). D-Luciferin substrate was applied to animals by intraperitoneal and intranasal application before they were euthanized 4 hours after application. Luciferase activity was measured in explanted organs using a Lumlna XR In Vivo Imaging system (Perkin Elmer, USA). In this context Luciferase activity was only detected in the lungs but not In the liver or the spleen. Luciferase radiance is measured as p/sec/cm2/sr, indicating the number of photons per second per square centimeter per steradian. Accordingly, the scale represents radiance in p/sec/cm2/sr.
Figure 2 and 3: BALB/c laboratory mice were short time anesthetized through inhalation of Isoflurane (2-3 %). Formulation I formulated Luciferase coding modified-RNA (i.e., Formulation I) was injected at a dose level of 20 μg/20 μL into the cranial tibial muscle of both hind limbs using 0.3 mL Insulin injection syringes (BD, Germany). Luciferase activity was measured 1 and 4 days (Fig 2 and 3, respectively) subsequently under full anesthesia and following the application of 3 mg D-Luciferin/100 μL PBS which was applied intraperitoneally. Luciferase activity was measured using a Lumina XR In Vivo Imaging System (Perkin Elmer, USA). Local Luciferase activity was measured using a defined Region-Of-lnterest (ROI) which was drawn over the injection site. A ROI over the chest region served as internal control. Scale represents radiance in p/sec/cm2/sr.
Results showed expression of Luciferase at a comparable level In both hind limbs as soon as 1 day (Figure 2) following application of the test item. Luciferase activity remained stable for at least 4 days (Figure 3) following application. No Luciferase activity was observed in any other region of the body, indicating that there was no systemic distribution of the test item.
Figure 4 Intramuscular injection using Formulation I formulated Firefly Luciferase with chemically modified RNA. BALB/c laboratory mice were short time anesthetized through inhalation of Isoflurane (2-3 %). Formulation I formulated Luciferase coding chemically modified RNA was injected at a dose level of 20 μg/20 μL into the cranial tibial muscle of both hind limbs using 0.3 mL Insulin injection syringes (BD, Germany). Luciferase activity was measured 1 and 4 days subsequently under full anesthesia and following the application of 3 mg D-Luciferin/100 μL PBS which was applied intraperitoneally. Luciferase activity was measured using a Lumina XR In Vivo Imaging System (Perkin Elmer, USA). Local Luciferase activity was measured using a defined Region-Of-lnterest (ROI) which was drawn over the injection site. A ROI over the chest region served as internal control. Scale represents radiance in p/sec/cm2/sr.
Results showed expression of Luciferase at a comparable level in both hind limbs as soon as 1 day following application of the test item. Luciferase activity remained stable for 4 days following application. No Luciferase activity was observed in any other region of the body, indicating that there was no systemic distribution of the test item.
Figure 5 and 6: BALB/c laboratory mice were short time anesthetized through inhalation of Isoflurane (2-3 %). Formulation I formulated Luciferase coding chemically modified RNA was injected at a dose level of 20 μg either alone (diluted in 300 μL 0.9% NaCI) (Figure 5) or combined with hyaluronidase (Hylase "Dessau" 150 I.U. diluted in 300 μL 0.9% NaCI which was prepared according to the manufacturer's instructions) (Figure 6) subcutaneously at the back of the animal using 0.3 mL Insulin injection syringes (BD, Germany). Hyaluronidase (Hylase) is an enzyme which facilitates degradation of the extracellular matrix, and which is typically used in dermatology for the aesthetic treatment of scar tissue. Due to its capacity for the degradation of the extracellular matrix hyaluronidase enables the locally applied Formulation I formulated Luciferase chemically modified RNA to spread into the systemic circulation. The Luciferase activity was measured 6 hours after application of the test item under full anesthesia and following the application of 3 mg D-Luciferin/100 μL PBS which was applied intraperitoneally. Luciferase activity was measured using a Lumina XR In Vivo Imaging System (Perkin Elmer, USA). Local Luciferase activity was measured using a defined Region- Of-lnterest (ROI) which was drawn over the injection site. Scale represents radiance in p/sec/cm2/sr.
Results showed Luciferase activity only at the injection site when no hyaluronidase was added to the test item (Figure 5). Addition of hyaluronidase (Figure 6) however, contributed to increased Luciferase activity (as indicated by a wider luciferase activity spread) following administration (2-fold) compared to treatment using Formulation I which was diluted in 0.9% NaCI.
Figure 7 and 8: Four hours following application of formulated Luciferase mRNA as Formulation I either diluted in 0.9% NaCI or hyaluronidase solution animals underwent necropsy. Subsequent ex vivo in vivo imaging using the Lumina XR In Vivo Imaging System (Perkin Elmer, USA) showed Luciferase activity explanted liver and spleen of animals being treated with hyaluronidase dissolved Formulation I (see organs at top and very left of Figure 8; spleen and liver, respectively). In contrast to this, no activity in liver and spleen was observed following treatment using Formulation I which was dissolved in 0.9% NaCI only (see organs at top and very left of Figure 7). Independent from the dilution agent, no activity was observed in the heart, the right kidney, and the lungs. Scale represents radiance in p/sec/cm2/sr.
Figure 9: Effect of phospholipid length. The figure shows the effect of phospholipid length on spike protein of SARS-Cov2 expression level measured as the area under the curve of antibody production for antibodies targeting the expressed protein (coronavirus spike protein receptor binding domain (RBD)). The tested phospholipid lengths were 14, 16 and 18 carbon atoms. Each point represents an individual mouse. PE: Phosphoethanolamine. PC: Phosphocholine (see Table 5). The dot diameter corresponds to the value of the area under the curve (AUC).
Figure 10: immunization effect of phospholipid type: PC (Phosphocholine) or PE (Phosphoethanolamine). The chart illustrates how different types of phospholipids impact the immunization level against the expressed SARS-CoV-2 spike protein. This impact is quantified by measuring the average area under the curve (AUC) for antibodies targeting the expressed protein, 36 days post-immunization. The tested phospholipid lengths were 14, 16 and 18 carbon atoms. Each dot in the figure represents data from an individual mouse. PE: Phosphoethanolamine. PC: Phosphocholine. The dot diameter corresponds to the value of the area under the curve (AUC).
Figure 11 : Effect of stealth PEG length (14 to 18 C PEG 2000 or 5000 Da). The figure presents the impact of stealth PEG length on the expression of the SARS-CoV-2 spike protein, quantified by the area under the curve (AUC) of antibodies targeting the protein 36 days after the initial immunization in mice. Additionally, the effect of stealth lipid chain lengths — comprising 14, 16, or 18 carbon atoms — was evaluated. Each dot in the figure represents data from an individual mouse. G: Dimyristoylglycerin-polyethylenglycol PE: Dipalmitoylglycerin- polyethylenglycol Phosphoethanolamine. The dot diameter corresponds to the value of the area under the curve (AUC).
Figure 12: Effect of stealth PEG %. The figure presents the impact of the % of PEG-lipid on the expression of the SARS-CoV-2 spike protein, quantified by the area under the curve (AUC) of antibodies targeting the protein 36 days after the initial immunization in mice. G: Dimyristoylglycerin-polyethylenglycol-2000 or Dimyristoylglycerin-polyethylenglycol-5000, PE: Dimyristoylphosphoethanolamin-polyethylenglycol-2000 or Dimyristoylphosphoethanolamin- polyethylenglycol-5000 (see Table 5). Figure 13: Effect of sterol percentage. The figure presents the impact of the % of sterol lipid on the expression/immunization against the SARS-CoV-2 spike protein, quantified by the area under the curve (AUC) of antibodies targeting the RBD of the spike protein 36 days after the initial immunization in mice.
Figure 14: effect cationic lipidoid %. The figure presents the impact of the amount of cationic lipidoid in % on the expression of/immunization against SARS-CoV-2 spike protein, quantified as the area under the curve (AUC) of antibodies targeting the RBD of the spike protein 36 days after the initial immunization in mice.
Figure 15: Effect of N/P ratio. The figure presents the impact of the ratio of nitrogen to phosphorous atoms on the expression of the SARS-CoV-2 spike protein, quantified by the area under the curve (AUC) of antibodies targeting the RBD of the spike protein 36 days after the initial immunization in mice.
Figure 16 (A-D): hlFNλ1 -mRNA levels in lungs homogenates following single inhaled administration measured by qPCR. (A) copies of hlFNλ1 mRNA in lung homogenates 24 hours after administration of 0.024 mg/m2of hlFNλ1-LiNP; (B) copies of hlFNλ1 mRNA in lung homogenates 24 hours after administration of 0.048 mg/m2of hlFNλ1-LiNP, (C) copies of hlFNλ1 mRNA in lung homogenates 24 hours after administration of 0.096 mg/m2 of hlFNλ1- LiNP; (D) copies of hlFNλ1 mRNA in lung homogenates 24 to 144 hours after administration of 0.024 mg/m2of hlFNλ1-LiNP. hlFNλ1-mRNA was quantified in lung homogenates by RT- qPCR. Control is a non-translatable mRNA-LiNP. Calculation of mRNA concentrations was done using a standard curve. Each dot represents one animal. Mean values and standard deviation are shown. “Vehicle” does not contain any LiNP.
Figure 17: Biodistribution of hlFNλ1-mRNA in lungs homogenates 24h after single (1x) and repeated (4x) inhaled administration in rats. hlFNλ1 LNP-mRNA was measured in serum and organ homogenates by RT-qPCR 24 h after single or repeated inhaled administration of test product hlFNλ1 LNP. Vehicle group was derived from study WP2_3_14 (see Table 10). Values were quantified by interpolating a standard curve of mRNA in spiked-in neat matrix of untreated animals. Signals in vehicle-treated animals are considered background levels of the assay. Each dot represents one animal. Columns and error bars represent mean values and standard deviation. “Vehicle” does not contain any LiNP.
Figure 18: Biodistribution of hlFNλ1-mRNA in lungs, serum, and key organs 24h after single (1x) (Fig 18 A) and multiple (3x) (Fig 18 B) administration in rats. Test product hlFNλ1 LNP- mRNA was measured in serum and organ homogenates by RT-qPCR 24 h after single or repeated inhaled administration of test product hlFNλ1 LNP. Vehicle group was derived from study WP2_3_14. Values were quantified by interpolating a standard curve of mRNA in spiked- in neat matrix of untreated animals. Each dot represents one animal. Columns and error bars represent mean values and standard deviation. No mRNA could be detected in the serum, liver, brain, heart, spleen, and kidney after single or repeated administration. "Vehicle” does not contain any LiNP.
Figure 19: Biodistribution of the ionizable lipidoid dL_05 in lungs, serum and key organs following single(1x) and multiple (3x) inhaled administration in rats. The lipid fractions of serum and organ lysates were analyzed by a validated LC-MS/MS method for the lipidoid dL_05 following single or repeated inhaled administration of test product hlFNλ1 LNP. Values were quantified by interpolating a standard curve of dL_05 spiked in neat matrix of untreated animals. The values of all matrices, except for lungs, are below the limit of detection. Each dot represents one animal. Columns and error bars represent mean values and standard deviation. “Vehicle” does not contain any LiNP.
Figure 20: hlFNλ1 protein concentrations in lung homogenates following single dose administration by nasal sniffing of test product hlFNλ1 LNP in Formulation I in mice. Fig 20 A shows results after administration of hlFNλ1 LNP at 0.020 mg/m2. Fig 20 B shows results after administration of hlFNλ1 LNP at 0.056 mg/m2 indicating that hlFNλ1 protein was produced dose-dependently in lung homogenates at 5 h after administration. Lungs of animals necropsied at later time points (24 to 96 h) showed no evidence of hlFNλ1 protein concentration above LLOQ. Mean and standard deviation of three animals is shown. “Vehicle” does not contain any LiNP.
Figure 21 (A-D): hlFNλ1 protein concentrations in lung homogenates following single inhaled administration of hlFNλ1 LNP in rats after administration of 0.012 mg/m2 (A), 0.024 mg/m2 (B), 0.048 mg/m2(C), 0.096 mg/m2 (D), and 0.103 mg/m2(E) according to study WP2_3_10 (Table 6). Administration resulted in highest hlFNλ1 protein concentration measured at 6 h post administration which returned to baseline at 48 h. Error bars indicate mean values and standard deviation. Animals for dosages 0.012 mg/m2 (A), 0.024 mg/m2 (B), 0.096 mg/m2 (D), and 0.103 mg/m2 (E) were sacrificed after 24 hours. For dosage 0.048 mg/m2 (C), satellite animals were sacrificed at 6, 24, 48, 72, 96 and 144 hours. “Vehicle” does not contain any LiNP.
Figure 22 (A-C): hlFNλ1 protein levels in lung homogenate and serum of rats following single and repeated inhaled administration of test product hlFNλ1 LiNP in the GLP Toxicology study. Serum and lung homogenate were analyzed with a validated hlFNλ1 ECL assay. Measured values are reported per lung tissue mass or per mL serum. Each dot represents one animal, error bars indicate mean values and standard deviation. hlFNλ 1 concentrations in all vehicle- treated animals were BLQ for both matrices. hlFNλ 1was detected in both lung and serum, but at much lower levels in the serum. hlFNλ1 was detected in both lung and serum, but at much lower levels in the serum. These results confirm the localized expression after administration to the lungs. “Vehicle” does not contain any LINP.
