WO2023144193A1

WO2023144193A1 - Mrnas for treatment of hereditary tyrosinemia type i

Info

Publication number: WO2023144193A1
Application number: PCT/EP2023/051783
Authority: WO
Inventors: Christine WEINL-TENBRUCK; Frédéric CHEVESSIER-TÜNNESEN; Nigel HORSCROFT
Original assignee: CureVac SE
Priority date: 2022-01-25
Filing date: 2023-01-25
Publication date: 2023-08-03

Abstract

The present invention relates to mRNA medicines for use in treating, attenuating or inhibiting Hereditary Tyrosinemia Type I, and more particularly to mRNA medicines encoding fumarylacetoacetate Hydrolase (FAH) which can exhibit excellent therapeutic effects.

Description

mRNAs for treatment of Hereditary Tyrosinemia Type I TECHNICAL FIELD The present invention relates to mRNA medicines for use in treating, attenuating or inhibiting hlereditary Tyrosinemia Type I, and more particularly to mRNA medicines encoding fumarylacetoacetate Hydrolase (FAH) which can exhibit excellent therapeutic effects. INTRODUCTION Hereditary Tyrosinemia Type I is the most severe form amongst different types of Tyrosinemia. hlereditary Tyrosinemia Type I is a metabolic disease caused by the absence of functional fumarylacetoacetate hydrolase (FAH). FAH deficiency results in the accumulation of toxic and carcinogenic metaboiites, such as succinylacetone (SA), Tyrosine (TYR), maleylacetoacetate (MAA) and fumarylacetoacetate (FAA). A newborn screening was implemented for Tyrosinemia, the disease occurring with 1:100.000 birth incidence. Acute presentation of symptoms include liver failure, vomiting, bleeding, hypoglycemia and tubulopathy; chronic manifestations present as hepatomegaly, cirrhosis, growth retardation, rickets, tubulopathy, and neuropathy. There is an increased risk in Tyrosinemia patients for the development of metabolic crisis, renal failure, and early-onset hepatocellular carcinoma (HCC). Standard-of-care treatment of patients includes the daily supplementation with NTBC (Nitisinone, 2-(2-nitro-4-trifluoromethyl benzoyl) cyclohexane-1, 3- dione, trade name: Orfadin, oral administration twice daily as suspension) acting as a strong inhibitor of4-Hydroxyphenyl-pyruvatdioxigenase to avoid accumulation of toxic metabolites. In turn, NTBC supplementation results in an upstream accumulation of tyrosine, leading to eye symptoms and neurocognitive defects, and patients develop Tyrosinemia Type II, therefore a strict life-long diet low in Tyrosine and Phenylalanine remains an essential part of disease management. NTBC has significantly improved management of the disease, especially when started early in life of affected patients, however some patients might still develop liver cancer, other patients might not respond to NTBC treatment, and a long-term risk assessment has not been completed; most importantly, children do not develop normally possibly facing neurocognitive problems despite treatment due to altered metabolites and amino acid deficiencies. It is essential that patients adhere strictly to continuous NTBC supplementation; it was reported that discontinuation of NTBC will result in neurological, in some instances life-threatening, crisis requiring hospitalization, even in young adults, which could have been prevented by strictly adhering to the daily treatment. The treatment option for severe cases (i.e. patients do not respond to NTBC treatment, patients developed liver cancer) is liver transplantation, accompanied by known risks of shortage of donor organs (especially for babies and young children), organ rejection, and side effects of immune suppressive combination therapy. Therefore, there is a high medical need of developing alternative treatment options, especially for babies and young children preventing early death due to hlCC. One further critical indication in connection with Hereditary Tyrosinemia Type I is the "neurologic crisis" of tyrosinemia type I, which is a rare complication seen after discontinuation of treatment characterized with anorexia, vomiting, and hyponatremia in the initial phase continuing with paresthesia and paralysis of the extremities and the diaphragm (PMID 27188289). One further disadvantage of the standard-of-care treatment with Nitisinone/Orfadin is, that the medication can only be used to slow the effects of hereditary tyrosinemia type 1 (HT-1) in combination with a dietary restriction of tyrosine and phenylalanine. Accordingly, object of the present invention is to provide an alternative and/or superior therapy than the standard-of-care treatment with NTBC. A further object of the present invention is to provide a fast and reliable emergency therapy, which can be used e.g. by patients going through a "neurologic crisis" as described above. The objects underlying the present invention are solved by the claimed subject matter. The inventors surprisingly found that an isolated mRNA encoding fumarylacetoacetate hydrolase (FAH) can be used for treating, attenuating or inhibiting Hereditary Tyrosinemia Type I. Preferably, the mRNA comprises an open reading frame (ORF) encoding FAN comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:100, or a fragment or variant of said sequences having the biological activity of a FAH protein. Further, preferably, the mRNA has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any single SEQ ID NO-element of SEQ ID NO:112 to SEQ ID NO:144 or SEQ ID NO:101 to SEQ ID NO:111, or a fragment or variant of said sequences, wherein the encoded protein has the biological activity of a FAH protein. Further surprisingly, the inventors found that the objects underlying the present invention are solved by the methods of the invention relating to the mRNA of the invention, or the LNPs of the invention, or the pharmaceutical compositions of the invention or the kit or kit of parts of the invention, being administered to the subject by intramuscular administration for treating, attenuating or inhibiting Hereditary Tyrosinemia Type I. This is in contrast to the expectation in the art, which was related nearly exclusively to intravenous administration for treating defects based on a genetic or enzyme defect. Accordingly, said intramuscular administration is able to provide the desired fast and reliable emergency therapy, which can be used e.g. by patients to avoid or diminish a "neurologic crisis" and/or to avoid dietary restrictions of tyrosine and phenylalanine during the inventive treatment. BRIEF DESCRIPTION OF THE FIGURES Figure 1A: Luciferase expression in FAH mice after single intravenous injection of PpLuc mRNA- LNPs. Luciferase signal was predominantly detected in FAH mouse livers (cf. Example 1). Figure 1B: Luciferase expression in FAhl mice after intramuscular injection of PpLuc mRNA-LNPs into both M. tibialis muscles. Luciferase signal was detected in FAhl mouse muscles, however, a substantial amount was also detected in FAH mouse livers suggesting transport of PpLuc mRNA- LNPs to the liver via blood stream, resulting in successful uptake and expression at this ectopic site (cf. Example 1). Figure 1C: Luciferase signal was undetectable in FAH mice after intravenous and intramuscular injection of PBS (cf. Example 1). Figure 1 D: Quantitation of Luciferase signals in liver and muscle tissue of FAN mice. Data are shown as mean +/- standard error of the mean (SEM). P values were obtained from two-tailed Student's t- test. *** P<0,001; ns not significant P>0,05 (cf. Example 1). Figure 2A: Measurement of Succinylacetone (SA) in FAhl mouse serum one day before treatment (PB = pre-bleeding, pre-treatment levels one day before single intravenous injections on day 0) and on assigned termination time points (Days 1, 2, and 4) (n = 4 mice per condition). Dotted line represents the mean value obtained from n = 5 wildtype (WT) age-matehed C57BL6 mice after single IV injection of PBS (cf. Example 2). Figure 2B: Measurement of Tyrosine (TYR) in FAH mouse serum one day before treatment (pre- treatment levels) and on assigned termination time points (Days 1, 2, and 4) (n = 4 mice per condition). Dotted line represents the mean value obtained from n = 5 wildtype (WT) age-matched C57BL6 mice after single IV injection of PBS. Data are shown as mean +/- SEM. P values were obtained from two-tailed Student's t-test. * P<0,05; ** P<0,01 ; *** P<0,001; ns not significant P>0,05 (cf. Example 2). Figure 3A: Livers of mice (from FAH and wildtype (WT) mice) were collected 24 hours after single injection (Sl), lysed and analysed via Western Blot (representative examples shown for n = 3 mouse ivers per condition). FAH protein in liver lysates was detected via anti-FAhl antibody (ab151998, abeam), beta-actin served as the loading control. FAH mice (lacking functional FAN protein) served as controls (two independent cohorts with continuous NTBC supplementation or with stop of NTBC supplementation 5 days before start of single IV injections) (cf. Example 2). Figure 3B: Quantitation of FAN protein bands normalized to beta-actin loading control. After single IV injection of FAH mRNA-LNPs in FAH mice, FAhl protein was detected in livers in substantial amount (~50% of the endogenous FAhl level observed in wildtype (WT) mice) (n = 3 mouse livers per condition) (cf. Example 2). Figure 3C: Livers of mice (from FAhl and wildtype (WT) mice) were collected 4 days after single intravenous injection (cf. Example 2). Figure 3D: Quantitation of FAH protein bands normalized to beta-actin loading control. After single IV injection of FAH mRNA-LNPs in FAN mice, FAH protein was detected in livers in substantial amount (~30% of the endogenous FAH level observed in wildtype (WT) mice) 4 days after treatment (n = 3 mouse livers per condition). Data are shown as mean +/- SEM. P values were obtained from

5 two-tailed Student's t-test. * P<0,05; ** P<0,01; *** P<0,001; ns not significant P>0,05 (cf. Example 2). Figure 4A: Survival of mRNA-injected FAhl mice was prolonged compared to PBS-injected FAN mice as shown by Kaplan Meier survival plots. FAH mice on continuous NTBC supplementation and FAH mice repeatedly injected with FAhl mRNA-LNPs survived until the end of the study (100% survival at the end of the study on day 21 -thecurvesof both groups are congruent), whereas NTBC (-) PBS (+) treated FAH mice had to be terminated due to body weight loss and health decline on day 12, leading to 0% survival of the untreated group at the end of the study on day 21 (cf. Example 3). Figure 4B: Body weight of FAH mice during life-phase. NTBC (-) PBS (+) treated FAH mice lost body weight and had to be terminated due to body weight loss of 20%. FAhl mRNA-LNP treated FAH mice did not lose weight; most importantly, their weight was not different from standard-of-care NTBC (+) PBS (+) treated FAH mice throughout life-phase and at the end of the study suggesting successful therapy (cf. Example 3). Figure 4C: Body weight of FAH mice at termination (n = 5 FAH mice per condition). NTBC (-) PBS (+) treated FAH mice had to be terminated due to significant body weight loss, whereas body weight of RNA-injected FAhl mice was not statistically different from standard-of-care-NTBC treated mice. Data are shown as mean +/- SEM. P values were obtained from two-tailed Student's t-test. ** P<0,01; ns not significant P>0,05 (cf. Example 3). Figure 5A: Succinylacetone (SA) levels in FAH mouse serum during life phase (n = 5 mice per condition). Dotted line represents the mean value obtained from n = 5 wildtype (WT) age-matched C57BL6 mice after single IV injection of PBS (cf. Example 3). Figure SB: Succinylacetone (SA) levels at termination on day 21 (cf. Example 3). Figure 5C: Tyrosine (TYR) levels in FAhl mouse serum during life phase (n = 5 mice per condition). Dotted line represents the mean value obtained from n = 5 wildtype (WT) age-matched C57BL6 mice after single IV injection of PBS (cf. Example 3). Figure 5D: Tyrosine (TYR) levels in FAhl mouse serum at termination on day 21. Data are shown as mean +/- SEM. P values were obtained from two-tailed Student's t-test. * P<0,05; ** P<0,01; ns not significant P>0,05 (cf. Example 3). Figure 6A: WB analysis of FAH protein in FAH mouse livers collected at termination on day 21.FAH protein was detected in mouse liver lysates via anti-FAH antibody (ab151998, abeam), beta-actin served as the loading control (Rl repeated injection; Sl single injection of PBS in wildtype (WT) mice or comparison of endogenous FAH protein level in mouse liver) (n = 4 mouse livers for Rl NTBC (+) PBS (+) and Rl (NTBC (-) PBS (+); n = 5 mouse livers for Rl NTBC (-) RNA (+) and WT Sl NTBC (- ) PBS (+)) (cf. Example 3). Figure 6B: Quantitation of FAH protein bands normalized to beta-actin loading control. Data are shown as mean +/- SEM. P values were obtained from two-tailed Student's t-test. *** P<0,001 (cf. Example 3). Figure 7A: Body weight of FAH mice during life phase. No significant difference in body weight of FAhi mice treated either via repeated IV or IM injections was detected. Treatments via both administration routes rescued FAhl mice from body weight decrease, health decline and death (cf. Example 4). Figure 7B: Body weight at termination on day 21. Data are shown as mean +/- SEM. P values were obtained from two-tailed Student's t-test. ns P>0,05 (cf. Example 4). Figure 8A: Succinylacetone (SA) levels in FAH mouse serum during life phase. Dotted line represents the mean value obtained from n = 5 wildtype (WT) age-matched C57BL6 mice after single IV injection of PBS (cf. Example 4). Figure 8B: Succinylacetone (SA) levels in FAH mouse serum at termination on day 21. SA levels at termination were not significantly different between the IM and IV route (cf. Example 4). Figure 8C: Tyrosine (TYR) levels in FAH mouse serum during life phase. Dotted line represents the mean value obtained from n = 5 wildtype (WT) age-matched C57BL6 mice after single IV injection of PBS (cf. Example 4). Figure 8D: Tyrosine (TYR) levels in FAH mouse serum at termination on day 21. TYR levels at termination were not significantly different between the IM and IV route. Data are shown as mean +/- SEM. P values were obtained from two-tailed Student's t-test. ns P>0,05 (cf. Example 4). Figure 9A: WB analysis of FAhl protein in FAH mouse livers collected at termination on day 21 after injection via IV (n = 4 FAhl mice) and IM (n = 5 mice) administration route. FAH protein in liver lysates was detected via anti-FAhl antibody (ab151998, abeam), beta-actin served as the loading control (cf. Example 4). Figure 9B: Quantitation of FAhl protein bands normalized to beta-actin loading control. When normalizing the FAH liver expression after repeated IV injection to 100%, the amount of FAN liver expression after repeated IM injection was ~6% of the amount detected in liver after IV injection. This small amount of FAH liver expression after IM injection was sufficient to save animals from health decline after NTBC withdrawal. Data are shown as mean +/- SEM. P values were obtained from two-ailed Student's t-test. * P<0,05 (cf. Example 4). Figure 10A: Normalized body weight of FAH mice during experimental life phase until termination of the study on day 21 (cf. Example 5). Figure 10B: Succinylacetone (SA) levels (normalized to pre-treatment levels) in FAH mouse serum during experiment life phase until termination of the study on day 21 (cf. Example 5). Figure 10C: Tyrosine (TYR) levels (normalized to pre-treatment levels) in FAhl mouse serum during experiment life phase until termination of the study on day 21 (cf. Example 5). Figure 11 A: Survival of FAhl mice upon repeated intramuscular injections of lower doses 0,5 mg/kg and 0,1 mg/kg of human FAH-mRNA LNPs (cf. Example 6). Figure 11 B: Normalized body weight of FAH mice during experimental life phase and at termination of the study on day 21 (cf. Example 6). Figure 11C: Succinylacetone (SA) levels (normalized to pre-treatment levels) in FAN mouse serum during experiment life phase until termination of the study on day 21 (cf. Example 6). Figure 11 D: Tyrosine (TYR) levels (normalized to pre-treatment levels) in FAH mouse serum during experiment life phase until termination of the study on day 21 (cf. Example 6). Figure 12A: Normalized body weight of FAH mice upon repeated intravenous and intramuscular injections of 1 mg/kg doses of human FAH-mRNA LNPs in a one-week interval schedule (cf. Example 7). Figure 12B: Succinylacetone (SA) levels (normalized to pre-treatment levels) in P/W mouse serum during experiment life phase until termination of the study on day 29 in a one-week interval schedule (cf. Example 7). Figure 12C: Tyrosine (TYR) levels (normalized to pre-treatment levels) in FAhl mouse serum during experiment life phase until termination of the study on day 29 in a one-week interval schedule (cf. Example 7). Figure 12D: Normalized body weight of FAH mice upon repeated intravenous and intramuscular injections of 1 mg/kg doses of human FAH-mRNA LNPs in a two-weeks interval schedule (cf. Example 7). Figure 12E: Succinylacetone (SA) levels (normalized to pre-treatment levels) in FAN mouse serum during experiment life phase until termination of the study on day 57 in a two-weeks interval schedule (cf. Example 7). Figure 12F: Tyrosine (TYR) levels (normalized to pre-treatment levels) in FAhl mouse serum during experiment life phase until termination of the study on day 57 in a two-weeks interval schedule (cf. Example 7).

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel mRNAs and to compositions and kits comprising the mRNA. Furthermore, several uses, in particular medical uses, of the mRNA according to the invention and of the pharmaceutical compositions and kits are provided. Said novel mRNAs encode fumarylacetoacetate hydrolase (FAhl), being beneficial when administered to a patient in need. In a first aspect, the present invention relates to an isolated mRNA encoding fumarylacetoacetate hydrolase (FAH) for use in treating, preventing, attenuating or inhibiting hlereditary Tyrosinemia Type I(HT1). In a second aspect, the present invention provides pharmaceutical compositions and/or kits as described herein for use in the treatment, prevention, attenuation, inhibition, or prophylaxis of Hereditary Tyrosinemia Type I (HT1). In a third aspect, the present invention provides a method of treating, attenuating or inhibiting hlereditary Tyrosinemia Type I (HT1), comprising administering to a human subject in need the mRNA, the LNP, the pharmaceutical composition, or the kit or kit of parts as described herein, wherein administration results in treatment, prevention, attenuation, inhibition, or prophylaxis of Hereditary Tyrosinemia Type I (HT1). As used herein, the term "fumarylacetoacetate hydrolase" or "FAH" relates to an FAH mRNA or FAhl protein having the biological activity ofaFAH protein (OMIM entry No: 613871; HGNC: 3579, HGNC Approved Gene Symbol: FAhl, alternative names fumarylacetoacetase). In the context of the present invention, the terms "FAhl", "WT FAH" or "human FAH" refer to the human unmodified FAH protein (or respectively an mRNA encoding said WT FAH). Naturally, it is to be understood, that FAH isoforms, e.g. produced by alternative promoter usage or alternative splicing, are comprised within the scope of the current invention. In other words, sequences, i.e., fragments or variants of the sequences as described herein, "having the biological activity of a FAN protein" refer to sequences having the biological activity of a FAH protein (OMIM entry No: 613871; HGNC: 3579, HGNC Approved Gene Symbol: FAH, alternative names fumarylacetoacetase). I.e., any sequence, which has the biological activity ofaFAH protein (OMIM entry No: 613871; HGNC: 3579, HGNC Approved Gene Symbol: FAN, alternative names fumarylacetoacetase), would fall within the scope of the claims. The term "increased expression", "enhanced expression" or "overexpression" as used herein means any form of expression which occurs in addition to the original wild type mRNA expression level. The term "preventing" refers to decreasing the probability that an organism contracts or develops an abnormal condition, like e.g., Hereditary Tyrosinemia Type I (HT1). The term "treating" refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism, like Hereditary Tyrosinemia Type I (h!T1). Treating includes inhibition of Hereditary Tyrosinemia Type I (h!T1), maintenance of hlereditary Tyrosinemia Type I (h!T1), or reducing, curing and induction of remission of hlereditary Tyrosinemia Type I (HT1). The term "attenuation" (attenuate: weaken, mitigate) of a disease means in principle the reduction, mitigation or lessening of the negative / disadvantageous symptoms, impacts or effects of hlereditary Tyrosinemia Type I (HT1) on the patient or subject in need. The term "therapeutic effect" or "therapeutic activity" refers to the inhibition of an abnormal condition, like Hereditary Tyrosinemia Type I (h!T1) upon administration of an mRNA of the present invention, encoding FAhl. A therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition, like Hereditary Tyrosinemia Type I (h!T1). In reference to the treatment of abnormal conditions, a therapeutic effect can refer to one or more of the following: (a) a decrease in the proliferation, growth, and/or progression of Hereditary Tyrosinemia Type I (h!T1); (b) inhibition (i.e., slowing or stopping) of Hereditary Tyrosinemia Type I (h!T1) in viva; and (c) relieving to some extent one or more of the symptoms associated with the abnormal condition e.g. Hereditary Tyrosinemia Type I (HT1). The admistration of the mRNAofthe invention encoding FAH as described herein effectuate the therapeutic effect. The term "therapeutic effect" as used herein, also refers to the effective provision of protection effects to prevent, inhibit, or arrest the symptoms and/or progression of Hereditary Tyrosinemia Type I (HT1) as described herein. The term "a therapeutically effective amount" as used herein means a sufficient amount of the FAhl protein of the invention to produce a therapeutic effect, as defined above, in a subject or patient in need of treatment. The terms "subject" or "patient" are used herein mean any mammal, including but not limited to human beings, including a human patient or subject to which the pharmaceutical compositions of thenvention can be administered. The term mammals include human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Thus, in some embodiments, the disclosure provides mRNAs encoding fumarylacetoacetate hydrolase (FAH) having an amino acid sequence that is at least about 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%, 99%, or 100% identical to the amino acid sequence corresponding to anyone ofSEQ ID NO:100. The mRNA according to the invention is preferably suitable for use in the treatment or prophylaxis of Hereditary Tyrosinemia Type I (HT1) as described herein. More preferably, the mRNA according to the present invention is preferably suitable for use in safe and effective treatment or prophylaxis of Hereditary Tyrosinemia Type I (HT1) as described herein in mammals, preferably in human. In the following, the mRNA according to the invention is described. The disclosure concerning the ventive mRNA as such also applies to the mRNA for (medical) use as described herein as well as to the mRNA in the context of the pharmaceutical composition or the mRNA in the context of the kit or kit of parts comprising the mRNA according to the invention. In particular, when referring to an "RNA according to the invention" or "mRNA according to the invention", the present disclosure also relates to an "RNA for use according to the invention" or "mRNA for use according to the invention" and vice versa. FAH mRNA sequences In preferred embodiments, the present invention relates to an mRNA for use in treating, preventing, attenuating or inhibiting Hereditary Tyrosinemia Type I (HT1), wherein said mRNA comprises an open reading frame (ORF) encoding fumarylacetoacetate hydrolase (FAH). In preferred embodiments, the present invention relates to an mRNA for use in treating, preventing, attenuating or inhibiting hlereditary Tyrosinemia Type I (h!T1), wherein said mRNA comprises an open reading frame (ORF) encoding fumarylacetoacetate hydrolase (FAH) preferably marylacetoacetate hydrolase (FAH) comprising an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO:100, or a fragment or variant of said sequences having the biological activity of a FAH protein. other preferred embodiments, the present invention relates to an mRNA comprising an open reading frame (ORF) encoding fumarylacetoacetate hydrolase (FAhl) for use in treating, preventing, attenuating or inhibiting hlereditary Tyrosinemia Type I (HT1), wherein said mRNA preferably has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO:112-144, wherein thencoded protein has the biological activity of a FAH protein. In further preferred embodiments, the present invention relates to an mRNA encoding fumarylacetoacetate hydrolase (FAH) according to SEQ ID NO:100, preferably wherein said mRNA has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,5%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO:112-144, or a fragment or variant of said sequences, wherein the encoded protein has the biological activity of a FAhl protein, further, wherein the mRNA further comprises an UTR combination selected from the group consisting of (i) a 5'-UTR derived from a mouse solute carrier family 7 (cationic amino acid transporter, y+ system) (SLC7A3) and a 3'-UTR derived from PSMB3; (ii) a 5'-UTR derived from ouse ribosomal protein L31 (RPL31) and a 3'-UTR derived from a human ribosomal protein S9 (RPS9); (iii) a 5'-UTR derived from ubiquilin 2 (Ubqln2) and a 3'-UTR derived from Guanine nucleotide-binding protein G(s) subunit alpha isoforms short (Gnas); and (iv) a 5'-UTR derived from a hydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4) and a 3'-UTR derived from a proteasome subunit beta type-3 (PSMB3) UTR. 5 In other embodiments, the (i) G/C content of the FAhl coding sequence is increased compared to the coding sequence of the corresponding wild type FAhl coding sequence of SEQ ID NO:101; (ii) C content of the FAhl coding sequence is increased compared to the coding sequence of the corresponding wild type FAH coding sequence ofSEQ ID NO:101; and/or (iii) at least one codon of the FAH coding sequence is adapted to human codon usage, wherein the codon adaptation index (CAI) is preferably increased or maximised in the corresponding FAN coding sequence compared to the coding sequence of the corresponding wild type FAH coding sequence of SEQ ID NO:101. 5 In even further embodiments, the mRNA of the invention comprises a 5'-cap structure, a poly(A) sequence comprising at least 70 A nucleotides, preferably about 100 A nucleotides, a poly(C) sequence, preferably comprising 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides, and/or at least one histone stem-loop, preferably, wherein the mRNA comprises a 3'- terminal A nucleotide. In other embodiments, the mRNA of the invention comprises, preferably in 5' to 3' direction, the following elements: a) a 5'-cap1 structure; b) a 5'-UTR element comprising a nucleic acid sequence, preferably derived from a 5'-UTR of a HSD17B4 gene, comprising the nucleic acid sequence according to SEQ ID NO:1 or SEQ ID NO:2, or a homolog, a fragment or a variant thereof; c) at least one coding sequence as defined herein above or below; d) a 3'-UTR element comprising a nucleic acid sequence, preferably derived from a 3'-UTR of a PSMB3 gene, comprising the nucleic acid sequence according to SEQ ID NO:33 or SEQ ID NO:34, or a homolog, a fragment or a variant thereof; e) a poly(A) sequence comprising about 100 adenosine nucleotides, preferably, wherein the mRNA comprises a S'-terminal A nucleotide; f) an optional poly(C) tail, preferably comprising 10 to 40 cytosine nucleotides; and/or g) an optional histone stem-loop, preferably comprising the nucleic acid sequence according to SEQ ID NO:63 or SEQ ID NO:64. In more preferred embodiments, the nucleic acid of the invention comprises a coding sequence that comprises at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences according to any one of SEQ ID NO:112-144 or a fragment or variant of any of these sequences, encoding fumarylacetoacetate hydrolase (FAN) according to SEQ ID NO:100. Preferred nucleic acid sequences, preferably mRNA sequences of the invention are provided in Table A. Therein, each row represents a specific suitable FAhl construct of the invention (for column designation: see herein below and above), wherein the description of the FAhl construct is indicated in column A of Table A and the SEQ ID NOs of the amino acid sequence of the respective FAhl construct is provided in column B. The corresponding SEQ ID NOs of the coding sequences encoding the respective FAhl constructs are provided in Table A. Further detailed information on the specific sequences is provided under <223> identifier of the respective SEQ ID NOs in the sequence listing. Table A: preferred mRNA sequences and constructs of the invention

