IVT messenger RNA and methods of treating or preventing PFIC type iv

Abstract

Disclosed are in vitro transcribed (IVT) messenger RNAs (mRNAs) comprising a polynucleotide that encodes human TJP2 gene, a 5' terminal cap, and a poly-adenylation (poly- A) tail at the 3' terminal end. In aspects, the polynucleotide is substantially free of uridine bases and comprises pseudouridine bases. Methods, compositions and uses employing the IVT mRNA for treating or preventing progressive familial intrahepatic Cholestasis type IV (PFIC type 4) in a patient in need thereof is also disclosed.

Description

IVT MESSENGER RNA AND METHODS OF TREATING OR PREVENTING PFIC TYPE IV CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is an international application which claims priority to and benefit of U.S. provisional application Serial No. 63 / 574,142, filed April 3, 2024, the contents of which are incorporated in their entirety for all purposes. REFERENCE TO SEQUENCE LISTING

[0002] A Sequence Listing submitted as an ST.26 XML file via Patent Center is hereby incorporated by reference. The name of the XML file for the Sequence Listing is 800560_SL.xml, the date of the creation of the XML file is March 31, 2025, and the size of the XML file is 20,881 bytes. FIELD OF THE INVENTION

[0003] The present invention is directed to in vitro transcribed (IVT) messenger RNA (mRNA), i.e., synthetic mRNA, and methods for treating or preventing Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4). BACKGROUND OF THE INVENTION

[0004] Progressive Familial Intrahepatic Cholestasis (PFIC) refers to a diverse group of rare autosomal- recessive disorders having an incidence ranging from 1 / 50,000-1 / 100,000, and which is characterized by defects in proteins involved in bile flow and formation within the liver that affect bile production and the secretion of bile acids. Bile flow originates at the hepatocyte canalicular membrane, which forms the bile canaliculus (BC), a specialized bile draining “channel” between hepatocytes demarcated by tight junctions built with scaffolding proteins, TJP1 and TJP2, and transmembrane junction proteins including claudins and occludins. Newborns with deficient tight junction protein 2 (TJP2) develop a subtype of progressive familial intrahepatic cholestasis (PFIC-TJP2, also known as PFIC type IV), which manifests as neonatalcholestasis that progresses to cirrhosis, while other organs often remain unaffected, requiring a liver transplant.

[0005] Progressive familial intrahepatic cholestasis (PFIC) manifests with a varying spectrum of clinical features, with some variants progressing rapidly into end stage liver disease. Current therapies for include off-label treatments such as ursodeoxycholic acid (UDCA) or rifampicin, as well as invasive approaches with different kinds of surgical biliary diversion (SBD) techniques. (See, e.g., Antonia Felzen, Henkjan J. Verkade, The spectrum of Progressive Familial Intrahepatic Cholestasis diseases: Update on pathophysiology and emerging treatments, European Journal of Medical Genetics, Volume 64, Issue 11, 2021, 104317, ISSN 1769-7212, doi.org / 10.1016 / j.ejmg.2021.104317.) While these treatments seek to improve bile flow and / or to manage symptoms and complications, there is no cure for PFIC, and transplantation remains the primary treatment, often required before adulthood. Liver transplantation may be necessary in severe cases to prevent or treat end-stage liver disease. The prognosis for PFIC varies depending on the severity of the disease and the effectiveness of treatment. Some individuals with PFIC may have a relatively mild disease course, while others experience rapid progression to liver failure and require liver transplantation. However, liver transplantation involves major surgery and requires lifelong immunosuppression. Thus, a need exists to provide early therapeutic intervention in the form of treatment, prevention, or amelioration of symptoms of PFIC type IV. The instant disclosure seeks to address one or more of the aforementioned needs in the art. SUMMARY OF THE INVENTION

[0006] It is an object of the present invention to provide means for treatment or prevention of PFIC type IV.

[0007] In embodiments, the means for treatment or prevention of PFIC type IV is administered to an individual having, or suspected of having PFIC type IV.

[0008] In embodiments, the means for treatment or prevention of PFIC type IV is administered to a pediatric individual having, or suspected of having PFIC type IV. In embodiments, the means for treatmentor prevention of PFIC type IV is administered to a neonate having, or suspected of having PFIC type IV. In one embodiment, an in vitro transcribed (IVT) messenger RNA (mRNA) is disclosed, the mRNA comprising a polynucleotide that encodes human TJP2 gene. In embodiments, the mRNA further comprises a 5’ terminal cap. In embodiments the mRNA further comprises a poly-adenylation (poly-A) tail at the 3’ terminal. In embodiments, the polynucleotide is substantially free of uridine bases and comprises pseudouridine bases.

[0009] In embodiments, a DNA template (IVT template) for synthesizing an IVT mRNA is disclosed, the DNA template comprising a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence homology to SEQ ID NO: 1. In embodiments, the DNA template is capable of producing an mRNA that is translated to the protein sequence of at least a portion of, or at least 95% of, or 100% of, SEQ ID NO: 2.

[0010] In embodiments, an in vitro transcribed (IVT) messenger RNA (mRNA) comprising, consisting of, or consisting essentially of SEQ ID NO: 3 is disclosed. In embodiments, the mRNA comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence homology to SEQ ID NO: 3.

[0011] In embodiments, an in vitro transcribed (IVT) messenger RNA (mRNA) comprising, consisting of, or consisting essentially of SEQ ID NO: 4 is disclosed. In embodiments, the mRNA comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence homology to SEQ ID NO: 4. In embodiments, mRNA is translated to the protein sequence of at least a portion of, or at least 95% of, or 100% of, SEQ ID NO: 2.

[0012] In embodiments, an in vitro transcribed (IVT) messenger RNA (mRNA) comprising, consisting of, or consisting essentially of SEQ ID NO: 5 is disclosed. In embodiments, the mRNA comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence homology to SEQ ID NO: 5. Inembodiments, mRNA is translated to the protein sequence of at least a portion of, or at least 95% of, or 100% of, SEQ ID NO: 2.

[0013] In embodiments, the invention is directed to a composition comprising the disclosed IVT mRNA, formulated in a lipid nanoparticle (LNP) carrier. In further embodiments, the IVT mRNA is formulated in a lipid nanoparticle (LNP) carrier that further comprises a pharmaceutically acceptable excipient.

[0014] Further disclosed are methods of treating, preventing, or otherwise ameliorating at least one symptom of Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4) in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the disclosed IVT mRNA or comprising administering to the patient a therapeutically effective amount of a composition comprising the disclosed IVT mRNA, formulated in a lipid nanoparticle (LNP) carrier.

[0015] In embodiments, a method of identifying an individual having a mutation in TJP2 is disclosed. In embodiments, the method includes identifying an individual having a decrease in functional TJP2 protein. In embodiments, the method includes identifying an individual having a mutation in TJP2 that results in a truncation of TJP2 protein. In embodiments, the method includes identifying an individual having a mutation in TJP2 that results in expression of a non-functional TJP2 protein. In embodiments, the method includes identifying an individual having TJP2 deficiency. In embodiments, the method includes identifying an individual having a disruption in TJP2 expression and / or activity.

[0016] In embodiments, the method includes identifying an individual having disrupted bile canaliculus (BC) formation. In embodiments, the method includes identifying an individual having disrupted bile canaliculus (BC) formation due to a mutation in TJP2.

[0017] In embodiments, the method comprises administering an mRNA as described herein to a cell of an individual. In embodiments, the method comprises administering an mRNA as described herein to a hepatic cell of an individual.

[0018] In yet another embodiment, the invention is directed to a composition comprising a therapeutically effective amount of the IVT mRNA for use in a method of treating or Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4).

[0019] In embodiments, a use of a composition comprising a therapeutically effective amount of the disclosed IVT mRNA for the manufacture of a medicament for use in a method of treating or preventing Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4) is disclosed.

[0020] Surprisingly, introduction of the IVT mRNA to human hepatocyte cells with TJP2 deficiency is shown to rectify characteristic and defective phenotypes of such human hepatocyte cells, providing effective therapy for prevention or treatment of PFIC type IV caused by TPJ2 deficiency. Additional advantages of the various aspects of the invention will be apparent form the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The detailed description and Examples will be more fully understood in view of the drawing in which:

[0022] FIGS. 1, A and B show TJP2 deficient hepatocytes derived from human induced pluripotent stem cells (TJP2-iHeps) as described in the Examples lack canalicular network and display impaired bile excretion mirroring patient presentation. FIG. 1, A shows MDR3 staining of PFIC-TJP2 patient liver exhibit pseudo-acini, compared to canaliculi of healthy liver. TJP2-iHeps immunostained with TJP1 (green, tight junction marker) and Ecadherin (purple, basolateral marker) form pseudo-acini compared to WT- iHeps. FIG. 1, B shows both WT- and TJP2-iHeps excrete fluorescent bile acid (green, CGamF) into the canalicular lumen (red, actin dense).

[0023] FIGS.2, A - C show TJP2 mRNA induces TJP2 protein expression in both wild type (WT) and TJP2 deficient cells in a dose dependent manner. FIG.2, A shows the result of treating wild type (WT) and TJP2 knockout (TJP2 KO) hepatocytes derived from human induced pluripotent stem cells with 1 ug of in vitro transcribed TJP2 mRNA induced protein expression 24 hours post mRNA lipofection in both cells types. The graph is normalized to untreated WT TJP2 expression. FIG. 2, B shows the result of one 1 ugTJP2 mRNA treatment increases TJP2 protein expression for ~72 hours in WT hepatocytes derived from human induced pluripotent stem cells. The graph is normalized to untreated, control cells TJP2 expression. FIG. 2, C shows TJP2 protein expression 24 hours post mRNA treatment is dose dependent in TJP2 KO hepatocytes derived from human induced pluripotent stem cells. The graph is normalized to untreated, 0 ug TJP2 KO TJP2 expression.