Figure 23 (A-l): IVIS imaging of BALB/c mice (left column) and ex vivo lungs, heart, liver, spleen and right kidney of the same animals (top to bottom, right column), 6 hours after intratracheal instillation of 50 μL (0.06 mg Luciferase mRNA/mL) for carrier formulations with different ratios for the same components as Formulation I (dL_05(R), DPPC, Cholesterol, DMG-PEG2000). (A): Formulation I, (b) Formulation M01-007, (C): Formulation M01-009, (D): Formulation M01-011 , (E): Formulation M01-017, (F): Formulation M01-034, (G): Formulation M01-006, (H): Formulation M01-021, (I): Formulation M01-027. Circles indicate radiance values measured in regions of interest (ROIs). Scale represents radiance in p/sec/cm2/sr.
Figure 24: Ex vivo luciferase activity measurement in whole lungs, 6 hours after intratracheal instillation. Normal distributed data were analyzed using an Ordinary One-Way ANOVA with correction for multiple comparison by Dunnett’s test (** p = 0.0010). Each data set is shown as a Box-Whiskers plot (minimum to maximum and median, for n = 5).
Figure 25 (A-D): IVIS imaging of BALB/c mice (left col.) and ex vivo lungs, heart, liver, spleen and right kidney of the same animals (right coL), 6 hours after intratracheal instillation of 50 μL (0.06 mg Luciferase mRNA/mL) for formulations with dL_05(R) (formula b-VI) and Cholesterol but different helper and stealth lipid, as well as different lipid-to-lipid ratios. (A): Formulation M02-102-006, (B) Formulation M02-103-003, (C) Formulation M02-002-003, and (D) Formulation M02-109-006. Scale represents radiance in p/sec cm2/sr. All tested formulations result in a localized luciferase signal in the lungs of BALB/c mice. No expression could be detected in other tissues, such as explanted hearts, livers, spleen, and right kidneys. This is indicative of a localized retention of the administered formulations at the site of administration (i.e., in the lungs).
Figure 26 (A and B): IVIS imaging of BALB/c mice (left col.) and ex vivo heart, lungs, right kidney, liver, and spleen (right coL), 6 hours after intratracheal instillation of 50 μL (0.06 mg of Luciferase mRNA/mL) for a formulation (Formulation M03-076-3) comprising a combination of two ethyl-propyl-ethyl lipidoids different to dL_05(R) (used in Formulation I), and also different helper lipids, as well as different lipid-to-lipid ratios. (A): experiment with three mice (B): experiment with two mice. Formulation M03-076-3 comprises lipidoids LG2C (formula b-IX) and LE1 D (formula b-X). Scale represents radiance in p/sec/cm2/sr. All tested formulations result in a localized luciferase signal in the lungs of BALB/c mice. No expression could be detected in other tissues, such as explanted hearts, livers, spleen, and right kidneys. This is indicative of a localized retention of the administered formulations at the site of administration (i.e., in the lungs).
Figure 27: IVIS imaging of BALB/c mice (left) and corresponding organs (lungs, liver, spleen), 6 hours after intratracheal microspray of 50 μL (0.2 mg of Luciferase mRNA/mL) for formulations with EPE-based dL but different than dL_05(R), otherwise same lipids and lipid- to-lipid molar ratios as Formulation I. (A): Formulation LF110, (B): Formulation LF181, (C): Formulation LF53. The figure scale represents radiance, measured in photons per second per square centimeter per steradian (p/sec/cm2/sr).
Figure 28: IVIS imaging of C57BL6&JRj female adult mice, 4 hours after intranasal delivery showing localized expression in the nasal airways when administered with 0.15 mg/mL (A), 0.5 mg/mL (B), 1.5 mg/mL (C) modified mRNA coding for luciferase formulated with Formulation I (i.e., comprising dL_05(R)). Left images show color image and right images show the same mice in grey scale. Circles indicate radiance values measured in regions of interest (ROIs). Scale represents radiance in p/sec/cm2/sr.
List of SEQ ID NOs:
SEQ ID NO: Description
1 spike protein of CoV
11 5’-UTR sequence of an mRNA of the invention (Min UTR C)
12 DNA/mRNA sequence coding for 5’-GAAG-MinUTR-CT
13 DNA/mRNA sequence coding for 5’-MinUTR-CT
14 5’-UTR sequence in the vector of the invention (Min UTR CT)
15 5’-UTR sequence in the vector of the invention (5’-GAAG-Min UTR CT)
16 5’-UTR sequence in the vector of the invention (5’-GGGA-Min UTR CT)
17. DNA sequence of T7 promoter + MinUTR-C +Kozak
18. DNA sequence of T3 promoter + Min UTR-C + Kozak
19. DNA sequence of SP6 promoter + Min UTR-C + Kozak
20 DNA sequence of K11 promoter + Min UTR-C + Kozak 21 DNA sequence of T7 promoter + Min UTR-CT + Kozak
22 DNA sequence of T3 promoter + Min UTR-CT + Kozak
23 DNA sequence of SP6 promoter + Min UTR-CT + Kozak
24 DNA sequence of K11 promoter + Min UTR-CT + Kozak
25 DNA/mRNA sequence of 5'CYBA UTR
26 DNA/mRNA sequence of 3'CYBA UTR
35 sequence of segmented polyA (1 )
36 sequence of segmented polyA (2)
37 sequence of segmented polyA (3)
38 sequence of segmented polyA (4)
39 sequence of segmented polyA (5)
40 sequence of segmented polyA (6)
41 Sequence of codon optimized hlFN-lambda1 (DNA and RNA)
42 mRNA coding for hlFN-lambda1
43 mRNA coding for hlFN-lambda1 (without 3’ UTR)
44 Elongated Kozak sequence
45 Luciferase mRNA
46 Luciferase protein
47 CCDC39 sequence with CYBA UTRs (ETH047T04) - codon optimized sequence encoding a functional version of a human CCDC39 protein
48 ETH047T02 (CCDC39 with Ethris minimal UTR) - codon optimized sequence encoding a functional version of a human CCDC39 protein
49 CCDC40 sequence with CYBA 5 and 3 UTR (ETH031T09) - codon optimized sequence encoding a functional version of a human CCDC40 protein
50 Sequence with Ethris minimal 5 UTR (ETH031T06) - codon optimized sequence encoding a functional version of a human CCDC40 protein Certain embodiments of the invention are described with reference to the following examples, which are intended for the purpose of illustration only and are not intended to limit the scope of the generality of the description hereinbefore.
Example 1 - Evaluation of the in vivo transfection efficiency of Formulation I after intra tracheal administration
The aim of this task was to investigate if Formulation I is able to transfect respiratory tissue after intratracheal administration and whether the transfected mRNA remains local or moves systematically in the patient body. For this, an mRNA encoding firefly luciferase formulated into lipid nanoparticles (LNP) identical to the hlFNλ1 LNP test drug product used in Examples 3 to 7 was used (composition I (herein also referred to as Formulation I or LF92)). The only difference between this tool product and the therapeutic hlFNλ1 LNP test drug is the mRNA of Examples 3 to 7 is the coding sequence of hlFNλ1 , whereas in Example 1 the mRNA encodes luciferase, an easily measurable reporter protein.
Summary & Conclusion Intra tracheal administration of mRNA formulated in Composition I results in efficient delivery of the mRNA to the respiratory tissue followed by translation of the encoded target gene (Luciferase).
Furthermore, absence of target protein detection in liver and spleen (target organs after systemic administration) suggests that the formulation remains local and does not translocate into the systemic circulation.
1. Methods
The test composition I (herein also referred to as Formulation I or LF92) comprising mRNA encoding luciferase was tested in vivo via intratracheal instillation in mice (1 μg/50 μL]
Euthanasia and luciferase detection in organs (IVIS measurement using an IVIS 100 In Vivo Imaging System (Perkin Elmer, USA) using the parameters Binnning: High, FOV, f1, 1 min).
Data collection/Evaluation The luciferase signals were recorded 6 hours post- administration, following exposure of the organs to luciferin, and bioluminescence was quantified using an in vivo imaging system (IVIS) as shown in Fig 1. Bioluminescence [p/s/cm2/sr] in explanted mouse lungs 1.1. Test Items and T est System
Test Item: Formulation I with chemically modified mRNA coding for luciferase and modified during in-vitro transcription to comprise 30 % I5U/ 3% I5C at a final concentration of 0.02 mg/mL.
Female mice of the C57BL/6J strain, aged between 8 and 10 weeks, were obtained from Charles River Laboratories for use in this study.
1.2. Formulation of lipidoid nanoparticle
Formulation I consists of the excipient mix dL_05(R) (i.e., the (R)-enantiomer of a compound according to formula (b-V) in the context of the present invention), DPPC, Cholesterol and DMG-PEG200 at a molar mixing ratio of 8: 5.29: 4.41 :0.88 at an N/P ratio of 8 (referring to the ratio of nitrogen atoms presents in the lipids to the amount of phosphorous atoms present in the therapeutic agent, preferably a nucleic acid, being packed; a lipid/lipidoid having a high N/P ratio, such as an N/P ratio of 8, allows to pack the same amount of RNA in an LiNP/LNP as compared to a lipid/lipidoid having a lower N/P ratio).
1.3. LNP production method
The mRNA sequences encoding the sequences luciferase were synthesized by in vitro transcription (IVT) from linearized plasmid DNA templates using modified nucleotides to generate partial modified mRNAs. IVT was performed for 120 min at 37°C using T7-RNA polymerase including co-transcriptional capping via Anti-Reverse Cap Analog (ARCA), followed by digestion of the template using DNAse I. After IVT, mRNAs were dephosphorylated for 15 min at 37°C using alkaline phosphates and enzymatically polyadenylated for 10-30 min at 37°C using PolyA polymerase to produce a Poly A tail of approximately 120 nucleotides. Purification steps were performed by precipitation and subsequently formulated in water for injection at a concentration of 1 mg/mL. mRNAs were stored at -80°C until LNP encapsulation. Each mRNA was LNP encapsulated via nanoprecipitation (NanoAssemblr lgnite+, Precision Nanosystems, Vancouver) by microfluidic mixing of mRNA in citrate buffer (pH 4.5) with ionizable-, structural-, helper- and polyethylene glycol (PEG) lipids in ethanol, followed by buffer exchange and concentration via tangential flow filtration. mRNA LNPs were filtered through a 0.2pm membrane and stored at -20°C until use. After manufacturing and freezing the drug product was analytically characterized for particle size (<100nm), particle polydispersity (< 0.2), encapsulation efficiency (≥90%), mRNA integrity (90-105%) and mRNA identity (confirmed length). The products were evaluated as acceptable for in vivo use. 1.4. Animal housing
All procedures were approved by the local animal welfare authorities (Regierung von Oberbayern) and were conducted according to the German animal protection law (Tierschutzgesetz). Mice were housed under specific pathogen free conditions (facility tested negative for any FELASA listed pathogens according to the annual health and hygiene survey 2017) in individually ventilated cages under a circadian light cycle (lights on from 7 a.m. to 7 p.m.). Food and drinking water were provided ad libitum. Animals were given at least 7 days for acclimatization until they entered the study.
1.5. Intratracheal instillation
Animals were anesthetized by the inhalation of pure oxygen containing 4% Isoflurane (Isothesia, Henry Shine, Germany). Unconscious animals were intubated using a 20 gauge catheter shortened to 37 mm. Test item in a final volume of 50μL was applied as one drop at the proximal tip of the tubus and thereby aspirated during the physiological inspiratory movement of the animal. Finally, 150 μL of air was applied, to assure that no liquid remained within the catheter.
1.6. Euthanasia and necropsy
Animals were set under full anesthesia through intraperitoneal injection of Fentanyl/Midazolam/Medetomidin (0.05/5.0/0.5 mg/kg bw). D-Luciferin (3 mg/100 μL PBS) was applied by intraperitoneal injection and intranasally using the “sniffing” method (1.5 mg/5 μL PBS). After 10 minutes mice were killed by cervical dislocation, immediately after blood withdrawal. Blood samples were centrifuged at 2.000 x g for 5 min at 4°C. The abdominal cavity was opened in the median axis. A careful cut was made in the diaphragm, which led to atmospheric pressure in the thoracal cavity and immediate collapse of the lungs. All rips were dissected, and the trachea was exposed. The left kidney artery was dissected. The small circulation was perfused using 5 mL PBS which was applied through the right ventricle. The lungs, livers and spleens were explanted, placed on a petri dish on their ventral surface. Ex vivo imaging was performed using an MS 100 In Vivo Imaging System (Perkin Elmer, USA) using the parameters Binnning: High, FOV, f1, 1 min.
1.7. Results
The aim of this task was to investigate whether mRNA encapsulated in Formulation I is able to transfect cells of the respiratory tissue after intratracheal administration and whether said formulation remains local to the administered organ or tissue. Results demonstrate that a clear signal of the reporter protein (Luciferase) encoded by the mRNA was detectable after treatment with a dose of 1 μg per animal. While a clear signal was detectable in the lung no protein could be detected in liver and spleen (Figure 1 ). This confirms that Formulation I (M01-001) surprisingly remains localized to the administered organ.
1.8. Discussion and Conclusion
Experiments have demonstrated that Formulation I (M01-001) efficiently deliver mRNA to respiratory cells resulting in translation into the target protein. The formulation remains local, and no expression is detectable in the liver or spleen.