Thus, SEQ ID NO:1 to SEQ ID NO:145 are preferred sequences useful in the context of the present invention. Detailed information related to the preferred sequences of the present invention is disclosed in the ST.25 sequence listing under <223> Other Information, which is easily accessible for a skilled artisan. According to preferred embodiments, the nucleic acid of the invention comprises at least one coding sequence encoding at least one FAN protein, preferably as defined above or below, or fragments and variants thereof. In that context, any coding sequence encoding at least one FAH protein as defined herein, or fragments and variants thereof may be understood as suitable coding sequence and may therefore be comprised in the nucleic acid of the invention. As used herein, "wild type" and "naturally-occurring" refer to the form found in nature. For example, a wild type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation. As used herein, "recombinant, "engineered, "variant" and "non-naturally occurring" when used with reference to a cell, nucleic acid, polypeptide or protein, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature. In some embodiments, the cell, nucleic acid, polypeptide or protein is identical a naturally occurring cell, nucleic acid, polypeptide or protein, but is produced or derived from synthetic materials and/or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non recombinant) form of the cell or express native genes that are otherwise expressed at a different level. The terms "the same biological activity", "essentially the same biological activity", "similar biological activity" or "increased biological activity" in connection with fumarylacetoacetate hydrolase (FAH) all refer to a FAH proteins having (essentially) the same, similar or increased structural, regulatory, biochemical functions or biological activity as compared to a wild type FAH having WT FAH biological activity. Accordingly, the term "biological activity" in connection with FAhl as used herein means the biological properties characteristic for a FAH protein. Further, a polynucleotide comprising a fragment of any of the aforementioned nucleic acid sequences is also encompassed as a polynucleotide of the present invention. The fragment shall encode a polypeptide which still has a biological activity as specified herein. In the context of the present invention, the at least one coding sequence of the mRNA according to the invention preferably comprises a nucleic acid sequence encoding a peptide or protein comprising or consisting of a peptide or protein as defined herein. In some embodiments, a peptide or protein substantially comprises the entire amino acid sequence of the reference peptide or protein, such as the naturally occuring peptide or protein (e.g. FAH). Alternatively, the at least one coding sequence of the mRNA according to the invention may also comprise a nucleic acid sequence encoding a peptide or protein comprising or consisting of a fragment of a peptide or protein or a fragment of a variant of a peptide or protein as defined herein. In the context of the present invention, a "fragment" of a peptide or protein or of a variant thereof may comprise a sequence of a peptide or protein or of a variant thereof as defined above, which is, with regard to its amino acid sequence (or its encoded nucleic acid sequence), N-terminally, C-terminally and/or intrasequentially truncated compared to the reference amino acid sequence, such as the amino acid sequence of the naturally occuring protein or a variant thereof (or its encoded nucleic acid sequence). Such truncation may occur either on the amino acid level or on the nucleic acid level, respectively. A sequence identity with respect to such a fragment as defined herein therefore preferably refers to the entire peptide or protein or a variant thereof as defined herein or to the entire (coding) nucleic acid sequence of such a peptide or protein or of a variant thereof. According to some embodiments of the invention, the mRNA comprises at least one coding sequence encoding a peptide or protein comprising or consisting of a variant of a peptide or protein as defined herein, or a fragment of a variant of a peptide or protein. In certain embodiments of the present invention, a "variant" of a peptide or protein or a fragment thereof as defined herein may be encoded by the mRNA comprising at least one coding sequence as defined herein, wherein the amino acid sequence encoded by the at least one coding sequence differs in at least one amino acid residue from the reference amino acid sequence, such as a naturally occuring amino acid sequence. In this context, the "change" in at least one amino acid residue may consist, for example, in a mutation of an amino acid residue to another amino acid, a deletion or an insertion. More preferably, the term "variant" as used in the context of the amino acid sequence encoded by the at least one coding sequence of the mRNA according to the invention comprises any homolog, isoform or transcript variant of a peptide or protein or a fragment thereof as defined herein, wherein the homolog, isoform or transcript variant is preferably characterized by a degree of identity or homology, respectively, as defined herein. In the context of the present invention, a "fragment" or a "variant" of a protein or peptide may have at least 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% amino acid identity over a stretch of at least 10, at least 20, at least 30, at least 50, at least 75 or at least 100 amino acids of such protein or peptide. More preferably, a "fragment" or a "variant" of a protein or peptide as used herein is at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% identical to the protein or peptide, from which the variant is derived. Preferably, a variant of a peptide or protein or a fragment thereof may be encoded by the mRNA comprising at least one coding sequence as defined herein, or may be provided simply by provision of the peptide or protein sequence per se, wherein at least one amino acid residue of the amino acid sequence encoded by the at least one coding sequence is substituted. Substitutions, wherein amino acids, which originate from the same class, are exchanged for one another, are called conservative substitutions or respectively conservative amino acid substitutions. In particular, these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups inhe side chains or amino acids, the side chains of which can form hydrogen bridges, e.g. side chains which have a hydroxyl function. By conservative constitution, e.g. an amino acid having a polar side chain may be replaced by another amino acid having a corresponding polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain may be substituted by another amino acid having a corresponding hydrophobic side chain (e.g. serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)). In a preferred embodiment, a variant of a peptide or protein or a fragment thereof may be encoded by the mRNA according to the invention, wherein at least one amino acid residue of the amino acid sequence encoded by the at least one coding sequence comprises at least one conservative substitution compared to a reference sequence, such as the respective naturally occuring sequence. These amino acid sequences as well as their encoding nucleic acid sequences in particular are comprised by the term "variant" as defined herein. Further, as used herein, the phrase "conservative amino acid substitutions" refers to the interchangeability of residues having similar side chains, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, in some embodiments, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively. Exemplary conservative substitutions are provided in Table (i). Table (i): Exemplary conservative amino acid substitutions

Insertions, deletions and/or non-conservative substitutions are also possible, in particular, at those sequence positions, which preferably do not cause a substantial modification of the three- dimensional structure. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g. using CD spectra (circular dichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, in: Modern Physical Methods in Biochemistry, Neuberger et al. (ed.), Elsevier, Amsterdam). In order to determine the percentage, to which two sequences (nucleic acid sequences, e.g. RNA or mRNA sequences as defined herein, or amino acid sequences, preferably the amino acid sequence encoded by the mRNA according to the invention) are identical, the sequences can be aligned in order to be subsequently compared to one another. For this purpose, e.g. gaps can be inserted into the sequence of the first sequence and the component at the corresponding position of the second sequence can be compared. If a position in the first sequence is occupied by the same component as is the case at a corresponding position in the second sequence, the two sequences are identical at this position. The percentage, to which two sequences are identical, is a function of the number of identical positions divided by the total number of positions. The percentage, to which two sequences are identical, can be determined using a mathematical algorithm. A preferred, but not limiting, example of a mathematical algorithm, which can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res, 25:3389-3402. Such an algorithm is integrated, for example, in the BLAST program. Sequences, which are identical to the sequences of the present invention to a certain extent, can be identified by this program. In the context of the present invention, a fragment of a peptide or protein or a variant thereof encoded by the at least one coding sequence of the mRNA according to the invention may typically comprise an amino acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with a reference amino acid sequence, preferably with the amino acid sequence of the respective naturally occuring full-length peptide or protein or a variant thereof. In certain embodiments, the mRNA according to the invention, preferably the at least one coding sequence of the mRNA according to the invention, may comprise or consist of a fragment of a nucleic acid sequence encoding a peptide or protein or a fragment or variant thereof as defined herein. Preferably, the at least one coding sequence of the mRNA according to the invention comprises or consists of a fragment or variant, preferably as defined herein, of any one of the nucleic acid sequences according to any single element from the group consisting of SEQ ID NO:112-144, or a variant of any one of these sequences. In this context, a fragment or variant of a nucleic acid (sequence) is preferably a nucleic acid sequence encoding a fragment of a peptide or protein or of a variant thereof as described herein. More preferably, the expression "fragment of a nucleic acid sequence" refers to a nucleic acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with a respective full- length nucleic acid sequence, preferably with a nucleic acid sequence selected from any single element from the group consisting of SEQ ID NO:112-144, era variant of any of these nucleic acid sequences. In another preferred embodiment, the mRNA according to the invention, preferably the at least one coding sequence of the mRNA according to the invention, may comprise or consist of a variant of a nucleic acid sequence as defined herein, preferably of a nucleic acid sequence encoding a peptide or protein or a fragment thereof as defined herein. The expression "variant of a nucleic acid sequence" as used herein in the context of a nucleic acid sequence encoding a peptide or protein as described herein or a fragment thereof, typically refers to a nucleic acid sequence, which differs by at least one nucleic acid residue from the respective reference nucleic acid sequence, for example from the respective naturally occuring nucleic acid sequence or from a full-length nucleic acid sequence as defined herein, or from a fragment thereof. More preferably, the expression "variant of a nucleic acid sequence" as used in the context of the present invention refers to a nucleic acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with a nucleic acid sequence, from which it is derived. Preferably, the mRNA according to the invention, more preferably the at least one coding sequence of the mRNA according to the invention, encodes a variant of a peptide or protein or a fragment thereof, preferably as defined herein. In a preferred embodiment, the mRNA according to the invention, more preferably the at least one coding sequence of the mRNA according to the invention, comprises or consists of a variant of a nucleic acid sequence encoding a peptide or protein or a fragment thereof as defined herein, wherein the variant of the nucleic acid sequence encodes an amino acid sequence comprising at least one conservative substitution of an amino acid residue. In another embodiment, the mRNA according to the invention, more preferably the at least one coding sequence of the mRNA according to the invention, comprises or consists of a variant of a nucleic acid sequence encoding a a peptide or protein or a fragment thereof as defined herein, wherein the nucleic acid sequence of the variant differs from a reference nucleic acid sequence, preferably from the respective naturally occuring nucleic acid sequence in at least one nucleic acid residue, more preferably without resulting - due to the degenerated genetic code - in an alteration of the encoded amino acid sequence, i.e. the amino acid sequence encoded by the variant or at least part thereof may preferably not differ from the naturally occuring amino acid sequence in one or more mutation(s) within the above meaning. Furthermore, a "variant" of a nucleic acid sequence encoding a peptide or protein or a "fragment or variant" thereof as defined herein, may also comprise mRNA or DNA sequences, which correspond to RNA or mRNA sequences as defined herein and may also comprise further RNA or mRNA sequences, which correspond to DNA sequences as defined herein. Those skilled in the art are familiar with the translation of an RNA or mRNA sequence into a DNA sequence (or vice versa) or with the creation of the complementary strand sequence (i.e. by substitution of U residues with T residues and/or by constructing the complementary strand with respect to a given sequence). A "fragment" refers to a portion of the mRNA or nucleotide sequence encoding an FAH protein or a portion of the amino acid sequence of the FAhl protein of the invention. A fragment of an FAhl mRNA or nucleotide sequence of the invention may encode a biologically active portion of an FAN protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods known to skilled persons in the art. A fragment of an FAH polypeptide may encompass a biologically active fragment of the FAH protein. The term "biologically active fragments or variants" refers to fragments or variants of the exemplified nucleic acid molecules and polypeptides that comprise or encode FAN activity. MRNAs or nucleic acid molecules that are "variants" of the mRNAs or nucleotide sequences disclosed herein are also encompassed by the present invention. "Variants" of the FAH nucleotide sequences of the invention include those sequences that encode the FAhl proteins disclosed herein but that differ conservatively because of the degeneracy of the genetic code. These naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the FAhl protein disclosed in the present invention as discussed herein above or below. Generally, nucleotide sequence variants of the invention will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a particular nucleotide sequence disclosed herein. A variant FAN mRNA or nucleotide sequence will encode a FAH protein, respectively, that has an aminoacid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of a FAH protein disclosed herein. According to certain embodiments, the mRNA according to the invention is mono-, bi-, or multicistronic, preferably as defined herein. The coding sequences in a bi- or multicistronic RNA preferably encode a distinct peptide or protein as defined herein or a fragment or variant thereof. Preferably, the coding sequences encoding two or more peptides or proteins may be separated in the bi- or multicistronic RNA by at least one IRES (internal ribosomal entry site) sequence, as defined below. Thus, the term "encoding two or more peptides or proteins" may mean, without being limited thereto, that the bi- or even multicistronic RNA, may encode e.g. at least two, three, four, five, six or more (preferably different) peptides or proteins as described herein or their fragments or variants within the definitions provided herein. More preferably, without being limited thereto, the bi- or even multicistronic mRNA, may encode, for example, at least two, three, four, five, six or more (preferably different) peptides or proteins as defined herein or their fragments or variants as defined herein. In this context, a so-called IRES (internal ribosomal entry site) sequence as defined above can function as a sole ribosome binding site, but it can also serve to provide a bi- or even multicistronic mRNA as defined above, which encodes several peptides or proteins, which are to be translated by the ribosomes independently of one another. Examples of IRES sequences, which can be used according to the invention, are those from picornaviruses (e.g. FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot and mouth disease viruses (FMDV), hepatitis C viruses (hlCV), classical swine fever viruses (CSFV), mouse leukoma virus (MLV), simian immunodeficiency viruses (SIV) or cricket paralysis viruses (CrPV). According to a further embodiment the at least one coding sequence of the mRNA according to the invention may encode at least two, three, four, five, six, seven, eight, nine and more peptides or proteins (or fragments or variants thereof) as defined herein linked with or without an amino acid linker sequence, wherein said linker sequence can comprise rigid linkers, flexible linkers, cleavable linkers (e.g., self-cleaving peptides) or a combination thereof. Therein, the peptides or proteins (or fragments or variants thereof) may be identical or different or a combination thereof. Preferably, the at least one coding sequence of the mRNA according to the invention comprises at least two, three, four, five, six, seven, eight, nine or more nucleic acid sequences identical to or having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with a nucleic acid sequence selected from any single element from the group consisting of SEQ ID NO:112-144, or a fragment or variant of any one of these nucleic acid sequences, wherein each mRNA may encode a different FAH protein, i.e. a cocktail of different FAhl proteins. Preferably, the mRNA comprising at least one coding sequence as defined herein typically comprises a length of about 50 to about 20000, or 100 to about 20000 nucleotides, preferably of about 250 to about 20000 nucleotides, more preferably of about 500 to about 10000, even more preferably of about 500 to about 5000. The mRNA according to the invention may further be single stranded or double stranded. When provided as a double stranded RNA, the mRNA according to the invention preferably comprises a sense and a corresponding antisense strand. In a preferred embodiment, the mRNA comprising at least one coding sequence as defined herein is an mRNA, a viral RNA or a replicon RNA. Preferably, the mRNA is an artificial nucleic acid, more preferably as described herein. According to a further embodiment, the RNA, preferably an mRNA, according to the invention is a modified RNA, preferably a modified RNA as described herein. A modified RNA as used herein does preferably not comprise a chemically modified sugar, a chemically modified backbone or a chemically modified nucleobase. More preferably, a modified RNA as used herein does not comprise a chemically modified nucleoside or a chemically modified nucleotide. It is further preferred that a modified RNA as used herein does not comprise a chemical modification as described in international patent application WO 2014158795. In the context of the present invention, a modification as defined herein preferably leads to a stabilization of the mRNA according to the invention. More preferably, the invention thus provides a stabilized RNA comprising at least one coding sequence as defined herein. According to one embodiment, the mRNA of the present invention may thus be provided as a "stabilized mRNA", that is to say as an RNA that is essentially resistant to in vivo degradation (e.g. by an exo- or endo-nuclease). Stabilization of an RNA can be achieved, for example, by a modified phosphate backbone of the mRNA of the present invention. A backbone modification in connection with the present invention is a modification, in which phosphates of the backbone of the nucleotides contained in the mRNA are chemically modified. Nucleotides that may be preferably used in this connection contain e.g. a phosphorothioate-modified phosphate backbone, preferably at least one of the phosphate oxygens contained in the phosphate backbone being replaced by a sulfur atom. Stabilized RNAs may further include, for example: non-ionic phosphate analogues, such as, for example, alkyl and aryl phosphonates, in which the charged phosphonate oxygen is replaced by an alkyl or aryl group, or phosphodiesters and alkylphosphotriesters, in which the charged oxygen residue is present in alkylated form. Such backbone modifications typically include, without implying any limitation, modifications from the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g. cytidine-5'-O-(1-thiophosphate)). In the following, specific modifications are described, which arepreferably capable of "stabilizing" the mRNA as defined herein. RNA constructs The mRNA according to the invention, which comprises at least one coding sequence as defined herein, may preferably comprise a 5'-UTR and/or a 3'-UTR preferably containing at least one histone stem-loop. Where, in addition to the peptide or protein as defined herein or a fragment or variant thereof, a further peptide or protein is encoded by the at least one coding sequence of the mRNA according to the invention, the encoded peptide or protein is preferably no histone protein, no reporter protein and/or no marker or selection protein, as defined herein. The 3'-UTR of the mRNA according to the invention preferably comprises also a poly(A) and/or a poly(C) sequence as defined herein. The single elements of the 3'-UTR may occur therein in any order from 5' to 3' along the sequence of the mRNA of the present invention. In addition, further elements as described herein, may also be contained, such as a stabilizing sequence as defined herein (e.g. derived from the UTR of a globin gene), IRES sequences, etc. Each of the elements may also be repeated in the mRNA according to the invention at least once (particularly in di- or multicistronic constructs), preferably twice or more. As an example, the single elements may be present in the mRNA according to the invention in the following order (wherein the mRNA may optionally comprise a 5'-UTR element as described herein 5' of the coding region/CDS and/or a 3'-UTR element as described herein 3' of the coding region/CDS): 5'- coding region histone stem-loop - poly(A)/(C) sequence - 3'; or 5' - coding region poly(A)/(C) sequence - histone stem-loop - 3'; or 5' - coding region histone stem-loop - polyadenylation signal - 3'; or 5' - coding region polyadenylation signal - histone stem-loop - 3'; or 5' - coding region histone stem-loop - histone stem-loop - poly(A)/(C) sequence - 3'; or 5'-coding region histone stem-loop - histone stem-loop - polyadenylation signal - 3'; or 5' -coding region poly(A)/(C) sequence - histone stem-loop - 3'; or 5' - coding region poly(A)/(C) sequence - poly(A)/(C) sequence - histone stem-loop - 3'; or 5' - coding region poly(A)/(C) sequence - histone stem-loop - 3' etc. According to a further embodiment, the mRNA of the present invention preferably comprises at least one of the following structural elements: a 5'- and/or S'-untranslated region element (UTR element), particularly a 5'-UTR element, which preferably comprises or consists of a nucleic acid sequence which is derived from the 5'-UTR of a TOP gene or from a fragment, homolog or a variant thereof, or a 5'- and/or 3'-UTR element which may preferably be derivable from a gene that provides a stable mRNA or from a homolog, fragment or variant thereof; a histone-stem-loop structure, preferably a histone-stem-loop in its S'-untranslated region; a 5'-cap structure; a poly(A) tail; or a poly(C) sequence. Preferably, the mRNA of the invention comprises a S'-terminal A nucleotide. According to some embodiments, it is particularly preferred that - if, in addition to a peptide or protein as defined herein or a fragment or variant thereof, a further peptide or protein is encoded by the at least one coding sequence as defined herein - the encoded peptide or protein is preferably no histone protein, no reporter protein (e.g. Luciferase, GFP, EGFP, p-Galactosidase, particularly EGFP) and/or no marker or selection protein (e.g. alpha-Globin, Galactokinase and Xanthine:Guanine phosphoribosyl transferase (GPT)). In a preferred embodiment, the mRNA according to the invention does not comprise a reporter gene or a marker gene. Preferably, the mRNA according to the invention does not encode, for instance, luciferase; green fluorescent protein (GFP) and its variants (such as EGFP, RFP or BFP); a-globin; hypoxanthine-guanine phosphoribosyltransferase (hlGPRT); P-galactosidase; galactokinase; alkaline phosphatase; secreted embryonic alkaline phosphatase (SEAP)) or a resistance gene (such as a resistance gene against neomycin, puromycin, hygromycin and zeocin). In a preferred embodiment, the mRNA according to the invention does not encode luciferase. In another embodiment, the mRNA according to the invention does not encode GFP or a variant thereof. In another embodiment, the mRNA according to the present invention comprises, preferably in 5' to 3' direction, the following elements: a) a 5'-cap structure, preferably m7GpppN, b) a 5'-UTR element, which comprises or consists of a nucleic acid sequence, which is derived from the 5'-UTR of a TOP gene, preferably comprising a nucleic acid sequence according to SEQ ID NO:1 or 2 (HSD17B4), or a homolog, a fragment or a variant thereof or a 5'-UTR element, which comprises or consists of a nucleic acid sequence according to SEQ ID NO:27 or 28 (Slc7a3) or SEQ ID NO:23 or 24 (Rpl31), or a homolog, a fragment or a variant thereof, c) at least one coding sequence as defined herein, d) a 3'-UTR element comprising a nucleic acid sequence, which is derived from a PSMB3 gene, preferably comprising a nucleic acid sequence according to SEQ ID NO:33 or 34, or a homolog, a fragment or a variant thereof; and/or a 3'-UTR element comprising a nucleic acid sequence, which is derived from a RPS9 gene, preferably comprising a nucleic acid sequence according to SEQ ID NO:51 or 52, or a homolog, a fragment or a variant thereof, e) a poly(A) tail, about 10 to about 200,about 10 to about 100,about 40 to about 80 or about 50 to about 70 adenosine nucleotides, more preferably about 100 adenosine nucleotides, preferably, wherein the mRNA comprises a S'-terminal A nucleotide, f) a poly(C) tail, preferably consisting of 10 to 200,10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytidine nucleotides, and g) a histone stem-loop, preferably comprising a nucleic acid sequence according to SEQ ID NO:630 or 64. In a further embodiment, the mRNA according to the present invention comprises, preferably in 5' to 3' direction, the following elements: a) a 5'-cap structure, preferably m7GpppN, b) a 5'-UTR element, which comprises or consists of a nucleic acid sequence, which is derived from the 5'-UTR of the HISD17B4 gene, preferably comprising a nucleic acid sequence according to SEQ ID NO:1 or 2, or a homolog, a fragment or a variant thereof, c) at least one coding sequence as defined herein, d) a 3'-UTR element comprising a nucleic acid sequence, which is derived from an PSMB3 gene, preferably comprising a nucleic acid sequence according to SEQ ID NO:33 or 34, or a homolog, a fragment or a variant thereof; e) optionally, a histone stem-loop selected from SEQ ID NOs:63 or 64; and/or f) a poly(A) tail, preferably consisting of about 10 to about 200, about 10 to about 100, about 40 to about 80 or about 50 to about 70 adenosine nucleotides, more preferably about 100 adenosine nucleotides. The mRNA according to the present invention may be prepared using any method known in the art, including synthetic methods such as e.g. solid phase RNA synthesis, as well as in vitro methods, such as RNA in vitro transcription reactions. Preferred sequences are shown herein below in Table C1/C2. Table C1: Preferred sequences of the present invention - as apparent, different construct designs were applied. More information on the sequences is disclosed in the ST.25 sequence listing under <223> Other Information. Each construct as shown in the sequence listing resembles a preferred construct of the invention.

In another embodiment, the nucleic acid of the present invention are DNA sequences, comprising a coding sequence that comprises at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the sequences selected from the group consisting of SEQ ID NO:112-144 or to any one of the sequences as disclosed in the Table C2 ("Constructs of the invention") herein below, wherein all Uracils (U) in the respective sequences are substituted by Thymidines (T), or a fragment or variant of any of these sequences. T_{able C2:_"Constructs of the invention"}

In a preferred embodiment, a sequence as shown in SEQ ID NO:114 (R9261) contains no chemically modified uracil. In another preferred embodiment, a sequence as shown in SEQ ID NO:114 (R9261) contains chemically modified uracil, preferably pseudouridine (psi-uridine), more preferably N1- methylpseudouridine (N1MPU). Modifications Chemical_modifications The term "RNA modification" as used herein may refer to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications. In this context, a modified RNA as defined herein may contain nucleotide analogues/modifications, e.g. backbone modifications, sugar modifications or base modifications. A backbone modification in connection with the present invention is a modification, in which phosphates of the backbone of the nucleotides contained in an RNA as defined herein are chemically modified. A sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides of the mRNA as defined herein. Furthermore, a base modification in connection with the present invention is a chemical modification of the base moiety of the nucleotides of the mRNA. In this context, nucleotide analogues or modifications are preferably selected from nucleotide analogues, which are applicable for transcription and/or translation. Base Modifications In certain embodiments, the open reading frame of the mRNA of the invention does not comprise any chemically modified uracil or cytosine nucleotides. In other embodiments, the mRNA of the invention is chemically modified, preferably wherein the mRNA comprises pseudouridine (psi-uridine), Nl-methylpseudouridine (N1MPU), 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosme, 7 -deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and/or 2-thiocytidine, more preferably wherein all uridine bases of the mRNA are fully chemically modified, even more preferably wherein all uridine bases of the mRNA are pseudouridine or N1- methylpseudouridine (N1 MPU) bases, most preferably wherein all uridine bases of the mRNA are N1 -methylpseudouridine (N1MPU) bases.

In some preferred embodiments, the nucleotide mixture comprises at least one modified nucleotide and/or at least one nucleotide analogue or nucleotide derivative. The modified nucleosides and nucleotides, which may be incorporated into a modified RNA as described herein can further be modified in the nucleobase moiety. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine and uracil. For example, the nucleosides and nucleotides described herein can be chemically modified on the major groove face. In some embodiments, the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group. In preferred embodiments the nucleotide mixture comprises least one modified nucleotide and/or at least one nucleotide analogues is selected from a backbone modified nucleotide, a sugar modified nucleotide and/or a base modified nucleotide, or any combination thereof.

In particularly preferred embodiments of the present invention, the nucleotide analogues/modifications are selected from base modifications, which are preferably selected from 2- amino-6-chloropurineriboside-5’-triphosphate, 2-Aminopurine-riboside-5’-triphosphate; 2-amino- adenosine-5’-triphosphate, 2’-Amino-2’-deoxycytidine-triphosphate, 2-thiocytidine-5’-triphosphate, 2-thiouridine-5’-triphosphate, 2’-Fluorothymidine-5’-triphosphate, 2’-O-Methyl-inosine-5’- triphosphate 4-thiouridine-5’-triphosphate, 5-aminoallylcytidine-5’-triphosphate, 5-aminoallyluridine- 5’-triphosphate, 5-bromocytidine-5’-triphosphate, 5-bromouridine-5’-triphosphate, 5-Bromo-2’-de- oxycytidine-5’-triphosphate, 5-Bromo-2’-deoxyuridine-5’-triphosphate, 5-iodocytidine-5’-triphos- phate, 5-lodo-2’-deoxycytidine-5’-triphosphate, 5-iodouridine-5'-triphosphate, 5-lodo-2’-deoxy- uridine-5'-triphosphate, 5-methylcytidine-5’-triphosphate, 5-methyluridine-5’-triphosphate, 5- Propynyl-2’-deoxycytidine-5’-triphosphate, 5-Propynyl-2’-deoxyuridine-5’-triphosphate, 6-aza- cytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurineriboside-5'-triphosphate, 7-de- azaadenosine-5'-triphosphate, 7-deazaguanosine-5’-triphosphate, 8-azaadenosine-5’-triphosphate, 8-azidoadenosine-5’-triphosphate, benzimidazole-riboside-5’-triphosphate, N 1 -methyladenosine-5’- triphosphate, N1-methylguanosine-5’-triphosphate, N6-methyladenosine-5’-triphosphate, 06- methylguanosine-5’-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5’-triphosphate, xanthosine-5’-triphosphate. Particular preference is given to nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5’-triphosphate, 7-deazaguanosine-5’-triphosphate, 5-bromocytidine-5’-triphosphate, and pseudouridine-5’- triphosphate. In some embodiments, modified nucleosides include pyridin-4-one ribonucleoside, 5-aza-uridine, 2- thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1- propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2- thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1- methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-1- methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl- cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl- cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-1-methyl- 1-deaza- pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidine. In other embodiments, modified nucleosides include 2-aminopurine, 2, 6-diaminopurine, 7-deaza- adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza- 2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, NS-methyladenosine, NS-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, NS-glycinylcarbamoyladenosine, NS-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2- methylthio-adenine, and 2-methoxy-adenine. In other embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio- 7-deaza-S-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6- methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo- guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. In some embodiments, the nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group. In specific embodiments, a modified nucleoside is 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1- thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine or5'-O-(1-thiophosphate)-pseudouridine. In further specific embodiments, a modified RNA may comprise nucleoside modifications selected from Nl-methyl-pseudouridine (N1MPU), 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, Pseudo-iso- cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, 5,6-dihydrouridine, a-thio-uridine, 4-thio-uridine, 6-aza- uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, a-thio- guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine, 7-deaza-guanosine, N1- methyl-adenosine, 2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso-cytidine, 6- Chloro-purine, N6-methyl-adenosine, a-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine. In other preferred embodiments, the open reading frame from any mRNA of the invention does not comprise any chemically modified nudeotides, more preferably does not comprise any chemically modified uracil or cytosine nucleotides. Sugar Modifications: The modified nucleosides and nucleotides, which may be incorporated into a modified RNA as described herein, can be modified in the sugar moiety. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. Examples of "oxy"- 2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R = hi, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), - 0(CH2CH20)nCH2CH20R; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; and amino groups (-0-amino, wherein the amino group, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino) or aminoalkoxy. "Deoxy" modifications include hydrogen, amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or the amino group can be attached to the sugar through a linker, wherein the linker comprises one or more of the atoms C,N,andO. The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified RNA can include nucleotides containing, for instance, arabinose as the sugar. Backbone Modifications: The phosphate backbone may further be modified in the modified nucleosides and nucleotides, which may be incorporated into a modified RNA as described herein. The phosphate groups of the backbone can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the full replacement of an unmodified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene- phosphonates). Lipid modification According to a further embodiment, a modified RNA as defined herein can contain a lipid modification. Such a lipid-modified RNA typically comprises an RNA as defined herein. Such a lipid- modified RNA as defined herein typically further comprises at least one linker covalently linked with that RNA, and at least one lipid covalently linked with the respective linker. Alternatively, the lipid- modified RNA comprises at least one RNA as defined herein and at least one (bifunctional) lipid covalently linked (without a linker) with that RNA. According to a third alternative, the lipid-modified RNA comprises an RNA molecule as defined herein, at least one linker covalently linked with that RNA, and at least one lipid covalently linked with the respective linker, and also at least one (bifunctional) lipid covalently linked (without a linker) with that RNA. In this context, it is particularly preferred that the lipid modification is present at the terminal ends of a linear RNA sequence. G/C content modification According to another embodiment, the mRNA of the present invention, may be modified, and thus stabilized, by modifying the guanosine/cytosine (G/C) content of the mRNA, preferably of the at least one coding sequence of the mRNA of the present invention. In a particularly preferred embodiment of the present invention, the G/C content of the coding sequence (coding region or CDS) of the mRNA of the present invention is modified, particularly increased, compared to the G/C content of the coding region of the respective wild type RNA, i.e. the unmodified RNA. The amino acid sequence encoded by the mRNA is preferably not modified as compared to the amino acid sequence encoded by the respective wild type RNA. This modification of the mRNA of the present invention is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that RNA. Thus, the pharmaceutical composition of the mRNAand the sequence of various nucleotides are important. In particular, sequences having an increased G (guanosine)/C (cytosine) content are more stable than sequences having an increased A (adenosine)/U (uracil) content. According to the invention, the codons of the mRNA are therefore varied compared to the respective wild type RNA, while retaining the translated amino acid sequence, such that they include an increased amount of G/C nucleotides. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favourable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the mRNA, there are various possibilities for modification of the mRNA sequence, compared to its wild type sequence. In the case ofamino acids, which are encoded by codons, which contain exclusively G or C nucleotides, no modification of the codon is necessary. Thus, the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) require no modification, since no A or U is present. In contrast, codons which contain A and/or U nucleotides can be modified by substitution of other codons, which code for the same amino acids but contain no A and/or U. Examples of these are: the codons for Pro can be modified from CCU or CCA to CCC or CCG; the codons for Arg can be modified from CGU or CGA or AGA or AGG to CGC or CGG; the codons for Ala can be modified from GCU or GCA to GCC or GCG; the codons for Gly can be modified from GGU or GGA to GGC or GGG. In other cases, although A or U nucleotides cannot be eliminated from the codons, it is however possible to decrease the A and U content by using codons which contain a lower content of A and/or U nucleotides. Examples of these are: the codons for Phe can be modified from UUU to UUC; the codons for Leu can be modified from UUA, UUG, CUU or CUA to CUC or CUG; the codons for Ser can be modified from UCU or UCA or AGU to UCC, UCG or AGC; the codon for Tyr can be modified from UAU to UAC; the codon for Cys can be modified from UGU to UGC; the codon for His can be modified from CAU to CAC; the codon for Gin can be modified from CAA to CAG; the codons for lie can be modified from AUU or AUA to AUC; the codons for Thr can be modified from ACU or ACA to ACC or ACG; the codon for Asn can be modified from AAU to AAC; the codon for Lys can be modified from AAA to AAG; the codons for Vai can be modified from GUU or GUA to GUC or GUG; the codon for Asp can be modified from GAU to GAC; the codon for Glu can be modified from GAA to GAG; the stop codon UAA can be modified to UAG or UGA. In the case of the codons for Met (AUG) and Trp (UGG), on the other hand, there is no possibility of sequence modification. The substitutions listed above can be used either individually or in all possible combinations to increase the G/C content of the at least one mRNA of the pharmaceutical composition of the present invention compared to its particular wild type mRNA (i.e. the original sequence). Thus, for example, all codons for Thr occurring in the wild type sequence can be modified to ACC (or ACG). Preferably, however, for example, combinations of the above substitution possibilities are used:

• substitution of all codons originally coding for Thr in the original (WT mRNA) sequence to ACC (or ACG) and/or

• substitution of all codons originally coding for Ser to UCC (or UCG or AGC); and/or

• substitution of all codons coding for IIe in the original sequence to AUC; and/or

• substitution of all codons originally coding for Lys to AAG and/or

• substitution of all codons originally coding for Tyr to UAC; and/or

• substitution of all codons coding for Vai in the original sequence to GUC (or GUG) and/or

• substitution of all codons originally coding for Glu to GAG and/or

• substitution of all codons originally coding for Ala to GCC (or GCG) and/or

• substitution of all codons originally coding for Arg to CGC (or CGG); and/or

• substitution of all codons originally coding for Glu to GAG and/or

• substitution of all codons originally coding for Ala to GCC (or GCG); and/or

• substitution of all codons originally coding for Gly to GGC (or GGG); and/or substitution of all codons originally coding forAsn to AAC; and/or substitution of all codons coding forVal in the original sequence to GUC (orGUG) and/or substitution of all codons originally coding for Phe to UUC and/or substitution of all codons originally coding for Cys to UGC and/or • substitution of all codons originally coding for Leu to CUG (or CUC) and/or substitution of all codons originally coding for Gin to CAG and/or • substitution of all codons originally coding for Pro to CCC (or CCG); etc. Preferably, the G/C content of the coding region of the mRNA of the present invention is increased by at least 7%, more preferably by at least 15%, particularly preferably by at least 20%, compared to the G/C content of the coding region of the wild type RNA, which codes for a peptide or protein as defined herein or a fragment or variant thereof. According to a specific embodiment at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, even more preferably at least 80% and most preferably at least 90%, 95% or even 100% of the substitutable codons in the region coding for a peptide or protein as defined herein or a fragment or variant thereof or the whole sequence of the wild type RNA sequence are substituted, thereby increasing the GC/content of said sequence. In this context, it is particularly preferable to increase the G/C content of the mRNA of the present invention, preferably of the at least one coding region of the mRNA according to the invention, to the maximum (i.e.100% of the substitutable codons) as compared to the wild type sequence. According to the invention, a further preferred modification of the mRNA of the present invention is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, if so-called "rare codons" are present in the mRNA of the present invention to an increased extent, the corresponding modified RNA sequence is translated to a significantly poorer degree than in the case where codons coding for relatively "frequent" tRNAs are present. According to the invention, in the modified RNA of the present invention, the region which codes for a peptide or protein as defined herein or a fragment or variant thereof is modified compared to the corresponding region of the wild type RNA such that at least one codon of the wild type sequence, which codes for a tRNA which is relatively rare in the cell, is exchanged for a codon, which codes for a tRNA which is relatively frequent in the cell and carries the same amino acid as the relatively rare tRNA. By this modification, the sequences of the mRNA of the present invention is modified such that codons for which frequently occurring tRNAs are available are inserted. In other words, according to the invention, by this modification all codons of the wild type sequence, which code for a tRNA which is relatively rare in the cell, can in each case be exchanged for a codon, which codes for a tRNA which is relatively frequent in the cell and which, in each case, carries the same amino acid as the relatively rare tRNA. Which tRNAs occur relatively frequently in the cell and which, in contrast, occur relatively rarely is known to a person skilled in the art; cf. e.g. Akashi, Curr. Opin. Genet. Dev.2001, 11(6): 660-666. The codons, which use for the particular amino acid the tRNA which occurs the most frequently, e.g. the Gly codon, which uses the tRNA, which occurs the most frequently in the (human) cell, are particularly preferred. According to the invention, it is particularly preferable to link the sequential G/C content which is increased, in particular maximized, in the modified RNA of the present invention, with the "frequent" codons without modifying the amino acid sequence of the peptide or protein encoded by the coding region of the mRNA. This preferred embodiment allows provision of a particularly efficiently translated and stabilized (modified) RNA of the present invention. The determination of a modified RNA of the present invention as described above (increased G/C content; exchange of tRNAs) can be carried out using the computer program explained in W02002098443 - the disclosure content of which is included in its full scope in the present invention. Using this computer program, the nucleotide sequence of any desired RNA can be modified with the aid of the genetic code or the degenerative nature thereof such that a maximum G/C content results, in combination with the use of codons which code for tRNAs occurring as frequently as possible in the cell, the amino acid sequence coded by the modified RNA preferably not being modified compared to the non-modified sequence. Alternatively, it is also possible to modify only the G/C content or only the codon usage compared to the original sequence. The source code in Visual Basic 6.0 (development environment used: Microsoft Visual Studio Enterprise 6.0 with Servicepack 3) is also described in WO2002098443. In a further preferred embodiment of the presentnvention, the A/U content in the environment of the ribosome binding site of the mRNA of the present invention is increased compared to the A/U content in the environment of the ribosome binding site of its respective wild type mRNA. This modification (an increased A/U content around the ribosome binding site) increases the efficiency of ribosome binding to the mRNA. An effective binding of the ribosomes to the ribosome binding site (Kozak sequence as shown herein below; AUG forms the start codon) in turn has the effect of an efficient translation of the mRNA. In this regard, due to the conversion to a sequence listing according to ST.26 standard, SEQ ID NO:57, 58, 61, and 62 as shown in the priority applications are now presented herein as part of the disclosure: formerSEQIDN057(RNA) Artificial Sequence mRNA 5'-end GGGAGA formerSEQIDN058(RNA) Artificial Sequence mRNA 5'-end AGGAGA • formerSEQ IDN061 (DNA) Artificial Sequence Kozak seq ACC former SEQ ID NO 62 (RNA) Artificial Sequence Kozak seq mRNA ACC According to a further embodiment of the present invention, the mRNA of the present invention may be modified with respect to potentially destabilizing sequence elements. Particularly, the coding region and/or the 5'- and/or S'-untranslated region of this RNA may be modified compared to the respective wild type RNA such that it contains no destabilizing sequence elements, the encoded amino acid sequence of the modified RNA preferably not being modified compared to its respective wild type RNA. It is known that, for example in sequences of eukaryoticRNAs, destabilizing sequence elements (DSE) occur, to which signal proteins bind and regulate enzymatic degradation of RNA in vivo. For further stabilization of the modified RNA, optionally in the region which encodes a peptide or protein as defined herein or a fragment or variant thereof, one or more such modifications compared to the corresponding region of the wild type RNA can therefore be carried out, so that no or substantially no destabilizing sequence elements are contained there. According to the invention, DSE present in the untranslated regions (3'- and/or 5'-UTR) can also be eliminated from the mRNA of the present invention by such modifications. Such destabilizing sequences are e.g. AU-rich sequences (AURES), which occur in 3'-UTR sections of numerous unstable RNAs (Caput et al., Proc. Natl. Acad. Sci. USA 1986, 83: 1670 to 1674). The mRNAofthe present invention is therefore preferably modified compared to the respective wild type RNA such that the mRNA of the present invention contains no such destabilizing sequences. This also applies to those sequence motifs which are recognized by possible endonucleases, e.g. the sequence GAACAAG, which is contained in the 3'-UTR segment of the gene encoding the transferrin receptor (Binder et al., EMBO J.1994, 13: 1969-1980). These sequence motifs are also preferably removed in the mRNA of the present invention. In preferred embodiments, the nucleic acid may be modified, wherein the G/C content of the at least one coding sequence may be optimized compared to the G/C content of the corresponding wild type or reference coding sequence (herein referred to as "G/C content optimized coding sequence"). "Optimized" in that context refers to a coding sequence wherein the G/C content is preferably increased to the essentially highest possible G/C content. The amino acid sequence encoded by the G/C content optimized coding sequence of the nucleic acid is preferably not modified as compared to the amino acid sequence encoded by the respective wild type or reference coding sequence. The generation of a G/C content optimized nucleic acid sequence (RNA or DNA) may be carried out using a method according to W02002098443. In this context, the disclosure of W02002/098443 is included in its full scope in the present invention. Throughout the description, including the <223>dentifier of the sequence listing, G/C optimized coding sequences are indicated by the abbreviations "opt1"or"gc". Seauences adapted to human codon usage According to the invention, a further preferred modification of the mRNA of the present invention is based on the finding that codons encoding the same amino acid typically occur at different frequencies. According to the invention, in the modified RNA of the present invention, the coding sequence (coding region) as defined herein is preferably modified compared to the corresponding region of the respective wild type RNA such that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to the human codon usage as e.g. shown in Table D. For example, in the case of the amino acid alanine (Ala) present in an amino acid sequence encoded by the at least one coding sequence of the mRNA according to the invention, the wild type coding sequence is preferably adapted in a way that the codon "GCC" is used with a frequency of 0.40, the codon "GCT" is used with a frequency of 0.28, the codon "GCA" is used with a frequency of 0.22 and the codon "GCG" is used with a frequency of 0.10 etc. (see Table D). TABLE D: HUMAN CODON USAGE TABLE

In preferred embodiments, the nucleic acid may be modified, wherein the codons in the at least one coding sequence may be adapted to human codon usage (herein referred to as "human codon usage adapted coding sequence"). Codons encoding the same amino acid occur at different frequencies in humans. Accordingly, the coding sequence of the nucleic acid is preferably modified such that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to the human codon usage. For example, in the case of the amino acid Ala, the wild type or reference coding sequence is preferably adapted in a way that the codon "GCC" is used with a frequency of 0.40, the codon "GCT" is used with a frequency of 0.28, the codon "GCA" is used with a frequency of 0.22 and the codon "GCG" is used with a frequency of 0.10 etc. (see Table D). Accordingly, such a procedure (as exemplified for Ala) is applied for each amino acid encoded by the coding sequence of the nucleic acid to obtain sequences adapted to human codon usage. Throughout the description, including the <223> identifier of the sequence listing, human codon usage adapted coding sequences are indicated by the abbreviation "opt3" or "human". Codon-optimized sequences As described above it is preferred according to the invention, that all codons of the wild type sequence which code for a tRNA, which is relatively rare in the cell, are exchanged for a codon which codes for a tRNA, which is relatively frequent in the cell and which, in each case, carries the same amino acid as the relatively rare tRNA. Therefore it is particularly preferred that the most frequent codons are used for each encoded amino acid (see Table D, most frequent codons are marked with asterisks). Such an optimization procedure increases the codon adaptation index (CAI) and ultimately maximises the CAI. In the context of the invention, sequences with increased or maximized CAI are typically referred to as "codon-optimized" sequences and/or CAI increased and/or maximized sequences. According to a preferred embodiment, the mRNA of the present invention comprises at least one coding sequence, wherein the coding sequence is codon-optimized as described herein. More preferably, the codon adaptation index (CAI) of the at least one coding sequence is at least 0.5, at least 0.8, at least 0.9 or at least 0.95. Most preferably, the codon adaptation index (CAI) of the at least one coding sequence is 1. For example, in the case of the amino acid alanine (Ala) present in the amino acid sequence encoded by the at least one coding sequence of the mRNA according to the invention, the wild type coding sequence is adapted in a way that the most frequent human codon "GCC" is always used for said amino acid, or for the amino acid Cysteine (Cys), the wild type sequence is adapted in a way that the most frequent human codon "TGC" is always used for said amino acid etc. C-optimized sequences According to another embodiment, the mRNA of the present invention may be modified by modifying, preferably increasing, the cytosine (C) content of the mRNA, preferably of the coding region of the mRNA. In a particularly preferred embodiment of the present invention, the C content of the coding region of the mRNA of the present invention is modified, preferably increased, compared to the C content of the coding region of the respective wild type RNA, i.e. the unmodified RNA. The amino acid sequence encoded by the at least one coding sequence of the mRNA of the present invention is preferably not modified as compared to the amino acid sequence encoded by the respective wild type mRNA. In a preferred embodiment of the present invention, the modified RNA is modified such that at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, or at least 90% of the theoretically possible maximum cytosine-content or even a maximum cytosine-content is achieved. In further preferred embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% of the codons of the target RNA wild type sequence, which are "cytosine content optimizable" are replaced by codons having a higher cytosine-content than the ones present in the wild type sequence. In a further preferred embodiment, some of the codons of the wild type coding sequence may additionally be modified such that a codon for a relatively rare tRNA in the cell is exchanged by a codon for a relatively frequent tRNA in the cell, provided that the substituted codon for a relatively frequent tRNA carries the same amino acid as the relatively rare tRNA of the original wild type codon. Preferably, all of the codons for a relatively rare tRNA are replaced by a codon for a relatively frequent tRNA in the cell, except codons encoding amino acids, which are exclusively encoded by codons not containing any cytosine, or except for glutamine (Gln), which is encoded by two codons each containing the same number ofcytosines. In a further preferred embodiment of the present invention, the modified target RNA is modified such that at least 80%, or at least 90% of the theoretically possible maximum cytosine-content or even a maximum cytosine-content is achieved by means of codons, which code for relatively frequent tRNAs in the cell, wherein the amino acid sequence remains unchanged. Due to the naturally occurring degeneracy of the genetic code, more than one codon may encode a particular amino acid. Accordingly, 18 out of 20 naturally occurring amino acids are encoded by more than one codon (with Tryp and Met being an exception), e.g. by 2 codons (e.g. Cys, Asp, Glu), by three codons (e.g. lie), by 4 codons (e.g. Al, Gly, Pro) or by 6 codons (e.g. Leu, Arg, Ser). However, not all codons encoding the same amino acid are utilized with the same frequency under in vivo conditions. Depending on each single organism, a typical codon usage profile is established. The term "cytosine content-optimizable codon" as used within the context of the present invention refers to codons, which exhibit a lower content of cytosines than other codons encoding the same amino acid. Accordingly, any wild type codon, which may be replaced by another codon encoding the same amino acid and exhibiting a higher number of cytosines within that codon, is considered to be cytosine-optimizable (C-optimizable). Any such substitution ofaC-optimizable wild type codon by the specific C-optimized codon within a wild type coding region increases its overall C-content and reflects a C-enriched modified mRNA sequence. According to a preferred embodiment, the mRNA of the present invention, preferably the at least one coding sequence of the mRNA of the present invention, comprises or consists of a C-maximized RNA sequence containing C-optimized codons for all potentially C-optimizable codons. Accordingly, 100% or all of the theoretically replaceable C- optimizable codons are preferably replaced by C-optimized codons over the entire length of the coding region. In this context, cytosine-content optimizable codons are codons, which contain a lower number of cytosines than other codons coding for the same amino acid. Any of the codons GCG, GCA, GCU codes for the amino acid Ala, which may be exchanged by the codon GCC encoding the same amino acid, and/or the codon UGU that codes for Cys may be exchanged by the codon UGC encoding the same amino acid, and/or the codon GAD which codes for Asp may be exchanged by the codon GAC encoding the same amino acid, and/or the codon that UUU that codes for Phe may be exchanged for the codon UUC encoding the same amino acid, and/or any of the codons GGG, GGA, GGU that code Gly may be exchanged by the codon GGC encoding the same amino acid, and/or the codon CAU that codes for His may be exchanged by the codon CAC encoding the same amino acid, and/or any of the codons AUA, AUU that code for lie may be exchanged by the codon AUG, and/or any of the codons DUG, UUA, CUG, CUA, CUU coding for Leu may be exchanged by the codon CUC encoding the same amino acid, and/or the codon ML) that codes for Asn may be exchanged by the codon AAC encoding the same amino acid, and/or any of the codons CCG, CCA, CCU coding for Pro may be exchanged by the codon CCC encoding the same amino acid, and/or anyofthecodonsAGG, AGA, CGG, CGA, CGU coding for Arg may be exchanged by the codon CGC encoding the same amino acid, and/or anyofthecodonsAGU,AGC, UCG, UCA, UCU coding for Ser may be exchanged by the codon UCC encoding the same amino acid, and/or any of the codons ACG, ACA, ACU coding for Thr may be exchanged by the codon ACC encoding the same amino acid, and/or any of the codons GUG, QUA, GUU coding for Val may be exchanged by the codon GUC encoding the same amino acid, and/or the codon DAD coding for Tyr may be exchanged by the codon UAC encoding the same amino acid. In any of the above instances, the number of cytosines is increased by 1 per exchanged codon. Exchange of all non C-optimized codons (corresponding to C-optimizable codons) of the coding region results in a C-maximized coding sequence. In the context of the invention, at least 70%, preferably at least 80%, more preferably at least 90%, of the non C-optimized codons within the at least one coding region of the mRNA according to the invention are replaced by C-optimized codons.

It may be preferred that for some amino acids the percentage of C-optimizable codons replaced by C-optimized codons is less than 70%, while for other amino acids the percentage of replaced codons is higher than 70% to meet the overall percentage of C-optimization of at least 70% of all C- optimizable wild type codons of the coding region.

Preferably, in a C-optimized RNA of the invention, at least 50% of the C-optimizable wild type codons for any given amino acid are replaced by C-optimized codons, e.g. any modified C-enriched RNA preferably contains at least 50% C-optimized codons at C-optimizable wild type codon positions encoding any one of the above mentioned amino acids Ala, Cys, Asp, Phe, Gly, His, IIe, Leu, Asn, Pro, Arg, Ser, Thr, Val and Tyr, preferably at least 60%.

In this context codons encoding amino acids, which are not cytosine content-optimizable and which are, however, encoded by at least two codons, may be used without any further selection process. However, the codon of the wild type sequence that codes for a relatively rare tRNA in the cell, e.g. a human cell, may be exchanged for a codon that codes for a relatively frequent tRNA in the cell, wherein both code for the same amino acid. Accordingly, the relatively rare codon GAA coding for Glu may be exchanged by the relative frequent codon GAG coding for the same amino acid, and/or the relatively rare codon AAA coding for Lys may be exchanged by the relative frequent codon AAG coding for the same amino acid, and/or the relatively rare codon CAA coding for Gin may be exchanged for the relative frequent codon CAG encoding the same amino acid.

In this context, the amino acids Met (AUG) and Trp (UGG), which are encoded by only one codon each, remain unchanged. Stop codons are not cytosine-content optimized, however, the relatively rare stop codons amber, ochre (UAA, UAG) may be exchanged by the relatively frequent stop codon opal (UGA).

The single substitutions listed above may be used individually as well as in all possible combinations in order to optimize the cytosine-content of the modified RNA compared to the wild type mRNA sequence.

Accordingly, the at least one coding sequence as defined herein may be changed compared to the coding region of the respective wild type RNA in such a way that an amino acid encoded by at least two or more codons, of which one comprises one additional cytosine, such a codon may be exchanged by the C-optimized codon comprising one additional cytosine, wherein the amino acid is preferably unaltered compared to the wild type sequence.

According to a particularly preferred embodiment, the invention provides an mRNA, comprising at least one coding sequence as defined herein, wherein the G/C content of the at least one coding sequence of the mRNA is increased compared to the G/C content of the corresponding coding sequence of the corresponding wild type RNA, and/or wherein the C content of the at least one coding sequence of the mRNA is increased compared to the C content of the corresponding coding sequence of the corresponding wild type RNA, and/or wherein the codons in the at least one coding sequence of the mRNA are adapted to human codon usage, wherein the codon adaptation index (CAI) is preferably increased or maximised in the at least one coding sequence of the mRNA, and wherein the amino acid sequence encoded by the mRNA is preferably not being modified compared to the amino acid sequence encoded by the corresponding wild type RNA.

5’-cap

According to another preferred embodiment of the invention, a modified RNA as defined herein, can be modified by the addition of a so-called “5’-cap" structure, which preferably stabilizes the mRNA as described herein. A 5’-cap is an entity, typically a modified nucleotide entity, which generally “caps" the 5’-end of a mature mRNA. A 5’-cap may typically be formed by a modified nucleotide, particularly by a derivative of a guanine nucleotide. Preferably, the 5’-cap is linked to the 5’-terminus via a 5’-5’-triphosphate linkage. A 5’-cap may be methylated, e.g. m7GpppN, wherein N is the terminal 5’ nucleotide of the nucleic acid carrying the 5’-cap, typically the 5'-end of an mRNA. m7GpppN is the 5’-cap structure, which naturally occurs in mRNA transcribed by polymerase II and is therefore preferably not considered as modification comprised in a modified mRNA in this context. Accordingly, a modified RNA of the present invention may comprise a m7GpppN as 5’-cap, but additionally the modified RNA typically comprises at least one further modification as defined herein.

Further examples of 5’-cap structures include glyceryl, inverted deoxy abasic residue (moiety), 4', 5’ methylene nucleotide, l-(beta-D-erythrofuranosyl) nucleotide, 4’-thio nucleotide, carbocyclic nucleotide, 1 ,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3’,4’-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3’-3'-inverted nucleotide moiety, 3’-3’-inverted abasic moiety, 3’-2’-inverted nucleotide moiety, 3’-2’-inverted abasic moiety, 1 ,4-butanediol phosphate, 3’-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3’-phosphate, 3’phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moiety. These modified 5'-cap structures are regarded as at least one modification in this context.

Particularly preferred modified 5’-cap structures are cap1 (methylation of the ribose of the adjacent nucleotide of m7G), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7G), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7G), cap4 (methylation of the ribose of the 4th nucleotide downstream of the m7G), ARCA (anti-reverse cap analogue, modified ARCA (e.g. phosphothioate modified ARCA), inosine, N1-methyl-guanosine, 2’-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. Accordingly, the mRNA according to the invention preferably comprises a 5’-cap structure.

In a preferred embodiment, the mRNA according to the invention comprises at least one 5’- or 3’- UTR element. In this context, an UTR element comprises or consists of a nucleic acid sequence, which is derived from the 5’- or 3’-UTR of any naturally occurring gene or which is derived from a fragment, a homolog or a variant of the 5’- or 3’-UTR of a gene. Preferably, the 5’- or 3'-UTR element used according to the present invention is heterologous to the at least one coding sequence of the mRNA of the invention. Even if 5’- or 3’-UTR elements derived from naturally occurring genes are preferred, also synthetically engineered UTR elements may be used in the context of the present invention.

The term “3’-UTR element” typically refers to a nucleic acid sequence, which comprises or consists of a nucleic acid sequence that is derived from a 3'-UTR or from a variant of a 3’-UTR. A 3’-UTR element in the sense of the present invention may represent the 3’-UTR of an RNA, preferably an mRNA. Thus, in the sense of the present invention, preferably, a 3’-UTR element may be the 3’-UTR of an RNA, preferably of an mRNA, or it may be the transcription template for a 3 -UTR of an RNA. Thus, a 3’-UTR element preferably is a nucleic acid sequence which corresponds to the 3’-UTR of an RNA, preferably to the 3’-UTR of an mRNA, such as an mRNA obtained by transcription of a genetically engineered vector construct. Preferably, the 3’-UTR element fulfils the function of a 3’- UTR or encodes a sequence which fulfils the function of a 3’-UTR.

According to a preferred embodiment, CleanCap® Reagent AG from TriLink is used as co- transcriptional capping reagent for in vitro transcription of 5’-capped mRNA resulting in a cap1 structure. CleanCap AG requires an AG initiator and use yields in a naturally occurring cap1 structure.

According to a preferred embodiment, the mRNA according to the invention comprises a 5’-cap structure and/or at least one 3’-untranslated region element (3’-UTR element), preferably as defined herein. More preferably, the mRNA further comprises a 5'-UTR element as defined herein.

UTRs

In a preferred embodiment, the pharmaceutical composition comprises an mRNA compound comprising at least one 5'- or 3’-UTR element. In this context, an UTR element comprises or consists of a nucleic acid sequence, which is derived from the 5’- or 3’-UTR of any naturally occurring gene or which is derived from a fragment, a homolog or a variant of the 5’- or 3’-UTR of a gene. Preferably, the 5’- or 3'-UTR element used according to the present invention is heterologous to the at least one coding region of the mRNA sequence of the invention. Even if 5’- or 3 -UTR elements derived from naturally occurring genes are preferred, also synthetically engineered UTR elements may be used in the context of the present invention.

The term “3’-UTR element” typically refers to a nucleic acid sequence, which comprises or consists of a nucleic acid sequence that is derived from a 3 -UTR or from a variant of a 3’-UTR. A 3’-UTR element in the sense of the present invention may represent the 3'-UTR of an RNA, preferably an mRNA. Thus, in the sense of the present invention, preferably, a 3’-UTR element may be the 3’-UTR of an RNA, preferably of an mRNA, or it may be the transcription template for a 3’-UTR of an RNA. Thus, a 3’-UTR element preferably is a nucleic acid sequence which corresponds to the 3’-UTR of an RNA, preferably to the 3’-UTR of an mRNA, such as an mRNA obtained by transcription of a genetically engineered vector construct. Preferably, the 3’-UTR element fulfils the function of a 3’- UTR or encodes a sequence which fulfils the function of a 3'-UTR.