[0024] FIGS. 3, A-C show TJP2 mRNA elongates pseudo-acinar BC structures in TJP2 deficient iHeps. In FIG. 3, A, tight junction protein 1 (TJP1) immunofluorescent staining shows that TJP2 mRNA treatment of TJP2-iHeps lengthens pseudo-acinar structures. FIG. 3, B shows measurements of BC structure length of untreated and TJP2 mRNA treated WT- and TJP2-iHeps. FIG. 3, C shows the number of nonbranching BC structures in untreated and TJP2 mRNA treated WT- and TJP2-iHeps. ∗P < 0.05, ∗∗P < 0.01.

[0025] FIG. 4 shows a cohort of induced human hepatocytes treated with TJP2 mRNA at day 1, harvested at various time points. Human hepatocytes derived from iPSC (pluripotent stem cells), or induced hepatocytes were used. Healthy induced hepatocytes and TJP2-KO induced hepatocytes cells were treated with 0.5 µg TJP2 mRNA via lipofection. The mRNA sequence used is codon optimized TJP2 + self- amplifying mechanism encoding a replicase. Cells were lysed and analyzed via western blot with anti-TJP2 antibody. Following TJP2 mRNA treatment, TJP2-KO hepatocytes successfully expressed TJP2 for 11 days following a single treatment. DETAILED DESCRIPTION

[0026] As noted, bile flow originates at the hepatocyte canalicular membrane, which forms the bile canaliculus (BC), a specialized bile draining “channel” between hepatocytes demarcated by tight junctions built with scaffolding proteins, TJP1 and TJP2, and transmembrane junction proteins. Newborns with deficient tight junction protein 2 (TJP2) develop a subtype of progressive familial intrahepatic cholestasis (PFIC-TJP2, also known as PFIC type IV), which manifests as neonatal cholestasis that progresses to cirrhosis, while other organs often remain unaffected. The TJP2 gene is located in chromosome 9q21. TheTJP2 gene encodes a protein called tight junction protein 2 or zona occludens-2, which, while named as a tight junction protein, is not present in the tight junction. Rather, TJP2 is a cytosolic protein that plays a role in maintaining cell-to-cell adhesion by linking the transmembrane tight junction proteins like claudin with the actin cytoskeleton. In TJP2 mutations, the claudin 1 (CLDN1) protein fails to localize to the bile canalicular membrane, resulting in reduced integrity of the canalicular membrane and reflux of toxic bile acids through the paracellular spaces into hepatocytes, causing hepatocyte damage and cholestasis. (See, e.g., Vinayagamoorthy V, Srivastava A, Sarma MS. Newer variants of progressive familial intrahepatic cholestasis. World J Hepatol. 2021 Dec 27;13(12):2024-2038. doi: 10.4254 / wjh.v13.i12.2024. PMID: 35070006; PMCID: PMC8727216.)

[0027] The inventors found that PFIC-TJP2 patients have disrupted BC structures in liver biopsies and increased bile concentrations in both liver tissue and blood serum, demonstrating that TJP2 plays a critical role in BC formation during hepatocyte differentiation.

[0028] Applicant has discovered a novel therapy for PFIC Type 4, in which mRNA coding TJP2 in lipid particles can reverse the disease status of human hepatocytes caused by the lack of TJP2. Applicant found that PFIC-TJP2 patients have disrupted BC structures in liver biopsies, and increased bile concentrations in both liver tissue and blood serum. A treatment that could elongate existing BC in PFIC- TJP2 patient livers would be greatly beneficial to patients and would mitigate the need for liver transplants in these patients.

[0029] The in vitro transcribed messenger RNA (IVT mRNA) of the invention has been shown to improve bile canaliculus (BC) formation to allow the BC to allow bile flow and resolve or prevent increased bile concentrations in both liver tissue and blood serum which result in PFIC type IV conditions. In embodiments, the IVT mRNA comprises a polynucleotide that encodes human TJP2 gene. In embodiments, the IVT mRNA further comprises additional features such as a 5’ terminal cap, and a poly-adenylation (poly-A) tail at the 3’ terminal end. In embodiments, the IVT mRNA is substantially free of uridine bases and comprises at least one modified nucleoside. In embodiments, the IVT mRNA is substantially free of uridine bases and comprises pseudouridine (abbreviated by the Greek letter “psi” or “ψ”) in place of at leasta portion (e.g., including substantially all) of the corresponding unmodified canonical nucleoside. In embodiments, all or nearly all of the uridine nucleosides in the IVT mRNA are replaced by pseudouridine nucleosides. In embodiments, the IVT mRNA includes a modified nucleobase selected from pseudouridine (ψ), 1-methylpseudouridine (m1ψ), 5-methoxyuridine (mo5U), 5-methylcytidine (m5C), α-thio-guanosine and α-thio-adenosine. In some embodiments, a RNA transcript (e.g., mRNA transcript) includes a combination of at least two (e.g., 2, 3, 4 or more) of the foregoing modified nucleobases.

[0030] The polynucleotide that encodes human TJP2 gene is TJP2-202 (transcript ID: ENST00000377245.9), or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. The nucleotide sequence of TJP2-202 can be found at www.ncbi.nlm.nih.gov / CCDS / CcdsBrowse.cgi?REQUEST=CCDS&DATA=CCDS6627. The human TJP2 gene may be used to synthesize an IVT mRNA for use in the disclosed compositions and methods. In embodiments, the DNA template (IVT template) for synthesizing IVT mRNA comprises, consists of, or consists essentially of SEQ ID NO: 1: ATGCCGGTGCGAGGAGACCGCGGGTTTCCACCCCGGCGGGAGCTGTCAGGTTGGCTCCGCGCCCCAGGCA TGGAAGAGCTGATATGGGAACAGTACACTGTGACCCTACAAAAGGATTCCAAAAGAGGATTTGGAATTGC AGTGTCCGGAGGCAGAGACAACCCCCACTTTGAAAATGGAGAAACGTCAATTGTCATTTCTGATGTGCTC CCGGGTGGGCCTGCTGATGGGCTGCTCCAAGAAAATGACAGAGTGGTCATGGTCAATGGCACCCCCATGG AGGATGTGCTTCATTCGTTTGCAGTTCAGCAGCTCAGAAAAAGTGGGAAGGTCGCTGCTATTGTGGTCAA GAGGCCCCGGAAGGTCCAGGTGGCCGCACTTCAGGCCAGCCCTCCCCTGGATCAGGATGACCGGGCTTTT GAGGTGATGGACGAGTTTGATGGCAGAAGTTTCCGGAGTGGCTACAGCGAGAGGAGCCGGCTGAACAGCC ATGGGGGGCGCAGCCGCAGCTGGGAGGACAGCCCGGAAAGGGGGCGTCCCCATGAGCGGGCCCGGAGCCG GGAGCGGGACCTCAGCCGGGACCGGAGCCGTGGCCGGAGCCTGGAGCGGGGCCTGGACCAAGACCATGCG CGCACCCGAGACCGCAGCCGTGGCCGGAGCCTGGAGCGGGGCCTGGACCACGACTTTGGGCCATCCCGGG ACCGGGACCGTGACCGCAGCCGCGGCCGGAGCATTGACCAGGACTACGAGCGAGCCTATCACCGGGCCTA CGACCCAGACTACGAGCGGGCCTACAGCCCGGAGTACAGGCGCGGGGCCCGCCACGATGCCCGCTCTCGGGGACCCCGAAGCCGCAGCCGCGAGCACCCGCACTCACGGAGCCCCAGCCCCGAGCCTAGGGGGCGGCCGG GGCCCATCGGGGTCCTCCTGATGAAAAGCAGAGCGAACGAAGAGTATGGTCTCCGGCTTGGGAGTCAGAT CTTCGTAAAGGAAATGACCCGAACGGGTCTGGCAACTAAAGATGGCAACCTTCACGAAGGAGACATAATT CTCAAGATCAATGGGACTGTAACTGAGAACATGTCTTTAACGGATGCTCGAAAATTGATAGAAAAGTCAA GAGGAAAACTACAGCTAGTGGTGTTGAGAGACAGCCAGCAGACCCTCATCAACATCCCGTCATTAAATGA CAGTGACTCAGAAATAGAAGATATTTCAGAAATAGAGTCAAACCGATCATTTTCTCCAGAGGAGAGACGT CATCAGTATTCTGATTATGATTATCATTCCTCAAGTGAGAAGCTGAAGGAAAGGCCAAGTTCCAGAGAGG ACACGCCGAGCAGATTGTCCAGGATGGGTGCGACACCCACTCCCTTTAAGTCCACAGGGGATATTGCAGG CACAGTTGTCCCAGAGACCAACAAGGAACCCAGATACCAAGAGGACCCCCCAGCTCCTCAACCAAAAGCA GCCCCGAGAACTTTTCTTCGTCCTAGTCCTGAAGATGAAGCAATATATGGCCCTAATACCAAAATGGTAA GGTTCAAGAAGGGAGACAGCGTGGGCCTCCGGTTGGCTGGTGGCAATGATGTCGGGATATTTGTTGCTGG CATTCAAGAAGGGACCTCGGCGGAGCAGGAGGGCCTTCAAGAAGGAGACCAGATTCTGAAGGTGAACACA CAGGATTTCAGAGGATTAGTGCGGGAGGATGCCGTTCTCTACCTGTTAGAAATCCCTAAAGGTGAAATGG TGACCATTTTAGCTCAGAGCCGAGCCGATGTGTATAGAGACATCCTGGCTTGTGGCAGAGGGGATTCGTT TTTTATAAGAAGCCACTTTGAATGTGAGAAGGAAACTCCACAGAGCCTGGCCTTCACCAGAGGGGAGGTC TTCCGAGTGGTAGACACACTGTATGACGGCAAGCTGGGCAACTGGCTGGCTGTGAGGATTGGGAACGAGT TGGAGAAAGGCTTAATCCCCAACAAGAGCAGAGCTGAACAAATGGCCAGTGTTCAAAATGCCCAGAGAGA CAACGCTGGGGACCGGGCAGATTTCTGGAGAATGCGTGGCCAGAGGTCTGGGGTGAAGAAGAACCTGAGG AAAAGTCGGGAAGACCTCACAGCTGTTGTGTCTGTCAGCACCAAGTTCCCAGCTTATGAGAGGGTTTTGC TGCGAGAAGCTGGTTTCAAGAGACCTGTGGTCTTATTCGGCCCCATAGCTGATATAGCAATGGAAAAATT GGCTAATGAGTTACCTGACTGGTTTCAAACTGCTAAAACGGAACCAAAAGATGCAGGATCTGAGAAATCC ACTGGAGTGGTCCGGTTAAATACCGTGAGGCAAATTATTGAACAGGATAAGCATGCACTACTGGATGTGA CTCCGAAAGCTGTGGACCTGTTGAATTACACCCAGTGGTTCCCAATTGTGATTTTTTTCAACCCAGACTC CAGACAAGGTGTCAAAACCATGAGACAAAGGTTAAATCCAACGTCCAACAAAAGTTCTCGAAAGTTATTT GATCAAGCCAACAAGCTTAAAAAAACGTGTGCACACCTTTTTACAGCTACAATCAACCTAAATTCAGCCA ATGATAGCTGGTTTGGCAGCTTAAAGGACACTATTCAGCATCAGCAAGGAGAAGCGGTTTGGGTCTCTGAAGGAAAGATGGAAGGGATGGATGATGACCCCGAAGACCGCATGTCCTACTTAACCGCCATGGGCGCGGAC TATCTGAGTTGCGACAGCCGCCTCATCAGTGACTTTGAAGACACGGACGGTGAAGGAGGCGCCTACACTG ACAATGAGCTGGATGAGCCAGCCGAGGAGCCGCTGGTGTCGTCCATCACCCGCTCCTCGGAGCCGGTGCA GCACGAGGAGAGCATAAGGAAACCCAGCCCAGAGCCACGAGCTCAGATGAGGAGGGCTGCTAGCAGCGAT CAACTTAGGGACAATAGCCCGCCCCCAGCATTCAAGCCAGAGCCGCCCAAGGCCAAAACCCAGAACAAAG AAGAATCCTATGACTTCTCCAAATCCTATGAATATAAGTCAAACCCCTCTGCCGTTGCTGGTAATGAAAC TCCTGGGGCATCTACCAAAGGTTATCCTCCTCCTGTTGCAGCAAAACCTACCTTTGGGCGGTCTATACTG AAGCCCTCCACTCCCATCCCTCCTCAAGAGGGTGAGGAGGTGGGAGAGAGCAGTGAGGAGCAAGATAATG CTCCCAAATCAGTCCTGGGCAAAGTCAAAATATTTGAGAAGATGGATCACAAGGCCAGGTTACAGAGAAT GCAGGAGCTCCAGGAAGCACAGAATGCAAGGATCGAAATTGCCCAGAAGCATCCTGATATCTATGCAGTT CCAATCAAAACGCACAAGCCAGACCCTGGCACGCCCCAGCACACGAGTTCCAGACCCCCTGAGCCACAGA AAGCTCCTTCCAGACCTTATCAGGATACCAGAGGAAGTTATGGCAGTGATGCCGAGGAGGAGGAGTACCG CCAGCAGCTGTCAGAACACTCCAAGCGCGGTTACTATGGCCAGTCTGCCCGATACCGGGACACAGAATTA TAG (SEQ ID NO: 1).