This is surprising, as Formulation I (M01-001) has demonstrated transfection of liver and spleen after intravenous administration in previous studies. Therefore, the absence of reporter protein in liver and spleen allows the conclusion that Formulation I (M01-001) remains local in the administered organ or tissue, specifically the lung tissue and does not translocate into the systems circulation. Both observations allow the conclusion that Formulation I (M01-001) is a good candidate for further development into clinical administration for localized expression.
Example 2 - Long term monitoring of localized expression
BALB/c laboratory mice were short time anesthetized through inhalation of Isoflurane (2-3 %). Formulation I formulated Luciferase coding chemically modified mRNA was injected at a dose level of 20 μg/20 μL into the cranial tibial muscle of both hind limbs using 0.3 mL Insulin injection syringes (BD, Germany). Luciferase activity was measured 1 and 4 days subsequently under full anesthesia and following the application of 3 mg D-Luciferin/100 μL PBS which was applied intraperitoneally. Luciferase activity was measured using a Lumina XR In Vivo Imaging System (Perkin Elmer, USA). Local Luciferase activity was measured using a defined Region-Of-lnterest (ROI) which was drawn over the injection site. A ROI over the chest region served as internal control.
Results (Figures 2-4) showed expression of Luciferase at a comparable level in both hind limbs as soon as 1 day following application of the test item. Luciferase activity remained stable for 4 days following application. No Luciferase activity was observed in any other region of the body, indicating that there was no systemic distribution of the test item.
Example 3 - Localized expression in the subdermal tissue following subcutaneous application
Luciferase mRNA (20 μg) formulated as Formulation I was dissolved either in 300 μL physiological saline (0.9% NaCI) or in 300 μL hyaluronidase solution (Hylase “Dessau” 150 LU., HWI Pharma Services GmbH, Germany) and was subcutaneously injected in the cervical back of female BALB/c mice which were short time anesthetized by Isofluran inhalation. Luciferase activity was measured 4 hours following application using a Lumina XR In Vivo Imaging System (Perkin Elmer, USA). Luciferase activity was observed for both, following application of saline dissolved and following application of hyaluronidase dissolved LF92 (Formulation I) LUC mRNA. However, Luciferase activity was substantially higher (2-fold) following application of hyaluronidase dissolved LF92 (Formulation I) LUC mRNA compared to mRNA dissolved in saline. Subsequent to in vivo measurement of Luciferase activity (Figures 5 and 6), the animals were euthanized, and organs were explanted for ex vivo measurement of Luciferase activity. Here, no Luciferase activity above background level was observed in any organ measured (liver, spleen, heart, lungs, right kidney) following application of saline dissolved LF92 (Formulation I) LUC mRNA. However, substantial Luciferase activity was observed following application of LF92 (Formulation I) LUC mRNA which was dissolved in hyaluronidase (37-fold higher in the liver and 52-fold higher in the spleen) (Figures 7 and 8).
In Figure 7 there is no detectable luciferase signal in any of the organs. The values correspond to background signal and the image corresponds to the visible light image taken from the organs. In Figure 8, there is a significant signal coming from the luciferase. In order to detect that signal, the camera was set to the highest sensitivity. This causes the generation of a pixelated image. Thus, the comparison of figures 7 and 8 shows that after intramuscular administration together with Hylase a significative luciferase expression in at least the liver and spleen, which is indicative of reduced localized retention (or increased systemic distribution) of the mRNA encoding the luciferase.
Example 4
4.1. Scope
To understand the effect of the molar ratio of the different components of the Formulation of the invention on activity/expression level and immunogenicity, multiple alternatives to all components were provided as summarized in Table 3 and Table 4 and administered intramuscularly to mice as summarized in Figures 9 to 15. Each formulation was administered to a different mouse. Fig 9 summarizes the effect of Effect of phospholipid length. Fig 10 summarizes the effect of phospholipid type: PC (Phosphocholine) or PE (Phosphoethanolamine). Fig 11 summarizes the effect of stealth PEG length (14 to 18 C PEG 2000 or 5000 Da). The figure presents the impact of stealth PEG length on the expression of the SARS-CoV-2 spike protein, quantified by the area under the curve (AUG) of antibodies targeting the protein 36 days after the initial immunization in mice. Additionally, the effect of stealth lipid chain lengths — comprising 14, 16, or 18 carbon atoms — was evaluated. All tested stealth lipids provided excellent immunization effect. Especially the use of 14 C PEG 2000 Da provided the best results (i.e. , the highest immunization level). Fig 12 also highlights the effects of using G (glycerol DMG PEG) or PE (DMG PEG 2000phosphoethanolamine) as shown in Fig 11. Both provided good immunization levels. Dot size is indicative of immunization AUG after 36 days.
The screening was performed in a mouse model. LINPs were complexed with mRNA encoding for the spike protein of SARS-Cov2 (Wuhan variant) and injected intramuscular at day 1 and day 21. Animals subjected to treatment were evaluated for spike protein receptor binding domain (RBD)-specific antibodies following the first and second immunizations and for the cytokine expression profile of splenocytes using ELISpot analysis
In summary, all tested ratios provide significant expression and significant immunization in the test. M01-001 is depicted in Figures 9 to 15 as an open circle (O). M04-001 comprises the same LNP components as M01-001 (also referred to as Formulation I or LF92 herein above) and further comprises a poloxamer (P188); M04-001 is depicted as a cross (+). The addition of Poloxamer provided a higher immunogenicity.
Fig 13, Fig 14 and Fig 15 show that a range of cholesterol, dL_05(R) and different N/P, respectively, can be used to induce high immunization level, recorded as AUG of antibodies targeting the protein, 36 days after the initial immunization.
In summary all provided alternatives provide significative immunization measured as area under the curve (AUC) at day 36 (see Example 4.6).
4.2. LiNP preparation
Carriers were mixed using a NanoAssemblr™ benchtop system as described in Example 1 . Lipid mixes were prepared according to Table 3 and Table 4. Downstream processing (buffer exchange) was performed via dialysis in Slide-A-Lyzer MINI dialysis devices. The devices were prepared according to the manual of the manufacturer. 2 mL of formulation were transferred into the sample reservoir and dialyzed against water by shaking on an orbital shaker at 300 rpm. After 1.5 - 2 h the dialysis buffer was replaced by fresh water and the formulations were dialyzed overnight (14 - 18 h). Afterwards, 920 μL of each formulation were transferred to reaction tubes and concentrated via speed vac (at 45 °C in the V-AQ mode (vacuum- aqueous)) down to 0.5 mL (c = 0.3125 mg/mL). Aggregates were removed by centrifugation (30 min at 16,000 x g). 400 μL of the supernatant were transferred in a new reaction tube, 100 μL of 5 % (w/v) P188, 50 % (w/v) sucrose and 250 mM NaCI were added and pipetted up and down four to five times. In the case of M04-001 100 μL of 5 % (w/v) P188, 50 % (w/v) sucrose and 125 mM NaCI is added. Formulations were stored at 4 °C until further use.
4.3. Immunization
On the first day of immunization, a total of 0.020 mL of the respective test item was administered to the animals by intramuscular (i.m.) injection to the right leg into the musculus quadriceps. All animals were administered a second time on study day 22 with 0.020 mL of the respective test item also via intramuscular (i.m.) injection into the same leg.
4.4. Blood Sampling
Approximately 100 μL blood was sampled from the vena facialis into serum separator tubes from each animal on study days 1 (pre values prior to test item application) and 15. As part of the sacrifice, blood was sampled via cardiac puncture into serum separator tubes on study day 36 from each animal.
All blood samples collected from all animals were left standing at room temperature for ca. 30 minutes for blood coagulation. Subsequently, samples were centrifuged for 10 min at 4°C at approx. 1000 g. The serum was transferred after the centrifugation into separate tubes. Samples collected at study day 36 will were split into two tubes.
4.5. Necropsy
On the day of the scheduled euthanasia (study day 36), all animals were sacrificed using anesthesia (ketamine /xylazin). The spleen of all animals was removed and transferred to 50 mL Falcons filled with 5 mL pre-warmed 1x C.T.L. wash buffer. The spleens were kept at 37°C until further processing shortly thereafter.
4.6. Quantification of RBD-specific antibodies
SARS-CoV-2 RBD-specific antibodies were quantified with ELISA. For this purpose, a 384- well plate was coated over night at 4 °C with 2 μg/mL recombinant SARS-CoV-2 spike protein RBD (Sino Biological, 40592-V08B), diluted in CBB buffer, using 20 μL per well. The plate was washed 3x with PBST using a microplate washer. The plate was blocked 1 h at RT and 600 rpm with 50 μL 1 % casein blocking solution (ThermoFisher Scientific, Cat. Nr. 37528) per well. The plate was washed 3x with PBST using a microplate washer. An anti-RBD IgG standard curve ranging from 0.06 -1,000 ng/mL was prepared in PBST using mouse anti-SARS-CoV-2 RBD antibody (R&D Systems, Cat. Nr. MAB105808-100). Standard curve and samples (Serum was applied in serial 1 :4-dilution starting at 1 :50) were applied to the plate using 20 μL per well and incubated for 1 h at RT and 600 rpm. The plate was washed 3x with PBST using a microplate washer. The detection antibody (Abeam, ab205719) was diluted 1 :2000 in PBST, applied to the plate using 20 μL per well and incubated for 1 h at RT and 600 rpm. The plate was washed 3x with PBST using a microplate washer. Lastly, 20 μL TMB was added to each well and the plate was incubated for 1.5 min at RT and 600 rpm. The reaction was stopped using 10 μL H2SO4 per well. Absorption is measured at 450/650 nm using a microplate reader. To obtain IgG concentrations in samples, the results were interpolated using the standard curve. The lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) of the assay are 0.5 ng/mL and 100 ng/mL, respectively. Serum MRD is 1 :800. In addition to quantification of antibody levels, also reciprocal titers were determined. Titers are defined as highest measured concentrations that obtains results > limit of detection (LOD). The LOD of the assay is defined as 3*Mean OD of pure PBST. Calculation of the area under the curve (AUC): For each sample collected, the ELISA optical density (OD) was measured over a dilution range. A non-linear fit was confirmed for the OD data: log (inhibitor) vs response. R2 values of >95 indicated a very high degree of correlation between the dilution of the inhibitor and the optical density measured. The AUC was computed for the OD data over the dilution factors 1.69897, 2.30103, 2.90308999, 3.50514998, 4.10720997 and 4.70926996. This data was used as AUC for all downstream analysis.
Table 3
Figure imgf000115_0001
Table 4
Figure imgf000116_0001
Table 5 -
Figure imgf000117_0001
Table 5 discloses the standard names of helper lipids and stealth lipids used in all formulations. Abbreviations for Examples 4-6
Figure imgf000118_0001
Figure imgf000119_0001
Example 4: Local delivery of Formulation l-based LiNPs carrying an mRNA encoding a therapeutic protein - human interferon lambda 1 (hlFNλ1 ).
4.1. Introduction
A frozen LiNP Formulation I suspension at a concentration of 2.5 mg/mL comprising a modified messenger RNA (mRNA) encoding human interferon lambda 1 (hlFNλ1), referred in the following as the test drug product hlFNλ1 LNP was used to test the localized delivery of a therapeutic polypeptide of interest.
Interferons (IFNs) are potent cytokines that play a key role as first line defense in viral infections by controlling inflammation and directly inducing anti-viral mechanisms. Of those, type III interferons such as interferon lambda 1 (IFNλ1) are critically important for viral defense directly at the respiratory epithelium, the common entry site for respiratory viruses. The advantages of localized expression of a therapeutic protein such as IFNλ1 is that side effects caused by systemic activation of downstream targets can be avoided. Here we show localized expression when using the lipidoids of the invention.
4.2. Test mRNA
The mRNA coding for a human codon optimized Interferon Lambda 1 according to SEQ ID NO: 41 with an average length of 788 nucleotides was manufactured by in-vitro transcription (IVT) from a linearized plasmid DNA template using a T7 RNA polymerase using in the mix 25% of modified bases 5-methylcytosine and 75% cytidine and 25% 2-thiouridine and 75% uridine to generate a partially modified mRNA. The use of modified nucleosides in the mRNA reduces the affinity and recognition of mRNA by intracellular like toll-like receptors (TLRs), retinoid-inducible gene 1 (RIG-1), and others and substantially decrease activation of the innate immune system in vitro and in vivo and concomitantly increase the stability of the mRNA, allowing for prolonged, high-level cellular translation. The LiNPs comprise the lipidoid component (dL_05 (R)) and excipients optimized for aerosol delivery of LNP-formulated mRNA. The complete structure of the mRNA includes a 5’ Cap, a 5’ minimal UTR, a human codon-optimized IFNλ1 coding sequence, and a poly(A) tail (SEQ ID NO: 42). The poly(A) might be an encoded poly A or might be added enzymatically. In the tested mRNA, the poly(A) was added enzymatically. 4.3. Generation of the hlFNλ1 Test LiNP