Preferably, the at least one 3’-UTR element comprises or consists of a nucleic acid sequence derived from the 3’-UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene, or from a variant of the 3’-UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene.

Preferably, the pharmaceutical composition comprises an mRNA compound that comprises a 3’- UTR element, which may be derivable from a gene that relates to an mRNA with an enhanced half- life (that provides a stable mRNA), for example a 3’-UTR element as defined and described below. Preferably, the 3’-UTR element comprises or consists of a nucleic acid sequence derived from a 3’- UTR of a gene, which preferably encodes a stable mRNA, or from a homolog, a fragment or a variant of said gene.

In one preferred embodiment, the UTR-combinations which are disclosed in Table 1 , claims 1 and claim 4, claims 6-8 and claim 9 of WO2019077001 are preferred UTR-combinations for mRNA compounds of the present invention. Further, preferably, the UTR-combinations as disclosed on page 24, second full paragraph after Table 1 and page 24, last paragraph to page 29, second paragraph of WO2019077001 are preferred UTR-combinations for mRNA compounds of the present invention. WO2019077001 is incorporated herein by reference in its entirety.

In a further preferred embodiment, that 3’-UTR element comprises or consists of a nucleic acid sequence which is derived from a 3’-UTR of a gene selected from the group consisting of a 3’-UTR of a gene selected from PSMB3 (see Table 1 - 5’-UTRs and 3’-UTRs herein below), ALB/albumin (see Table 1 - 5’-UTRs and 3’-UTRs herein below), alpha-globin (referred to as “muag” i.e. a mutated alpha-globin 3’-UTR; see Table 1 - 5’-UTRs and 3’-UTRs herein below), CASP1 (see Table 1 - 5’- UTRs and 3’-UTRs herein below), COX6B1 (see Table 1 - 5’-UTRs and 3’-UTRs herein below), GNAS (see Table 1 - 5’-UTRs and 3’-UTRs herein below), NDUFA1 (see Table 1 - 5’-UTRs and 3'- UTRs herein below) and RPS9 (see Table 1 - 5’-UTRs and 3’-UTRs herein below), or from a homolog, a fragment or a variant of any one of these genes (for example, human albumin/alb 3’-UTR as disclosed in SEQ ID NO:1369 of WO2013143700, which is incorporated herein by reference), or from a homolog, a fragment or a variant thereof. In a further preferred embodiment, the 3’-UTR element comprises the nucleic acid sequence derived from a fragment of the human albumin gene according to SEQ ID NO:1376 of WO2013143700 (albumin/alb S’-UTR). In a further preferred embodiment, the 3’-UTR element comprises or consists of a nucleic acid sequence which is derived from a 3’-UTR of an albumin gene, preferably a vertebrate albumin gene, more preferably a mammalian albumin gene, most preferably a human albumin gene such as from the 3’-UTR of the human albumin gene according to GenBank Accession number NMJD00477.5, or a fragment or variant thereof. In another preferred embodiment, the 3’-UTR element comprises or consists of the center, a-complex-binding portion of the 3'-UTR of an a-globin gene, such as of a human a-globin gene, or an a-complex-binding portion of the 3’-UTR of an a-globin gene (also named herein as “muag”), corresponding to SEQ ID NO:1393 of patent application WO2013143700.

Table 1 - 5’-UTRs and 3’-UTRs

In this context it is very preferred that the 3’-UTR element of the mRNA sequence according to the invention comprises or consists of a corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO:33 or 34, or a homolog, a fragment or variant thereof.

UTR-combination SLC7A3 (5’-UTR of mouse solute carrier family 7 (cationic amino acid transporter, y+ system), member 3)/PSMB3: in another preferred embodiment, the mRNA compound comprises a 5’-UTR element, which comprises or consists of a nucleic acid sequence which is derived from a cationic amino acid transporter 3 (solute carrier family 7 member 3, SLC7A3) gene, wherein said 5’- UTR element comprises or consists of a DNA sequence according to SEQ ID NO: 15 as disclosed in WO2019077001 or respectively a RNA sequence according to SEQ ID NO: 16 as disclosed in WO2019077001. In another preferred embodiment, the mRNA compound comprises a 3’-UTR element, which comprises or consists of a nucleic acid sequence which is derived from a proteasome subunit beta type-3 (PSMB3) gene, wherein said 3’-UTR element comprises or consists of a DNA sequence according to SEQ ID NO:23 as disclosed in WO2019077001 or respectively a RNA sequence according to SEQ ID NO:24 as disclosed in WO2019077001. In further preferred embodiments, the mRNA compound comprises an UTR-combination as disclosed in WO2019077001 , i.e. both a 5’-UTR element, which comprises or consists of a nucleic acid sequence which is derived from a SLC7A3 gene and a 3’-UTR element, which comprises or consists of a nucleic acid sequence which is derived from a PSMB3 gene. For direct reference to the sequence listing of the present invention, see Table 1 - 5’-UTRs and 3’-UTRs.

UTR-combination RPL31 (5’-UTR of mouse ribosomal protein L31)/RPS9 (3 -UTR of human ribosomal protein S9 (RPS9): In another preferred embodiment, the mRNA compound comprises a 5’-UTR element, which comprises or consists of a nucleic acid sequence which is derived from a 60S ribosomal protein L31 (RPL31 ) gene, wherein said 5’-UTR element comprises or consists of a DNA sequence according to SEQ ID NO:13 as disclosed in WO2019077001 or respectively a RNA sequence according to SEQ ID NO:14 as disclosed in WO2019077001. In another preferred embodiment, the mRNA compound comprises a 3’-UTR element, which comprises or consists of a nucleic acid sequence which is derived from a 40S ribosomal protein S9 (RPS9) gene, wherein said 3’-UTR element comprises or consists of a DNA sequence according to SEQ ID NO:33 as disclosed in WO2019077001 or respectively a RNA sequence according to SEQ ID NO:34 as disclosed in WO2019077001. In further preferred embodiments, the mRNA compound comprises an UTR- combination as disclosed in WO2019077001 , i.e. both a 5’-UTR element, which comprises or consists of a nucleic acid sequence which is derived from a RPL31 gene and a 3’-UTR element, which comprises or consists of a nucleic acid sequence which is derived from a RPS9 gene (preferably SEQ ID NO:51/52). For direct reference to the sequence listing of the present invention, see Table 1 - S’-UTRs and 3’-UTRs.

In another preferred embodiment, the UTR-combiantion 5’-UTR Ubqln2 (ubiquitin 2, see Table 1 - 5’- UTRs and 3’-UTRs) and 3’-UTR Gnas (Guanine nucleotide-binding protein G(s) subunit alpha isoforms short, see Table 1 - 5’-UTRs and 3’-UTRs) is used.

In a very preferred embodiment, the 5’-UTR element of the mRNA sequence according to the invention comprises or consists of a corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO:1 or SEQ ID NO:2, i.e. HSD17B4. Also, in a very preferred embodiment, the 3’-UTR element of the mRNA sequence according to the invention comprises or consists of a corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO.33 or SEQ ID NO:34, i.e. PSMB3. In also a very preferred embodiment, the 5’-UTR element of the mRNA sequence and the 3’-UTR-element according to the invention comprises or consists of a combination of aforementioned HSD17B4 and PSMB3-UTRs.

The term “a nucleic acid sequence which is derived from a variant of the 3’-UTR of a [...] gene" preferably refers to a nucleic acid sequence, which is based on a variant of the 3’-UTR sequence of a gene, such as on a variant of the 3’-UTR of an albumin gene, an a-globin gene, a p-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, or a collagen alpha gene, such as a collagen alpha 1 (1) gene, or on a part thereof as described above. This term includes sequences corresponding to the entire sequence of the variant of the 3’-UTR of a gene, i.e. the full length variant 3’-UTR sequence of a gene, and sequences corresponding to a fragment of the variant 3’-UTR sequence of a gene. A fragment in this context preferably consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length variant 3’-UTR, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length variant 3’-UTR. Such a fragment of a variant, in the sense of the present invention, is preferably a functional fragment of a variant as described herein.

According to a preferred embodiment, the mRNA compound comprising an mRNA sequence according to the invention comprises a 5’-cap structure and/or at least one 3’-untranslated region element (3’-UTR element), preferably as defined herein. More preferably, the RNA further comprises a 5’-UTR element as defined herein. In a further preferred embodiment, the pharmaceutical composition comprises an mRNA compound comprising at least one 5’-untranslated region element (5’-UTR element). Preferably, the at least one 5’-UTR element comprises or consists of a nucleic acid sequence, which is derived from the 5'-UTR of a TOP gene or which is derived from a fragment, homolog or variant of the 5’-UTR of a TOP gene. It is preferred that the 5’-UTR element does not comprise a TOP motif or a 5’-TOP, as defined above.

In some embodiments, the nucleic acid sequence of the 5’-UTR element, which is derived from a 5’- UTR of a TOP gene, terminates at its 3’-end with a nucleotide located at position 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 upstream of the start codon (e.g. A(U/T)G) of the gene or mRNA it is derived from. Thus, the 5’-UTR element does not comprise any part of the protein coding region. Thus, preferably, the only protein coding part of the at least one mRNA sequence is provided by the coding region.

The nucleic acid sequence derived from the 5’-UTR of a TOP gene is preferably derived from a eukaryotic TOP gene, preferably a plant or animal TOP gene, more preferably a chordate TOP gene, even more preferably a vertebrate TOP gene, most preferably a mammalian TOP gene, such as a human TOP gene.

For example, the 5’-UTR element may be selected from 5’-UTR elements comprising or consisting of a nucleic acid sequence, which is derived from a nucleic acid sequence selected from the group consisting of SEQ ID NO.1-1363, SEQ ID NO:1395, SEQ ID NO:1421 and SEQ ID NO:1422 of patent application WO2013143700, whose disclosure is incorporated herein by reference, from the homologs of SEQ ID NO:1-1363, SEQ ID NO:1395, SEQ ID NO:1421 and SEQ ID NO:1422 of patent application WO2013143700, from a variant thereof, or preferably from a corresponding RNA sequence. The term “homologs of SEQ ID NO:1-1363, SEQ ID NO:1395, SEQ ID NO:1421 and SEQ ID NO:1422 of patent application WO2013143700” refers to sequences of other species than homo sapiens, which are homologous to the sequences according to SEQ ID NO:1-1363, SEQ ID NO:1395, SEQ ID NO:1421 and SEQ ID NO:1422 of patent application WO2013143700. For direct reference to the sequence listing of the present invention, see Table 1 - 5’-UTRs and 3’-UTRs.

In a preferred embodiment, the 5’-UTR element of the mRNA compound comprises or consists of a nucleic acid sequence, which is derived from a nucleic acid sequence extending from nucleotide position 5 (i.e. the nucleotide that is located at position 5 in the sequence) to the nucleotide position immediately 5’ to the start codon (located at the 3’-end of the sequences), e.g. the nucleotide position immediately 5’ to the ATG sequence, of a nucleic acid sequence selected from SEQ ID NO:1-1363, SEQ ID NO:1395, SEQ ID NO:1421 and SEQ ID NO:1422 of patent application WO2013143700, from the homologs of SEQ ID NO:1-1363, SEQ ID NO:1395, SEQ ID NO:1421 and SEQ ID NO:1422 of patent application WO2013143700 from a variant thereof, or a corresponding RNA sequence. It is particularly preferred that the 5’-UTR element is derived from a nucleic acid sequence extending from the nucleotide position immediately 3’ to the 5’-TOP to the nucleotide position immediately 5’ to the start codon (located at the 3’-end of the sequences), e.g. the nucleotide position immediately 5’ to the ATG sequence, of a nucleic acid sequence selected from SEQ ID NO:1-1363, SEQ ID NO:1395, SEQ ID NO:1421 and SEQ ID NO:1422 of patent application WO2013143700, from the homologs of SEQ ID NO:1-1363, SEQ ID NO:1395, SEQ ID NO:1421 and SEQ ID NO:1422 of patent application WO2013143700, from a variant thereof, or a corresponding RNA sequence. For direct reference to the sequence listing of the present invention, see Table 1 - 5’-UTRs and 3'-UTRs.

In a further preferred embodiment, the 5'-UTR element comprises or consists of a nucleic acid sequence, which is derived from a 5’-UTR of a TOP gene encoding a ribosomal protein or from a variant of a 5’-UTR of a TOP gene encoding a ribosomal protein. For example, the 5’-UTR element comprises or consists of a nucleic acid sequence, which is derived from a 5’-UTR of a nucleic acid sequence according to any of SEQ ID NO:67, 170, 193, 244, 259, 554, 650, 675, 700, 721 , 913, 1016, 1063, 1120, 1138, and 1284-1360 of patent application W02013143700, a corresponding RNA sequence, a homolog thereof, or a variant thereof as described herein, preferably lacking the 5’-TOP motif. As described above, the sequence extending from position 5 to the nucleotide immediately 5’ to the ATG (which is located at the 3’-end of the sequences) corresponds to the 5'-UTR of said sequences. For direct reference to the sequence listing of the present invention, see Table 1 - 5’- UTRs and 3’-UTRs.

In further preferred embodiments, the preferred 5'-UTR or 3’-UTR element comprises or consists of a nucleic acid sequence, which is disclosed in Table 1 - 5'-UTRs and 3’-UTRs.

Accordingly, in a preferred embodiment, the 5’-UTR element comprises or consists of a nucleic acid sequence, which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID NO: 1368, or SEQ ID NO: 1412-1420 of patent application WO2013143700, or a corresponding RNA sequence, or wherein the at least one 5’-UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID NO:1368, or SEQ ID NO:1412-1420 of patent application WO2013143700, wherein, preferably, the fragment is as described above, i.e. being a continuous stretch of nucleotides representing at least 20% etc. of the full-length 5’-UTR. Preferably, the fragment exhibits a length of at least about 20 nucleotides or more, preferably of at least about 30 nucleotides or more, more preferably of at least about 40 nucleotides or more. Preferably, the fragment is a functional fragment as described herein.

Preferably, the at least one 5’-UTR element and the at least one 3’-UTR element act synergistically to increase protein production from the at least one mRNA sequence as described above. According to a preferred embodiment, the pharmaceutical composition of the invention comprises an mRNA compound that comprises, preferably in 5’- to 3’-direction: a) a 5’-cap structure, preferably m7GpppN, more preferably cap1 or m7G(5’)ppp(5’)(2’OMeA)pG; b) optionally, a 5’-UTR element which preferably comprises or consists of a nucleic acid sequence which is optionally derived from the 5’-UTR of a TOP gene, more preferably comprising or consisting of the corresponding RNA sequence of a nucleic acid sequence according to any of the 5’-UTRs as disclosed in Table 1 - 5’-UTRs and 3’-UTRs, a homolog, a fragment or a variant thereof, most preferably according to SEQ ID NO:1 or 2 (HSD17B4), or a 5’-UTR element which preferably comprises or consists of a nucleic acid sequence which is derived from a solute carrier family 7 cationic amino acid transporter, y+ system), member 3 (SLC7A3) gene (SEQ ID NO: 15 as disclosed in WO2019077001 or respectively a RNA sequence according to SEQ ID NO:16 as disclosed in WO2019077001) or a 60S ribosomal protein 131 (RPL31) gene (SEQ ID NO: 13 as disclosed in WO2019077001 or respectively a RNA sequence according to SEQ ID NO: 14 as disclosed in WO2019077001); c) (i) at least one coding region encoding at least one FAH protein according to SEQ ID NO:100 as described herein above or below or as disclosed in the sequence listing of the present invention; d) optionally, a 3’-UTR element which preferably comprises or consists of a nucleic acid sequence which is derived from a gene providing a stable mRNA, preferably comprising or consisting of the corresponding RNA sequence of a nucleic acid sequence according to any of the 3’-UTRs as disclosed in Table 1 - 5’-UTRs and 3'-UTRs, most preferably according to SEQ ID NO:33 or 34 (PSMB3) or a 3’-UTR element which preferably comprises or consists of a nucleic acid sequence which is derived from a 40S ribosomal protein S9 (RPS9) gene (SEQ ID NO:33 as disclosed in WO2019077001 or respectively a RNA sequence according to SEQ ID NO:34 as disclosed in WO2019077001 ); e) optionally, a histone stem-loop selected from SEQ ID NOs:63 or 64; and/or f) optionally, a poly(A) sequence preferably comprising 64 adenosines or 100 adenosines; and g) optionally, a poly(C) sequence, preferably comprising 30 cytosines; h) optionally, a histone stem-loop selected from SEQ ID NOs:63 or 64; and/or i) optionally, a 3'-terminal sequence element selected from SEQ ID NOs:65-99.

According to one embodiment, the mRNA compound comprises an miRNA binding site located in the 5’ or 3’ UTR. A miRNA (microRNA) is typically a small, non-coding single stranded RNA molecules of about 20 to 25 nucleotides in length which may function in gene regulation, for example, but not limited to, by mRNA degradation or translation inhibition or repression. miRNAs are typically produced from hairpin precursor RNAs (pre-miRNAs), and they may form functional complexes with proteins. Furthermore, miRNAs may bind to 5’ and/or 3'-UTR regions of target mRNAs. Preferably, the microRNA binding site is for a microRNA selected from the group consisting of miR-126, miR- 142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21 , miR-223, miR-24, miR-27, miR-26a binding site, preferably a miR-122 or miR-142 binding site, or any combination of the aforementioned miRNA binding sites.

In one embodiment, the miRNA binding site is a naturally occurring miRNA binding site. In another embodiment, the miRNA binding site may be a mimetic, or a modification of a naturally-occurring miRNA binding site.

According to one preferred embodiment, the mRNA compound comprising an mRNA sequence according to the invention may further comprise, as defined herein: a) a 5’-cap structure, preferably m7GpppN, more preferably cap1 or m7G(5’)ppp(5’)(2’OMeA)pG; b) at least one miRNA binding site, preferably wherein the microRNA binding site is selected from the group consisting of a miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21 , miR-223, miR-24, miR-27, miR-26a binding site, preferably a miR-122 or miR-142 binding site, or any combination of the aforementioned miRNA binding sites; c) at least one 5’-UTR element; d) at least one 3’-UTR element; e) at least one poly(A) sequence; f) optionally at least one poly(C) sequence; g) optionally, a histone stem-loop selected from SEQ ID NOs:63 or 64; h) optionally, a 3'-terminal sequence element selected from SEQ ID NOs:65-99; or any combinations of these.

In some embodiments, the artificial nucleic acid molecule according to the invention may comprise UTR elements according to a-2 (NDUFA4/PSMB3); a-5 (MP68/PSMB3); c-1 (NDUFA4/RPS9); a-1 (HSD17B4/PSMB3); e-3 (MP68/RPS9); e-4 ( NOSIP/RPS9); a-4 (NOSIP/PSMB3); e-2 (RPL31/RPS9); e-5 (ATP5A1/RPS9); d-4 (HSD17B4/NUDFA1); b-5 (NOSIP/COX6B1); a-3 (SLC7A3/PSMB3); b-1 (UBQLN2/RPS9); b-2 (ASAH1/RPS9); b-4 (HSD17B4/CASP1); e-6 (ATP5A1/COX6B1); b-3 (HSD17B4/RPS9); g-5 (RPL31/CASP1); h-1 (RPL31/COX6B1); and/or c-5 (ATP5A1/PSMB3) in accordance with the disclosure of W02019077001 , the aforementioned UTR combinations and the subject-matter of claim 4 and subject-matter related thereto in WO2019077001 is incorporated herein by reference. poly(A) tail

According to a further preferred embodiment, the mRNA of the present invention may contain a poly(A) tail on the 3'-terminus of typically about 10 to 200 adenosine nucleotides, preferably about 10 to 100 adenosine nucleotides, more preferably about 40 to 80 adenosine nucleotides or even more preferably about 50 to 70 adenosine nucleotides, more preferably about 100 adenosine nucleotides. In one embodiment, the poly(A) tail does not consist of exclusively adenosine nucleotides but is interrupted by a stretch of 3, 4, 5, 6, 7, or 8 non-adenosine nucleotides. Preferably, the mRNA of the invention comprises a 3’-terminal A nucleotide. Preferably, the poly(A) sequence in the mRNA of the present invention is derived from a DNA template by RNA in vitro transcription. Alternatively, the poly(A) sequence may also be obtained in vitro by common methods of chemical-synthesis without being necessarily transcribed from a DNA- progenitor. Moreover, poly(A) sequences, or poly(A) tails may be generated by enzymatic polyadenylation of the mRNA according to the present invention using commercially available polyadenylation kits and corresponding protocols known in the art.

Alternatively, the mRNA as described herein optionally comprises a polyadenylation signal for enzymatic polyadenylation, which is defined herein as a signal, which conveys polyadenylation to a (transcribed) RNA by specific protein factors (e.g. cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factors I and II (CF I and CF II), poly(A) polymerase (PAP)). In this context, a consensus polyadenylation signal for enzymatic polyadenylation is preferred comprising the NN(U/T)ANA consensus sequence. In a particularly preferred aspect, the polyadenylation signal comprises one of the following sequences: AA(U/T)AAA or A(U/T)(U/T)AAA (wherein uridine is usually present in RNA and thymidine is usually present in DNA). It is clear for a skilled artisan, that said consensus sequence is not mandatory for enzymatic or non-enzymatic polyadenylation. polv(C) tail

According to a further preferred embodiment, the mRNA of the present invention may contain a poly(C) tail on the 3’-terminus of typically about 10 to 200 cytidine nucleotides, preferably about 10 to 100 cytidine nucleotides, more preferably about 20 to 70 cytidine nucleotides or even more preferably about 20 to 60 or even 10 to 40 cytidine nucleotides.

Histone stem-loop

In a further preferred embodiment, the pharmaceutical composition comprises an mRNA compound comprising a histone stem-loop sequence/structure (HSL, hSL, histoneSL, preferably according to SEQ ID NO:3 or SEQ ID NO:4). In said embodiment, the mRNA sequence may comprise at least one (or more) histone stem loop sequence or structure. Such histone stem-loop sequences are preferably selected from histone stem-loop sequences as disclosed in W02012019780, the disclosure of which is incorporated herewith by reference. A histone stem-loop sequence that may be used within the present invention may preferably be derived from formulae (I) or (II) of WO2012019780. According to a further preferred embodiment the coding RNA may comprise at least one histone stem-loop sequence derived from at least one of the specific formulae (la) or (Ila) of patent application WO2012019780. According to a further preferred embodiment the coding RNA may comprise at least one histone stem-loop sequence derived from a Histone stem-loop as disclosed in patent application WO2018104538 under formula (I), formula (II), formula (la) or on pages 49-52 under section “Histone stem-loop” and WO2018104538-SEQ ID NOs:1451 or WO2018104538-SEQ ID NO:1452; WO2018104538A1 which is herein incorporated by reference in its entirety, also especially SEQ ID NOs:1451-1452 (herein SEQ ID NO:63 or 64).

In particularly preferred embodiment, the RNA of the invention comprises at least one histone stem- loop sequence, wherein said histone stem-loop sequence comprises a nucleic acid sequence being identical or at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs:63 or 64, or fragments or variants thereof.

Signal peptide

According to another particularly preferred embodiment, the mRNA according to the invention may additionally or alternatively encode a secretory signal peptide. Such signal peptides are sequences, which typically exhibit a length of about 15 to 30 amino acids and are preferably located at the N- terminus of the encoded peptide, without being limited thereto. Signal peptides as defined herein preferably allow the transport of the peptide or protein as encoded by the at least one mRNA of the pharmaceutical composition into a defined cellular compartiment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartiment. Examples of secretory signal peptide sequences as defined herein include, without being limited thereto, signal sequences of classical or non-classical MHC-molecules (e.g. signal sequences of MHC I and II molecules, e.g. of the MHC class I molecule HLA-A*0201 ), signal sequences of cytokines or immunoglobulines as defined herein, signal sequences of the invariant chain of immunoglobulines or antibodies as defined herein, signal sequences of Lampl , Tapasin, Erp57, Calretikulin, Calnexin, and further membrane associated proteins or of proteins associated with the endoplasmic reticulum (ER) or the endosomal- lysosomal compartiment. Most preferably, signal sequences of MHC class I molecule HLA-A*0201 may be used according to the present invention. For example, a signal peptide derived from HLA-A is preferably used in order to promote secretion of the encoded peptide or protein as defined herein or a fragment or variant thereof. More preferably, an HLA-A signal peptide is fused to an encoded peptide or protein as defined herein or to a fragment or variant thereof:

Any of the above modifications may be applied to the mRNA of the present invention, and further to any RNA as used in the context of the present invention and may be, if suitable or necessary, be combined with each other in any combination, provided, these combinations of modifications do not interfere with each other in the respective at least one mRNA. A person skilled in the art will be able to take his choice accordingly. miRNA binding sites

In further preferred embodiments, the mRNA comprises a 5’- or 3’-untranslated region (UTR) comprising at least one microRNA-binding site, preferably not being a microRNA-122 (miR-122) binding site, more preferably being miR-16, miR-21 , miR-24, miR-27, miR-30c, miR-132, miR-133, miR-149, miR-192, miR-194, miR-204, miR-206, miR-208, or miR-223, most preferably being miRNA-148a, miRNA-101 , miRNA-192 or miRNA-194, miR-126, miR-142-3p, or miR-142-5p.

In other preferred embodiments, the nucleic acid sequences of the invention comprise at least one miRNA binding site, which is substantially complementary to miRNA sequences selected from at least one or more of the group of Table I consisting of miRNA-148a, miRNA-101 , miRNA-192 or miRNA-194. In further embodiments wherein a preferred expression in immune cells has to be avoided such as for protein replacement therapy the miRNA binding site sequence according to the invention preferably comprises at least one miRNA-148a, miRNA-101 , and/or optionally a miRNA- 192 binding site (depending on the target tissue), preferably at least one miRNA-148a binding site.

Table I: miRNA binding sites

In another embodiment, the method of the invention of treatming or preventing Tyrosinemia Type I (HT1 ) involves a single administration of the mRNA, the LNP, the pharmaceutical composition or the kit or kit of parts.

In other embodiments, the mRNA, the LNP, the pharmaceutical composition or the kit or kit of parts of the invention are the being administered in the method of the invention

(a) once, preferably more than once, more preferably wherein administration is repeated for a period of at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least one year, or lifelong; or

(b) about once a day, about once a week, about twice a week, about three times a week, about four times a week, about six or seven times a week, about once every two weeks, about once every three weeks, about once a month, about twice a month, about three times a month, or about four times a month.

In another aspect, the present invention relates to an isolated mRNA of the invention, the LNP of the invention or the pharmaceutical composition of the invention, or the kit or kit of parts of the invention, for use as a medicament.

According to one specific aspect, the present invention is directed to the first medical use of the mRNA according to the invention, of the pharmaceutical composition or of the kit or kit of parts comprising the mRNA according to the invention or a plurality of inventive RNAs as defined herein as a medicament, particularly in gene therapy, preferably for the treatment or prophylaxis of Hereditary Tyrosinemia Type I (HT1) as defined herein.

According to another aspect, the present invention is directed to the second medical use of the mRNA according to the invention, of the pharmaceutical composition, or of the kit or kit of parts comprising the mRNA according to the invention or a plurality of inventive RNAs as defined herein, for the treatment or prophylaxis of Hereditary Tyrosinemia Type I (HT1 ) as defined herein, preferably to the use of the mRNA as defined herein, of the pharmaceutical composition, or the kit or kit of parts comprising the mRNA according to the invention as defined herein, for the preparation of a medicament for the prophylaxis, treatment and/or amelioration of Hereditary Tyrosinemia Type I (HT1) as defined herein. Preferably, the pharmaceutical composition is used on or to be administered to a patient in need thereof for this purpose.

According to a further aspect, the mRNA according to the invention or the pharmaceutical composition comprising the mRNA according to the invention is used in the manufacture of a medicament, wherein the medicament is preferably for treatment or prophylaxis of Hereditary Tyrosinemia Type I (HT1 ) as defined herein.

In yet a further aspect, the present invention relates to an isolated mRNA comprising an open reading frame (ORF) encoding fumarylacetoacetate hydrolase (FAH), comprising one or more amino acid exchange(s), leading to an increased FAH activity, stability, longer-lasting FAH half-life and/or therapeutic effect as compared to the unmodified human wild type FAH protein according to SEQ ID NO: 100, preferably fumarylacetoacetate hydrolase (FAH) comprising an amino acid sequence having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 100.