[0031] The translated sequence of SEQ ID NO: 1 is a 1190 amino acid, having the sequence of SEQ ID NO: 2, as follows: MPVRGDRGFPPRRELSGWLRAPGMEELIWEQYTVTLQKDSKRGFGIAVSGGRDNPHFENGETSIVISDVL PGGPADGLLQENDRVVMVNGTPMEDVLHSFAVQQLRKSGKVAAIVVKRPRKVQVAALQASPPLDQDDRAF EVMDEFDGRSFRSGYSERSRLNSHGGRSRSWEDSPERGRPHERARSRERDLSRDRSRGRSLERGLDQDHA RTRDRSRGRSLERGLDHDFGPSRDRDRDRSRGRSIDQDYERAYHRAYDPDYERAYSPEYRRGARHDARSR GPRSRSREHPHSRSPSPEPRGRPGPIGVLLMKSRANEEYGLRLGSQIFVKEMTRTGLATKDGNLHEGDII LKINGTVTENMSLTDARKLIEKSRGKLQLVVLRDSQQTLINIPSLNDSDSEIEDISEIESNRSFSPEERR HQYSDYDYHSSSEKLKERPSSREDTPSRLSRMGATPTPFKSTGDIAGTVVPETNKEPRYQEDPPAPQPKA APRTFLRPSPEDEAIYGPNTKMVRFKKGDSVGLRLAGGNDVGIFVAGIQEGTSAEQEGLQEGDQILKVNT QDFRGLVREDAVLYLLEIPKGEMVTILAQSRADVYRDILACGRGDSFFIRSHFECEKETPQSLAFTRGEVFRVVDTLYDGKLGNWLAVRIGNELEKGLIPNKSRAEQMASVQNAQRDNAGDRADFWRMRGQRSGVKKNLR KSREDLTAVVSVSTKFPAYERVLLREAGFKRPVVLFGPIADIAMEKLANELPDWFQTAKTEPKDAGSEKS TGVVRLNTVRQIIEQDKHALLDVTPKAVDLLNYTQWFPIVIFFNPDSRQGVKTMRQRLNPTSNKSSRKLF DQANKLKKTCAHLFTATINLNSANDSWFGSLKDTIQHQQGEAVWVSEGKMEGMDDDPEDRMSYLTAMGAD YLSCDSRLISDFEDTDGEGGAYTDNELDEPAEEPLVSSITRSSEPVQHEESIRKPSPEPRAQMRRAASSD QLRDNSPPPAFKPEPPKAKTQNKEESYDFSKSYEYKSNPSAVAGNETPGASTKGYPPPVAAKPTFGRSIL KPSTPIPPQEGEEVGESSEEQDNAPKSVLGKVKIFEKMDHKARLQRMQELQEAQNARIEIAQKHPDIYAV PIKTHKPDPGTPQHTSSRPPEPQKAPSRPYQDTRGSYGSDAEEEEYRQQLSEHSKRGYYGQSARYRDTEL (SEQ ID NO: 2)