The drug product tested was a preservative-free, sterile dispersion of mRNA encoding hlFNλ1 formulated in Formulation I lipidoid nanoparticles (LiNP) in an aqueous cryoprotectant solution. The lipidoid nanoparticles were generated by mixing the mRNA with a solution of one lipidoid and three lipid excipients (lipidoid dL_05(R), and the lipids DPPC [1 ,2-dipalmitoyl-sn-glycero- 3-phosphocholine], cholesterol, and DMG-PEG 2000 [1 ,2-Dimyristoyl-rac-glycero-3- methyl polyoxyethylene glycol 2000]). The excipients associate with the mRNA, protect it from degradation and aid its delivery to the target cells in the respiratory epithelium. Downstream processing after mixing lipids and mRNA included buffer exchange and concentration. After compounding into buffer containing sucrose, poloxamer P188 and sodium chloride in water for injection and sterile filtration, the drug was aseptically filled at a nominal filling volume of 1 .0 mL with an mRNA concentration of 2.50 ± 0.50 mg/mL and stored frozen at -20 ± 5°C. The drug was characterized as a white to off-white suspension.
4.4 Calculation of lung surface exposure
The delivered dose in inhalation toxicology studies was calculated using the recommendations of the Association of Inhalation Toxicologists considering aerosol concentration, respiratory minute volume and the inhalation duration (Alexander et al. 2008). The resulting nasal and lung surface exposures are dependent on the droplet size of the aerosol. Based on the measured mass median aerodynamic diameter (MMAD) of hlFNλ1 LNP aerosol droplets of 1.5-2 pm a fraction of 60% of the delivered dose was deposited in the upper respiratory tract while a fraction of 10% of the delivered dose was deposited in the lower respiratory tract (Snipes, M. B., R. O. McClellan, J. L. Mauderly, and R. K. Wolff. 1989. 'Retention patterns for inhaled particles in the lung: comparisons between laboratory animals and humans for chronic exposures', Health Phys, 57 Suppl 1 : 69-77; discussion 77-8; Wong, B. A. 2007. 'Inhalation exposure systems: design, methods and operation', Toxicol Pathol, 35: 3-14). This deposition pattern was confirmed by modelling using the Multiple Path Particle Dosimetry (MPPD) model (Anjilvel, S., and B. Asgharian. 1995. 'A multiple-path model of particle deposition in the rat lung', Fundam Appl Toxicol, 28: 41-50).
Example 5: Pharmacokinetics of hlFNλ1 mRNA in vivo
5.1. hlFNλ1 mRNA biodistribution in Mice and Rats - Methods
The biodistribution of hlFNλ1 LNP-mRNA (drug substance) was analyzed by RT-qPCR in lung, serum, and liver in the inhalation DRF study in rats and in the GLP-toxicology study in rats in serum and all major perfused organs (i.e. lung, liver, spleen, heart, kidney, brain). For DRF study, tissues were pulverized in liquid nitrogen and homogenized with Lysing Matrix D Tubes (MP Biomedicals). RNA was isolated using RNeasy Mini Kit® (QIAGEN®). RNA from serum was isolated using miRNeasy Serum/Plasma Advanced Kit® (QIAGEN). cDNA synthesis was performed with Transcriptor First Strand cDNA Synthesis Kit (Roche®) using Oligo(dT) priming. qPCR was run with customized TaqMan Primer/Probe using the TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific). For the GLP toxicology study hlFNλ1 LNP- mRNA was extracted from rat tissues using QIAGEN RNeasy Mini Kit (QIAGEN) and from serum using miRNeasy Serum/Plasma Advanced Kit (QIAGEN). Rat tissue and serum RNA extracts were quantified by a 2-step reverse-transcription RT-qPCR using the Roche Transcriptor High Fidelity cDNA synthesis Kit with Oligo(dT) priming followed by qPCR using TaqMan™ Environmental Master Mix (2.0) (Thermo Fisher Scientific). mRNA extraction and RT-qPCR for the GLP study were validated methods.
5.2. Pharmacokinetics of mRNA of test product hlFNλ1 LNP
The systemic bioavailability of the hlFNλ1 LNP-mRNA was assessed through quantitative PGR (qPCR) following its inhalation administration in rat Dose Range Finding (DRF) and Good Laboratory Practice (GLP) toxicology studies. (Study WP2_3_10, WP2_3_13, and WP2_3_15 summarized respectively in Table 6, Table 7 and Table 8 below and in Fig 16).
In the non-GLP Dose Range Finding (DRF) study involving Wistar Han rats, the highest concentrations of hlFNλ1 LNP-mRNA from the test product were detected in lung homogenates at 6 hours post single inhaled administration (the earliest measurement after drug administration). Lung levels at 24 hours were lower than at 6 hours but remained consistent up to 48 hours. Beyond 48 hours, hlFNλ1 LNP-mRNA levels in the lung further decreased (see Fig 16D). Notably, 24 hours post-administration, hlFNλ1 LNP-mRNA levels in the lungs showed a dose-dependent increase, aligning with the dose-dependent rise in hlFNλ1 protein concentrations in the lung (see Fig 21A-E).
Following a single inhaled dose in rats (designated as WP2_3_15, detailed in Table 7 below), Test product hlFNλ1 LNP-mRNA was measured in serum and homogenates of major perfused organs by RT-qPCR within 2h following single inhaled administration of test product hlFNλ1 LNP. Values were quantified by interpolating a standard curve of mRNA in spiked-in neat matrix of untreated animals. Signals in vehicle-treated animals are considered background levels of the assay. Each dot represents one animal. Columns and error bars represent mean values and standard deviation. The hlFNλ1 coding mRNA from the test product was not detected in the serum, and its liver copy numbers matched the background levels of vehicle- treated animals (Fig 17). The reported vehicle copies/ng or copies/mL above the LOD are considered an artifact, as the vehicle does not contain any mRNA. These results confirm that the drug substance mRNA, coding for therapeutic protein such as hlFNλ, does not exhibit a substantial systemic bioavailability when inhaled.
Following single inhaled administration in the GLP-tox study test product hlFNλ1 LNP-mRNA was detected in the lung. Low signals in the range of background levels of vehicle treated animals (i.e. at Ct values >35) were observed in serum and major perfused organs. The hypothetical copy numbers in these organs correspond to less than 0.01 % of the copy numbers detected in lung and are considered as not biologically relevant.
The data confirm the results of the biodistribution assessment in the non-GLP DRF studies and demonstrate that test product hlFNλ1 LNP is not systemically bioavailable to a relevant extent following inhaled administrations.
Table 6
Figure imgf000122_0001
Figure imgf000123_0002
Table 7
Figure imgf000123_0001
Table 8
Figure imgf000124_0001
Example 6: Pharmacokinetics of ionizable lipidoid dL_05 in hlFNλ1 LNP
To test the biodistribution of the LiNPs lipid components, the biodistribution of defined ionizable lipidoid 05 (dL_05) was determined in lungs, serum, and liver in a GLP-toxicology study in rats (Study WP2_3_13, see Table 7 above) by a validated HPLC-MS/MS (High Performance Liquid Chromatography Tandem Mass Spectrometry). Detection of LNPs using dL_05 is particularly suitable as a specific biomarker for the LNP biodistribution as no endogenous levels are expected. The other lipid components of the LNP are cholesterol (occurs endogenously), DPPC and DMG-PEG. DPPC is naturally occurring and the main phospholipid of pulmonary surfactant, while detection of DMG-PEG in humans is potentially biased by the exposure to PEG from other sources as it is a widely used excipient in many pharmaceutical and cosmetical products. This renders dL_05 as a very specific translational biomarker for systemic detection of the drug product. When using HPLC-MS/MS, the lowest limit of quantification using (LLOQ) to detect dL_05 in serum and lung was 100 ng/mL and in liver 200 ng/ml.
The focus of the in vivo biodistribution assessment was serum and liver as known LNPs and LNP-formulated mRNA typically distribute to the liver if they become systemically bioavailable. In line with the distribution pattern of the test product hlFNλ1 LNP-mRNA (Fig 18A and Fig 18B, biodistribution of dL_05 was only detected in the lung following inhalation of test product hlFNλ1 LNP while no systemic bioavailability in the serum or any of the major perfused organs was observed after single (Fig 19A) or multiple administration (Fig 19B). Lack of exposure in serum and liver therefore supports lack of systemic bioavailability of LiNPs comprising the lipidoid of the invention.
Example 7 Pharmacokinetics of hlFNλ1 protein in vivo
7.1 hlFNλ1 protein expression in mice
Pulmonary hlFNλ1 production following local delivery of hlFNλ1 LNP to the lungs was confirmed in several studies in mice and rats by measuring hlFNλ1 in lung homogenates.
In Study WP2_2_4 (Table 9) wild-type C57BL/6 mice received a single dose of test product hlFNλ1 LNP through nasal sniffing at lung surface exposure of 0.020 or 0.056 mg/m2. A control group that received 0.020 mg/m2 untranslatable control-mRNA was included. Animals were euthanized at 5, 24, 48, 72 and 96 h post administration and lungs were harvested for endpoint measurements. Study results demonstrate that test product hlFNλ1 LNP was well tolerated following administration to the lungs of mice and resulted in dose-dependent pulmonary production of hlFNλ1 at the 5 h time (Fig 20A and Fig 20B). Protein levels returned to background level from 24 h onwards. Formulated control-mRNA did not result in increased hlFNλ1 protein level, confirming that increased hlFNλ1 concentration resulted from translation of test product hlFNλ1 LNP-mRNA. hlFNλ1 protein was produced dose-dependently in lung homogenates at 5 h after administration. Lungs of animals necropsied at later time points (24 to 96 h) showed no evidence of hlFNλ1 protein concentration above lower limit of quantification (LLOQ). Mean and standard deviation of three animals is shown.
Table 9 Summary of in vivo pharmacokinetics / pharmacodynamics studies
Figure imgf000126_0001
7.2. hlFNλ1 protein expression in rats
7.2.1. Study WP2_3_10
Single and multiple dose-range-finding inhalation studies in rats were conducted in which hlFNλ1 production was analyzed. Additionally, a single dose DRF study conducted in rats in which serum and lung levels of hlFNλ1 were measured following exposure to test product hlFNλ1 LNP only to the nasal cavity. These DRF studies in rats are summarized in Tables 5 and 6 above and Table 11 below.
In an inhalation study (Study WP2_3_10, summarized in Table 6 above) Wistar Han rats received test product hlFNλ1 LNP via nose-only inhalation at lung exposures of 0.012, 0.024, 0.048, 0.096 mg/m2 or formulated untranslatable control-mRNA at a lung exposure of 0.024 mg/m2 (Fig 21 A to 21 E). A control group receiving only a vehicle solution containing all components of the drug product formulation was included. Animals of all dose groups were euthanized 24h post administration. In the 0.024 mg/m2 dose group satellite animals were also sacrificed at 6, 24, 48, 72, 96 and 144 h post administration. Single-dose inhalation administration of test product hlFNλ1 LNP in rats led to dose- and time-dependent hlFNλ1 production in the lungs at 24 h, whereas hlFNλ1 protein concentrations in animals treated with untranslatable control-mRNA and vehicle were below the lower limit of quantification. Highest hlFNλ1 protein levels were measured at 6 h post administration and returned to baseline at 48 hours following administration of test product hlFNλ1 LNP at a dose of 0.024 mg/m2 (Fig 19B). No hlFNλ1 was detected in serum.
7.2.2. Study WP2_3_13
In a GLP-compliant inhalation toxicology study involving Wistar Han rats (Study WP2_3_13, see Table 7 above), a single dose of LiNP hlFNλ1 was administered to each subject. The dosage was 2.05 mg/kg of body weight, delivered via nose-only inhalation. The study included 10 males and 10 females in the main study group, and an additional subgroup comprising 5 males and 8 females for biodistribution and lung safety assessment. The calculated nasal exposure for this administration was 176 mg/m2 of surface area as explained below.
The doses are specified as surface exposure [mg/m2] in lung or nose for better comparability of studies conducted in different species following different routes of administration. The surface exposures were calculated based on deposited hlFNλ1 LNP following nasal sniffing or inhalation and the respective surface area in the animal models (Fernandes, C. A., and R. Vanbever. 2009. 'Preclinical models for pulmonary drug delivery', Expert Opin Drug Deliv, 6: 1231-45; Gizurarson, S. 1990. Animal models for intranasal drug delivery studies. A review article', Acta Pharm Nord, 2: 105-22). Principles of calculation of surfaces exposures and extrapolation across species are further detailed in Section 7.1.
Following nasal administration in mice, the deposited dose and therefore surface exposures in nasal cavity and the lungs depend on the applied volume (Southam, D. S., M. Dolovich, P. M. O'Byrne, and M. D. Inman. 2002. 'Distribution of intranasal instillations in mice: effects of volume, time, body position, and anesthesia', Am J Physiol Lung Cell Mol Physiol, 282: L833- 9). Volumes relevant to studies described herein are 15, 20 and 50 pl of drug product, resulting in lung deposition of hlFNλ1 LNP of 35%, 40%, and 55.7% of the delivered dose, respectively.