In yet a further aspect, the present invention relates to an isolated mRNA of the invention, LNP of the invention, composition of the invention, or kit or kit of parts of the invention, for use in treating, preventing, attenuating or inhibiting Hereditary Tyrosinemia Type I (HT1) in a human subject in need, comprising administering to a human subject in need the wherein the administration results in treatment, prevention, attenuation, inhibition, or prophylaxis of Hereditary Tyrosinemia Type I (HT1).

In a further aspect, the present invention relates to an isolated nucleic acid construct comprising a nucleic acid sequence encoding the mRNA of the invention, preferably an isolated nucleic acid construct having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the sequences selected from the group consisting of SEQ ID NO: 101 -144 or to any one of the sequences as disclosed in the Table “Constructs of the invention”. In yet another aspect, the present invention relates to a vector comprising any one of the isolated mRNAs of the invention or to a host cell carrying said vector.

Lipid Nanoparticles (LNPs)

Lipid nanoparticles as used herein preferably have the structure of a liposome. A liposome is a structure having lipid-containing membranes enclosing an aqueous interior. Liposomes preferably have one or more lipid membranes. Liposomes are preferably single-layered, referred to as unilamellar, or multi-layered, referred to as multilamellar. When complexed with nucleic acids, lipid particles may also be lipoplexes, which are composed of cationic lipid bilayers sandwiched between DNA layers. Liposomes can further be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50nm and 500nm in diameter. Liposome design preferably includes, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.

As a non-limiting example, liposomes such as synthetic membrane vesicles may be prepared by the methods, apparatus and devices described in US Patent Publication No. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373 and US20130183372, the contents of each of which are herein incorporated by reference in its entirety. The nucleic acid may be encapsulated by the liposome and/or it may be contained in an aqueous core which may then be encapsulated by the liposome (see International Pub. Nos. W02012031046, W02012031043, W02012030901 and WO2012006378 and US Patent Publication No. US20130189351 , US20130195969 and

US20130202684; the contents of each of which are herein incorporated by reference in their entirety).

In another embodiment, the mRNA is preferably formulated in a cationic oil-in-water emulsion where the emulsion particle comprises an oil core and a cationic lipid which can interact with the polynucleotide anchoring the molecule to the emulsion particle (see International Pub. No. WO2012006380; herein incorporated by reference in its entirety). In one embodiment, the mRNA may be formulated in a water-in-oil emulsion comprising a continuous hydrophobic phase in which the hydrophilic phase is dispersed.

In one embodiment, the mRNA pharmaceutical compositions is formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SMARTICLES® (Marina Biotech, Bothell, WA), neutral DOPC (l,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

In another embodiment, the lipid nanoparticles have a median diameter size of from about 50nm to about 300nm, such as from about 50nm to about 250nm, for example, from about 50nm to about 200nm.

In some embodiments, the mRNA is delivered using smaller LNPs. Such particles may comprise a diameter from below 0.1μm up to 100nm such as, but not limited to, less than 0.1μm, less than 1.0μm, less than 5μm, less than 10μm, less than 15μm, less than 20μm, less than 25μm, less than 30μm, less than 35μm, less than 40μm, less than 50μm, less than 55μm, less than 60μm, less than 65μm, less than 70μm, less than 75μm, less than 80μm, less than 85μm, less than 90μm, less than 95μm, less than 100μm, less than 125μm, less than 150μm, less than 175μm, less than 200μm, less than 225μm, less than 250μm, less than 275μm, less than 300μm, less than 325μm, less than 350μm, less than 375μm, less than 400μm, less than 425μm, less than 450μm, less than 475μm, less than 500μm, less than 525μm, less than 550μm, less than 575μm, less than 600μm, less than 625μm, less than 650μm, less than 675μm, less than 700μm, less than 725μm, less than 750μm, less than 775μm, less than 800μm, less than 825μm, less than 850μm, less than 875μm, less than 900μm, less than 925μm, less than 950μm, less than 975μm, In another embodiment, RNA is delivered using smaller LNPs which may comprise a diameter from about 1 nm to about 10Onm, from about 1 nm to about 10nm, about 1 nm to about 20nm, from about 1 nm to about 30nm, from about 1 nm to about 40nm, from about 1 nm to about 50nm, from about 1 nm to about 60nm, from about 1 nm to about 70nm, from about 1 nm to about 80nm, from about 1 nm to about 90nm, from about 5nm to about from 100nm, from about 5nm to about 10nm, about 5nm to about 20nm, from about 5nm to about 30nm, from about 5nm to about 40nm, from about 5nm to about 50nm, from about 5nm to about 60nm, from about 5nm to about 70nm, from about 5nm to about 80nm, from about 5nm to about 90nm, about 10nm to about 50nm, from about 20nm to about 50nm, from about 30nm to about 50nm, from about 40nm to about 50nm, from about 20nm to about 60nm, from about 30nm to about 60nm, from about 40nm to about 60nm, from about 20nm to about 70nm, from about 30nm to about 70nm, from about 40nm to about 70nm, from about 50nm to about 70nm, from about 60nm to about 70nm, from about 20nm to about 80nm, from about 30nm to about 80nm, from about 40nm to about 80nm, from about 50nm to about 80nm, from about 60nm to about 80nm, from about 20nm to about 90nm, from about 30nm to about 90nm, from about 40nm to about 90nm, from about 50nm to about 90nm, from about 60nm to about 90nm and/or from about 70nm to about 90nm.

In one embodiment, the lipid nanoparticle has a diameter greater than 100nm, greater than 150nm, greater than 200nm, greater than 250nm, greater than 300nm, greater than 350nm, greater than 400nm, greater than 450nm, greater than 500nm, greater than 550nm, greater than 600nm, greater than 650nm, greater than 700nm, greater than 750nm, greater than 800nm, greater than 850nm, greater than 900nm, greater than 950nm or greater than 1000nm. In yet another embodiment, the lipid nanoparticles in the formulation of the present invention have a single mode particle size distribution (i.e., they are not bi- or poly-modal).

The lipid nanoparticles preferably further comprise one or more lipids and/or other components in addition to those mentioned above. Other lipids may be included in the liposome compositions for a variety of purposes, such as to prevent lipid oxidation or to attach ligands onto the liposome surface. Any of a number of lipids may be present in lipid particles, including amphipathic, neutral, cationic, and anionic lipids. Such lipids can be used alone or in combination.

Additional components that may be present in a lipid particle include bilayer stabilizing components such as polyamide oligomers (see, e.g., U.S. Patent No. 6,320,017, which is incorporated by reference in its entirety), peptides, proteins, and detergents.

Different lipid nanoparticles having varying molar ratios of cationic lipid, non-cationic (or neutral) lipid, sterol (e.g., cholesterol), and aggregation reducing agent (such as a PEG- modified lipid) on a molar basis (based upon the total moles of lipid in the lipid nanoparticles) are provided in Table D herein below.

TABLE D: EXEMPLARY LIPID NANOPARTICLE COMPOSITIONS

In one embodiment, the weight ratio of lipid to RNA is at least about 0.5:1, at least about 1:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 11:1, at least about 20:1, at least about 25:1 , at least about 27: 1, at least about 30:1, or at least about 33:1. In one embodiment, the weight ratio of lipid to RNA is from about 1:1 to about 35:1, about 3:1 to about 15:1, about 4:1 to about 15:1, or about 5:1 to about 13:1 or about 25:1 to about 33:1. In one embodiment, the weight ratio oflipid to RNA is from about 0.5:1 to about 12:1. In one embodiment, the mRNA of the present invention may be encapsulated in a therapeutic anoparticle, referred to herein as "therapeutic nanoparticle nucleic acids". Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, International Pub Nos. WO2010/005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923, US Pub. Nos. US20110262491, US20100104645, US20100087337, US20100068285, US20110274759, US20100068286, US20120288541, S20130123351 and US20130230567 and US Pat No.8,206,747, 8,293,276, 8,318,208 and 8,318,211; the contents of each of which are herein incorporated by reference in their entirety. In another embodiment, therapeutic polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, the contents of which is herein incorporated by reference in its entirety. In one embodiment, the mRNA according to the invention may be encapsulated in, linked to and/or associated with synthetic nanocarriers. Synthetic nanocarriers include, but are not limited to, those described in International Pub. Nos. WO2010005740, WO2010030763, WO2012135010, WO2012149252, WO2012149255, WO2012149259, WO2012149265, WO2012149268, WO2012149282, WO2012149301, WO2012149393, WO2012149405, WO2012149411, WO2012149454 and WO2013019669, and US Pub. Nos. US20110262491, US20100104645, US20100087337 and US2012244222, each of which is herein incorporated by reference in their entirety. The synthetic nanocarriers may be formulated using methods known in the art and/or described herein. As a non-limiting example, the synthetic nanocarriers may be formulated by the ethods described in International Pub Nos. WO2010005740, and WO2010030763 and WO2012135010 and US Pub. Nos. US20110262491, US20100104645, US20100087337 and US2012244222, each of which is herein incorporated by reference in their entirety. In another embodiment, the synthetic nanocarrier formulations may be lyophilized by methods described in International Pub. No. W02011072218 and US Pat No.8,211,473; the content of each of which is rein incorporated by reference in their entirety. In yet another embodiment, formulations of the present invention, including, but not limited to, synthetic nanocarriers, may be lyophilized or reconstituted by the methods described in US Patent Publication No. US20130230568, the contents of which are herein incorporated by reference in its entirety. In one embodiment, the mRNA of the invention is formulated for delivery using the drug encapsulating microspheres described in International Patent Publication No. WO2013063468 or U.S. Patent No. 8,440,614, each of which is herein incorporated by reference in its entirety.

According to preferred embodiments, the mRNA according to the invention may be formulated in order to target a specific tissue or organ. In particular, the mRNA according to the invention or a pharmaceutical carrier formulated together with the mRNA preferably forms a conjugate with a targeting group. Said targeting group preferably targets the conjugate, preferably the conjugate comprising the mRNA, to a specific tissue or organ. In other words, by conjugating the mRNA according to the invention with a target group (either directly by forming an RNA-target group conjugate or indirectly by forming a conjugate of a target group of a pharmaceutical carrier that is present in a complex with the mRNA according to the invention), the mRNA is delivered to a specific tissue or organ due to the targeting of said target group to that specific tissue or organ. Most preferably, the targeting group provides for delivery to liver tissue, preferably to liver macrophages, hepatocytes and or liver sinusoidal endothelial cells (LSEC). In this context, a targeting group is preferably selected from the group consisting of folate, GalNAc, galactose, mannose, mannose-6P, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL ligands and HDL ligands. Suitable approaches for targeted delivery to the liver, which may be applied to the mRNA of the invention, are also described in Bartneck et al. (Bartneck et al: Therapeutic targeting of liver inflammation and fibrosis by nanomedicine. Hepatobiliary Surgery and Nutrition 2014;3(6):364-376), the disclosure of which is incorporated herein in its entirety.

In another embodiment, liposomes or LNPs may be formulated for targeted delivery. Preferably, the liposome or LNP is formulated for targeted delivery of the mRNA according to the invention to the liver, preferably to liver macrophages, hepatocytes and/or liver sinusoidal endothelial cells (LSEC). The liposome or LNP used for targeted delivery may include, but is not limited to, the liposomes or LNPs described herein.

In a preferred embodiment, the pharmaceutical composition according to the invention comprises the mRNA according to the invention that is formulated together with a cationic or polycationic compound and/or with a polymeric carrier. Accordingly, in a further embodiment of the invention, it is preferred that the mRNA as defined herein or any other nucleic acid comprised in the inventive pharmaceutical composition is associated with or complexed with a cationic or polycationic compound or a polymeric carrier, optionally in a weight ratio selected from a range of about 6:1 (w/w) to about 0.25:1 (w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1 :1 (w/w) or of about 3: 1 (w/w) to about 1 :1 (w/w), and most preferably a ratio of about 3:1 (w/w) to about 2:1 (w/w) of mRNA or nucleic acid to cationic or polycationic compound and/or with a polymeric carrier; or optionally in a nitrogen/phosphate (N/P) ratio of mRNA or nucleic acid to cationic or polycationic compound and/or polymeric carrier in the range of about 0.1-10, preferably in a range of about 0.3-4 or 0.3-1 , and most preferably in a range of about 0.5-1 or 0.7-1 , and even most preferably in a range of about 0.3-0.9 or 0.5-0.9. More preferably, the N/P ratio of the mRNA to the one or more polycations is in the range of about 0.1 to 10, including a range of about 0.3 to 4, of about 0.5 to 2, of about 0.7 to 2 and of about 0.7 to 1 .5.

Therein, the mRNA as defined herein or any other nucleic acid comprised in the pharmaceutical composition according to the invention can also be associated with a vehicle, transfection or complexation agent for increasing the transfection efficiency and/or the expression of the mRNA according to the invention or of optionally comprised further included nucleic acids.

Cationic or polycationic compounds, being particularly preferred agents in this context include protamine, nucleoline, spermine or spermidine, or other cationic peptides or proteins, such as poly- L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV- binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1 , L- oligomers, Calcitonin peptide(s), Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, or histones. More preferably, the mRNA according to the invention is complexed with one or more polycations, preferably with protamine or oligofectamine, most preferably with protamine. In this context protamine is particularly preferred.

Additionally, preferred cationic or polycationic proteins or peptides may be selected from the following proteins or peptides having the following total formula (III):

wherein I + m + n +o + x = 8-15, and I, m, n, o, or y independently of each other may be any number selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn represents at least 50% of all amino acids of the oligopeptide; and Xaa may be any amino acid selected from native (= naturally occurring) or non-native amino acids except of Arg, Lys, His or Orn; and x may be any number selected from 0, 1 , 2, 3 or 4, provided, that the overall content of Xaa does not exceed 50% of all amino acids of the oligopeptide. Particularly preferred cationic peptides in this context are e.g. Arg₇, Arg₈, Arg₉, H₃R₉, R₉H₃, H₃R₉H₃, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc. In this context the disclosure of WO 2009/030481 is incorporated herewith by reference. According to another embodiment, the pharmaceutical composition of the present invention comprises the mRNA as defined herein and a polymeric carrier. A polymeric carrier used according to the invention might be a polymeric carrier formed by disulfide-crosslinked cationic components. The disulfide-crosslinked cationic components may be the same or different from each other. The polymeric carrier can also contain further components. It is also particularly preferred that the polymeric carrier used according to the present invention comprises mixtures of cationic peptides, proteins or polymers and optionally further components as defined herein, which are crosslinked by disulfide bonds as described herein. In this context, the disclosure of WO2012/013326 is incorporated herewith by reference. Also in this context, the disclosure of WO2011/026641 is incorporated herewith by reference. Further preferred cationic or polycationic compounds, which can be used as transfection or complexation agent may include cationic polysaccharides, for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g. DOTMA: [1-(2,3-sioleyloxy)propyl)]- N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: 0,0- ditetradecanoyl-N-(a-trimethylammonioacetyl)diethanolamine chloride, CLIP1: rac-[(2,3- dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIPS: rac-[2(2,3- dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium, CLIP9: rac-[2(2,3- dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic or polycationic polymers, e.g. modified polyaminoacids, such as (3-aminoacid-polymers or reversed polyamides, etc., modified polyethylenes, such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates, such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc., modified amidoamines such as pAMAM (poly(amidoamine)), etc., modified polybetaaminoester PBAE), such as diamine end modified 1,4 butanediol diacrylate-co-5-amino-l-pentanol polymers, etc., dendrimers, such as polypropylamine dendrimers or pAMAM based dendrimers, etc., polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine), etc., polyallylamine, sugar backbone based polymers, such as cyclodextrin based polymers, dextran based polymers, chitosan, etc., silan backbone based polymers, such as PMOXA-PDMS copolymers, etc., blockpolymers consisting of a combination of one or more cationic blocks (e.g. selected from a cationic polymer as mentioned above) and of one or more hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole); etc. Preferably, the inventive composition comprises at least one RNA as defined herein, which is complexed with one or more polycations, and at least one free RNA, wherein the at least one complexed RNA is preferably identical to the at least one free RNA. In this context, it is particularly preferred that the pharmaceutical composition of the present invention comprises the mRNA according to the invention that is complexed at least partially with a cationic or polycationic compound and/or a polymeric carrier, preferably cationic proteins or peptides. In this context, the disclosure of WO2010037539 and WO2012113513 is incorporated herewith by reference. Partially means that only a part of the mRNA as defined herein is complexed in the pharmaceutical composition according to the invention with a cationic compound and that the rest of the mRNA as defined herein is (comprised in the inventive pharmaceutical composition) in uncomplexed form ("free"). Preferably, the molar ratio of the complexed RNA to the free RNA is selected from a molar ratio of about 0.001:1 to about 1:0.001 , including a ratio of about 1:1. More preferably the ratio of complexed RNA to free RNA (in the pharmaceutical composition of the present invention) is selected from a range of about 5:1 (w/w) to about 1 :10 (w/w), more preferably from a range of about 4:1 (w/w) to about 1 :8 (w/w), even more preferably from a range of about 3:1 (w/w) to about 1 :5 (w/w) or 1 :3 (w/w), and most preferably the ratio of complexed mRNA to free mRNA in the inventive pharmaceutical composition is selected from a ratio of about 1:1 (w/w).

The complexed RNA in the pharmaceutical composition according to the present invention, is preferably prepared according to a first step by complexing the mRNA according to the invention with a cationic or polycationic compound and/or with a polymeric carrier, preferably as defined herein, in a specific ratio to form a stable complex. In this context, it is highly preferable, that no free cationic or polycationic compound or polymeric carrier or only a negligibly small amount thereof remains in the component of the complexed RNA after complexing the mRNA. Accordingly, the ratio of the mRNA and the cationic or polycationic compound and/or the polymeric carrier in the component of the complexed RNA is typically selected in a range so that the mRNA is entirely complexed and no free cationic or polycationic compound or polymeric carrier or only a negligibly small amount thereof remains in the pharmaceutical composition.

Preferably the ratio of the mRNA as defined herein to the cationic or polycationic compound and/or the polymeric carrier, preferably as defined herein, is selected from a range of about 6:1 (w/w) to about 0,25:1 (w/w), more preferably from about 5:1 (w/w) to about 0,5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1 :1 (w/w) or of about 3:1 (w/w) to about 1 :1 (w/w), and most preferably a ratio of about 3: 1 (w/w) to about 2: 1 (w/w). Alternatively, the ratio of the mRNA as defined herein to the cationic or polycationic compound and/or the polymeric carrier, preferably as defined herein, in the component of the complexed mRNA, may also be calculated on the basis of the nitrogen/phosphate ratio (N/P-ratio) of the entire complex. In the context of the present invention, an N/P-ratio is preferably in the range of about 0.1 to 10, preferably in a range of about 0.3 to 4 and most preferably in a range of about 0.5 to 2 or 0.7 to 2 regarding the ratio of RNA : cationic or polycationic compound and/or polymeric carrier, preferably as defined herein, in the complex, and most preferably in a range of about 0.7 to 1.5, 0.5 to 1 or 0.7 to 1 , and even most preferably in a range of about 0.3 to 0.9 or 0.5 to 0.9, preferably provided that the cationic or polycationic compound in the complex is a cationic or polycationic cationic or polycationic protein or peptide and/or the polymeric carrier as defined above.

In other embodiments, the pharmaceutical composition according to the invention comprising the mRNA as defined herein may be administered naked without being associated with any further vehicle, transfection or complexation agent.

It has to be understood and recognized, that according to the present invention, the inventive composition may comprise at least one mRNA, and/or at least one formulated/complexed mRNA as defined herein, wherein every formulation and/or complexation as disclosed above may be used. In embodiments, wherein the pharmaceutical composition comprises more than one RNA species, these RNA species may be provided such that, for example, two, three, four, five, six, seven, eight, nine or more separate compositions, which may contain at least one RNA species each (e.g. three distinct mRNA species), each encoding a distinct peptide or protein as defined herein or a fragment or variant thereof, are provided, which may or may not be combined. Also, the pharmaceutical composition may be a combination of at least two distinct compositions, each composition comprising at least one mRNA encoding at least one of the peptides or proteins defined herein. Alternatively, the pharmaceutical composition may be provided as a combination of at least one mRNA, preferably at least two, three, four, five, six, seven, eight, nine or more mRNAs, each encoding one of the peptides or proteins defined herein. The pharmaceutical composition may be combined to provide one single composition prior to its use or it may be used such that more than one administration is required to administer the distinct mRNA species encoding a certain combination of the proteins as defined herein. If the pharmaceutical composition contains at least one mRNA molecule, typically at least two mRNA molecules, encoding of a combination of peptides or proteins as defined herein, it may e.g. be administered by one single administration (combining all mRNA species), by at least two separate administrations. Accordingly, any combination of mono-, bi- or multicistronic mRNAs encoding a peptide or protein or any combination of peptides or proteins as defined herein (and optionally further proteins), provided as separate entities (containing one mRNA species) or as combined entity (containing more than one mRNA species), is understood as a pharmaceutical composition according to the present invention. According to a particularly preferred embodiment of the pharmaceutical composition, the at least one peptide or protein, preferably a combination of at least two, three, four, five, six, seven, eight, nine or more peptides or proteins encoded by the pharmaceutical composition as a whole, is provided as an individual (monocistronic) mRNA, which is administered separately.

The pharmaceutical composition according to the present invention may be provided in liquid and or in dry (e.g. lyophilized) form.

The pharmaceutical composition typically comprises a safe and effective amount of the mRNA according to the invention as defined herein, encoding a peptide or protein as defined herein or a fragment or variant thereof or a combination of peptides or proteins, preferably as defined herein. As used herein, “safe and effective amount” means an amount of the mRNA that is sufficient to significantly induce a positive modification of a disease or disorder as defined herein. At the same time, however, a “safe and effective amount” is small enough to avoid serious side-effects, that is to say to permit a sensible relationship between advantage and risk. The determination of these limits typically lies within the scope of sensible medical judgment. In relation to the pharmaceutical composition of the present invention, the expression “safe and effective amount" preferably means an amount of the mRNA (and thus of the encoded peptide or protein) that is suitable for obtaining an appropriate expression level of the encoded protein(s). Such a “safe and effective amount” of the mRNA of the pharmaceutical composition as defined herein may furthermore be selected in dependence of the type of RNA, e.g. monocistronic, bi- or even multicistronic RNA, since a bi- or even multicistronic RNA may lead to a significantly higher expression of the encoded protein(s) than the use of an equal amount of a monocistronic RNA. A "safe and effective amount" of the mRNA of the pharmaceutical composition as defined above may furthermore vary in connection with the particular condition to be treated and also with the age and physical condition of the patient to be treated, the severity of the condition, the duration of the treatment, the nature of the accompanying therapy, of the particular pharmaceutically acceptable carrier used, and similar factors, within the knowledge and experience of the accompanying doctor. The pharmaceutical composition according to the invention can be used according to the invention for human and also for veterinary medical purposes. In a preferred embodiment, the mRNA of the pharmaceutical composition or kit of parts according to the invention is provided in lyophilized form. Preferably, the lyophilized RNA is reconstituted in a suitable buffer, advantageously based on an aqueous carrier, prior to administration, e.g. Ringer- Lactate solution, which is preferred, Ringer solution, a phosphate buffer solution. In a preferred embodiment, the pharmaceutical composition or the kit of parts according to the invention contains at least two, three, four, five, six, seven, eight, nine or more RNAs, preferably mRNAs, which are provided separately in lyophilized form (optionally together with at least one further additive) and which are preferably reconstituted separately in a suitable buffer (such as Ringer-Lactate solution) prior to their use so as to allow individual administration of each of the (monocistronic) RNAs. The pharmaceutical composition according to the invention may typically contain a pharmaceutically acceptable carrier. The expression "pharmaceutically acceptable carrier" as used herein preferably includes the liquid or non-liquid basis of the pharmaceutical composition. If the pharmaceutical composition is provided in liquid form, the carrier will be water, typically pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g phosphate, citrate etc. buffered solutions. Particularly for injection of the pharmaceutical composition, water or preferably a buffer, more preferably an aqueous buffer, may be used, containing a sodium salt, preferably at least 50mM of a sodium salt, a calcium salt, preferably at least 0,01 mM of a calcium salt, and optionally a potassium salt, preferably at least 3mM of a potassium salt. According to a preferred embodiment, the sodium, calcium and, optionally, potassium salts may occur in the form of their halogenides, e.g. chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc. Without being limited thereto, examples of sodium salts include e.g. NaCI, Nal, NaBr, Na₂CO₃, NaHCO₃, Na₂SO₄, examples of the optional potassium salts include e.g. KCI, Kl, KBr, K₂CO₃, KHCO₃, K₂SO₄, and examples of calcium salts include e.g. CaCl2, Cal2, CaBr2, CaCOs, CaSO4, Ca(OH)2. Furthermore, organic anions of the aforementioned cations may be contained in the buffer. According to a more preferred embodiment, the buffer suitable for injection purposes as defined above, may contain salts selected from sodium chloride (NaCI), calcium chloride (CaCl2) and optionally potassium chloride (KCI), wherein further anions may be present additional to the chlorides. CaCh can also be replaced by another salt like KCI. Typically, the salts in the injection buffer are present in a concentration of at least 50 mM sodium chloride (NaCI), at least 3 mM potassium chloride (KCI) and at least 0,01 mM calcium chloride (CaCl₂). The injection buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects. Reference media are e.g. in “in vivo” methods occurring liquids such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in “in vitro" methods, such as common buffers or liquids. Such common buffers or liquids are known to a skilled person. Ring er- Lactate solution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents or encapsulating compounds may be used as well, which are suitable for administration to a person. The term “compatible” as used herein means that the constituents of the pharmaceutical composition according to the invention are capable of being mixed with the mRNA according to the invention as defined herein, in such a manner that no interaction occurs, which would substantially reduce the pharmaceutical effectiveness of the pharmaceutical composition according to the invention under typical use conditions. Pharmaceutically acceptable carriers, fillers and diluents must, of course, have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a person to be treated. Some examples of compounds which can be used as pharmaceutically acceptable carriers, fillers or constituents thereof are sugars, such as, for example, lactose, glucose, trehalose and sucrose; starches, such as, for example, corn starch or potato starch; dextrose; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.

The choice of a pharmaceutically acceptable carrier is determined, in principle, by the manner, in which the pharmaceutical composition according to the invention is administered. The pharmaceutical composition can be administered, for example, systemically or locally. Routes for systemic administration in general include, for example, transdermal, oral, parenteral routes, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injections and/or intranasal administration routes. Routes for local administration in general include, for example, topical administration routes but also intradermal, transdermal, subcutaneous, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, and sublingual injections. More preferably, the pharmaceutical composition according to the present invention may be administered by an intradermal, subcutaneous, or intramuscular route, preferably by injection, which may be needle-free and/or needle injection. The pharmaceutical composition is therefore preferably formulated in liquid or solid form. The suitable amount of the pharmaceutical composition according to the invention to be administered can be determined by routine experiments, e.g. by using animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog and non-human primate models. Preferred unit dose forms for injection include sterile solutions of water, physiological saline or mixtures thereof. The pH of such solutions should be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid and collagen matrices. Suitable pharmaceutically acceptable carriers for topical application include those which are suitable for use in lotions, creams, gels and the like. If the pharmaceutical composition is to be administered perorally, tablets, capsules and the like are the preferred unit dose form. The pharmaceutically acceptable carriers for the preparation of unit dose forms which can be used for oral administration are well known in the prior art. The choice thereof will depend on secondary considerations such as taste, costs and storability, which are not critical for the purposes of the present invention, and can be made without difficulty by a person skilled in the art. In some very preferred embodiments, the pharmaceutical composition according to the present invention is administered through an intramuscular route, preferably by injection, which may be needle-free and/or needle injection.

Further additives which may be included in the pharmaceutical composition are emulsifiers, such as, for example, Tween; wetting agents, such as, for example, sodium lauryl sulfate; colouring agents; taste-imparting agents, pharmaceutical carriers; tablet-forming agents; stabilizers; antioxidants; preservatives.

In preferred embodiments, the pharmaceutical composition according to the invention comprises a further pharmaceutically active ingredient in addition to the mRNA according to the invention. Preferably, the further pharmaceutically active ingredient is selected from compounds suitable for use in the treatment or prophylaxis of Hereditary Tyrosinemia Type I (HT1 ) as defined herein.

The pharmaceutical composition as defined herein may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.

The pharmaceutical composition may also be administered topically. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the pharmaceutical composition may be formulated in a suitable ointment, containing the mRNA according to the invention suspended or dissolved in one or more carriers.