[0032] In embodiments, the DNA template (IVT template) for synthesizing IVT mRNA for use in the disclosed compositions and methods comprises, consists of, or consists essentially of SEQ ID NO: 3, as follows: ATGCCCGTCAGGGGCGATCGCGGATTCCCCCCCCGGAGGGAACTGAGCGGCTGGCTGCGCGCCCCCGGGA TGGAGGAGCTGATCTGGGAGCAGTACACCGTGACCCTGCAGAAGGACAGCAAACGGGGCTTCGGAATCGC CGTCAGCGGAGGCAGGGACAATCCTCACTTTGAAAACGGCGAGACCAGCATCGTGATCTCCGACGTGCTG CCCGGCGGCCCTGCCGATGGCCTGCTGCAGGAGAATGATAGGGTGGTCATGGTGAACGGCACCCCCATGG AAGACGTGCTCCACAGCTTTGCCGTGCAGCAGCTGCGCAAGAGCGGCAAGGTCGCCGCCATTGTGGTGAA GAGACCTAGGAAGGTGCAGGTGGCTGCCCTGCAGGCTTCTCCCCCACTCGACCAGGACGACAGGGCCTTC GAAGTGATGGATGAGTTCGACGGAAGGTCTTTCAGGTCTGGATACAGCGAACGGAGTCGGCTGAATTCAC ACGGAGGAAGGTCCCGCAGTTGGGAGGATTCCCCAGAGAGAGGACGGCCCCATGAGAGGGCTCGCTCCAG AGAACGGGACCTGAGCCGGGACCGGAGTCGGGGCAGGAGCCTGGAGAGAGGCCTGGATCAGGATCACGCC AGAACACGGGATCGGTCCAGAGGGAGGAGCCTGGAAAGAGGACTGGACCACGATTTCGGGCCTTCAAGAG ACCGCGACAGGGATCGCAGCCGGGGACGCAGTATCGACCAGGATTACGAGCGAGCCTACCACAGGGCCTA CGACCCAGACTATGAAAGAGCCTACTCCCCCGAGTACCGCAGGGGCGCCCGGCACGATGCCCGGAGCAGG GGCCCCAGGAGTCGGAGCCGGGAACACCCCCACAGCCGGTCCCCCAGCCCCGAGCCCAGGGGCCGCCCCG GCCCTATTGGCGTGCTGCTGATGAAGAGCAGGGCCAATGAGGAGTATGGGCTGAGGCTGGGATCTCAGAT TTTCGTGAAGGAGATGACCAGAACTGGCCTGGCTACAAAGGACGGGAACCTGCATGAGGGCGATATCATC CTGAAGATTAATGGCACCGTGACCGAGAACATGAGCCTGACCGACGCCCGTAAGCTGATCGAGAAGAGCA GAGGCAAACTGCAGCTCGTGGTGCTGAGAGACTCTCAGCAGACCCTGATCAACATCCCATCGCTGAATGATTCCGATTCCGAAATCGAAGATATCTCCGAAATCGAGTCCAACAGATCGTTCAGCCCTGAAGAGAGAAGA CATCAGTACAGCGACTACGACTACCACTCCAGCTCCGAGAAACTGAAGGAGAGGCCAAGCTCACGCGAGG ATACACCCTCCAGGCTGTCTAGGATGGGGGCCACCCCCACCCCCTTCAAGTCCACTGGCGATATCGCCGG CACCGTGGTGCCCGAGACCAACAAAGAGCCCAGGTACCAGGAAGACCCACCTGCCCCTCAGCCCAAGGCT GCACCTAGGACATTCTTGCGCCCCTCACCTGAGGACGAGGCTATTTACGGCCCTAACACCAAAATGGTGA GGTTTAAGAAGGGGGACTCCGTGGGCCTGAGGCTCGCCGGCGGCAATGACGTGGGAATTTTTGTGGCCGG GATTCAGGAGGGGACATCCGCTGAACAGGAGGGCCTGCAGGAAGGGGATCAGATCCTGAAGGTTAACACA CAGGATTTTCGCGGCCTGGTGCGGGAGGACGCCGTGCTGTATCTGCTGGAGATCCCCAAGGGGGAGATGG TGACAATCCTGGCCCAGAGCCGGGCTGATGTGTACAGAGACATCCTGGCCTGTGGCCGGGGCGACTCCTT CTTCATCCGGAGCCACTTCGAGTGCGAAAAAGAGACCCCTCAGAGCCTGGCCTTCACCAGGGGCGAGGTG TTCAGAGTGGTGGATACCCTGTACGATGGTAAGCTGGGCAACTGGCTCGCCGTGCGGATCGGAAATGAGC TGGAGAAAGGCCTGATCCCTAATAAATCTAGGGCTGAGCAGATGGCCAGCGTGCAGAATGCTCAGAGAGA CAACGCCGGCGACCGTGCCGATTTTTGGAGAATGCGGGGCCAGCGCAGTGGTGTGAAGAAAAACCTGCGA AAGAGTAGAGAGGATCTGACCGCCGTCGTGAGTGTGAGCACTAAGTTTCCCGCCTACGAGCGGGTGCTGC TGCGGGAGGCCGGCTTTAAGCGGCCCGTGGTGCTGTTCGGCCCTATTGCCGATATTGCTATGGAAAAACT GGCTAACGAACTGCCCGACTGGTTCCAGACCGCAAAGACTGAGCCCAAGGACGCCGGATCCGAAAAGAGC ACCGGCGTCGTGCGCCTGAATACTGTGCGGCAGATTATCGAGCAGGATAAGCACGCCCTGCTGGACGTGA CTCCAAAGGCCGTGGACCTGCTGAACTACACCCAGTGGTTCCCCATCGTGATCTTTTTCAATCCTGACAG CAGACAGGGTGTGAAAACAATGCGGCAGAGGCTGAACCCAACATCCAACAAAAGCAGCCGGAAACTGTTC GACCAGGCCAATAAGCTGAAGAAGACATGCGCCCATCTGTTTACAGCCACCATCAACCTCAACAGTGCCA ACGATTCATGGTTCGGCTCCTTGAAGGACACAATCCAGCATCAGCAGGGCGAGGCCGTGTGGGTGAGCGA GGGCAAAATGGAGGGAATGGATGACGACCCAGAGGATAGAATGAGCTACCTGACCGCAATGGGAGCCGAT TACCTGTCCTGCGACAGCCGGCTGATCTCCGATTTCGAGGACACTGATGGGGAGGGGGGCGCCTACACAG ACAACGAGCTGGACGAGCCCGCCGAAGAACCTCTGGTGTCTAGCATCACAAGATCAAGTGAACCAGTGCA GCATGAGGAGTCTATTAGGAAACCCTCTCCTGAGCCTCGGGCCCAGATGCGGCGCGCTGCCAGCTCCGAC CAGCTGAGAGATAACAGCCCTCCCCCTGCCTTTAAACCCGAGCCCCCTAAGGCGAAAACCCAGAACAAAG AGGAATCCTATGATTTCAGTAAGAGCTATGAGTATAAGTCCAATCCCAGCGCCGTGGCCGGCAACGAGAC TCCTGGCGCTAGCACCAAGGGATATCCCCCCCCCGTGGCCGCTAAGCCAACCTTCGGCAGGTCCATCCTG AAGCCTAGCACCCCCATCCCTCCTCAGGAGGGCGAGGAGGTGGGTGAGAGCAGCGAAGAGCAGGATAATG CCCCCAAGAGCGTGCTGGGGAAAGTGAAGATCTTTGAGAAGATGGACCACAAGGCTAGGCTGCAGCGCAT GCAGGAGCTGCAGGAAGCTCAGAATGCTAGAATTGAGATAGCCCAGAAGCACCCTGACATTTATGCAGTG CCCATCAAGACCCACAAGCCTGACCCTGGCACTCCACAGCACACCAGCTCCCGGCCCCCTGAGCCACAGA AGGCCCCTAGCAGACCATACCAGGACACCAGAGGAAGCTACGGATCTGATGCAGAAGAGGAAGAGTACCGGCAGCAGTTGTCCGAGCATTCCAAGCGGGGCTATTATGGCCAGTCTGCCCGGTACAGGGACACAGAGCTG TGA (SEQ ID NO: 3). SEQ ID NO: 3 is a codon-optimized TJP2 sequence, referred to as “TJP2-202”.

[0033] Generation of IVT mRNA from a DNA template (IVT Template) sequence is known in the art. See, for example, U.S. Patent Pub. No. 20240229109, to Rabideau et al, filed March 31, 2022. In embodiments, a promoter sequence (e.g., T7, T3, or SP6) is added upstream of the coding sequence in the template DNA (e.g., SEQ ID NO: 1 or SEQ ID NO: 3) to enable RNA polymerase binding and initiation of transcription. The template DNA sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 3) is transcribed in vitro using a suitable RNA polymerase, such as T7 RNA polymerase, which binds to the promoter sequence incorporated into the DNA template. The IVT can be performed using any RNA polymerase as long as synthesis of the mRNA from the DNA template that encodes the RNA is specifically and sufficiently initiated from a respective cognate RNA polymerase promoter and full-length mRNA is obtained. In embodiments, the RNA polymerase is selected from T7 RNA polymerase, SP6 RNA polymerase and T3 RNA polymerase.

[0034] This process results in the synthesis of an RNA strand complementary to the DNA template (wherein uridine (U) is incorporated in place of thymine (T)). The RNA polymerase transcribes the DNA template strand in the 3' to 5' direction, generating a complementary mRNA strand in the 5' to 3' direction. In embodiments, uridine is replaced with pseudouridine (Ψ) during transcription to further modify the mRNA. In embodiments, all, or substantially all uridine is replaced with pseudouridine (Ψ) during transcription to further modify the mRNA. This substitution can be achieved, for example, either by directly using pseudouridine in the nucleotide pool during transcription or by post-transcriptionally modifying the RNA to replace uridine with pseudouridine.

[0035] Following transcription, the resulting mRNA may undergo additional modifications, such as the addition of a 5' cap structure and a 3' poly-A tail, to further optimize its stability, translation, and cellular uptake. The 5′ cap structure of a natural mRNA is involved in nuclear export. The 5’ terminal cap of the inventive IVT mRNA may be any such structure known in the art, examples of which include but are notlimited to, CapO, Capl, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8- oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5′ methylG cap, or an analog thereof. In a specific embodiment, the IVT mRNA 5’ terminal cap comprises Capl. Multiple distinct 5′-cap structures can be used to generate the 5′-cap of a nucleic acid molecule, such as a polynucleotide that functions as an mRNA molecule. Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5′-caps in their chemical structure, while retaining cap function. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or linked to the polynucleotides. Non-limiting examples of more authentic 5′cap structures are those that, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5′ endonucleases and / or reduced 5′decapping, as compared to synthetic 5′cap structures known in the art (or to a wild-type, natural or physiological 5′cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2′-O-methyltransferase enzyme can create a canonical 5′-5′-triphosphate linkage between the 5′-terminal nucleotide of a polynucleotide and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5′-terminal nucleotide of the mRNA contains a 2′-O-methyl. Such a structure is termed the Cap1 structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5′cap analog structures known in the art. In some embodiments, a 5′ terminal cap can comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2′fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido- guanosine.

[0036] The poly-A tail, comprising multiple adenosine residues, is added to the 3' end of the mRNA, enhancing mRNA stability and translation efficiency. The poly-A tail of the inventive IVT mRNA may be of any length desire. In a specific embodiment, the poly-A tail is about 50-150 nucleotides in length, or about 75-150, 85-150, 90-150, 90-150, 90-140, or 90-130 nucleotides in length. In some instances. In additional embodiments, the poly-A tail is about 100-150 nucleotides in length. In embodiments, the lengthof a polyA tail, when present, is greater than 30 nucleotides in length. In embodiments, the polyA tail is greater than 35 nucleotides in length (e.g., at least or greater than about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, or 3,000 nucleotides). In embodiments, the polyA tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the nucleic acid or feature thereof. The polyA tail can also be designed as a fraction of the nucleic acid to which it belongs. In this context, the polyA tail can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the polyA tail.

[0037] Following synthesis, the mRNA is purified (e.g., by chromatography, such as oligo-dT chromatography) to remove any remaining template DNA, RNA polymerase, or other reaction components.

[0038] The IVT mRNA polynucleotide may be modified to replace a substantial number of uridine bases occurring in the human mRNA encoding the TJP2 gene with pseudouridine bases in the polynucleotide so that the polynucleotide is substantially free of uridine bases. For example, at least 75%, 80%, or 90% of uridine bases in the human mRNA encoding the TJP2 gene are replaced with pseudouridine bases in the polynucleotide. In another embodiment, of uridine bases in the human mRNA encoding the TJP2 gene are replaced with pseudouridine bases in the polynucleotide. For example, in one embodiment, the mRNA is produced from a DNA template including SEQ ID NO: 1, a promoter sequence and a polyA tail. In one embodiment, the mRNA is produced from a DNA template including SEQ ID NO: 3, a promoter sequence and a polyA tail. The mRNA is transcribed in vitro using an RNA polymerase that recognizes the promoter as described above and as known in the art. During transcription, pseudouridine (Ψ) is incorporated into the mRNA in place of uridine (U) through the use of modified nucleotide pools or post- transcriptional modification.