Animals in the main study group and those in the biodistribution and lung safety groups were sacrificed 24 hours after their last administration. The recovery group animals were sacrificed 14 days post their final exposure. The test product, hlFNλ1 LNP, was administered via nose- only inhalation. This included a single-dose group that received the highest dose once, and a vehicle group that underwent three administrations of the vehicle solution, which contains all components of the drug product formulation. Additionally, satellite animals designated for pharmacokinetic (PK) analysis were euthanized immediately after plethysmography on the day of their last administration, which was on day 1 for the single-dose group. hlFNλ1 protein levels in lung homogenates and serum were measured using a validated ECL assay. Within 2 hours of a single administration of hlFNλ1 LNP, lung concentrations reached up to 60,000 ng/g of tissue (Fig 22A), while serum levels were below 600 μg/mL (see Fig 22B and Fig 22C). By normalizing these data to the absolute amounts of hlFNλ1 - using the average lung weight of 1.7 g and the total blood volume of approximately 18 mL - it was found that less than 0.01% of the translated hlFNλ1 in the lungs reached the circulation on average approximately 2 hours after administration (between 33 minutes and 5 hours 22 minutes, see Fig 22). After repeated administrations of hlFNλ1 LNP, both lung and serum hlFNλ1 concentrations were lower than those observed with a single dose. The lungs showed a dose- dependent increase in dL_05 concentrations, but serum and liver levels remained below the limit of quantification. No significant observations were noted in clinical examinations, clinical chemistry, or urine analysis.
Table 10 - sample recollection times after last dose and quantification of lipidoid dL_05 and mRNA copies.
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
7.2.3
In an intranasal dose range finding study (Study WP2_3_14, below, see Fig 22) test product hlFNλ1 LNP was administered to Wistar Han rats at doses that result in a nasal surface exposure of 3, 11 and 44 mg/m2, respectively. Nasal administration was as a single droplet of 13 μL per nostril (max. volume retained in the nasal cavity). Single dose administration (all dose levels of hlFNλ1 LNP, vehicle, and control-mRNA and rat IFNλ1 -encoding mRNAat 0.075 mg/kg bw). Vehicle is the buffer in which the drug product hlFNλ1 LNP is diluted after purification and contains 10 % Sucrose w/v, 1% P188w/v, 50 mM NaCI, and water for Injection. Control-mRNA is a mRNA in which the ATGs have been scrambled and thus does not express any protein. Repeated dose administration (dose level 0.075 mg/kg bw; day 1 , 3, 5, 7). Control groups received untranslatable control-mRNA or mRNA coding for rat IFNλ at a dose that results in a nasal surface exposure of 11 mg/m2. Animals were sacrificed one day after the last administration. The two highest dose groups also included animals that were sacrificed 5 days after the last administration. An additional multiple dose group received four administrations of test product hlFNλ1 LNP at a dose resulting in 11 mg/m2 nasal surface exposure. Animals of this group were sacrificed 1 day after the last exposure. No hlFNλ1 protein was measured in serum and lungs confirming the absence of systemic bioavailability of hlFNλ1 protein encoded by test product hlFNλ1 LNP upon nasal administration.
Table 11
Figure imgf000131_0001
Figure imgf000132_0002
Example 8
8.1. Scope
To understand the effect of the molar ratio of the different components of the Formulation I on the capacity of Formulation I to remain localized, multiple alternatives to all components present in Formulation I were provided as summarized in Table 12 and tested in vivo using an in Vivo Imaging System (IVIS) in mice. The results are shown in Fig 23 A) to I).
8.2 Experimental design:
The following Formulations were prepared as previously described for Formulation I.
Table 12
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000133_0002
Similarly to Example 1, 50 μL of LINPs formulations comprising 0.06 mg of chemically modified mRNA coding for luciferase mRNA per mL were administered to five BALB/c mice by intratracheal instillation of encapsulated formulation. D-Luciferin substrate was applied to animals by intraperitoneal and intranasal application before they were euthanized 6 hours after application. Luciferase activity was measured in explanted organs using a Lumina XR In vivo Imaging system (Perkin Elmer, USA).
The Formulations of Table 12 were tested in five BALB/c mice for each treatment group carrier formulations with different ratios for the same components as Formulation I (dL_05(R), DPPC, Cholesterol, DMG-PEG2000). Fig. 23 shows the results for (A): Formulation I, (B) Formulation
M01-007, (C): Formulation M01-009, (D): Formulation M01-011 , (E): Formulation M01-017, (F): Formulation M01-034, (G): Formulation M01-006, (H): Formulation M01-021, and (I): Formulation M01-027 in whole BALB/c mice (left column) and ex vivo lungs, heart, liver, spleen and right kidney of the same animals (right column). All tested animals showed the same localization restricted to the lungs.
All tested formulations showed high expression in the organ to which they were delivered (lungs). A remarkable high expression was found for Formulations M01-006, M01-021 and M01-027 which presented up to an order of magnitude higher luciferase activity than Formulation I (see Fig. 23 (G-l), measured as average radiance as p/s/cm2/ sr). Comparative values for the tested formulations are shown in Fig 24 showing a radiance with average above 107 p/s/cm2/ sr for formulation M01-006, M01-021 and M01-027.
8.3 - Summary and conclusions
All tested ratios of lipidoid to helper, sterol, stealth lipid or N/P Ratios worked efficiently, showing a high luciferase expression. Additionally, all tested formulation showed the same restricted localization as Formulation I. This is highly surprising and supports the concept that the localization of the formulation is not caused by a specific ratio of lipids or mRNA but is caused by the lipidoid of the invention. A surprisingly higher expression was found for formulation M01-006, M01-021 and M01-027, compared to the already good expression of Formulation I.
Example 9
9.1. Scope
To understand the effect of using different helper and stealth lipids to those used in Formulation
I, when combined with lipidoid DL05, components were provided as summarized in Table 13 below and administered to BALC mice by intratracheal administration as described in Example
8. Results are summarized in Fig 25.
Table 13
Figure imgf000134_0001
Table 13 (Cont.)
Figure imgf000134_0002
Figure imgf000135_0001
9.2. Methods and results
Five mice were sacrificed six hours after intratracheal instillation of 50 μL (0.06 mg Luciferase mRNA/mL) for formulations with dL_05(R) and cholesterol but different helper and stealth lipid , as well as different lipid-to-lipid ratios. Results are show in Fig 25 (A-D): IVIS imaging of BALB/c mice (left col.) and ex vivo lungs, heart, liver, spleen, and right kidney of the same animals (right col.), (A): Formulation M02-102-006, (B) Formulation M02-103-003, (C) Formulation M02-002-003, and (D) Formulation M02-109-006. Scale represents radiance in p/sec/cm2/sr.
9.3. Conclusion
All tested additional helper (DSPC or DOPC) and stealth lipids (MPG-PEG 2000 or poly sarcosine based stealth lipid N-TETAMINE-pSar25 provided the same restricted localization after intratracheal instillation, supporting that good expression and localized expressions can be achieved with other helper and stealth lipids than those used in Formulation I. These results also support that the tissue restriction is caused by the lipidoid of the invention.
Example 10 - Localization of lipidoid combinations
10.1. Scope
To determine whether other lipidoids as defined in Formula (b-1 ) also show the same restricted localization after delivery, and also to test whether combination of lipidoids show restricted localization the following components were formulated as summarized in Table 14. The results after IVIS in mice are shown in Fig 26 (A) and (B).
10.2 Test formulation
A test formulation comprising a combination of LG2C and LE1D was formulated with mRNA coding for luciferase and administered as shown in Table 14 to five BALC mice by instillation as previously described in Example 8 and 9. LG2C: N1,N17-Didecyl-4,7,11,14-tetrakis(3-(decylamino)-3-oxopropyl)-4,7,11,74- tetraazaheptadecanediamide
Figure imgf000136_0001
LE1D: (13R,27R)-15, 18,22-Tris((R)-2-hydroxytetradecyl)-15,18,22,25- tetraazanonatriacontane-13,27-diol
Figure imgf000136_0002
Figure imgf000136_0003
Figure imgf000136_0004
Result Results are summarized for three mice in Fig 26 (A and B): showing localized expression of luciferase in BALB/c mice (left col.) and after ex vivo analysis of heart, lungs, right kidney, liver, and spleen (right col.) 6 hours after intratracheal instillation Formulation M03-076-3 comprising a combination of two ethyl-propyl-ethyl lipidoids different to dL_05(R), and also different helper lipids, as well as different lipid-to-lipid ratios also showed a surprising localized expression of luciferase. The results support that the lipidoids of the invention in general have the capacity to remain localized to the tissue to which they are administered. Figure 26 (A) shows an experiment with three mice and Fig 26 (B) an experiment with two mice. Formulation M03-076-3 comprises lipidoids DL_F (LG2C) (formula b-IX) and LE1D (formula b-X). Scale represents radiance in p/sec/cm2/sr.
Example 11 - Localization of other DL lipidoids
11.1. Scope
To determine whether other lipidoids as defined in Formula (b-l) also show the same restricted localization/localized retention after delivery, and also to test whether combination of lipidoids show restricted localization the lipidoids DL_F (formula b-IX above), DL_L (formula b-XI) and DL_N (formula b-XI I) were formulated as summarized in Table 15 below. The results after I VIS in mice and their excised organs are shown in Fig 27 (A), (B) and (C).
DL_L
Figure imgf000137_0001
DL_N
Figure imgf000138_0001
11.2 Test formulation The test formulation was formulated with mRNA coding for luciferase and administered as shown in Table 15 to three BALC mice by instillation as previously described in Example 8 and 9. The tested lipidoid comprises amide bonds in its lipid side chains that are potentially biodegradable which is thought to be advantageous regarding long-term tolerability after administration to a subject. This study aimed at exploring a library of lipidoid structures varying the substitution degree of alkyl chains per oligo amine as well as the alkyl chain length. For this purpose, complexes were formed with modified RNA generated in vitro in the presence of 25% m5C and 25% S2U, and encoding for firefly luciferase. The test mRNA 50 μL of the test item was applied to mice using an intratracheal spray application device (MicroSprayer 1A device (PennCentury, USA) under Isofluran inhalation anesthesia. The efficiency of delivery was quantified after 6 h via measurement of reporter protein level (as quantified by a luciferase luminescence signal) in the lung.
Table 15 (Cont.)
Figure imgf000139_0001
Table 15
Figure imgf000139_0002
For the luciferase analysis, mice were anesthetized through intraperitoneal injection of medetomidine/midazolam/fentanyl (0.15/2/0.005 mg/kg bw). 50 μL D-Luciferin (1.5 mg dissolved in PBS) were applied intranasally and 100μL (3 mg) intraperitoneally. Bioluminescence Imaging was conducted using an MS Lumina XR in vivo imaging system (Perkin Elmer, USA). After in vivo imaging mice were killed by cervical dislocation. The left kidney artery was dissected, and the circulation was flushed through injection of 5 mL PBS through the right heart ventricle. Lung, liver and spleen were placed on a petri dish and were as well imaged ex vivo.
11.3 Results are summarized for three mice in Fig 27 (A to C): showing localized expression of luciferase in BALB/c mice (left col.) and after ex vivo analysis of lungs, liver, and spleen (right col.) 6 hours after intratracheal spray application. Formulations LF110, LF181 and LF53, comprising each an ethyl-propyl-ethyl lipidoid different to dL_05(R), showed, surprisingly, a localized expression of luciferase. The figure scale represents radiance, measured in photons per second per square centimeter per steradian (p/sec/cm2/sr). The results support that the lipidoids of the invention in general have the capacity to remain localized to the tissue to which they are administered.
Example 12 - Localized retention of Formulation I after nasal delivery
12.1 Scope
The test whether the formulation of the invention remains localized after intranasal delivery, Formulation I comprising DL_05(R) as described in Example 3 was delivered into the nasal cavity. Results are shown in Fig 28 in color on the left and greyscale on the right.
12.2 Test formulation and methods
Each C57BL6&JRj female adult mice received 20 μL of test Formulation I with an mRNA coding for luciferase. 4 hours after test item application, 1 .5 mg D-Luciferin dissolved in 50 μL PBS was applied intranasally (sniffing method), 3 mg D-Luciferin dissolved in 100 μL PBS was also applied via intraperitoneal injection. The mRNA coded for Firefly luciferase was generated in vitro in the presence of 25% s2U and 25% m5C.
12.3 Results are summarized for three mice in Fig 28 (A to C):
Figure 28 shows MS imaging of C57BL6&JRj female adult mice, 4 hours after intranasal delivery showing localized expression in the nasal airways when administered with 0.15 mg/mL (A), 0.5 mg/mL (B), 1.5 mg/mL (C) modified mRNA coding for luciferase formulated with Formulation I (i.e. , comprising dL_05(R)). Left images show color image and right images show the same mice in grey scale. Circles indicate radiance values measured in regions of interest (ROIs). Scale represents radiance in p/sec/cm2/sr. Formulations LF110, LF181 and LF53, each comprising an ethyl-propyl-ethyl lipidoid different to dL_05(R), showed, surprisingly, a localized expression of luciferase. The results further support that the lipidoids of the invention (as well as compositions comprising the same) in general have the capacity to remain localized to the tissue to which they are administered. APPLICATION SEQUENCES
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
 