According to a preferred embodiment of the aspects of the invention, the pharmaceutical composition according to the invention is administered via a parenteral route, preferably by injection. Preferably, the inventive composition is administered by intradermal, subcutaneous, intramuscular or intravenous injection, most preferably by intravenous injection. Any suitable injection technique known in the art may be employed, for example conventional needle injection or needle-less injection techniques, such as jet-injection, or intravenous infusion or respectively intravenous therapy (IV therapy).

In one embodiment, the pharmaceutical composition comprises at least two, three, four, five, six, seven, eight, nine or more RNAs as defined herein, each of which is preferably injected separately, preferably by needle-less injection. Alternatively, the pharmaceutical composition comprises at least two, three, four, five, six, seven, eight, nine or more RNAs, wherein the at least two, three, four, five, six, seven, eight, nine or more RNAs are administered, preferably by injection as defined herein, as a mixture.

Administration of the mRNA as defined herein or the pharmaceutical composition according to the invention may be carried out in a time staggered treatment. A time staggered treatment may be e.g. administration of the mRNA or the pharmaceutical composition prior, concurrent and/or subsequent to a conventional therapy of a disease or disorder, preferably as described herein, e.g. by administration of the mRNA or the pharmaceutical composition prior, concurrent and/or subsequent to a therapy or an administration of a therapeutic agent suitable for the treatment or prophylaxis of a disease or disorder as described herein, preferably Tyrosinemia Type I (HT1 ). Such time staggered treatment may be carried out using e.g. a kit, preferably a kit of parts as defined herein. The term disease and disorder are used interchangeably herein.

Time staggered treatment may additionally or alternatively also comprise an administration of the mRNA as defined herein or the pharmaceutical composition according to the invention in a form, wherein the mRNA encoding a peptide or protein as defined herein or a fragment or variant thereof, preferably forming part of the pharmaceutical composition, is administered parallel, prior or subsequent to another RNA encoding a peptide or protein as defined above, preferably forming part of the same inventive composition. Preferably, the administration (of all RNAs) occurs within an hour, more preferably within 30 minutes, even more preferably within 15, 10, 5, 4, 3, or 2 minutes or even within 1 minute. Such time staggered treatment may be carried out using e.g. a kit, preferably a kit of parts as defined herein.

According to a further aspect, the present invention also provides kits, particularly kits of parts. Such kits, particularly kits of parts, typically comprise as components alone or in combination with further components as defined herein at least one inventive RNA species as defined herein, or the inventive pharmaceutical composition comprising the mRNA according to the invention. The at least one RNA as defined herein, is optionally in combination with further components as defined herein, whereby the at least one RNA is provided separately (first part of the kit) from at least one other part of the kit comprising one or more other components. The pharmaceutical composition may occur in one or different parts of the kit. As an example, e.g. at least one part of the kit may comprise at least one RNA as defined herein, and at least one further part of the kit at least one other component as defined herein, e.g. at least one other part of the kit may comprise at least one pharmaceutical composition or a part thereof, e.g. at least one part of the kit may comprise the mRNA as defined herein, at least one further part of the kit at least one other component as defined herein, at least one further part of the kit at least one component of the pharmaceutical composition or the pharmaceutical composition as a whole, and at least one further part of the kit e.g. at least one pharmaceutical carrier or vehicle, etc. In case the kit or kit of parts comprises a plurality of RNAs as described herein, one component of the kit can comprise only one, several or all RNAs comprised in the kit. In an alternative embodiment every/each RNA species may be comprised in a different/separate component of the kit such that each component forms a part of the kit. Also, more than one RNA as defined herein may be comprised in a first component as part of the kit, whereas one or more other (second, third etc.) components (providing one or more other parts of the kit) may either contain one or more than one RNA as defined herein, which may be identical or partially identical or different from the first component. The kit or kit of parts may furthermore contain technical instructions with information on the administration and dosage of the mRNA according to the invention, the pharmaceutical composition of the invention or of any of its components or parts, e.g. if the kit is prepared as a kit of parts.

In a further aspect, the present invention furthermore provides several applications and uses of the mRNA, of the pharmaceutical composition or the kit of parts according to the invention. In particular, the present invention provides medical uses of the mRNA according to the invention. Moreover, the use of the mRNA according to the invention, of the pharmaceutical composition or the kit of parts according to the invention is envisaged in gene therapy.

In a preferred embodiment of the present invention, the mRNA, the pharmaceutical composition or the kit or kit of parts as described herein is provided for use in the treatment or prophylaxis of Hereditary Tyrosinemia Type I (HT1). Accordingly, the present invention concerns an mRNA comprising at least one coding sequence, wherein the coding sequence encodes at least one peptide or protein as described herein, preferably comprising or consisting of a FAH protein, or a fragment or a variant of any of these peptides or proteins having the biological activity of a wild type FAH protein, or a pharmaceutical composition or kit or kit of parts comprising the mRNA according to the invention, for use in the treatment or prophylaxis of Hereditary Tyrosinemia Type I (HT1).

In a preferred embodiment, the mRNA as described herein or the pharmaceutical composition is provided for treatment or prophylaxis of Hereditary Tyrosinemia Type I (HT1 ), which comprises targeted delivery of the mRNA. Preferably, the mRNA is targeted to the liver upon administration to a mammalian subject. Targeted delivery of the mRNA according to the invention is preferably achieved by formulating the mRNA in a suitable manner (e.g. as a liposome or lipid nanoparticle as described herein) and/or by administering the mRNA or the pharmaceutical composition, respectively, according to the invention via a suitable route. Preferably, the treatment or prophylaxis of Hereditary Tyrosinemia Type I (HT1) as described herein comprises administration of the mRNA or the pharmaceutical composition according to the invention in any suitable manner, preferably as described herein with respect to the pharmaceutical composition. The description of the pharmaceutical composition, where appropriate, also applies to the medical use of the mRNA according to the invention.

In preferred embodiments, the treatment or prophylaxis comprises administration of a further pharmaceutically active ingredient in combination with the mRNA according to the invention or the pharmaceutical composition according to the invention. Preferably, the further pharmaceutically active ingredient is selected from compounds suitable for use in the treatment or prophylaxis of Hereditary Tyrosinemia Type I (HT1) as defined herein.

Also comprised by the present invention are methods of treating or preventing a disease or disorder, preferably Hereditary Tyrosinemia Type I (HT1) as defined herein, by administering to a subject in need thereof a pharmaceutically effective amount of the mRNA or the pharmaceutical composition according to the invention. Such a method typically comprises an optional first step of preparing the mRNA or the pharmaceutical composition of the present invention, and a second step, comprising administering (a pharmaceutically effective amount of) said composition to a patient/subject in need thereof. A subject in need thereof will typically be a mammal. In the context of the present invention, the mammal is preferably selected from the group comprising, without being limited thereto, e.g. goat, cattle, swine, dog, cat, donkey, monkey, ape, a rodent such as a mouse, hamster, rabbit and, particularly, human, wherein the mammal typically suffers from a disease or disorder, preferably from Hereditary Tyrosinemia Type I (HT1) as defined herein.

According to a further aspect, the present invention also provides a method for increasing the expression of a peptide or protein as described herein comprising the steps, e.g. a) providing the mRNA as defined herein or the pharmaceutical composition as defined herein, b) applying or administering the mRNA or the pharmaceutical composition to an expression system, e.g. to a cell- free expression system, a cell (e.g. an expression host cell or a somatic cell), a tissue or an organism. The method may be applied for laboratory, for research, for diagnostic, for commercial production of peptides or proteins and/or for therapeutic purposes. In this context, typically after preparing the mRNA or the pharmaceutical composition, it is typically applied or administered to a cell-free expression system, a cell (e.g. an expression host cell or a somatic cell), a tissue or an organism, e.g. in naked or complexed form or as a pharmaceutical composition as described herein, preferably via transfection or by using any of the administration modes as described herein. The method may be carried out in vitro, in vivo or ex vivo. The method may furthermore be carried out in the context of the treatment of a specific disease, preferably as defined herein.

In this context in vitro is defined herein as transfection or transduction of the mRNA or the pharmaceutical composition according to the invention into cells in culture outside of an organism; in vivo is defined herein as transfection or transduction of the mRNA or the pharmaceutical composition according to the invention into cells by application of the mRNA or the pharmaceutical composition to the whole organism or individual and ex vivo is defined herein as transfection or transduction of the mRNA or the pharmaceutical composition according to the invention into cells outside of an organism or individual and subsequent application of the transfected cells to the organism or individual.

Likewise, according to another aspect, the present invention also provides the use of the mRNA or the pharmaceutical composition according to the invention, preferably for diagnostic or therapeutic purposes, for increasing the expression of a peptide or protein as described herein, particularly in gene therapy e.g. by applying or administering the mRNA or the pharmaceutical composition, e.g. to a cell-free expression system, a cell (e.g. an expression host cell or a somatic cell), a tissue or an organism. The use may be applied for laboratory, for research, for diagnostic for commercial production of peptides or proteins and/or for therapeutic purposes, preferably for gene therapy. In this context, typically after preparing the mRNA or the pharmaceutical composition according to the invention, it is typically applied or administered to a cell-free expression system, a cell (e.g. an expression host cell or a somatic cell), a tissue or an organism, preferably in naked form or complexed form, or as a pharmaceutical composition as described herein, preferably via transfection or by using any of the administration modes as described herein. The use may be carried out in vitro, in vivo or ex vivo. The use may furthermore be carried out in the context of the treatment of a specific disease, preferably Hereditary Tyrosinemia Type I (HT1) as defined herein.

In yet another aspect the present invention also relates to an inventive expression system comprising the mRNA according to the invention or an expression vector or plasmid comprising a corresponding nucleic acid sequence according to the aspects of the present invention. In this context the expression system may be a cell-free expression system (e.g. an in vitro transcription/translation system), a cellular expression system (e.g. mammalian cells like CHO cells, insect cells, yeast cells, bacterial cells like E. coli) or organisms used for expression of peptides or proteins (e.g. plants or animals like cows).

In a further aspect, the present invention relates to a lipid nanoparticle (LNP), comprising the mRNA of the invention, wherein the LNP comprises an ionizable or cationic lipid, a phospholipid, a structural lipid, and a polymer conjugated lipid.

In a preferred embodiment, the lipids comprised in the LNP of the invention have a molar ratio of about 20-60% cationic or ionizable lipid, about 5-25% non-cationic lipid, about 25-55% sterol and about 0.5-15% polymer conjugated lipid.

In other embodiments, the LNP of the invention does not comprise polyethylene glycol (PEG) or a PEG-modified lipid. In another aspect, the present invention relates to a pharmaceutical composition, comprising the mRNA of the invention or the LNP of the invention.

In yet another aspect, the present invention related to a kit, preferably kit of parts, comprising at least one mRNA of the invention, the LNP of the invention, or the pharmaceutical composition of the invention, and optionally a liquid vehicle for solubilising and optionally technical instructions with

Cationic Lipids

The lipid nanoparticle may include any cationic lipid suitable for forming a lipid nanoparticle. Preferably, the cationic lipid carries a net positive charge at about physiological pH.

The cationic lipid is preferably an amino lipid. As used herein, the term “amino lipid” is meant to include those lipids having one or two fatty acid or fatty alkyl chains and an amino head group (including an alkylamino or dialkylamino group) that may be protonated to form a cationic lipid at physiological pH.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N- distearyl-N,N-dimethylammonium bromide (DDAB), 1 ,2-dioleoyltrimethyl ammonium propane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and

1.2-Dioleyloxy-3-trimethylaminopropane chloride salt), N-(1-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1 ,2- DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), 1 ,2-di-y-linolenyloxy-N,N-dimethylaminopropane (y- DLenDMA), 1 ,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1 ,2-Dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),

1 .2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1 ,2-Dilinoleylthio-3-dimethylaminopropane

(DLin-S- DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1 ,2- Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI), 1 ,2-Dilinoleoyl-3- trimethylaminopropane chloride salt (DLin-TAP.CI), 1 ,2-Dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1 ,2-propanediol (DLinAP), 3- (N,N-Dioleylamino)-1 ,2-propanedio (DOAP), 1 ,2-Dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DM A), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1 ,3]- dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca- 9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1 ,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1 ,1’-(2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)- didodecan-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3]- dioxolane (DLin-K-C2- DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA), (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-M-C3-DMA), 3- ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,3 1-tetraen-19-yloxy)-N,N-dimethylpropan-1 -amine (MC3

Ether), 4-((6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 Ether), or any combination of any of the foregoing.

Other cationic lipids include, but are not limited to, N,N-distearyl-N,N- dimethylammonium bromide (DDAB), 3P-(N-(N’,N’-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(1-(2,3- dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1 ,2-dileoyl-sn-3- phosphoethanolamine (DOPE), 1 ,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(1 ,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), and 2,2- Dilinoleyl-4- dimethylaminoethyl-[1 ,3]-dioxolane (XTC). Additionally, commercial preparations of cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECT AMINE (comprising DOSPA and DOPE, available from GIBCO/BRL).

Other suitable cationic lipids are disclosed in International Publication Nos. WO09/086558, WO09/127060, WO10/048536, WO10/054406, WO10/088537, WO10/129709, and WO2011/153493; U.S. Patent Publication Nos. 2011/0256175, 2012/0128760, and 2012/0027803; U.S. Patent Nos. 8,158,601 ; and Love et al, PNAS, 107(5), 1864-69, 2010. [51] Other suitable amino lipids include those having alternative fatty acid groups and other dialkylamino groups, including those in which the alkyl substituents are different (e.g., N-ethyl-N-methylamino-, and N-propyl-N- ethylamino-). In general, amino lipids having less saturated acyl chains are more easily sized, particularly when the complexes must be sized below about 0.3 microns, for purposes of filter sterilization. Amino lipids containing unsaturated fatty acids with carbon chain lengths in the range of C14 to C22 may be used. Other scaffolds can also be used to separate the amino group and the fatty acid or fatty alkyl portion of the amino lipid.

In certain embodiments, amino or cationic lipids of the invention have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Lipids that have more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded from use in the invention.

In certain embodiments, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11 , e.g., a pKa of about 5 to about 7.

Lipid particles preferably include two or more cationic lipids. The cationic lipids are preferably selected to contribute different advantageous properties. For example, cationic lipids that differ in properties such as amine pKa, chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue, or toxicity can be used in the lipid nanoparticle. In particular, the cationic lipids can be chosen so that the properties of the mixed-lipid particle are more desirable than the properties of a single-lipid particle of individual lipids.

The cationic lipid preferably comprises from about 20mol% to about 70mol% or 75mol% or from about 45mol% to about 65mol% or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or about 70mol% of the total lipid present in the particle. In another embodiment, the lipid nanoparticles include from about 25% to about 75% on a molar basis of cationic lipid, e.g., from about 20 to about 70%, from about 35 to about 65%, from about 45 to about 65%, about 60%, about 57.5%, about 57.1%, about 50% or about 40% on a molar basis (based upon 100% total moles of lipid in the lipid nanoparticle). In one embodiment, the ratio of cationic lipid to nucleic acid is from about 3 to about 15, such as from about 5 to about 13 or from about 7 to about 11 .

Non-Cationic Lipids

The non-cationic lipid is preferably a neutral lipid, an anionic lipid, or an amphipathic lipid. Neutral lipids, when present, can be any of a number of lipid species which exist either in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids for use in the particles described herein is generally guided by consideration of, e.g., lipid particle size and stability of the lipid particle in the bloodstream. Preferably, the neutral lipid is a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine). In one embodiment, the neutral lipids contain saturated fatty acids with carbon chain lengths in the range of C10 to C20. In another embodiment, neutral lipids with mono or diunsaturated fatty acids with carbon chain lengths in the range of C10 to C2o are used. Additionally, neutral lipids having mixtures of saturated and unsaturated fatty acid chains can be used.

Suitable neutral lipids include, but are not limited to, distearoylphosphatidylcholine or 1,2-distearoyl- sn-glycero-3-phosphocholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoyl- phosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dimyristoyl phosphatidylcholine (DMPC), distearoyl-phosphatidyl-ethanolamine (DSPE), SM, 16-0-monomethyl PE, 16-O-dimethyl PE, 18-1- trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. Anionic lipids suitable for use in lipid particles of the invention include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids.

The term “amphipathic lipid(s)” refers to any suitable material, wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Such compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids. Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatdylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and (3-acyloxyacids, can also be used.

The non-cationic lipid is preferably from about 5mol% to about 90mol%, about 5mol% to about 10mol%, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or about 90mol% of the total lipid present in the particle. In one embodiment, the lipid nanoparticles include from about 0% to about 15% or 45% on a molar basis of neutral lipid, e.g., from about 3% to about 12% or from about 5% to about 10%. For instance, the lipid nanoparticles may include about 15%, about 10%, about 7.5%, or about 7.1 % of neutral lipid on a molar basis (based upon 100% total moles of lipid in the lipid nanoparticle).

Sterols

A preferred sterol is cholesterol. Further sterols as known in the art are further envisaged for use in the context of the present invention.

The sterol preferably constitutes about 10mol% to about 60mol% or about 25mol% to about 40mol% of the lipid particle. In one embodiment, the sterol is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60mol% of the total lipid present in the lipid particle. In another embodiment, the lipid nanoparticles include from about 5% to about 50% on a molar basis of the sterol, e.g., about 15% to about 45%, about 20% to about 40%, about 48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31.5% or about 31% on a molar basis (based upon 100% total moles of lipid in the lipid nanoparticle).

Polymer conjugated lipid - aggregation reducing agent

The aggregation reducing agent is preferably a lipid capable of reducing aggregation. Examples of such lipids include, but are not limited to, polyethylene glycol (PEG)-modified lipids, monosialoganglioside Gml, and polyamide oligomers (RAO) such as those described in U.S. Patent No. 6,320,017, which is incorporated by reference in its entirety. Other compounds with uncharged, hydrophilic, steric-barrier moieties, which prevent aggregation during formulation, like PEG, Gml or ATTA, can also be coupled to lipids. ATTA-lipids are described, e.g., in U.S. Patent No. 6,320,017, and PEG-lipid conjugates are described, e.g., in U.S. Patent Nos. 5,820,873, 5,534,499 and 5,885,613, each of which is incorporated by reference in its entirety.

The aggregation reducing agent may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkylglycerol, a PEG- dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof (such as PEG-Cerl4 or PEG-Cer20). The PEG-DAA conjugate may be, for example, a PEG- dilauryloxypropyl (C12), a PEG- dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG- distearyloxypropyl (C18). Other pegylated-lipids include, but are not limited to, polyethylene glycol-didimyristoyl glycerol (C14- PEG or PEG-C14, where PEG has an average molecular weight of 2000 Da) (PEG-DMG); (R)-2,3- bis(octadecyloxy)propyl-1 -(methoxy polyethylene glycol)2000)propylcarbamate) (PEG-DSG); PEG- carbamoyl-1 ,2- dimyristyloxypropylamine, in which PEG has an average molecular weight of 2000 Da (PEG- eDMA); N-Acetylgalactosamine-((R)-2,3-bis(octadecyloxy)propyl-1- (methoxypoly(ethylene glycol)2000)propylcarbamate)) (GalNAc-PEG-DSG); mPEG (mw2000)- diastearoylphosphatidyl-ethanolamine (PEG-DSPE); and polyethylene glycol - dipalmitoylglycerol (PEG-DPG). In one embodiment, the aggregation reducing agent is PEG- DMG. In another embodiment, the aggregation reducing agent is PEG-c-DMA.

The liposome formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. In one example by Semple et al. (Semple et al. Nature Biotech. 2010 28: 172-176; herein incorporated by reference in its entirety), the liposome formulation was composed of 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA. As another example, changing the pharmaceutical composition of the cationic lipid could more effectively deliver siRNA to various antigen presenting cells (Basha et al. Mol Ther. 2011 19:2186-2200; herein incorporated by reference in its entirety). In some embodiments, liposome formulations may comprise from about 35 to about 45% cationic lipid, from about 40% to about 50% cationic lipid, from about 50% to about 60% cationic lipid and/or from about 55% to about 65% cationic lipid. In some embodiments, the ratio of lipid to mRNA in liposomes may be from about 5: 1 to about 20: 1 , from about 10: 1 to about 25: 1 , from about 15: 1 to about 30: 1 and/or at least 30: 1 .

The average molecular weight of the PEG moiety in the PEG-modified lipids preferably ranges from about 500 to about 8,000 Daltons (e.g., from about 1 ,000 to about 4,000 Daltons). In one preferred embodiment, the average molecular weight of the PEG moiety is about 2,000 Daltons.

The concentration of the aggregation reducing agent preferably ranges from about 0.1 mol% to about 15mol%, based upon the 100% total moles of lipid in the lipid particle. In one embodiment, the formulation includes less than about 3, 2, or 1 mole percent of PEG or PEG-modified lipid, based upon the total moles of lipid in the lipid particle.

In another embodiment, the lipid nanoparticles include from about 0.1 % to about 20% on a molar basis of the PEG-modified lipid, e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 10%, about 5%, about 3.5%, about 1.5%, about 0.5%, or about 0.3% on a molar basis (based on 100% total moles of lipids in the lipid nanoparticle).

In some embodiments, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a PEGylated lipid. The term “PEGylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s-DMG) and the like.

A polymer conjugated lipid as defined herein, e.g. a PEG-lipid, may serve as an aggregation reducing lipid.

In certain embodiments, the LNP comprises a stabilizing-lipid which is a polyethylene glycol-lipid (PEGylated lipid). Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g. PEG- CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG- modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c- DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxy polyethylene glycol)2000)carbamyl]-1 ,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In a preferred embodiment, the polyethylene glycol-lipid is PEG-2000-DMG. In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a PEGylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-0-(2’,3'-di(tetradecanoyloxy)propyl-1 -0-(ω -methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as ω- methoxy(polyethoxy)ethyl-N-(2,3di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl- N-(ω)-methoxy(polyethoxy)ethyl)carbamate.

In preferred embodiments, the PEGylated lipid is preferably derived from formula (IV) of published PCT patent application WO2018078053. Accordingly, PEGylated lipids derived from formula (IV) of published PCT patent application WO2018078053, and the respective disclosure relating thereto, are herewith incorporated by reference. In a particularly preferred embodiments, the at least one nucleic acid (e.g. RNA or DNA) of the composition is complexed with one or more lipids thereby forming LNPs, wherein the LNP comprises a PEGylated lipid, wherein the PEG lipid is preferably derived from formula (IVa) of published PCT patent application WO2018078053. Accordingly, PEGylated lipid derived from formula (IVa) of published PCT patent application WO2018078053, and the respective disclosure relating thereto, is herewith incorporated by reference.

In a particularly preferred embodiment, the at least one nucleic acid, preferably the at least one RNA is complexed with one or more lipids thereby forming lipid nanoparticles (LNP), wherein the LNP comprises a PEGylated lipid / PEG lipid. Preferably, said PEG lipid is of formula (IVa):

wherein n has a mean value ranging from 30 to 60, such as about 30±2, 32±2, 34±2, 36±2, 38±2, 40±2, 42±2, 44±2, 46±2, 48±2, 50±2, 52±2, 54±2, 56±2, 58±2, or 60±2. In a most preferred embodiment n is about 49. In further preferred aspects said PEG lipid is of formula (IVa) wherein n is an integer selected such that the average molecular weight of the PEG lipid is about 2000g/mol to about 3000 g/mol or about 2300g/mol to about 2700g/mol, even more preferably about 2500g/mol.

The lipid of formula IVa as suitably used herein has the chemical term 2[(polyethylene glycol)-2000]- N,N-ditetradecylacetamide, also referred to as ALC-0159.

Further examples of PEG-lipids suitable in that context are provided in US20150376115A1 and WO2015199952, each of which is incorporated by reference in its entirety.

In some embodiments, LNPs include less than about 3, 2, or 1 mole percent of PEG or PEG-modified lipid, based on the total moles of lipid in the LNP. In further embodiments, LNPs comprise from about 0.1 % to about 20% of the PEG-modified lipid on a molar basis, e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 10%, about 5%, about 3.5%, about 3%, about 2,5%, about 2%, about 1.5%, about 1%, about 0.5%, or about 0.3% on a molar basis (based on 100% total moles of lipids in the LNP). In preferred embodiments, LNPs comprise from about 1.0% to about 2.0% of the PEG- modified lipid on a molar basis, e.g., about 1.2 to about 1.9%, about 1.2 to about 1.8%, about 1.3 to about 1 .8%, about 1 .4 to about 1 .8%, about 1 .5 to about 1 .8%, about 1 .6 to about 1 .8%, in particular about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, most preferably 1.7% (based on 100% total moles of lipids in the LNP). In various embodiments, the molar ratio of the cationic lipid to the PEGylated lipid ranges from about 100:1 to about 25:1. Encapsulation/Complexation in LNPs:

In preferred embodiments of the aspects of the invention, the at least one nucleic acid (e.g. DNA or RNA), preferably the at least one RNA, and optionally the at least one further nucleic acid, is complexed, encapsulated, partially encapsulated, or associated with one or more lipids (e.g. cationic lipids and/or neutral lipids), thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes.

The liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes - incorporated nucleic acid (e.g. DNA or RNA) may be completely or partially located in the interior space of the liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes, within the lipid layer/membrane, or associated with the exterior surface of the lipid layer/membrane. The incorporation of a nucleic acid into liposomes/LNPs is also referred to herein as "encapsulation" wherein the nucleic acid, e.g. the RNA is entirely contained within the interior space of the liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes. The purpose of incorporating nucleic acid into liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes is to protect the nucleic acid, preferably RNA from an environment which may contain enzymes or chemicals or conditions that degrade nucleic acid and/or systems or receptors that cause the rapid excretion of the nucleic acid. Moreover, incorporating nucleic acid, preferably RNA into liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes may promote the uptake of the nucleic acid, and hence, may enhance the therapeutic effect of the nucleic acid, e.g. the mRNA medicine(s) for use in the therapy and prevention of Hereditary Tyrosinemia Type I (HT 1 ), and more particularly to mRNA medicines of this kind which can exhibit excellent therapeutic and preventive effects with respect to Hereditary Tyrosinemia Type I (HT1). Accordingly, incorporating a nucleic acid, e.g. RNA or DNA, into liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes may be particularly suitable for mRNA medicines for use in the therapy and prevention of Hereditary Tyrosinemia Type I (HT1), and more particularly to mRNA medicines of this kind which can exhibit excellent therapeutic and preventive effects with respect to Hereditary Tyrosinemia Type I (HT1 ) individually developed or to complications resulting from diseases of these organs, e.g. for intravenous administration.

In this context, the terms “complexed" or “associated" refer to the essentially stable combination of nucleic acid with one or more lipids into larger complexes or assemblies without covalent binding.

The term “lipid nanoparticle”, also referred to as “LNP”, is not restricted to any particular morphology, and include any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g. in an aqueous environment and/or in the presence of a nucleic acid, e.g. an RNA. For example, a liposome, a lipid complex, a lipoplex and the like are within the scope of a lipid nanoparticle (LNP).

Liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50nm and 500nm in diameter.

LNPs of the invention are suitably characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers. Bilayer membranes of LNPs are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains. Bilayer membranes of the liposomes can also be formed by amphophilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, an LNP typically serves to transport the at least one nucleic acid, preferably the at least one RNA to a target tissue.

Accordingly, in preferred embodiments of all aspects of the invention, the at least one nucleic acid, preferably the at least one RNA is complexed with one or more lipids thereby forming lipid nanoparticles (LNP). Preferably, said LNP is particularly suitable for intramuscular, intradermal administration, subcutaneous, or intravenous injection, most preferably for intravenous injection i.e. intravenous infusion or respectively intravenous therapy (as IV therapy).

Alternatively, the pharmaceutical composition may be provided in solid form. In particular, it may be provided as a sterile solid composition for reconstitution with a sterile liquid carrier; the solid composition may in this case further comprise one or more inactive ingredients selected from pH- modifying agents, bulking agents, stabilizers, non-ionic surfactants and antioxidants. In this embodiment, the sterile liquid carrier is preferably an aqueous carrier.