[0039] In embodiments, the IVT mRNA generated sequence is codon-optimized and is that of SEQ ID NO: 4, as follows:

[0040] AUGCCCGUCAGGGGCGAUCGCGGAUUCCCCCCCCGGAGGGAACUGAGCGGCUGG CUGCGCGCCCCCGGGAUGGAGGAGCUGAUCUGGGAGCAGUACACCGUGACCCUGCAGAAGGACAGCAAACGGGGCUUCGGAAUCGCCGUCAGCGGAGGCAGGGACAAUCCUCACUUUGAA AACGGCGAGACCAGCAUCGUGAUCUCCGACGUGCUGCCCGGCGGCCCUGCCGAUGGCCUG CUGCAGGAGAAUGAUAGGGUGGUCAUGGUGAACGGCACCCCCAUGGAAGACGUGCUCCAC AGCUUUGCCGUGCAGCAGCUGCGCAAGAGCGGCAAGGUCGCCGCCAUUGUGGUGAAGAGA CCUAGGAAGGUGCAGGUGGCUGCCCUGCAGGCUUCUCCCCCACUCGACCAGGACGACAGG GCCUUCGAAGUGAUGGAUGAGUUCGACGGAAGGUCUUUCAGGUCUGGAUACAGCGAACG GAGUCGGCUGAAUUCACACGGAGGAAGGUCCCGCAGUUGGGAGGAUUCCCCAGAGAGAG GACGGCCCCAUGAGAGGGCUCGCUCCAGAGAACGGGACCUGAGCCGGGACCGGAGUCGGG GCAGGAGCCUGGAGAGAGGCCUGGAUCAGGAUCACGCCAGAACACGGGAUCGGUCCAGAG GGAGGAGCCUGGAAAGAGGACUGGACCACGAUUUCGGGCCUUCAAGAGACCGCGACAGG GAUCGCAGCCGGGGACGCAGUAUCGACCAGGAUUACGAGCGAGCCUACCACAGGGCCUAC GACCCAGACUAUGAAAGAGCCUACUCCCCCGAGUACCGCAGGGGCGCCCGGCACGAUGCC CGGAGCAGGGGCCCCAGGAGUCGGAGCCGGGAACACCCCCACAGCCGGUCCCCCAGCCCC GAGCCCAGGGGCCGCCCCGGCCCUAUUGGCGUGCUGCUGAUGAAGAGCAGGGCCAAUGAG GAGUAUGGGCUGAGGCUGGGAUCUCAGAUUUUCGUGAAGGAGAUGACCAGAACUGGCCU GGCUACAAAGGACGGGAACCUGCAUGAGGGCGAUAUCAUCCUGAAGAUUAAUGGCACCG UGACCGAGAACAUGAGCCUGACCGACGCCCGUAAGCUGAUCGAGAAGAGCAGAGGCAAAC UGCAGCUCGUGGUGCUGAGAGACUCUCAGCAGACCCUGAUCAACAUCCCAUCGCUGAAUG AUUCCGAUUCCGAAAUCGAAGAUAUCUCCGAAAUCGAGUCCAACAGAUCGUUCAGCCCUG AAGAGAGAAGACAUCAGUACAGCGACUACGACUACCACUCCAGCUCCGAGAAACUGAAGG AGAGGCCAAGCUCACGCGAGGAUACACCCUCCAGGCUGUCUAGGAUGGGGGCCACCCCCA CCCCCUUCAAGUCCACUGGCGAUAUCGCCGGCACCGUGGUGCCCGAGACCAACAAAGAGC CCAGGUACCAGGAAGACCCACCUGCCCCUCAGCCCAAGGCUGCACCUAGGACAUUCUUGC GCCCCUCACCUGAGGACGAGGCUAUUUACGGCCCUAACACCAAAAUGGUGAGGUUUAAGA AGGGGGACUCCGUGGGCCUGAGGCUCGCCGGCGGCAAUGACGUGGGAAUUUUUGUGGCC GGGAUUCAGGAGGGGACAUCCGCUGAACAGGAGGGCCUGCAGGAAGGGGAUCAGAUCCUGAAGGUUAACACACAGGAUUUUCGCGGCCUGGUGCGGGAGGACGCCGUGCUGUAUCUGC UGGAGAUCCCCAAGGGGGAGAUGGUGACAAUCCUGGCCCAGAGCCGGGCUGAUGUGUAC AGAGACAUCCUGGCCUGUGGCCGGGGCGACUCCUUCUUCAUCCGGAGCCACUUCGAGUGC GAAAAAGAGACCCCUCAGAGCCUGGCCUUCACCAGGGGCGAGGUGUUCAGAGUGGUGGA UACCCUGUACGAUGGUAAGCUGGGCAACUGGCUCGCCGUGCGGAUCGGAAAUGAGCUGG AGAAAGGCCUGAUCCCUAAUAAAUCUAGGGCUGAGCAGAUGGCCAGCGUGCAGAAUGCU CAGAGAGACAACGCCGGCGACCGUGCCGAUUUUUGGAGAAUGCGGGGCCAGCGCAGUGGU GUGAAGAAAAACCUGCGAAAGAGUAGAGAGGAUCUGACCGCCGUCGUGAGUGUGAGCAC UAAGUUUCCCGCCUACGAGCGGGUGCUGCUGCGGGAGGCCGGCUUUAAGCGGCCCGUGGU GCUGUUCGGCCCUAUUGCCGAUAUUGCUAUGGAAAAACUGGCUAACGAACUGCCCGACUG GUUCCAGACCGCAAAGACUGAGCCCAAGGACGCCGGAUCCGAAAAGAGCACCGGCGUCGU GCGCCUGAAUACUGUGCGGCAGAUUAUCGAGCAGGAUAAGCACGCCCUGCUGGACGUGAC UCCAAAGGCCGUGGACCUGCUGAACUACACCCAGUGGUUCCCCAUCGUGAUCUUUUUCAA UCCUGACAGCAGACAGGGUGUGAAAACAAUGCGGCAGAGGCUGAACCCAACAUCCAACAA AAGCAGCCGGAAACUGUUCGACCAGGCCAAUAAGCUGAAGAAGACAUGCGCCCAUCUGUU UACAGCCACCAUCAACCUCAACAGUGCCAACGAUUCAUGGUUCGGCUCCUUGAAGGACAC AAUCCAGCAUCAGCAGGGCGAGGCCGUGUGGGUGAGCGAGGGCAAAAUGGAGGGAAUGG AUGACGACCCAGAGGAUAGAAUGAGCUACCUGACCGCAAUGGGAGCCGAUUACCUGUCCU GCGACAGCCGGCUGAUCUCCGAUUUCGAGGACACUGAUGGGGAGGGGGGCGCCUACACAG ACAACGAGCUGGACGAGCCCGCCGAAGAACCUCUGGUGUCUAGCAUCACAAGAUCAAGUG AACCAGUGCAGCAUGAGGAGUCUAUUAGGAAACCCUCUCCUGAGCCUCGGGCCCAGAUGC GGCGCGCUGCCAGCUCCGACCAGCUGAGAGAUAACAGCCCUCCCCCUGCCUUUAAACCCG AGCCCCCUAAGGCGAAAACCCAGAACAAAGAGGAAUCCUAUGAUUUCAGUAAGAGCUAU GAGUAUAAGUCCAAUCCCAGCGCCGUGGCCGGCAACGAGACUCCUGGCGCUAGCACCAAG GGAUAUCCCCCCCCCGUGGCCGCUAAGCCAACCUUCGGCAGGUCCAUCCUGAAGCCUAGC ACCCCCAUCCCUCCUCAGGAGGGCGAGGAGGUGGGUGAGAGCAGCGAAGAGCAGGAUAAUGCCCCCAAGAGCGUGCUGGGGAAAGUGAAGAUCUUUGAGAAGAUGGACCACAAGGCUAG GCUGCAGCGCAUGCAGGAGCUGCAGGAAGCUCAGAAUGCUAGAAUUGAGAUAGCCCAGA AGCACCCUGACAUUUAUGCAGUGCCCAUCAAGACCCACAAGCCUGACCCUGGCACUCCAC AGCACACCAGCUCCCGGCCCCCUGAGCCACAGAAGGCCCCUAGCAGACCAUACCAGGACA CCAGAGGAAGCUACGGAUCUGAUGCAGAAGAGGAAGAGUACCGGCAGCAGUUGUCCGAG CAUUCCAAGCGGGGCUAUUAUGGCCAGUCUGCCCGGUACAGGGACACAGAGCUGUGA (SEQ ID NO: 4).

[0041] In embodiments, the resulting IVT mRNA composition used for the disclosed compositions and methods comprise a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO: 4.

[0042] In embodiments, the IVT mRNA generated sequence is not codon-optimized and is that of SEQ ID NO: 5, as follows:

[0043] AUGCCGGUGCGAGGAGACCGCGGGUUUCCACCCCGGCGGGAGCUGUCAGGUUG GCUCCGCGCCCCAGGCAUGGAAGAGCUGAUAUGGGAACAGUACACUGUGACCCUACAAAA GGAUUCCAAAAGAGGAUUUGGAAUUGCAGUGUCCGGAGGCAGAGACAACCCCCACUUUG AAAAUGGAGAAACGUCAAUUGUCAUUUCUGAUGUGCUCCCGGGUGGGCCUGCUGAUGGG CUGCUCCAAGAAAAUGACAGAGUGGUCAUGGUCAAUGGCACCCCCAUGGAGGAUGUGCU UCAUUCGUUUGCAGUUCAGCAGCUCAGAAAAAGUGGGAAGGUCGCUGCUAUUGUGGUCA AGAGGCCCCGGAAGGUCCAGGUGGCCGCACUUCAGGCCAGCCCUCCCCUGGAUCAGGAUG ACCGGGCUUUUGAGGUGAUGGACGAGUUUGAUGGCAGAAGUUUCCGGAGUGGCUACAGC GAGAGGAGCCGGCUGAACAGCCAUGGGGGGCGCAGCCGCAGCUGGGAGGACAGCCCGGAA AGGGGGCGUCCCCAUGAGCGGGCCCGGAGCCGGGAGCGGGACCUCAGCCGGGACCGGAGC CGUGGCCGGAGCCUGGAGCGGGGCCUGGACCAAGACCAUGCGCGCACCCGAGACCGCAGC CGUGGCCGGAGCCUGGAGCGGGGCCUGGACCACGACUUUGGGCCAUCCCGGGACCGGGAC CGUGACCGCAGCCGCGGCCGGAGCAUUGACCAGGACUACGAGCGAGCCUAUCACCGGGCCUACGACCCAGACUACGAGCGGGCCUACAGCCCGGAGUACAGGCGCGGGGCCCGCCACGAU GCCCGCUCUCGGGGACCCCGAAGCCGCAGCCGCGAGCACCCGCACUCACGGAGCCCCAGCC CCGAGCCUAGGGGGCGGCCGGGGCCCAUCGGGGUCCUCCUGAUGAAAAGCAGAGCGAACG AAGAGUAUGGUCUCCGGCUUGGGAGUCAGAUCUUCGUAAAGGAAAUGACCCGAACGGGU CUGGCAACUAAAGAUGGCAACCUUCACGAAGGAGACAUAAUUCUCAAGAUCAAUGGGAC UGUAACUGAGAACAUGUCUUUAACGGAUGCUCGAAAAUUGAUAGAAAAGUCAAGAGGAA AACUACAGCUAGUGGUGUUGAGAGACAGCCAGCAGACCCUCAUCAACAUCCCGUCAUUAA AUGACAGUGACUCAGAAAUAGAAGAUAUUUCAGAAAUAGAGUCAAACCGAUCAUUUUCU CCAGAGGAGAGACGUCAUCAGUAUUCUGAUUAUGAUUAUCAUUCCUCAAGUGAGAAGCU GAAGGAAAGGCCAAGUUCCAGAGAGGACACGCCGAGCAGAUUGUCCAGGAUGGGUGCGA CACCCACUCCCUUUAAGUCCACAGGGGAUAUUGCAGGCACAGUUGUCCCAGAGACCAACA AGGAACCCAGAUACCAAGAGGACCCCCCAGCUCCUCAACCAAAAGCAGCCCCGAGAACUU UUCUUCGUCCUAGUCCUGAAGAUGAAGCAAUAUAUGGCCCUAAUACCAAAAUGGUAAGG UUCAAGAAGGGAGACAGCGUGGGCCUCCGGUUGGCUGGUGGCAAUGAUGUCGGGAUAUU UGUUGCUGGCAUUCAAGAAGGGACCUCGGCGGAGCAGGAGGGCCUUCAAGAAGGAGACC AGAUUCUGAAGGUGAACACACAGGAUUUCAGAGGAUUAGUGCGGGAGGAUGCCGUUCUC UACCUGUUAGAAAUCCCUAAAGGUGAAAUGGUGACCAUUUUAGCUCAGAGCCGAGCCGA UGUGUAUAGAGACAUCCUGGCUUGUGGCAGAGGGGAUUCGUUUUUUAUAAGAAGCCACU UUGAAUGUGAGAAGGAAACUCCACAGAGCCUGGCCUUCACCAGAGGGGAGGUCUUCCGA GUGGUAGACACACUGUAUGACGGCAAGCUGGGCAACUGGCUGGCUGUGAGGAUUGGGAA CGAGUUGGAGAAAGGCUUAAUCCCCAACAAGAGCAGAGCUGAACAAAUGGCCAGUGUUC AAAAUGCCCAGAGAGACAACGCUGGGGACCGGGCAGAUUUCUGGAGAAUGCGUGGCCAG AGGUCUGGGGUGAAGAAGAACCUGAGGAAAAGUCGGGAAGACCUCACAGCUGUUGUGUC UGUCAGCACCAAGUUCCCAGCUUAUGAGAGGGUUUUGCUGCGAGAAGCUGGUUUCAAGA GACCUGUGGUCUUAUUCGGCCCCAUAGCUGAUAUAGCAAUGGAAAAAUUGGCUAAUGAG UUACCUGACUGGUUUCAAACUGCUAAAACGGAACCAAAAGAUGCAGGAUCUGAGAAAUCCACUGGAGUGGUCCGGUUAAAUACCGUGAGGCAAAUUAUUGAACAGGAUAAGCAUGCAC UACUGGAUGUGACUCCGAAAGCUGUGGACCUGUUGAAUUACACCCAGUGGUUCCCAAUU GUGAUUUUUUUCAACCCAGACUCCAGACAAGGUGUCAAAACCAUGAGACAAAGGUUAAA UCCAACGUCCAACAAAAGUUCUCGAAAGUUAUUUGAUCAAGCCAACAAGCUUAAAAAAA CGUGUGCACACCUUUUUACAGCUACAAUCAACCUAAAUUCAGCCAAUGAUAGCUGGUUUG GCAGCUUAAAGGACACUAUUCAGCAUCAGCAAGGAGAAGCGGUUUGGGUCUCUGAAGGA AAGAUGGAAGGGAUGGAUGAUGACCCCGAAGACCGCAUGUCCUACUUAACCGCCAUGGGC GCGGACUAUCUGAGUUGCGACAGCCGCCUCAUCAGUGACUUUGAAGACACGGACGGUGAA GGAGGCGCCUACACUGACAAUGAGCUGGAUGAGCCAGCCGAGGAGCCGCUGGUGUCGUCC AUCACCCGCUCCUCGGAGCCGGUGCAGCACGAGGAGAGCAUAAGGAAACCCAGCCCAGAG CCACGAGCUCAGAUGAGGAGGGCUGCUAGCAGCGAUCAACUUAGGGACAAUAGCCCGCCC CCAGCAUUCAAGCCAGAGCCGCCCAAGGCCAAAACCCAGAACAAAGAAGAAUCCUAUGAC UUCUCCAAAUCCUAUGAAUAUAAGUCAAACCCCUCUGCCGUUGCUGGUAAUGAAACUCCU GGGGCAUCUACCAAAGGUUAUCCUCCUCCUGUUGCAGCAAAACCUACCUUUGGGCGGUCU AUACUGAAGCCCUCCACUCCCAUCCCUCCUCAAGAGGGUGAGGAGGUGGGAGAGAGCAGU GAGGAGCAAGAUAAUGCUCCCAAAUCAGUCCUGGGCAAAGUCAAAAUAUUUGAGAAGAU GGAUCACAAGGCCAGGUUACAGAGAAUGCAGGAGCUCCAGGAAGCACAGAAUGCAAGGA UCGAAAUUGCCCAGAAGCAUCCUGAUAUCUAUGCAGUUCCAAUCAAAACGCACAAGCCAG ACCCUGGCACGCCCCAGCACACGAGUUCCAGACCCCCUGAGCCACAGAAAGCUCCUUCCA GACCUUAUCAGGAUACCAGAGGAAGUUAUGGCAGUGAUGCCGAGGAGGAGGAGUACCGC CAGCAGCUGUCAGAACACUCCAAGCGCGGUUACUAUGGCCAGUCUGCCCGAUACCGGGAC ACAGAAUUAUAG (SEQ ID NO: 5).

[0044] In embodiments, the resulting IVT mRNA composition used for the disclosed compositions and methods comprise a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO: 5.

[0045] In embodiments, a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 is used as a template for mRNA transcription. In embodiments, a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO: 3 is used as a template for mRNA transcription.

[0046] The disclosed IVT mRNA may be formulated in any desirable carrier for administration in vivo, for example administration by injection. In a specific embodiment, the IVT mRNA is packed into particles for delivery in vivo, specifically for deliver to the liver of a patient. In a specific embodiment, a composition comprising the IVT mRNA of the invention is formulated in a lipid nanoparticle (LNP) carrier. Accordingly, the present disclosure also relates to nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent, and (ii) an IVT mRNA. The lipid composition can encapsulate the nucleic acid. Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less. Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes. In some embodiments, nanoparticle compositions are vesicles including one or more lipid bilayers. In embodiments, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers can be functionalized and / or crosslinked to one another. Lipid bilayers can include one or more ligands, proteins, or channels. In some embodiments, a lipid nanoparticle comprises an ionizable lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNP comprises an ionizable lipid, a PEG-modified lipid, a phospholipid and a structural lipid. Various lipid compositions which are known in the art may be employed for delivering the inventive IVT mRNA to the liver. In a specific embodiment, the lipid forming the LNP comprises an ionizable amino lipid as disclosed, for example, in US 2022 / 0054653 A1.

[0047] In additional embodiments, disclosed are methods of synthesizing an in vitro-transcribed RNA molecule comprising combining an isolated RNA polymerase, a template nucleic acid sequence comprising SEQ ID NO: 1, unmodified nucleotides, and pseudouridine modified nucleotides under conditions such that an in vitro-transcribed RNA molecule encoding the TJP2 gene is generated that comprises at least one pseudouridine residue.

[0048] In additional embodiments, disclosed are methods of synthesizing an in vitro-transcribed RNA molecule comprising combining an isolated RNA polymerase, a template nucleic acid sequence comprising SEQ ID NO: 3, unmodified nucleotides, and pseudouridine modified nucleotides under conditions such that an in vitro-transcribed RNA molecule encoding the TJP2 gene is generated that comprises at least one pseudouridine residue.

[0049] The inventive IVT mRNA may be used in various methods and compositions. In one embodiment, a method of treating or preventing Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4) in a patient in need thereof comprises administering to the patient a therapeutically effective amount of the IVT mRNA as described herein, for example in an amount sufficient to increase expression of the TJP2 in the individual being administered the IVT mRNA. In embodiments, a method for decreasing the severity of Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4) in a patient in need thereof comprises administering to the patient a therapeutically effective amount of the IVT mRNA as described herein, for example in an amount sufficient to increase expression of the TJP2 in the individual being administered the IVT mRNA..

[0050] In another embodiment, a method of treating or preventing Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4) in a patient in need thereof comprises administering to the patient a therapeutically effective amount of a composition comprising the inventive IVT mRNA, for example in an amount sufficient to increase expression of the TJP2 in the individual being administered the IVT mRNA. Thus, the invention also encompasses compositions comprising a therapeutically effective amount of the IVT mRNA use in a method of treating or preventing Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4), as well as use of a composition comprising a therapeutically effective amount of the IVTmRNA for the manufacture of a medicament for use in a method of treating or preventing Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4). The methods, in general, include contacting the IVT mRNA, or a composition comprising the IVT mRNA, with a target cell, for a sufficient time and in an amount sufficient to achieve expression of TJP2 in the cell.