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001

Claims

1 . A composition for use in the treatment and/or prevention of a disease or disorder, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein said composition, remains localized at the site of administration and/or essentially does not exhibit systemic distribution throughout the patient's body.
2. A composition for use in the treatment and/or prevention of a disease, the treatment comprising local administration of the composition, the composition comprising:
(a) one or more therapeutic agent(s); and
(b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) and/or diluent(s); wherein said composition has a prolonged retention at the site of administration; and/or wherein said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration.
3. A composition for use in the treatment and/or prevention of a disease, the treatment comprising local administration of the composition, the composition comprising: a) one or more therapeutic agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration; and/or wherein the patient has less side-effects compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
4. The composition for use according to claim 1 or 2, wherein a reduced amount of the composition or of the therapeutic agent is to be administered to achieve a similar therapeutic effect compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration; and/or wherein the patient has less side-effects compared to the same therapeutic agent formulated in a composition that does not have prolonged retention at the site of administration.
5. The composition for use according to claim 1 or 3, wherein said composition has a prolonged retention at the site of administration; and/or wherein said therapeutic agent exerts its effect at the site of administration by prolonged retention at the site of administration.
6. The composition for use according to claim 2 or 3, wherein said composition, when administered to said site of administration, remains localized and essentially does not exhibit systemic distribution throughout the patient's body.
7. A cosmetic composition comprising: a) one or more active agent(s); and b) a carrier, wherein said carrier comprises: i. an ionizable lipid and/or an ionizable lipidoid; ii. optionally one or more helper lipid(s); and iii. optionally one or more pharmaceutically acceptable excipient(s) or diluent(s); wherein said cosmetic composition is an ointment, a creme, a foam, a gel, a lotion, an aqueous liquid, or a powder, or wherein said cosmetic composition is formulated as an ointment, a creme, a foam, a gel, a lotion, an aqueous liquid, or a powder.
8. The composition for use according to any one of claims 1 to 6, or the cosmetic composition according to claim 7, wherein the carrier is a lipid nanoparticle (LNP), a lipidoid nanoparticle (LiNP), a liposome, a micelle, an emulsion, a Nanostructured Lipid Carrier (NLCs), or a Lipid-Drug Conjugate (LDC), preferably an LNP or an LiNP, and/or wherein the agent is formulated as a lipid nanoparticle (LNP), a lipidoid nanoparticle (LiNP), a liposome, a micelle, an emulsion, a Nanostructured Lipid Carrier (NLCs), or a Lipid-Drug Conjugate (LDC), preferably as an LNP or as an LiNP.
9. The composition for use or the cosmetic composition according to claim 8, wherein said ionizable lipidoid is a compound of formula (b-l):
Figure imgf000157_0001
formula (b-l), wherein a is 1 or 2 and b is an integer of 1 to 4, or a is an integer of 1 to 4 and b is 1 or 2, p is 1 or 2, m is 1 or 2, n is 0 or 1 , m+n is ≥ 2, and
R1A to R6A are independently of each other selected from hydrogen, -CH2-CH(OH)-R7A, -CH(R7A)-CH2-OH, -CH2-CH2-C(=O)-O-R7A, -CH2-CH2-C(=O)-NH-R7A, and -CH2-R7A, wherein R7A is selected from C3-C18 alkyl, C3-C18 alkenyl having one C-C double bond, a protecting group for an amino group, -C(NH)-NH2, a poly(ethylene glycol) chain, and a receptor ligand; and wherein at least two residues among R1A to R6A are a group selected from -CH2-CH(OH)-R7A, -CH(R7A)-CH2OH, -CH2-CH2-C(=O)-O-R7A, -CH2-CH2-C(=O)-NH-R7A, and -CH2R7A, wherein R7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms comprised or contained in the compound of formula (b-l) are optionally protonated to provide a compound carrying one or more positive charges, preferably wherein the variables a, b, p, m, n and R1A to R6A are defined as follows: a is 1 and b is an integer of 2 to 4, or a is an integer of 2 to 4 and b is 1 , p is 1 or 2, m is 1 or 2, n is 0 or 1 , m+n is ≥ 2, and R1A to R6A are independently of each other selected from hydrogen, -CH2-CH(OH)-R7A,
-CH(R7A)-CH2-OH, -CH2-CH2-C(=O)-O-R7A, -CH2CH2C(=O)-NH-R7A, and -CH2-R7A, wherein R7A is selected from C3-C18 alkyl, C3-C18 alkenyl having one C-C double bond, a protecting group for an amino group, -C(NH)-NH2, a poly(ethylene glycol) chain, and a receptor ligand; and wherein at least two residues among R1A to R6A are a group selected from -CH2- CH(OH)-R7A, -CH(R7A)-CH2OH, -CH2CH2-C(=O)-O-R7A, -CH2CH2-C(=O)-NH- R7A, and -CH2R7A, wherein R7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms comprised or contained in the compound of formula (b-l) are optionally protonated to provide a compound carrying one or more positive charges.
10. The composition for use or the cosmetic composition according to claim 8 or 9, wherein said ionizable lipidoid is a compound of formula (b-ll):
Figure imgf000158_0001
wherein a is 1 or 2, preferably 1 , b is 1 or 2, preferably 2,
R1A to R6A are defined as in claim 9, and wherein one or more of the nitrogen atoms comprised or contained in the compound of formula (b-ll) are optionally protonated to provide a compound carrying one or more positive charges.
11. The composition for use or the cosmetic composition according to any one of claims 8 to 10, wherein R1A to R6A are independently of each other selected from hydrogen, -CH2-CH(OH)-R7A, -CH2-CH2-C(=O)-O-R7A, -CH2CH2-C(=O)-NH-R7A, wherein R7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond, and wherein at least three, preferably at least four of R1A to R6A are selected from -CH2-CH(OH)-R7A, -CH2-CH2-C(=O)-O-R7A, and -CH2-CH2-C(=O)-NH-R7A, wherein R7A is selected from C3-C18 alkyl and C3-C18 alkenyl having one C-C double bond.
12. The composition for use or the cosmetic composition according any one of claims 9 to 11 , wherein said ionizable lipidoid comprises or consists of a compound of formula (b- V), formula (b-XI) and/or formula (b-XII), preferably a compound of formula (b-V):
Figure imgf000159_0001
13. The composition for use or the cosmetic composition according to any one of claims 8 to 12, wherein said ionizable lipidoid comprises or consists of a compound of formula (b-IX) and/or a compound of formula (b-X), preferably a compound of formula (b-X):
Figure imgf000160_0001
14. The composition for use or the cosmetic composition according to any one of claims 8 to 13, wherein said carrier comprises at least two ionizable lipids and/or at least two ionizable lipidoids.
15. The composition for use or the cosmetic composition according to claim 14, wherein said at least two ionizable lipids and/or said at least two ionizable lipidoids are as defined in any one of claims 9 to 14.
16. The composition for use or the cosmetic composition according to claim 15, wherein said carrier comprises a lipidoid according to formula (b-IX) and a lipidoid according to formula (b-X).
17. The composition for use or the cosmetic composition according to any one of claims 8 to 11 , wherein said ionizable lipidoid comprises or consists of a compound of formula (b-VII) or a compound of formula (b-VIII), preferably a compound of formula (b-VII):
Figure imgf000161_0001
18. The composition for use or the cosmetic composition according to any one of claims 8 to 12, wherein said ionizable lipidoid is a compound of formula (b-V) and preferably: a) is an R isomer of the compound of formula (b-V), and/or b) is present at a molar ratio of about 22 mol% to about 65 mol%, preferably about 34 mol% to about 52 mol%, more preferably about 36 mol% to about 50 mol%, and most preferably about 43.1 mol%.
19. The composition for use or the cosmetic composition according to any one of claims 8 to 18, wherein said one or more helper lipid(s) are selected from the group consisting of a) to c): a) a phospholipid; b) a sterol; and/or c) a stealth lipid.
20. The composition for use or the cosmetic composition according to claim 19, wherein said composition comprises said ionizable lipid and/or said ionizable lipidoid, said phospholipid, said sterol, and said stealth lipid, preferably at a molar ratio of about 8.0 : about 5.3 : about 4.4 : about 0.9,
21. The composition for use or the cosmetic composition according to claim 19 or 20, wherein said phospholipid: a) is selected from phosphocholine (PC) or phosphoethanolamine (PE), preferably PC; b) has a carbon chain length of about 14 to about 18, most preferably about 16; and/or c) is present in a molar ratio of about 10 mol% to about 45 mol%, preferably about 18 mol% to about 39 mol%, more preferably about 24 mol% to about 33 mol%, and most preferably about 28.5 mol%.
22. The composition for use or the cosmetic composition according to any one of claims 19 to 21 , wherein said sterol: a) is cholesterol; and/or b) is present at a molar ratio of about 12 mol% to about 38.5 mol%, preferably about 15 mol% to about 32 mol%, more preferably about 19 mol% to about 29 mol%, and most preferably about 23.7 mol%.
23. The composition for use or the cosmetic composition according to any one of claims 19 to 22, wherein said stealth lipid: a) is glycerolipid-based or PE lipid-based; b) has a carbon chain length of about 14 to about 18, most preferably about 14; c) comprises polyethylene glycol (PEG), and wherein said PEG has a molar mass of about 2000 to about 5000 Daton, most preferably about 2000 Dalton; and/or d) is present molar ratio of about 1.5 mol% to about 7 mol%, preferably about 3 mol% to about 6 mol%, more preferably about 4 mol% to about 5 mol%, and most preferably about 4.7 mol%.
24. The composition for use or the cosmetic composition according to any one of claims 19 to 23, wherein: a) the phospholipid is preferably a phospholipid with a carbon chain length of about 12 to about 18, more preferably phospholipid with a carbon chain length of about 16, most preferably DPPC; b) the sterol is cholesterol; and/or c) the stealth lipid is a PEGylated lipid, preferably a PEGylated lipid with a molar mass of the PEG chain between about 2000 to about 5000 Dalton, more preferably a PEGylated lipid with a molar mass of the PEG chain of about 2000 Dalton, most preferably the PEGylated lipid is DMG-PEG2000.
25. The composition for use or the cosmetic composition according to any one of claims 8 to 24, wherein said composition further comprises a triblock copolymer as component (p) preferably wherein said triblock copolymer comprises about one polypropylene oxide) block and about two poly(ethylene oxide) blocks.
26. The composition for use according to any one of claims 8 to 25, wherein said one or more therapeutic agent(s) is/are a) an anionic therapeutical substance and/or b) a nucleic acid, preferably an RNA, more preferably an mRNA, a miRNA, and/or an siRNA, even more preferably an mRNA, most preferably an mRNA comprising an open reading frame (ORF) encoding one or more polypeptide(s).
27. The composition for use according to claim 26, wherein said nucleic acid is an RNA encoding a microRNA or wherein said nucleic acid is an mRNA comprising an ORF encoding one or more polypeptides, preferably wherein said one or more polypeptide(s) are one or more functional protein(s) and/or one or more antigen(s).
28. The composition for use according to claim 27, wherein said one or more antigen(s) is/are selected from the group consisting of a viral antigen, a bacterial antigen, a cancer, and/or tumor associated antigen, and/or an allergen.
29. The composition for use according to any one of claims 26 to 28, wherein the mRNA comprises one or more features selected from the group consisting of the following: a) a CAP, preferably an anti-Reverse Cap Analog (ARCA) at its 5’ end, b) a 5’-untranslated region (5’-UTR) upstream of the ORF encoding said one or more polypeptide(s), c) a 5’-UTR comprising an elongated Kozak sequence (GCCACCAUG; SEQ ID NO: 44) upstream of the initiation codon of the ORF, d) a 5’-UTR comprising proximately upstream of an initiation codon of the ORF any one of the following sequences: i. GGGAGACGCCACC (SEQ ID NO: 11), ii. GAAGCGCCACC (SEQ ID NO: 12), iii. GGGACGCCACC (SEQ ID NO: 13), iv. GGGAGACTGCCACC (SEQ ID NO: 14), v.GAAGCTGCCACC (SEQ ID NO: 15), vi. GGGACTGCCACC (SEQ ID NO:16). e) a 3’-untranslated region (3’-UTR) downstream of the ORF encoding said one or more polypeptide(s), and f) a 3’-UTR sequence downstream of the ORF encoding said one or more polypeptide(s) selected from: i. GAAUU, and ii.
CCTCGCCCCGGACCTGCCCTCCCGCCAGGTGCACCCACCTGCAA TAAATGCAGCGAAGCCGGGA (SEQ ID NO:26).
30. The composition for use according to any one of claims 26 to 29, wherein the mRNA is a product of in-vitro transcription (IVT).
31. The composition for use according to any one of claims 26 to 30, wherein the mRNA comprises a polyadenylation signal or a (poly(A)) tail downstream of the ORF encoding said one or more polypeptide(s).
32. The composition for use according to any one of claims 26 to 31 , wherein the mRNA comprises one or more modified nucleosides.
33. The composition for use according to claim 26 to 32, wherein the one or more modified nucleosides are selected from the group consisting of the following:
N -methylpseudouridine (m1ψ ), pseudouridine, N -ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1- methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-iodo-uridine, 5-methoxyuridine, 2'-O-methyl uridine, 5- iodocytidine, 5-methylcytosine, 5-methylcytidine, N -methyladenosine, and NQ- methyladenosine, preferably N1 -methylpseudouridine.
34. The composition for use according to claim 32 or 33, wherein any one or more of the following applies: a) wherein up to 100% of the uridines comprised in the ORF have been modified, preferably wherein at least about 50 mol% of the uridines comprised in the ORF have been modified, more preferably wherein any value between 50 and 100% has been modified, even more preferably wherein 100% of the uridines have been modified; b) wherein at least about 50 mol% of the uridines comprised in the mRNA have been modified; c) wherein at least about 50 mol% of the uridines comprised in the ORF have been modified to m1ψ ; d) wherein at least about 50 mol% of the uridines comprised in the mRNA have been modified to m1ψ ; e) wherein about 5 mol% to about 50 mol% of the uridines comprised in the mRNA are 5-iodouridines and about 5 mol% to about 50 mol% of the cytidines comprised in the mRNA are 5-iodocytidines; and/or f) wherein about 0.5 mol% to about 50 mol% of the uridines comprised in the mRNA are 2-thiouridine, preferably about 1 mol% to about 50 mol%, more preferably 1 mol% to 5 ml% of the uridines are 2-thiouridine, and of the cytidines comprised in the mRNA 0.5 mol% to about 50 mol% are 5-methylcytidines.
35. The composition for use according to any one of claims 8 to 34, wherein the composition is to be administered to a patient in need thereof.