The zeta potential of a nanoparticle composition may be used to indicate the electrokinetic potential of the pharmaceutical composition. For example, the zeta potential may describe the surface charge of a nanoparticle composition. The lipid nanoparticles according to the invention may, due to the presence of both negatively and positively charged compounds, exhibit a relatively neutral zeta potential. The zeta potential (sometimes abbreviated as “charge") may be determined along with the particle size of the particles, for example, by dynamic light scattering and Laser Doppler Microelectrophoresis, for example using a Malvern Zetasizer Nano (Malvern Instruments Ltd.; Malvern, UK). Depending on the amount and nature of charged compounds in the lipid nanoparticles, the nanoparticles may be characterized by a zeta potential. In a preferred embodiment, the zeta potential is in the range from about -50mV to about +50mV. In other preferred embodiments, the zeta potential is in the range from about -25mV to about +25mV. In some embodiments, the zeta potential of a lipid nanoparticle of the invention may be from about -1 OmV to about +20mV, from about -1 OmV to about +15mV, from about -10mV to about +10mV, from about -10mV to about +5mV, from about -10mV to about OmV, from about -10mV to about -5mV, from about -5mV to about +20mV, from about -5mV to about +15mV, from about -5mV to about +10mV, from about -5mV to about +5mV, from about -5mV to about OmV, from about OmV to about +20mV, from about OmV to about +15mV, from about 0mV to about +10mV, from about 0mV to about +5mV, from about +5mV to about +20mV, from about +5mV to about +15mV, or from about +5mV to about +10mV.

In certain embodiments, the LNP comprises one or more targeting moieties which are capable of targeting the LNP to a cell or cell population. For example, in one embodiment, the targeting moiety is a ligand which directs the LNP to a receptor found on a cell surface.

In certain embodiments, the LNP comprises one or more internalization domains. For example, in one embodiment, the LNP comprises one or more domains which bind to a cell to induce the internalization of the LNP. For example, in one embodiment, the one or more internalization domains bind to a receptor found on a cell surface to induce receptor-mediated uptake of the LNP. In certain embodiments, the LNP is capable of binding a biomolecule in vivo, where the LNP-bound biomolecule can then be recognized by a cell-surface receptor to induce internalization. For example, in one embodiment, the LNP binds systemic ApoE, which leads to the uptake of the LNP and associated cargo. In certain embodiments of the invention, ApoE may be supplemented to the medium or pharmaceutical composition used.

Further preferred polymer conjugated lipids are described in PCT/EP2022/074439, the full disclosure herewith incorporated by reference. In particular, the disclosure relating to polymer conjugated lipids as shown in any one of claims 1 to 8 the disclosure relating to polymer conjugated lipids as shown in any one of claims 9 to 46 of PCT/EP2022/074439 are incorporated by reference.

Preferably, in one embodiment, the pharmaceutical compositions of the invention further comprise a biologically active ingredient.

Pharmaceutical composition

In a further aspect, the present invention concerns a composition or a pharmaceutical composition comprising the mRNA according to the invention as described herein. The pharmaceutical composition according to the invention thus comprises an RNA comprising at least one coding sequence, wherein the coding sequence encodes at least one peptide or protein as described herein, preferably a FAH protein selected, or a fragment or a variant of any of a FAN protein, having the biological activity of a wild type FAH protein, as defined herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition according to the invention is preferably provided as a pharmaceutical composition.

With respect to the mRNA comprised in the pharmaceutical composition, reference is made to the description of the mRNA according to the invention, which applies to the pharmaceutical composition. The pharmaceutical composition according to the invention preferably comprises at least one RNA according to the invention as described herein. In alternative embodiments, the pharmaceutical composition comprises at least two species of the mRNA according to the invention.

In a preferred embodiment, the pharmaceutical composition of the present invention may comprise at least one RNA according to the invention, wherein the at least one RNA encodes at least two, three, four, five, six, seven, eight, nine or more distinct peptides or proteins as defined herein or a fragment or variant thereof. Preferably, the pharmaceutical composition comprises several species, more preferably at least two, three, four, five, six, seven, eight, nine or more species, of the mRNA according to the invention, wherein each RNA species encodes one of the peptides or proteins or a fragment or variant thereof as defined herein. In another embodiment, the mRNA comprised in the pharmaceutical composition is a bi- or multicistronic RNA as defined herein, which encodes the at least two, three, four, five, six, seven, eight, nine or more distinct peptides or proteins. Mixtures between these embodiments are also envisaged, such as compositions comprising more than one RNA species, wherein at least one RNA species may be monocistronic, while at least one other RNA species may be bi- or multicistronic.

The pharmaceutical composition according to the present invention, preferably the at least one coding sequence of the mRNA comprised therein, may thus comprise any combination of the nucleic acid sequences as defined herein.

In a preferred embodiment of the pharmaceutical composition according to the invention, the mRNA as described herein is complexed with one or more cationic or polycationic compounds, preferably with cationic or polycationic polymers, cationic or polycationic peptides or proteins, e.g. protamine, cationic or polycationic polysaccharides and/or cationic or polycationic lipids.

In some embodiments, the mRNA may be formulated as saline or lipid formulation. According to a preferred embodiment, the mRNA according to the present invention may be complexed with lipids to form one or more liposomes, lipoplexes, or lipid nanoparticles. Therefore, in one embodiment, the inventive composition comprises liposomes, lipoplexes, and/or lipid nanoparticles comprising the mRNA according to the invention. In one embodiment the mRNA according to the present invention is complexed with cationic lipids and/or neutral lipids and thereby forms liposomes, lipid nanoparticles, lipoplexes or neutral lipid-based nanoliposomes.

In a preferred embodiment, the lipid formulation is thus selected from the group consisting of liposomes, lipoplexes, copolymers such as RIGA and lipid nanoparticles.

In one preferred embodiment, a lipid nanoparticle (LNP) comprises: a) an RNA comprising at least one coding sequence as defined herein, b) a cationic lipid, c) an aggregation reducing agent (such as polyethylene glycol (PEG) lipid or PEG-modified lipid), d) optionally a non-cationic lipid (such as a neutral lipid), and e) optionally, a sterol.

In one embodiment, the lipid nanoparticle formulation consists of (i) at least one cationic lipid; (ii) a neutral lipid; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid.

In one embodiment, the nucleic acids may be formulated in an aminoalcohol lipidoid. Aminoalcohol lipidoids which may be used in the present invention may be prepared by the methods described in U.S. Patent No. 8,450,298, herein incorporated by reference in its entirety.

In another aspect, the present invention relates to a method of treating, preventing, attenuating or inhibiting Hereditary Tyrosinemia Type I (HT1), comprising administering to a human subject in need the mRNA of the invention, the LNP of the invention, the pharmaceutical composition of the invention, or the kit or kit of parts of the invention, wherein the administration results in treatment, prevention, attenuation, inhibition, or prophylaxis of the disease.

In another embodiment, an isolated mRNA encoding fumarylacetoacetate hydrolase (FAH), a lipid nanoparticle (LNP) comprising said mRNA, a pharmaceutical composition or a kit or kit of parts according to the disclosure are provided for reducing pathologically increased Succinylacetone (SA) and/or Tyrosine (TYR) levels in a patient in need.

Antagonists of RNA sensing pattern recognition receptors (single stranded oligonucleotides)

In preferred embodiments, the pharmaceutical composition comprises at least one antagonist of at least one RNA sensing pattern recognition receptor.

In preferred embodiments in that context, the pharmaceutical composition comprises at least one antagonist of at least one RNA sensing pattern recognition receptor selected from a Toll-like receptor, preferably a TLR7 antagonist and/or a TLR8 antagonist.

Suitable antagonist of at least one RNA sensing pattern recognition receptor are disclosed in published PCT patent application WO2021028439, the full disclosure herewith incorporated by reference. In particular, the disclosure relating to suitable antagonist of at least one RNA sensing pattern recognition receptors as defined in any one of the claim 1 to claim 94 of WO2021028439 and SEQ ID NOs: 85-212 of WO2021028439 are incorporated by reference.

In preferred embodiments, the at least one antagonist of at least one RNA sensing pattern recognition receptor is a single stranded oligonucleotide that comprises or consists of a nucleic acid sequence being identical or at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 85-212 of WO2021028439, or fragments of any of these sequences. A particularly preferred antagonist in that context is 5’-GAG CGmG CCA-3’ (SEQ ID NO: 85 of WO2021028439), or a fragment or variant thereof, whereby “Gm” in 5’-GAG CGmG CCA-3’ (SEQ ID NO: 85 of WO2021028439) preferably is a 2-O-methylated guanosine.

In preferred embodiments, the molar ratio of the at least one antagonist of at least one RNA sensing pattern recognition receptor to the at least one RNA suitably ranges from about 20:1 to about 80:1.

In preferred embodiments, the weight to weight ratio of the at least one antagonist of at least one RNA sensing pattern recognition receptor to the at least one RNA suitably ranges from about 1 :2 to about 1 :10.

In embodiments, the at least one antagonist of at least one RNA sensing pattern recognition receptor and the at least one RNA are separately formulated (e.g. in LNPs) as defined herein or co-formulated (e.g. in LNPs) as defined herein.

Routes of administration

In a further embodiment, the method of the invention relates to the mRNA of the invention, or the LNP of the invention, or the pharmaceutical composition of the invention or the kit or kit of parts of the invention, being administered to the subject by subcutaneous, intramuscular or intravenous administration, preferably by intramuscular or intravenous administration. In a specific embodiment, the method of the invention relates to the mRNA of the invention, or the LNP of the invention, or the pharmaceutical composition of the invention or the kit or kit of parts of the invention, being administered to the subject by intravenous administration. In another specific and inventive embodiment, the method of the invention relates to the mRNA of the invention, or the LNP of the invention, or the pharmaceutical composition of the invention or the kit or kit of parts of the invention, being administered to the subject by intramuscular administration.

As described above, in contrast to the expectation in the art (which was related nearly exclusively to intravenous administration for treating defects based on a genetic or enzyme defect), surprisingly, the inventors found that the objects underlying the present invention were solved by the methods of the invention relating to the mRNA of the invention, or the LNPs of the invention, or the pharmaceutical compositions of the invention or the kit or kit of parts of the invention, being administered to the subject by intramuscular administration for treating, attenuating or inhibiting Hereditary Tyrosinemia Type I. The choice of a pharmaceutically acceptable carrier is determined, in principle, by the manner, in which the pharmaceutical composition according to the invention is administered. The pharmaceutical composition of the invention can be administered, for example, systemically or locally. Routes for systemic administration in general include, for example, transdermal, oral, parenteral routes, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injections and/or intranasal administration routes.

Routes for local administration in general include, for example, topical administration routes but also intradermal, transdermal, subcutaneous, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, intratumoral and sublingual injections. Administration to the respiratory system can be performed by spray administration or inhalation may in particular be performed by aerosol administration to the lungs, bronchi, bronchioli, alveoli, or paranasal sinuses.

In further preferred embodiments, the route of administration is selected from the group consisting of extravascular administration to a subject, such as by extravascular injection, infusion or implantation; topical administration to the skin or a mucosa; inhalation such as to deliver the pharmaceutical composition to the respiratory system; or by transdermal or percutaneous administration. In even further preferred embodiments, the pharmaceutical composition of the invention can be administered via local or locoregional injection, infusion or implantation, in particular intradermal, subcutaneous, intramuscular, intracameral, subconjunctival, suprachoroidal injection, subretinal, subtenon, retrobulbar, topical, posterior juxtascleral administration, or intrapulmonal inhalation, interstitial, locoregional, intravitreal, intratumoral, intralymphatic, intranodal, intra-articular, intrasynovial, periarticular, intraperitoneal, intra-abdominal, intracardial, intralesional, intrapericardial, intraventricular, intrapleural, perineural, intrathoracic, epidural, intradural, peridural, intrathecal, intramedullary, intracerebral, intracavernous, intracorporus cavernosum, intraprostatic, intratesticular, intracartilaginous, intraosseous, intradiscal, intraspinal, intracaudal, intrabursal, intragingival, intraovarian, intrauterine, periocular, periodontal, retrobulbar, subarachnoid, subconjunctival or suprachoroidal injection, infusion or implantation.

Preferably, compositions according to the present invention may be administered by an intradermal, subcutaneous, intramuscular or intravenous route, preferably by injection, which may be needle-free and/or needle injection. Compositions according to the present invention are therefore preferably formulated in liquid or solid form. The suitable amount of the composition according to the invention to be administered can be determined by routine experiments, e.g. by using animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog and non-human primate models.

Preferred unit dose forms for injection include sterile solutions of water, physiological saline or mixtures thereof. The pH of such solutions should be adjusted to a physiologically tolerable pH, such as about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid and collagen matrices. Suitable pharmaceutically acceptable carriers for topical application include those which are suitable for use in lotions, creams, gels and the like. If the inventive composition is to be administered perorally, tablets, capsules and the like are the preferred unit dose form. The pharmaceutically acceptable carriers for the preparation of unit dose forms which can be used for oral administration are well known in the prior art. The choice thereof will depend on secondary considerations such as taste, costs and storability, which are not critical for the purposes of the present invention, and can be made without difficulty by a person skilled in the art.

The present application is filed together with a sequence listing in electronic format, which is part of the description of the present application (WIPO standard ST.26). The information contained in the sequence listing is incorporated herein by reference in its entirety. Where reference is made herein to a “SEQ ID NO”, the corresponding nucleic acid sequence or amino acid (aa) sequence in the sequence listing having the respective identifier is referred to. For many sequences, the sequence listing also provides additional detailed information, e.g. regarding certain structural features, sequence optimizations, GenBank (NCBI) or GISAID (epi) identifiers, or additional detailed information regarding its coding capacity. In particular, such information on the specific sequences is provided under "feature key”, i.e. “source” (for nucleic acids or proteins) or “misc_feature” (for nucleic acids) or "REGION” (for proteins).

Here, and throughout the whole specification, it has to be noted that the priority application(s) were filed with a sequence listing in accordance with the WIPO Standard ST.25, which then was converted into a sequence listing according to WIPO Standard ST.26 - information which was comprised within line <223> in ST.25 now was added to the respective SEO ID NO: as a note under “feature key”, i.e. “misc_feature” (for nucleic acids) or “REGION” (for proteins)]. Sequences, which are disclosed in the sequence listing as chemically unmodified sequences comprising uracil may also be used in conjunction with chemically modified nucleic acids as described above, e.g. preferably modified uridine/uracil, most preferably N1 MPU. I.e. all sequences as disclosed in the sequence listing may be used in a preferred embodiment or preferred aspect of the invention as N1MPU-modified sequences.

ITEMS

Additional items of the present disclosure include the following:

Item 1. An isolated mRNA encoding fumarylacetoacetate hydrolase (FAH) for use in treating, attenuating or inhibiting Hereditary Tyrosinemia Type I (HT1 ). item 2. The mRNA of Item 1 , wherein said mRNA comprises an open reading frame (ORF) encoding FAN comprising an amino acid sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 100, or a fragment or variant of said sequences having the biological activity of a FAH protein.

Item 3. The mRNA according to any one of Item 1 to Item 2, wherein said mRNA preferably has at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any single SEQ ID NO-element of SEQ ID NO:112 to SEQ ID NO:144 or SEQ ID NQ: 101 to SEQ ID NO: 111 , or a fragment or variant of said sequences, wherein the encoded protein has the biological activity of a FAH protein.

Item 4. The mRNA of any one of Item 1 to Item 3 for use according to Item 1 , further comprising an UTR combination selected from the group consisting of (i) a 5 -UTR derived from a mouse solute carrier family 7 (cationic amino acid transporter, y+ system) (SLC7A3) and a 3'-UTR derived from PSMB3; (ii) a 5 -UTR derived from mouse ribosomal protein L31 (RPL31) and a 3 -UTR derived from a human ribosomal protein S9 (RPS9); (iii) a 5'-UTR derived from ubiquilin 2 (Ubqln2) and a 3’-UTR derived from Guanine nucleotide-binding protein G(s) subunit alpha isoforms short (Gnas); and (iv) a 5’-UTR derived from a hydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4) and a 3 -UTR derived from a proteasome subunit beta type-3 (PSMB3) UTR.

Item 5. The mRNA according to any one of Item 1 to item 4, wherein the

(i) G/C content of the FAH coding sequence in said mRNA is increased compared to the coding sequence of the corresponding wild type FAH coding sequence of SEQ ID NO: 101 ;

(ii) C content of the FAH coding sequence in said mRNA is increased compared to the coding sequence of the corresponding wild type FAH coding sequence of SEQ ID NO: 101 ; and/or wherein

(iii) at least one codon of the FAH coding sequence in said mRNA is adapted to human codon usage, wherein the codon adaptation index (CAI) is preferably increased or maximised in the corresponding FAH coding sequence compared to the coding sequence of the corresponding wild type FAH coding sequence of SEQ ID NO:101.

Item 6. The mRNA according to any one of Item 1 to Item 5, wherein the mRNA comprises a 5’-cap structure, a poly(A) sequence comprising at least 70 A nucleotides, preferably about 100 A nucleotides, a poly(C) sequence, preferably comprising 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides, and/or at least one histone stem-loop, preferably, wherein the mRNA comprises a 3’-terminal A nucleotide.

Item 7. The mRNA according to any one of Item 1 to Item 6, wherein the mRNA comprises, preferably in 5’ to 3’ direction, the following elements: a) a 5’-cap1 structure; b) a 5’-UTR element comprising a nucleic acid sequence, preferably derived from a 5’-UTR of a HSD17B4 gene, comprising the nucleic acid sequence according to SEQ ID NO:1 or 2, or a homolog, a fragment or a variant thereof; c) at least one coding sequence as defined in any one of Item 1 to Item 10; d) a 3’-UTR element comprising a nucleic acid sequence, preferably derived from a 3’-UTR of a PSMB3 gene, comprising the nucleic acid sequence according to SEQ ID NO:33 or 34, or a homolog, a fragment or a variant thereof; e) a poly(A) sequence comprising about 100 adenosine nucleotides, preferably, wherein the mRNA comprises a 3’-terminal A nucleotide; f) an optional poly(C) tail, preferably comprising 10 to 40 cytosine nucleotides; and/or g) an optional histone stem-loop, preferably comprising the nucleic acid sequence according to

SEQ ID NO:63 or 64.

Item 8. The mRNA according to any one of Item 1 to Item 7, wherein the open reading frame does not comprise any chemically modified uracil or cytosine nucleotides.

Item 9. The mRNA according to any one of Item 1 to Item 7, wherein the mRNA is chemically modified, preferably wherein the mRNA comprises pseudouridine (psi-uridine), N1- methylpseudouridine (N1 MPU), 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3- methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5- methylcytidine, 2-aminoadenosine, 7-deazaadenosme, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, O(6)-methylguanine, and/or 2-thiocytidine, more preferably wherein all uridine bases of the mRNA are fully chemically modified, even more preferably wherein all uridine bases of the mRNA are pseudouridine or N1-methylpseudouridine (N1 MPU) bases, most preferably wherein all uridine bases of the mRNA are N1-methylpseudouridine (N1 MPU) bases.

Item 10. A lipid nanoparticle (LNP) comprising the mRNA according to any one of Item 1 to Item 9, wherein the LNP comprises an ionizable or cationic lipid, a phospholipid, a structural lipid, and a polymer conjugated lipid.

Item 11 . The LNP according to Item 10, wherein the lipids comprised in the LNP have a molar ratio of about 20-60% cationic or ionizable lipid, about 5-25% non-cationic lipid, about 25-55% sterol and about 0.5-15% polymer conjugated lipid. Item 12. The LNP according to anyone of Item 10 to Item 11 , wherein the LNP does not comprise polyethylene glycol (PEG) or a PEG-modified lipid.

Item 13. A pharmaceutical composition, comprising the mRNA according to any one of Item

1 to Item 9 or the LNP according to any one of Item 10 to Item 12.

Item 14. A kit, preferably kit of parts, comprising at least one mRNA according to any one of Item 1 to Item 9, the LNP according to any one of Item 10 to Item 12, or the pharmaceutical composition according to Item 13, and optionally a liquid vehicle for solubilising and optionally technical instructions with information on the administration and dosage of the pharmaceutical composition.

Item 15. A method of treating, attenuating or inhibiting Hereditary Tyrosinemia Type I (HT1 ), comprising administering to a human subject in need the mRNA according to any one of Item 1 to Item 9, the LNP according to any one of Item 10 to Item 12, the pharmaceutical composition according to Item 13, or the kit or kit of parts according to Item 14, wherein the administration results in treatment, prevention, attenuation, inhibition, or prophylaxis of Hereditary Tyrosinemia Type I (HT1 ). item 16. The method according to Item 15, wherein the mRNA according to any one of Item 1 to Item 9, or the LNP according to any one of item 10 to Item 12, or the pharmaceutical composition according to Item 13 or the kit or kit of parts according to Item 14 is administered to the subject by subcutaneous, intramuscular or intravenous administration, preferably by intramuscular or intravenous administration.

Item 17. The method according to any one of Item 15 to Item 16, wherein the mRNA comprises a 5’- or 3’-untranslated region (UTR) comprising at least one microRNA-binding site, preferably not being a microRNA-122 (miR-122) binding site, more preferably being miR-16, miR- 21 , miR-24, miR-27, miR-30c, miR-132, miR-133, miR-149, miR-192, miR-194, miR-204, miR-206, miR-208, or miR-223, most preferably being miRNA-148a, miRNA-101 , miRNA-192 or miRNA-194, miR-126, miR-142-3p, or miR-142-5p.

Item 18. The method according to any one of Item 15 to Item 17, wherein the method of treating, attenuating or inhibiting Hereditary Tyrosinemia Type I (HT1), involves a single administration of the mRNA, the LNP, the pharmaceutical composition or the kit or kit of parts.

Item 19. The method according to any one of Item 15 to Item 18, wherein the mRNA, the

LNP, the pharmaceutical composition or the kit or kit of parts is administered (a) once, preferably more than once, more preferably wherein administration is repeated for a period of at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least one year, or lifelong; or

Item 20. An isolated mRNA according to any one of Item 1 to Item 9, or LNP according to any one of Item 10 to Item 12 or pharmaceutical composition according to Item 13 or a kit or kit of parts according to Item 14, for use as a medicament.

Item 21. A vector comprising the isolated mRNA according to any one of Item 1 to Item 9, preferably a DNA vector.

Item 22. A host celi carrying the vector of Item 21 .

Item 23. An isolated mRNA encoding fumarylacetoacetate hydrolase (FAN) according to any one of Item 1 to Item 9, a lipid nanoparticle (LNP) according to any one of Item 10 to Item 12, a pharmaceutical composition according to Item 13 or a kit or kit of parts according to Item 14, for reducing pathologically increased Succinylacetone (SA) and/or Tyrosine (TYR) levels in a patient in need.

EXAMPLES

In the following section, particular examples illustrating various embodiments and aspects of the invention are presented. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the claims as disclosed herein.

Table of contents for Examples

Example 1 : Single intravenous and intramuscular injections of PpLuc mRNA-LNPs in a mouse model of Hereditary Tyrosinemia Type I

Experimental Setup:

The FAH mutant mouse model was chosen as a mouse model representing the human disease Hereditary Tyrosinemia Type I. This mouse model was

available upon cryorecovery (abbreviated FAH mice). The mutation present in this Tyrosinemia mouse model is a G-to-A transition at the last base of exon 7 leading to the splicing of exon 6 to exon 8, and resulting in a transcript that lacks exon 7. The absence of exon 7 in the transcript results in a frameshift and subsequently the introduction of a premature stop codon at amino acid position 303 (https://www.jax.org/strain/018129). To avoid early postnatal lethality of FAH mice, NTBC was supplemented via drinking water to pregnant and nursing females and to homozygotes throughout life until otherwise stated. Both female and male FAH mice at 10-12 weeks of age were included in the in vivo studies. To induce the disease phenotype in FAH mice, NTBC supplementation was withdrawn 5 days before start of treatment in all experimental cohorts. To improve animal's health after NTBC withdrawal, water-soaked food and water bottles with long neck were provided to FAH mice.

An RNA sequence encoding Luciferase (PpLuc mRNA; SEQ ID NO:145, see Table C2: “Constructs of the invention”) was designed and synthesized in vitro. PpLuc mRNA, as well as all other mRNAs described herein, was formulated into lipid nanoparticles (LNPs) as described herein below for single intravenous or intramuscular injections into FAH mice.

LNP Formulation

Lipid nanoparticles, cationic lipids and polymer conjugated lipids (PEG-lipid) were prepared and tested according to the general procedures described in PCT Pub. Nos. WO 2015/199952, WO 2017/004143 and WO 2017/075531 , the full disclosures of which are incorporated herein by reference. Lipid nanoparticle (LNP)-formulated mRNA was prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. LNPs were prepared as follows. Cationic lipid, DSPC, cholesterol and PEG-lipid were solubilized in ethanol at a molar ratio of approximately 47.4 : 10 : 40.9 : 1 .7.

LNPs for the Examples included, cationic lipid compound III-3

(compound III-3), a PEG lipid according to formula (IV)

and the other foregoing components like cholesterol and DSPC.

Lipid nanoparticles (LNP) comprising compound III-3 were prepared at a ratio of mRNA to Total Lipid of 0.03-0.04 w/w. Briefly, the mRNA was diluted to 0.05 to 0.2mg/mL in 10 to 50mM citrate buffer, pH 4. Syringe pumps were used to mix the ethanolic lipid solution with the mRNA aqueous solution at a ratio of about 1 :5 to 1 :3 (vol/vol) with total flow rates above 15ml/min. The ethanol was then removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2pm pore sterile filter. Lipid nanoparticle particle diameter size was 60-90 nm as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK).

Alternatively, LNPs were prepared using the NanoAssemblr™ microfluidic system (Precision NanoSystems Inc., Vancouver, BC) according to standard protocols which enables controlled, bottom-up, molecular self-assembly of nanoparticles via custom-engineered microfluidic mixing chips that enable millisecond mixing of nanoparticle components at a nanolitre scale. Briefly, mRNA as indicated in the working examples, was diluted to 0.05 to 0.2 mg/ml in 50 mM acetate buffer, pH 4. Syringe pumps were installed into inlet parts of the NanoAssemblr™ (Precision NanoSystems Inc., Vancouver, BC) and used to mix the ethanolic lipid solution with the mRNA aqueous solution at a ratio of about 1 :5 to 1 :3 (vol/vol) with total flow rates from about 14 ml/min to about 18 ml/min. The ethanol was then removed and the external buffer replaced with PBS/sucrose buffer (pH 7.4, 75 mM NaCI, 10mM phosphate, 150 mM sucrose) by dialysis (Slide- A-Lyzer™ Dialysis Cassettes, ThermoFisher). Finally, the lipid nanoparticles were filtered through a 0.2 pm pore sterile filter. Lipid nanoparticle particle diameter size was from about 90 nm to about 140 nm as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern Instruments Ltd.; Malvern, UK). For other cationic lipid compounds mentioned in the present specification, the formulation process is similar. For all LNPs, the ethanol was then removed and buffer replaced by 10 mM PBS, pH 7.4 comprising 9% Sucrose.

For in vivo imaging, substrate Luciferin was administered intraperitoneally into FAH mice 10 minutes before start of image recording. In vivo imaging was performed six hours after single intravenous injection. FAH mice were terminated, and livers and muscles were harvested for analysis of Luciferase expression in tissue lysates.

The detailed Study design of the present working example is depicted in Table Ex-1 .

Table Ex-1 : Study design.

The biodistribution of PpLuc mRNA and subsequent Luciferase protein expression was assessed upon single intravenous or intramuscular injections of PpLuc mRNA-LNPs in FAH mice. For evaluation of Luciferase expression, PpLuc mRNA (formulated in Lipid Nanoparticles (LNPs) (diluted in Phosphate Buffered Saline (PBS)) was injected into FAH mice (n=5 FAH mice) intravenously (iv) or intramuscularly (im) on day D0. In vivo images of Luciferase expression in livers and muscles were obtained 6 hours after single injections. Animals in group 1 (n=5): Single intravenous administration of PpLuc mRNA formulated in Lipid Nanoparticles (diluted in PBS). FAH mice were terminated 6 hours after single injection.

Animals in group 2 (n=5): Single intramuscular administration of PpLuc mRNA formulated in Lipid Nanoparticles (diluted in PBS). FAH mice were terminated 6 hours after single injection.

Animals in group 3 (n=5): Single intravenous and intramuscular administration of PBS. FAH mice were terminated 6 hours after single injection.