[0051] In embodiments, a gene therapy vector comprising a DNA sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 is disclosed. In embodiments, a gene therapy vector comprising a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO: 3 is disclosed.

[0052] In embodiments, a gene therapy vector comprising an RNA sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO: 4 is disclosed. In embodiments, a gene therapy vector comprising a sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to SEQ ID NO: 5 is disclosed. In this aspect, the RNA gene therapy vector has all, or substantially all uridines replaced with pseudouridine.

[0053] In embodiments, the gene therapy vector further comprises a promoter, for example a constitutive promoter, a tissue-specific promoter, or an inducible promoter. In embodiments, the gene therapy vector further comprises a polyA signal. In embodiments, the gene therapy vector further comprises a vector backbone, for example a plasmid, a viral vector (such as adenovirus or lentivirus), or a lipid nanoparticle (LNP). In further aspects, the gene therapy vector is a replication-defective vector. In further aspects, the vector is a viral vector and further comprises a packaging signal.

[0054] In embodiments, disclosed are methods for inducing a mammalian cell to produce TJP2, comprising contacting the mammalian cell with an in vitro-synthesized RNA molecule encoding TJP2 asdescribed herein, the in vitro-synthesized RNA molecule comprising a pseudouridine, thereby inducing a mammalian cell to produce TJP2.

[0055] In embodiments, a method comprising mixing the RNA, oligoribonucleotide, or polyribonucleotide molecule with a transfection reagent prior to the step of contacting with a target cell is disclosed. In embodiments, a method comprising administering the RNA, oligoribonucleotide, or polyribonucleotide molecule together with the transfection reagent is disclosed. In embodiments, the transfection reagent is a cationic lipid reagent.

[0056] The RNA may be administered via one or more routes to an individual in need thereof. For example, in one aspect, the route of administration is intravenous (IV) injection. In this aspect, the IV administration allows for direct entry into the bloodstream and efficient delivery to the liver via hepatic circulation. In one aspect, the route of administration is intramuscular (IM) injection. In one aspect, the route of administration is intraperitoneal (IP) injection. In one aspect, the route of administration is a liver- directed delivery method, such as, for example portal vein infusion and / or transhepatic arterial injection (directly into the liver via interventional radiology). Such methods are within the skill of one of ordinary skill in the art.

[0057] In embodiments, the transfection reagent is a lipid-based transfection reagent. In embodiments, the transfection reagent is a protein-based transfection reagent. In embodiments, the transfection reagent is a polyethyleneimine based transfection reagent. In embodiments, the transfection reagent is calcium phosphate. In embodiments, the transfection reagent is Lipofectin® or Lipofectamine®. In embodiments, the transfection reagent is any other transfection reagent known in the art.

[0058] In embodiments, the transfection reagent forms a liposome. Liposomes can increase intracellular stability, increase uptake efficiency and improve biological activity. In embodiments, liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane an can have an internal aqueous space for entrapping water soluble compounds and range in size from 0.05 to several microns in diameter.

[0059] In embodiments, the target cell is a hepatic cell, and the disorder to be treated via delivery of the oligonucleotide and transfection agent is PFIC Type 4.

[0060] In another embodiment, the dosage is a daily dose. In another embodiment, the dosage is a weekly dose. In another embodiment, the dosage is a monthly dose. In another embodiment, the dosage is an annual dose. In another embodiment, the dose is one is a series of a defined number of doses. In another embodiment, the dose is a one-time dose. As described below, in another embodiment, an advantage of RNA, oligoribonucleotide, or polyribonucleotide molecules of the present invention is their greater potency, enabling the use of smaller doses.

[0061] Formulations of the IVT mRNA compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) (e.g., mRNA as described herein) into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi-dose unit. In embodiments, the compositions comprise a pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable carriers or diluents are well known to those skilled in the art, and may be, for example, a solid carrier or diluent for solid formulations, a liquid carrier or diluent for liquid formulations, or mixtures thereof. In embodiments, solid carriers / diluents include, but are not limited to, a gum, a starch (e.g. com starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g. microcrystalline cellulose), an acrylate (e.g. polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof. In embodiments, the carrier or diluent for liquid formulations may be aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.

[0062] In embodiments, parenteral vehicles (for subcutaneous, intravenous, intra-arterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.

[0063] In embodiments, the compositions further comprise binders (e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris-HCI., acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents(e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g. aspartame, citric acid), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g. ethyl cellulose, acrylates, polymethacrylates) and / or adjuvants. Each of the above excipients represents a separate embodiment of the present invention.

[0064] Definitions

[0065] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The methods may comprise, consist of, or consist essentially of the elements of the compositions and / or methods as described herein, as well as any additional or optional element described herein or otherwise useful in in vitro transcribed (IVT) messenger RNA (mRNA) compositions and methods of making and using same.

[0066] As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0067] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0068] As used herein “substantially all,” in reference to single-stranded complete mRNAs comprising a pseudouridine residue, means that of all the single-stranded complete mRNAs present in a sample, at least 95% have a pseudouridine residue.

[0069] As used herein “essentially all,” in reference to single-stranded complete mRNAs comprising a pseudouridine residue, means that of all the single-stranded complete mRNAs present in a sample, at least 99% have a pseudouridine residue.

[0070] The terms “comprising”, “containing”, “having”, “include”, and “including” are to be construed as “including, but not limited to” unless otherwise noted. The terms “a,” “an,” and “the” and similar referents in the context of describing the invention and, specifically, in the context of the appended claims, are to be construed to cover both the singular and the plural unless otherwise noted. The use of any and all examples or exemplary language (“for example”, “e.g.”, “such as”) is intended merely to illustrate aspects or embodiments of the invention, and is not to be construed as limiting the scope thereof, unless otherwise claimed.

[0071] As used herein, the term “effective amount” means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0072] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and / or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some aspects, the terms refer to humans. In further aspects, the terms may refer to children.

[0073] An “in vitro transcription template” (IVT template), or “input DNA” as used herein, refers to deoxyribonucleic acid (DNA) suitable for use in an IVT reaction for the production of messenger RNA (mRNA). In some embodiments, an IVT template encodes a 5′ untranslated region, contains an open reading frame, and encodes a 3′ untranslated region and a polyA tail.

[0074] “Effective amount” of the RNA, oligoribonucleotide, or polyribonucleotide molecule refers, in another embodiment, to an amount sufficient to exert a therapeutic effect. In another embodiment, the term refers to an amount sufficient to elicit expression of a detectable amount of the recombinant protein.

[0075] The terms “isolated” or “purified” when used in relation to a polynucleotide or nucleic acid, as in “isolated RNA” or “purified RNA” refers to a nucleic acid that is identified and separated from at least one contaminant with which it is ordinarily associated in its source. Thus, an isolated or purified nucleic acid (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome together with other genes as well as structural and functional proteins, and a specific RNA (e.g., a specific mRNA encoding a specific protein), is found in the cell as a mixture with numerous other RNAs and other cellular components. The isolated or purified polynucleotide or nucleic acid may be present in single-stranded or double-stranded form.

[0076] As used herein “pharmaceutically acceptable carriers or diluents” are well known to those skilled in the art. The carrier or diluent may be, in various embodiments, a solid carrier or diluent for solid formulations, a liquid carrier or diluent for liquid formulations, or mixtures thereof.

[0077] “Pseudouridine” refers, in another embodiment, to m1acp3ψ (1-methyl-3-(3-amino-3- carboxypropyl) pseudouridine. In another embodiment, the term refers to m1ψ (1-methylpseudouridine). In another embodiment, the term refers to ψm (2′-O-methylpseudouridine. In another embodiment, the term refers to m5D (5-methyldihydrouridine). In another embodiment, the term refers to m3ψ (3- methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not further modified. In another embodiment, the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the present invention.

[0078] Additional aspects and advantages of the invention are apparent from the following Examples.EXAMPLES

[0079] To investigate TJP2’s role in BC formation the inventors developed an innovative in vitro model. By combining a novel induced pluripotent stem cell (iPSC) differentiation similar to that described in WO 2020 / 097555 A1, incorporated herein by reference, and the culturing method described herein, the induced hepatocytes (iHeps) self-organize, undergoing dynamic remodeling, to form bi-layers that generate canalicular tubes between opposing iHeps. The differentiation is very similar to that described in WO 2020 / 097555 A1 with the addition an overlay of the cells with Matrigel to induce the cells to self-organize to form the bile canalicular structure. The canalicular tubes transport fluorescent bile acids into the lumen of these canaliculi (FIG. 1). A set of isogenic iPSC pairs was then developed using CRISPR / Cas9 gene- editing of healthy (WT) human induced pluripotent stem cells (iPSCs) to induce TJP1 knockout or TJP2 truncation and precisely model hepatocytes of a PFIC-TJP2 patient.

[0080] In contrast to control iHeps, TJP2 deficient iHeps (TJP2-iHeps) failed to make canalicular tubes, instead forming pseudo-acini, which mimics the pseudo-acini BC seen in PFIC-TJP2 patient livers (FIG. 1, A).

[0081] After establishing the novel in vitro BC model, the inventive IVT mRNA were tested as a potential therapeutic for TJP2 deficiency in hepatocytes. Treating wild type (WT) and TJP2-deficient iHeps, respectively, with the inventive IVT human TJP2 mRNA increased TJP2 protein levels within the both WT- and TJP2-iHeps cells, although the increase in protein expression was not as robust in the TJP2-iHeps (FIG. 2, A). TJP2 protein expression was determined to be dose dependent and a single dose of TJP2 mRNA increased TJP2 protein levels for approximately 72 hours in WT-iHeps (FIGS. 2, B and C). After determining the optimal dose and treatment regime for the iHeps, treating iHeps with 2 doses of 2 ug of TJP2 mRNA every other day over 5 days was found to significantly increase length of BC structures when compared to untreated TJP2-iHeps. The treatment did not affect BC length of WT-iHeps (FIGS. 3, A and 3). Additionally, significantly fewer pseudo-acinar BC structures were seen in TJP2-iHeps after TJP2mRNA treatment, indicating that the longer BC structures observed are lengthened pre-existing pseudo- acinar structures, rather than forming de novo BC structures (FIG.3, C).