36. The composition for use according to any one of claims 8 to 35, wherein said site of administration comprises a tissue, an organ, and/or an anatomical region, preferably said solid tissue, organ, and/or anatomical region is a solid tissue, organ and/or anatomical region, more preferably said solid tissue, organ, and/or anatomical region is selected from the group consisting of the lungs, the nose, the heart, the brain, the spleen, the lymph nodes, the bones, the tendons, the skeletal muscles, joints, the stomach, the small intestine, the large intestine, the kidneys, the bladder, the breast, the testes, the ovaries, the uterus, the spleen, the thymus, the brainstem, the cerebellum, the spinal cord, the eye, the ear, the tongue, the skin and/or tumors present in said solid tissues, organs and/or anatomical regions.
37. The composition for use according to any one of claims 8 to 36, further comprising one or more stabilizing agent(s), adjuvant(s), and/or immunomodulator(s).
38. The composition for use according to any one of claims 8 to 37, wherein said therapeutic agent or carrier is encapsulated within a hydrogel or a biocompatible matrix.
39. A method for preventing, treating, and/or ameliorating a disease, wherein the method comprises administering an effective amount of the composition as defined in any one of claims 1 to 6 and 8 to 38 to a subject.
40. Use of a composition as defined in any one of claims 1 to 6 and 8 to 38 in the manufacture of medicament for the prevention, treatment, and/or amelioration of a disease.
41. The composition for use according to any one of claims 1 to 6 and 8 to 38, the method of treatment according to claim 39, or the use of the composition according to claim 40, wherein the prevention of said disease comprises prevention by immunization, even more preferably in the prevention by local immunization.
42. The composition for use according to any one of claims 1 to 6, 8 to 38, and 41 , the method of treatment according to claim 39 or 41 , or the use of the composition according to claim 40 or 41 , wherein said disease is selected from: genetic mutations, autoimmune diseases, metabolic imbalances, neurodegenerative disorders, degenerative disorders of the joints, arthrosis, arthritis, bone fractures, non-union fractures, solid tumor diseases (including soft tissue tumors, tumors of the heart, the lungs, the liver, the spleen, the kidneys, the brain, the oral cavity, the intestine, the skin, the pancreas, the prostate gland, the mammary glands, the ovaries, the urinary bladder, the bones (including osteosarcoma, chondrosarcoma, Ewing sarcoma)), tumors of the pleural and the peritoneal cavity, diseases of the respiratory system including rhinitis and lung diseases such as asthma, viral induced asthma, COPD, including lung autoimmune diseases and ciliopathies, bone fractures or lesions thereof, tendon fractures or lesions thereof, joint infections, ligament ruptures, resistant Staphylococcus Aureus (MRSA) and/or Multidrug resistant Tuberculosis), viral infections, preferably a viral infection, more preferably a viral infection selected from enterovirus, rhinovirus, Influenza (Flu), respiratory syncytial virus (RSV) Hepatitis A, Hepatitis B, Hepatitis C, Human Papillomavirus (HPV), Measles, Mumps, Rubella, Polio, Rabies, Varicella (Chickenpox), Shingles (Herpes Zoster), Rotavirus, Yellow Fever, Smallpox, Japanese Encephalitis, Tick-Borne Encephalitis (TBE), Dengue Fever, West Nile Virus, Chikungunya Virus, Ebola Virus, Marburg Virus, Human Immunodeficiency Virus (HIV), a coronavirus infection (including COVID-19), most preferably a coronavirus infection.
43. The composition for use according to any one of claims 1 to 6, 8 to 38, 41 , and 42, the method of treatment according to any one of claims 39, 41 , and 42, or the use of the composition according to any one of claims 40 to 42, wherein said composition is to be administered to one or more solid tissue(s), solid organ(s) and/or solid anatomical region(s), preferably wherein said one or more solid tissue(s), solid organ(s) and/or solid anatomical region(s) are selected from the group consisting of the lungs, the nose, the heart, the brain, the spleen, the lymph nodes, the bones, the tendons, the skeletal muscles, the joints, the stomach, the small intestine, the large intestine, the kidneys, the bladder, the breast, the testes, the ovaries, the uterus, the spleen, the thymus, the brainstem, the cerebellum, the spinal cord, the eye, the ear, the tongue, the skin and/or tumors present in said one or more solid tissue(s), solid organ(s) and/or solid anatomical region(s).
44. The composition for use according to any one of claims 1 to 6, 8 to 38, and 41 to 43, the method of treatment according to any one of claims 39, and 41 to 43, or the use of the composition according to any one of claims 40 to 43, wherein at least about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of said one or more therapeutic agent(s) are restricted to the treated tissue, organ, and/or anatomical region, as quantified by a method selected from the group consisting of qPCR, HPLC, mass spectrometry, combinations of HPLC and mass spectrometry.
45. The composition for use according to any one of claims 1 to 6, 8 to 38, and 41 to 44, the method of treatment according to any one of claims 39, and 41 to 44, or the use of the composition according to any one of claims 40 to 44, wherein said restriction of the one or more therapeutic agent(s) to the treated tissue, organ, and/or anatomical region enables a reduction in the dose of said one or more therapeutic agent(s) to be administered by up to 20%, including any and all ranges within this limit, such as but not limited to reductions of 1-20%, 5-15%, or 10-20%.
46. The composition for use according to any one of claims 1 to 6, 8 to 38, and 41 to 45, the method of treatment according to any one of claims 39, and 41 to 45, or the use of the composition according to any one of claims 40 to 45, wherein said restriction of said one or more therapeutic agent(s) to the treated tissue, organ, and/or anatomical region reduces the number of administrations of said one or more therapeutic agent(s), preferably the number of administrations is reduced by about or less than about 25%.
47. The composition for use according to any one of claims 1 to 6, 8 to 38, and 41 to 46, the method of treatment according to any one of claims 39, and 41 to 46, or the use of the composition according to any one of claims 40 to 46, wherein said restriction of said one or more therapeutic agent(s) to the treated tissue, organ, and/or anatomical region reduces toxicity caused by and/or associated with the accumulation of said one or more therapeutic agents in off-target organs, preferably wherein toxicity caused by and/or associated with said one or more therapeutic agent(s) is reduced in the liver, the brain, the kidneys, the heart, and/or the spleen.
48. The composition for use according to any one of claims 1 to 6, 8 to 38, and 41 to 47, the method of treatment according to any one of claims 39, and 41 to 47, or the use of the composition according to any one of claims 40 to 47, wherein said restriction of said one or more therapeutic agent(s) to the treated tissue, organ, and/or anatomical region reduces and/or avoids off-target effects caused by and/or associated with said one or more therapeutic agent(s), preferably wherein off-target effects caused by and/or associated with said one or more therapeutic agent(s) are reduced in the liver, the brain, the kidneys, the heart, and/or the spleen.
49. The composition for use according to any one of claims 1 to 6, 8 to 38, and 41 to 48, the method of treatment according to any one of claims 39, and 41 to 48, or the use of the composition according to any one of claims 40 to 48, wherein said composition is not to be co-administered with one or more hyaluronidase(s) and/or enzyme(s) comprising hyaluronidase activity.
50. The composition for use according to any one of claims 1 to 6, 8 to 38, and 41 to 49, the method of treatment according to any one of claims 39, and 41 to 49, or the use of the composition according to any one of claims 40 to 49, wherein the subject to be treated is a mammal, preferably a human.
51. The composition for use according to any one of claims 1 to 6, 8 to 38, and 41 to 50, the method of treatment according to any one of claims 39, and 41 to 50, or the use of the composition according to any one of claims 40 to 50, wherein said composition is to be administered via intradermal, subcutaneous, intramuscular, or intratumoral injection, aerosol delivery such as into the respiratory system including intranasal or lung delivery, or topic application.
52. A method of inducing an immune response in a subject, which comprises administering to said subject an effective amount of the composition as defined in or according to any one of claims 1 to 6, 8 to 38, and 41 to 50.
53. A method of immunizing a subject against a pathogen, which comprises administering to said subject an effective amount of an mRNA vaccine in a pharmaceutical composition, wherein said pharmaceutical composition comprises the composition as defined in or according to any one of claims 1 to 6, 8 to 38, and 41 to 50.
54. The method according to claim 53, wherein said mRNA vaccine is administered via intradermal, subcutaneous, intramuscular, or intratumoral injection.
55. The method according to claim 53 or 54, wherein said mRNA vaccine elicits an immune response predominantly at the site of administration, thereby reducing the risk of systemic adverse effects.
56. The method according to any one of claims 53 to 55, wherein said mRNA vaccine is designed to promote local production of antigen-specific antibodies or cellular immune responses at the site of administration.
57. The method according to any one of claims 53 to 56, wherein said mRNA vaccine further comprises a polymeric coating or encapsulation to enhance local retention and prevent systemic dissemination.
58. The composition for use according to any one of claims 8 to 38, and 41 to 51 , or the method according to any one of claims 53 to 57, wherein said carrier further comprises a targeting moiety or ligand that specifically binds to cells or receptors present at the site of interest, thereby enhancing the specificity and efficacy of said therapeutic agent.
59. The composition for use according to any one of claims 8 to 38, 41 to 51 , and 58, or the method according to any one of claims 53 to 58, wherein said pharmaceutically acceptable excipient or diluent further comprises a biodegradable or bioresorbable material, facilitating gradual release and local persistence of said therapeutic agent at the site of interest.
60. The composition for use according to any one of claims 8 to 38, 41 to 51 , 58, and 59, or the method according to any one of claims 53 to 59, wherein said mRNA further comprises a tissue-specific promoter and/or enhancer element to enhance the expression of the antigen at the site of interest.
61. The composition for use according to any one of claims 8 to 38, 41 to 51 , and 58 to 60, or the method according to any one of claims 53 to 60, wherein said therapeutic agent is encapsulated within a biocompatible microneedle patch or implantable device, facilitating controlled and/or sustained release of said therapeutic agent at the site of interest.
62. The composition for use according to any one of claims 8 to 38, 41 to 51 , and 58 to 61 , or the method according to any one of claims 53 to 61 , wherein said mRNA further comprises a self-amplifying mRNA (saRNA) molecule, enabling enhanced protein or antigen production at the site of interest.
63. The composition for use according to any one of claims 8 to 38, 41 to 51 , and 58 to 62, or the method according to any one of claims 53 to 62, wherein said one or more mRNA molecules comprise an ORF encoding CFTR, Erythropoietin (EPO), Factor VIII, Factor IX, Chimeric Antigen Receptor (CAR) T-cell, Survivin (BIRC5) or a dominant-negative form thereof, P53, Vascular Endothelial Growth Factor (VEGF), Insulin, SARS-CoV-2 Spike protein, Alpha-synuclein, Dystrophin, Glucocerebrosidase (GCase), a cytokine such as lnterleukin-2 (IL-2), Interleukin- 10 (IL-10), Interleukin- 12 (IL-12), an interferon, Interferon-alpha (IFN-a), Interferon-beta (IFN-β), Interferon-gamma (IFN-y), interferon lambda (IFNλ), such as interferon lambda 1 (IFN-λ1 , also known as IL-29), IFN-λ2 (also known as IL-28A), IFN-λ3 (also known as IL-28B), and/or IFN-λ4, human interferon lambda 1 (hlFNλ1 ), Tumor Necrosis Factor-alpha (TNF-α), Granulocyte-macrophage colony-stimulating factor (GM-CSF), a primary ciliary dyskinesia protein or factor such as DNAH5, DNAH11 , CCDC39, DNAI1 , CCDC40, CCDC103, SPAG1 , ZMYND10, ARMC4, CCDC151 , DNAI2, RSPH1 , CCDC114, RSPH4A, DNAAF1 (LRRC50), DNAAF2 (KTU), LRRC6, C21orf59, CCDC65 (DRC2), CCNO, DNAAF3, DNAH1 , DNAH8, DNAL1 , DRC1 (CCDC164), DYX1C1 , DNAAF5 (HEATR2), HYDIN, MCIDAS, NME8 (TXNDC3), RSPH3, RSPH9, or FOXJ1 , preferably wherein said one or more mRNA molecules comprise an ORF encoding interferon lambda 1 (IFNλ1), more preferably human interferon lambda 1 (hl FNλ1)
64. The cosmetic composition according to any one of claims 7 to 25, wherein said active ingredient is selected from the group consisting of the following: a growth factor, a peptide, an antioxidant, a retinoid, a cytokine, a siRNA, a miRNA, a mRNA, and an asRNA.
65. Use of the cosmetic composition according to any one of claims 7 to 25, and 64, in the amelioration of cutaneous condition.
66. A method for the amelioration of a cutaneous condition, wherein said method comprises the administration of the cosmetic composition according to any one of claims 7 to 25, and 64.
67. A kit comprising the cosmetic composition according to any one of claims 7 to 25, and 64.
68. A drug conjugate comprising an ionizable lipidoid as defined in any one of claims 9 to 18 and one or more therapeutic agent(s), preferably wherein said one or more therapeutic agent(s) is/are as defined in any one of claims 26 to 34.
69. In vitro use of an ionizable lipidoid for the restriction of the dissemination of one or more to be administered therapeutic agent(s), wherein said ionizable lipidoid is co-formulated with said one or more therapeutic agent (s), preferably wherein said ionizable lipidoid is as defined in any one of claims 9 to 18, preferably wherein said one or more therapeutic agent(s) is/are as defined in any one of claims 26 to 34.
70. An (in vitro) method for the restriction of the dissemination of one or more to be administered therapeutic agent(s), wherein said method comprises the step of co- formulating an ionizable lipidoid with said one or more therapeutic agent(s), wherein said ionizable lipidoid is as defined in any one of claims 9 to 18.
71. The drug conjugate according to claim 68, the in vitro use according to claim 69, or the in vitro method according to claim 70, wherein said ionizable lipidoid comprises a compound of formula (b-l), more preferably a compound of formula (b-V) or formula (b- VII), even more preferably a compound of formula (b-V).
PCT/EP2024/072949 2023-08-14 2024-08-14 Non-systemic mrna administration Pending WO2025036956A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP23020381 2023-08-14
EP23020381.2 2023-08-14
EP24184146 2024-06-24
EP24184146.9 2024-06-24