Results:

Luciferase expression was predominantly expressed in FAH mouse livers upon intravenous injection of PpLuc mRNA-LNPs. Luciferase expression was detected in muscles after intramuscular injection of PpLuc mRNA-LNPs, however a substantial amount of Luciferase was also detected in FAH mouse livers, suggesting transport of PpLuc mRNA-LNPs to the liver via blood stream, resulting in successful uptake and expression in liver tissue. Further details can be found in Figures 1A to 1 D and the respective figure legend(s).

Example 2: Single intravenous injection of human FAH mRNA-LNPs in a mouse model of Hereditary Tyrosinemia Type I

Experimental Setup:

An RNA sequence encoding human fumarylacetoacetate hydrolase (FAH mRNA; SEQ ID NO:136; see Table 02: “Constructs of the invention") was designed and synthesized in vitro. Each mRNA contained a cap structure (CleanCap), 5’ untranslated region (UTR), an open-reading frame (ORF) encoding human FAH, a 3’-UTR and an enzymatically added poly-A tail. Human FAH mRNA was formulated into lipid nanoparticles (LNPs) for in vivo injections into FAH mice as described above. Aliquots of FAH mRNA-LNPs were provided at 1g/l and diluted in PBS before injection into FAH mice. Physicochemical characterization of LNPs resulted in an encapsulation efficiency of FAH mRNA in LNPs of 93%, a particle diameter of 70 nm, and a homogenous size distribution (as judged by polydispersity index 0,047).

The FAH mutant mouse model was chosen as a mouse model representing the human disease Hereditary Tyrosinemia Type I. This mouse model

) was available upon cryorecovery (abbreviated FAH mice). The mutation present in this Tyrosinemia mouse model is a G-to-A transition at the last base of exon 7 leading to the splicing of exon 6 to exon 8, and resulting in a transcript that lacks exon 7. The absence of exon 7 in the transcript results in a frameshift and subsequently the introduction of a premature stop codon at amino acid position 303 (https://www.jax.org/strain/018129). To avoid early postnatal lethality of FAH mice, NTBC was supplemented via drinking water to pregnant and nursing females and to homozygotes throughout life until otherwise stated. Both female and male FAH mice at 10-12 weeks of age were included in the in vivo studies. To induce the disease phenotype in FAH mice, NTBC supplementation was withdrawn 5 days before start of treatment in all experimental cohorts. Body weight of FAH mice was determined before start of injections, during life phase and at termination. Blood was collected one day before treatment to determine pre-treatment metabolite levels, and on day of termination. FAH mice were assigned to the following cohorts: (1) A standard-of-care (SOC)-treated cohort of FAH mice received NTBC supplementation throughout life and injection of PBS (NTBC (+) PBS (+)), (2) a negative control group of FAH mice stopped NTBC supplementation 5 days before start of treatment and was injected with PBS on day 0 (NTBC (-) PBS (+)), and (3) a third group of FAH mice stopped NTBC treatment 5 days before start of treatment and received intravenous (IV) or intramuscular (IM) injections of LNP-formulated therapeutic FAH mRNA (NTBC (-) RNA (+)). Body weight of FAH mice was measured throughout life-phase and at termination. FAH mice were terminated on designated days after single and repeated IV or IM injections, and liver and serum were collected. Succinylacetone (SA) and Tyrosine (TYR) levels were measured in FAH mouse serum, and FAH protein expression was quantified by Western Blot analysis detecting FAH protein in liver lysates.

The detailed Study design of the present working example is depicted in Table Ex-2.

Table Ex-2: Study design.

The efficacy of mRNA treatment was assessed by its ability to reduce pathologically increased Succinylacetone (SA) and Tyrosine (TYR) levels in FAH mouse serum after single intravenous injections. For evaluation of therapeutic efficacy, 1 mg/kg dose of therapeutic FAH mRNA (formulated in LNPs (diluted in Phosphate Buffered Saline (PBS)) were injected into mice (n=4 FAH mice) intravenously (iv) on day DO. Bleeding of FAH mice was performed on day D-1 and on days of termination (D1 , D2, and D4).

Animals in group 1 (n=4) served as standard-of-care treated FAH mice (i.e. intravenous administration of PBS and continuous supplementation with NTBC) = NTBC (+) PBS (+).

Animals in group 2 (n=4) served as a negative control (i.e. intravenous administration of PBS and stop of NTBC supplementation 5 days before start of single iv injections = NTBC (-) PBS (+).

Animals in group 3 (n=4): Single intravenous administration of 1 mg/kg dose of therapeutic mRNA formulated in Lipid Nanoparticles (diluted in PBS) = NTBC (-) RNA (+). FAH mice were terminated on day 1. Animals in group 4 (n=4): Single intravenous administration of 1 mg/kg dose of therapeutic mRNA formulated in Lipid Nanoparticles (diluted in PBS) = NTBC (-) RNA (+). FAH mice were terminated on day 2.

Animals in group 5 (n=4): Single intravenous administration of 1 mg/kg dose of therapeutic mRNA formulated in Lipid Nanoparticles (diluted in PBS) = NTBC (-) RNA (+). FAH mice were terminated on day 4.

Results:

Succinylacetone (SA) and Tyrosine (TYR) levels were significantly reduced upon single IV injection of FAH mRNA-LNPs up to 4 days after injection. Succinylacetone (SA) levels in FAH mRNA-LNPs treated FAH mice were not significantly different compared to standard-of-care NTBC-treated FAH mice. Most importantly, Tyrosine (TYR) levels upon single IV FAH mRNA-LNP injections were lowered to physiological levels observed in wildtype mice, which was not achieved upon standard- of-care NTBC supplementation. Further details can be found in Figures 2A to 2B and the respective figure legend(s).

Results:

FAH protein expression was detected in FAH mouse livers up to 4 days after single intravenous injection of human FAH mRNA-LNPs. On day 1 , approximately 50% of the endogenous liver FAH protein expression in wildtype mice was detected in FAH mouse livers after single intravenous injection of FAH mRNA-LNPs. On day 4, approximately 30% of the endogenous liver FAH protein expression in wildtype mice was detected in FAH mouse livers after single intravenous injection of FAH mRNA-LNPs. Further details can be found in Figures 3A to 3D and the respective figure legend(s).

Example 3: Repeated intravenous injection of human FAH mRNA-LNPs in a mouse model of Hereditary Tyrosinemia Type I

Experimental Setup:

NTBC supplementation was withdrawn 5 days before start of treatment to induce the disease phenotype. Blood was collected one day before treatment (pre-bleeding on day -1 ), at intermediate bleeding time points 24 hours after injections (B1-B3 on days 6, 11 , and 16), and on day of termination (day 21). A standard-of-care-treated cohort of mice received NTBC supplementation throughout their life and repeated PBS injections (NTBC (+) PBS (+)). A negative control group of FAH mice stopped NTBC supplementation 5 days before start of treatment and was injected with PBS repeatedly (NTBC (-) PBS (+)). The third group of FAH mice stopped NTBC treatment 5 days before start of treatment and received repeated IV injections of LNP-formulated therapeutic FAH mRNA 5 times every 5 days at a dose of 1 mg/kg (adjusted to weight of individual FAH mice) (NTBC (-) RNA (+)). On day of termination (day 21 ), final bleeding was performed and livers were harvested for further analysis. The detailed Study design of the present working example is depicted in Table Ex-3.

Table Ex-3: Study design.

The efficacy of mRNA treatment was assessed by its ability to prolong survival of FAH mice, to stabilize their body weight, to prevent health decline, and to reduce pathologically increased Succinylacetone (SA) and Tyrosine (TYR) levels in FAH mouse serum. For evaluation of therapeutic efficacy, 1 mg/kg dose of therapeutic FAH mRNA (formulated in LNPs (diluted in Phosphate Buffered Saline (PBS)) were injected into mice (n=5 FAH mice) intravenously (iv) on days DO, D5, D10, D15, and D20. Bleeding of FAH mice was performed on day D-1 , D6, D11 , D16, and D21. FAH mice were terminated on day 21 or sacrificed dependent on body weight loss.

Animals in group 1 (n=5) served as standard-of-care treated FAH mice (i.e. intravenous administration of PBS and continuous supplementation with NTBC) = NTBC (+) PBS (+).

Animals in group 2 (n=5) served as a negative control (i.e. intravenous administration of PBS and stop of NTBC supplementation 5 days before start of repeated iv injections = NTBC (-) PBS (+).

Animals in group 3 (n=5): Repeated intravenous administration of 1mg/kg dose of therapeutic mRNA formulated in Lipid Nanoparticles (diluted in PBS) = NTBC (-) RNA (+).

Results (survival, body weight):

Survival of human FAH mRNA-injected FAH mice was significantly prolonged compared to PBS- injected FAH mice (100% survival of NTBC- and mRNA-treated FAH mice compared to 0% survival of NBTC (-) PBS(+) treated FAH mice at the end of the study). Body weight of NTBC- and FAH mRNA-LNPs-treated mice did not drop during the experimental life-phase, whereas body weights of NTBC (-) PBS (+) FAH mice dropped significantly; NTBC (-) PBS (+) FAH mice had to be terminated due to body weight loss. Most importantly, body weights of FAH mice on mRNA treatment were not significantly different compared to standard-of-care NTBC-treated FAH mice throughout experimental life-phase and at the end of the study (day 21). Further details can be found in Figures 4A to 4C and the respective figure legend(s).

Results (Succinylacetone (SA) and Tyrosine (TYR) levels):

Succinylacetone (SA) and Tyrosine (TYR) levels of mRNA-treated FAH mice were significantly reduced throughout life-phase and at termination, whereas PBS-treated FAH mice after withdrawal of NTBC showed pathologically increased Succinylacetone (SA) and Tyrosine (TYR) levels and had to be terminated due to body weight loss and health decline before the end of the study. Most importantly, TYR levels were reduced to physiological levels observed in wildtype mice, which was not achieved upon standard-of-care NTBC supplementation. Further details can be found in Figures 5A to 5D and the respective figure legend(s).

Results (FAH expression/detection):

FAH protein was detected in FAH mouse livers on day 21 at termination, approximately 20% of the endogenously expressed FAH protein in wildtype mouse livers. This amount of liver FAH protein expression in FAH mice upon repeated intravenous FAH mRNA-LNP injection was sufficient to rescue FAH mice from body weight loss, health decline and death, and to reduce pathologically increased serum Succinylacetone (SA) and Tyrosine (TYR) level. Further details can be found in Figures 6A to 6B and the respective figure legend(s).

Example 4: Repeated intramuscular injections in direct comparison to repeated intravenous injection of human FAH mRNA-LNPs in a mouse model of Hereditary Tyrosinemia Type I Experimental Setup:

NTBC supplementation was withdrawn 5 days before start of treatment to induce the disease phenotype. Blood was collected one day before treatment (PB = pre-bleeding), at intermediate bleeding time points 24 hours after injections (B1-B3), and on day of termination (T). FAH mice were repeatedly injected intravenously (IV) or intramuscularly (IM) to compare administration routes. 20pg of FAH mRNA-LNPs were administered, irrespective of the weight of FAH mice for IV route, and 10pg of FAH mRNA-LNPs were administered into both M. tibialis muscles to result in a total dose of 20pg FAH mRNA-LNPs comparable to the dose of IV treated FAH mice.

The detailed Study design of the present working example is depicted in Table Ex-4.

Table Ex-4: Study design. Animals in group 1 (n=5): Intramuscular administration of 10pg of therapeutic FAH mRNA formulated in Lipid Nanoparticles (diluted in PBS) into each M. tibialis muscle. Animals in groups 2 (n=4): Intravenous administration of 20pg of therapeutic FAH mRNA formulated in Lipid Nanoparticles (diluted in PBS).

The efficacy of mRNA treatment was assessed by its ability to stabilize body weight of FAH mice, to prevent death of FAH mice, and to reduce pathologically increased Succinylacetone (SA) and Tyrosine (TYR) levels in FAH mouse serum. For evaluation of therapeutic efficacy, 20pg doses of therapeutic FAH mRNA (formulated in Lipid Nanoparticles (LNPs) diluted in Phosphate Buffered Saline (PBS)) were injected into the animals (n=4-5 FAH mice) intravenously (IV) and intramuscularly (IM) on days DO, D5, D10, D15, and D20. Bleeding of FAH mice was performed on day D-1 (minus 1), D6, D11 , D16, and D21. FAH mice were terminated on day 21. Results (body weight):

Body weight of FAH mice repeatedly injected with FAH mRNA-LNPs either via IM or IV administration route had stable and indistinguishable body weight throughout the experimental life-phase and at termination. Further details can be found in Figures 7 A to 7B and the respective figure legend(s).

Results (Succinylacetone (SA) and Tyrosine (TYR) levels):

Succinylacetone (SA) and Tyrosine (TYR) levels were reduced upon mRNA-based therapy after repeated injections either via IM or IV administration route compared to pathologically increased pre- treatment levels. Reductions in Succinylacetone (SA) and Tyrosine (TYR) levels were comparable after IM and IV administration routes both during life-phase and at termination. Further details can be found in Figures 8A to 8D and the respective figure legend(s).

Results (FAH expression):

After repeated intramuscular injections of FAH mRNA-LNPs, a substantial amount of FAH mRNA- LNPs were transported to the liver via blood stream, resulting in uptake into hepatocytes and successful liver FAH protein expression. The amount expressed in FAH mouse livers after repeated IM injections was ~6% of the amount expressed after repeated IV injections, however sufficient to rescue FAH mice from body weight loss, health decline and death, and to decrease pathologically increased Succinylacetone (SA) and Tyrosine (TYR) levels in FAH mouse serum. Further details can be found in Figures 9A to 9B and the respective figure legend(s).

Example 5: Dose finding studies for repeated intravenous injections of human FAH mRNA-

LNPs in a mouse model of Hereditary Tyrosinemia Type I

Experimental Setup:

For dose finding studies for repeated intravenous injections of human FAH mRNA-LNPs in a mouse model of Hereditary Tyrosinemia Type I, NTBC supplementation was withdrawn 5 days before start of treatment to induce the disease phenotype. Blood was collected one day before treatment (pre- bleeding on day -1), at intermediate bleeding time points 24 hours after injections (B1-B3 on days 6, 11 , and 16), and on day of termination (day 21). FAH mice received repeated intravenous injections of lower doses of human FAH mRNA formulated in LNPs (0,5 mg/kg and 0,1 mg/kg). On day of termination (day 21), final bleeding was performed and livers were harvested for further analysis.

The detailed Study design of the present working example is depicted in Table Ex-5.

Table Ex-5: Study design.

The efficacy of mRNA treatment was assessed by its ability to prolong survival of FAH mice, to stabilize their body weight, and to reduce pathologically increased Succinylacetone (SA) and Tyrosine (TYR) levels in FAH mouse serum. For evaluation of therapeutic efficacy, different doses (0,5 mg/kg and 0,1 mg/kg) of therapeutic FAH mRNA (formulated in Lipid Nanoparticles (LNPs) (diluted in Phosphate Buffered Saline (PBS)) were injected into mice (n=5 FAH mice) intravenously (iv) on days DO, D5, D10, D15, and D20. Bleeding of FAH mice was performed on day D-1 , D6, D11 , D16, and D21 . FAH mice were terminated on day 21 or sacrificed dependent on body weight loss.

Results (body weight, Succinylacetone (SA) and Tyrosine (TYR) levels):

FAH mice repeatedly injected intravenously with lower doses of 0,5 mg/kg and 0,1 mg/kg survived until the end of the study (day 21). Body weight of all FAH mRNA-LNPs-treated mice was stable throughout experimental life phase until the end of the experiment (day 21 ). Pathologically increased Succinylacetone and Tyrosine levels in FAH mouse serum were lowered upon mRNA-LNP based therapy at lower doses. Further details can be found in Figures 10A to 10C and the respective figure legend(s).

Example 6: Dose finding studies for repeated intramuscular injections of human FAH mRNA-

LNPs in a mouse model of Hereditary Tyrosinemia Type I

Experimental Setup:

For dose finding studies for repeated intramuscular injections of human FAH mRNA-LNPs in a mouse model of Hereditary Tyrosinemia Type I, NTBC supplementation was withdrawn 5 days before start of treatment to induce the disease phenotype. Blood was collected one day before treatment (pre-bleeding on day -1 ), at intermediate bleeding time points 24 hours after injections (BI- B3 on days 6, 11 , and 16), and on day of termination (day 21). FAH mice received repeated intramuscular injections of lower doses of human FAH mRNA formulated in LNPs (0,5 mg/kg and 0,1 mg/kg). On day of termination (day 21), final bleeding was performed and livers were harvested for further analysis.

The detailed Study design of the present working example is depicted in Table Ex-6.

Table Ex-6: Study design.

The efficacy of mRNA treatment was assessed by its ability to prolong survival of FAH mice, to stabilize their body weight, and to reduce pathologically increased Succinylacetone (SA) and Tyrosine (TYR) levels in FAH mouse serum. For evaluation of therapeutic efficacy, different doses (0,5 mg/kg and 0,1 mg/kg) of therapeutic FAH mRNA (formulated in Lipid Nanoparticles (LNPs) (diluted in Phosphate Buffered Saline (PBS)) were injected into mice (n=5 FAH mice) intramuscularly (im) on days DO, D5, D10, D15, and D20. Bleeding of FAH mice was performed on day D-1 , D6, D11 , D16, and D21. FAH mice were terminated on day 21 or sacrificed dependent on body weight loss.

Results (Survival, body weight, Succinylacetone (SA) and Tyrosine (TYR) levels):

FAH mice repeatedly injected intramuscularly with doses of 0,5 mg/kg survived until the end of the study (day 21 ). In contrast, FAH mice repeatedly injected intramuscularly with doses of 0,1 mg/kg had to be terminated due to body weight loss before the end of the study. Body weight of FAH mRNA- LNPs-treated mice at dose of 0,5 mg/kg was stable throughout experimental life phase until the end of the experiment (day 21), whereas FAH mice injected with doses of 0,1 mg/kg had to be terminated due to body weight loss. Succinylacetone and Tyrosine levels in FAH mouse serum were reduced compared to pre-treatment levels upon administration of 0,5 mg/kg doses, however were not reduced upon repeated intramuscular injections of 0,1 mg/kg doses. Further details can be found in Figures 11 A to 11 D and the respective figure legend(s).

Example 7: Interval finding studies for repeated intravenous and intramuscular injections of human FAH mRNA-LNPs in a mouse model of Hereditary Tyrosinemia Type I

Experimental Setup:

For interval finding studies for repeated intravenous and intramuscular injections of human FAH mRNA-LNPs in a mouse model of Hereditary Tyrosinemia Type I (one week and two weeks schedules), NTBC supplementation was withdrawn 5 days before start of treatment to induce the disease phenotype. Blood was collected one day before treatment (pre-bleeding on day -1), at intermediate bleeding time points and on days of termination. FAH mice received repeated intravenous and intramuscular injections of human FAH mRNA formulated in LNPs at doses of 1 mg/kg. On day of termination, final bleeding was performed and livers were harvested for further analysis.

The detailed Study design of the present working example is depicted in Table Ex-7. Table Ex-7: Study design.

The efficacy of mRNA treatment was assessed by its ability to prolong survival of FAH mice, to stabilize their body weight, and to reduce pathologically increased Succinylacetone (SA) and Tyrosine (TYR) levels in FAH mouse serum. For evaluation of therapeutic efficacy, different intervals (one week and two weeks intervals) between 1 mg/kg doses of therapeutic FAH mRNA (formulated in Lipid Nanoparticles (LNPs) (diluted in Phosphate Buffered Saline (PBS)) were tested in FAH mice (n=5 FAH mice). Repeated injections were administered both intravenously (iv) and intramuscularly (im). Weekly injections were administered on days D0, D7, D14, D21 , and D28. Injections for the two weeks interval schedule were administered on days D0, D14, D28, D42, and D56. Termination of mice and collection of final serum and livers was done on day D29 for the one-week schedule and on day D57 for the two-weeks schedule.

Results

All FAH mice survived until termination of the studies. Repeated intravenous injections in both one- week and two-weeks schedules rescued mice, stabilized body weight of mice, and reduced Succinylacetone and Tyrosine levels compared to pre-treatment levels. Repeated intramuscular injections in the one week schedule rescued mice, stabilized body weight of mice, and reduced Succinylacetone and Tyrosine levels compared to pre-treatment levels. Repeated intramuscular injections in the two-weeks schedule also rescued mice, however body weight, Succinylacetone and Tyrosine levels fluctuated during life phase. Further details can be found in Figures 12A to 12F and the respective figure legend(s).

Claims

1. An isolated mRNA encoding fumarylacetoacetate hydrolase (FAH) for use in treating, attenuating or inhibiting Hereditary Tyrosinemia Type I (HT1 ).

2. The mRNA of claim 1 , wherein said mRNA comprises an open reading frame (ORF) encoding FAH comprising an amino acid sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 100, or a fragment or variant of said sequences having the biological activity of a FAH protein.

3. The mRNA according to any one of claim 1 to claim 2, wherein said mRNA preferably has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any single SEQ ID NO-element of SEQ ID NO:112 to SEQ ID NO:144 or SEQ ID NQ:101 to SEQ ID NO:111 , or a fragment or variant of said sequences, wherein the encoded protein has the biological activity of a FAH protein.

4. The mRNA of any one of claim 1 to claim 3 for use according to claim 1 , further comprising an UTR combination selected from the group consisting of (i) a 5'-UTR derived from a mouse solute carrier family 7 (cationic amino acid transporter, y+ system) (SLC7A3) and a 3'-UTR derived from PSMB3; (ii) a 5 -UTR derived from mouse ribosomal protein L31 (RPL31) and a 3'-UTR derived from a human ribosomal protein S9 (RPS9); (iii) a 5 -UTR derived from ubiquilin 2 (Ubqln2) and a 3 -UTR derived from Guanine nucleotide-binding protein G(s) subunit alpha isoforms short (Gnas); and (iv) a 5’-UTR derived from a hydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4) and a 3 -UTR derived from a proteasome subunit beta type-3 (PSMB3) UTR.

5. The mRNA according to any one of claim 1 to claim 4, wherein the

(i) G/C content of the FAH coding sequence in said mRNA is increased compared to the coding sequence of the corresponding wild type FAH coding sequence of SEQ ID NQ:101 ;

(iii) at least one codon of the FAH coding sequence in said mRNA is adapted to human codon usage, wherein the codon adaptation index (CAI) is preferably increased or maximised in the corresponding FAH coding sequence compared to the coding sequence of the corresponding wild type FAH coding sequence of SEQ ID NQ:101.

6. The mRNA according to any one of claim 1 to claim 5, wherein the mRNA comprises a 5’- cap structure, a poly(A) sequence comprising at least 70 A nucleotides, preferably about 100 A nucleotides, a poly(C) sequence, preferably comprising 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides, and/or at least one histone stem-loop, preferably, wherein the mRNA comprises a 3’-terminal A nucleotide.

7. The mRNA according to any one of claim 1 to claim 6, wherein the mRNA comprises, preferably in 5’ to 3’ direction, the following elements: a) a 5’-cap1 structure; b) a 5’-UTR element comprising a nucleic acid sequence, preferably derived from a 5’-UTR of a HSD17B4 gene, comprising the nucleic acid sequence according to SEQ ID NO:1 or 2, or a homolog, a fragment or a variant thereof; c) at least one coding sequence as defined in any one of claim 1 to claim 10; d) a 3’-UTR element comprising a nucleic acid sequence, preferably derived from a 3’-UTR of a PSMB3 gene, comprising the nucleic acid sequence according to SEQ ID NO:33 or 34, or a homolog, a fragment or a variant thereof; e) a poly(A) sequence comprising about 100 adenosine nucleotides, preferably, wherein the mRNA comprises a 3’-terminal A nucleotide; f) an optional poly(C) tail, preferably comprising 10 to 40 cytosine nucleotides; and/or g) an optional histone stem-loop, preferably comprising the nucleic acid sequence according to SEQ ID NO:63 or 64.

8. The mRNA according to any one of claim 1 to claim 7, wherein the open reading frame does not comprise any chemically modified uracil or cytosine nucleotides.

9. The mRNA according to any one of claim 1 to claim 7, wherein the mRNA is chemically modified, preferably wherein the mRNA comprises pseudouridine (psi-uridine), N1- methylpseudouridine (N1 MPU), 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3- methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5- methylcytidine, 2-aminoadenosine, 7-deazaadenosme, 7 -deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, O(6)-methylguanine, and/or 2-thiocytidine, more preferably wherein all uridine bases of the mRNA are fully chemically modified, even more preferably wherein all uridine bases of the mRNA are pseudouridine or N1 -methylpseudouridine (N1 MPU) bases, most preferably wherein all uridine bases of the mRNA are N1 -methylpseudouridine (N1 MPU) bases.

10. A lipid nanoparticle (LNP) comprising the mRNA according to any one of claim 1 to claim 9, wherein the LNP comprises an ionizable or cationic lipid, a phospholipid, a structural lipid, and a polymer conjugated lipid.

11. The LNP according to claim 10, wherein the lipids comprised in the LNP have a molar ratio of about 20-60% cationic or ionizable lipid, about 5-25% non-cationic lipid, about 25-55% sterol and about 0.5-15% polymer conjugated lipid.

12. The LNP according to anyone of claim 10 to claim 11 , wherein the LNP does not comprise polyethylene glycol (PEG) or a PEG-modified lipid.

13. A pharmaceutical composition, comprising the mRNA according to any one of claim 1 to claim 9 or the LNP according to any one of claim 10 to claim 12.

14. A kit, preferably kit of parts, comprising at least one mRNA according to any one of claim 1 to claim 9, the LNP according to any one of claim 10 to claim 12, or the pharmaceutical composition according to claim 13, and optionally a liquid vehicle for solubilising and optionally technical instructions with information on the administration and dosage of the pharmaceutical composition.

15. A method of treating, attenuating or inhibiting Hereditary Tyrosinemia Type I (HT1 ), comprising administering to a human subject in need the mRNA according to any one of claim 1 to claim 9, the LNP according to any one of claim 10 to claim 12, the pharmaceutical composition according to claim 13, or the kit or kit of parts according to claim 14, wherein the administration results in treatment, prevention, attenuation, inhibition, or prophylaxis of Hereditary Tyrosinemia Type I (HT1).

16. The method according to claim 15, wherein the mRNA according to any one of claim 1 to claim 9, or the LNP according to any one of claim 10 to claim 12, or the pharmaceutical composition according to claim 13 or the kit or kit of parts according to claim 14 is administered to the subject by subcutaneous, intramuscular or intravenous administration, preferably by intramuscular or intravenous administration.

17. The method according to any one of claim 15 to claim 16, wherein the mRNA comprises a 5'- or 3'-untranslated region (UTR) comprising at least one microRNA-binding site, preferably not being a microRNA-122 (miR-122) binding site, more preferably being miR-16, miR-21 , miR-24, miR- 27, miR-30c, miR-132, miR-133, miR-149, miR-192, miR-194, miR-204, miR-206, miR-208, or miR- 223, most preferably being miRNA-148a, miRNA-101 , miRNA-192 or miRNA-194, miR-126, miR- 142-3p, or miR-142-5p.

18. The method according to any one of claim 15 to claim 17, wherein the method of treating, attenuating or inhibiting Hereditary Tyrosinemia Type I (HT1), involves a single administration of the mRNA, the LNP, the pharmaceutical composition or the kit or kit of parts.

19. The method according to any one of claim 15 to claim 18, wherein the mRNA, the LNP, the pharmaceutical composition or the kit or kit of parts is administered

20. An isolated mRNA according to any one of claim 1 to claim 9, or LNP according to any one of claim 10 to claim 12 or pharmaceutical composition according to claim 13 or a kit or kit of parts according to claim 14, for use as a medicament.

21. A vector comprising the isolated mRNA according to any one of claim 1 to claim 9, preferably a DNA vector.

22. A host cell carrying the vector of claim 21 .

23. An isolated mRNA encoding fumarylacetoacetate hydrolase (FAH) according to any one of claim 1 to claim 9, a lipid nanoparticle (LNP) according to any one of claim 10 to claim 12, a pharmaceutical composition according to claim 13 or a kit or kit of parts according to claim 14, for reducing pathologically increased Succinylacetone (SA) and/or Tyrosine (TYR) levels in a patient in need.

24. The LNP according to any one of claim 10 to claim 12, or the pharmaceutical composition according to claim 13 or the kit or kit of parts according to claim 14, additionally comprising at least one antagonist of at least one RNA sensing pattern recognition receptor, preferably wherein the at least one antagonist of at least one RNA sensing pattern recognition receptor is a single stranded oligonucleotide.