[0082] These findings indicate that TJP2 deficiency disrupts BC network formation and likely causes a build-up of bile in the disrupted BC, resulting in cholestatic injury and ultimate liver failure observed in PFIC-TJP2 patients. Treating TJP2-deficient iHeps with the inventive IVT TJP2 mRNA rescues disrupted BC structure, thereby offering a novel method for therapy this ultra-rare liver disease.

[0083] In conclusion, TJP2-iHeps with truncated TJP2, found in PFIC-TJP2 patients, mimic patient bile canalicular structure in vitro. Treating TJP2-iHeps with the inventive IVT mRNA increases TJP2 protein levels and significantly elongates existing bile canalicular structures in vitro. TJP2 deficiency disrupts BC network formation in vitro, which causes a buildup of bile in the disrupted BC in vivo resulting in cholestatic injury and ultimate liver failure observed in PFIC-TJP2 patients. Finally, TJP2 mRNA treatment of TJP2 deficient hepatocytes is a therapy for treatment or prevention of TJP2-PFIC (PFIC type IV) to induce elongation of bile canaliculi after formation has completed.

[0084] The following description provides embodiments consistent with the claims and should be interpreted as illustrative rather than limiting. The claimed invention encompasses these and other variations.

[0085] Embodiment 1

[0086] An in vitro transcribed messenger RNA (IVT mRNA) comprising a polynucleotide that encodes a human TJP2 gene, a 5’ terminal cap, and a poly-adenylation (poly-A) tail at the 3’ terminal, wherein the polynucleotide is substantially free of uridine bases and comprises pseudouridine bases.

[0087] Embodiment 2

[0088] The IVT mRNA of Embodiment 1, the IVT mRNA, the polynucleotide comprising a sequence having at least 90% sequence homology, or at least 91% sequence homology, or at least 91% sequence homology, or at least 92% sequence homology, or at least 93% sequence homology, or at least 94% sequence homology, or at least 95% sequence homology, or at least 96% sequence homology, or at least97% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology to SEQ ID NO: 4.

[0089] Embodiment 3

[0090] The IVT mRNA of Embodiment 1, the IVT mRNA, the polynucleotide consisting of SEQ ID NO: 4.

[0091] Embodiment 4

[0092] The IVT mRNA of Embodiment 1, the IVT mRNA, the polynucleotide comprising a sequence having at least 90% sequence homology, or at least 91% sequence homology, or at least 91% sequence homology, or at least 92% sequence homology, or at least 93% sequence homology, or at least 94% sequence homology, or at least 95% sequence homology, or at least 96% sequence homology, or at least 97% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology to SEQ ID NO: 5.

[0093] Embodiment 5

[0094] The IVT mRNA of Embodiment 1, the IVT mRNA, the polynucleotide consisting of SEQ ID NO: 5.

[0095] Embodiment 6

[0096] The IVT mRNA of any preceding embodiment, wherein at least 75%, 80%, or 90%, , or 95%, or 100%, of uridine bases in the mRNA are replaced with pseudouridine bases in the polynucleotide.

[0097] Embodiment 7

[0098] The IVT mRNA of any preceding embodiment, wherein all uridine bases in the polynucleotide are replaced with pseudouridine bases.

[0099] Embodiment 8 [000100] The IVT mRNA of any preceding claim, wherein the 5’ terminal cap is selected from CapO, Capl, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2- amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5′ methylG cap, or an analog thereof. [000101] Embodiment 9[000102] The IVT mRNA of embodiment 1, wherein the 5’ terminal cap comprises Capl. [000103] Embodiment 10 [000104] The IVT mRNA of any preceding claim, wherein the poly-A tail is from about 50 to about 150 nucleotides in length. [000105] Embodiment 11 [000106] The IVT mRNA of any preceding claim, wherein the poly-A tail is from about 100 to about 150 nucleotides in length. [000107] Embodiment 12 [000108] A composition comprising the IVT mRNA of any preceding embodiment formulated in a lipid nanoparticle (LNP) carrier. [000109] Embodiment 13 [000110] The composition of embodiment 12, the LNP comprising an ionizable amino lipid. [000111] Embodiment 14 [000112] A method of treating or preventing Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4) in an individual in need thereof comprising administering to the individual a therapeutically effective amount of the IVT mRNA of any one of embodiments 1 to 13. [000113] Embodiment 15 [000114] The method of embodiment 14 wherein the individual is a pediatric individual. [000115] Embodiment 16 [000116] The method of embodiment 14 wherein the individual is a neonate. [000117] Embodiment 17 [000118] The method of any one of embodiments 14 through 16, further comprising identifying a defect in the bile canaliculus (BC) in the individual. [000119] Embodiment 18 [000120] The method of any one of embodiments 14 through 17, further comprising identifying a decrease in TJP2 protein in the individual.[000121] Embodiment 19 [000122] The method of any one of embodiments 14 through 18, further comprising identifying a mutation in TJP2 in the individual.20 [000123] Embodiment 21 [000124] The method of embodiment 19 wherein the mutation results in a decrease in functional TJP2 protein. [000125] Embodiment 22 [000126] The method of any one of embodiments 14 through 20, further comprising administering the IVT mRNA to a cell of the individual. [000127] Embodiment 23 [000128] The method of any one of embodiments 14 through 21, further comprising administering the IVT mRNA to a hepatic cell of the individual. [000129] Embodiment 24 [000130] A method of treating or preventing Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4) in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the composition of any one of embodiments 1 to 13. [000131] Embodiment 25 [000132] A composition comprising a therapeutically effective amount of the IVT mRNA of any one of embodiments 1 to 13 for use in a method of treating or preventing Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4). [000133] Embodiment 26 [000134] Use of a composition comprising a therapeutically effective amount of the IVT mRNA of any one of embodiments 1 to 13 for manufacture of a medicament for use in a method of treating or preventing Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4). [000135] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be madewithout departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention. [000136] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. All accessioned information (e.g., as identified by PUBMED, PUBCHEM, NCBI, UNIPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

Claims

CLAIMS What is claimed is:

1. An in vitro transcribed messenger RNA (IVT mRNA) comprising a polynucleotide that encodes a human TJP2 gene, a 5’ terminal cap, and a poly-adenylation (poly-A) tail at the 3’ terminal, wherein the polynucleotide is substantially free of uridine bases and comprises pseudouridine bases.

2. The IVT mRNA of claim 1, the IVT mRNA, the polynucleotide comprising a sequence having at least 90% sequence homology, or at least 91% sequence homology, or at least 91% sequence homology, or at least 92% sequence homology, or at least 93% sequence homology, or at least 94% sequence homology, or at least 95% sequence homology, or at least 96% sequence homology, or at least 97% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology to SEQ ID NO:

4.

3. The IVT mRNA of claim 1, the IVT mRNA, the polynucleotide consisting of SEQ ID NO:

4.

4. The IVT mRNA of claim 1, the IVT mRNA, the polynucleotide comprising a sequence having at least 90% sequence homology, or at least 91% sequence homology, or at least 91% sequence homology, or at least 92% sequence homology, or at least 93% sequence homology, or at least 94% sequence homology, or at least 95% sequence homology, or at least 96% sequence homology, or at least 97% sequence homology, or at least 98% sequence homology, or at least 99% sequence homology to SEQ ID NO:

5.

5. The IVT mRNA of claim 1, the IVT mRNA, the polynucleotide consisting of SEQ ID NO:

5.

6. The IVT mRNA of any preceding claim, wherein at least 75%, 80%, or 90%, , or 95%, or 100%, of uridine bases in the mRNA are replaced with pseudouridine bases in the polynucleotide.

7. The IVT mRNA of any preceding claim, wherein all uridine bases in the polynucleotide are replaced with pseudouridine bases.

8. The IVT mRNA of any preceding claim, wherein the 5’ terminal cap is selected from CapO, Capl, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5′ methylG cap, or an analog thereof.

9. The IVT mRNA of claim 1, wherein the 5’ terminal cap comprises Capl.

10. The IVT mRNA of any preceding claim, wherein the poly-A tail is from about 50 to about 150 nucleotides in length.

11. The IVT mRNA of any preceding claim, wherein the poly-A tail is from about 100 to about 150 nucleotides in length.

12. A composition comprising the IVT mRNA of any preceding claim formulated in a lipid nanoparticle (LNP) carrier.

13. The composition of claim 12, the LNP comprising an ionizable amino lipid.

14. A method of treating or preventing Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4) in an individual in need thereof comprising administering to the individual a therapeutically effective amount of the IVT mRNA of any one of claims 1 to 13.

15. The method of claim 14 wherein the individual is a pediatric individual.

16. The method of claim 14 wherein the individual is a neonate.

17. The method of any one of claims 14 through 16, further comprising identifying a defect in the bile canaliculus (BC) in the individual.

18. The method of any one of claims 14 through 17, further comprising identifying a decrease in TJP2 protein in the individual.

19. The method of any one of claims 14 through 18, further comprising identifying a mutation in TJP2 in the individual.

20. The method of claim 19 wherein the mutation results in a decrease in functional TJP2 protein.

21. The method of any one of claims 14 through 20, further comprising administering the IVT mRNA to a cell of the individual.

22. The method of any one of claims 14 through 21, further comprising administering the IVT mRNA to a hepatic cell of the individual.

23. A method of treating or preventing Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4) in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the composition of any one of claims 1 to 13.

24. A composition comprising a therapeutically effective amount of the IVT mRNA of any one of claims 1 to 13 for use in a method of treating or preventing Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4).

25. Use of a composition comprising a therapeutically effective amount of the IVT mRNA of any one of claims 1 to 13 for manufacture of a medicament for use in a method of treating or preventing Progressive Familial Intrahepatic Cholestasis Type IV (PFIC type 4).

Images