Publications (1)

Publication Number Publication Date
WO2025036956A1 true WO2025036956A1 (en) 2025-02-20

Family

ID=92456791

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/072949 Pending WO2025036956A1 (en) 2023-08-14 2024-08-14 Non-systemic mrna administration

Country Status (1)

Country Link
WO (1) WO2025036956A1 (en)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8158601B2 (en) 2009-06-10 2012-04-17 Alnylam Pharmaceuticals, Inc. Lipid formulation
WO2014207231A1 (en) 2013-06-28 2014-12-31 Ethris Gmbh Compositions for introducing rna into cells
WO2019204451A1 (en) * 2018-04-17 2019-10-24 Carnegie Mellon University Enhanced lipid nanoparticle drug delivery using a negatively charged polymer
WO2020074642A1 (en) 2018-10-11 2020-04-16 Ethris Gmbh Plasmid containing a sequence encoding an mrna with a segmented poly(a) tail
US10702600B1 (en) 2015-10-22 2020-07-07 Modernatx, Inc. Betacoronavirus mRNA vaccine
WO2020165352A1 (en) 2019-02-14 2020-08-20 Ethris Gmbh Treatment of ciliopathies
WO2022180213A1 (en) * 2021-02-26 2022-09-01 Ethris Gmbh Formulations for aerosol formation and aerosols for the delivery of nucleic acid
US11510937B2 (en) * 2013-03-14 2022-11-29 Translate Bio, Inc. CFTR MRNA compositions and related methods and uses

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8158601B2 (en) 2009-06-10 2012-04-17 Alnylam Pharmaceuticals, Inc. Lipid formulation
US11510937B2 (en) * 2013-03-14 2022-11-29 Translate Bio, Inc. CFTR MRNA compositions and related methods and uses
WO2014207231A1 (en) 2013-06-28 2014-12-31 Ethris Gmbh Compositions for introducing rna into cells
EP3013964B1 (en) 2013-06-28 2020-05-06 ethris GmbH Compositions for introducing rna into cells
US10702600B1 (en) 2015-10-22 2020-07-07 Modernatx, Inc. Betacoronavirus mRNA vaccine
WO2019204451A1 (en) * 2018-04-17 2019-10-24 Carnegie Mellon University Enhanced lipid nanoparticle drug delivery using a negatively charged polymer
WO2020074642A1 (en) 2018-10-11 2020-04-16 Ethris Gmbh Plasmid containing a sequence encoding an mrna with a segmented poly(a) tail
WO2020165352A1 (en) 2019-02-14 2020-08-20 Ethris Gmbh Treatment of ciliopathies
WO2022180213A1 (en) * 2021-02-26 2022-09-01 Ethris Gmbh Formulations for aerosol formation and aerosols for the delivery of nucleic acid

Non-Patent Citations (25)

* Cited by examiner, † Cited by third party
Title
"Pharmaceutical dosage forms and drug delivery", 25 October 2011, CRC PRESS, ISBN: 978-1-4398-4918-7, article MAHATO RAM I. ET AL: "Inserts, Implants, and Devices", pages: 388 - 388, XP093220620 *
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, pages 403 - 410
ALTSCHUL ET AL., NATURE GENET, vol. 6, 1994, pages 119 - 129
ANJILVEL, S.B. ASGHARIAN: "A multiple-path model of particle deposition in the rat lung", FUNDAM APPL TOXICOL, vol. 28, 1995, pages 41 - 50
CAMPANELLA ET AL., BMC BIOINFORMATICS, vol. 4, 2003, pages 29
CORPET ET AL., NUCLEIC ACIDS' RESEARCH, vol. 16, 1988, pages 10881 - 10890
E. W. MARTIN: "Remington's Pharmaceutical Sciences", 1975, MACK PUBLISHING CO.
FERNANDES, C. A.R. VANBEVER: "Preclinical models for pulmonary drug delivery", EXPERT OPIN DRUG DELIV, vol. 6, 2009, pages 1231 - 45
FERRARESSO ET AL., MOLECULAR THERAPY: METHODS & CLINICAL DEVELOPMENT, 2014, Retrieved from the Internet <URL:https://doi.org/10.1016/j.omtm.2024.101314>
GIZURARSON, S: "Animal models for intranasal drug delivery studies", ACTA PHARM NORD, vol. 2, 1990, pages 105 - 22, XP055524098
HIGGINSSHARP, CABIOS, vol. 5, 1989, pages 151 - 153
HIGGINSSHARP, GENE, vol. 73, 1988, pages 237 - 244
HOU ET AL., NATURE REVIEWS MATERIALS, 2021
LARKIN ET AL., BIOINFORMATICS, vol. 23, 2007, pages 2947 - 2948
NEEDLEMANWUNSCH, J. MOL. BIOL, vol. 48, 1970, pages 443
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
PARD ET AL., NATURE REVIEWS DRUG DISCOVERY, vol. 17, 2018, pages 261 - 279
PEARSONLIPMAN, PROC. NATL. ACAD. SCI. U.S.A, vol. 85, 1988, pages 2444
PEARSONLIPMAN, PROC. NATL. ACAD. SCI. U.S.A., vol. 85, 1988, pages 2444
SMITHWATERMAN: "Adv. Appl. Math", vol. 2, 1981, pages: 482
SNIPES, M. B.R. O. MCCLELLANJ. L. MAUDERLYR. K. WOLFF: "Retention patterns for inhaled particles in the lung: comparisons between laboratory animals and humans for chronic exposures", HEALTH PHYS, vol. 1, 1989, pages 69 - 77
SOUTHAM, D. S.M. DOLOVICHP. M. O'BYRNEM. D. INMAN: "Distribution of intranasal instillations in mice: effects of volume, time, body position, and anesthesia", AM J PHYSIOL LUNG CELL MOL PHYSIOL, vol. 282, 2002, pages L833 - 9
WANG ET AL., MOLECULAR CANCER, vol. 20, 2021, pages 33
WONG, B. A: "Inhalation exposure systems: design, methods and operation", TOXICOL PATHOL, vol. 35, 2007, pages 3 - 14

Similar Documents

Publication Publication Date Title
KR102087643B1 (en) Pulmonary delivery of messenger rna
US12433941B2 (en) Exosomal nucleic acid vaccine modularly configured to harness multiple antigen presentation mechanisms
KR102380363B1 (en) Ionizable cationic lipid for rna delivery
US20210316002A1 (en) Compositions and methods for drug delivery and treating viral infections
US20230173104A1 (en) Lnp compositions comprising an mrna therapeutic and an effector molecule
CN116710118A (en) Treatment for viral respiratory infections
JP2009542582A (en) Substances and complexes for delivering bioactive substances to cells
IL323798A (en) Reconstitutable dry powder formulations and methods of use thereof
WO2025036956A1 (en) Non-systemic mrna administration
WO1999061063A1 (en) Stable gene preparations
US20250352632A2 (en) Hpv infectious disease vaccine
CN117821468A (en) Lung-targeted soluble PD-L1 mRNA lipid nanoparticles and their applications
WO2026017917A1 (en) Rnas with reduced ribosomal frameshift expression products
CN111909246B (en) AAV mutants highly efficient in infecting supporting cells
WO2022170396A1 (en) Agents and methods for therapy and prophylaxis
WO2021211552A1 (en) Compositions and methods for drug delivery and treating viral infections
CN117838832B (en) Application of RBM25 and its related substances in antiviral infection
EP4269424A1 (en) Novel antiviral compounds and use thereof
RU2746362C9 (en) Combined drug with antiviral effect against new sars-cov-2 coronavirus
WO2026006276A1 (en) Materials and methods for the prevention and treatment of viral diseases
Hou Lipid Nanoparticle-Messenger RNA Formulations against Infections
EA049858B1 (en) COMBINATION DRUG WITH ANTIVIRAL EFFECT AGAINST THE NEW CORONAVIRUS SARS-CoV-2
WO2024131726A1 (en) Broad-spectrum influenza mrna vaccine
WO2025086091A1 (en) Mrna vaccine against rabies viruses and preparation method therefor
WO2025021922A1 (en) A pharmaceutical formulation that combines hydroxychloroquine or its analogs with nucleic acid-based therapeutic ingredients to suppress excessive inflammation triggered by the introduction of nucleic acids

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24757921

Country of ref document: EP

Kind code of ref document: A1